新疆特殊生物功能基因克隆及应用研究
|
摘要
新疆特殊生物功能基因克隆及应用研究
随着人类基因组计划和其它模式生物如拟南芥、水稻等基因组计划的完成和人们对基因认识的不断深入，基于同源序列克隆候选基因的方法成为了一条快捷克隆基因家族成员的新途径。土壤盐分是制约作物产量的主要因素，它可使作物尤其是灌溉农业的作物减产。土壤盐分过高还可造成盐碱地，限制土壤的利用。目前，世界耕种土地的大约20%和接近一半的灌溉土地都遭到了盐胁迫的危害。随着分子遗传学和植物转基因技术的发展，采用生物技术提高作物的耐盐性，使作物在盐胁迫环境中能正常生长并提高作物的产量，正受到越来越多的关注并取得了一定的成果。NHX1是液泡膜中的一种Na+/H+反向运输体，可促进钠离子在液泡中的区隔化效应，跨液泡膜的pH为其提供能量。钠离子和氯离子区隔化在液泡中，不仅可作为有效的渗透调节剂，还可减小细胞毒性，从而提高植物的耐盐能力。本研究以同源序列克隆候选基因的方法主要开展新疆野生耐盐碱植物犁苞滨藜NHX1基因的克隆、盐角草雄性不育基因orf25基因的克隆、大肠杆菌nhaA和nhaB基因的克隆、拟南芥AtNHX2 基因启动子的克隆、表达模式分析及其分子检测的研究。
研究工作首先根据GenBank上已发表的各种物种的NHX1基因序列，分析其保守性并根据保守区设计了7条引物（上游4条，下游3条），分别组合经RT-PCR扩增出新疆野生耐盐碱植物犁苞滨藜NHX1基因的核心片段，连接上pMD18-T载体，经DNA序列测定后，结果与GenBank中NHX1基因序列进行同源性比较，确定已克隆出的核心片段为NHX1基因的核心片段。根据已获得的核心序列设计3`-RACE和5`-RACE引物，扩增出NHX1基因的3`末端和5`末端，拼出全长序列。根据已拼接出的NHX1全长cDNA序列设计基因特异性引物，利用RT-PCR方法扩增NHX1全长cDNA。将扩增产物连接到pMD18-T载
体上并进行测序。序列分析表明已克隆出AdNHX1全长cDNA，已在GeneBank注册为新基因（基因注册号为：AY211397）。将其与AtNHX1进行同源性比较后，发现氨基酸具有很大差别，其后续研究可能有助于阐明耐盐植物和甜土植物耐盐能力的不同。从而为解决我国新疆地区大面积的盐碱荒地提供可行的办法。
根据已发表的orf25基因、nhaA基因和nhaB基因设计基因特异性引物，采用RT-PCR或PCR的方法扩增相应的基因片段，序列分析显示所克隆的基因均含有完整的编码框，分别是orf25基因、nhaA基因和nhaB基因。
为了检测AtNHX2 启动子是否受盐胁迫诱导，根据拟南芥的基因组DNA序列，设计一对引物扩增AtNHX2基因起始密码子ATG上游一段约2800bp的片段，序列分析显示克隆的片段与发表的一致。将该片段构建到植物表达载体pCAMBIA1301-1中，序列分析显示重组质粒构建正确。通过基因枪轰击洋葱表皮瞬时表达进行初步检测，检测结果说明该启动子具有活性。pCAMBIA1301-1/ AtNHX2 promoter重组质粒通过Floral dip method转染拟南芥，收获种子，筛选具有抗性的转基因植株，收获T1代转基因种子。T1代种子萌发后采用GUS染色的方法初步鉴定转基因是否成功，然后采用PCR扩增技术进一步验证。T2代转基因植株GUS表达分析显示，AtNHX2 在所有的组织中均有表达。在保卫细胞中检测到了强烈的GUS表达，这一结果表明AtNHX2 对特殊细胞的pH调控和K+自身稳定方面起着重要的作用。AtNHX2 启动子的活性可被NaCl和ABA调控，说明盐和ABA是在转录水平上调控AtNHX2的表达的。在老叶中GUS活性比在新叶中GUS活性强，这说明了AtNHX2在耐盐方面的作用。在根毛细胞中也观测到了强烈的GUS活性，这就暗示了AtNHX2 在扩大的液泡中储存Na+。这为利用该基因提高植物耐盐水平，改良农作物品种奠定了坚实的基础。
Studies on Molecular Cloning of Functional Genes from Special Organisms in Xinjing and Their Application
With the accomplishment of the human genome project and others mode organisms such as Arabidopsis thaliana and rice, it has become a shortcut way to clone new genes in a gene family via homology sequences. Salt stress is the primary effect that limits and decreases the output of crops in many parts of the world, particularly irrigated land. Overly high soil salinity can cause salina and limit the utility of soil. Today, ~20% of the world’s cultivated land and nearly half of all irrigated land are affected by salinity. Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the salt tolerance of crops, keeping steady-state growth and increasing the output in the saline environment. NHX1 , a tonoplast Na+/H+ antiporter energized by the (pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na+ into the vacuole, which results in avoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis. This research contains: cloning of NHX1 gene from Atriplex dimorphostegia in Xinjiang by RT-PCR and RACE; cloning of orf25 gene, nhaA gene, nhaB gene and AtNHX2 promoter; expression analysis and molecular testing.
