小麦WRKY基因的克隆、表达分析及其遗传转化
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摘要
WRKY转录因子是植物转录调控子家族成员之一,它可以特异地与W-box的结合,并调控启动子中含W-box元件的抗病、损伤、衰老相关基因的表达,进而广泛参与植物对生物胁迫和非生物胁迫的响应。
本研究首先利用植物WRKY蛋白的保守序列在小麦基因组数据库中搜索出大量高相似性的ESTs,并拼接获得多条全长序列。通过设计特异引物,采用PCR技术成功克隆到一条小麦WRKY基因的cDNA全长序列。对该基因的序列比对结果显示:它与小麦的TaWRKY53a和TaWRKY53b的序列有较高的相似度,说明此基因确实为小麦中的WRKY基因,并命名为TaWRKY53c。进而对其进行生物信息学分析发现,TaWRKY53c蛋白具有两个完整的WRKY结构域和锌指结构,属于典型的WRKY转录因子Ⅰ类成员。且在与拟南芥Ⅰ类WRKY蛋白的多序列比对中发现,该蛋白与AtWRKY33亲缘进化关系较近,文献报道AtWRKY33参与拟南芥的抗盐响应,因此认为它在小麦中可能参与该蛋白相似的信号通路。小麦幼苗经高盐胁迫处理后,TaWRKY53c表达的组织及时空特异性的荧光定量PCR分析结果表明,该基因确实参与对高盐胁迫的响应,且短时间内表达量增加九倍,并且在叶片中的表达量最高。
为获得具有抗盐特性的转基因小麦品种,构建了TaWRKY53c的过表达载体,并利用基因枪技术将其转化到小麦盾片中。再通过组织培养,获得了大量再生植株,以备后续转基因植株筛选。
本论文的研究结果对阐明小麦中TaWRKY53c基因的生物学功能奠定了初步的基础。
WRKY transcription factors are one of the largest families of transcriptional regulaters in plant, which can specifically bind to W-box, and modulate the expression of disease, wound and senescence-related genes expression whose promoters contain W-box elements, thereby extensively involved in the responses to biotic and abiotic stresses in plant.
In the present research, based on the conserved protein sequence of WRKY transcription factor, ESTs with high similarity to WRKY in wheat genome sequence database were researched, collected and then assembled into several uni-genes. A full-length cDNA sequence encoding WRKY in wheat was successufully cloned by using gene specific primers via PCR approach. The alignment of this gene in NCBI showed that it had high similarity with that of TaWRKY53a and TaWRKY53b, suggesting that it should be a novel WRKY gene in wheat, and thus designated TaWRKY53c. The bioinformatic analysis of this gene showed that TaWRKY53c has two conserved WRKY domains and zinc-finger structures, indicating that it belongs to GroupⅠin WRKY family. A phylogenetic tree was made by multiple sequence alignment with WRKY GroupⅠproteins in Arabidopsis and TaWRKY53c protein, and it was showed that TaWRKY53c was closely related to AtWRKY33, therefore we could infer that they maybe involved in the similar signaling pathway. It has been reported that AtWRKY33 participated in the salt resistance in Arabidopsis. Spatiotemporal expression pattern and fluorescent quantitation RT-PCR analysis showed that TaWRKY53c was strongly expressed in leaf, moderately in stem and weakly in root. The expression of TaWRKY53c was greatly enhanced after one hour of salt stress in wheat seedlings and returned to the normal level thereafter. Thus AtWRKY33 was considered as one of the important signalling component in response to salt stress in wheat.?
In order to verify its function, TaWRKY53c was transformed into wheat by particle bombardment, and a large number of regenerated plants were obtained. These results of the present study provided the bases to clarify the regulation mechanisms and biological functions of WRKY proteins and to breed new transgenetic wheat variety with salt resistance.
本研究首先利用植物WRKY蛋白的保守序列在小麦基因组数据库中搜索出大量高相似性的ESTs,并拼接获得多条全长序列。通过设计特异引物,采用PCR技术成功克隆到一条小麦WRKY基因的cDNA全长序列。对该基因的序列比对结果显示:它与小麦的TaWRKY53a和TaWRKY53b的序列有较高的相似度,说明此基因确实为小麦中的WRKY基因,并命名为TaWRKY53c。进而对其进行生物信息学分析发现,TaWRKY53c蛋白具有两个完整的WRKY结构域和锌指结构,属于典型的WRKY转录因子Ⅰ类成员。且在与拟南芥Ⅰ类WRKY蛋白的多序列比对中发现,该蛋白与AtWRKY33亲缘进化关系较近,文献报道AtWRKY33参与拟南芥的抗盐响应,因此认为它在小麦中可能参与该蛋白相似的信号通路。小麦幼苗经高盐胁迫处理后,TaWRKY53c表达的组织及时空特异性的荧光定量PCR分析结果表明,该基因确实参与对高盐胁迫的响应,且短时间内表达量增加九倍,并且在叶片中的表达量最高。
为获得具有抗盐特性的转基因小麦品种,构建了TaWRKY53c的过表达载体,并利用基因枪技术将其转化到小麦盾片中。再通过组织培养,获得了大量再生植株,以备后续转基因植株筛选。
本论文的研究结果对阐明小麦中TaWRKY53c基因的生物学功能奠定了初步的基础。
WRKY transcription factors are one of the largest families of transcriptional regulaters in plant, which can specifically bind to W-box, and modulate the expression of disease, wound and senescence-related genes expression whose promoters contain W-box elements, thereby extensively involved in the responses to biotic and abiotic stresses in plant.
