水稻OsMADS1基因异位表达的功能研究

Abstract MADS-domain-containing transcription fac-tors play diverse roles in plant development. The proto-typic members of this gene family are the floral organ identity genes of the model dicotyledonous plant,Arabidopsis thaliana . Sequence relatedness and function ascribe them to AP1/AGL9,AG ,AP3and PI gene groups. The rice MADS-box gene,OsMADS1, is a mem-ber of the AP1/ AGL9sub-group. Tomato and Petunia members of this sub-group specify floral meristem iden-tity and control organ development in three inner whorls.Reported here are phylogenetic analyses that show OsMADS1to form a distinct clade within the AGL9gene family. This sub-group currently has only three other monocot genes. We have studied the expression pattern of OsMADS1and determined the consequences of its ec-topic expression in transgenic rice plants.OsMADS1is not expressed during panicle branching; earliest expres-sion is in spikelet meristems where it is excluded from the outer rudimentary/sterile glumes. During organogen-esis,OsMADS1expression is confined to the lemma and palea, with weak expression in the carpel. Ectopic OsMADS1expression results in stunted panicles with ir-regularly positioned branches and spikelets. Additional-ly, in spikelets, the outer rudimentary glumes are trans-formed to lemma/palea-like organs. Together, these data suggest a distinct role for OsMADS1and its monocot rel-atives in assigning lemma/palea identity.

Keywords OsMADS1· MADS-box genes · Flower development · Lemma/palea · Phylogeny

Introduction

The shoot apical meristem of higher plants contains un-differentiated progenitor cells that give rise to both vege-tative and reproductive structures. Transition from vege-tative to reproductive phase is controlled by both genetic and environmental factors and is responsible for specify-ing floral identity to newly arising meristems. The flow-ers formed hence are complex reproductive structures wherein the invariant ordering of floral organs – sepals,petals, stamens and carpels from the periphery to the center – implies a common ground plan for organ pat-terning in all flowering plants. In the model dicot plant Arabidopsis thaliana , conferment of floral fate requires the activity of meristem identity genes:LFY ,AP1,AP2and CAL (Weigel et al. 1992; Bowman et al. 1993; Weigel 1995). In addition, these floral meristem identity genes are transcription activators of floral organ identity genes that pattern organs in four concentric whorls (Parcy et al. 1998; Wagner et al. 1999). The latter genes include AP1,AP2,AP3/PI and AG , identified first on the basis of loss-of-function homeotic mutations that alter cell fate in two adjacent whorls of the flower (reviewed by Ma 1994; Irish 1999). Thus, their combined and indi-vidual domains of action are proposed to dictate organ fate. All but one of these A. thaliana floral organ identity genes belong to the MADS-domain-containing group of evolutionarily conserved transcription factors that regu-late gene expression in plant, yeast and animal cells (Reichmann and Meyerowitz 1997). The temporal and spatial expression pattern of the floral organ identity genes is consistent with their function in two adjacent whorls (Yanofsky et al. 1990; Jack et al. 1992; Mandel et al. 1992b; Goto and Meyerowtiz 1994). For instance,AG is expressed in whorls 3 and 4 of the flower and loss-of-AG function results in altered cell fate in these whorls alone (Yanofsky et al. 1990). The sufficiency of these

Edited by G. Jürgens

K. Prasad · P. Sriram · K. Kushalappa · U. Vijayraghavan (?)Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India e-mail: uvr@mcbl.iisc.ernet.in

Tel.: +91-80-3092681, Fax: +91-80-3602697

C.S. Kumar

The Institute for Molecular Agrobiology,

The National University of Singapore, 117604, Singapore Present address :

K. Kushalappa, Department of Botany,

University of British Columbia, Vancouver, Canada

Dev Genes Evol (2001) 211:281–290DOI 10.1007/s004270100153

Kalika Prasad · P. Sriram · C. Santhosh Kumar Kumuda Kushalappa · Usha Vijayraghavan

Ectopic expression of rice OsMADS1reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals

Received: 28 August 2000 / Accepted: 14 February 2001 / Published online: 25 April 2001?Springer-Verlag 2001

genes to alter cell fate, when expressed ectopically in floral whorls where they are not normally found, has also been tested (Mandel et al. 1992a; Mizukami and Ma 1992; Jack et al.1994). All of these data are, to a large extent, explained and accommodated by the ABC model for floral organ specification, originally proposed based on the phenotypes of loss-of-function mutations in these floral organ identity genes (Coen and Meyerowitz 1991; Ma 1994; Irish 1999).

The MADS-box genes have provided a gateway to probing the function of regulatory transcription factors in patterning floral organs in diverse species. They also provide tools to study mechanisms by which sequence homologues and/or orthologues could contribute to spe-cies-specific differences in floral organ patterns. Modern day monocotyledonous (monocot) plants shared ances-tors with dicotyledonous (dicots) plants more than 120million years ago (Doyle 1973). The divergent and reduced flowers of cereal grasses, a large group among the monocots, provide an excellent model to examine the above mentioned hypotheses. Typical of grasses, rice flowers are borne on a branched inflorescence called the panicle, where the developmental progress from inflores-cence specification to flower meristem allocation occurs in steps. Upon floral induction, the inflorescence meri-stem generates primary branch primordia on its flanks, which in turn elongate and allocate basally second order branch primordia. Subsequently, spikelet primordia are specified apically on the primary as well as on the sec-ondary branch primordia (Hoshikawa 1989). In sum, the inflorescence/panicle meristem, primary branch primor-dia, secondary branch primordia, and floral primordia comprise the reproductive meristems in rice. A rice spikelet primordium bears a single flower in the axil of rudimentary glumes and the flower contains the organs-lemma, palea, lodicules, stamens and carpel. Recently, several rice MADS-box genes have been partially char-acterized (Chung et al. 1994, 1995; Kang et al. 1995, 1998; Kang and An 1997). However, their precise role during panicle development remains largely unknown. One such MADS-box gene is OsMADS1, whose predict-ed product has overall 68.4% identity to AGL9, 56.2% identity to AGL2, and 44.4% identity to AP1 proteins of Arabidopsis(Chung et al. 1994). All of these Arabidop-sis genes are initially expressed throughout the floral meristem. Their later expression patterns vary; they are either expressed in all four floral organ primordia, or on-ly in the perianth, or in three inner whorls. Preliminary studies on OsMADS1reveal it to be temporally and spa-tially regulated, with features both similar to and differ-ent from AP1/AGL9-like factors. Early expression is uni-form throughout the floral meristem and its later high level expression is restricted to the lemma and palea (modified first whorl organs), with weak expression in the carpel (Chung et al. 1994; Vijayraghavan 1996). We have examined the phylogenetic relationships of OsM-ADS1and its closely related homologue OsMADS5. We find that they form a distinct clade within the AP1/AGL9 group with no members as yet from any dicot species that can be assigned to this sub-group. Also, we have studied in detail the expression pattern of OsMADS1and have determined that it is not expressed in the spikelet organs, i.e. the glumes, which are modified bracts pe-ripheral to the flower. Additionally, by adopting a re-verse genetics strategy, we have studied the consequenc-es of ectopic OsMADS1expression to understand its role in organ fate specification. We show that OsMADS1has a role in assigning cells to a lemma and palea fate, and that its ectopic expression affects panicle branch differ-entiation.

Materials and methods

Phylogenetic analysis

The nucleotide and protein sequence of OsMADS1was retrieved from the GenBank sequence database (Accession Number L34271). The predicted protein from the full-length cDNA se-quence was used as query in BLAST and FASTA searches for re-lated sequences in the GenBank and SwissProt non-redundant se-quence databases. The high scoring sequences were retrieved from the databases. Plant MADS-box genes used for phylogenetic anal-ysis are given below, with their accession numbers given within brackets. Rice genes,OsMADS1(L34271),OsMADS2(L37526), OsMADS3(L37528),OsMADS4(L37527),OsMADS5(U78890), OsMADS6(U78782),OsMADS14(AF058697),OsMADS15 (AF058698),OsMADS16(AF077760),OsMADS17(AF109153), OsMADS45(U31994),RAP1b(AB041020), rice MADS-like (RML; (AB003324);Arabidopsis genes AGL2(B39534),AGL3 (P29383),AGL4(P29384),AGL6(M55554),AGL9(AF015552), AGL13(U20183),AP1(Z16421),AGAMOUS(S10933); and maize genes ZAP1(L46400),ZAG1(L18924),ZAG2(X80206), ZMM1(X81199),ZMM2(L81162),ZMM3(Y09301),ZMM8 (Y09308) and SILKY1(AF181479) were retrieved from GenBank. The sequences for tomato TDR5(X60480), petunia FBP2 (M91666) and Antirrhinum PLENA(S53900) genes were also re-trieved from GenBank. Yeast MCM1gene (P11746) and human MEF2(L16794) gene sequences were also similarly retrieved.

Alignment and sequence analysis

For alignment and sequence analysis the following approaches were taken. The full-length protein sequences were aligned using the PILEUP of the UWGCG package. This was refined using CLUSTALW 1.7. Phylogenetic analysis, with the full-length pro-tein sequence, employing maximum parsimony methods per-formed using the experimental version of PAUP* program, (4.0.0d55 Version for UNIX; D.L. Swofford, Laboratory of Mo-lecular Systematics, Smithsonian Institute, Washington, D.C). For maximum parsimony analysis 33 taxa were analyzed, with 100 replicates, keeping all optimal trees in each replicate. Gaps were treated as missing residues. Tree construction was undertaken us-ing the Bootstrap method with Neighbor-Joining (NJ) or Heuristic search. The NJ analysis was done using the software default set-tings and the optimality criterion was set to distance. The trees were examined using the PAUPDISPLAY program of the UWGCG package. The tree obtained using Bootstrap method with heuristic search algorithm was essentially the same as that ob-tained with NJ. The MCM1 protein sequence was used as out-group in this analysis, to study the branching order of the gene lin-eages relative to the main group. In addition, a tree was also gen-erated with the plant floral organ identity gene AG as the out-group. Distance based criteria (Kimura protein distance) and NJ were used to generate the tree.

