水稻长芒基因AN-1的克隆

An-1Encodes a Basic Helix-Loop-Helix Protein That Regulates Awn Development,Grain Size,and Grain Number

in Rice C W OPEN

Jianghong Luo,a,1Hui Liu,a,1Taoying Zhou,a,1Benguo Gu,a,1Xuehui Huang,a Yingying Shangguan,a Jingjie Zhu,a Yan Li,a Yan Zhao,a Yongchun Wang,a Qiang Zhao,a Ahong Wang,a Ziqun Wang,a Tao Sang,b Zixuan Wang,a,c

and Bin Han a,2

a National Center for Gene Research,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai200233,China

b State Key Laboratory of Systemati

c an

d Evolutionary Botany,Key Laboratory of Plant Resources,Institut

e o

f Botany,Chinese Academy of Sciences,Beijing100093,China

c National Institute of Genetics,Mishima,Shizuoka411-8540,Japan

ORCID ID:0000-0001-8695-0274(B.H.).

Long awns are important for seed dispersal in wild rice(Oryza ru?pogon),but are absent in cultivated rice(Oryza sativa).The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown.We report here the molecular cloning of a major quantitative trait locus,An-1,which regulates long awn formation in O.ru?pogon.An-1encodes a basic helix-loop-helix protein,which regulates cell division.The nearly-isogenic line(NIL-An-1)carrying a wild allele An-1in the genetic background of the awnless indica Guangluai4produces long awns and longer grains,but signi?cantly fewer grains per panicle compared with Guangluai4.Transgenic studies con?rmed that An-1positively regulates awn elongation,but negatively regulates grain number per panicle.Genetic variations in the An-1locus were found to be associated with awn loss in cultivated rice.Population genetic analysis of wild and cultivated rice showed a signi?cant reduction in nucleotide diversity of the An-1locus in rice cultivars, suggesting that the An-1locus was a major target for arti?cial selection.Thus,we propose that awn loss was favored and strongly selected by humans,as genetic variations at the An-1locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.

INTRODUCTION

Modern genetics and archaeological studies have revealed that the Asian cultivated rice Oryza sativa was domesticated from the ancestor of the wild rice species Oryza ru?pogon;8000years ago(Zong et al.,2007;Fuller et al.,2009;Izawa et al.,2009; Huang et al.,2012).Wild rice exhibits a number of traits,such as easy seed shattering,prostrate growth,long awns,black hulls, and few grains per panicle.These unique characteristics have important roles in wild rice and are strongly associated with seed dispersal,dormancy,and survival in harsh environmental conditions.In comparison to its wild progenitor,cultivated rice typically displays reduced seed shattering and dormancy,a re-duction of outcrossing rate and awn length,erect growth,and pericarp and hull color conversion(Kovach et al.,2007;Sweeney and McCouch,2007).All of these traits that distinguish cultivated rice from its wild progenitor are called domestication traits.

To date,the well-characterized rice domestication genes in-clude shattering4(sh4),QTL of seed shattering in chromosome1 (qSH1),PROSTRATE GROWTH1(prog1),Black hull4(Bh4),Red pericarp(Rc),QTL for seed width on chromosome5(qSW5),the rice ortholog of maize C1gene,and Waxy.An examination of domestication genes reveals that the molecular mechanisms underlying the evolution of phenotypes are varied.Natural var-iations have been found to directly disrupt the functions of some genes and to be associated with phenotype changes,such as those in prog1,Bh4,and Rc(Sweeney et al.,2006;Jin et al., 2008;Tan et al.,2008;Zhu et al.,2011).Other domestica-tion genes have been identi?ed as being involved in protein modi?cation,regulatory changes,or both.qSH1eliminated its expression at the provisional abscission layer to confer non-shattering to japonica rice(Konishi et al.,2006),while sh4 differs both in the coding region and59regulatory sequence between wild rice and cultivated rice(Li et al.,2006a).Fur-thermore,domestication genes often have pleiotropic effects on multiple traits.Prog1resulted in erect growth and an in-creased number of grains per panicle(Jin et al.,2008;Tan et al.,2008).Another aspect that increases the intricacy of rice domestication is that O.sativa is divided into japonica and indica subspecies.Previous research showed that the do-mestication alleles of sh4,Prog1,qSW5,and Bh4were?xed

1These authors contributed equally to this work.

2Address correspondence to bhan@https://www.wendangku.net/doc/4516661047.html,.

The author responsible for distribution of materials integral to the?ndings

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Instructions for Authors(https://www.wendangku.net/doc/4516661047.html,)is:Bin Han(bhan@ncgr.ac.

cn).

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The Plant Cell,Vol.25:3360–3376,September2013,https://www.wendangku.net/doc/4516661047.html,?2013American Society of Plant Biologists.All rights reserved.

in both japonica and indica,while qSH1,Waxy,and Rc were ?xed only in the japonica population(Huang et al.,2012).

A recent study revealed the origins of O.sativa ssp japonica and ssp indica by massive analyses of the55regions of se-lective sweeps and genome-wide patterns(Huang et al.,2012). This study demonstrated that japonica rice was?rst domesti-cated from the wild populations of O.ru?pogon in the middle Pearl River regions in southern China and that indica rice was subsequently domesticated through introgression of domestica-tion genes from japonica into wild populations toward south-eastern and southern Asia(Huang et al.,2012).This research provides an important resource and an effective genomics ap-proach for identifying genes that regulate key differences in plant structure and physiology that distinguish cultivated rice from its wild progenitors.

The awn is one of the morphological characteristics of rice seeds and is also found in other species,such as wheat(Triti-cum aestivum),barley(Hordeum vulgare),oats(Avena sativa), and sorghum(Sorghum bicolor).Awn is an extension of apex of the lemma of spikelet.As a characteristic of seeds in wild plants, long awns are reported to aid seed dispersal and seed burial and protect cereal grains from animal predation(Elbaum et al.,2007). However,long awns are not favorable during harvest and stor-age;hence,this trait was arti?cially selected during domesti-cation.Even so,long awns are retained in some cereal crops, such as wheat and barley,because long awns contribute sig-ni?cantly to photosynthesis and yield(Abebe et al.,2010).On the contrary,most cultivated rice bear no awns or very short awns because the round rice awn contains only one vascular bundle and may not contribute to photosynthesis(Toriba et al., 2010).

Genetic analysis has shown that the awn is a complex trait and many awn-related quantitative trait loci(QTLs)have been identi?ed in rice(Cai and Morishima,2002;Thomson et al., 2003;Gu et al.,2005a,2005b;Wang et al.,2011),but no genes have been molecularly identi?ed to date.Therefore,the molec-ular mechanism that transforms the long awns of wild rice to the awnless trait of cultivated rice is still unknown.

In this study,we investigated genetic variations involved in awn loss in domesticated rice.We cloned the major QTL Awn-1 (An-1)that encodes a basic helix-loop-helix(bHLH)protein and regulates the long-awn trait in wild rice.An-1is intensely ex-pressed at the apex of the lemma primordia,speci?cally causing continuous cell division to form a long awn.Upregulation of An-1expression could induce long awns and grain elonga-tion as well as reduce grain number per panicle in awnless cultivated rice,whereas RNA interference(RNAi)of An-1in a long-awn indica variety Kasalath generated shorter awns and shorter grains but produced more grains per panicle com-pared with the control plants.Our population genetic analysis of wild and cultivated rice showed a signi?cant reduction in nu-cleotide diversity of the An-1locus and revealed that the An-1 locus was a major target for arti?cial selection.We thus propose that awn loss was also favored and selected by humans,in addition to easy harvest and storage,as the genetic variation causing awn loss would increase grain number and sub-sequently improve yield in cultivated rice during the long course of domestication.RESULTS

Cloning and Characterization of the Wild Rice Allele of An-1, Which Regulates Long-Awn Development

We previously constructed a wild rice chromosome4substitution line(SL4)derived from a cross between cultivated rice variety O.sativa ssp indica cv Guangluai4(GLA4)(awnless)as the re-current parent and wild rice accession O.ru?pogon Griff W1943 (W1943)(long awn)as the donor parent(Figures1A and1B)(Zhu et al.,2011;Zhou et al.,2012).SL4had long awns of36.456 11.32-mm long in Sanya(N18.2°,E109.5°in China)and 24.8164.17-mm long in Shanghai(N31.2°,E121.4°in China) (see Supplemental Table1online).The awn length was affected to a certain degree by some environmental factors,such as photoperiod and temperature during the?oral differentiation stage,which was consistent with a previous study(Aspinall, 1969).However,the proportion of the awned plants(awn rate) of SL4remained stable and ranged from;70to90%in Sanya and Shanghai,whereas GLA4had no awns in either area(see Supplemental Table1online).

Using the F2population derived from the cross between SL4 and GLA4,two QTLs for awn length,designated as Awn-1(An-1) and Awn-2(An-2),were mapped between the markers M6298 and M6285(;5.88Mb)and the markers M1108and M1160on the long arm of chromosome4,respectively(Figure1C).The wild alleles of An-1and An-2showed positive effects and ac-counted for52.5and12%of the phenotypic variations,re-spectively.To further map the loci and clone the genes for awn length,we constructed a set of single chromosome segment substitution lines(CSSLs).Among the CSSLs,CSSL-Z3(Z3) contained the major QTL,the An-1locus(see Supplemental Figure1online),and displayed a stable awn phenotype in both Sanya and Shanghai.The awn length of Z3was18.8263.25 mm in Sanya(Figures1A and1B)and15.7462.16mm in Shanghai.The awn rate of Z3was67.16%69.12%in Sanya and60.55%68.30%in Shanghai(see Supplemental Table1 online).The F1plants from the cross between Z3and GLA4were awned.An examination of awn phenotypes in126F2plants re-vealed that87plants were awned but39plants were awnless, which?tted a3:1segregation ratio.This indicated that An-1is a single dominant gene that regulates the awn phenotype in this population.Then,a larger F2population containing10,500plants was genotyped using the?anking markers M6298and M6285. By progressively examining the insertion or deletion(indel)and single nucleotide polymorphism(SNP)markers between M6298 and M6285,An-1was?nally delimited between FM3and FM6, representing a70-kb genomic region on chromosome4(Figure1D) according to the Build4.0pseudomolecules of the Nipponbare genome(Feng et al.,2002;International Rice Genome Sequencing Project,2005).

To clone the wild allele An-1,a BAC library of W1943was screened using the FM3and FM6probes.The BAC(ORW1943Ba0047B01) containing the An-1locus was identi?ed,sequenced,and anno-tated.Meanwhile,the BAC(OSIGBa0144C23)corresponding to the collinear FM3-FM6regions from GLA4had been sequenced before,and the same gene content was identi?ed within this region between GLA4and Nipponbare.However,a sequence

An-1Controls Awn Development3361

Figure 1.Map-Based Cloning and Identi ?cation of An-1.

(A)Panicle comparison among SL4,GLA4,and CSSL-Z3.Bar =10mm.(B)Awn length comparison among CSSL-Z3,GLA4,and SL4.Bar =10mm.

(C)Two QTLs for awn length were identi ?ed on chromosome 4in SL4,and An-1was ?rst mapped to the interval between the markers M6298and RM6285,while An-2localized between the markers M1108and M1160.

(D)An-1was further delimited to a 70-kb genomic region between the markers FM3and FM6,which corresponded to two Nipponbare BACs and one W1943BAC.FM1to FM9are primers used for ?ne mapping.The numbers underneath the bars indicate the number of recombinants between An-1and the molecular markers.The black arrows indicate gene direction.Bar =10kb.

