丹参SPL基因分子解析

Genome ‐wide analysis and molecular dissection of the SPL gene family in Salvia miltiorrhiza

Linsu Zhang 1–3,Bin Wu 2,Degang Zhao 1,Caili Li 2,Fenjuan Shao 2and Shanfa Lu 2*

1

Center for Research and Development of Fine Chemicals,Guizhou University,Guiyang 550025,China,2Institute of Medicinal Plant Development,the Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China,3Qiannan Medical College for Nationalities,Duyun 558003,China.*Correspondence:s ?u@https://www.wendangku.net/doc/e15813780.html,

Abstract SQUAMOSA promoter binding protein ‐likes (SPLs)are plant ‐speci ?c transcription factors playing vital regulatory roles in plant growth and development.There is no information about SPLs in Salvia miltiorrhiza (Danshen),a signi ?cant medicinal plant widely used in Traditional Chinese medicine (TCM)for >1,700years and an emerging model plant for TCM studies.Through genome ‐wide identi ?cation and subsequent molecular cloning,we identi ?ed a total 15SmSPLs with divergent sequence features,gene structures,and https://www.wendangku.net/doc/e15813780.html,parative analysis showed sequence conservation between SmSPLs and their Arabidopsis counterparts.A phylogenetic tree clusters SmSPLs into six groups.Many of the motifs identi ?ed commonly exist in a group/subgroup,implying their functional redundancy.Eight SmSPLs were predicted and experimentally validated to be targets of miR156/157.SmSPLs were differen-tially expressed in various tissues of https://www.wendangku.net/doc/e15813780.html,ltiorrhiza .The expression of miR156/157‐targeted SmSPLs was increased with

the maturation of https://www.wendangku.net/doc/e15813780.html,tiorrhiza ,whereas the expression of miR156/157was decreased,con ?rming the regulatory roles of miR156/157in SmSPLs and suggesting the functions of SmSPLs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza development.The expression of miR156/157was negatively correlated with miR172during the maturation of https://www.wendangku.net/doc/e15813780.html,tiorrhiza .The results indicate the signi ?cance and complexity of SmSPL ‐,miR156‐,and miR172‐mediated regula-tion of developmental timing in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .

Keywords:miR156;miR172;Salvia miltiorrhiza ;SQUAMOSA promoter binding protein domain;SQUAMOSA promoter binding protein ‐likes Citation:Zhang L,Wu B,Zhao D,Li C,Shao F,Lu S (2014)Genome ‐wide analysis and molecular dissection of the SPL gene family in Salvia miltiorrhiza .J Integr Plant Biol 56:38–50.doi:10.1111/jipb.12111Edited by:Jianmin Wan,Institute of Crop Science,CAAS,China Received Jul.26,2013;Accepted Sept.22,2013

Available online on Sept.30,2013at https://www.wendangku.net/doc/e15813780.html,/journal/jipb

?2013Institute of Botany,Chinese Academy of Sciences

INTRODUCTION

Plant gene expression is regulated by various factors,such as environmental signals,transcription factors (TFs),microRNAs (miRNAs),etc.TFs are a large class of regulators controlling gene expression at the transcriptional level,and usually serve as an on ‐off switch in the developmental process of eukaryotic organisms (Sun and Oberley 1996).The typical structure of TFs includes the DNA ‐binding domain,transcriptional regulatory domain,oligomerization domain,and the nuclear localization signal (NLS)(Liu et al.1999).The DNA ‐binding domain is relatively conserved and can speci ?cally bind to cis ‐elements.SPL (SQUAMOSA promoter binding protein ‐like)is a family of plant ‐speci ?c transcription factors (Chen et al.2010).It was ?rst identi ?ed in snapdragon (Klein et al.1996)and later found to be conserved in plants,such as Arabidopsis (Cardon et al.1999),rice (Xie et al.2006),and Populus trichocarpa (Lu et al.2011).SPL contains a conserved SBP (SQUAMOSA promoter binding protein)domain having two zinc ‐binding sites assembled as Cys ‐Cys ‐Cys ‐His and Cys ‐Cys ‐His ‐Cys,respec-tively (Yamasaki et al.2004),and a NLS partially overlapping with the second Zn ‐?nger located at the C ‐terminal of the SBP domain.SPL is encoded by a large gene family in plants.For instance,there are 16SPL genes in Arabidopsis thaliana (Cardon et al.1999),19in rice (Xie et al.2006),and at least 17in P.trichocarpa (Lu et al.2011).These genes play signi ?cant regulatory roles in various plant developmental processes,such as vegetative phase change (Cardon et al.1997),male fertility (Xing et al.2010),GA biosynthesis (Zhang et al.2007),plant architecture (Stone et al.2005),and in plant response to stress (Stone et al.2005).

MiRNAs are the other class of vital regulators of gene expression in plants,animals,and viruses (Lee et al.1993).They are small RNA molecules of about 21nucleotides in length derived from long primary transcripts (pri ‐miRNAs).In plants,miRNAs regulate plant development and response to biotic and abiotic stresses through direct cleavage of transcripts,translational repression,or epigenetic modi ?cation (Jones ‐Rhoades et al.2006;Sunkar and Zhu 2007;Chen 2012).miR156/157is one of the miRNA families highly conserved in plants (Axtell and Bowman 2008).It regulates plant development through direct cleavage of SPL transcripts.Among 16Arabidopsis SPLs ,10are miR156/157targets.It includes AtSPL2,AtSPL3,AtSPL4,AtSPL5,AtSPL6,AtSPL9,AtSPL10,AtSPL11,AtSPL13,and AtSPL15(Schwab et al.2005;Wu and Poethig 2006;Gandikota et al.2007;Addo ‐Quaye et al.2008).These miR156/157‐regulated AtSPLs play divergent and redundant roles in the morphology and development of Arabidopsis .For instance,AtSPL2,AtSPL10,and AtSPL11are closely related members of the SPL gene family.They redundantly control lateral organ development in the reproductive phase (Shikata et al.2009).AtSPL3,AtSPL4,and AtSPL5regulate ?oral transition (Cardon et al.1997;Jung et al.2011;Yu et al.2012).AtSPL6is able to activate the defense transcriptome and is a positive regulator in the TIR ‐NB ‐LRR receptor ‐mediated plant innate immunity (Padmanabhan et al.2013).AtSPL9and AtSPL15control shoot maturation (Schwarz et al.2008).In addition,AtSPL9is also involved in the production

and

F r e e A c c e s

s

R e s e a r c h A r t i c l

e

distribution of trichomes and the biosynthesis of anthocyanin (Yu et al.2010;Gou et al.2011).

Salvia miltiorrhiza,known as Danshen,has been widely used in Traditional Chinese medicine(TCM)for the treatment of dysmenorrhea,amenorrhea,and cardiovascular diseases (Cheng2006).It belongs to the Labiatae family and is an emerging model plant for TCM studies(Ma et al.2012). Transcription factors and their regulation roles in https://www.wendangku.net/doc/e15813780.html,tiorrhiza are poorly understood.In order to elucidate the regulatory networks of SPLs in DanShen growth and development,we performed a genome‐wide analysis and molecular dissection of the SmSPL gene family.It resulted in identi?cation of the?rst set of SmSPLs,providing a great example for understanding transcription factors and their regulatory roles in https://www.wendangku.net/doc/e15813780.html,tiorrhiza. RESULTS

Genome‐wide identification,molecular cloning and sequence feature analysis of SmSPL genes

In order to identify SPL genes in https://www.wendangku.net/doc/e15813780.html,tiorrhiza,we performed a genome‐wide prediction of SmSPLs by BLAST analysis of16 AtSPLs against the working draft of the https://www.wendangku.net/doc/e15813780.html,tiorrhiza genome (Chen S.et al.unpubl.data2010)using the tBLASTn algorithm (Altschul et al.1997).It resulted in the identi?cation of15SmSPL genes,which were named SmSPL1–SmSPL15,respectively.The number of SPL genes in https://www.wendangku.net/doc/e15813780.html,tiorrhiza is comparable with that in A.thaliana(Cardon et al.1999),rice(Xie et al.2006),and P. trichocarpa(Lu et al.2011),which contain16,19,and at least17 SPLs,respectively.It suggests that the number of duplication events of SPLs occurring in these plant species is similar.The length of SmSPL genes varies from757(SmSPL14)to5,201bp (SmSPL13)(Table1).The wide size range of SmSPL genes is consistent with that of AtSPL genes,varying from984(AtSPL5) to4,798bp(AtSPL14)(Table1).We further predicted gene models of15SmSPLs using Genscan(https://www.wendangku.net/doc/e15813780.html,/ GENSCAN.html),and manually corrected the predicted models by comparing with known plant SPLs using the BLASTx algorithm(https://www.wendangku.net/doc/e15813780.html,/BLAST).All of the deduced SmSPL proteins contain the conserved SBP domain, implying they are authentic SPLs.

To further verify the results from computational prediction of SmSPL gene models,we ampli?ed,cloned,and sequenced the coding region of all15SmSPL cDNAs using polymerase chain reaction(PCR)technology.All cloned SmSPL cDNAs have been submitted to GenBank.The accession numbers in GenBank are shown in Table1.Analysis of the experimentally con?rmed cDNA sequence of SmSPLs revealed that the length of SmSPL cDNAs varied between402(SmSPL15)and3,216bp (SmSPL1),which is consistent with the length of AtSPL cDNAs widely ranging from396(AtSPL3)to3,108bp(AtSPL14) (Figure1,Table1).Moreover,the number of introns varies between1and10in both SmSPL and AtSPL genes(Figure1, Table1).These results suggest the diverse of SPL gene structures in a plant species.