Furthermore, in the aligned predicted protein sequences, the 525nucleotides coding for the MADS-intermediate-keratin-like

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(MIK) region of OsMADS1 and related proteins were visually se-lected, manually refined and analyzed. These nucleotide sequenc-es were re-aligned using the PILEUP program. The 120nucleo-tides corresponding to the MADS box and 233nucleotides corre-sponding to the K box sequences were thus deciphered. Sequence divergence at the nucleotide level was then derived using the DISTANCES program of the UWGCG package. The Tajima and Nei method (Tajima and Nei 1984) was used to estimate rate of nucleotide substitution. The resulting distance matrix was pro-cessed using the GROWTREE program of the UWGCG package. The tree was constructed using the NJ method. The trees were vi-sualized with TREEVIEW. The DIVERGE program of the UWGCG package based on the methods described by Li et al. (1985) and Li (1993) was employed to determine the rates of non-synonymous substitution in the MADS box, K box and MIK re-gion.

Isolation of OsMADS1cDNA clone

A λcDNA library in the vector λUniZAP (Stratagene), represent-ing mRNAs from wild-type rice panicles (length 2–5cm; Kusha-lappa 1999), was screened to obtain full-length OsMADS1 cDNAs. The probe used was a partial cDNA, previously cloned in the laboratory that encoded amino acid residues 175–257 of the predicted OsMADS1 protein (Chung et al. 1994; Vijayraghavan 1996). From screening 106plaques, 12 positives were obtained, one of which contained the full-length cDNA as determined by se-quence analysis of the excised phagemid.

Construction of transgene to ectopically express OsMADS1

Binary vector pCAMBIA1300 was modified for ectopic expres-sion of OsMADS1. The complete maize ubiquitin (Ubi1) promoter (Christensen et al. 1992) was cloned in as a Hin dIII-Bam HI frag-ment. Subsequently, the nopaline synthase (nos) terminator was cloned as a Sac I-Eco RI fragment downstream of the Ubi1 promot-er. Lastly,OsMADS1full-length cDNA was blunt-end cloned into the Sac I site between the promoter and terminator sequences. The sense orientation of the cDNA was verified by Kpn I digestion. The recombinant was called pUbi-OsMADS1.

Rice transformation

The embryogenic calli from rice seeds of the japonica variety TP309 were co-cultivated with Agrobacterium tumefaciens(Hiei et al. 1994) carrying the pUbi-OsMADS1plasmid. Co-cultivation was for 2–3days in the dark (at 25°C) on 2N6-AS medium sup-plemented with 100μM acetosyringone. The co-cultivated calli were washed with water containing 250mg/l cefotaxime and then transferred onto N6 medium containing 50mg/l hygromycin and 250mg/l cefotaxime for 8weeks. Actively proliferating calli were further transferred onto regeneration medium (3mg/l BAP and 0.5mg/l NAA) for 4–8weeks and subsequently onto 1/2 MS me-dium (0.05mg/l NAA) for 3weeks. A light/dark cycle of 16/8h was provided during regeneration and rooting. Plantlets were then transferred to soil and grown in a greenhouse.

Light microscopy and scanning electron microscopy

Morphological features of transgenic and wild-type panicles and flowers were observed by stereomicroscopy. For SEM, panicles were dissected away from the enveloping leaf sheaths, fixed, de-hydrated and then critical point dried. The dried panicles were mounted onto stubs, sputter coated with gold and then viewed in a Joel JSM-5310 LV microscope (Skandinaviska AB, Sundbyerg, Sweden).RT-PCR of OsMADS1transcripts

Total RNA from young leaves of transgenic plants or control plants was isolated by phenol extraction and then treated with RNAse-free DNAse. RT-PCR experiments were performed using this total RNA (Wang and Tobin 1998; Nandi et al. 2000). First strand cDNA was made from 7.5μg RNA in 20μl reaction mix-ture using the primer OSM2-1 (5′CGGGATCCGCCAATTAATT-GTTACC 3′). The primer used in control cDNA synthesis reac-tions was Act2 (5′GATGGATCCTCCAATCCAGACACTGTA 3′). Standard PCR reactions of 50μl were then performed with 5μl RT product generated for OsMADS1or ACT1. PCRs con-tained, as the forward primer, OSM4 (5′GAGAATGTGCTCCAT-ATG 3′) in the OsMADS1RT-PCR or Act1 (5′GTATTGTGTTG-GACTCTGGTGATGGTGT 3′) in the ACT1RT-PCR reactions. PCR products were visualized on 1% agarose gel after loading 10μl from each PCR reaction.

In situ hybridization for OsMADS1transcripts

Preparation of rice tissues and hybridization conditions included only minor modifications of the protocol as described by Drews et al. (1991) for Arabidopsis. The OsMADS135S-labeled anti-sense mRNA probe was synthesized with T3 RNA polymerase from a Bam HI digest of a plasmid containing 500bp of the 3′gene spe-cific region of OsMADS1. The slides were exposed for 1week.

Results and discussion

Phylogenetic relationships of OsMADS1

A preliminary sequence alignment of the predicted OsM-ADS1 protein to a few other plant MADS-domain-con-taining factors had suggested relatedness to the AGL2 and AP1genes of Arabidopsis(Chung et al. 1994). This homology was consistent with its flower-specific expres-sion in the lemma, palea and carpel (Chung et al. 1994; Vijayraghavan 1996). Members of the plant MADS-domain gene family can be distinguished by phylogenet-ic relationships, and such analyses show them to cluster into distinct groups:AP3/PI,AG,AP1/AGL9and orphan gene clades (Purugganan et al. 1995; Theissen et al. 1996; Alvarez-Buylla et al. 2000). Unlike members of the AP3or PI groups, the precise function of many of the molecules belonging to the AP1/AGL9clades remains unknown. Additionally, this group bears within it multi-ple lineages, the most obvious being AP1,AGL9and the AGL6/AGL13clades.

Recently, several MADS-domain-containing genes have been isolated, their expression patterns studied and in some instances in vivo functions deciphered through reverse genetic approaches (Reichmann and Meyerowitz 1997; Schmidt and Ambrose 1998). We have attempted to obtain a hint of OsMADS1function by examining its evolutionary relationship with other genes, particularly those of the AP1/AGL9gene clades. The related evolu-tionary histories of individual gene groups could reflect their distinct functional specificity in regulating floral cell fate. The amino acids corresponding to the full length predicted OsMADS1 protein align well with predicted proteins for most members of this group particularly in the following domains: MADS box, intermediate linker

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region and the K box. These regions comprise the DNA binding domain, connecting linker or intermediate do-main and the predicted coiled-coil domain with structural similarity to the fibrous protein keratin, respectively. The alignment was unambiguous for protein as well as the nu-cleotide sequences that code for the MIK region of the proteins (data not shown). The phylogenetic relatedness of OsMADS1to members of the AP1and AGL9clades from Arabidopsis ,Petunia , tomato, rice and maize was examined. In addition, relationship to members of the AG clade from Arabidopsis ,Antirrhinum , rice and maize was also determined. We employed the maximum parsimony method (MP) and phylogenetic trees were generated by NJ to determine its position within this gene family. A single tree was derived from parsimony analysis with high bootstrap support in 100replicates [consistency in-dex CI=0.6952, retention index =0.6732, rescaled consis-tency index (RC)=0.4680]. This analysis included the yeast MCM1 protein as the outgroup (Fig.1A). The tree thus generated was largely similar to one where the Arab-idopsis AGAMOUS protein was used as the outgroup (data not shown). Interestingly, while OsMADS1belongs to the AP1/AGL9lineage, it clearly forms a distinct sub-group with the maize genes ZMM8and ZMM3. Further-more,OsMADS1and ZMM8are the two rice and maize genes that are most closely related in sequence (80.97%overall identity and 100% identity in MADS box). In ad-dition,OsMADS5, a closely related rice gene, also be-longs to this sub-group. Notably, this sub-group is distinct from the previously designated orphan group of genes typified by Arabidopsis AGL6and AGL13genes. The OsMADS1sub-group seems to be unique to the monocots with no sequence homologues as yet from any dicot spe-cies, despite the near complete sequence information from the Arabidopsis genome. Phylogenetic relationships using distance-based criteria were also determined and this was performed for nucleotide sequences correspond-ing to the MIK region of the derived proteins. The data was again used to generate a tree with NJ where bootstrap values were used to indicate the reliability of the branch-es (see Materials and methods). This analysis deduced a lineage similar to that obtained from analysis of the pre-dicted protein (Fig.1B). Thus, both analyses place OsM-ADS1and its likely maize homologues in a monophyletic sub-group that has diverged from the AGL9clade. The to-pology of the tree derived with the distance matrix corre-lates well with the cladogram derived using protein se-quences for the entire gene.