(E)Comparison of BAC sequence and annotation between W1943and Nipponbare.Black bars represent genomic sequences of W1943and Nip-ponbare.Red bars represent transposon-like and repeat sequences.Green/blue bars represent predicated ORFs.Green bars represent ORFs with a forward direction.Blue bars represent ORFs with a reverse direction.Gray represents regions sharing sequence collinearity between W1943BAC and Nipponbare BAC.Genes annotated on the BACs are listed in Supplemental Table 2online.

(F)Gene structure of An-1and constructs used in An-1function investigation.pCPL represents the 10-kb W1943genomic fragment used for the complementation test;pOX contains W1943An-1ORF used for ectopic expression and overexpression;pRNAi denotes the RNA interference con-struct.UBI is a maize Ubiquitin promoter.

3362The Plant Cell

comparison between W1943and Nipponbare revealed that mul-tiple reciprocal insertions and deletions existed within this region (Figure1E).Most of those indels were repeat sequences(Figure1E), which might make further recombination dif?cult.Within the col-linear FM3-FM6regions between W1943and Nipponbare,we found that there were only two unique genes,Os04g0350700and Os04g0351333,that were likely candidates for awn length ac-cording to the Rice Annotation Project Database(Figure1E) (http://rapdb.dna.affrc.go.jp/viewer/gbrowse/build4/).Other pre-dicted open reading frames(ORFs)within this region were all transposon element–like sequences or highly repetitive sequence-related genes(see Supplemental Table2online).Furthermore, Os04g0350700encodes a bHLH transcription factor with a few polymorphisms in the coding region and59upstream region(see Supplemental Table3online),while Os04g0351333encodes an RF1-like gene and contains polymorphisms in the promoter re-gion between W1943and GLA4.

To determine which one is the casual gene,we generated two complementary constructs,pCPL and pCPL-RF.pCPL con-tained a10,244-bp W1943genomic sequence covering the entire Os04g0350700gene region and6-kb59upstream and 500-bp39downstream sequences(Figure1F).The pCPL-RF contained a10,501-bp W1943genomic sequence covering the entire Os04g0351333gene region and4-kb59upstream and2-kb 39downstream sequences(see Supplemental Figure2A online). Since we were unable to regenerate shoots from the callus of awnless indica variety GLA4,we transformed an awnless japonica Nipponbare to determine the function of An-1.The vector pCAMBIA1301was used as a control.Eighty-two percent of the T0plants transformed with pCPL produced long awns,whereas none of T0plants transformed with pCPL-RF produced any awns(see Supplemental Table4and Supplemental Figures2B and2C online).The long awn phenotypes were stably inherited in the T1and T2progeny of different pCPL transgenic lines,which indicated that Os04g0350700indeed regulates awn length.

We further transformed Nipponbare with an ectopic and overexpression construct of pOX and the indica Kasalath,which produces long awns,with the pRNAi construct(Figure1F).The pTCK303vector was used as the control for RNAi plants.The phenotypes of T0transgenic plants revealed that the pOX con-struct could also produce long awns,whereas the pRNAi con-struct could induce shortened awns in transgenic plants.Control plants transformed with either pCAMBIA1301or pTCK303did not show any morphological changes during both vegetative and reproductive stages(see Supplemental Table4online).Therefore, the results demonstrate that Os04g0350700is An-1and that this gene regulates awn development in SL4.

Analysis of Functional Allelic Variations in the An-1Locus Associated with Awn Loss in Cultivated Rice

A1978-bp An-1cDNA,which encodes a262–amino acid pro-tein containing a typical bHLH domain,was obtained in W1943 by59and39rapid ampli?cation of cDNA ends(RACE)(see Supplemental Figure3online).The1979-bp cDNA cloned in GLA4was almost identical to An-1cDNA but for a3-bp indel causing an insertion of Ala and an SNP causing a substitution of Gly by Ala in the?rst exon,and two1-bp deletions and a SNP in the39untranslated region(see Supplemental Figure4online). The C terminus of An-1was found to contain transcriptional activation activity in a yeast one-hybrid activation assay(see Supplemental Figure5A online).The green?uorescent protein–An-1fusion was predominantly located in the nucleus(see Supplemental Figures5B and5C online).Thus,An-1is a tran-scription factor with transactivation activity.

Based on a BLAST search of a public database using the An-1protein sequence(https://www.wendangku.net/doc/4516661047.html,/Blast.cgi), we constructed a phylogenetic tree of An-1and its homologs. An-1clustered with three grass bHLH proteins in a small branch with100%bootstrap support(see Supplemental Figure6

and

Figure2.Typical Haplotypes of An-1/an-1in O.sativa and O.ru?pogon.

(A)The an-1(Tn+)subhaplotype in Nipponbare,GLA4,and most japonica varieties.

(B)The an-1(G-)subhaplotype in HP228and most indica varieties.

(C)The An-1haplotype in W1943and most accessions of wild rice.

The common variations between cultivated rice and wild rice are indicated in this?gure.Black bars represent59upstream regions and introns.Light-gray bars represent59and39untranslated regions.Dark-gray bars represent coding regions.The triangles represent insertions.The short dashes represent single base pair deletions.The star in(B)represents the premature stop codon site in an-1(G-).Bar=1kb.

An-1Controls Awn Development3363

Supplemental Data Set 1online).This group contained bHLH proteins from Brachypodium distachyon ,Aegilops tauschii ,and barley,all of which have grains with long awns,indicating that those proteins might also be responsible for awn formation in those species.

As the An-1allele from W1943could induce long-awn forma-tion,the corresponding allele in awnless O.sativa was denoted as

an-1.We compared sequence variations between An-1of W1943and an-1of Nipponbare or GLA4.Except for two SNPs in the 59upstream region,the sequence of an-1in Nipponbare was nearly identical to that in GLA4.Sequence analysis of An-1and an-1revealed the existence of a 3-bp indel,one SNP in the coding region,and several variants in the promoter region (see Supplemental Table 3online).

To identify possible functional variations,we sequenced about an 8-kb genomic region of An-1covering the 4.5-kb promoter region and the 3.5-kb gene region in 27accessions of wild rice and 43cultivars,including 21japonica varieties and 22indica varieties.By comparative sequence analysis of An-1and an-1alleles,12common SNPs,four 1-bp indels,and one 4.4-kb mutator-like transposon polymorphism were detected.According to those common variants,we identi ?ed two major haplotypes in cultivars (Figures 2A and 2B)and wild rice (Figure 2C).The cultivar haplotypes could be further divided into two subhaplotypes based on a transposon-like indel in the promoter region and a

1-bp

Figure 3.Awn Length Comparison.

(A)Photograph of apical grains of primary branches in GLA4and NIL-An-1.(B)Photograph of apical grains of primary branches in Nipponbare,CPL-1,and OX-1.

(C)Photograph of apical grains of primary branches in Kasalath and RNAi-6.

(D)Awn length comparison between GLA4and NIL-An-1.Whereas GLA4lacked awns,the awns of NIL-An-1were 10to 25mm long.

(E)Awn length comparison among Nipponbare,CPL,and OX.There was no visible awn in Nipponbare,but the awns of both the An-1comple-mentation plants and An-1–overexpressing plants were 30to 50mm long.

(F)Awn length comparison among RNAi lines and Kasalath.The awns of Kasalath were 15to 40mm long and shorter in the RNAi plants.

CPL,An-1complementation plants;OX,An-1–overexpressing plants;RNAi,An-1suppression plants.Bars =10mm.In (D)to (F),for GLA4,NIL-An-1,Nipponbare,and Kasalath,the sample size was n =60.For transgenic lines,the sample size was n =30.The statistical signi ?cance was at P <0.05based on a two-tailed Student ’s t test.Error bars rep-resent the SD .

[See online article for color version of this ?

gure.]

Figure 4.Grain Length Comparison.

(A)Photograph of 10grains of GLA4and NIL-An-1.

(B)Photograph of 10grains of Nipponbare,CPL-1,and OX-1.(C)Photograph of 10grains of Kasalath,RNAi-2,and RNAi-6.

(D)Grain length comparison between GLA4and NIL-An-1;grains in NIL-An-1are slightly longer than those in GLA4.

(E)Grain length comparison among CPL,OX,and Nipponbare;grains in CPL and OX are much longer than those in Nipponbare.

(F)Grain length comparison among RNAi lines and Kasalath;grains in RNAi lines are shorter than those in Kasalath.

CPL,An-1complementation plants;OX,An-1–overexpressing plants;RNAi,An-1suppression plants.Bars =10mm.In (D)to (F),the sample size for all analyses was n =100.The statistical signi ?cance was at P <0.05based on a two-tailed Student ’s t test.Error bars represent the SD .[See online article for color version of this ?gure.]

3364The Plant Cell

deletion in the second exon(Figures2A and2B).The Nippon-bare subhaplotype an-1(Tn+)harbored the transposon-like indel in the promoter region(Figure2A),while another typical sub-haplotype an-1(G-)(Figure2B)contained a1-bp nucleotide-G deletion in the second exon of An-1that led to a frame shift and generated a premature stop codon.The truncated protein only consists of97amino acids,which does not contain the bHLH domain and probably loses its functions(see Supplemental Figure7 online).Twelve of21japonica varieties displayed the an-1(Tn+) subhaplotype,while17of22indica varieties displayed the an-1 (G-)subhaplotype(see Supplemental Table5online).Among those43cultivars,awn length was examined in30cultivars; 25were found to be awnless and?ve awned.Among the 25awnless cultivars,nine japonica varieties displayed the an-1 (Tn+)subhaplotype,while13of16indica cultivars displayed the an-1(G-)subhaplotype,but GLA4displayed the an-1(Tn+) subhaplotype.The two exceptions,HP219and HP188,dis-played neither the an-1(Tn+)nor the an-1(G-)subhaplotype (see Supplemental Table5online).Except for two major sub-haplotypes,another haplotype was also observed in a small proportion of cultivars(see Supplemental Tables3and5online). However,sequence variations were more diverse in wild rice. Although some accessions displayed the indica genotype(see Supplemental Table5online),;70%of wild rice accessions shared the same haplotype with W1943(see Supplemental Table3online).Thus,sequence variations between the An-1 and an-1haplotypes might generate awn differences between wild and cultivated rice.

An-1Promotes Awn Development and Grain Length

To investigate An-1functions,we developed a near-isogenic line, NIL-An-1,that contained only a120-kb W1943genomic fragment of the An-1locus in GLA4and exhibited a stable and similar awn length and awn rate as Z3(Figure3A;see Supplemental Figure1 online).The awn length of NIL-An-1was14.6862.45mm,

while

Figure5.Grain Number per Panicle and Yield per Plant Comparison.

(A)Photograph of the panicles in GLA4and NIL-An-1.

(B)Photograph of the panicles in Kasalath and RNAi-6.

(C)Grain number per panicle comparison between GLA4and NIL-An-1;the grain number per panicle in NIL-An-1is slightly lower than that in GLA4.

(D)Grain number per panicle comparison among RNAi lines and Kasalath;grain number per panicle was greater in RNAi lines than in Kasalath.

(E)Photograph of the panicles in Nipponbare,CPL-1,and OX-1.

(F)Grain number per panicle comparison among Nipponbare,CPL,and OX lines;grain number was less in CPL and OX than in Nipponbare.

(G)Yield per plant comparison among Nipponbare,CPL-1,and CPL-2.