Comparative analysis of conserved domains and motifs in SmSPLs and AtSPLs

BLAST analysis of SmSPLs against the National Center for Biotechnology Information(NCBI)conserved domain database (https://www.wendangku.net/doc/e15813780.html,/Structure/cdd/wrpsb.cgi)showed that all SmSPLs contained an SBP domain,which was located in a region close to the N‐terminus and was encoded by the?rst two exons of SmSPL genes(Figure1).The location of SBP domains in SmSPLs is similar to that of Arabidopsis SPLs (Figure1),suggesting the conservation of SBP domain in two plant species.Sequence alignment of the SBP domain in SmSPLs showed many highly conserved amino acids (Figure S1),most of which were located in two zinc‐binding sites,Zn1and Zn2(Figure2).Zn1contains the CX4CX16CX2H (CCCH)or CX4CX16CX2C(CCCC)signature sequence,whereas Zn2contains the CX2CX3HX11C(CCHC)signature(Figure2).It is consistent with the results from Arabidopsis(Yamasaki et al.2004),indicating high conservation of zinc‐binding sites in the SBP domain in plants.In addition to zinc‐binding sites,a NLS overlapping with Zn2was also found in the SBP domain (Figure2).It is located in the region close to the C terminus of the SBP domain,and is a bipartite nuclear targeting sequence with the consensus sequence of KRX11RRRK(Figure2) (Robbins et al.1991).Similarly,many SPLs in Arabidopsis and rice also contain the NLS with KRX11RRRK consensus sequence (Birkenbihl et al.2005;Xie et al.2006).The conservation of SBP domain location,zinc‐binding sites and the NLS appear to be important for speci?c recognition and binding to cis‐elements in the promoter of nuclear genes(Birkenbihl et al.2005).

Four https://www.wendangku.net/doc/e15813780.html,tiorrhiza SPLs(SmSPL1,SmSPL9,SmSPL10,and SmSPL13)and four A.thaliana SPLs(AtSPL1,AtSPL12,AtSPL14, and AtSPL16)contain an ANK or Ank‐2domain with four or three ankyrin repeats(Table1),each of which usually contain about33amino acid residues forming two antiparallel helices separated by a beta‐hairpin(Michaely et al.2002).Ankyrin repeats mediate protein–protein interactions(Michaely and Bennett1992),indicating these SPLs may interact with other proteins for functions in plant cells.Although the ANK or Ank‐2 domain of https://www.wendangku.net/doc/e15813780.html,tiorrhiza and A.thaliana SPLs is less conserved compared with the SBP domain(Figures S1,S2),there are several highly conserved amino acid residues among SmSPLs and AtSPLs(Figure3).The signi?cance of these conserved amino acid residues is currently unknown and need to be further investigated(Michaely et al.2002).

In addition to the conserved SBP domain and ankyrin repeats,other conserved motifs were found in rice and Arabidopsis SPLs(Xie et al.2006;Guo et al.2008).Although the actual function of these motifs is currently unknown,they may be structural units and play important roles in plants.Thus,we performed a search of motifs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza and A.thaliana SPLs using the motif discovery tool MEME version3.0.14(Bailey and Elkan1994).It resulted in the identi?cation of20conserved motifs(Figure4,Table2).Motif1is actually the SBP domain. Motif2and motif8correspond to the ANK domain.Functions of the other motifs need to be further investigated. Phylogenetic analysis of SPLs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza and A.thaliana In order to analyze the evolutionary relationship of15SmSPLs and16AtSPLs,we constructed an unrooted neighbor‐joining tree using MEGA4(Tamura et al.2007).SmSPLs and AtSPLs were clustered into six groups,G1–G6(Figure5).G1–G3and G5 to include short SPLs with no more than465amino acid residues,whereas the members of G4and G6are longer and vary from774to1,040amino acids.G1is the largest group including four SmSPLs and six AtSPLs(Figure5,Table1).Based on the phylogenetic tree,it may be further divided into two subgroups,G1a(SmSPL2,SmSPL5,AtSPL2,AtSPL10,and AtSPL11)and G1b(SmSPL6,SmSPL11,AtSPL6,AtSPL9,and

The SPL gene family in Salvia miltiorrhiza39

AtSPL15)(Figure5,Table1).Except AtSPL6,all of SPLs in G1 contain motifs1and12,whereas G1a and G1b SPLs have an additional motif14and motif19,respectively.It suggests the signi?cance of these motifs and implies functional conserva-tion of SPLs within a subgroup.G1a includes two highly related SmSPLs,SmSPL2,and SmSPL5,and three Arabidopsis homologs, AtSPL2,AtSPL10,and AtSPL11,all of which contain three introns with intron phase2,1,and1,respectively(Figure1). AtSPL10and AtSPL11are tandem repeats in the Arabidopsis genome(Yang et al.2008),whereas the arrangement of SmSPL2and SmSPL5genes in the https://www.wendangku.net/doc/e15813780.html,tiorrhiza genome is currently unknown.Similarly,four of the?ve members of G1b, including SmSPL6,AtSPL6,AtSPL9,and AtSPL15,have the same number of intron and patterns of intron phase(Figure1).It suggests the close evolutionary relationship of SPLs in a subgroup.

Based on the phylogenetic relationship,eight members of G4were further grouped into G4a and G4b,each of which contains two SmSPLs and two AtSPLs(Figure5,Table1). Except SmSPL13having10introns,the other seven SPLs in G4 contain nine introns and share the same intron phase patterns (Figure1).G4SPLs commonly share10motifs,which include the ankyrin repeats(motifs2and8)involved in protein–protein interactions(Michaely and Bennett1992),suggesting it is important to interact with other proteins for the function of SPLs in this group.In addition to the common motifs in G4,there are another three(motifs5,17,and20)commonly found in G4a and four(motifs9,13,15,and18)in G4b(Figure4).The existence of group‐common motifs suggests functional redundancy of G4SPLs in some cellular processes,whereas subgroup‐speci?c motifs indicate the speci?city of G4a and G4b SPLs in the other plant cellular processes.It provides useful clues for elucidating the function of SPLs in G4.

All G5members,including SmSPL3,SmSPL8,SmSPL15,and AtSPL3–AtSPL5,are short SPLs with only one conserved motif (motif1)(Figure4).Furthermore,all of them contain an intron with the intron phase2(Figure1).The results suggest the conserved evolutionary relationship among G5SPLs and indicate functional conservation of these SPLs in plants. Consistently,in Arabidopsis,AtSPL4and AtSPL5are located in the duplicated genomic regions(Bowers et al.2003)and AtSPL3,AtSPL4,and AtSPL5redundantly regulate?owering time and phase change(Cardon et al.1997;Jung et al.2011;Yu et al.2012).

There are two SPLs in G2(SmSPL14and AtSPL13)and G3 (SmSPL12and AtSPL8),three(SmSPL4,SmSPL7,and AtSPL7) in G6(Figure5).The number of SPLs is relatively small compared with G1,G4,and G5,suggesting less duplication events occurred for G2,G3,and G6SPLs.G3members contain motif12conserved among9of10G1SPLs(Figure5). Consistently,AtSPL8plays redundant roles in male fertility with AtSPL2,AtSPL9,and AtSPL15,which belong to G1(Xing

Table1.Gene feature,structure and classi?cation of SPLs in Salvia mitirrohiza and Arabidopsis

Gene name Accession number No.intron Gene length(bp)CDS length(bp)No.domain Group miR156target SmSPL1KF43787794,2843,21624a

SmSPL2KF43787832,1811,38011a√SmSPL3KF43787911,53256715√SmSPL4KF43788095,1352,32516

SmSPL5KF43788132,1351,39811a√SmSPL6KF43788223,0511,08311b√SmSPL7KF43788394,1372,32516

SmSPL8KF437884194186115√SmSPL9KF43788594,8063,12314a

SmSPL10KF43788695,0672,91624b

SmSPL11KF43788712,8051,06511b√SmSPL12KF43788821,32093913

SmSPL13KF437889105,2012,85924b

SmSPL14KF437890175767812√SmSPL15KF437891191840215√AtSPL1AT2G4707094,1602,64624b

AtSPL2AT5G4327033,4691,26011a√AtSPL3AT2G3381011,07239615√AtSPL4AT1G5316011,19052515√AtSPL5AT3G15270198454615√AtSPL6AT1G6917022,1961,21811b√AtSPL7AT5G18830104,5782,45716

AtSPL8AT1G0206522,1321,00213

AtSPL9AT2G4220022,2241,12811b√AtSPL10AT1G2737033,0801,19111a√AtSPL11AT1G2736031,8991,18211a√AtSPL12AT3G6003093,9192,78424b

AtSPL13AT5G5057022,4771,08012√AtSPL14AT1G2098094,7983,10824a

AtSPL15AT3G5792021,7151,06511b√AtSPL16AT1G7658094,6842,96724a

40Zhang et al.

et al.2010).The members of G6belong to long SPLs with multiple conserved motifs (Figure 4).AtSPL7can directly bind to the conserved Cu ‐responsive element (CuRE)containing a core sequence of GTAC and is a signi ?cant regulator of Cu homeostasis in Arabidopsis (L?nnenp??et al.2004;Yamasaki et al.2009).It indicates SmSPL4and SmSPL7may be the key regulators for Cu homeostasis in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .miR156/157‐mediated posttranscriptional regulation of SPL genes in https://www.wendangku.net/doc/e15813780.html,tiorrhiza

A subset of SPL genes in Arabidopsis has been con ?rmed to be targets of miR156/157(Schwab et al.2005;Wu and Poethig 2006;Gandikota et al.2007;Addo ‐Quaye et al.2008).They belong to G1,G2,and G5,all of which are short SPLs (Figure 4,Table 1).With the online plant target prediction tool,known

as

Figure 1.Gene structures of SPLs in Salvia miltiorrhiza and Arabidopsis

Exons,introns,SQUAMOSA promoter binding protein (SPB)domains,ANK/ANK ‐2domains,and intron phases are

shown.