Different domains of plant MADS-box genes show differing rates of non-synonymous substitutions, with the

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Fig.1A Phylogenetic tree for the rice OsMADS1,OsMADS5and AP1,AGL9-like MADS-box genes from other genera. A selected number of AG -related genes were also used in the analysis. Maximum parsimony based analyses was performed for the predicted full-length protein sequences. The yeast MCM1 protein was used as the outgroup.Numbers (written above each node) give the

Bootstrap values from 100rep-licates. Nodes with <50% boot-strap support are collapsed. The tree length is 2,408 steps. The topology of the tree remained the same even when the Heu-ristic search algorithm was em-ployed. The shaded box indi-cates the OsMADS1clade.B Neighbor-Joining-based tree showing evolutionary distances for plant MADS-box-contain-ing genes. The scale bar indi-cates the number of nucleotide substitutions. The distance ma-trix was determined for nucleo-tide sequences corresponding to the MADS-intermediate-ker-atin-like (MIK) domains of the indicated proteins, by deter-mining the Tajima-Nei nucleo-tide substitution rate

lowest degree of change occurring in the MADS box which is constrained by its DNA binding property. Com-paring the levels of non-synonymous substitutions can provide clues on functional divergence of specific do-mains for members of this family. We have compared the proportion of non-synonymous substitution in the gene pairs OsMADS1versus ZMM8,OsMADS5,ZMM3,AP1 or AGL9. In this analysis we have taken the maize ZAP1, rice RAP1b and OsMADS3, tomato TDR5and Arabidop-sis AGL13as divergent sequences (Table1). Despite ac-counting for the constraints on the MADS box, these an-alyses suggest OsMADS1to be distinct from other mole-cules of the AP1or AGL9clade and its divergence possi-bly reflects its differing functional specificity. Signifi-cantly, all of these analyses place this sub-group as one that is different from that of AGL13and AGL6. Collec-tively, these data hint at a unique function for OsMADS1 and OsMADS5in rice panicle and flower development. Expression profile of OsMADS1during panicle development

AP1,AGL9and its likely orthologues from Antirrhinum, Petunia and tomato are the best characterized among the genes in this group.AP1is a floral meristem identity gene and the earliest amongst the MADS-box genes to be expressed exclusively in floral tissues. Its temporally early expression in very young floral primordia is uni-form and precedes the onset of expression of the other floral organ identity genes. The temporally later expres-sion of AP1is only in the perianth structures: whorls 1 and 2 (Mandel et al. 1992b). This spatial restriction occurs concomitant to the expression of the organ identi-ty MADS-box gene AG in presumptive whorls 3 and 4 (Gustafson-Brown et al. 1994). In contrast,AGL9as well as its putative Petunia and tomato orthologues FBP2and TM5, are expressed uniformly in the young floral meri-stem. However, the onset of AGL9expression is after that of AP1. In more mature flowers undergoing organo-genesis the AGL9group of genes are expressed in three inner whorls: petals, stamens and carpels (Angenent et al. 1992; Pneuli et al.1994; Mandel and Yanofsky 1997).Other members of this clade are expressed in all four whorls of the flower. Not surprisingly, the rice genes such as OsMADS24and OsMADS45that are closely related to AGL9(Fig.1A) share similarities in expres-sion profiles with these dicot genes.OsMADS45and OsMADS24are expressed in lodicules, stamens and car-pel of rice flowers (Greco et al. 1997).

Initial studies on OsMADS1expression revealed high levels of expression in the flower primordium, a feature similar to that of AP1-and AGL9-like factors (Chung et al. 1994; Vijayraghavan 1996). The developmentally la-ter expression pattern of OsMADS1differs in that it is expressed strongly in lemma, palea and weakly in the carpel. The lemma and palea are modified glumes, sug-gested by certain criteria to be analogous to sepals of di-cot flowers (Schmidt and Ambrose 1998). The rice flow-er bears additional pair of glumes peripheral to the lem-ma and palea. We have examined whether OsMADS1is a likely determinant of lemma/palea cell fate by examining in detail its expression pattern in the developing panicle, particularly in very early flower primordia where the outer glumes are established, but the lemma and palea are yet to be formed. We observe that OsMADS1RNA is first detected in the incipient floral primordium where it is excluded from outer sterile glumes that subtend the flower primordia (Fig.2A, B, and C, D).OsMADS1is not expressed in earlier stages of panicle development, i.e. during primary or secondary rachis branch formation (Fig.2A, B). Its earliest expression occurs well after dif-ferentiation of the panicle branches. Therefore,OsM-ADS1and RAP1A are currently the earliest markers to be expressed exclusively in the rice floral meristem(Fig.2 and Kyozuka et al. 2000). As reported earlier, the early uniform expression of OsMADS1in the wild-type floral meristem (Fig.2C, D) is later confined to strong expres-sion in the lemma and palea primordia at about the stage of their initiation (Fig.2E, F).OsMADS1RNA is com-pletely excluded from the lodicule and stamen primordia and spikelets where organ differentiation is occurring (Fig.2E–J). Notably, weak but clearly detectable levels of OsMADS1RNA are seen in the carpel primordia (Fig.2E, F, and G, H). In contrast,RAP1A is expressed in the lemma, palea and lodicules, with low levels of RNA in the sterile glumes (Kyozuka et al. 2000). These differences in expression patterns reinforce the differ-ences between these two genes hinted at by our phyloge-netic analysis (Fig.1). Prior suggestions of distinctions between the lemma (bract) and palea (prophyll; reviewed in Schmidt and Ambrose 1998) are not reflected in the expression pattern of OsMADS1which is indistinguish-able in these two structures. In this aspect,OsMADS1 and RAP1A are similar. The developmentally late expres-sion pattern of OsMADS1in the lemma, palea and car-pels of rice flowers varies from the known pattern of ex-pression for AGL9group of MADS-domain proteins, in-cluding those known in rice.

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Table1Estimates of non-synonymous nucleotide substitution rates within MADS, I and K domains of pairs of genes.OsMADS1 is compared to AGL9, or AG-related factors (MIK MADS interme-diate keratin-like domain)

MADS K-box MIK

OsMADS50.0200.0950.091 ZMM80.0000.0360.050 ZMM30.0150.0790.077

TDR50.0910.3530.290

ZAP10.0800.4580.361

RAP1b0.0910.3920.328

AP10.1360.4990.401

AGL90.0600.3370.278

AGL130.0790.5100.370 OsMADS30.1230.7370.459

Ectopic expression of OsMADS1affects the panicle morphology

In wild-type plants (TP309), the inflorescence stem de-velops as a central rachis bearing 6–8 nodes. Each of these nodes produces primary rachis branches (prb) that in turn bear 6–9 nodes. Basal nodes of the prb bear sec-ondary rachis branches (srb), while apical nodes bear in-dividual spikelet primordia. A spikelet primordia in rice bears a single bisexual flower, and these primordia are also generated on the srb (Hoshikawa 1989). A single spikelet in rice contains a pair of sterile glumes that re-main small and underdeveloped (Fig.4A) compared to that observed in other cereal flowers, for example, sor-ghum, wheat or oat. Internal to these glumes, the rice spikelet bears a single flower with a lemma, a palea, two

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Fig.2A–J The RNA expression pattern of OsMADS1in second-ary rachis branch primordia (srb ), very young flower primordia,and flower primordia with differentiating organs. In situ hybrid-ization of longitudinal sections of wild-type tissues with an OsM-ADS1-specific mRNA probe is shown . A ,C ,E ,G, I Bright-field photographs; the corresponding dark field images are B ,D ,F ,H and J . RNA expression was detected as the silver grains in the dark field images.A, B Young panicle with developing srb (white arrowhead ) and young spikelet primordia (blue arrowhead ).C, D Spikelet primordium at the stage of lemma specification; outer ru-dimentary glumes (white arrowheads ) and lemma primordium (yellow arrowhead ).E, F Spikelets during organ primordia speci-fication; young spikelet primordium (red star ) slightly later in de-velopment than that in C , lemma/palea (white arrowhead ), and carpel primordium in an older spikelet (green arrowhead ).G, H Spikelet with differentiating organ primordia; lemma and palea (white arrowhead ), lodicule (black arrowhead ), stamen (red ar-rowhead ), and carpel (green arrowhead ).I, J Spikelet undergoing anther differentiation; lemma and palea (white arrowhead ), lodic-ule (black arrowhead), and anther lobe (red arrowhead

)

Fig.3Morphology of panicles from wild-type plants (A ) versus pUBI-OsMADS1transgenic plants (B ) at the stage of panicle heading for the wild-type plant. The stunted panicle in the T0 transgenic plant remained embedded in leaf sheath. The disorganized positioning of the spikelets gives a severely twisted phenotype (white arrowhead in B ).C A fully emerged wild-type panicle where seeds/spikelets are removed to display the rachis branches.White arrow central rachis,yellow arrow primary rachis branch,yellow arrowhead secondary ra-chis branch.D T0OsMADS1transgenic panicle with spikelets re-moved to show the severely shortened central rachis and branches thereof.E RT-PCR analysis of OsMADS1transcripts in transgenic lines. Total RNA from leaves of transgenic plants were used in RT-PCR with primers for either the 541-bp C-terminal fragment of OsM-ADS1(upper panel ) or the 588-bp fragment of the constitutively ex-pressed endogenous rice ACT1(lower panel ).Lane 1RT-PCR prod-uct from vector pCAMBIA 1381Xc-transformed https://www.wendangku.net/doc/ea12427304.html,nes 2–5RT-PCR products from four independent Ubi-OsMADS1lines

Fig.4A–J Homeotic phenotypes of OsMADS1transgenic spike-lets.A Wild-type spikelet with rudimentary outer glumes (red ar-rowheads ), lemma (l ) and palea (p ).B A portion of a T0 transgenic panicle, with one spikelet partially dissected open; the outer glume (red arrowhead ) is converted into a lemma/palea-like organ. The other rudimentary glume, while enlarged, is smaller than the first one. Inner to these glumes are the lemma, palea, lodicule (lo ),

stamens (st ) and carpel (ca ). A deformed spikelet with prema-ture termination of differentiation is shown on the same panicle branch (blue arrowhead ).C Spikelet where both the glumes (arrow-heads ) are enlarged and transformed into lemma/palea-like organs.D Spikelet with one outer glume, converted into a lemma/palea-like structure. Internal to this are found the normal lemma and palea, but

there are no further internal organs.E –G Scanning electron micro-graphs (SEM) of the outer surface of rudimentary glume (E ), lemma (F), and palea (G ) of a wild-type spikelet.H SEM of the epidermal cell surface of a transformed outer glume in a T0 spikelet.White ar-rows in F ,G , and H point to several epidermal cells with protru-sions typical of lemma/palea and white arrowheads indicate trich-omes.I SEM of a wild-type seed enclosed by the lemma and palea.J Transgenic seed partially enclosed by the lemma/palea and trans-formed glumes.Scale bars 50μM in E –H and 1mm in I ,J

lodicules, six stamens and a central carpel. For the sake of convenience, we have used the term rice flower pri-mordium and rice spikelet primordium interchangeably here. We have used ectopic expression studies as a tool to examine the functional contribution of OsMADS1to rice panicle and spikelet formation.