At first, the conservative region of the variable NHX1 gene sequences published in Genbank were analyzed, and seven primers (4 upstream, 3 downstream) were designed for amplifying core fragment of NHX1 according to these
conservative region. Total RNA was isolated from the plant tissue and cDNAs were synthesized by reverse transcription. The different combinations of these primers were used to amplify the core fragment of NHX1. The core fragments were cloned into pMD18-T cloning vector, positive clones were checked with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the core fragments from Atriplex dimorphostegia were similar with the AtNHX1 (short for NHX1 from Arabidopsis thaliana) sequence (GI: 30690553). With a set of primers designed according to the sequence of the cloned core fragments, the 3’ and 5’ ends of the cDNAs were obtained by 5’ and 3’ rapid amplification of cDNA ends (RACE), respectively. Combining the sequences of the 3’ ends, 5’ ends and cloned core cDNA, the full-length sequence of the NHX1 cDNA was assembled. To obtain the open reading frame (ORF) of the NHX1, a pair of primers was designed according to the assembled cDNA sequence. PCR product was cloned into pMD18-T cloning vector and sequenced. The result shows that ORF of the AdNHX1 was obtained. And then it was registered in Genbank (GI: AY211397). The deductive amino acid sequence of AdNHX1 (short for NHX1 from Atriplex dimorphostegia) is significantly different from AtNHX1. The result may have important implications for the elucidation of the different abilities of halophytes and glytophytes for salt tolerance, and may present a feasible method to utilize much more salina area in Xinjiang.
Specific primers for orf25 gene, nhaA gene and nhaB gene were designed according to published sequences and RT-PCR or PCR were conducted to amplify orf25 gene, nhaA gene and nhaB gene. Sequence analyses suggest that cloned genes contain entire coding frame of orf25 gene, nhaA gene and nhaB gene, respectively.
In order to detect whether AtNHX2 promoter can be induced by salt stress, a
pair of primers were designed to amplify a 2.8kb DNA fragment upstream of ATG start codon of AtNHX2 gene according to the sequence of Arabidopsis thaliana genomic DNA. Sequence analysis shows that the amplified fragment conform to the published sequence. AtNHX2 promoter was cloned into the plant expression vector pCAMBIA1301-1. The promoter activity was detected by transient e
随着人类基因组计划和其它模式生物如拟南芥、水稻等基因组计划的完成和人们对基因认识的不断深入，基于同源序列克隆候选基因的方法成为了一条快捷克隆基因家族成员的新途径。土壤盐分是制约作物产量的主要因素，它可使作物尤其是灌溉农业的作物减产。土壤盐分过高还可造成盐碱地，限制土壤的利用。目前，世界耕种土地的大约20%和接近一半的灌溉土地都遭到了盐胁迫的危害。随着分子遗传学和植物转基因技术的发展，采用生物技术提高作物的耐盐性，使作物在盐胁迫环境中能正常生长并提高作物的产量，正受到越来越多的关注并取得了一定的成果。NHX1是液泡膜中的一种Na+/H+反向运输体，可促进钠离子在液泡中的区隔化效应，跨液泡膜的pH为其提供能量。钠离子和氯离子区隔化在液泡中，不仅可作为有效的渗透调节剂，还可减小细胞毒性，从而提高植物的耐盐能力。本研究以同源序列克隆候选基因的方法主要开展新疆野生耐盐碱植物犁苞滨藜NHX1基因的克隆、盐角草雄性不育基因orf25基因的克隆、大肠杆菌nhaA和nhaB基因的克隆、拟南芥AtNHX2 基因启动子的克隆、表达模式分析及其分子检测的研究。
