甘蔗水分胁迫响应相关醛脱氢酶基因的克隆及其表达特征分析

 【目的】筛选并克隆与水分胁迫相关的基因,通过对目的基因的表达分析进一步解析植物的抗旱机制,为甘蔗抗逆育种提供候选基因和理论依据。【方法】应用消减文库技术结合cDNA芯片技术筛选水分胁迫诱导的基因的EST序列,根据筛选到感兴趣的上调表达基因的EST序列,用RACE技术获得SSADH的全长cDNA序列,并通过实时荧光RT-PCR技术对该基因的表达特征进行分析。【结果】通过消减文库技术结合cDNA芯片技术筛选的EST中含有4个推测为基因SSADH 的EST,其在水分胁迫处理的斑茅中均明显上调表达。通过RACE扩增获得甘蔗SSADH全长cDNA序列为1 914 bp,其中5′端非编码区25 bp,开放阅读框为1 581 bp,3′端非编码区306 bp,在1 749处有终止加A信号AATAA。克隆的甘蔗全长cDNA序列进行NCBI比对分析显示,所克隆的甘蔗SSADH全长cDNA与拟南芥（NM_106592.3）的ALDH5F1同源性为73%,表明克隆的cDNA序列可以归为甘蔗的醛脱氢酶家族基因ALDH5F1。通过荧光实时PCR分析SSADH表达表明,该基因参与干旱胁迫下的全程响应,并证明水分胁迫下该基因表达与Ca2+的存在调控关系。【结论】克隆了甘蔗基因SSADH,该基因为一个水分胁迫响应基因;SSADH的植物在体表达与Ca2+存在调控关系。克隆的基因SSADH可用于植物抗逆性调控研究。

Cloning and Expression Analysis of a Water Stress-Induced ALDH Gene from Sugarcane

【Objective】 In this study, the objective is to screen and clone the drought stress resistance-related genes, and analyze the expression pattern of those genes which aim to probe into the molecular mechanism of drought resistance and provide candidate genes for drought-resistance plant breeding. 【Method】 Combining SSH and cDNA microarray to screen of up-regulated ESTs in the water stress Erianthus arundinaceus based on the analysis of up-regulated ESTs, a full-length cDNA sequence was cloned from sugarcane through Rapid Amplification of cDNA Ends (RACE) method, and the gene expression character was analyzed by real time RT-PCR. 【Result】 Four obvious up-regulated ESTs of SSADH were obtained in the drought stress Erianthus arundinaceus througth cDNA microarray hybridization. The full-length cDNA of SSADH termed as sc-SSADH from sugarcane was 1 914 bp in length, contains a 1 581 bp open reading frame (ORF) encoding a 527 amino acid protein, with 25 bp in the 5′ UTR and 306 bp in the 3′ UTR, including a poly (A) signal at 1 749 position. GenBank Blast analysis showed that SSADH was 73% identical to Arabidopsis thaliana SSADH (NM_106592.3). Real time RT-PCR analysis revealed that the sc-SSADH was a whole range PEG-stress responsive gene, and it could also be regulated by Ca2+ in sugarcane. 【Conclusion】 It is the first report of the cloning and expression analysis of SSADH in sugarcane, and the sc-SSADH is a dehydration-responsive gene. At the same time, Ca2+ has been proposed to play a role in the regulation of SSADH