CPL,An-1complementation plants;OX,An-1–overexpressing plants;RNAi,An-1suppression plants.Bars=50mm.In(C),(D),and(F),for GLA4, NIL-An-1,Nipponbare,and Kasalath,sample size was n=60.For transgenic lines,sample size was n=30.In(G),sample size was n=48.The statistical signi?cance was at P<0.05based on a two-tailed Student’s t test.Error bars represent the SD.

[See online article for color version of this?gure.]

An-1Controls Awn Development3365

the awn rate was52.81%612.8%(Figure3D;see Supplemental Table6online).

For complementation and RNAi experiments,we performed the analysis on single-copy T2homozygotes of CPL and RNAi transgenic lines.For the overexpression study,we just analyzed the T0OX transgenic plants.The awn length and awn rate were greatly increased in CPL and OX plants compared with those in Nipponbare.The awn length was31.8466.36mm and31.906 6.98mm,while the awn rate was54.48%614.41%and 53.21%615.14%,respectively,in CPL-1and CPL-2plants. The awn length was33.8265.81mm and34.0466.99mm, while the awn rate was63.41%614.19%and65.74%6 16.82%,respectively,in OX-1and OX-5plants(Figures3B and 3E;see Supplemental Table6online).

Kasalath harboring the pRNAi construct exhibited shortened awns and a reduced awn rate(Figure3C).The awn length of RNAi plants ranged from0.8261.29mm to9.1461.87mm, while the awn length of Kasalath was23.3063.05mm(Figure 3F).The awn rate of RNAi plants ranged from4.25%63.44%to 23.84%69.49%,which was lower than the56.32%64.11% awn rate in Kasalath(see Supplemental Table6online).

In addition to awn elongation,An-1also regulated grain length. The grains of NIL-An-1were3.19%longer than those of GLA4 (Figures4A and4D).The grain length in CPL-1and CPL-2plants was13.90and11.97%longer,respectively,than that in Nip-ponbare.The grain length in OX-1and OX-5plants increased 19.99and16.74%,respectively,compared with that in Nip-ponbare(Figures4B and4E).By contrast,the grain length in RNAi lines was decreased,being3.30to9.60%shorter than those of Kasalath(Figures4C and4F).Therefore,An-1is a major regulator of awn length and awn rate and also plays a role in grain elongation.

An-1Negatively Regulates Grain Number per Panicle and, Thus,an-1Increases Yield per Plant

Except for awn elongation and grain elongation,An-1plays an additional role in regulating grain number per panicle.NIL-An-1 generated10.58%fewer grains per panicle than GLA4(Figures 5A and5C).However,the RNAi plants generated more grains per panicle.The different RNAi lines produced13.60to38.4% more grains per panicle than Kasalath(Figures5B and5D).The examination of complementary and overexpressing plants re-vealed similar changes in grain number per panicle as found in NIL-An-1(Figure5E).Nipponbare generated9.676 1.57 primary branches,19.766 3.95secondary branches,and 111.56619.29grains per panicle.Both primary branches and secondary branches were reduced in CPL plants and over-expressing plants(see Supplemental Table6online).The CPL-1 and CPL-2plants produced63.6169.17and68.47612.19 grains per panicle separately,while OX-1and OX-5plants pro-duced51.7969.57and55.82614.34grains per panicle separately(Figure5F).The grain number per panicle decreased by42.08,38.62,54.58,and49.96%in CPL-1,CPL-2,OX-1,and OX-5plants,respectively,compared with that in Nipponbare. The panicle phenotypes of CPL plants were more severe than those of NIL-An-1,which might be due to the different genetic backgrounds.

An analysis of yield per plant was performed on Nipponbare and CPL plants.The analysis showed that yield per plant de-creased by34.83and30.80%in CPL-1and CPL-2plants,re-spectively,compared with that in Nipponbare(Figure5G).Thus, introducing the W1943allele An-1into cultivated rice reduced grain number per panicle and yield per plant.

An-1Expression Level Changes in NIL-An-1and Transgenic Plants

To characterize the An-1expression level changes underlying the panicle phenotypes,we collected young panicles<4cm and analyzed An-1expression using real-time quantitative RT-PCR (qRT-PCR).The results showed that An-1expression of young panicles was increased twofold in NIL-An-1compared with that in GLA4(Figure6A).For complementary plants,An-1expression increased nearly40-to45-fold in CPL-1and CPL-2plants compared with that in Nipponbare(Figure6B).Considering both CPL lines in analysis contained only a single-copy transgene, expression level having been raised thus high might be mainly due to insertion of W1943An-1allele in different genetic back-ground.In RNAi plants,the expression of endogenous An-1 gene decreased(Figure6C).The extent of An-1

downregulation

Figure6.An-1Expression Level Comparison.

(A)The An-1transcript level comparison between GLA4and NIL-An-1 panicles.

(B)The An-1transcript level comparison among Nipponbare,CPL-1,and CPL-2panicles.

(C)The An-1transcript level comparison between Kasalath and RNAi panicles.

Young panicles<4cm high were collected and subjected to qRT-PCR. The data represent the average of three independent biological replicates and were normalized to the EF1a gene as a reference.The statistical signi?cance was at P<0.05based on a two-tailed Student’s t test.Error bars represent the SD.CPL,An-1complementation plants;RNAi,An-1 suppression plants.

3366The Plant Cell

was highly correlated with panicle phenotypes in RNAi plants.The qRT-PCR results and panicle phenotypes suggested An-1expression level was directly proportional to awn length and grain length but inversely proportional to grain number per panicle.Speci ?c Expression of An-1Induces Cell Division and Awn Development

To identify the speci ?c stage when the awn differentiated,we compared the spikelet development between GLA4and NIL-An-1using scanning electron microscopy.According to the rice spikelet development (Sp)stages de ?ned before (Itoh et al.,2005),lemma primordia initiated at the Sp3stage and palea primordia formed at the Sp4stage.No difference was found on spikelet primordia until the Sp6stage between NIL-An-1and GLA4(Figures 7A1and 7B1).At the Sp6stage,the lemma primordia in NIL-An-1grew faster than that in GLA4(Figures 7A2and 7B2).At the Sp7stage,the apex of the lemma in NIL-An-1protruded and the awn primordia formed,whereas no awn primordia formed in GLA4(Figures 7A3and 7B3).At the Sp8early stage (Sp8e)when lemma and palea were gradually closed,awn primordia extended further in NIL-An-1,while the apex of lemma formed a round tip in GLA4(Figures 7A4and 7B4).In the Sp8late stage (Sp8l),the awn primordia kept extending,but the apex of lemma stopped growth in GL A4(Figures 7A5and 7B5).

To determine how An-1regulates awn development,we ex-amined the expression pattern of An-1by RNA in situ

hybridization.

Figure 7.Awn Development and Expression Analysis of An-1and Histone Hs in GLA4and NIL-An-1.

(A1)to (A5)Scanning electron microscopy images of spikelets at dif-ferent developmental stages in GLA4.Arrows point to the apex of lemma primordia.

(B1)to (B5)Scanning electron microscopy images of spikelets at dif-ferent developmental stages in NIL-An-1.Arrows point to awn primordia.(C1)to (C5)Expression patterns of An-1during spikelet development in GLA4.Arrow points to the apex of lemma primordia.

(D1)to (D5)Expression patterns of An-1during spikelet development in NIL-An-1.Arrows point to awn primordia.

(E1)A longitudinal histological section of a spikelet at the Sp8e stage in GLA4.The arrow points to the apex of the lemma primordium.The cells above the black line were quanti ?ed.

(E2)to (E5)Expression patterns of Histone H4during spikelet de-velopment in GLA4.Arrows point to the apex of lemma primordia.

(F1)A longitudinal histological section of a spikelet at the Sp8e stage in NIL-An-1.The arrow points to awn primordium.The cells above the black line were quanti ?ed.

(F2)to (F5)Expression patterns of Histone H4during spikelet de-velopment in NIL-An-1.Arrows point to awn primordia.

(G1)Cell number comparison between the apices of spikelets in GLA4and awn primordia in NIL-An-1.

(G2)A comparison of An-1expression in the 1-to 3-cm young panicles of NIL-An-1and GLA4.

(G3)A comparison of Histone H1expression in the young panicles of NIL-An-1and GLA4.

(A1),(B1),(C1),and (D1)Sp4-Sp5,formation of lemma and palea pri-mordia stage.

(A2),(B2)(C2),(D2),(E2),and (F2)Sp6,formation of stamen primordia stage.

(A3),(B3),(C3),(D3),(E3),and (F3)Sp7,formation of carpel primordia stage.

(A4),(B4),(C4),(D4),(E4),and (F4)Sp8e (Sp8early),differentiation of ovule and pollen stage.

(A5),(B5),(C5),(D5),(E5),and (F5)Sp8l (Sp8late),differentiation of ovule and pollen stage.

In (G1),sample size was as follows:GLA4(n =8)and NIL-An-1(n =8).In (G2)to (G3),the data represent the average of three independent bi-ological replicates and were normalized to the EF1a gene as a reference.The statistical signi ?cance was at P <0.05based on a two-tailed Stu-dent ’s t test.Error bars represent the SD .Bars =100m m.

An-1Controls Awn Development 3367

During spikelet development,An-1transcripts were?rst de-tected at the two rudimentary glume primordia and two empty glume primordia,next at the lemma and palea primordia(Figures 7C1and7D1),and then at the stamen(Figures7C2and7D2) and carpel primordia(Figures7C3and7D3).During the early stages of spikelet development,the An-1expression pattern in NIL-An-1was the same as that in GLA4.Differences emerged in the Sp6stage,when the lemma and palea elongated;An-1 expression increased gradually at the apices of lemma primor-dia,which would subsequently form awn primordia in NIL-An-1 (Figure7D2).Then,An-1expression strongly increased in awn primordia of Sp6(Figure7D3),was maintained until Sp8e(Figure 7D4),and gradually faded during the Sp8l stage(Figure7D5).By contrast,An-1was consistently weakly expressed in all?oral organ primordia in GLA4(Figures7C2and7C3)but not expressed in the apex of the lemma(Figure7C4).Finally,An-1expression almost ceased in the lemma and palea during the Sp8l stage in GLA4(Figure7C5).The speci?c expression of An-1at the apices of lemma and awn primordia in NIL-An-1demonstrated its role in awn initiation and formation.

We further examined An-1expression level in young panicles at different developmental stages using qRT-PCR.The results showed that within1-to3-cm young panicles,An-1expres-sion was about2times higher in NIL-An-1than in GLA4in each pair of samples(Figure7G2).In summary,both the pattern and level of An-1expression contributed to the awn phenotypes in NIL-An-1.

On historical sections of spikelets at the Sp8e stage,we quan-ti?ed the number of cells in awn primordia and spikelet apices (Figures7E1and7F1,above the black lines).The awn primordia in NIL-An-1contained3times more cells than the apex of lemma in GLA4(Figures7E1,7F1,and7G1),indicating that cell division plays a crucial role in awn formation.As HISTONE Hs are expressed during the G1-S phase,their expression is usually used as a marker for cell division(Marzluff and Duronio,2002). RNA in situ hybridization indicated that Histone H4was evenly expressed at the early?oral development stages,and no dif-ference was found between NIL-An-1and GLA4(Figures7E2 and7F2).However,from the Sp7stage,when awn primordia formed,Histone H4was highly expressed in the awn

primordia,

Figure8.Epidermis Cell Number Comparison and Histone Hs Expression Analysis.