研究工作首先根据GenBank上已发表的各种物种的NHX1基因序列，分析其保守性并根据保守区设计了7条引物（上游4条，下游3条），分别组合经RT-PCR扩增出新疆野生耐盐碱植物犁苞滨藜NHX1基因的核心片段，连接上pMD18-T载体，经DNA序列测定后，结果与GenBank中NHX1基因序列进行同源性比较，确定已克隆出的核心片段为NHX1基因的核心片段。根据已获得的核心序列设计3`-RACE和5`-RACE引物，扩增出NHX1基因的3`末端和5`末端，拼出全长序列。根据已拼接出的NHX1全长cDNA序列设计基因特异性引物，利用RT-PCR方法扩增NHX1全长cDNA。将扩增产物连接到pMD18-T载
体上并进行测序。序列分析表明已克隆出AdNHX1全长cDNA，已在GeneBank注册为新基因（基因注册号为：AY211397）。将其与AtNHX1进行同源性比较后，发现氨基酸具有很大差别，其后续研究可能有助于阐明耐盐植物和甜土植物耐盐能力的不同。从而为解决我国新疆地区大面积的盐碱荒地提供可行的办法。
根据已发表的orf25基因、nhaA基因和nhaB基因设计基因特异性引物，采用RT-PCR或PCR的方法扩增相应的基因片段，序列分析显示所克隆的基因均含有完整的编码框，分别是orf25基因、nhaA基因和nhaB基因。
为了检测AtNHX2 启动子是否受盐胁迫诱导，根据拟南芥的基因组DNA序列，设计一对引物扩增AtNHX2基因起始密码子ATG上游一段约2800bp的片段，序列分析显示克隆的片段与发表的一致。将该片段构建到植物表达载体pCAMBIA1301-1中，序列分析显示重组质粒构建正确。通过基因枪轰击洋葱表皮瞬时表达进行初步检测，检测结果说明该启动子具有活性。pCAMBIA1301-1/ AtNHX2 promoter重组质粒通过Floral dip method转染拟南芥，收获种子，筛选具有抗性的转基因植株，收获T1代转基因种子。T1代种子萌发后采用GUS染色的方法初步鉴定转基因是否成功，然后采用PCR扩增技术进一步验证。T2代转基因植株GUS表达分析显示，AtNHX2 在所有的组织中均有表达。在保卫细胞中检测到了强烈的GUS表达，这一结果表明AtNHX2 对特殊细胞的pH调控和K+自身稳定方面起着重要的作用。AtNHX2 启动子的活性可被NaCl和ABA调控，说明盐和ABA是在转录水平上调控AtNHX2的表达的。在老叶中GUS活性比在新叶中GUS活性强，这说明了AtNHX2在耐盐方面的作用。在根毛细胞中也观测到了强烈的GUS活性，这就暗示了AtNHX2 在扩大的液泡中储存Na+。这为利用该基因提高植物耐盐水平，改良农作物品种奠定了坚实的基础。
Studies on Molecular Cloning of Functional Genes from Special Organisms in Xinjing and Their Application
With the accomplishment of the human genome project and others mode organisms such as Arabidopsis thaliana and rice, it has become a shortcut way to clone new genes in a gene family via homology sequences. Salt stress is the primary effect that limits and decreases the output of crops in many parts of the world, particularly irrigated land. Overly high soil salinity can cause salina and limit the utility of soil. Today, ~20% of the world’s cultivated land and nearly half of all irrigated land are affected by salinity. Advances in molecular genetics and plant transformation have made it feasible to assess biotechnological strategies in improving the salt tolerance of crops, keeping steady-state growth and increasing the output in the saline environment. NHX1 , a tonoplast Na+/H+ antiporter energized by the (pH across the tonoplast, facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance, an active antiporter would function to sequester Na+ into the vacuole, which results in avoidance of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis. This research contains: cloning of NHX1 gene from Atriplex dimorphostegia in Xinjiang by RT-PCR and RACE; cloning of orf25 gene, nhaA gene, nhaB gene and AtNHX2 promoter; expression analysis and molecular testing.
