摘要
目的:克隆阳春砂3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因的编码区c DNA.方法:根据阳春砂叶片转录组得到的HMGR基因序列自主设计引物.用改良的CTAB法提取阳春砂叶片总RNA,反转录得到c DNA,以c DNA为模板通过PCR反应扩增阳春砂HMGR基因并克隆到p MD-18T载体,将获得的阳性克隆进行测序分析.运用生物信息学方法分析得到的c DNA序列.结果:成功扩增出全长1 716 bp的c DNA片段,将其成功克隆进入p MD-18T载体,测序后将序列提交至Gen Bank得到其编号KU572444.生物信息分析结果显示,该基因片段编码1个由571个氨基酸残基组成HMGR蛋白.HMGR二级结构主要由43.95%的α-螺旋和47.29%的β-转角组成,同时还含有8.76%的β-折叠.与其他植物该蛋白质序列同源性及进化树分析结果表明,阳春砂HMGR与小米(Setaria italic)的同源性最高,且阳春砂与单子叶植物聚为一类.结论:从阳春砂叶片中成功克隆HMGR基因并进行了生物信息学分析.
Aim：To clone 3-hydroxyl-3-methylglutaricacyl coenzyme A reductase gene from Amomum villosum and provide the data for further studies on the function of AvHMGR protein as well as the biosynthesis of terpenes in cells and tissues of Amomum villosum. Methods： Primers were designed according to the sequence of AvHMGR from leaf transcriptomics and total RNA was extracted from Amomum villosum leaves using the modified CTAB method. The cDNA was synthesized by reverse transcription and then was used as a template for AvHMGR gene amplification by PCR. The recovered AvHMGR fragment was cloned to pMD-18T vector. The plasmids harboring the target gene were subjected to sequence. Bioinformatic analysis was performed for the AvHMGR sequence. Results： Sequence analysis showed that the ORF of AvHMGR was 1716 bp, which encoded a protein of 571 amino acid residues. The sequence was submitted to GenBank with the accession number KU572444. The secondary structure of AvHMGR protein was predicted to be composed of α-helix （43.95%）,β-loop （47.29%）, and β-strand （8.76%）. The highest sequence identity was found between AvHMGR and SiHMGR （Setaria italic） when HMGR sequences of selected plants were compared. Phylogenetic analysis showed that Amomum villosum was grouped in monocots.Conclusion：The AvHMGR gene from Amomum villosum was successfully cloned and bioinformatically analyzed.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2016年第4期285-292,共8页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
广东省教育厅科技创新项目(2012KJCX0017)
广东省中医药管理局项目(20141065)