An OsMADS1cDNA clone was isolated from the rice panicle cDNA library and then sub-cloned under maize ubiquitin promoter to generate pUbi-OsMADS1con-struct (see Materials and methods). This construct was transformed by Agrobacterium -mediated transformation into embryogenic rice calli. Plants from four independent hygromycin-resistant calli were regenerated. During veg-etative development, plants transgenic for OsMADS1did not differ from control vector transformed plants. The parameters of plant height, leaf development and number of tillers per plant (branches from basal vegetative nodes) remained similar to those in controls. Panicle heading was also comparable. A reduction in flowering time by a few days and overall dwarfing upon OsMADS1expression was recently reported by Jeon et al. (2000).The differences between our observations may lie in the photoperiods during transgenic plant growth. This possi-bility can be tested to examine if OsMADS1contributes to flowering time in a photoperiod-sensitive pathway.All the plants from four independent Ubi-OsMADS1

lines show defects in panicle development. The panicles are shorter in length, severely defective for internode elongation, and have abnormally positioned branch pri-mordia or lateral primordia on the main axis (Fig.3C vs D). The panicle length is reduced to about 60% of that in control plants (Fig.3C vs D, and Table 2). This is one of the two general features noted in the recent study of Jeon et al. (2000). We detail here our further characterization of the panicle defects. Significant reduction in length of prb, which are in average only 65% of the length of con-trol branches, was observed (Table 2). Additionally, a striking finding is that the prb are also highly deformed in shape with lateral primordia (i.e. srb primordia and flower primordia) arising at irregular and atypical inter-vals (Fig.3D). While the panicle rachis branches are atypical in these plants the number of nodes remains the same as in control panicles (Table 2). The disorganized

287

Table 2Panicle characteristics in control vector-transformed plants (1381Xc ) or pUbi-OsMADS1-transformed plants (prb primary ra-chis branches)Transgene

Length of Number of nodes Length of prb Number of nodes Number of

panicle in on central in cm±SD on prb±SD spikelets on each cm±SD a rachis±SD panicle±SD 1381Xc no. 111.5±1.3 6.0±0.8 5.6±0.48.0±0.568.0±5.91381Xc no. 210.9±1.0 6.7±0.7 4.7±0.67.2±0.464.0±6.01381Xc no. 312.1±1.0 6.2±0.8 6.0±0.67.6±0.462.0±5.81381Xc no. 4

11.3±0.9 6.4±0.8 6.3±1.07.0±0.571.0±5.6pUbi-OsMADS1 no. 1 5.9±0.9 6.0±0.7 2.1±0.5 6.0±0.761.2±5.2pUbi-OsMADS1 no. 2 4.7±1.0 5.6±0.6 2.7±0.57.2±0.859.1±6.0pUbi-OsMADS1 no. 3 5.2±0.9 6.6±0.7 2.4±0.4 5.9±0.664.2±5.8pUbi-OsMADS1 no. 4

4.9±0.8

5.9±0.8

2.6±0.4

6.7±0.8

66.2±5.1

a

Panicle length is represented as length of central rachis. At least seven panicles of each line were examined

arrangement of the flowers on the shortened panicle and rachis branches contributes to its compact appearance (Fig.3B). Spikelet morphology was also affected; these data are discussed in the following section. To establish that the panicle phenotype results from ectopic expres-sion of OsMADS1and not co-suppression (silencing) of the endogenous OsMADS1gene, we have determined the steady state levels of OsMADS1RNA in vegetative tis-sues of these transgenic lines. The ubiquitin promoter is expected to direct constitutive expression of OsMADS1 RNA in these transgenic lines. RT-PCR was carried out to measure OsMADS1RNA levels as compared to those of the endogenous rice actin mRNA. We found a high level and stable expression of OsMADS1RNA that was quantitatively comparable to that of actin (ACT1) mRNA (Fig.3E). Together, these results suggest that ectopic ex-pression of OsMADS1is responsible for the stunted transgenic panicles with their distinct phenotypes. Re-cently, it has been suggested that floral homeotic genes differentially regulate the cell division patterns during various stages of Arabidopsis flower development (Jenik and Irish 2000). Possibly, the ectopically expressed OsMADS1exerts its effects on the early stage of panicle branching by altering patterns of cell proliferation. OsMADS1ectopic expression results in homeotic transformation of outer glumes into lemma/palea-like organ

As mentioned earlier, rice spikelet has a pair of rudimenta-ry outer glumes at its base, inner to which are the larger, well developed modified glumes: the lemma and palea. These latter organs enclose the internal three whorls of floral organs. By the criteria of OsMADS1gene expression pattern, and also that of rice RAP1A or the maize ZAP1, it is inferred that lemma and palea define floral organs analogous to first whorl sepals of other flow-ers (Chung et al. 1994; Mena et al. 1995; Vijayraghavan 1996; Ambrose et al. 2000; Kyozuka et al. 2000). A dif-ferent line of support for this hypothesis comes from phe-notypic analysis of a maize mutant:Silky1, where lodic-ules are replaced by lemma/ palea structures; a transfor-mation consistent with a class B loss-of-function mutation (Ambrose et al. 2000). The reasonable prediction is that in grass flowers orthologues of the dicot ABC floral organ identity genes pattern the lemma/palea, lodicules, stamens and carpels. Since the OsMADS1gene, by sequence relat-edness and by expression pattern differs from the above-mentioned molecules, we anticipated that the consequenc-es of its ectopic expression on spikelet structure would be informative vis-a-vis its function.

A consistent phenotype in a significant proportion (~42%) of flowers in all independently generated OsMADS1transgenic lines was a drastic enlargement of one pair of the outer glumes. The normally rudimentary glumes immediately peripheral to the lemma and palea now approach the size of the lemma or the palea (Fig.4C). The normal lemma and palea are found inner to these transformed glumes. A weaker phenotype ob-served was flowers wherein only one glume was trans-formed to a lemma/palea like structure; the other glume remained at its normal size. Internal to the transformed glume was the lemma and palea, yet many of such flow-ers did not bear any stamen or carpel (Fig.4D). Howev-er, a small proportion of the flowers displayed transfor-mation of one glume to a lemma/palea like structure, with otherwise normal floral organ patterning (Fig.4B). Thus, the most notable phenotype was the presence of over-developed outer glumes, a characteristic also seen by Jeon et al. (2000). We have examined further if these over-developed glumes are merely the result of increase in glume size, or whether they originate from homeotic transformation of a pair of normally rudimentary glumes to lemma/palea. We have determined the epidermal cell surface characteristics of the rudimentary glume, lemma and palea in wild-type flowers and then used their distin-guishing features to determine the organ and cellular identities in the over-developed glumes of transgenic flowers. The rudimentary glume bears epidermal cells organized as long files of smooth cells with few trich-omes present only on the edges (Fig.4E). The lemma is typified by epidermal cells with rounded projections and also by a high density of trichomes (Fig.4F). The wild-type palea bears similar cell morphology as the lemma but has fewer trichomes (Fig.4G). The epidermal cells of the transformed glume of transgenic flowers has char-acteristics of both lemma and palea, i.e. cells with round-ed epidermal projections and interspersed trichomes (Fig.4H). These features prove the homeotic transforma-tion of glumes to lemma/palea. Thus OsMADS1expres-sion in rudimentary outer glumes is sufficient for their conversion into lemma-palea like organs. Possibly,OsM-ADS1achieves this function by targeting downstream genes needed for differentiation of lemma and palea cell types. A speculative function for the Arabidopsis AGL9 like genes is that they act as cofactors for

B and

C floral organ identity genes (Pelaz et al. 2000). The closely re-lated rice genes OsMADS24and OsMADS45(Fig.1) could function similarly and contribute to lodicule, sta-men and carpel differentiation in rice. In a similar man-ner, a plausible hypothesis for OsMADS1action in the sterile glumes is as a partner/cofactor for gene/s ex-pressed in these glumes.RAP1A, which is expressed at low levels in outer sterile glumes, could be one such can-didate partner. In this scenario, ectopic expression of OsMADS1possibly facilitates the transformation of glu-mes to lemma/palea like organs. However, we do not ob-serve any transformation of the lodicule in transgenic Ubi-OsMADS1plants. Thus, it is probable that in lodic-ules, interaction of RAP1A with other homeotic genes precludes the effects of OsMADS1ectopic expression therein. Studying the consequences of loss-of-function and gain-of-function of RAP1A in Ubi-OsMADS1trans-genic lines can test these hypotheses.