At first, the conservative region of the variable NHX1 gene sequences published in Genbank were analyzed, and seven primers (4 upstream, 3 downstream) were designed for amplifying core fragment of NHX1 according to these
conservative region. Total RNA was isolated from the plant tissue and cDNAs were synthesized by reverse transcription. The different combinations of these primers were used to amplify the core fragment of NHX1. The core fragments were cloned into pMD18-T cloning vector, positive clones were checked with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the core fragments from Atriplex dimorphostegia were similar with the AtNHX1 (short for NHX1 from Arabidopsis thaliana) sequence (GI: 30690553). With a set of primers designed according to the sequence of the cloned core fragments, the 3’ and 5’ ends of the cDNAs were obtained by 5’ and 3’ rapid amplification of cDNA ends (RACE), respectively. Combining the sequences of the 3’ ends, 5’ ends and cloned core cDNA, the full-length sequence of the NHX1 cDNA was assembled. To obtain the open reading frame (ORF) of the NHX1, a pair of primers was designed according to the assembled cDNA sequence. PCR product was cloned into pMD18-T cloning vector and sequenced. The result shows that ORF of the AdNHX1 was obtained. And then it was registered in Genbank (GI: AY211397). The deductive amino acid sequence of AdNHX1 (short for NHX1 from Atriplex dimorphostegia) is significantly different from AtNHX1. The result may have important implications for the elucidation of the different abilities of halophytes and glytophytes for salt tolerance, and may present a feasible method to utilize much more salina area in Xinjiang.
Specific primers for orf25 gene, nhaA gene and nhaB gene were designed according to published sequences and RT-PCR or PCR were conducted to amplify orf25 gene, nhaA gene and nhaB gene. Sequence analyses suggest that cloned genes contain entire coding frame of orf25 gene, nhaA gene and nhaB gene, respectively.
In order to detect whether AtNHX2 promoter can be induced by salt stress, a
pair of primers were designed to amplify a 2.8kb DNA fragment upstream of ATG start codon of AtNHX2 gene according to the sequence of Arabidopsis thaliana genomic DNA. Sequence analysis shows that the amplified fragment conform to the published sequence. AtNHX2 promoter was cloned into the plant expression vector pCAMBIA1301-1. The promoter activity was detected by transient e
引文
[1] LIANGP , PARADEE A B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J ] . Science , 1992 ,257 :9672970.
[2] SANG J , THOMPSON N J . An efficient procedure for obtaining long cDNA clones fromphage library screening[J ] . Biotechnique , 1994 ,17 :4472451.
[3] LISITSYN N , WIGLEr M. Cloning the difference between two complex genomes[J ] .
Science ,1993 ,259 :9462951.
[4] Diatchenko L , Chris Lau Y F , Campbell A P , et al. Suppression subtractive hybridization : a method for generating differentially reg2 ulated or tissue2specific cDNA probes. Proc Natl Acad Sci USA , 1996 , 93 (12) : 6025～6030
[5] KANGD C , FRANCE RL ,SU Z Z , et al . Reciprocal subtraction differential RNA display : an efficient rapid procedure for isolation differential expressed gene sequences [ J ] . Proc Natl Acad Sci USA ,1998 ,95 (23) :13788213793.
[6] 王石平,刘克德,王江等. 用同源序列的染色体定位寻找水稻抗病基因DNA 片段. 植物学报,1998 ,40 (1) :42～50
[7] 李子银,陈受宜. 水稻抗病基因同源序列的克隆、定位及表达. 科学通报,1999 ,44 (7) :727 - 733
[8] 薛勇彪,唐定中,张燕生等. 水稻基因组中R 类抗病基因同源序列的分离. 科学通报, 1998 , 43 (3) : 277～281
[9] Rousley S D , Glodek A , Sutton G, et al. The construction of A rabi dopsis expressed sequence tag assembles : a new resource to
facillitate gene identification. Plant Physiol. , 1996 , 112 : 1177 ～1183
[10] Adams M D , Kelley J M , Gocayne J D , et al. Complementary DNA sequencing : expressed sequence tags and Human Genome Project . Science , 1991 , 252 : 1651-1656
[11] 田 芳,陈主初. 运用生物信息方法快速获取与肿瘤基因同源的 EST 及其新基因克隆的策略. 生命科学. 2000 , 12 (2) : 72～75
[12] Okano M , Xie S P , Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine25) methyltransferases. Nature Genetics , 1998 , 19 (3) : 219～220
[13] Dymock P S , Dymock D , Hooley R. A higher plant seven-transmembrane receptor
that influences sensitivity to cytokinins. Current Biology , 1998 , 8 (16) : 315-324
[14] Vasmatzis G, Essand M , Brinkmann U , et al. Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis. Proc Natl Acad Sci USA , 1998 , 95 (8) : 300～304
[15] Carstea E D , Morris J A , Coleman K G, et al. Niemann2pick type C ( N P2C) disease gene : homology to mediators of cholesterol homeostasis. Science , 1997 , 277 : 228～231
[16] Ziegelhoffer E C , Medrano L J , Meyerowitz E M. Cloning of the arabidopsis WIGGUM gene identifies a role farnesylation in meristem development . Proc Natl Acad Sci USA , 2000 , 97 (13) : 7633～7638
[17] Yang Z N , Mirkow T E. Isolation of large terminal sequences of BAC inserts based on double2restriction enzyme digestion followed by anchored PCR. Genome , 2000 , 43 : 412～415
[18] GIRAUDATJ ,HAUGE B M,VALONC C. Isolation of the Arabidosis ABI3 gene by positional cloning [J ] . Plant Cell ,1992 ,4 :1251-1261.