(A)Scanning electron microscopy photograph of a whole grain.Arrow1indicates the magni?ed area on the outer epidermis of the lemma,and arrow2 represents the longitudinal axis along which cell number was quanti?ed.Bar=1mm.

(B)and(C)Scanning electron microscopy photographs of lemma outer epidermis cells in Kasalath and RNAi-6grains.Bars=100m m.

(D)Comparison of average cell number along the longitudinal axis between Kasalath and RNAi-6grains.

(E)qRT-PCR analysis of Histone H1expression in3-to4-cm young panicles of Kasalath and RNAi-6.

(F)and(G)Scanning electron microscopy photographs of lemma outer epidermis cells in Nipponbare and CPL-1grains.Bars=100m m.

(H)Comparison of the average cell number along the longitudinal axis between Nipponbare and CPL-1grains.

(I)qRT-PCR analysis of Histone H1expression in the3-4cm young panicles of Nipponbare and CPL-1.

(J)Expression pattern of Histone H4in spikelets at the Sp8l stage in Nipponbare.

(K)Expression pattern of Histone H4in spikelets at the Sp8l stage in CPL-1.Bars=100m m.

CPL,An-1complementation plants;RNAi,An-1suppression plants.In(D)and(H),sample size was n=8for all analyses.In(E)and(I),the data represented the average of three independent biological replicates and were normalized to the EF1a gene as a reference.The statistical signi?cance was at P<0.05based on two-tailed Student’s t test.Error bars represent the SD.

3368The Plant Cell

lemma,and palea primordia of young spikelets in NIL-An-1(Figures 7F3and 7F4),whereas Histone H4expression at the lemma and palea primordia gradually weakened in GLA4(Figures 7E3and 7E4).In Sp8l spikelets,Histone H4transcript was al-most absent from the lemma and palea and only stayed in pollen and ovule tissues in both NIL-An-1and GLA4(Figures 7E5and 7F5).A qRT-PCR analysis further revealed that Histone H1expres-sion in NIL-An-1young panicles was almost 2times higher than that in GLA4(Figure 7G3),which indicated that cell di-vision was more active in NIL-An-1primordia than in the apices of GLA4spikelets.

Therefore,the speci ?c expression of An-1could induce con-tinually rapid cell division at the apex of the lemma to lead to awn primordia formation,which was con ?rmed by Histone Hs ex-pression.By contrast,genetic variations of an-1(Tn+)eliminated its expression at the apex of lemma,resulting in loss of awns in GLA4.

An-1Drives Grain Elongation through Cell Division

The cell number and cell size of the outer epidermis of grains were examined to determine what caused the changes in grain length in transgenic plants.The cell number of a single line was quanti ?ed along the long axis of the lemma using scanning electron microscopy images (Figure 8A).Scanning electron mi-croscopy images revealed that the outer epidermal cell size at the lemma did not change obviously in RNAi-6grains compared with that in Kasalath grains (Figures 8B and 8C),whereas quan-ti ?cation of epidermal cells in the lemma showed that cell number decreased by 9.68%in RNAi-6grains compared with that in Kasalath grains (Figure 8D).The qRT-PCR results con ?rmed that Histone H1expression in the young panicles in RNAi plants decreased to about half of that in Kasalath (Figure 8E).

The epidermal cell size in the lemma was nearly the same in CPL-1grains as in Nipponbare grains (Figures 8F and 8G).However,the epidermal cell number in CPL-1was 14.16%more than that in Nipponbare (Figure 8H).Accordingly,Histone H1expression in CPL young panicles was more than twofold up-regulated compared with that in Nipponbare (Figure 8I).RNA in situ hybridization also revealed that Histone H4was expressed in the lemma and palea primordia until the Sp8l stage in the CPL spikelet,whereas no Histone H4transcript was detected in the Nipponbare spikelet at the same stage (Figures 8J and 8K).

We

Figure 9.Expression Analysis of An-1,OSH1,LOG ,and Os CKX2during Panicle Development in NIL-An-1,GLA4,and CPL-1.(A)to (D)Expression patterns of An-1in young NIL-An-1panicles.(E)to (H)Expression patterns of An-1in young GLA4panicles.(I)to (L)Expression patterns of An-1in young CPL-1panicles.

(M)to (P)Expression patterns of OSH1in young NIL-An-1panicles.

(A),(E),(I),and (M)Rachis meristem stage.

(B),(F),(J),and (N)Primary branch formation stage.(C),(G),(K),and (O)Secondary branch formation stage.

(D),(H),(L),and (P)Spikelet primordia formation stage.Bars =100m m.(Q)A comparison of An-1expression in the 1-to 3-cm young panicles of Nipponbare,CPL-1,and CPL-2plants.

(R)A comparison of LOG expression in young panicles <1cm of Nip-ponbare,CPL-1,and CPL-2.

(S)A comparison of OsCKX2expression in young panicles <1cm of Nipponbare,CPL-1,and CPL-2.

In (Q)to (S),the data represent the average of three independent bi-ological replicates and were normalized to the EF1a gene as a reference.The statistical signi ?cance was at P <0.05based on two-tailed Student ’s t test.Error bars represent the SD .

An-1Controls Awn Development 3369

conclude that prolonged cell division led to an increase in both cell number and grain length in CPL plants.

Upregulation of An-1in the In?orescence Reduces Meristem Activity and Grain Number per Panicle

To investigate how An-1regulates grain number per panicle,we characterized the pattern and level of An-1expression during in?orescence development.As de?ned before,rice in?orescence development was divided into nine stages(Itoh et al.,2005). During the early stages of in?orescence development,An-1shared a similar expression pattern in GLA4,NIL-An-1,and CPL-1.At the In1stage,when bract primordia formed,An-1began to be weakly expressed at the surface layers of the rachis meristem in both NIL-An-1and GLA4(Figures9A and9E),while An-1ex-pression in CPL-1increased and expanded to the pith and procambium of the rachis meristem(Figure9I).At the In2-3stage, An-1was moderately expressed at the primary branch meristem in NIL-An-1and GLA4,but strongly expressed in CPL-1(Figures 9B,9F,and9J).Once the secondary branch meristem initiated at the In5stage,An-1transcript was detected at the secondary branch meristem in NIL-An-1,GLA4,and CPL-1(Figures9C,9G, and9K).From stage In6,when spikelet development started, An-1expression appeared at the spikelet primordia(Figures9D, 9H,and9L).No speci?c signal was detected at any stage of in?orescence development on An-1sense control sections(see Supplemental Figures8A to8C online).Then,we quanti?ed the An-1expression level in young panicles at different developmental stages.Within1-to3-cm young panicles,the An-1expression was highly increased in CPL transgenic plants compared with in Nipponbare in each group of samples(Figure9Q).

We further compared the expression patterns between OSH1 and An-1in NIL-An-1.At the In1-5stages,OSH1was highly expressed in the rachis meristem,primary branch meristem,and secondary branch meristem(Figures9M to9O),where An-1 expression was relatively low.The differences in expression pat-tern between An-1and OSH1began at In6,when spikelet pri-mordia initiated.OSH1was only expressed in the?oral meristem, but An-1started its expression at?oral organ primordia(Figures 9D and9P).Although both genes were coexpressed during the early stages of in?orescence development,phenotypes in NIL-An-1and transgenic plants indicated that An-1might have an opposite role in meristem maintenance compared with OSH1. Considering the expression of An-1in the rachis meristem, the primary branch meristem,and the secondary branch meri-stem,we predicted that the increased An-1expression level in NIL-An-1and CPL might in?uence the expression of genes that are involved in meristem maintenance and grain number per panicle.LONELY GUY(LOG)and Grain number1a(Gn1a)are known to regulate cytokinin concentration and grain number per panicle in rice.LOG encodes a key enzyme that converts in-active cytokinin to the biologically active form,whereas Gn1a encodes cytokinin oxidase(Os CKX2),which degrades cytokinin (Ashikari et al.,2005;Kurakawa et al.,2007).The two genes directly determined the concentration of active cytokinin,which plays an important role in meristem maintenance(Leibfried et al., 2005;Chickarmane et al.,2012).We analyzed the expression level of LOG and Os CKX2in CPL plants,which exhibit a sig-ni?cant reduction in grain number per panicle.The qRT-PCR results showed that LOG expression in the young panicles (<1cm)of both CPL-1and CPL-2plants was downregulated to about half of that in Nipponbare(Figure9R),while Os CKX2ex-pression did not change markedly between CPL and Nipponbare (Figure9S).Therefore,upregulation of An-1expression during the early stages of in?orescence formation could downregulate LOG expression to reduce meristem activity and then reduce grain number per panicle and yield per plant in CPL plants. Neutrality Test and Phylogenetic Tree of An-1

To investigate whether An-1was subjected to arti?cial selection, nucleotide diversity was calculated and a neutrality test was performed based on total polymorphic sites of An-1sequences (;7kb,including its?anking genomic region).The nucleotide diversity of An-1in cultivated rice(p=0.00073;u w=0.00229) was signi?cantly reduced compared with that of An-1in wild rice (p=0.01012;u w=0.01174)(Table1).The Tajima’D test results revealed that only An-1in cultivated rice deviated signi?cantly from the neutral expectation(P<0.01),which indicated that An-1might have been subjected to arti?cial directional selection (Table1).We also noticed that An-1was located around a major domestication sweep(see Supplemental Figure9online)that showed the third strongest selection signal across the rice ge-nome(Huang et al.,2012).A combination of the above analysis of An-1and its chromosome location suggest that An-1was probably an important target during rice domestication.More-over,we constructed a phylogenetic tree based on the An-1 sequence from43cultivated rice varieties and27wild rice ac-cessions.Two accessions,W3106(Oryza barthii)and W3104 (Oryza glaberrima),were selected as outgroup.A phylogenetic analysis of multiple An-1sequences showed that nearly all of the cultivars grouped together tightly,which revealed that these cultivars might have originated from a common progenitor in a single domestication event(Figure10).

Table1.Nucleotide Diversity and Tajima’s D Test

Taxon N L H S p u w Tajima’s D

O.sativa82679118780.000730.0022922.28439P<0.01

O.ru?pogon506789173630.010120.0117420.55367P>0.10

N,total number of sequences;L,average length(bp)of the sequences per taxon;H,total number of haplotypes per taxon;S,total number of polymorphic sites per taxon;p,average number of pairwise nucleotide differences per site calculated based on the total number of polymorphic sites; u w,Watterson’s estimator of per base pair calculated based on the total number of polymorphic sites.Kasalath and GP69were not included in the analysis.

3370The Plant Cell

DISCUSSION

An-1Has Pleiotropic Effects on Awn Development,Grain Length,and Grain Number per Panicle in Rice

Since long awns are a vital trait for seed dispersal and implan-tation in wild rice,many important genes are involved in awn development.Twenty QTLs for awn length were identi?ed on nine chromosomes by different mapping populations(http://www. https://www.wendangku.net/doc/4516661047.html,).However,genes involved in awn development have not hitherto been molecularly identi?ed in rice.Recently,Lks2, which regulates awn length in barley,was cloned and natural variation in the conserved domain of Lks2led to short awns in some varieties.The putative ortholog of Lks2in rice is Os06g0712600, but no QTL for awn length has been mapped within this region in rice(Yuo et al.,2012).

In our study,two QTLs,An-1and An-2,were mapped on chromosome4.We cloned and functionally characterized the major QTL,An-1.When a single W1943An-1allele was introduced into an awnless variety,GLA4or Nipponbare,long awns were induced in NIL-An-1and the transgenic plants.RNAi of An-1in long-awned Kasalath led to shorter and fewer awns. Scanning electron microscopy revealed that awn primordia formed in NIL-An-1from the Sp6stage.Speci?c expression of An-1at the apex of lemma at the Sp6stage promoted contin-uous and rapid cell division at the apex of lemma,which induced awn primordia formation.Conversely,the an-1(Tn+)allele elim-inated its expression at the apex of lemma and caused loss of awn initiation in GLA4.Unlike Lks2in barley that only regulates awn length(Yuo et al.,2012),An-1regulated both awn initiation and awn length.Therefore,An-1is a major regulator of awn de-velopment in rice.

Furthermore,we found long awn induction accompanied by grain elongation and reduction of grain number per panicle in both NIL-An-1and transgenic plants.The An-1expression level was highly correlated with grain length and grain number per panicle.Therefore,An-1is a multifunctional gene with pleiotro-pic roles in rice development.

An-1Promotes Cell Division

An-1encodes a typical bHLH transcription factor,which con-tains a bHLH DNA binding domain and transcriptional activation activity.The bHLH transcription factors belong to a large gene family,which contains147members in Arabidopsis thaliana and 167members in O.sativa(Li et al.,2006b).The bHLH genes in O.sativa have been reported to be involved in grain size,leaf and root hair development,pericarp color,and axillary meristem formation(Sweeney et al.,2006;Ding et al.,2009;Zhang et al., 2009;Heang and Sassa,2012;Yang et al.,2012).As a member of the bHLH gene family in rice,An-1regulates awn development, seed elongation,and grain number.

Awn formation is dependent on cell division,which was con-?rmed by RNA in situ hybridization of Histone H4expression. Grain elongation is also promoted by enhancing cell division.The upregulation of An-1in CPL plants resulted in a corresponding increase in Histone H1expression,whereas suppression of An-1 downregulated Histone H1expression in RNAi plants.Therefore,An-1might directly or indirectly promote cell division in rice. SPATULA(SPT)and BIGPETALp(BPEp)are two members of the bHLH family in Arabidopsis.SPT plays pleiotropic roles in carpel development,root growth,and leaf size(Heisler et al.,2001; Ichihashi et al.,2010;Makkena and Lamb,2013).In the spt loss-of-function mutant,root meristem size and leaf size increased due to increased cell number(Ichihashi et al.,2010;Makkena and Lamb,2013).BPEp interacts with AUXIN RESPONSE FAC-TOR8to limit cell division during the early stages of petal de-velopmental and limit cell expansion during later stages(Varaud et al.,2011).Both SPT and BPEp are negative regulators of cell division,whereas An-1is a positive regulator of cell division.

An-1Downregulates Meristematic Activity and Grain Number per Panicle

Except for regulating awn development and grain elongation, An-1also affects grain number per panicle.Cytokinin concen-tration has been reported to affect grain number per panicle and yield in rice(Ashikari et al.,2005;Kurakawa et al.,2007;Li et al., 2013).Grain number per panicle depends on the meristematic activity of the in?orescence.Both OSH1and cytokinin are key players in maintaining meristem identity(Sato et al.,1996;Leibfried et al.,2005;Tsuda et al.,2011;Chickarmane et al.,2012).There is reciprocal positive regulation between OSH1and cytokinin(Rupp et al.,1999;Frank et al.,2000;Yanai et al.,2005).RNA in situ hybridization analysis showed that An-1is coexpressed with OSH1 in the rachis meristem and primary and secondary branch mer-istems during earlier stages of in?orescence development.Based on the panicle phenotypes in NIL-An-1and transgenic plants,we considered that An-1functions as a negative regulator in main-taining meristematic activity.Upregulation of An-1led to down-regulation of LOG expression,which might reduce the active cytokinin level as well as meristem identity.Although in the vivo cytokinin concentration was not measured in our study,the re-duction in grain number per panicle and yield per plant coincided with the downregulation of LOG in transgenic plants.Therefore, we propose that An-1is a negative regulator of meristem identity. In cultivated rice,an-1(Tn+)reduced An-1expression and an-1 (G-)abolished An-1function,which increased meristematic activity of the in?orescence and subsequently increased grain number per panicle and yield per plant.

An-1Is a Major Target of Arti?cial Selection

The results in this study revealed that formation of long awns was accompanied by a reduction of grain number per panicle and yield per plant.In contrast with long awns increasing yield in barley and wheat(Abebe et al.,2010),long-awn formation re-duced yield per plant in rice,which might partly explain why the long awn trait was under strong arti?cial selection during rice domestication.

Tajima’s D statistic is widely used as a neutrality test.Positive values of Tajima’s D arise from an excess of intermediate fre-quency alleles,indicating a decrease in population size and/or balancing selection.Negative values of Tajima’s D arise from an excess of low-frequency alleles and indicate population ex-pansions(e.g.,after a bottleneck or a selective sweep)and/or

An-1Controls Awn Development3371

Figure10.Phylogenetic Tree of An-1in Rice.

Maximum parsimony phylogenetic tree of An-1in rice cultivars and wild relatives.Bootstrap values were estimated(with1000replicates)to assess the relative support for each branch.The bootstrap values of50%and above are indicated on the tree.ND,not detected.Alignments used to generate the

phylogeny are presented in Supplemental Data Set2online.

purifying selection(Tajima,1989).The reduced nucleotide di-versity of an-1in cultivated rice and negative Tajima’D statistics indicated that An-1in cultivated rice might have been subjected to arti?cial selection.According to a recent large genomic in-vestigation of cultivated rice and wild rice,55selective sweeps occurred during rice domestication(Huang et al.,2012).Like all well-characterized domestication genes,An-1locates in one of the selective sweeps on chromosome4,which is the third stron-gest selective sweep.This indicates that An-1is a major regulator of awn and grain number and is a main target of arti?cial selection with strong selection pressure.

The phylogenic tree revealed that nearly all cultivars of O.sativa grouped together.This suggested that An-1might originate from a common progenitor with a single domestication event.Although phylogenic analysis revealed that an-1of cultivated rice origi-nated from a single domestication event,we also detected two major haplotypes in cultivated rice.The?anking sequence com-parison between An-1and an-1loci revealed that this gene is located in a complicated genomic context,comprised of multiple reciprocal indels of transposon-like or repeat sequence.This demonstrates that the evolution process of the An-1locus during domestication might have undergone multiple recombination events or genetic drifts.

METHODS

Plant Materials

In QTL mapping,map-based cloning,and subsequent analysis of An-1, we used an awnless cultivar GLA4and Nipponbare,awned Kasalath,and

awned SL4,Z3,and NIL-An-1.Z3was screened from the BC

5F

3

pop-

ulation of SL4and GLA4.NIL-An-1was identi?ed in a BC

1F

3

population

derived from the cross between Z3and GLA4.The43varieties of cul-tivated rice(Oryza sativa)and the28accessions of wild rice used in the phylogenetic analysis are listed in Supplemental Table7online. Primers

The primers used in this study are listed in Supplemental Table8online. QTL Mapping and Fine Mapping of An-1

An F2population,containing255plants,from the cross between SL4and GLA4was constructed for QTL mapping.The total28indel primers were used to construct a genetic linkage map of chromosome4by Joinmap4.0 (Stam,1993).QTL Cartographer V2.5software was used to detect two QTLs of An-1and An-2on chromosome4(Wang et al.,2007).An F2 population containing10,500plants was raised from the cross between Z3and GLA4for?ne mapping of An-1.The molecular markers used in QTL mapping and?ne mapping are listed in Supplemental Table8online. The An-1locus was?nally narrowed down to an interval between SNP markers FM3and FM6.

BAC Screening,Sequencing,Assembly,and Annotation

The BAC clone ORW9143Ba0047B01,containing An-1,was identi?ed from the W1943BAC library and sequenced by shotgun sequencing.The BAC sequence was assembled by Phred/Phrap and the Gap4software package(Ewing and Green,1998;Ewing et al.,1998)and was annotated by Genescan(Burge and Karlin,1997).The annotated genes were compared with gene annotations on RAP-DB(http://rapdb.dna.affrc.go.jp/viewer/ gbrowse/build4).Constructs and Transformation

A10244-kb fragment from W1943BAC harboring the entire An-1gene was inserted into the binary vector pCAMBIA1301to form the pCPL construct.A10,501-kb fragment from W1943BAC harboring the entire Os04g0351333gene was inserted into the binary vector pCAMBIA1301to form the pCPL-RF construct.The pOX construct contained an ORF of W1943An-1under the control of the maize Ubiquitin promoter with pCAMBIA1300as the backbone.The RNAi construct contained an inverted repeat harboring the282-bp An-1cDNA fragment in vector pTCK303(Wang et al.,2004).All plasmid constructs were introduced to Agrobacterium tumefaciens strain EHA105and subsequently trans-ferred into the japonica cultivar Nipponbare or Aus variety Kasalath. Phenotypic Evaluation

For the near-isogenic line NIL-An-1and the recipient parent GLA4,we used20plants to measure awn length and the number of primary branches, secondary branches,and grains per panicle on the main panicles.Three main panicles of each plant were collected for analysis.The awn length of apical spikelets of all primary branches was measured,which was used to represent the awn length of the whole panicle.To avoid gene dosage effects caused by multiple copies of transgenes,T2homozygotes with a single-copy transgene were identi?ed by a hygromycin resistance test. Phenotypic measurements were performed using two independent com-plementary T2homozygous lines(CPL-1and CPL-2),two independent overexpressing T0lines(OX-1and OX-5),four RNAi T2homozygous lines (RNAi-1,-2,-5,and-6),and10plants from each line.Twenty Nipponbare and Kasalath plants were used as the control.The100fully matured seeds from NIL-An-1,GLA4,Nipponbare,Kasalath,and independent transgenic lines were randomly selected for grain length measurement.The seeds with their awns removed were mounted on a scanner for scanning.Scanning images were analyzed using software to calculate the average grain length and width.

Yield Analysis

The average yield per CPL-1,CPL-2,and Nippponbare plant was de-termined at the experimental farm in Shanghai during the growing season (May to September)of2012.The germinated seeds were sown in a seedling bed,and seedlings were transplanted to a paddy?eld30d later with a single plant per hill spaced at20325cm.Each plot included eight rows with12plants per row.The48plants in the middle of each plot were selected to investigate grain yield per plant.After harvest and awn re-moval,all fully mature seeds of each plant were collected and weighed. The average weight of48plants was used in the data analysis.

Quanti?cation of Cell Number

The cell number in the awn primordia or apex of the spikelet was quanti?ed in histological sections.The quanti?cation was performed on three serial sections of three samples,and average cell numbers were compared.The outer epidermal cells of lemmas were quanti?ed on scanning electron microscopy images.The quanti?cation was performed using eight grains,and the average cell numbers were compared.

59and39RACE and qRT-PCR

Total RNA was extracted using TRIzol reagent,treated with DNaseI,and reverse transcribed with oligo(dT

20

)primer using SuperScript II reverse transcriptase(Life Technologies).First-strand cDNAs were used as templates in qRT-PCR using SYBR Green PCR Master Mix(Takara).The qRT-PCR was performed and analyzed using the ABI Prism7500se-quence detection system and software(PE Applied Biosystems).The

An-1Controls Awn Development3373

qRT-PCR was performed in triplicate,and the rice gene eEF-1a(AK061464) was used as a control to normalize all data.The59and39RACE reactions were performed using the GeneRacer kit(Life Technologies),following the manufacturer’s instructions.

