割手密醛脱氢酶ScALDH基因的克隆与表达分析

Cloning and expression analysis on the ScALDH gene of Saccharum spontaneum

该研究从甘蔗细茎野生种(割手密Saccharum spontaneum)中克隆抗旱相关的基因Sc ALDH,并分析其在干旱处理条件下的表达情况和序列特征。利用RT-PCR技术克隆甘蔗的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析。使用NCBI Blastx、ORF finder、Mega、NCBI Conserved Domain Search等程序对其分别进行不同物种氨基酸比较、开放阅读框(ORF)寻找、进化树及保守序列分析,并用Real Time-PCR分析所克隆基因在干旱胁迫前后的表达差异。结果表明:克隆出甘蔗的ALDH基因片段,总长度为1996bp,其中蛋白质编码区(CDS)全长1524bp,编码508个氨基酸;与其它物种ALDH类蛋白氨基酸序列有很高的同源性,有ALDH家族的保守序列,并含有完整的开放阅读框,系统进化树分析显示与玉米的蛋白质亲缘关系最近;Real timePCR数据表明,在干旱胁迫下,随着干旱时间的延长,该基因的表达量呈持续积累的表达模式。总体上来说,该基因对干旱胁迫显著表达。利用RT-PCR技术克隆的甘蔗ALDH基因,属于ALDH蛋白家族的一员,具有其典型的功能域,该基因在干旱胁迫过程中参与抗旱作用。该研究结果为野生种资源开发、优良抗旱亲本选择和培育抗旱性强甘蔗品种提供了参考依据。

We cloned a drought resistant ALDH gene with RT-PCR gene from Saccharum spontaneum,detected the gene expression level under different drought conditions,and analyzed the nucleotide and amino acid sequences with Blastx,Conserved Domain Search,ORF finder and MEGA. We compared the amino acids among different species,and tried to find reading frames in the sequence,to search conserved Domain and to make evolutionary tree. We analyzed the gene expression level of the cloned gene in drought stress with real time-PCR. The total length of the ALDH gene fragment cloned was 1996bp,including 1524bp coding sequence,which was encoded 508 amino acids. The cloned sequence had a very high homology with other species＇ amino acid sequences and had the conserved sequences of ALDH family,a complete open reading frame as well. Phylogenetic tree analysis showed that protein had the closest relationship with corn. The real time-PCR data showed that the expression of the gene was expressed in a continuous accumulation mode. On the whole,the gene responds to drought stress. The cloned gene belong to the ALDH protein family,which has its typical function domain. The expression pattern analysis showed that the gene was involved in the process of drought stress. The results of this study aimed at developing the excellent resources of wild species,as well as to explore the drought resistance of these resources. The study provides the basis for the selection of the parents of the fine and drought resistance in the future.