[19] ARONDEL V ,LEMIEUX B ,HWANG I , et al . Map2based cloning of a gene controlling omega23 fatty acid desaturation in Arabidosis [J ] . Science ,1992 ,258 :135321355.
[20] Martin G B , Brommonschenkel S H , Chumwongse J , et al. Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science , 1993 , 262 : 1432～1436
[21] Song W Y, Wang G L , Chen L L , et al. A receptor kinase2like protein encoded by the rice disease resistance gene Xa21. Science ,1995 , 270 : 1804-1806
[22] Lagudah E S , Moullet O , Apples R. Map2based cloning of a gene sequence encoding a nucleotide binding domain and a leucine2rich region at the Cre3 nematode
resistance locus of wheat . Genome , 1997 , 40 (5) : 659～665
[23] SARMA R N ,FISH L ,GILL B S , et al . Physical characterization of the homologous groups chromosome of wheat in terms of rice linkage blocks , and physical mapping of some important genes[J ] . Gemone , 2000 ,43 :1912198.
[24] McClintock. Cytogenetic studies of maize neurospora [J ] . Carnegie Inst WashYork ,1974 ,46 :1462152.
[25] JOHAL GS , BRIDGS S P. Reductase activity encoded by the Hm1 disease resistance gene in maize[J ] . Science , 1992 , 258 :9852987.
[26] JONES D A ,HAMMOND K E. Isolation of the tomato cf29 for resistance to cladoporium fulvum by transposon tagging[J ] . Science , 1994 ,266 :7892793.
[27] AARTS M G,DIRKS W G,STIEKEMA W T. Transposon tagging of male sterility gene in Arabidosis [J ] . Nature , 1993 ,363 :715-717.
[28] CUI X Q , WISE R. The rf2 nuclear restore gene of male sterile T2cytoplasm maize[J ] . Science ,1996 ,272 :133421336.
[29] 谢道昕, 范云六, 倪丕冲. 苏云金杆菌杀虫蛋白基因导入中国栽培水稻品种中花11 号获得转基因植株. 中国科学B, 1991 , 8: 830～ 838
[30] 蒋红, 朱玉贤, 王雅平. 蜘蛛杀虫肽基因的合成及其在植物中表达质粒的构建. 植物学报, 1995, 37: 323～325
[2] SANG J , THOMPSON N J . An efficient procedure for obtaining long cDNA clones fromphage library screening[J ] . Biotechnique , 1994 ,17 :4472451.
[3] LISITSYN N , WIGLEr M. Cloning the difference between two complex genomes[J ] .
Science ,1993 ,259 :9462951.
[4] Diatchenko L , Chris Lau Y F , Campbell A P , et al. Suppression subtractive hybridization : a method for generating differentially reg2 ulated or tissue2specific cDNA probes. Proc Natl Acad Sci USA , 1996 , 93 (12) : 6025～6030
[5] KANGD C , FRANCE RL ,SU Z Z , et al . Reciprocal subtraction differential RNA display : an efficient rapid procedure for isolation differential expressed gene sequences [ J ] . Proc Natl Acad Sci USA ,1998 ,95 (23) :13788213793.
