野生夏葡萄ERF基因的分离克隆、序列分析及转基因植株的筛选
摘要
干旱、高盐和低温等非生物环境以及生物胁迫严重影响了植物的生长发育和限制作物的产量。在逆境胁迫条件下,植物首先要通过多种途径感应环境的变化,然后将环境变化转变为细胞内部的信号,在接受了细胞的内部信号后,转录因子等反式作用因子与顺式作用元件结合来启动抗逆基因的表达,从而提高植物的抗逆性。许多植物逆境胁迫诱导基因的表达都在转录水平上受到转录因子和顺式作用成分的调控,因此转录因子在植物抗逆信号传递过程中起着中心调节的作用。
本文采用电子拼接结合的RT-PCR,获得了野生夏葡萄的另外一个转录因子,命名为VaERF4-1;序列分析表明,VaERF4-1、VaERF4-2两个基因大小分别为825bp、837bp,分别编码由274个、278个氨基酸组成的分别含有一个AP2结构域的蛋白质。另外,通过GENBANK获得两个基因的启动子区域的顺式作用成分,发现在基因的启动子区含有许多与生物和非生物胁迫有关的顺式作用成分。通过同源性分析表明,VaERF4-1、VaERF4-2两个基因与AtERF5基因的同源性较高,我们预测这两个基因在调节方面与AtERF5转录因子具有相似的功能,也能促进下游基因的表达。最后通过表达载体的构建以及转基因植物的筛选为后续研究基因的生物学功能做准备。
总之,通过VaERF4-1、VaERF4-2两个基因的获得、序列分析、VaERF4-2基因表达载体的构建和转基因植物的获得,我们对这两个基因的结构有了进一步的认识,也为后续基因功能的鉴定做好了基础。
Abiotic stresses such as drought , salinity or low temperature are major environmental factors that have adverse effects on plant growth and limit crop production . Under stress conditions , plants firstly sense the environmental changes through many ways, and then transform these changes into the cell signals , finally, transription factors that accept these cell signals will act with cis-acting element and change the expression of stress-responsive genes , resulting in an enhancement of stress-tolerance in plants .Many genes were regulated at transcriotional level,and mostly by transcription factors .Therefore ,transciption factors play a pivotal role in strss tolerance of plants.
In this paper ,we describe the cloning of the other gene from Vitis-aestivalis,and designed VaERF4-1.The two genes are 825bp、837bp and encoded protein of 274、278amino acids with a typically plant specific AP2 domain in the center of the protein respectively.In addition ,we find and analyze the promoter region of VaERF4-1、VaERF4-2 gene and several cis-element involving biotic and abiotic stress response were found in this region. VaERF4-1、VaERF4-2、genes hold the high homologous with AtERF5 gene by the homologous analysis and we forecast they have the same function with AtERF5 in regulation to promote the expression of the backword genes .At last ,overexpression vectors constructs of VaERF4-2 gene were made and transformed into Arabidopsis by Agrobacterium-mediation preparing for the identification of biological functions .
In conclusion , by abtaining of these genes、analysis of the sequence、overexpression vectors constructs and transformation into Arabidopsis ,we know about the two genes futherly and prepare for the identification of biological functions .
本文采用电子拼接结合的RT-PCR,获得了野生夏葡萄的另外一个转录因子,命名为VaERF4-1;序列分析表明,VaERF4-1、VaERF4-2两个基因大小分别为825bp、837bp,分别编码由274个、278个氨基酸组成的分别含有一个AP2结构域的蛋白质。另外,通过GENBANK获得两个基因的启动子区域的顺式作用成分,发现在基因的启动子区含有许多与生物和非生物胁迫有关的顺式作用成分。通过同源性分析表明,VaERF4-1、VaERF4-2两个基因与AtERF5基因的同源性较高,我们预测这两个基因在调节方面与AtERF5转录因子具有相似的功能,也能促进下游基因的表达。最后通过表达载体的构建以及转基因植物的筛选为后续研究基因的生物学功能做准备。
总之,通过VaERF4-1、VaERF4-2两个基因的获得、序列分析、VaERF4-2基因表达载体的构建和转基因植物的获得,我们对这两个基因的结构有了进一步的认识,也为后续基因功能的鉴定做好了基础。
Abiotic stresses such as drought , salinity or low temperature are major environmental factors that have adverse effects on plant growth and limit crop production . Under stress conditions , plants firstly sense the environmental changes through many ways, and then transform these changes into the cell signals , finally, transription factors that accept these cell signals will act with cis-acting element and change the expression of stress-responsive genes , resulting in an enhancement of stress-tolerance in plants .Many genes were regulated at transcriotional level,and mostly by transcription factors .Therefore ,transciption factors play a pivotal role in strss tolerance of plants.
In this paper ,we describe the cloning of the other gene from Vitis-aestivalis,and designed VaERF4-1.The two genes are 825bp、837bp and encoded protein of 274、278amino acids with a typically plant specific AP2 domain in the center of the protein respectively.In addition ,we find and analyze the promoter region of VaERF4-1、VaERF4-2 gene and several cis-element involving biotic and abiotic stress response were found in this region. VaERF4-1、VaERF4-2、genes hold the high homologous with AtERF5 gene by the homologous analysis and we forecast they have the same function with AtERF5 in regulation to promote the expression of the backword genes .At last ,overexpression vectors constructs of VaERF4-2 gene were made and transformed into Arabidopsis by Agrobacterium-mediation preparing for the identification of biological functions .
In conclusion , by abtaining of these genes、analysis of the sequence、overexpression vectors constructs and transformation into Arabidopsis ,we know about the two genes futherly and prepare for the identification of biological functions .
引文
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3刘文奇,陈旭君徐晓晖凌建群郭泽建.ERF类转录因子OPBP1基因的超表达提高烟草的耐盐能力. J ournal of Plant Physiology and Molecular Biology 2002 , 28( 6) :473– 478.
4刘强,张贵友,陈受宜.植物转录因子的结构与调控作用[J].科学通报,2000,45(14):1465—1474.
5刘强,赵南明,Yamaguch-Shinozaki K,等.DREB转录因子在提高植物抗逆性中的作用[J].科学通报,2000, 45(1):11-16.
6王金生.分子植物病理学.[M].北京:中国农业出版社1999.212.
7张今今,王跃进,王西平,杨克强,杨进孝。葡萄总RNA提取方法的研究。果树学报,2003.20(3):178-181.
8 Abe,H,Rrao,T,Ito,T ,Shinozaki ,K, and Yamaguchi-Shinozaki,K. Arabidopsis AtMYVC2 and AtMYB2 function as transcriptional activators in abscisic acid signaling.Plant cell.2003,15:63-78.
9 Allen MD, Yamasaki K, Ohme-Takagi M, et al. 1998. A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J, 17: 5484-5496.
10 Aida ,M, et al .Genes involved in organ separation in Arabidopsis , analysis of the cup-shaped cotyledon mutant .Plant Cell.1997,9:841-857.
11 Alonso JM, Stepanova AN, Leisse TJ, et al. 2003. Genome-wide insertionalmutagenesis of Arabidopsis thaliana. Science, 301: 653-657.
12 Asai T ,Tena G, Plotnikova J ,et al. MAP kinase signaling cascade in Arabidopsis innate immunity .Nature,2002,415:977-983.
13 Banno H, Ikeda Y, Niu QW, et al. 2001. Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell, 13: 2609-2618.
14 Benhamou N, Belanger RR. 1998. Inductionh of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response. Plant J, 14: 13-21.
15 Berrocal-Lobo M, Molina A, Solano R. 2002. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant J, 29: 23-32.
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