Figure 2.Sequence logo of the SQUAMOSA promoter binding protein (SPB)‐domain in SmSPLs

Bits represent the conservation of sequence at a position.Two zinc ?nger structures (Zn ‐1and Zn ‐2)and the nuclear localization signal (NSL)are shown.The SPL gene family in Salvia miltiorrhiza 41

psRNATarget (Dai and Zhao 2011),we screened SPL transcripts targeted by miR156/157in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .A total of eight SmSPLs were predicted to be targets of miR156/157(Figure 6).The complementary sites of miR156/157are in coding regions for ?ve SmSPLs (SmSPL2,SmSPL5,SmSLP6,SmSPL11,and SmSPL14)belonging to G1and G2,and one (SmSPL8)belonging to G5,whereas it locates in the 30UTR for the other two (SmSPL3and SmSPL15)belonging to G5(Figure 6).It is consistent with the results from Arabidopsis (Rhoade et al.2002;Yang et al.2008).Using the modi ?ed 50‐rapid ampli ?cation of cDNA ends (Lu et al.2005),we experimentally validated all eight SmSPLs to be authentic targets of miR156/157(Figure 6).Similar to Arabidopsis SPLs ,the eight SmSPLs regulated by miR156/157belong to G1,G2,and G5and all members in these groups are targets of miR156/157,suggesting the conservation of miR156/157‐mediated regulation of SPLs in plants.

Expression patterns of SmSPLs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza at different developmental stages

SPLs play signi ?cant regulatory roles in various developmental processes of Arabidopsis .In order to obtain preliminary information on the functions of SPLs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza ,we investigated the expression levels of SmSPLs in roots,stems and leaves of https://www.wendangku.net/doc/e15813780.html,tiorrhiza at different developmental stages,including 1‐and 3‐month ‐old and ?owering (Figure 7).All miR156/157‐targeted SmSPLs ,including SmSPL2(Figure 7B ),SmSPL5(Figure 7E ),SmSPL6(Figure 7F ),and SmSPL11(Figure 7K )belonging to G1,SmSPL14(Figure 7N )belonging to G2,and SmSPL3(Figure 7C ),SmSPL8(Figure 7H ),and SmSPL15(Figure 7O )belonging to G5,showed the increased expression levels with the maturation of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants.It is particularly true for the expression in leaves.Moreover,the expression of G5SmSPLs showed the highest expression level in ?owers (Figure 7C,H,O ).These results are consistent with those for miR156/157‐regulated AtSPLs (Wu and Poethig 2006;Wang et al.2009;Yu et al.2010).The other short SmSPL ,SmSPL12,which belongs to G3,is also highly expressed in ?owers (Figure 7L ).It is consistent with the role of its Arabidopsis homolog,AtSPL8,in the development of ?owers (Unte et al.2003;Zhang et al.2007;Xing et al.2010).However,we observed a high expression of SmSPL12in 1‐month ‐old young stems (Figure 7L ),indicating the function of SmSPL12in the development of organs other than ?owers.SmSPL1(Figure 7A ),SmSPL9(Figure 7I ),SmSPL10(Figure 7J ),and SmSPL13(Figure 7M ),long SPLs belonging to G4,showed more complicated expression patterns compared with miR156/157‐regulated SmSPLs .All of them showed increased expression in leaves with the maturation of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants,whereas the expression in stems was decreased.Moreover,the expression of SmSPL1(Figure 7A )and SmSPL10(Figure 7J )were increased with the maturation of roots,whereas SmSPL9(Figure 7I )and SmSPL13(Figure 7M )were decreased.The expression of SmSPL4and SmSPL7in the other long SPL group,G6,were more constant in all the tissues analyzed compared with G4SmSPLs ,except the expression of SmSPL4in 1‐month ‐old roots and SmSPL7in 1‐month ‐old stems (Figure 7D,G ).These results provide useful information for further elucidating the functions of SPLs in the development of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants.

Negative correlation of miR156/157and miR172expression in https://www.wendangku.net/doc/e15813780.html,tiorrhiza

In Arabidopsis ,a subset of miR156/157‐regulated AtSPLs ,such as AtSPL9and AtSPL10,activate the expression of miR172(Wu et al.2009).In order to know the expression patterns of miR156/157and miR172in the development of https://www.wendangku.net/doc/e15813780.html,tiorrhiza ,the levels of two mature miR156/157,miR156a and miR156b,and two mature miR172,miR172a,and miR172b,were analyzed using the miRNA ‐speci ?c qRT ‐PCR method (Shi and Chiang 2005).The levels of miR156a and miR156b decreased in roots,stems and leaves with the development of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants.Conversely,the levels of miR172a and miR172b increased (Figure 8).Negative correlation of the expression of miR156and miR172was also observed in Arabidopsis (Wu et al.2009),suggesting that miR156‐and miR172‐mediated regulation of developmental timing is conserved between Arabidopsis and https://www.wendangku.net/doc/e15813780.html,tiorrhiza .

DISCUSSION

Salvia is a large plant genus widely distributed in the world.It includes about 900species,many of which have great economic and medicinal value.For instance,https://www.wendangku.net/doc/e15813780.html,tiorrhiza Bunge,known as Danshen in Chinese,has been widely used

in

Figure 3.Sequence logo of the ANK/ANK ‐2domain in SmSPLs and AtSPLs

It includes SmSPL1,SmSPL9,SmSPL10,SmSPL13,AtSPL1,AtSPL12,AtSPL14,and AtSPL16.Bits represent the conservation of sequence at a position.

42Zhang et al.

Traditional Chinese medicine (TCM)for more than 1,700years (Cheng 2006).It is also the ?rst Chinese medicinal material entering the international market.Because of the relatively small genome size ($600Mb),short life cycle,undemanding growth requirements and signi ?cant medicinal value,https://www.wendangku.net/doc/e15813780.html,tiorrhiza is being developed to be a model plant for TCM studies (Ma et al.2012).A working draft of the https://www.wendangku.net/doc/e15813780.html,tiorrhiza genome has been recently obtained (Chen S.et al.unpubl.data 2010).However,the progress of biological studies on https://www.wendangku.net/doc/e15813780.html,tiorrhiza is signi ?cantly less compared with other model plants,such as Arabidopsis and rice.The function of transcription factors is poorly understood in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .Through a genome ‐wide identi ?cation and subsequent molec-ular cloning,we obtained the ?rst set of SmSPLs (Table 1),showing the existence of at least 15SmSPLs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .It provides a great example for understanding transcription factors and their regulatory roles in the growth and development of https://www.wendangku.net/doc/e15813780.html,tiorrhiza .Phylogenetic tree analysis showed that SmSPLs are clustered into six groups (Figure 5).Many sequence features are conserved between SmSPLs and their Arabidopsis homologs in the same group or subgroup (Figure 1),suggesting the close evolutionary relationship between SmSPLs and AtSPLs.Additionally,using the motif discovery tool,we identi ?ed a total of 20conserved motifs in SmSPLs and AtSPLs (Figure 4).Although most of them are functionally unknown,many commonly exist in a group or subgroup of SPLs,implying functional redundancy of SPLs in a group or subgroup.Consistently,AtSPL2,AtSPL10,and AtSPL11,which belong to G1a,redundantly control proper development of lateral organs in association with shoot maturation in the reproductive phase (Shikata et al.2009).G5AtSPLs,including AtSPL3,AtSPL4,and AtSPL5,activate the expression of LFY ,FUL ,and AP1genes (Wang et al.2009)and play redundant roles in reproductive transition (Cardon et al.1997;Gandikota et al.2007;Wang et al.2009;Yamaguchi et al.2009;Jung et al.2012;

Porri

Figure 4.Conserved motifs predicted by MEME

The SPL gene family in Salvia miltiorrhiza 43

Table 2.Consensus sequences of twenty motifs identi ?ed in SmSPLs Motif Length (aa)Consensus sequence

180PRCQVEGCXADLSXAKDYHRRHKVCEVHSKAPKVLVGGLEQRFCQQCSRFHLLSEFDEGKRSCRRRLAGH-NERRRKPQPD

280FLFRPDAAGPGGLTPLHLAASADGSEDVIDALTEDPQEVGIEAWKSARDATGFTPEDYARLRGHHSYIHLVARK-LADKPN

355IFDWLSNSPSEMESYIRPGCIILTIYLAMPESAWEELECDLLSSLKRLLDSSDDP 440SDSASDQSPSSSNSDAQSRTGRIVFKLFDKDPSEFPGRLR

5113KIRLSKSWRTWNLPELISVSPVAVVAGEETSLLLRGRSLTADGTRIHCTHAGGYNIMEVTASECRDTAFDELNLSSFKVN-GAASNFLGRCFIEVENGFRGDNFPLIIANATIC 629LYRPAIHSMLAIAAVCVCVALFFKSCPEV

737DDFPLERFKFLLEFSVDHDWCALVKKLLDILFEGNLG 829AALSEICLLHRAVRRNCKPMVEMLLHYSP

941SIKPIAFAAGEKAQFVVKGSNLLQPGFRLLCSFEGKYLAQE 1024KRSDEWDLNDWKWDGDLFEATPLN 1125GPDSREEALDFIHELGWLFHRKQLS 1224SVASAESLLGLKLGKRTYFEDXGG

1329FWTTGWIYARVQNQLAFVYNGQVVLDTSL

1439MVSNFKWDWENLIAFGPSATENPKKLQLTEWEIEDDEEI 1525PALSGRGFIEVEDQGGLSSFFPFII 1615GVFSPFSWELLDYGS

1742GNAEDRNAGFPAIPDKDQLLQILNKINALPLPANLASKLNNI

1852LLKILSNIHSNMSDHTGDQDLLSHLLKSLASQAGEHIERNLVELLQEGGDLQ 1921YHELENSRAYDSSGHHFNWSL

20

44

QDTRPSLSLQLFSSSPEDESRPKVASSRKYLSSASSNPSEDRSP

Figure 5.Phylogeny relationship of SmSPLs and AtSPLs

The unrooted neighbor ‐joining tree was constructed using MEGA4(Tamura et al.2007).G1–G6indicate the six groups identi ?ed.44Zhang et al.

et al.2012;Yu et al.2012).Moreover,SmSPLs probably play similar biological functions as their Arabidopsis counterparts in the same group or subgroup because of high sequence homology and the presence of conserved motifs.

AtSPL7belonging to G6,activates the expression of various genes associated with Cu homeostasis in Arabidopsis .It directly binds to the promoter regions known as Cu ‐responsive elements (CuREs),and is considered to be a central regulator for copper homeostasis (L?nnenp??et al.2004;Yamasaki et al.2009;Lu et al.2011).Only one homolog of AtSPL7was found in rice (Yang et al.2008),tomato,grape,and Physcomitrella patens (Li et al.2013).In this study,we identi ?ed two SmSPLs (SmSPL4and SmSPL7)clustering with AtSPL7(Figure 5).These SPLs commonly contain motifs 1,3,4,9,and 15.However,SmSPL4and AtSPL7contain an additional motif 6,while SmSPL7has an additional motif 2(Figure 4).Consistently,the expression patterns of SmSPL4and SmSPL7were similar in most tissues analyzed,while the levels of SmSPL4in 1‐month ‐old roots and SmSPL7in 1‐month ‐old stems were relatively high (Figure 7).It implies the redundant and speci ?c roles of SmSPL4and SmSPL7in the development of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants.

Although SmSPL4,SmSPL7,AtSPL7,and members of the other long SPL group,G4,are not regulated by miR156/157,almost all short SPLs except AtSPL8and SmSPL12were con ?rmed to be targets of miR156/157(Figure 6),suggesting the existence of distinct regulatory mechanisms for SPLs .AtSPL8regulates microsporangia development,megaspora-genesis,trichome formation on sepals,stamen ?lament elongation (Unte et al.2003).It also acts as a local regulator in a subset of GA ‐dependent developmental processes (Zhang et al.2007).The regulatory roles of AtSPL8in the development of anther and gynoecium are functionally redundant with AtSPL2,AtSPL9,and AtSPL15,which belong to G1and target by miR156/157(Xing et al.2010,2013).SmSPL12is the homolog of AtSPL8in https://www.wendangku.net/doc/e15813780.html,tiorrhiza (Figure 5),indicating it probably regulates the development of reproductive organs in https://www.wendangku.net/doc/e15813780.html,tiorrhiza with miR156/157‐targeted SPLs belonging to G1.Functional redundancy of miR156/157‐targeted and nontar-geted SPLs suggests the complexity and signi ?cance of SPL ‐related regulatory network.

All of the members of G1,G2and G5are regulated by miR156/157(Figure 6).In Arabidopsis ,G5SPLs,including AtSPL3,AtSPL4,and AtSPL5,redundantly regulate ?owering time and phase change through direct activation of a subset of transcription factor genes,such as LEAFY (LFY ),FRUITFULL (FUL ),and APETALA1(AP1)(Yamaguchi et al.2009),whereas various G1SPLs,such as AtSPL9,AtSPL10,and probably AtSPL11and AtSPL15,act redundantly in controlling ?owering time and phase change by directly activating the transcription of MIR172genes,which further promote plant development and ?owering (Wu et al.2009).Due to the conservation of AtSPLs and SmSPLs this regulatory mechanism may also exist in https://www.wendangku.net/doc/e15813780.html,tiorrhiza .Consistently,the expression of miR156/157‐regulated SmSPLs increased with the maturation of https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants,and the expression of miR156/157was negatively correlated with miR172(Figures 7,8).

Further

Figure 6.Experimental validation of miR156‐directed cleavage of SmSPLs

The cleavage sites of SmSPLs (A –H )were determined by the modi ?ed 50RNA ligase ‐mediated rapid ampli ?cation of cDNA ends (RACE).Heavy grey lines represent open reading frames (ORFs).The lines ?anking ORFs represent nontranslated regions.SQUAMOSA promoter binding protein (SPB)domains are indicated in blue.MiRNA complementary sites with the nucleotide positions of SmSPL cDNAs are indicated in green.The RNA sequence of each complementary site from 50to 30and the predicted miRNA sequence from 30to 50are shown in the expanded regions.Watson ‐Crick pairing is indicated by vertical dashes.Vertical arrows indicate the 50termini of miRNA ‐guided cleavage products,as identi ?ed by 50‐RACE,with the frequency of clones shown.The SPL gene family in Salvia miltiorrhiza 45

Figure 7.Differential expression of SmSPLs in Salvia miltiorrhiza

Relative expression of SmSPL1–SmSPL15(A –O )was quanti ?ed in total RNA isolated from roots (Rf),stems (Sf),leaves (Lf)and ?owers (F)of ?eld nursery ‐grown plants with ?owers and roots (R1and R3),stems (S1and S3)and leaves (L1and L3)of 1‐and 3‐month ‐old plants cultivated in vitro by quantitative real ‐time reverse transcription ‐polymerase chain reaction (RT ‐PCR)and normalized to the level of SmUBQ10in the sample.Fold changes of transcript level are shown.Error bars represent the standard deviations of three technical replicates.Normalized mRNA levels in ?owers were arbitrarily set to 1.46Zhang et al.

analysis of transgenic https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants with up ‐or downregulated SPLs ,miR156/157or miR172will help us understand the regulatory network of SmSPLs.

MATERIALS AND METHODS

Plant materials

Salvia miltiorrhiza Bunge (line 993)was grown in a ?eld nursery of the Institute of Medicinal Plant Development.Roots,stems,leaves,and ?owers were collected from 1‐year ‐old https://www.wendangku.net/doc/e15813780.html,tior-rhiza in August when the plants were blooming.The tissues were named Rf (roots from plants with ?owers),Sf (stems from plants with ?owers),Lf (leaves from plants with ?owers),and F (?owers),respectively,and then stored in liquid nitrogen until use.Roots,stems and leaves were also collected from 1‐and 3‐month ‐old https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants cultivated in vitro and named R1,S1,and L1,respectively,for 1‐month ‐old plants,and R3,S3,and L3,respectively,for 3‐month ‐old plants.

Database search and gene prediction

The nucleotide and amino acid sequence information of Arabidopsis AtSPLs were obtained from the Arabidopsis Information Resource (TAIR,https://www.wendangku.net/doc/e15813780.html,/).SmSPL genes were identi ?ed by tBLASTn analysis (Altschul et al.1997)of AtSPL protein sequences against the current assembly of the https://www.wendangku.net/doc/e15813780.html,tiorrhiza genome (Chen S.et al.unpubl.data 2010).Gene models of SmSPLs were predicted on the Genscan web server (https://www.wendangku.net/doc/e15813780.html,/GENSCAN.html )(Burge and Karlin 1998).The predicted models were manually corrected by comparison with known plant SPLs using the BLASTx algorithm (https://www.wendangku.net/doc/e15813780.html,/BLAST ).Analysis of gene structures,conserved domains and motifs Gene structures were analyzed using the Gene Structure Display Sever (https://www.wendangku.net/doc/e15813780.html,/index.php ).Con-served domains were analyzed by BLAST analysis of SmSPL protein sequences against the Conserved Domain Database (CDD,https://www.wendangku.net/doc/e15813780.html,/Structure/cdd/wrpsb.cgi )with the expected E ‐value threshold of 0.01and the maximum size of hits to be 500amino acids (Marchler ‐Bauer et al.2011).Sequence logos were created using WebLogo (https://www.wendangku.net/doc/e15813780.html,/).Protein alignment of SBP and ANK domains were carried out using DNAMAN version 6(Lynnon BioSoft).Conserved motifs were predicted using MEME version 3.0.14(Bailey and Elkan 1994,Bailey et al.2009).

Phylogenetic analysis

A phylogenetic tree was constructed with the full ‐length SmSPL and AtSPL protein sequences using MEGA version 4.0by the neighbor ‐joining method with 1,000bootstrap replicates (Tamura et al.2007

).