[6] 王石平,刘克德,王江等. 用同源序列的染色体定位寻找水稻抗病基因DNA 片段. 植物学报,1998 ,40 (1) :42～50
[7] 李子银,陈受宜. 水稻抗病基因同源序列的克隆、定位及表达. 科学通报,1999 ,44 (7) :727 - 733
[8] 薛勇彪,唐定中,张燕生等. 水稻基因组中R 类抗病基因同源序列的分离. 科学通报, 1998 , 43 (3) : 277～281
[9] Rousley S D , Glodek A , Sutton G, et al. The construction of A rabi dopsis expressed sequence tag assembles : a new resource to
facillitate gene identification. Plant Physiol. , 1996 , 112 : 1177 ～1183
[10] Adams M D , Kelley J M , Gocayne J D , et al. Complementary DNA sequencing : expressed sequence tags and Human Genome Project . Science , 1991 , 252 : 1651-1656
[11] 田 芳,陈主初. 运用生物信息方法快速获取与肿瘤基因同源的 EST 及其新基因克隆的策略. 生命科学. 2000 , 12 (2) : 72～75
[12] Okano M , Xie S P , Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine25) methyltransferases. Nature Genetics , 1998 , 19 (3) : 219～220
[13] Dymock P S , Dymock D , Hooley R. A higher plant seven-transmembrane receptor
that influences sensitivity to cytokinins. Current Biology , 1998 , 8 (16) : 315-324
[14] Vasmatzis G, Essand M , Brinkmann U , et al. Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis. Proc Natl Acad Sci USA , 1998 , 95 (8) : 300～304
[15] Carstea E D , Morris J A , Coleman K G, et al. Niemann2pick type C ( N P2C) disease gene : homology to mediators of cholesterol homeostasis. Science , 1997 , 277 : 228～231
[16] Ziegelhoffer E C , Medrano L J , Meyerowitz E M. Cloning of the arabidopsis WIGGUM gene identifies a role farnesylation in meristem development . Proc Natl Acad Sci USA , 2000 , 97 (13) : 7633～7638
[17] Yang Z N , Mirkow T E. Isolation of large terminal sequences of BAC inserts based on double2restriction enzyme digestion followed by anchored PCR. Genome , 2000 , 43 : 412～415
[18] GIRAUDATJ ,HAUGE B M,VALONC C. Isolation of the Arabidosis ABI3 gene by positional cloning [J ] . Plant Cell ,1992 ,4 :1251-1261.
[19] ARONDEL V ,LEMIEUX B ,HWANG I , et al . Map2based cloning of a gene controlling omega23 fatty acid desaturation in Arabidosis [J ] . Science ,1992 ,258 :135321355.
[20] Martin G B , Brommonschenkel S H , Chumwongse J , et al. Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science , 1993 , 262 : 1432～1436
[21] Song W Y, Wang G L , Chen L L , et al. A receptor kinase2like protein encoded by the rice disease resistance gene Xa21. Science ,1995 , 270 : 1804-1806
[22] Lagudah E S , Moullet O , Apples R. Map2based cloning of a gene sequence encoding a nucleotide binding domain and a leucine2rich region at the Cre3 nematode
resistance locus of wheat . Genome , 1997 , 40 (5) : 659～665
[23] SARMA R N ,FISH L ,GILL B S , et al . Physical characterization of the homologous groups chromosome of wheat in terms of rice linkage blocks , and physical mapping of some important genes[J ] . Gemone , 2000 ,43 :1912198.
[24] McClintock. Cytogenetic studies of maize neurospora [J ] . Carnegie Inst WashYork ,1974 ,46 :1462152.
[25] JOHAL GS , BRIDGS S P. Reductase activity encoded by the Hm1 disease resistance gene in maize[J ] . Science , 1992 , 258 :9852987.
[26] JONES D A ,HAMMOND K E. Isolation of the tomato cf29 for resistance to cladoporium fulvum by transposon tagging[J ] . Science , 1994 ,266 :7892793.
[27] AARTS M G,DIRKS W G,STIEKEMA W T. Transposon tagging of male sterility gene in Arabidosis [J ] . Nature , 1993 ,363 :715-717.
[28] CUI X Q , WISE R. The rf2 nuclear restore gene of male sterile T2cytoplasm maize[J ] . Science ,1996 ,272 :133421336.
[29] 谢道昕, 范云六, 倪丕冲. 苏云金杆菌杀虫蛋白基因导入中国栽培水稻品种中花11 号获得转基因植株. 中国科学B, 1991 , 8: 830～ 838
[30] 蒋红, 朱玉贤, 王雅平. 蜘蛛杀虫肽基因的合成及其在植物中表达质粒的构建. 植物学报, 1995, 37: 323～325
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |