黄瓜CsDREB1的克隆及其在霜霉威胁迫下的表达分析

Cloning and Expression Analysis of CsDREB1 in Cucumber Under Propamocarb Stress

以高农药残留黄瓜品系‘D9320’和低农药残留黄瓜品系‘D0351’为试材,采用PCR技术克隆获得响应霜霉威(propamocarb)的黄瓜CsDREB1基因的全长,并对其进行生物信息学分析及表达分析,以期为低农残黄瓜品种的选育提供参考依据。结果表明:黄瓜CsDREB1基因全长603bp,与同属葫芦科的甜瓜CmDREB1D为同一个进化小分支,亲缘关系最近。启动子序列分析结果表明,CsDREB1的启动子区域含有5UTR Py-rich stretch高转录水平调节、O2-site蛋白代谢调节和CGTCA-motif茉莉酸甲酯响应元件等。实时荧光定量PCR结果表明在农药霜霉威胁迫下,低农残品系‘D0351’中CsDREB1的表达量显著下降;而在高农残品系‘D9320’中的CsDREB1的表达量与对照无显著差异。组织特异性表达分析表明,CsDREB1主要在果实表达;CsDREB1编码的蛋白质定位于细胞核上,属于核蛋白。黄瓜CsDREB1负响应霜霉威胁迫,过表达CsDREB1转基因拟南芥植株抵抗霜霉威胁迫能力明显下降。CsDREB1不能响应ABA、干旱和高盐类非生物胁迫及多主棒孢霉菌生物胁迫,能够响应JA胁迫。

High residue cucumber ‘D9320＇and low residue cucumber ‘D0351＇were selected as materials in this study,by PCR cloning method,the full length of cucumber CsDREB1 gene was obtained which could response to propamocarb,the gene characteristics and expression were analysed in order to provide reference for breeding of low residue cucumber variety.The results showed that CsDREB1 with 603 bp in cucumber and CmDREB1 D in melon both came from the same small evolutionary branches,with the closest relationship by the analysis of phylogenetic tree.Promoter sequence analysis showed that CsDREB1 promoter region contained 5 UTR Py-rich stretch which was a high transcription level cis-acting element,O2-site protein which could regulate zein metabolism and a MeJA responsiveness cis-acting regulatory element called CGTCA-motif.RT-PCR results showed that the expression of CsDREB1 in low residue cucumber ‘D0351＇decreased significantly under propamocarb stress compared to control,at the same time significant difference in high residue cucumber‘D9320＇between stress and control was not found.The analysis of tissue specific expression showed that CsDREB1 was mainly expressed in the fruit,the protein encoded by CsDREB1 was located on the nucleus which belonged to the nucleoprotein.CsDREB1 negative response to propamocarb stress.Overexpression CsDREB1 in Arabidopsis under propamocarb stress test showed that the resistance ability to propamocarb of transgenic Arabidopsis significantly decreased compared to wild Arabidopsis.CsDREB1 could not respond to ABA,drought and high salt stress,neither does Corynesporacassiicola,but CsDREB1 could respond to JA.