Scanning Electron Microscopy

The young panicles were?xed in formalin–acetic acid–alcohol?xative solution,dehydrated through an ethanol series,and dried using a carbon dioxide critical-point dryer.Mature seeds were cleaned with1%Tween20 and dried at45°C.The dry panicles and seeds were gold plated and observed using a Hitachi S-2460scanning electron microscope at15kV. RNA in Situ Hybridization

Young panicles of GLA4,NIL-An-1,and CPL-1were?xed in4%para-formaldehyde,dehydrated through an ethanol series,and embedded in Paraplast.A gene-speci?c region of An-1,OSH1,and OsHistone H4cDNA was used to generate digoxigenin-labeled RNA probes(Roche).RNA in situ hybridization with sense and antisense probes was performed on8-m m sections of young panicles,as described by Luo et al.(1996). Neutrality Test and Phylogenetic Tree

Multiple sequences were aligned with ClustalX(Thompson et al.,1997). The program DnaSP,version5.0(Rozas et al.,2003),was used to cal-culate nucleotide diversity and perform Tajima’s D test.These values were calculated after?rst excluding all regions of alignment gaps and missing data.A phylogenetic tree of the sequenced accessions was reconstructed by the Maximum parsimony method.MEGA version5.1was used to perform the phylogenetic reconstruction(Tamura et al.,2011).Bootstrap values were estimated(with1000replicates)to assess the relative support for each branch,and bootstrap values were labeled with cutoff=50. Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers:An-1cDNA from W1943 (FO681395),an-1cDNA from GLA4(FO681475),ORW1943Ba0047B01 (FO681399),and OSIGBa0144C23(CR855121).The accession numbers of An-1/an-1genomic sequences in43cultivated and27wild rice vari-eties are listed in Supplemental Table7online.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure1.Genotypes of Guangluai-4,SL4,CSSL-Z3, and NIL-An-1Lines.

Supplemental Figure2.The T0Transgenic Phenotypes in Comple-mentation Tests.

Supplemental Figure3.Full-Length cDNA of An-1in W1943.

Supplemental Figure4.Full-Length cDNA of an-1in GLA4.

Supplemental Figure5.Transcription Activation Activity Assay and Nuclear Localization of An-1.

Supplemental Figure6.Phylogenetic Tree of An-1and Its Homologs.

Supplemental Figure7.Full-Length cDNA of an-1in HP228.

Supplemental Figure8.In Situ Hybridization of An-1Sense Probe at Different Developmental Stages in NIL-An-1.

Supplemental Figure9.Screening of Domestication Sweeps on Chromosome4.

Supplemental https://www.wendangku.net/doc/4516661047.html,parison of Awn Length and Awn Rate among SL4,CSSSL-Z3,and GLA4.

Supplemental Table 2.Gene Annotation Summary of Genomic Region between FM3and FM6in W1943and Nipponbare.

Supplemental Table3.Summary of An-1Genotypes and Sequence Variations in Cultivated and Wild Rice.

Supplemental Table4.Phenotype Summary of T0Transgenic Plants.

Supplemental Table5.Summary of An-1Haplotypes and Pheno-types in Cultivated and Wild Rice.

Supplemental Table6.Panicle Phenotype Comparison in NIL-An-1/ CPL/OX/RNAi Plants.

Supplemental Table7.The List of Accession Numbers and Names in 43Cultivars and28Wild Rice Varieties Used in This Study.

Supplemental Table8.Summary of Primers Used in This Study.

Supplemental Data Set1.Alignments Used to Generate the Phylog-eny in Supplemental Figure6Online.

Supplemental Data Set2.Alignments Used to Generate the Phylog-eny in Figure10.

ACKNOWLEDGMENTS

We thank Jiqin Li and Xiaoyan Gao for scanning electron microscopy experiments,Xiaoshu Gao for the confocal microscopy experiment,and Lizhen Si for the neutrality test and transcriptional activity analysis in yeast.We thank Kang Chong for providing the vector pTCK303and Hongquan Yang for the vector pA7.This work was supported by the grants from the Ministry of Science and Technology of China(2011CB100205, 2012AA10A302,and2012AA10A304),the Ministry of Agriculture of China (2011ZX08009-002and2011ZX08001-004),and the National Natural Science Foundation of China(31121063).

AUTHOR CONTRIBUTIONS

B.H.conceived the project and its components.H.L.,T.Z.,B.G.,T.S., and Zixuan W.performed map-based cloning and construction of the introgression lines.J.L.,T.Z.,Y.S.,and J.Z.performed transgenic,cell biological,and other functional analyses.Y.W.,Ziqun W.,A.W.,and B.G. performed?eldwork.X.H.,Y.Z.,Q.Z.,and Y.L.performed evolutionary study.J.L.,H.L.,and T.Z analyzed the data.J.L.and B.H.wrote the article.

Received May10,2013;revised August14,2013;accepted September 6,2013;published September27,2013.

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克隆构建目的基因的简便方法

克隆构建 一、 PCR 1、PCR 20ul体系，4个Mix，3个回收，1个对照 H2O 10.8ul 94℃3min KOD Buffer 2ul 94℃30s DNTPs 2ul 5X℃30s MgSO4 2ul 68℃Xs Primer F 2ul 68℃10min Primer R 2ul cycle 34 KOD 0.2ul Plasmid 1ul 试剂确保融化完全，枪头触到表面打净，最后用白枪头反复打几次混合溶液，离心3-4s，分装20ul至PCR管。 2、加尾 0.1ul Taq 酶，加入到20ul，微弹，离心，PCR仪上放置15min。 3、回收 配中孔胶，电泳并回收试剂盒回收：熔胶后室温冷却再上柱，PW缓冲液离心过后于超净台上吹干7-9min，电泳检测回收产物。 二、连接 Solution 1 5ul 目的片段 4.5ul 16℃过夜 T-vector 0.5ul 三、转化 1、感受态细胞制备 取5ml无抗LB培养液，加入到灭菌试管中，取60-80ul新鲜菌液，37℃恒温摇床2h；取其中2.4ml倒入2.5ml EP管中，尽量保持4℃低温下12000g离心30s， 沿壁缓缓加入CaCl2溶液，冰上滑动EP管管底使菌体悬浮； 2、转化涂板 加入连接产物时边加边搅，热激时温度略微低于42℃，时间严格控制1min30s 之后冰上静置3min左右，加入300ul LB 无抗培养基，于37℃恒温箱复苏50min，涂板并37℃恒温箱过夜培养。 四、鉴定 1、质粒提取 挑去单克隆菌落，37℃过夜培养，次日提取质粒。 2、酶切鉴定 根据设计引物选择双切酶，20ul体系37℃酶切2h，电泳检测并送去测序。 3、PCR鉴定 将切出片段所属的菌液进行菌液PCR鉴定。

水稻表皮毛性状研究与相关基因的克隆

Open Journal of Nature Science 自然科学, 2018, 6(1), 14-16 Published Online January 2018 in Hans. https://www.wendangku.net/doc/4516661047.html,/journal/ojns https://https://www.wendangku.net/doc/4516661047.html,/10.12677/ojns.2018.61003 Studies on the Rice Trichome and Cloning of Related Gene Caifeng Lv, Shengjian Ma, Huilin Liang, Linqing Pan, Qianmei Li, Guanlong Cai, Xinqi Lin, Yongjian Huang, Huanying Chen, Jingchao Liu School of Life Science and Biotechnology, Lingnan Normal University, Zhanjiang Guangdong Received: Dec. 18th, 2017; accepted: Dec. 28th, 2017; published: Jan. 4th, 2018 Abstract Rice leaf epidermal hair mutants are important research materials for rice improvement. The de-velopment of rice epidermis hair is controlled by multiple genes. It is of great scientific value to study the development of rice epidermal hair and promote the cultivation of Kumito rice. This paper mainly introduces the types of rice epidermis and its related control genes. Keywords Rice, Epidermis Hairs, Glabrous 水稻表皮毛性状研究与相关基因的克隆 吕彩凤，马生健*，梁慧琳，潘琳清，黎倩美，蔡冠龙，林欣琪，黄永健， 陈浣滢，刘景超 岭南师范学院生命科学与技术学院，广东湛江 收稿日期：2017年12月18日；录用日期：2017年12月28日；发布日期：2018年1月4日 摘要 水稻(Oryza sativa L.)叶片表皮毛突变体是进行水稻改良的重要研究材料，水稻表皮毛的发育受多个基因的控制。研究水稻表皮毛发育和推广光身稻的种植具有较大的科学价值。本文主要对水稻表皮毛类型及其相关控制基因进行介绍。 *通讯作者。

基因克隆、假病毒操作步骤

实验名称：基因克隆 实验器材:荧光定量PCR仪、摇床、离心机、生工PCR产物纯化试剂盒、恒温加热器、 NEB连接体系、灭菌纯水、JM109感受态、冰、LB培养基、酒精灯、涂棒、氨苄、氨苄抗性平板、甘油等； 操作步骤： 1、可通过PCR进行拼接获得目的基因的，过柱纯化（生工试剂盒根据说明书进行纯化， 在最后一步的洗脱可以用预热的灭菌纯水洗脱，在加灭菌纯水洗脱的时候一定要加在纯化柱子的膜中间）； 2、选择合适的载体（EZ-T）用连接酶进行连接，NEB体系，16℃过夜连接 T4lages 1.0 10×T4buffer 2.0 EZ-T 1.0 目的基因8.0 DdH2O 8.0 ＿＿＿＿＿＿＿＿＿ 20ul 3、取100μl摇匀后的JM109感受态细胞悬浮液（如是冷冻保存液，则需化冻后马上进行下 面的操作），加入10μl连接产物，轻轻摇匀，冰上放置30min后，于42度水浴中保温90s，然后迅速在冰上冷却2min； 4、加入500μl LB液体培养基，混匀于37℃振荡培养45min使受体菌恢复正常生长状态并 使转化体产生抗药性； 5、将恢复培养的菌体5000rpm离心3min，移去上层LB培养基，用余下的200μl重悬菌体， 并用灭菌玻璃推子(酒精灯上烧后冷却)，均匀涂布于琼脂凝胶表面（氨苄抗性），37℃倒置培养12~16小时； 6、挑取多个单克隆菌落分别接种到1ml含有抗生素（氨苄）的LB液体培养基中，37℃振 荡培养3h； 7、培养1-2小时即可以利用PCR（定量或定性）进行鉴定； 8、选取初步鉴定阳性的菌液送测序，测序正确后甘油保存（甘油的浓度为30%-50%），充 分混匀，-80℃保存；

基因克隆及转基因方法

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based on escherichia coli segment of DNA (or genes), including the main course target DNA, restructuring of the carrier, transformation of receptor cells and reorganization of screening and reproductive cells. This paper mainly introduces the characteristics of plant gene and gene cloning and carrier, gene clone tool enzyme, gene cloning and plant gene strategy of separation cloning method, etc. Keywords Plant gene cloning tool enzyme gene cloning vector method of separation of cloning strategy 一、植物基因的结构和功能 基因（gene）是核酸分子中包含了遗传信息的遗传单位。一般来说，植物基因都可分为转录区和非转录的调控区两部分。 （一）植物基因的启动子 启动子（promoter）是指在位于结构基因上游决定基因转录起始的区域，植物积阴德启动子包括三个较重要的区域，一时转录起始位点，而是转录起始位点上游25~40bp的区域，三是转录起始位点上游-75bp处或更远些的区域。 （二）植物基因的增强子序列