Since the total number of the flowers in these panicles is similar to that in control panicles (Table2) we con-clude that the flowers which do not bear any internal or-

288

gans are not supernumerary flowers generated as a conse-quence of ectopic expression of OsMADS1. Instead, the data suggest that ectopic expression promotes the preco-cious assignment of floral meristems on the branches, perhaps at the expense of branch primordia differentia-tion. Additionally, because there is a sizeable fraction of flowers with underdeveloped floral organs, particularly those with no carpels, we hypothesize that increased ex-pression of OsMADS1(gain-of-function) promotes in-creased determinacy of fourth whorl cells. This specula-tion is supported by observations on phenotypic conse-quence of OsMADS1loss-of-function. The lhs loss-of-function mutant arises from mutations in the MADS box of OsMADS1, and the consequences are under-developed leafy lemma and palea, leafy lodicules, decreased sta-mens and occasional additional carpel or flower (Jeon et al. 2000). However these mutant flowers have normal ru-dimentary outer glumes as in wild type. Unlike lhs loss-of-function mutant, Ubi-OsMADS1spikelets show home-otic conversion of outer sterile glumes into lemma -palea like organs without affecting the lodicule morphology. These contrasting phenotypes are consistent with a role for OsMADS1and possibly a partner gene expressed in outer glumes in assigning lemma/palea fate. In strong loss-of-function lhs alleles, internal flowers are generated within a spikelet. These observations suggest a loss-of determinacy in the flower primordium in these mutants. These data, together with our observations of underdevel-oped carpels in pUbi-OsMADS1spikelets, implies that OsMADS1plays an important role in conferring deter-minacy to the floral meristem center i.e., the fourth whorl. They also suggest that OsMADS1possibly func-tions as a floral meristem as well as organ identity gene, though the mechanism by which it defines the floral mer-istem remains to be examined.

The closely related OsMADS5gene, whose product shares 72% identity with OsMADS1, perhaps shares some functions with OsMADS1(Kang and An 1997). Preliminary studies on the consequences of its ectopic expression in a heterologous system: tobacco, showed weak early flowering (Kang and An 1997). No data was gathered on floral organ types. The individual and com-bined contributions of OsMADS1and OsMADS5can be examined in rice plants that bear loss-of-func-tion/gain-of-function alleles for both loci. The genes ZMM8/ZMM3could be the orthologues in maize for the OsMADS1/OsMADS5genes, and may similarly be in-volved in functions distinct from that of previously de-scribed AP1or AGL9family of genes. Since no Arab-idopsis gene that is closely related to this sub-group has been found as yet, the role of such factors in dicot flow-ers remains to be investigated.

Acknowledgements We thank Prof. N.V. Joshi, Center for Eco-logical Sciences, IISc. for his critical thoughts and comments on the phylogenetic analysis of OsMADS1. We thank the Bioinforma-tics Center, IISc. for the computing facilities. U.V. acknowledges the grants from the Department of Science and Technology, Gov-ernment of India and The Rockefeller Foundation, USA, during the course of the study.References

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浅谈我国转基因水稻的研究(一)

浅谈我国转基因水稻的研究(一) 论文关键词]水稻转基因论文摘要]稻转基因研究是国内外植物分子遗传学研究的热点之一。目前，水稻转基因研究在我国已取得显著进展。详细介绍转基因技术，并阐明我国转基因技术在水稻上的应用及研究进展， 水稻是我国的重要经济作物和粮食作物。水稻分布极其广泛，由于生态环境的复杂性和所处地理环境的影响，水稻在漫长的进化过程中，形成了极其丰富的遗传多样性，染色体组型和数目复杂多样，成为研究稻种起源、演化和分化必不可少的材料。 植物转基因技术是利用遗传工程手段有目的地将外源基因或DNA构建，并导入植物基因组中，通过外源基因的直接表达，或者通过对内源基因表达的调控，甚至通过直接调控植物相关生物如病毒的表达，使植物获得新性状的一种品种改良技术。它是基因工程、细胞工程与育种技术的有机结合而产生的一种全新的育种技术体系。转基因技术可以将水稻基因库中不具备的各种抗性或抗性相关基因转入水稻，进一步拓宽了水稻抗病基因源，为抗病育种提供了一条新途径。 一、国内外的转基因技术 转基因技术自20世纪70年代诞生以来，已经取得迅速的发展。到目前为止，中国已经是全球第4大转基因技术应用国。 转基因生物技术的应用，大多分布在抗虫基因工程、抗病基因工程、抗逆基因工程、品质基因工程、品质改良基因工程、控制发育的基因工程等领域。中国是继美国之后育成转基因抗虫棉的第二个国家。现在河北省与美国孟山都合作育成33B抗虫棉（高抗棉铃虫、抗枯萎病、耐黄萎病）。由中国农科院生物中心、江苏省农科院导入Bt基因，由安徽省种子公司，安徽省东至县棉种场共同选育的抗虫棉“国抗1号”在安徽省已通过审定。国际水稻所将抗虫基因导入水稻，育成抗二化螟、纵卷叶螟的转基因水稻。中国农科院、中国农业大学、中国科学院、河南农科院等许多科研单位和高校将几丁质酶和葡聚糖酶双价基因导入小麦育成抗病转基因小麦、转基因烟草、转基因水稻等等。英国爱丁堡大学将水母发光基因导入烟草、芹菜、马铃薯等作物，获得发光作物，驱赶害虫。 至于油菜方面利用转基因工程培育雄性不育系及其恢复系的研究，亦取得了突破性的进展。比利时为了提高菜饼粗蛋白质的含量，将一种草控制的蛋白质基因转移到油菜上来，选出高蛋白质含量的转基因油菜品种。瑞典Svalow-Weibull等公司利用基因工程技术将外源基因导入甘蓝型油菜，培育成抗除草剂油菜新品种；比利时PGS公司采用基因工程手段创造出新的油菜授粉系统；法国应用原生质体融合技术将萝卜不育细胞质的恢复基因引入甘蓝型油菜，充分利用萝卜不育细胞质不育彻底的特性，实现了萝卜不育细胞质的三系配套，对推动全球杂交油菜育种具有革命性的影响。 二、我国转基因技术在水稻上的应用及研究进展 我国是农业超级国，因此，中国人吃饭问题的关键是水稻问题（高产和抗性问题），而水稻问题的核心便是转基因技术在水稻中的成功应用。 近年来，植物抗病毒基因工程的技术路线已趋向成熟，国内外相继开展了水稻东格鲁病、条纹叶枯病、黄矮病、矮缩病等8种病毒病的转基因育种研究，将各病原病毒的外壳蛋白基因、复制酶基因、编码结构或非结构蛋白基因干扰素CDNA等分别导入水稻，获得了抗不同病毒病的转基因株系或植株。在我国，转基因技术在水稻中的应用已经取得了惊人的成果。（一）转基因技术在提高水稻植株的抗Basra除草剂的成果 王才林等利用花粉管通道法将抗Basta除草剂的bar基因导入水稻品系“E32”，获得转基因植株。抗性鉴定表明，转基因植株能充分表达对Basta除草剂的抗性；通过对转基因植株后代PCR分析，证实bar基因已整合到受体植株的基因组中，遗传分析表明，bar基因能在有性生殖过程中传递给后代，并在T代开始分离出抗性一致的稳定株系。段俊等利用转基因技术，

转基因作物的研究进展

生物与环境工程学院课程论文 转基因作物的研究进展 学生姓名：魏斌聪 学号：200806016139 专业/班级：生物工程081班 课程名称：生物工程原理 指导教师：陈蔚青教授 浙江树人大学生物与环境工程学院 2011年5月

转基因作物的研究进展 魏斌聪 （浙江树人大学生物与环境工程学院生工081班浙江杭州310015） 摘要：人们将所需要的外源基因(如高产、抗病虫害优质基因) 定向导入作物细胞中, 使其在新的作物中稳定遗传和表现,产生转基因作物新品种, 是大幅度提高作物产量的一项新技术。本文先描述了转基因作物的发展进程，对其基因问题的研究作了讨论，并列出转基因作物目前存在的主要问题并作分析，最后对此项技术作出展望。 关键词：转基因作物；DNA技术；基因导入；安全性 前言 转基因植物（transgenic plant），是指基因工程中运用DNA 技术将外源基因整合于受体植物基因组、改变其遗传组成后产生的植物及其后代。转基因植物的研究主要在于改进植物的品质，改变生长周期等提高其经济价值或实用价值。[ 1 ]其主要范围是在作物方面，如可食用的大豆、玉米等，或者可投入生产的棉花等作物。 从表面上看来，转基因作物同普通植物似乎没有任何区别，它只是多了能使它产生额外特性的基因。从1983年以来，生物学家已经知道怎样将外来基因移植到某种植物的脱氧核糖核酸中去，以便使它具有某种新的特性：抗除莠剂的特性，抗植物病毒的特性，抗某种害虫的特性。[ 2 ]这个基因可以来自于任何一种生命体：细菌、病毒、昆虫等。这样，通过生物工程技术，人们可以给某种作物注入一种靠杂交方式根本无法获得的特性，这是人类9000年作物栽培史上的一场空前革命。[ 3 ] 1 转基因作物的发展进程 转基因作物的研究最早始于20世纪80年代初期。1983年，全球第一例转基因烟草在美国问世。1986年，首批转基因抗虫和抗除草剂棉花进入田间试验。1996年，美国最早开始商业化生产和销售转基因作物(包括大豆、玉米、油菜、

