马铃薯C-8,7甾醇异构酶基因(StSI1) cDNA克隆及表达

 以拟南芥C-8, 7甾醇异构酶的氨基酸序列为信息探针搜索GenBank数据库, 对高度同源的马铃薯EST序列进行拼接、引物设计和RT2PCR扩增, 扩增产物测序结果证实获得一个马铃薯C-8, 7甾醇异构酶基因(StSI1) 的全长cDNA序列。序列分析结果显示, StSI1全长886 bp, 包含59 bp的5′非编码序列、161 bp的3′非编码序列和一个长度为666 bp编码221个氨基酸的开放阅读框, 分子量约为25 kD。氨基酸结构分析显示该蛋白的N端含有一个长度由35个氨基酸残基组成的信号肽, C端成熟肽区域含有典型的类EBP结合域。氨基酸比对分析表明, StSI1与已知C-8,7甾醇异构酶同源性介于32.9% ～61.3%之间, 与拟南芥AtSI1相似性最(61.3% ) 。RT-PCR 表达谱分析显示, StSI1在马铃薯的块茎芽眼和表皮组织中均能表达, 并且该基因的表达水平受贮藏温度升高和光照增强的正向调节。

Cloning and Expression of C-8,7 Sterol Isomerase Gene (StSI1) cDNA from Potato

C-8,7 sterol isomerase plays a vital role in the sterol biosynthesis pathway. UsingA rabidopsis C-8, 7 sterol isomerase (GenBank accession No. AAD03489) amino acid sequence as a querying probe,many highly homologous EST sequenceswere obtained from GenBank and a putative cDNA sequence of potato C-8, 7 sterol isomerase was assembled. Futhermore, a putative C-8, 7 sterol isomerase gene cDNA, named StSI1 (GenBank accession No. EU103613 ) was cloned from potato (Solanum tuberosum L. ). StSI1 was 886 bp in full length, including a 5′untranslated region of 59 bp, 3′untranslated region of 161 bp with poly A, and a 666 bp length open reading frame (ORF) encoding 221 amino acids with the molecular weight of 25 kD. Software prediction results showed that StSI1 contained a 35 amino acid lenghth signal pep tide at the N terminal. Homology analysis showed that the deduced amino acid sequence of StSI1 shared 32.9% - 61.3% identity with sterol isomerase from other organisms. RT2PCR analysis showed that the StSI1 was highly expressed in skin and eyes of potato stem tuber. The exp ression profiling also showed that StSI1 mRNA was up regulated intensively both by temperature and light.