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水稻基因的图位克隆技术 吴自明 (江西农业大学,作物生理生态与遗传育种教育部重点实验室,农业部双季稻生理生态与栽培重点实验室,江西省作物生理生态与遗 传育种重点实验室,江西南昌330045) 摘要 综述了水稻图位克隆技术的原理、技术环节及其在水稻基因克隆上的应用,分析了当前存在的主要问题,并对其应用前景作出了展望。 关键词 水稻;图位克隆;基因 中图分类号 S 511 文献标识码 A 文章编号 0517-6611(2008)34-14905-02 Map based C lo ning T echnique of R ice Genes WU Zi m ing (Key Laboratory of C rop Ph ysiology,Ecol ogy and Genetic B reeding,Mi nistry of Ed ucation,Key Laboratory of Ph ysiology,Ecology and C ultivati on of Double C roppin g Rice,Mini stry of Agriculture,Key Lab oratory of Crop Physi ology,Ecology an d Genetic Breedin g of Jian gxi Province,Jiangxi Agricultural Uni versi ty,Nanchang,Jiangxi 330045)Abstract The p rinciple an d tech nical links of m ap based gene cloni ng techniq ue i n rice an d its applications in the gene cloni ng of rice were s um ma rized .And the mai n existing problems at present were analyzed .And its applicati on foregrou nd was p redicted.Key w ords Rice;Map based cloni ng;Gene 基金项目 江西省教育厅项目(GJJ09477);江西农业大学博士启动基 金;江西农业大学校自然科学基金。 作者简介 吴自明(1974-),男,江西鄱阳人,副教授,从事植物分子 生物学研究。 收稿日期 2008 10 06 近年来,水稻基因组研究进展非常迅速,高密度遗传图谱和物理图谱的构建,全基因组序列的公布,数以万计ES T 、全长c DN A 等序列及功能分析数据的释放以及新型水稻分子标记及其高效检测技术的发展等,为基因的图位克隆带来了新的思路和方法。同时,这些新的进展也能够使基因的精细定位和物理图谱构建等相关工作大大简化,使基因的图位克隆朝着更加简便、快速的方向发展。1 图位克隆技术原理 图位克隆(Ma p based Cloning)又称定位克隆(Positional Cloning),1986年首先由剑桥大学的Alan Co ulson 提出[1]。用该方法分离基因是根据目的基因在染色体上的位置进行,无需预先知道基因的D N A 序列,也无需预先知道其表达产物的有关信息,而是通过分析突变位点与已知分子标记的连锁关系来确定突变表型的遗传基础。实现基因图位克隆的关键是筛选与目标基因连锁的分子标记。 近几年来,水稻各种分子标记的日趋丰富和各种数据库的日趋完善,在很大程度上得益于以粳稻日本晴和籼稻9311为代表的基因组测序的完成[2-3]。目前已有几十种技术可用于分子标记筛选,其中最常用的是简单序列长度多态性(SSLPs)、单核苷酸多态性(SNPs)和插入缺失多态性(Inser tio n/Deletio n,InDel)[4-7]。Shen 等利用日本晴和9311基因组序列构建了水稻基因组水平的D NA 多态性数据库,其中包括1703176个单核苷酸多态性(Single Nucleo tide Polymor phis m,SNP)和479406个插入缺失多态性(InDel)[8]。Fe ltus 等通过对除去多重拷贝序列及低质量序列后的日本晴和9311基因组草图的比对分析,得到408898个D N A 多态性,包括SNP 和单碱基I nD el [9],这些差别的核苷酸通常位于非编码区[10]。 目前,常把SNP 多态性转化成基于P CR 的剪切扩增多态性(Cle ave d Amplified P olymorphic Se que nc es,CAPS)或CAPS 衍生的dCAPS 标记[11-12]。CAPS 标记是PCR 反应和酶切相结 合产生的一种分子标记。如果不同材料间在PCR 扩增区域有S NP 位点,且该位点是限制性内切酶作用位点,那么不同 材料的PCR 扩增产物经特定的酶切后,再进行琼脂糖凝胶电泳就会表现多态性。当SNP 恰好位于限制性酶切位点比较少时,这种情况可以在CAPS 标记的基础上通过在扩增引物中引入错配碱基,则可以结合SNP 位点引入新的限制性内切酶作用位点,产生和CAPS 标记类似的多态性,这就是dCAPS 的方法。用CAPS 或dCAPS 的方法则可以将几乎所有的SNP 位点转化成以P CR 为基础的分子标记[12] 。 2 图位克隆技术环节 2.1 构建遗传作图群体 对于基因图位克隆而言,构建特殊的遗传作图群体是筛选与目标基因紧密连锁分子标记的关键环节。常用的作图群体有F 2、近等基因系、重组自交系等群体,水稻常用F 2群体。创建F 2群体应注意优先选择基因组已测序的日本晴、9311和培矮64S 等品种为亲本之一。2.2 基因初定位 一般说来,当标记为显性遗传时,欲获得最大遗传信息量的F 2群体,需借助于进一步子代测验,以分辨F 2中的杂合体。为此,Mic helmo re 等发明分离群体分组分析法(Bulke d Se gre ga nt Ana lysis,BS A)以筛选目标基因所在局部区域的分子标记[13]。其原理是将目标基因的F 2(或BC)代分离群体各个体仅以目标基因所控制的性状按双亲表型分为2群,每一群中各个体D N A 等量混合,形成2个D N A 混合池(如抗病和感病、不育和可育),并且用目的基因附近的所有分子标记对混合D NA 样品池进行分析,根据所有池中包含有交换的DN A 池的比例来确定与目的基因连锁最紧密的分子标记和目的基因附近所有分子标记的顺序。混合样品作图可以极大提高分子标记分析效率,减小D NA 提取工作量。 2.3 基因精细定位 一旦把目标基因初步定位在2个标记之间后,就可以从国际水稻基因组测序计划(IRG SP)公布的序列中下载这2个标记区域的部分P AC/BAC 克隆序列。利用软件SSRI T 搜索克隆序列中的微卫星序列,然后选择合适的微卫星序列进行特异PCR 引物的设计。也可以直接借助于公共数据库的序列进行比较,如寻找R GP 基因组数据库(粳稻)与中国华大基因组数据库(籼稻)的单核苷酸多态性(SNP)序列差异,设计CAPS 或dCAPS 标记和插入/缺失多态 安徽农业科学,J ou rn al of An hui Agri.Sci.2008,36(34):14905-14906 责任编辑 张彩丽 责任校对 张士敏

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拟南芥基因克隆的策略与途径 拟南芥（Arabidopsis thaliana）是一种模式植物，具有基因组小（125 Mbp）、生长周期短等特点，而且基因组测序 已经完成（The Arabidopsis Genomic Initiative, 2000）。同时，拟南芥属十字花科（Cruciferae），具有高等植物 的一般特点，拟南芥研究中所取得成果很容易用于其它高等植物包括农作物的研究，产生重大的经济效益，特别是十字 花科中还有许多重要的经济作物，与人类的生产生活密切相关，因此目前拟南芥的研究越来越多地受到国际植物学及各 国政府的重视。 基因(gene)是遗传物质的最基本单位,也是所有生命活动的基础。不论要揭示某个基因的功能,还是要改变某个基因的功 能,都必须首先将所要研究的基因克隆出来。特定基因的克隆是整个基因工程或分子生物学的起点。本文就基因克隆的 几种常用方法介绍如下。 1、图位克隆 Map-based cloning, also known as positional cloning, first proposed by Alan Coulson of the University of Cambridge in 1986, Gene isolated by this method is based on functional genes in the genome has a relatively stable loci, in the use of genetic linkage analysis or chromosomal abnormalities of separate groups will queue into the chromosome of a specific location, By constructing high-density molecular linkage map, to find molecular markers tightly linked with the aimed gene, continued to narrow the candidate region and then clone the gene and to clarify its function and biochemical mechanisms. 图位克隆（map-based clonig）又称定位克隆（positoinal cloning），1986年首先由剑桥大学的Alan Coulson提出。用该方法分离基因是根据功能基因在基因组中都有相对较稳定的基因座，在利用分离群体的遗传连锁分析或染色体异常将基因伫到染色体的1个具体位置的基础上，通过构建高密度的分子连锁图，找到与目的基因紧密连锁的分子标记，不断缩小候选区域进而克隆该基因，并阐明其功能和生化机制。 用该方法分离基因是根据目的基因在染色体上的位置进行的，无需预先知道基因的DNA序列，也无需预先知道其表达产物的有关信息。它是通过分析突变位点与已知分子标记的连锁关系来确定突变表型的遗传基础。近几年来随着拟南芥基因组测序工作的完成，各种分子标记的日趋丰富和各种数据库的完善，在拟南芥中克隆一个基因所需要的努力已经大大减少了（图1）。