转基因育种研究进展

作物转基因育种研究进展 摘要：近年来，植物基因工程取得了辉煌的成就，而转基因技术由于其巨大的产业价值,特别是在作物品质改良、产量和抗逆性提高等方面的明显优势,一直是国际农业高新技术竞争的焦点和热点。本文主以棉花、玉米、水稻为例就转基因育种技术在作物上的研究进展进行相关的介绍。 关键词：作物，棉花，玉米，水稻，转基因育种，研究进展 植物转基因技术是指利用重组技术、细胞DNA培养技术或种质系统转化技术将目的基因导入植物基因组，并能在后代中稳定遗传，同时赋予植物新的农艺性状，如抗虫、抗病、抗逆、高产、优质等。常规育种常常受有性杂交亲和性的制约，而利用转基因技术可以打破物种界限、克服有性杂交障碍，快速有效地创造遗传变异，培育新品种、创造新类型，大大缩短新品种育成的时间。因此，随着现代生物技术的迅速发展，植物转基因技术也蓬勃发展［1］。 1 转基因棉花育种的研究与进展 近年来，随着基因工程技术的不断发展，利用生物技术来创新棉花种质资源和培育新品种是一条非常有效的途径，极大地推动了棉花遗传育种的发展［2］。中棉所是世界上唯一可以同时采用农杆菌介导法、花粉管通道法、基因枪轰击法快速获得转基因抗虫棉新材料的技术平台,能将植物嫁接技术成功应用于转基因棉花的快速移栽,成活率超过90%。未来3~5年,中棉所将挖掘、整合与优化抗病、抗除草剂等基因10个,筛选高产因子、高品质纤维等基因或分子标记150个,创造转基因棉花育种新材料100份以上,培育重大新品种(组合)3~5个。 1.1转抗虫基因 1991年成功将外源Bt基因导人棉株中，1992年人工合成了全长1824bp的CrylAb和CrylAc融合的GFMCry1A基因，并于1993年采用农杆菌介导法和外源基因胚珠直接注射法成功导入晋棉7号、中棉12、泗棉3号等主栽品种，获得了高抗棉铃虫的转基因棉花株系；包含CryIAc和AP基因双价抗虫基因载体，通过农杆菌介导转化冀合321胚性愈伤组织，经6代筛选后培育出抗棉铃虫90%的纯合品系，且农艺性状均优于对照。 1.2转抗黄萎病相关基因 利用花粉管通道法和农杆菌介导转化法将菜豆中的几丁质酶和烟草中的葡聚糖酶基因转入棉花，并从转基因高世代材料中筛选出了高抗黄萎病的品系；将天麻抗真菌蛋白基因用花粉管通道法转化天然彩色棉主栽品种，从高世代系中选育出既抗枯萎病又抗黄萎病的兼抗材料；将葡萄糖氧化酶基因(GO)转入棉花，转基因后代对枯萎病和黄萎病抗性均有显著提高，部分材料抗性达到抗病水平。1.3转抗除草剂基因 1997年由美国孟山都公司推出抗除草剂棉花抗性品种，他们从土壤农杆菌变种CP4中分离到编码抗草甘膦酶的基因，并通过农杆菌介导法转化珂字棉312，把该基因导入棉花植株，从而使其对草甘膦产生抗性。采用中棉35下胚轴为材料，将草甘膦突变基因aroAM12导入到棉花中，获得65棵再生植株，通过Southern及Western试验验证了该基因的导入和表达状况，结果表明，转化株对草甘膦具有很高的抗性；将抗草甘膦基因aroAM12和抗虫基因Btslm一起整合到一个载体中，并以抗草甘膦基因作为选择标记，通过转化棉花品种石远321后获得了抗草甘膦和抗棉铃虫的再生株。

转基因作物安全评价研究进展

转基因作物安全评价研究进展 转基因技术是现代生物技术的核心。推进转基因技 术研究与应用，是着眼于未来国际竞争和产业分工的重大发展战略，是解决粮食短缺、人口问题、确保国家粮食安全的必然要求和重要途径。温家宝总理2010年政府工作报告中 明确指出要重点抓好“以良种培育为重点，加快农业科技创新和推广，实施好转基因生物新品种培育科技重大专项”工作。“农业转基因生物新品种培育科技重大专项”的实施，标志着转基因技术已成为我国抢占科技制高点和增强农业国际竞争力的战略重点。转基因技术自诞生以来，生物安全问题相伴而生。在转基因作物的研究和产业化过程中，转基因作物的安全性成为亟待解决的关键问题。 1 国内外转基因作物安全评价原则 全球各国都加强了对转基因作物安全性评价的研究工作，主要国际组织和研究机构都制定了相关“基于实质等同性”的安全评价原则和标准，在遵循这一原则的基础上对转基因作物进行安全性评价…。 2转基因作物安全评价体外实验研究现状 目前，转基因作物食用安全性评价主要方法是实验研究法。实验研究法有体外实验和体内实验两种研究途径。体外实验是通过各种物理化学方法对转基因作物及其产品进行评价分析。主要有关键成分分析和营养学评价：如蛋白质及氨基酸、脂肪及脂肪酸、碳水

化合物、矿物质、维生素等营养成分分析；抗营养因子和酶抑制剂等抗营养成分和天然毒素分析；因基因修饰生成的新成分和其他可能产生的非预期成分分析等。还有转基因作物主要成分稳定性分析：如 加工贮存过程中转基因作物稳定性的研究；转基因作物在动物体内消化稳定性的研究等。 现有研究表明转基因大豆、豆粕中干物质、粗脂肪、粗蛋白、中性洗涤纤维、酸性洗涤纤维、灰分、钙和总磷8种普通营养成分与普通大豆含量较接近，无显著差异；转基因大豆中氨基酸、微量元素铁、铜、锰、锌含量与普通大豆相近。转基因大豆中转基因植酸磷、胰蛋白酶抑制因子、脲酶活性和蛋白溶解度等抗营养因子未发生变化，大豆异黄酮和大豆凝集素等在二者之间也具有实质等同性[10]。研究者 还认为尽管转基因大豆中转基因豆粕C14:1脂肪酸、C22:0 脂肪酸、共轭亚油酸含量存在差异，但二者差异没有实际意义，饱和脂肪酸、不饱和脂肪酸含量及各种脂肪酸含量与传统常规大豆间无显著差异。转基因大豆与常规大豆具有实质等同性。部分研究也表明转基因玉米、转基因大米与普通作物具有实质等同性。 3转基因作物安全评价体内实验研究现状 体内实验主要是通过先饲喂动物转基因产品，然后通过研究实验动物身体各方面机能参数（日常活动、体液指标、器官发育、病理检查等）来评价转基因作物的安全性。一些研究表明转基因作物对动物的影响与传统非转基因作物相同。如有研究证实：转基因大豆

水稻转基因步骤

在植物转基因过程中，为了有效地识别和筛选转化子，常将目的基因和标记基因构建在同一表达载体中。这种载体结构导致转基因植物中目的基因和标记基因始终共存，而标记基因（尤其是抗生素抗性基因）的存在可能给转基因植物的生物安全带来隐患。目前已研发了多种方法剔除转基因植物中的标记基因，其中最常见的是共转化法(Komari 1996，McCormac 等2001)。共转化系统是采用2个质粒或1个含有两套T—DNA表达盒的表达载体共同转化植物，其中一套表达盒含有抗性选择标记基因，另一套表达盒含有目的基因，它们转化植物时可能整合到植物基因组的不同位置。转基因植株在减数分裂过程中，标记基因和目的基因发生分离，从而可在转基因后代中筛选到只含目的基因而不含选择标记基因的个体。共转化从根本上排除了转基因植物中的选择标记，是保证人畜和环境安全的重要措施，因此受到了广泛的重视。Zhou 等（2003）认为，用分别含一个T-DNA区的两个载体共转化的效率低于双T-DNA区表达载体的共转化效率。目前关于利用双T-DNA区表达载体，获得无选择标记转基因阳性株系的研究已有不少报道（唐俐等2006，张秀春等2006，于恒秀等2005）。花药培养与遗传转化技术相结合，可以快速获得纯合转基因植株（斯华敏等，1999，付亚萍等，2001），但是应用花药培养快速获得只含目的基因而无选择标记的转基因研究尚未见报告。 水稻是最主要的粮食作物，转基因水稻的安全显得尤为重要。本实验室通过农杆菌介导的水稻转化体系，将包含人乳铁蛋白(hLF)、高赖氨酸(SB401)、高甲硫氨酸(RZ10)基因的表达载体p13HSR成功转化脆茎稻，由于该表达载体采用双T-DNA结构，将检测出含选择标记潮霉素磷酸转移酶基因（hpt）和目的基因的转基因阳性T0植株按单株直接进行花药培养。在189株二倍体花培植株中检出23株有目的基因没有选择标记hpt的转基因纯合植株，得率为9.87％。RT-PCR检测结果显示外源基因已整合到转基因水稻基因组中并转录。本文首次发现插入的外源基因间存在交换事件，从而改变了花培群体中无选择标记而目的基因阳性的转基因纯系的获得率。同时还对农杆菌介导的同一载体上多个基因转化水稻后，会出现个别基因丢失的情况进行了讨论。 基因转化方法参照Hiei等(1994)的方法并加以修改。取开花后12-15 d左右的稻穗脱粒，表面灭菌后接种在NB培养基上，26℃暗培养诱导愈伤组织。约5-7d后取愈伤组织在相同条件下继代培养，用于共培养。农杆菌于含50mg/L卡那霉素(Kam)的YM平板上划线,28℃黑暗培养3d，用金属匙收集农杆菌菌体,将其悬浮于共培养CM液体培养基中,调整菌体浓度至OD600为0.3-0.5，加入AS（终浓度为100mΜ）,即为共培养转化水稻用的农杆菌悬浮液。将继代培养4d后的愈伤组织浸于此菌液中，20min后取出并用无菌滤纸吸去多余菌液，随即转入铺有无菌滤纸的固体培养基上，于26℃下暗培养2~3d。共培养后的愈伤组织在含有50mg/l潮霉素的筛选培养基上，26℃暗培养14d，再转到新鲜配制的筛选培养基上继续筛选14d。然后选择生长旺盛的抗性愈伤组织转移到含有50mg/l潮霉素的分化培养基上，暗培养3天后转至15h/d 光照条件下培养,再生的小苗在1/2MS上生根壮苗两周左右。选择高约10cm、根系发达的小