Figure 8.Negative correlation of miR156and miR172expression in Salvia miltiorrhiza

Relative expression of miR156a (A ),miR156b (B ),miR172a (C )and miR172b (D )was quanti ?ed in total RNA isolated from roots (Rf),stems (Sf),leaves (Lf)of ?eld nursery ‐grown plants with ?owers and roots (R1and R3),stems (S1and S3)and leaves (L1and L3)of 1‐and 3‐month ‐old plants cultivated in vitro by quantitative real ‐time reverse transcription ‐polymerase chain reaction (RT ‐PCR)and normalized to the level of 5.8S rRNA in the sample.Fold changes of transcript level are shown.Error bars represent the standard deviations of three technical replicates.Normalized mRNA levels in R1were arbitrarily set to 1.

The SPL gene family in Salvia miltiorrhiza 47

RNA isolation

Total RNA was extracted from roots,stems,leaves and?owers of https://www.wendangku.net/doc/e15813780.html,tiorrhiza using the EASYspin Plant microRNA Extract kit (Aidlab Biotechnologies,Beijing,China).The quality and quantity of total RNA was analyzed with agarose gel electrophoresis and nanodrop2000spectrophotometer (Thermo Scienti?c,Wilmington,DE,USA).Genomic DNA was removed by treating total RNA with RNase‐free DNase (Promega,Madison,WI,USA).

Molecular cloning of SmSPLs

The50and30regions of SmSPL8and SmSPL14,showing low sequence homology with known plant SPLs,were ampli?ed by the50and30rapid ampli?cation of cDNA ends(RACE)method with the GeneRacer kit(Invitrogen,Carlsbad,CA,USA).Plant mRNA was isolated using the Oligotex mRNA Mini kit(Qiagen, Hilden,Germany).Total RNA was reverse‐transcribed into cDNA using Superscript III reverse transcriptase(Invitrogen).50 and30RACE was performed according to the instruction of GeneRacer kit using gene‐speci?c nesting and nested primers listed in Table S1.The full‐length coding regions of SmSPLs were ampli?ed by PCR using the gene‐speci?c forward and reverse primers listed in Table S1.PCR products were gel‐puri?ed, cloned,and then sequenced.

Prediction and experimental validation of SmSPLs targeted by miR156

SmSPLs targeted by miR156were predicted using psRNATarget (Dai and Zhao2011,https://www.wendangku.net/doc/e15813780.html,/psRNATarget/? function?3)with the maximum expectation of3and the target accessibility(UPE)of50.For experimental validation,total RNA was extracted from https://www.wendangku.net/doc/e15813780.html,tiorrhiza tissues using the EASYspin Plant microRNA Extract kit(Aidlab Biotechnologies).mRNA was isolated using the Oligotex mRNA mini kit(Qiagen).The modi?ed RNA ligase‐mediated rapid ampli?cation of50cDNAs method(50RLM‐RACE)was performed using the GeneRacer kit (Invitrogen)as described previously(Lu et al.2005).Gene‐speci?c primers used are listed in Table S2.

Quantitative real‐time PCR

RNase‐free DNase‐treated total RNA were reverse‐transcribed into cDNA using Superscript III reverse transcriptase(Invi-trogen).Quantitative real‐time PCR(qRT‐PCR)of SmSPLs was performed as described previously(Ma et al.2012).Gene‐speci?c primers were designed using the IDT online tools (https://www.wendangku.net/doc/e15813780.html,/site)and listed in Table S3.The expressions of miR156and miR172were analyzed using the miRNA‐speci?c qRT‐PCR method as described previously(Shi and Chiang2005).Primers are shown in Table S3. ACKNOWLEDGEMENTS

We thank Dr Shilin Chen and the sequencing group in our institute for kindly providing the Salvia miltiorrhiza genome sequence.We appreciate Professor Xian’en Li for providing https://www.wendangku.net/doc/e15813780.html,tiorrhiza plants.This work was supported by grants from the Beijing Natural Science Foundation(Grant No.5112026to S.L.),the Major Scienti?c and Technological Special Project for Signi?cant New Drugs Creation(Grant No.2012ZX09301002‐001‐031to S.L.),the Research Fund for the Doctoral Program of Higher Education of China(20111106110033to S.L.),the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,Grant No.IRT1150),and the Program for Xiehe Scholars in Chinese Academy of Medical Sciences&Peking Union Medical College(to S.L.). REFERENCES

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Additional supporting information can be found in the online version of this article:Figure S1.Protein sequence alignment of the SBP domain identi?ed in15SmSPLs.

Figure S2.Protein sequence alignment of the ANK domain identi?ed in SmSPL1,SmSPL9,SmSPL10,SmSPL13,AtSPL1, AtSPL12,AtSPL14,and AtSPL16.

Table S1.Gene‐speci?c primers used for PCR ampli?cation of SmSPLs.

Table S2.Primers used for50RLM‐RACE validation of SmSPLs targeted by miR156.

Table S3.Gene‐speci?c primers used for quantitative real‐time PCR of SmSPLs,miR156a/b and miR172a/b.

50Zhang et al.

基因工程在医学上的应用

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第五章 单基因遗传与单基因病(答案)

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(完整版)高中生物选修3第一章基因工程习题及答案

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核酸和蛋白质序列分析 在获得一个基因序列后，需要对其进行生物信息学分析，从中尽量发掘信息，从而指导进一步的实验研究。通过染色体定位分析、内含子／外显子分析、ORF分析、表达谱分析等，能够阐明基因的基本信息。通过启动子预测、CpG 岛分析和转录因子分析等，识别调控区的顺式作用元件，可以为基因的调控研究提供基础。通过蛋白质基本性质分析，疏水性分析，跨膜区预测，信号肽预测，亚细胞定位预测，抗原性位点预测，可以对基因编码蛋白的性质作出初步判断和预测。尤其通过疏水性分析和跨膜区预测可以预测基因是否为膜蛋白，这对确定实验研究方向有重要的参考意义。此外，通过相似性搜索、功能位点分析、结构分析、查询基因表达谱聚簇数据库、基因敲除数据库、基因组上下游邻居等，尽量挖掘网络数据库中的信息，可以对基因功能作出推论。上述技术路线可为其它类似分子的生物信息学分析提供借鉴。本路线图及推荐网址已建立超级链接，放在北京大学人类疾病基因研究中心网站 （https://www.wendangku.net/doc/e15813780.html,/science/bioinfomatics.htm）,可以直接点击进入检索网站。 下面介绍其中一些基本分析。值得注意的是，在对序列进行分析时，首先应当明确序列的性质,是mRNA序列还是基因组序列？是计算机拼接得到还是经过PCR扩增测序得到？是原核生物还是真核生物？这些决定了分析方法的选择和分析结果的解释。 （一）核酸序列分析 1、双序列比对（pairwise alignment） 双序列比对是指比较两条序列的相似性和寻找相似碱基及氨基酸的对应位置，它是用计算机进行序列分析的强大工具，分为全局比对和局部比对两类，各以Needleman-Wunsch算法和Smith-Waterman算法为代表。由于这些算法都是启发式（heuristic）的算法，因此并没有最优值。根据比对的需要，选用适当的比对工具，在比对时适当调整空格罚分（gap penalty）和空格延伸罚分（gap extension penalty），以获得更优的比对。 除了利用BLAST、FASTA等局部比对工具进行序列对数据库的搜索外，我们还推荐使用EMBOSS软件包中的Needle软件 （http://bioinfo.pbi.nrc.ca:8090/EMBOSS/），和Pairwise BLAST

基因工程在疾病治疗方面的应用

浅谈基因工程药物 基因工程药物是指用现代基因重组高科技对基因进行克隆，通过重组DNA导入大肠杆菌、酵母或动物细胞成功构建工程菌株或细胞株，在工程菌株、细胞中所表达生产的新型药物包括细胞因子、多肽类激素、溶血栓药物、疫苗、抗体、反义RNA及基因治疗药物等等多种难治疾病的基因工程药物. 基因工程药物因其疗效好、应用范围广泛、副作用小的特点成为新药研究开发的新宠。也是发展最迅速和最活跃的领域。自1982年美国Lilly公司上市了第一个基因工程产品——人胰岛素以来，至今已有基因工程药物大约140多种上市，尚处于临床试验或申报阶段的基因工程药物有500多种。当传统制药业的增长速度减慢时，基因工程制药正在加速发展，全世界基因工程药物持续6年销售额增长率都在l5％～33％，基因工程制药已成为制药业的一个新亮点[1-2]。 一.目前药物治疗的主要类型 1.胰岛素至今仍是临床上治疗糖尿病最有效的方法。 过去，胰岛素主要从猪等大家畜胰腺中提取。从一头猪的胰腺中只能提取出300单位胰岛素，而一个病人每天就需要40单位胰岛素，因此远远不能满足需要。 基因工程技术一问世，科学家就想到利用该技术来解决胰岛素药源不足的问题。他们首先要找到胰岛素基因，在人的胰岛细胞里有一段特定结构的DNA分子指挥着胰岛素的合成，然后又找到在人的大肠里存在对人体无害的大肠杆菌。把人的胰岛素基因转入到大肠杆菌的细胞中，随着大肠杆菌的繁殖，胰岛素基因

