中间锦鸡儿CiMYB31基因克隆及表达分析

Cloning and Expression Analysis of CiMYB31 Gene from Caragana intermedia

MYB类转录因子是植物中最大的转录因子家族之一,在植物的初生与次生代谢、细胞命运、生长发育及在生物与非生物胁迫应答中具有重要的作用。本研究以中间锦鸡儿为实验材料利用PCR技术分别以cDNA与gDNA为模板对CiMYB31基因进行了克隆。研究测序表明:CiMYB31基因的开放阅读框(ORF)为969 bp,编码323个氨基酸,其基因组DNA序列长度为1 724 bp,包含3个外显子与2个内含子。生物信息学分析显示:CiMYB31所编码蛋白的N端包含2个MYB结构域(14~64 aa和67~115 aa),属于R2R3-MYB类蛋白。预测该蛋白分子量为36.32 k D,等电点为5.6,蛋白整体上是亲水性的。利用染色体步移技术克隆CiMYB31基因启动子序列,得到902 bp ATG上游序列。分析显示启动子序列中包含一些非生物胁迫相关的顺式作用元件,如干旱响应元件(MBS)与低温响应元件(LTR)。利用实时荧光定量PCR技术对CiMYB31基因的表达进行分析,发现该基因受到低温的诱导。上述研究表明CiMYB31基因可能在中间锦鸡儿对非生物胁迫的响应过程中起作用。

MYB transcription factor constitutes one of the largest transcription families in plant, which plays crucial roles in the primary and secondary metabolism of plant, cell fate, growth development, and responses to biotic and abiotic stress. In this study CiMYB31 gene was cloned by PCR technique from C. intermedia, using cDNA and gDNA as cloned template. Sequencing results indicated that CiMYB31 had an open reading form of 969 bp, encoding 323-amino acids protein. The full-length gDNA of CiMYB31 was 1 724 bp, including two introns and three exons. Bioinformatics analysis showed that N-terminal of CiMYB31 contained two MYB domains （14-64 aa and 67-115 aa） and regarded as R2R3-MYB protein. The calculated molecular weight ofCiMYB31 was 36.32 kD and the isoionic point was 5.6. The CiMYB31 protein was a hydrophilic protein as a whole. The promoter sequence ofCiMYB31 was cloned by genome walking, and 902 bp nucleic acid sequence before ATG was obtained. It turned out that the promot- er sequence contained some abiotic stress cis-elements, such as drought response element （MBS） and low temperature response element. The expression of CiMYB31 was analyzed by qRT-PCR, which revealed it was induced by cold stress. These results showed that CiMYB31 might play a role in response to abiotic stress ofC. intermedia.