水稻转基因育种研究进展 7

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转基因水稻大规模生产重组人血清白蛋白

转基因水稻大规模生产重组人血清白蛋白 由武汉大学生命科学院教授、武汉禾元生物科技有限公司董事长杨代常领衔的研发团队从2006年开始进行植物源替代血浆来源的医药蛋白的 研究与开发，现已取得突破性进展并已跨入规模化生产的阶段，填补了国际上此项技术空白。相关论文于2011年10月31日在线发表于《美国 科学院院报》。该论文在线之际，受到国外Scientist ，Nature news, The Australian, Thomson Reuters, Fox News, Agence France Presse (AFP法新社)等美国、英国、俄罗斯、德国、巴西、印度各专业杂志及媒体的广泛关注和报道。 该研究表明由转基因水稻种子生产的重组人血清白蛋白(OsrHSA)在生理生化性质、物理结构，生物学功能、免疫原性与血浆来源的人血清白 蛋白一致；并建立了大规模生产重组人血清白蛋白的生产工艺，获得了高纯度和高产量重组人血清白蛋白产品。利用大量数据证明了转基因 水稻种子可取代现有基于发酵的表达技术来生产重组蛋白质是经济有效的。正如PNAS 审稿人对该文章的评价：“这篇文章解决了在科学上振 奋人心、在经济上都非常重要的议题－－即用转基因植物生产血浆产品或其他蛋白产品的技术平台，可代替其他基于发酵的表达技术，其重 要性也不言而喻……这篇文章近乎完美地证实了植物生产的医药蛋白和批准临床使用的血浆来源医药蛋白是完全相同的，并提供了翔实数据 证明植物系统规模化容易和成本优势。” 目前，人血清白蛋白(human serum albumin)广泛应用于临床治疗和细胞培养领域。常见的人血清白蛋白大多数从人的血浆中提取，这样的生 产方式不仅受到血浆供应的限制，而且还具有携带病毒传播的高风险性。国际上以重组人血白蛋白替代血源产品的应用已成为趋势，国内市 场需求也逐年扩大，2010年已达150吨。尽管市场广阔，但高纯度重组人血白蛋白的规模化生产技术和质量控制技术却是世界性难题。武汉禾 元历经多年的技术攻关，利用水稻胚乳表达技术平台，研发出国际先进水平的重组人血白蛋白产品生产技术，并成功实现重组人血白蛋白规 模化和产业化，完全摆脱了相关制约，具有纯度更高、无动物组分、安全、高效、绿色环保、廉价、无限量供应等优势。随着植物源重组人 血清白蛋白的发展，我国人血清白蛋白日益紧张的局面必将得到缓解。

我国转基因水稻现状及安全管理

我国转基因水稻现状及安全管理 环境与生化工程系食品生物技术 0901班刘文婷随着世界经济和科技的发展，转基因物质经本上已不再是天方夜谭，几乎可以说是家喻户晓了。 自从第一株转基因烟草问世以来，转基因技术日趋成熟，世界各国都应为转基因技术的发展，是国家的工农业的到发展，特别是发展落后国家和发展中国家，转基因技术使国家的经济得到发展，农民生活得到改善。 我国是一个人口众多，粮食短缺的国家，所以转基因技术是我国的粮食产量得到提高，玉米、小麦、水稻……都已涉及到转基因技术，而事实上转基因技术确实为我们带来了预想不到的喜悦，但是同时又带来了不可避免的问题和担忧。 水稻—13亿中国百姓的主食，转基因水稻必不可免的成为人们担忧的对象，虽然农民伯伯自己会种植它，但是他们却不会轻易的去以身试法。全国人大香港特别行政区代表蔡素玉接受《环球财经》记者采访时揭示了跨国公司通过种子盈利的奥秘:种子公司通过加收专利费抬高转基因种子的价格，农民在种植转基因水稻的时候必须多付2 倍～3 倍以上的价格来购买转基因的种子。而且，转基因的种子是不允许下一年再种植的，农民必须再购买新的种子，无疑提高了农民的生产成本，加重了农民的负担。报道同时指出，据绿色和平组织的有关调查，转基因作物并不能降低农药使用量，恰恰相反，孟山都转基因大豆所需的农药总量有增无减。我国的Bt 棉花也发现这样的问

题。美国学者在研究这个问题时发现，由于转基因种子不是每个国家都可以有的，如果弱小国家大量使用，几代下去，种子就必须向国外进口，购买的价格会越来越高，直到这些国家的粮食主权被大的国家控制。在转基因水稻商业种植之前，应该充分考虑到转基因食品的副作用，甚至不妨将转基因食品的副作用放大。而对转基因食品，当前不少人对其安全性表示了担忧。有专家表示，转基因至少存在三方面的不确定性：一是转基因对生命结构改变后的连锁反应不确定；二是转基因导致食物链“潜在风险”不确定；三是转基因污染、扩散及其清除途径不确定。 转基因水稻对中国人和中国社会的冲击是多方面的，但主要表现在人们对其安全性的怀疑。自从转基因作物诞生以来，对其安全性的争论就没有断绝过，而且有愈演愈烈的趋势，中国批准转基因水稻则是火上加油。就目前的研究而言，既没有转基因作物是绝对安全的研究结论，也没有转基因作物是绝对不安全的研究结论。 目前，转基因水稻的不确定性大于确定性。不确定性在专业领域的称谓是“非预期效应”，相当多的人认为这就是潜在的危险。转基因作物的“非预期效应”主要包括几个方面:一是外源DNA(基因)随机插入可能破坏宿主原有的功能基因，产生非预期效应。二是蛋白质表达发生改变或形成新的代谢产物，产生非预期效应。三是可能诱发突变，产生非预期效应。四是转基因产生高水平表达的酶可能引起继发性生化反应，产生非预期效应。五是其他非预期效应。能威胁到人们的健康，而且还会对生态造成极大的破坏。

转基因水稻简介

转基因水稻简介 水稻是世界上最重要的粮食作物之一，杂种优势的成功利用使得水稻产量得到了极大的提高，为解决世界范围内的粮食危机做出了极大的贡献。但是，自20世纪80年代以来，杂交水稻的产量就处于徘徊不前的局面。不断提高水稻产量和改良其品质是当前水稻育种的重要任务，这一任务的完成单纯依靠传统的遗传育种是不可能实现的。 80年代产生的转基因技术由于直接在基因水平上改造植物的遗传物质、可定向改造植物的遗传性状、外源基因的转入打破了物种之间的生殖隔离障碍、丰富了基因资源等优点而弥补了常规育种方法的不足，得到了前所未有的发展。许多学者在水稻的转基因研究上做了大量工作并取得了很大的进展，为水稻的遗传改良奠定了基础。 转抗虫基因： 害虫是危害我国农业生产的主要限制因素，大量化学农药的使用不但污染环境，而且也使得有益昆虫的数量锐减，害虫的抗药性不断加强。此外，化学杀虫剂使用后的农药残留对人畜都会有严重的危害。因而植物抗虫基因工程成为科学家的研究热点领域之一。由于水稻本身没有足够的抗虫基因，目前研究者利用人工合成或从其它生物中克隆的抗虫基因转化到水稻栽培品种中，提高品种的抗虫性。 在水稻抗虫转基因方面，使用得较多的基因有：苏云金杆菌毒蛋白基因(Bt)、蛋白酶抑制剂基因(Pin2，SKTI，OC—IAD86，Cp-Ti)、植物凝集素基因(GNA)等，将这些基因导入水稻，可使水稻产生对二化螟虫、三化螟虫、稻纵卷叶螟等鳞翅目害虫及蝗虫、褐飞虱、线虫的抗性。Bt毒蛋白基因是目前使用最广的基因，众多的研究都表明用转基因的方法将Bt毒蛋白基因导入常规水稻可使水稻对螟虫的抗性提高刚。 转抗病基因： 病害(包括真菌病、细菌病和病毒病)是影响我同农业生产的另一类重要限制因素。在我国，大面积发生且危害严重的病害有水稻稻瘟病、纹枯病、白叶枯病，因此，我国科学家在抗病基因工程方面也开展了大量的工作。 转抗逆基因： 逆境是限制植物生长、影响产量形成的重要因素之一。抗逆基因的分离、克隆、转化一直受到科学家们的高度重视。目前已分离出大量的抗逆相关基因，并在抗逆基因的遗传转化中取得了明显的成绩。Hossan等已分离克隆出3个与水稻耐淹能力有关的基因pdc I，pdcⅡ，pdcⅢ，并转入水稻中获得部分转基因植株．Rathinasabathi等将烟草中的CMO基因导入水稻，获得了具有很强抗旱性的转基因水稻．日本村田纪夫将甜菜碱生物合成酶基因codA导入水稻，获得了耐碱性的转基因水稻植株．高倍铁子等将编码大肠甜菜碱生物合成酶基因ktA导入水稻，获得了耐盐性强的转基因水稻植株。