也一代代的遗传下去。大肠杆菌繁殖速度相当快，大约20分钟就能繁殖一代，把它放到大型的发酵罐里进行人工培养，就可以大量繁殖，并且生产出大量人的胰岛素。 1981年，基因重组人胰岛素产品正式投入市场，大肠杆菌成了名副其实的生产胰岛素的“活工厂”，胰岛素供不应求的问题彻底解决了 胰岛素是治疗糖尿病的特效药，长期以来只能依靠从猪、牛等动物的胰腺中提取，100Kg胰腺只能提取4-5g的胰岛素，其产量之低和价格之高可想而知。将合成的胰岛素基因导入大肠杆菌，每2000L培养液就能产生100g胰岛素!大规模工业化生产不但解决了这种比黄金还贵的药品产量问题 2.干扰素： 是哺乳动物细胞在诱导下产生的一种淋巴因子，能够加强巨噬细胞的吞噬作用和对癌细胞的杀伤作用，抑制病毒在细胞内的增殖，用于肿瘤和其他病毒病的治疗。基因工程干扰素干扰素治疗病毒感染简直是“万能灵药”!过去从人血中提取，300L血才提取1mg!其“珍贵”程度自不用多说。基因工程人干扰素α-2b(安达芬)是我国第一个全国产化基因工程人干扰素α-2b，具有抗病毒，抑制肿瘤细胞增生，调节人体免疫功能的作用，广泛用于病毒性疾病治疗和多种肿瘤的治疗，是当前国际公认的病毒性疾病治疗的首选药物和肿瘤生物治疗的主要药物。 生长激素人体生长激素能够治疗侏儒症和促进伤口愈合，动物生长激素能够加速畜禽生长发育。目前，人和动物的生长激素基因都已经在大肠杆菌中成功表达．在医学和畜牧业领域取得了很好的应用效果。

基因序列分析word版

南开大学数学院“学而思”杯数学建模比赛 编号专用页 赛区评阅编号（由赛区组委会评阅前进行编号）： 全国统一编号（由赛区组委会送交全国前编号）： 全国评阅编号（由全国组委会评阅前进行编号）：

A 题：基因序列分析 摘要 本文通过对比HIV病毒基因序列，找出不同阶段的DNA基因序列的异同，进而分析基因位点的相关性，从而对比找出HIV病毒基因序列中较为重要的位点，为HIV病毒研究提供更多的研究方法与思路。 针对问题一：我们利用点矩阵分析及统计各碱基含量的百分比的方法，对比两文件中具有相同序列名的基因序列及具有不同序列名的基因序列，找出两者的异同，得出结论。两者的相似性表现在：同名序列具有子序列关系，不同名序列具有相当的相似性，各种碱基的含量具有稳定性。两者的不同点表现在：基因规模有很大差异，不同名序列出现了具有突变特点的基因序列差异。 针对问题二：我们首先利用DNAwalk法对HIV病毒基因序列位点进行分析，在分析的过程中发现由于基因和基因组序列中存在着高度的不均一性，即不同位置的碱基密度存在着很大的差异，因而DNAwalk法不太适合基因序列的分析，转而使用DFA模型对HIV 基因的相关性进行分析和度量，得出了与DNAwalk模型相同的结论。 针对问题三：在前两问的分析基础上，结合前两问的分析结果及HIV病毒高度变异性的特点，我们得出重要的基因位点应满足下列条件：1、该基因位点位于Ⅱ基因序列，2、该基因位点所在序列的序列名应不同于Ⅰ中的序列名，3、该基因位点在问题二的分析中具有较高的相关性。 关键字：矩阵分析 DNAwalk DFA模型

问题重述 人类免疫缺陷病毒(Human Immunodeficiency Virus，HIV)，简称艾滋病病毒，会造成人类免疫系统的缺陷, 导致艾滋病（AIDS）. HIV基因组翻译成蛋白的过程相对复杂, 它会重复交叉使用某些基因片段。病毒序列在进化和传播的过程中主要是envelope 基因变化很快。详细描述可见HIV的生活史。由于现有的抗艾滋病病毒药对HIV无法根治，因此就将“责任”归咎高变异性. 目前, 很多的HIV序列已经被测定出来, 附件给出了一些HIV的序列. 我们试图通过对HIV序列的分析来断定这些序列上哪些位置比较重要, 从而给艾滋病的研究一些帮助. 例如, 某些位置上的突变可能会影响到HIV的传播机制, 如果我们瞄准这些位置设计药物, 可能会对艾滋病的传播起到抑制作用. HIV基因组序列大约长10k，HIV1_GENOME_DNA.fasta包含了1400余条基因组的序列，因为在序列突变的过程中，有一些核酸会消失，这些消失的核酸在文件中使用”-“来表示。表示此处发生了一次删除突变。也就是说, 文件中所有序列都是”对齐”的. 这样, 我们可以知道这些序列中某一个特定位点上核酸的分布情况. 另外，HIV基因组中包含了若干个编码蛋白质的基因，编码后的蛋白质可以行使病毒传播，致病等功能。HIV1_ENV_DNA.fasta是其中一个编码蛋白质基因的序列，HIV1_ENV_PRO.fasta是编码后的蛋白序列。它们同样是已经比对好的。基于以上说明，我们来分析如下问题： (1)对于HIV1_ENV和HIV_GENOME的DNA序列，构造数学方法对序列的位点进行分析， 指出这两者之间的异同。 (2)HIV序列位点之间或者某些位点之间是否存在相关性？如果存在，那么如何去度 量这种相关性？ (3)对这些序列进行进一步的分析，找到你认为的HIV中较为重要的位点，并说明这 些位点为什么重要。 知识背景 本文通过对HIV病毒的基因信息进行分析，从而得出HIV病毒基因中比较重要的位点，由于本问题专业性较强，所以我们将先对其中相关知识做出阐述： 1、名词解释： 基因组：Genome,生物所携带的遗传信息的总和，即单倍体细胞中包括编码序列和非编码序列在内的全部DNA分子。 基因位点：基因在染色体上占有的特定位置。 染色体：由脱氧核糖核苷酸、蛋白质和少量核糖核酸组成的线状或棒状物，是生物主要遗传物质的载体。因是细胞中可被碱性染料着色的物质而得名。 核糖体：结合着辅助蛋白质因子的多个核糖体RNA(rRNA)亚基组成的细胞器。 碱基：指嘌呤和嘧啶的衍生物，是核酸、核苷、核苷酸的成分。 2、一般细胞遗传信息传递相关原理 DNA转录成RNA，RNA再被翻译成蛋白质执行相应的功能。DNA碱基的序列决定了蛋白质的结构，但DNA并非直接翻译成蛋白质，基因组DNA先通过转录生成信使RNA(mRNA)，单链的mRNA随后将离开细胞核，指导蛋白质的合成。这一过程称为翻译，由核糖体负责完成。构成蛋白质的20种氨基酸通过转运RNA（tRNA）的作用到达核糖体，在核糖体的作用下，mRNA分子的核苷酸序列被翻译成相应的氨基酸，形成肽键。

基因工程在医药工业中的的应用

基因工程及其在医学中的应用基因工程及其在医学中的应用基因工程及其在医学中的应用基因工程及其在医学中的应用 摘要: 作为生物工程技术的核心，及新工程的发展与应用，在医学方面有着非同凡响的影响。本文首先回顾了基因工程的发展简史，然后在基因工程制药，抗病毒疫苗，疾病治疗及基因诊病等方面综述了基因工程在医学中的应用。基因工程将给医药方面带来更美好的前景。关键词关键词关键词关键词: 基因工程医学应用1 前言前言前言前言：分子生物学主要是从分子水平上阐述生命现象和本质的科学，是现代生命科学的“共同语言”。分子生物学又是生命科学中进展迅速的前沿学科，它的理论和技术已经渗透到其他基础生物学科的各个领域，它的主要核心内容是通过生物的物质基础---核酸、蛋白、酶等生物大分子的结构、功能及其相互作用的运动规律的研究来阐明生命分子基础，从而探讨生命的奥秘。这门课与基因工程关系很大，主要讲了核酸、蛋白、酶等生物大分子的结构、功能以及它们之间的相互作用。近年来，随着生物技术的飞速发展，分子生物学在较多领域得以应用。其中在核酸，基因方面医学中的发展迅猛。基因工程在制药，抗病菌疫苗发展前景较广，在疾病治疗及诊断对人们生活影响较大。本文将对基因工程的发展及其在医学中的应用作简单的阐述。2 基因工程的发展基因工程的发展基因工程的发展基因工程的发展基因工程又叫遗传工程，是分子遗传学和工程技术相结合的产物，是生物技术的主体。基因工程是指用酶学方法将异源基因与载体DNA在体外进行重组，将形成的重组因子转入受体细胞，使异源基因在其中复制并表达，从而改造生物特性，生产出目标产物的高新技术。1857年至1864年，孟德尔通过豌豆杂交试验，提出了生物体的性状是由遗传基因子控制的。1909年，丹麦生物学家约翰生首先提出基因一词代替孟德尔的遗传因子。1910年至1915年，美国遗传学家摩尔根通过果蝇实验，首次将代表某一性状的基因同特定的染色体联系起来，创建了基因学说。直到1944年，美国微生物学家埃弗里等通过细菌转化研究，证明基因的载体是DNA 而不是蛋白质，从而确立了遗传的物质基础。1953年，美国的遗传学家华生和英国的生物学家克里克揭示了DNA分子双螺旋模型和半保留复制机理，解决了积阴德自我复制和传递问题。开辟了分子生物学的研究时代。之后，1958年克里克确立了中心法则。1961年雅各和莫诺德提出的操纵子学说以及说有64种密码子的破译，成功的揭示了遗传信息的流向和表达问题，为基因工程的发展奠定了坚实的基础。DNA分子的切除与连接，基因的转化技术，还有诸如核酸分子杂交，凝胶电泳，DNA序列结构分析等分子生物学试验方法的进步为基因的创立和发展奠定了强有力的技术基础。1972年，美国斯坦福大学的P.Berg构建了世界上第一个重组分子，发展了DNA重组技术，并因此获得了1980年的诺贝尔学奖。1983年，美国斯坦福大学的S.Chen等人也成功的进行了另一个体外DNA重组试验并发现了细菌间性状的转移。这是基因工程发展史上第一次成功实现重组转化成功的例子，基因工程从此诞生了。基因工程问世近30年，不论是基因理论研究领域，还是在生产实践中的应用，均已取得了惊人的成绩。给国民经济的发展和人类社会的发展带来了深远而广泛的影响。3 基因工程在药学方面的应用基因工程在药学方面的应用基因工程在药学方面的应用基因工程在药学方面的应用运用基因工程技术对基因的转导和整合来获取新的抗体，及新药的制取及研究都具有较高效益；基因技术在诊断疾病及刑事案件的侦破方面发挥着不可小觑的力量，因此基因工程在药学发展有着深远影响。 3.1 基因工程制药基因工程制药基因工程制药基因工程制药基因工程制药开创了制药工业的新纪元，解决了过去不能生产或者不能经济生产的药物问题。现在，人类已经可以按照需要，通过基因工程生产出大量廉价优质的新药物和诊断试剂，诸如人生长激素、人的胰岛素、尿激酶、红细胞生成素、白细胞介素、干扰素、细胞集落刺激因子、表皮生长因子等。令人振奋的是，具有高度特异性和针对性的基因工程蛋白质多肽药物的问世，不仅改变了制药工业的产品结构，而且为治疗各种疾病如糖尿病、肾衰竭、肿瘤、侏儒症等提供了有效的药物。 3.2 基因工程抗病毒疫苗基因工程抗