In the present research, based on the conserved protein sequence of WRKY transcription factor, ESTs with high similarity to WRKY in wheat genome sequence database were researched, collected and then assembled into several uni-genes. A full-length cDNA sequence encoding WRKY in wheat was successufully cloned by using gene specific primers via PCR approach. The alignment of this gene in NCBI showed that it had high similarity with that of TaWRKY53a and TaWRKY53b, suggesting that it should be a novel WRKY gene in wheat, and thus designated TaWRKY53c. The bioinformatic analysis of this gene showed that TaWRKY53c has two conserved WRKY domains and zinc-finger structures, indicating that it belongs to GroupⅠin WRKY family. A phylogenetic tree was made by multiple sequence alignment with WRKY GroupⅠproteins in Arabidopsis and TaWRKY53c protein, and it was showed that TaWRKY53c was closely related to AtWRKY33, therefore we could infer that they maybe involved in the similar signaling pathway. It has been reported that AtWRKY33 participated in the salt resistance in Arabidopsis. Spatiotemporal expression pattern and fluorescent quantitation RT-PCR analysis showed that TaWRKY53c was strongly expressed in leaf, moderately in stem and weakly in root. The expression of TaWRKY53c was greatly enhanced after one hour of salt stress in wheat seedlings and returned to the normal level thereafter. Thus AtWRKY33 was considered as one of the important signalling component in response to salt stress in wheat.?
In order to verify its function, TaWRKY53c was transformed into wheat by particle bombardment, and a large number of regenerated plants were obtained. These results of the present study provided the bases to clarify the regulation mechanisms and biological functions of WRKY proteins and to breed new transgenetic wheat variety with salt resistance.
引文
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[2] P. J. Rushton, H. Macdonald, A. K. Huttly et al. Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha-Amy2 genes. Plant Mol. Biol. 1995, 29: 691~702.
[3] P. J. Rushton, J. T. Torres, M. Parniske et al. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 1996, 15: 5690~5700.
[4] T. Eulgem, P. J. Rushton, S. Robatzek et al. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000, 5: 199~206.
[5] Z. Xie, Z. L. Zhang, X. Zou et al. Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol. 2005, 137: 176~189.
[6] P. J. Rushton, I. E. Somssich, P. Ringler et al. WRKY transcription factors. Trends in plant cell. 2010, 15: 247~258.?
[7] K. Yamasaki, T. Kigawa, M. Inoue et al. Solution structure of an Arabidopsis WRKY DNA binding domain. Plant Cell 2005, 17: 944~956.
[8] M. R. Duan, J. Nan, Y. H. Liang et al. DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Res. 2007, 35: 1145~1154.
[9] M. M. Babu, L. M. Iyer, S. Balaji et al. The natural history of the WRKY-GCM1 zinc fingers and the relationship between transcription factors and transposons. Nucleic Acids Res. 2006, 34: 6505~6520.
[10] I. Ciolkowski, D. Wanke, R. P. Birkenbihl et al. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKYdomain function. Plant Mol. Biol. 2008, 68: 81~92.
[11] K. Maleck, A. Levine, T. Eulgem et al. The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat. Genet. 2000, 26: 403~410.
[12] T. Eulgem, P. J. Rushton, E. Schmelzer et al. Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J. 1999, 18: 4689~4699.
[13] C. Marè, E. Mazzucotelli, C. Crosatti et al. Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol. Biol. 2004, 55: 399~416.
[14] M. Cai, D. Qiu, T. Yuan et al. Identification of novel pathogen-responsive ciselements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance. Plant Cell Environ. 2008, 31: 86~96.
[15] E. Mangelsen, J. Kilian, K. W. Berendzen et al. Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare) WRKY transcription factor family reveals putatively retained functions between monocots and dicots. BMC Genomics 2008, 9: 194.
[16] C. Grierson, J. S. Du, M de Torres Zabala et al. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J. 1994, 5: 815~826.
[17] C. Sun, S. Palmqvist, H. Olsson et al. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugarresponsive elements of the iso1 promoter. Plant Cell 2003, 15: 2076~2092.
[18] S. C. Popescu, G. V. Popescu, S. Bachan et al. MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev. 2009, 23: 80~92.
[19] Q. H. Shen, Y. Saijo, S. Mauch et al. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses. Science 2007, 315: 1098~1103.
[20] Z. Tao, H. Liu, D. Qiu et al. A pair of allelic WRKY genes play opposite roles inrice-bacteria interactions. Plant Physiol. 2009, 151: 936~948.
[21] M. Shimono, S. Sugano, A Nakayama et al. Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell 2007, 19: 2064~2076.
[22] M. Skibbe, N. Qu, I. Galis et al. Induced plant defenses in the natural environment: Nicotiana attenuata WRKY3 and WRKY6 coordinate responses to herbivory. Plant Cell 2008, 20: 1984~2000.
[23] W. Grunewald, M. Karimi, K. Wieczorek et al. A role for AtWRKY23 in feeding site establishment of plant-parasitic nematodes. Plant Physiol. 2008, 148: 358~368.
[24] S. B. Gelvin. Agrobacterium in the genomics age. Plant Physiol. 2009, 150: 1665~1676.
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