转基因水稻的进展

转基因水稻的进展 水稻是最重要的粮食作物之一,世界上约有一半以上的人口以稻米为主食。据专家预测,到2025年在现有稻谷产量的基础上再增加60%才能满足需要。随着人口的增长和耕地面积的减少,世界尤其是我国将面临粮食问题的严峻挑战,培育优良品种成为提高稻谷产量的主要途径。传统的育种技术已为培育水稻新品种做出了巨大贡献,并将继续发挥主导作用,但由于品种资源的贫乏,单靠传统育种已很难有太大的突破。 20世纪下半叶以来,随着分子生物学研究的不断发展,基因工程技术特别是转基因技术在植物遗传育种上得到了广泛的应用,并取得了显著的成就。转基因技术就是将外源基因通过生物、物理或化学手段导入其它生物基因组,以获得外源基因稳定遗传和表达的遗传改良体。自1983年世界上首例转基因植物———一种含有抗生素药类的烟草在美国成功培植以来,全球范围转基因作物的种植面积和销售收入均以倍数增长。2004年,转基因作物面积(主要是大豆、玉米、油菜和棉花)已达11250万hm2,已被批准可使用的产品有1000多种。水稻作为世界上最重要的粮食作物之一,自1988年首次获得可育的转基因水稻以来,转基因技术在水稻品种改良上得到了广泛的应用,已选育了一系列转基因水稻品系(组合)。本文简要介绍了近年来水稻转基因研究方面所取得的成就,并就存在的问题提出了一些看法。 1水稻转基因研究进展在植物分子生物学研究的众多材料中,水稻由于其

基因组较小、重复序列较少等优点而成为一种重要的模式植物。自1988年首次获得转基因水稻以来,水稻转基因技术已获得突飞猛进的发展,目前已成功获得籼稻、粳稻、爪哇稻的转基因水稻。 1.1抗虫转基因水稻研究 虫害是水稻生产中的一大害,化学药剂杀虫不仅成本较高,而且严重污染环境,抗虫转基因水稻的应用前景是不言而喻的。目前应用于水稻抗虫性改良的外源基因主要有苏云金杆杀虫结晶蛋白基因(Bt基因)、昆虫蛋白酶抑制剂(PI基因)和植物凝集素基因3种,其中Bt基因是当前应用最为广泛的杀虫基因。 1989年中国农业科学院生物技术中心杨虹等将Bt基因导入水稻品种台北309、中花8号的原生质体并获得再生植株;Maqbool等通过基因枪法将人工合成的CryIIA基因转入水稻,毒蛋白的表达量可高达1%,某些植株的杀虫率可达到100%。浙江大学舒庆尧等采用农杆菌介导法将密码子经优化Bt基因cryIA(b)导入到“秀水11”,获得抗性株系的Bt毒蛋白表达量占可溶性蛋白的0.5%~3%,对二化螟、稻纵卷叶螟和稻螟蛉1-5龄幼虫的毒杀作用达到100%,对8种鳞翅目害虫均表现高抗。中国科学院遗传与发育所朱祯等将豌豆胰蛋白酶抑制剂基因与信号肽和内质网定位信号KDEL的编码序列融合,得到融合基因,其编码的融合蛋白具有富积于内质网的特性,从而大大提高了转化植株的杀虫效果。转化该基因的水稻比转化未修饰的cpti基因的植株蛋白酶活性平均高出2倍。目前利用该基因已获得了包括明恢81和明恢86等高抗二化螟鳞翅目害虫的转基因水稻植株,用其配制的杂交组合已批准进入中试。

转基因水稻的商业化

转基因水稻：商业化前夜（发表于：2010-02-22 21:52:41） 两种转基因水稻首次获得农业部安全审批，距离普通中国公众的餐桌越来越近；但关于转基因水稻的争议正不断升级 这是一顿不同寻常的“年夜饭”。 2月5日晚，武汉市狮子山，华中农业大学作物遗传改良国家重点实验室50多位研究人员共聚一堂。寿司作为开胃点心放在头盘，红薯稀饭和武汉名小吃豆皮则是主食——这些米制品的原料，都是实验室研发的转基因水稻。 实际上，由中国科学院院士张启发带领的上述研究团队食用转基因大米已经多年，但此次意义非凡：他们研发的两种转基因水稻，率先获得了农业部颁发的安全证书。 1月31日公布的中央一号文件，明确提出“在科学评估、依法管理基础上，推进转基因新品种产业化”，这对于张启发及其他转基因水稻研发者来说，无疑也是利好消息。 转基因大米进入普通中国公众的餐桌，尚差“临门一脚”。由于安全证书是转基因作物品种上市之前最难的关口，剩下来的或许仅仅是时间问题。但关于转基因水稻的争议，正因此进一步升级。 神秘的安全证书 2009年10月22日，中国生物安全网公布了“2009年第二批农业转基因生物安全证书批准清单”。该网站由农业部农业转基因生物安全管理办公室主办。 清单中的两种转基因水稻，正是由华中农业大学团队所研发：在华恢1号和汕优63这两种水稻品种中转入具有Bt抗虫蛋白的基因Cry1Ab/Cry1Ac。这也是中国首次颁发转基因水稻的生产应用安全证书。 所谓Bt抗虫水稻，是将野生土壤细菌苏云金芽孢杆菌(下称Bt)中的基因经人工合成后，插入到水稻的遗传物质DNA 中，使水稻自己产生Bt抗虫蛋白，杀死多种以谷物为食的螟蛾科害虫。 两种转基因水稻的有效期是2009年8月17日至2014年8月17日，生产应用范围限在湖北省。 华中农业大学研究团队成员林拥军教授表示，安全证书应该是2009年8月就批了，但他们也是2009年11月才知道。 公众知道的时间则更晚。2009年12月初，很多媒体和公众才从一些专家和国际环保组织绿色和平那里获知消息。 林拥军表示，现在就说转基因水稻已经打开商业化种植的大门为时尚早，但有了农业部的安全认证，除了商业化应用所必需的品种证，不再需要其他的证书。 “是否有生产利用价值，要经过品种审定评价，我们已经向湖北省农技推广部门提交生产性能审定材料。”他解释说。 品种审定通常需要进行区域试验，时间可能会持续两年到三年。但在很多业内人士看来，安全证书是最难过的“关口”，之后更多的只是程序问题。 两种转基因水稻以近乎神秘的方式“闯关”成功，让人觉得有几分意外。而其他的转基因水稻品种，也正在排队等待安全审批。 第三次飞跃 与西方国家以小麦消费为主、稻米只作为补充不同，稻米是中国城乡居民最重要的口粮。随着人口的增长和消费水平的提升，中国水稻生产面临的压力也越来越大。根据中国水稻研

国内外转基因农作物研发进展_彭永刚

植物生理学报 Plant Physiology Journal 2013, 49 (7): 611~614611 收稿 2013-04-23 修定 2013-05-20 资助 转基因生物新品种培育重大专项(2011ZX08001-001和 2013ZX08012-002)。 * 通讯作者(E-mail: zzhu@https://www.wendangku.net/doc/ea12427304.html,; Tel: 010-********)。 国内外转基因农作物研发进展 彭永刚, 张磊, 朱祯* 中国科学院遗传与发育生物学研究所植物基因组学国家重点实验室, 国家植物基因研究中心(北京), 北京100101 摘要: 发展转基因技术可以更好地应对我国农业上面临的耕地减少、水资源缺乏等诸多问题, 然而转基因技术却引起了广泛的争议。本文综述了国内外转基因农作物的研发进展, 以及我国转基因产业化等问题, 阐述了应用先进技术对我国农业可持续发展以及确保粮食安全的重要作用。同时, 本文概括了我国在基因组学研究和基因挖掘上取得的重要进展, 以及我国转基因产业化已经具备的发展条件。本文还对未来我国种业尤其是生物技术种业的发展做出展望。关键词: 转基因作物; 转基因技术; 产业化 A Review on Research and Development of Transgenic Crops PENG Yong-Gang, ZHANG Lei, ZHU Zhen * State Key Laboratory of Plant Genomics, National Plant Gene Research Center (Beijing), Institute of Genetics and Developmen-tal Biology, Chinese Academy of Sciences, Beijing 100101, China Abstract: Development of transgenic technology helps to deal with China's agricultural challenges such as the reduction of arable land, and water scarcity etc. However, bio-safety involved in transgenic technology has aroused widespread controversy. This paper reviewed the progress in research and development of transgenic crops, and commercialization of transgenic crops both in China and abroad. Meanwhile, the paper brie ? y sum-marized the research progress that has been made in genome sequencing and functional genomics in China, and discussed future prospects of seed industry, especially biotechnology seed industry in China.Key words: transgenic crop; transgenic technology; commercialization 1 我国农业的主要问题及解决途径 目前, 我国农业面临着三方面的重大挑战。首先耕地锐减, 人均耕地面积不足世界平均水平的40%。其次, 水资源匮乏, 人均水资源占有率只有世界平均水平的25%左右。再次, 病虫害、旱涝等自然灾害频发, 环境恶化。 发展转基因技术可以有效地缓解或解决这些问题。第一, 转基因农作物品种能够显著提高农作物产量, 改善农产品的品质, 确保我国的粮食安全。第二, 进行抗性的转基因育种还可以降低农药、化肥的施用量, 确保我国农业的生态安全。第三, 通过开发功能性和治疗性的食品, 可以提高农产品的附加值, 增加农民的收入。第四, 通过对转基因技术的研究、创新, 可以建立我国自己的生物技术研发体系, 提高我国在这方面的国际竞争力。 2 转基因农作物国际研发进展 国际上对转基因农作物的研究已有30年历 史。1983年, 第一例转外源基因植物(烟草)获得成功(Zambryski 等1983); 1987年, 第一例转基因植物(转基因抗虫番茄)田间试验在美国进行; 1994年, 转基因番茄上市; 1996年, 全球转基因作物种植面积已达160万公顷; 到了2012年, 种植面积达1亿7千万公顷, 约30个国家正式批准种植转基因农作物, 从1996年到2012年, 累计种植面积达15亿公顷(International Service for the Acquisition of Agri-biotech Applications, https://www.wendangku.net/doc/ea12427304.html,/resources/publications/briefs/default.asp)。 目前, 美国是种植转基因作物面积最大的国家, 面积达6 950万公顷, 其后依次为巴西、阿根廷、加拿大、印度和中国。2012年我国转基因作