13个水稻WRKY基因的克隆及其表达谱分析1

第49卷 第18期 2004年9月 论 文 1860 https://www.wendangku.net/doc/4516661047.html, 13个水稻WRKY 基因的克隆及其表达谱分析 仇玉萍①②* 荆邵娟①②* 付 坚①②* 李 璐① 余迪求①? (①中国科学院西双版纳热带植物园昆明分部, 昆明 650223; ②中国科学院研究生院, 北京 100039. * 同等贡献. ? 联系人, E-mail: ydq@https://www.wendangku.net/doc/4516661047.html,) 摘要 转录调控因子WRKY 蛋白拥有高度保守的氨基酸序列WRKYGQK 和Cys 2His 2或Cys 2HisCys 锌指型结构. 利用WRKY 蛋白质的保守结构域, 搜索了整个水稻基因组编码WRKY 蛋白质的基因, 鉴定了97个WRKY 基因, 并从4℃胁迫的水稻植株cDNA 文库中获得13个WRKY 基因全长cDNA 克隆. Northern blotting 分析结果显示, 其中10个WRKY 基因的表达受到NaCl, PEG, 低温(4℃)和高温(42℃)等4种非生物逆境因子胁迫的影响, 但其诱导表达模式不论在逆境因子种类还是在诱导时间上均存在着很大的差异, 这种基因诱导表达模式的差异可能体现于它们之间的生物学功能的差异. 关键词 水稻 转录调控因子WRKY 基因 非生物逆境因子 基因表达谱 转录调控因子WRKY 基因家族是植物特有的超级基因家族, 在其编码蛋白的N-端含有高度保守的氨基酸序列WRKYGQK 和Cys 2His 2或Cys 2HisCys 锌指型结构[1,2]. WRKY 蛋白特异地结合靶基因启动子区域的特异序列(T)TGACC(A/T)(W 盒)来调节靶基因的表达[3]. 许多研究工作证实, WRKY 基因调控植物许多重要的生命活动. 首先, WRKY 基因调控植物抗病反应的建立[4~10]. 比如, 拟南芥WRKY 基因家族第3组WRKY 基因成员分别参与植物不同保护信号转导途径的抗病反应[8]. 拟南芥RRS1-R(WRKY16) 蛋白拥有抗病基因产物典型的核苷结合位点(NBS)、富含亮氨酸重复序列(LRR)结构域特征以及WRKY 基因产物典型的入细胞核信号及DNA-结合结构域(WRKY)特征. 因此, WRKY16基因既作为典型的抗病基因参与拟南芥植株抵抗病原菌Ralstonia so-lanacearum 的侵入, 又作为典型的WRKY 基因参与拟南芥植株的抗病信号转导[9], 并将植物保护反应的战场扩展至细胞核[11]. 拟南芥WRKY70蛋白质激活水杨酸介导的植株抗病信号转导途径而抑制茉莉酸介导的植株抗病信号转导途径, 是此二条抗病信号转导途径的调控交叉点[10]. 其次, WRKY 蛋白质还调控植株非生物抗逆性反应的建立和部分形态建成[12~14]. 比如, 拟南芥WRKY6蛋白质通过特异地结合到衰老诱导受体样激酶(SIRK)基因启动子区域的W 盒序列而调节植物衰老和植物抗病性建立[12], 但拟南芥WRKY53参与拟南芥叶片衰老早期发生[13]. 拟南芥 WRKY44蛋白质调控叶片表皮毛和种子外表皮的发育[14]. 另外, 马铃薯WRKY 基因与马铃薯抗病性数量性状的形成有关, 并受晚疫病病原菌诱导表达[15,16]; 豆科WRKY 基因参与豆科植物抗旱性的建 立和种子休眠的形成[17]; 大麦SUSIBA2(WRKY )基因产物通过特异地结合到异构淀粉酶1(iso1: isoamy-lase1)基因启动子区域的SURE (sugar responsive)和W 盒序列而调控糖代谢信号转导[18]. 水稻是重要的农作物. 有关水稻WRKY 基因家簇的基因功能研究, 目前尚未有详细的文献报道. 随着水稻基因组核苷酸序列测定完成, 通过搜索水稻基因组核苷酸序列, 已鉴定出77个WRKY 基因, 并证实水稻WRKY71蛋白是糊粉层细胞内的赤霉素信号转导途径的转录抑制因子[19]. 在本研究中, 我们重新搜索了整个水稻基因组核苷酸序列, 鉴定了20多个额外的WRKY 基因, 并综合汇总所有水稻WRKY 基因的信息(表1). 在此基础上, 通过综合利用RT-PCR 和cDNA 文库筛选等分子生物学技术, 从水稻基因组中克隆获得13个WRKY 基因全长cDNA. 为了阐明WRKY 基因的分子生物学功能, 我们首先开展所获得的13个基因表达谱分析, 其中10个WRKY 基因受不同的非生物逆境因子诱导表达. 1 材料与方法 (ⅰ) 材料. 滇旬8号水稻种子由云南农业大学水稻研究所提供. [32P]dATP(>3000 Ci/mmol)购自北京福瑞生物工程公司. cDNA 合成和文库构建试剂盒购自Clontech 公司. 其他化学试剂购自上海生工公司和大连TaKaRa 生物工程公司. (ⅱ) 序列分析. 燕麦(Avena sativa )的AsWRKY3作为外类群, 用Megalign 5.01的Cluster method 的方法形成矩阵[20]. 同时, 用PAUP4.0b10进行系统发生重建分析[21], 采用邻接法搜索形成系统树, 对分支的可靠性评价采用了靴带分析(Bootstrap). 通过上述系统的分析研究, 我们获得水稻WRKY 蛋白质家族97

目的基因克隆之欧阳光明创编

一、目的基因克隆的策略有哪些？其理论依据什么？如何根据具体条件，如目的性状的特点，已知控制目的性状的基因的信息合理选择基因克隆的方法？ 欧阳光明（2021.03.07） 1、主要有以下几个克隆的策略： （1）PCR法分离目的基因：从蛋白质的一级序列分析得到核酸序列的相关信息，设计简并引物，通过对mRNA进行反转录得到cDNA，以cDNA为模板，然后将目的基因通过PCR方法扩增，或者直接从基因组DNA扩增的方法。 （2）核酸杂交的方法：通过对蛋白质的氨基酸序列分析，设计简并引物，通过核酸杂交的方法从基因文库中筛选得到目的基因。（3）免疫学筛选法分离目的基因：利用免疫学原理，通过目的蛋白的特异抗体与目的蛋白的专一结合，从表达文库中分离目的蛋白基因。 2、若控制该性状的目的蛋白质不容易分离纯化，这PCR方法比较适宜，若蛋白质分离纯化容易，且有现成的基因文库，则后两种方法较为简单。 二、蛋白组学方法克隆目的基因的理论依据是什么？有哪些技术环节？要用到哪些技术？ 1、理论依据：以分离纯化的目的蛋白为研究起点，通过对目的蛋白的一级结构分析，获得起码的氨基酸序列信息后，反推可能的

DNA序列，然后设计引物，从cDNA中将目的基因扩增出来，或者设计核酸探针，通过杂交技术将目的基因从基因文库中筛选出来。或通过抗体抗原免疫反应从表达文库中将该基因分离出来。 2、技术环节是确定并制备出高纯度的蛋白质。 3、所需要的实验技术有：蛋白质的双向电泳技术，由第一向的等电聚焦电泳和第二向的SDS-PAGE电泳组成；蛋白质氨基酸序列分析。 三、基因组学方法克隆基因的策略有哪些？各有什么特点？如何选择恰当的基因组学方法克隆目的基因？ 1、基因文库筛选方法 通过对基因文库的筛选将目的基因分离出来，一般有两种方法：核酸杂交法，原理是分子杂交；PCR筛选法，通过ＰＣＲ方法将目的基因分离出来，对于以混合形式保存的文库，先将文库分成几份，每份为一个“反应池”进行PCR反应，待选出阳性池后，将阳性池的混合克隆稀释，然后等量分置96孔板中，进行横向池及纵向池的ＰＣＲ反应，然后将阳性菌落群进行稀释，重复上述工作，直到筛出阳性单克隆。 2、图位克隆目的基因 利用分子标记技术对目的基因进行精细定位，利用分子标记技术对目的基因进行精细定位，用获得的与目的基因紧密连锁的分子标记筛选基因文库的方法。主要有染色体步移法、染色体登陆、跳查和连接法；外显子捕捉和cDNA直选法等。 3、插入突变分离克隆目的基因

肌动蛋白的克隆与鉴定

β—肌动蛋白的克隆与验证 李亚楠邹曾阳孟冠奇李军张建忠沈彤 摘要：Actin即“肌动蛋白”，是细胞的一种重要骨架蛋白。Actin在不同物种之间高度保守，以至于很难获得较好的针对actin的抗血清。Actin大致可分为六种，其中四种是不同肌肉组织特异性的，包括α-skeletal muscle actin，α-cardiac muscle actin，α-smooth muscle actin，和γ-smooth muscle actin；其余两种广泛分布于各种组织中，包括β-acti n(β-non-muscle)和γ-non-muscle actin。[1]β-Actin是横纹肌肌纤维中的一种主要蛋白质成分，也是肌肉细丝及细胞骨架微丝的主要成分。具有收缩功能，分布广泛。β-Actin是PCR常用的内参，β-Actin抗体是Western Blot 很好的内参指数。内参即是内部参照（Internal Control），对于哺乳动物细胞表达来说一般是指由管家基因编码表达的蛋白。它们在各组织和细胞中的表达相对恒定，在检测蛋白的表达水平变化时常用它来做参照物。[2] 本次实验主要通过PCR的手法来扩增目标蛋白。通过组织细胞提取DNA，用琼脂凝胶电泳来验证是否得到目标DNA；回收后的目标DNA 用PCR仪扩增，并用电泳进行回收；与T载体（PUD-18T）连接；用培养的大肠杆菌DH-5a来制备感受态细胞；将制备完成的感受态细胞均匀的涂布于含有x-gal和IPTG的混合液的LB培养基平板中进行蓝白斑的筛选；最后提取质粒DNA，经验证后保藏菌种。 关键词：β-肌动蛋白横纹肌蛋白质PCR 1材料与方法 1.1 组织细胞DNA提取。[3] 1.1.1 试剂：细胞裂解缓冲液、蛋白酶K、TE缓冲液、酚：氯仿：异戊醇（25:24:1）、7.5mol/l乙酸铵。 器材：胶头滴管、离心机、烧杯、动物组织、研钵、水浴。 1.1.2 方法 取新鲜或冰冻动物组织块0.1g，尽量剪碎，置于石英研钵中，加入1ml的细胞裂解缓冲液匀浆至不见组织块，转入1.5ml离心管中，加入蛋白酶K20微升，混匀。在65℃恒温水浴锅中水浴20min，间歇震荡离心管数次。于台式离心机以

基因克隆的几种常见方法

基因克隆得几种常见方法 基因（gｅｎe)就是遗传物质得最基本单位,也就是所有生命活动得基础。不论要揭示某个基因得功能,还就是要改变某个基因得功能,都必须首先将所要研究得基因克隆出来。特定基因得克隆就是整个基因工程或分子生物学得起点。本文就基因克隆得几种常用方法介绍如下。 1 根据已知序列克隆基因 对已知序列得基因克隆就是基因克隆方法中最为简便得一种。获取基因序列多从文献中查取，即将别人报道得基因序列直接作为自己克隆得依据。现在国际上公开发行得杂志一般都不登载整个基因序列,而要求作者在投稿之前将文章中所涉及得基因序列在基因库中注册，拟发表得文章中仅提供该基因在基因库中得注册号(acｃｅssion numｂer）,以便别人参考与查询。目前，世界上主要得基因库有１）EMBL,为设在欧洲分子生物学实验室得基因库，其网上地址为; （2)Genbank，为设在美国国家卫生研究院(ＮIＨ）得基因库，其网上地址为;（3)Swｉssport与TＲEMBＬ,Swissport就是一蛋白质序列库,其所含序列得准确度比较高,而TREMBＬ只含有从EＭBＬ库中翻译过来得序列。目前,以Geｎbａnk得应用最频繁。这些基因库就是相互联系得,在Genbank注册得基因序列,也可能在Swisspoｒt注册。要克隆某个基因可首先通过Inｔeｒnet查询一下该基因或相关基因就是否已经在基因库中注存。查询所有基因文库都就是免费得,因而极易将所感兴趣得基因从库中拿出来,根据整个基因序列设计特异得引物，通过PＣＲ从基因组中克隆该基因,也可以通过RＴ-PCＲ克隆cＤNA。值得注意得就是,由于物种与分离株之间得差异,为了保证PＣR扩增得准确性,有必要采用两步扩增法,即neｓted PCR。 根据蛋白质序列也可以将编码该蛋白质得基因扩增出来。在基因文库中注册得蛋白质序列都可以找到相应得ＤNA或cDNＡ序列。如蛋白质序列就是自己测定得，那么需要设计至少１对简并引物(dｅgｅneｒated ｐrimeｒ),从cDNA文库中克隆该基因。以这种方法克隆得基因必须做序列测定才能鉴别所扩增产物得特异性。 另外,在基因克隆之后，如还要进一步做表达研究，所使用得PCR酶最好不用Taq DNA聚合酶，而采用其她有自我检测（reading proｏf)功能得酶,如pfｕ。这样可以避免由于扩增过程中出现得点突变或终止密码子而导致整个研究结论得错误。 ２根据已知探针克隆基因 这也就是基因克隆得一种较直接得方法。首先将探针作放射性或非放射性标记,再将其与用不同内切酶处理得基因组DNＡ杂交,最后将所识别得片段从胶中切下来，克隆到特定得载体（质粒、噬菌体或病毒)中作序列测定或功能分析。这种方法不但可以将基因克隆出来,还能同时观察该基因在基因组中得拷贝数。