单基因遗传病的遗传方式判断和概率计算

单基因遗传病的遗传方式判断和概率计算 【课标扫描】 1、 了解人类遗传病的类型（A ） 2、掌握单基因遗传病遗传方式的判断（C ） 3、掌握单基因遗传病遗传概率的计算（C ） 【复习内容】 一、单基因遗传病遗传方式的判断 例题1：根据遗传系谱图，分别判断下列遗传病的遗传方式(■●患病男女,□○正常男女)： A 的遗传方式为 。 B 的遗传方式为 。 C 的遗传方式为 。 D 的遗传方式为 。 E 的遗传方式为 。 方法归纳： 第一、判断遗传病的显隐性 ①双亲都正常，子代有患病→隐性（即“无中生有为隐性”） ②双亲都患病，子代有正常→显性（即“有中生无为显性”） 第二、判断是常染色体遗传还是伴性遗传 一般是通过先排除伴性遗传来确定为常染色体遗传 ①能排除：只能为常染色体遗传 ②不能排除：常染色体遗传、X 染色体遗传都有可能，可再结合题干信息进行判断 对应训练1：根据遗传系谱图，分别判断下列遗传病的遗传方式(其中乙图的7号个体不是携带者, ■●患病男女, □○正常男女)： 甲的遗传方式为 。 乙的遗传方式为 。 B A D C 乙 甲

高二生物复习教学案 江苏省昆山中学 赵姬 2008.12.26 对应训练2：根据遗传系谱图，分别判断下列遗传病的遗传方式： 甲的遗传方式为 。 乙的遗传方式为 。 丙的遗传方式为 。 丁的遗传方式为 。 对应训练3：下列最可能反映红绿色盲的遗传系谱图是 （ ） 二、单基因遗传病遗传概率的计算 例题2：A 图中5号个体患甲病（用A 、a 表示）的概率为多少？ 若2号不是乙病的携带者， B 图中5号个体患乙病（用B 、b 表示）的概率为多少？ 若2号不是乙病的携带者，C 图中5号个体患病的概率又为多少？ A 4 甲病女性 B 4 乙病男性 C 4 甲病女性 乙病男性

基因工程之基因治疗

基因治疗 摘要: 生物技术在生命科学领域扮演者重要得角色,基因治疗在治疗方面,将新得遗传物质转移到某个个体得体细胞内使其获得治疗效果;在基因工程方面,将正常得有功能得基因置换或增补缺陷基因。近些年来,已对若干人类单基因遗传病与肿瘤开展了临床得基因治疗。基因治疗作为治疗疾病得一种新手段,正愈来愈受到人们得重视与关注。 关键词:基因工程基因治疗基因 一、基因治疗得历史 随着DNA双螺旋结构得发现与以DNA重组技术为代表得现代分子生物学技术得发展以及人类对疾病认识得不断深入,越来越多得证据证明,多种疾病与基因得结构或功能改变有关,因而萌生了从基因水平治疗疾病得念头与梦想。 早在1968年,美国科学家发表了“改变基因缺损:医疗美好前景”得文章,首次在医学界提出了基因疗法得概念。1989年美国批准了世界上第一个基因治疗临床试验方案。1990年美国NIH得Frenuch Anderson博士开始了世界上第一个基因治疗临床试验,用腺苷酸脱氨酶基因治疗了一位ADA基因缺陷导致得严重免疫缺损得四岁女孩,并获得成功[1]。 1994年美国科学家利用经过修饰得腺病毒为载体,成功地将治疗遗传性囊性纤维化病得正常基因cfdr 转入患者肺组织中。2000年,法国巴黎内克尔儿童医院利用基因治疗,使数名有免疫缺陷得婴儿恢复了正常得免疫功能,取得了基因治疗开展近十年最大得成功[2]。 2004年1月,深圳赛百诺基因技术有限公司将世界第一个基因治疗产品重组人p53抗癌注射液正式推向市场,这就是全球基因治疗产业化发展得里程碑[3]。迄今报道已有数千例基因治疗患者,病种主要就是恶性肿瘤、艾滋病、血友病B、病毒性肝炎等等。 二、基因治疗得概念 基因治疗就是指向有功能缺陷得细胞补充相应得基因,以纠正或补偿其基因缺陷,从而达到治疗得目得。 广义得说,基因治疗就就是应用基因或基因产物治疗疾病得一种方法。狭义得说,基因治疗就是把外界得正常基因或治疗基因,通过载体转移到人体得靶细胞,进行基因修饰与表达,治疗疾病得一种手段。

《单基因遗传病遗传方式的判断技巧》

《单基因遗传病遗传方式的判断技巧》 进阶练习一 1 选项遗传方式遗传特点 A 伴X染色体隐性遗传病患者男性多于女性，常表现交叉遗传 B 伴X染色体显性遗传病患者女性多于男性，代代相传 C 伴Y染色体遗传病男女发病率相当，有明显的显隐性 D 常染色体遗传病男女发病率相当，有明显的显隐性 2、下列关于伴性遗传方式和特点的说法中，不正确的是( ) A．X染色体显性遗传：女性发病率高；若男性发病其母、女必发病 B．X染色体隐性遗传：男性发病率高；若女子发病其父、子必发病 C．Y染色体遗传：父传子、子传孙 D．Y染色体遗传：男女发病率相当；也有明显的显隐性关系 3.由于控制血友病的基因是隐性的，且位于X染色体上，以下不可能的是（） A.携带此基因的母亲把基因传给儿子 B.患血友病的父亲把基因传给儿子 C.患血友病的父亲把基因传给女儿 D.携带此基因的母亲把基因传给女儿 4.下列系谱图中一定能排除伴性遗传的是( ) 的性染色体只有一条X染色体，5、如图所示的红绿色盲患者家系中，女性患者Ⅲ 9 其他成员性染色体组成正常。Ⅲ 的红绿色盲致病基因来自于（） 9 A.Ⅰ1 B.Ⅰ2 C.Ⅰ3 D.Ⅰ4

进阶练习二 1、.福建省畲族儿童遗传病调查发现近亲结婚的子女患病率是非近亲结婚子女的 2.45倍，反映了近亲结婚的有害遗传效应。如图为某种遗传病的家族系谱图，有关该遗传病的分析错误的是（） A.Ⅲ5与正常男性结婚，生下一个患该病男孩的概率是1/4 B.若Ⅲ2与Ⅲ4结婚，后代患该遗传病的几率将大大增加 C.从优生的角度出发，建议Ⅲ1在怀孕期间进行胎儿的基因诊断 D.Ⅲ1与Ⅲ2为旁系血亲 2、某种遗传病受一对等位基因控制，下图为该遗传病的系谱图。下列叙述正确的是( ) A．该病为伴X染色体隐性遗传病，Ⅱ1为纯合子 B．该病为伴X染色体显性遗传病，Ⅱ4为纯合子 C．该病为常染色体隐性遗传病，Ⅲ2为杂合子 D．该病为常染色体显性遗传病，Ⅱ3为纯合子 3、下图为某家族遗传病系谱图，下列分析正确的是( )

关于基因工程在医药领域发展以及前景的若干思考

关于基因工程在医药领域双重性的若干思考 （政法1102班111070043 黄光志序号3）【摘要】基因工程作为一门理论性与实践性的较强的学科，其方法与技术已经渗透到现代生命科学的各个分支领域，成为生命科学的一门核心技术。基因工程包含许多独特的实验方法和技术，不仅内容丰富，涉及面广，实用性也强。基因工程技术广泛应用于农业、医学、食品工业等。本文主要阐述基因工程在制药领域的应用，包括生物制药、基因诊断、基因治疗的应用及其前景，同时事物具有双重性，认识基因工程的一些问题，趋利避害，让基因工程能带给我们更多福音。 【关键词】基因工程；医药领域；生物制药；基因诊断；基因治疗 [Abstract] Emphasizing both on practice and theory, genetic engineering has been applied to various fields of life science in terms of its methods and technologies and becomes the core technology of life science. Genetic engineering consists of many unique experiment methods and technologies and is highly and widely practiced in agriculture, medical science, and food industry and so on. This thesis will focus on its applications in pharmacy, including biopharming, genetic diagnose and gene therapy as well their prospects. As each coin has two sides, it is important to realize the deficiencies of genetic engineering. Only if the genetic engineering is made use of in the right way will it truly benefit us. [Key words] Genetic engineering; Medical field; Biological pharmaceutical; Gene diagnosis; Gene therapy 基因为DNA中的一部分，虽然只占整个DNA质量的2%～4%,却主宰着人类由生到死的整个生命旅程。基因工程是指人工使某特定基因（目的基因）经载体携带，进入某生物细胞并重组入其DNA分子中，经筛选、纯化、扩增，并使之表达出人类所需要的蛋白质或对人类有益的生物性状的技术。伴随“人类基因组序列”分析工作的完成，基因工程会在各领域给人类带来福音，目前基因工程已经应用在医学、农业生产、环境科学等诸多领域。本文主要探究基因工程在医学领域方面的发展以及其前景，面对其出现的一些问题，展望其未来，希望随着科学技术的发展，基因工程能给我们带来更多的福音。 一、基因工程的概况 基因工程(genetic engineering)又称基因拼接技术或DNA重组技术，是在在分子水平上对基因进行操作的复杂技术，按照人们的想法，以分子遗传学为理论基础，以分子生物学

浅谈单基因遗传病遗传方式的判定方法

浅谈单基因遗传病遗传方式的判定方法在近年的教师招聘考试试题中，遗传系谱图的分析所占分值比例逐年增大，试题呈现形式灵活多样，主要考查考生对人类遗传病方式的判定和有关概率的计算能力，掌握单基因遗传病遗传方式的判定方法是基础，尤其是两种遗传病同在一个系谱图中的分析。为了帮助广大考生顺利的解决此类问题，总结出一套简单、快速、准确的单基因遗传病方式的判定方法，以供大家参考。 1.单基因遗传病遗传方式的判定方法 2.遗传系谱图判定口诀 无中生有为隐性，有中生无为显性；隐性看女病，女病男正非伴性(男指患病女的父亲和儿子)，显性看男病，男病女正非伴性(女指患病男的母亲或女儿)。

3.应用 下面以一道例题为例来看看单基因遗传病遗传方式的判定方法在解题里面的应用。 例：下图是某家族的一种遗传系谱，请根据对图的分析回答问题： (1)该病属于_________性遗传病，致病基因位于_________染色体。 (2)Ⅲ4可能的基因型是_________，她是杂合体的几率为_________。 (3)如果Ⅲ2和Ⅲ4婚配，出现病孩的几率为_________。 【答案】(1)隐；常(2)AA或Aa；2/3(3)1/9 【解题思路】 (1)本小题主要考查显隐性的判定和致病基因的位置，根据遗传系谱图Ⅱ1、Ⅱ2不患病而他们的儿子Ⅲ1得病，由口诀“无中生有为隐性”推出该病为隐性遗传病；又根据遗传系谱图Ⅲ3女儿患病，其父亲Ⅱ3和母亲Ⅱ4表现正常，由口诀“隐性看女病，女病男正非伴性(男指患病女的父亲和儿子)”，推出该病为常染色体隐性遗传病。 (2)本小题主要考查相关个体的基因型及其概率的确定，由Ⅲ3是患者(aa)，其双亲表现正常，则他们都是杂合子(Aa)，Ⅲ4表现正常，其基因型可能是AA或者Aa，且AA:Aa=1:2，所以Ⅲ4为1/3AA，2/3Aa。 (3)本小题主要考查有关概率的计算，求后代某性状或某基因型概率，先必须求得能导致后代出现某性状或基因型的亲代基因型及其概率。由遗传系谱图容易推出Ⅲ2、Ⅲ4的可能的基因型概率，即Ⅲ2：2/3Aa；1/3AA。Ⅲ4：2/3Aa；1/3AA。他们的后代患者(aa)的概率为：2/3×2/3×1/4=1/9。

最新高中生物(人教版)同步习题：1-2基因诊断与基因治疗(选修2)及答案解析

第2节基因诊断与基因治疗 (时间：30分钟满分：50分) 难度及题号 考查知识点及角度 基础中档稍难 基因诊断 2 1 基因芯片3、7 4 基因治疗5、6 8 一、选择题(共6小题，每小题4分，共24分) 1．用DNA探针诊断疾病的具体方法是()。 A．与被测样品的DNA碱基序列做比较 B．与被测样品的DNA分子重组 C．与被测样品的DNA分子杂交 D．A、B、C三种方法均可 解析基因诊断是指用标记的DNA分子做探针，利用DNA分子杂交原理， 与待测样品DNA杂交，从而推测待测DNA序列。 答案 C 2．对某些传染性疾病(例如SARS)的诊断的困难在于病原体数量在初期极少，因此稳定、可靠而快捷的检测手段是()。 A．病毒的大量培养B．患者体内相关抗体的检测 C．PCR技术扩增D．临床症状确诊 解析对于病原体数量极少的待测样本，可利用PCR技术，对待测核酸进行 PCR技术扩增，获得大量核酸。 答案 C 3．基因芯片()。 A．是计算机上的微处理器 B．只能少量地对DNA分子的碱基序列进行测定和定量分析 C．是将少量DNA片段有序地固定在尼龙膜、玻片或硅片上 D．是一种高密度的DNA阵列 解析基因芯片是将大量特定序列的DNA片段(探针)有序地固定在尼龙膜、

玻片或硅片上，从而能大量、快速、平行地对DNA分子的碱基序列进行测 定和定量分析。基因芯片实际上是一种高密度的DNA阵列。 答案 D 4．下列对基因芯片的叙述中，错误的是()。 A．基因芯片可直接检测样品DNA和RNA B．基因芯片技术依据DNA分子杂交原理 C．基因芯片技术有助于发现不同个体对疾病易感性的差异 D．基因芯片技术不会造成社会负面效应 解析基因芯片技术也会造成负面效应，如基因歧视所引发的社会问题；婚姻、就业、保险等方面受到不公平的待遇；侵犯隐私权；对自己的心理、生活带来许多压力等。 答案 D 5．基因治疗的步骤是()。 ①治疗基因的表达②选择治疗基因③将治疗基因转入患者体内④选择 运输治疗基因的载体 A．②③①④B．②③④① C．③④②①D．②④③① 解析基因治疗的步骤包括选择治疗基因、选择运输治疗基因的载体、将治疗基因转入患者体内、治疗基因的表达。 答案 D 6．对基因治疗安全性的问题叙述不当的是()。 A．基因治疗中最常用的载体是病毒，它们能自我复制 B．在基因治疗中，科学家抑制逆转录病毒的某种活动防止它们引起疾病，使之能被安全地使用 C．使用病毒载体运载基因，它们可能更多地改变目标细胞 D．目的基因插入载体DNA的位置可能出现错误，导致癌症和其他损伤的产生解析基因治疗中最常用的载体为病毒，大多数基因治疗临床实验用小鼠逆转录病毒运送目的基因，其他病毒载体还包括腺病毒、痘病毒和疱疹病毒等。

基因工程之基因治疗

基因治疗 摘要： 生物技术在生命科学领域扮演者重要的角色，基因治疗在治疗方面，将新的遗传物质转移到某个个体的体细胞内使其获得治疗效果；在基因工程方面，将正常的有功能的基因置换或增补缺陷基因。近些年来，已对若干人类单基因遗传病和肿瘤开展了临床的基因治疗。基因治疗作为治疗疾病的一种新手段，正愈来愈受到人们的重视和关注。 关键词：基因工程基因治疗基因 一、基因治疗的历史 随着DNA双螺旋结构的发现和以DNA重组技术为代表的现代分子生物学技术的发展以及人类对疾病认识的不断深入，越来越多的证据证明，多种疾病与基因的结构或功能改变有关，因而萌生了从基因水平治疗疾病的念头和梦想。 早在1968年，美国科学家发表了“改变基因缺损：医疗美好前景”的文章，首次在医学界提出了基因疗法的概念。1989年美国批准了世界上第一个基因治疗临床试验方案。1990年美国NIH的Frenuch Anderson博士开始了世界上第一个基因治疗临床试验，用腺苷酸脱氨酶基因治疗了一位ADA基因缺陷导致的严重免疫缺损的四岁女孩，并获得成功[1]。 1994年美国科学家利用经过修饰的腺病毒为载体，成功地将治疗遗传性囊性纤维化病的正常基因cfdr 转入患者肺组织中。2000年，法国巴黎内克尔儿童医院利用基因治疗，使数名有免疫缺陷的婴儿恢复了正常的免疫功能，取得了基因治疗开展近十年最大的成功[2]。 2004年1月，深圳赛百诺基因技术有限公司将世界第一个基因治疗产品重组人p53抗癌注射液正式推向市场，这是全球基因治疗产业化发展的里程碑[3]。迄今报道已有数千例基因治疗患者，病种主要是恶性肿瘤、艾滋病、血友病B、病毒性肝炎等等。 二、基因治疗的概念 基因治疗是指向有功能缺陷的细胞补充相应的基因，以纠正或补偿其基因缺陷，从而达到治疗的目的。 广义的说，基因治疗就是应用基因或基因产物治疗疾病的一种方法。狭义的说，基因治疗是把外界的正常基因或治疗基因，通过载体转移到人体的靶细胞，进行基因修饰和表达，治疗疾病的一种手段。