枸杞LmP5CS基因的克隆及表达分析  

Cloning and Expression Analysis of LmP5CS Gene from Lycium chinense Miller

作　　者：冯远航[1] 王罡[1] 季静[1] 关春峰[1] 金超[1] 

机构地区：[1]天津大学遗传工程研究所,天津300072

出　　处：《中国生物工程杂志》2013年第1期33-40,共8页China Biotechnology

基　　金：国家自然科学基金(31271419;31271793);国家转基因生物新品种培育科技重大专项(2011zx08003-005);2010年度高等学校博士生导师专项科研基金(20100032110060)资助项目

摘　　要：目的:为进一步研究枸杞抗逆境胁迫的机制,并为转基因育种,提供理论依据。提高农作物的抗逆性提供优质的基因资源。方法:提选取盐胁迫后脯氨酸含量变化较大的耐盐植物枸杞为材料,用1.5%NaCl处理后,提取枸杞叶片总RNA,利用RT-PCR及3'RACE方法克隆获得吡咯啉-5-羧酸合成酶(delta 1-pyrroline-5-carboxylate synthetase,P5CS)基因的全长cDNA,命名为LmP5CS,构建pH7m24GW,3rc-LmP5CS植物表达载体。结果:LmP5CS基因的ORF长2 154 bp,编码1个等电点为6.07、分子量为77.5kDa、由717个氨基酸组成的蛋白。枸杞在200 mmol/LNaCl盐胁迫下,LmP5CS基因表达量随处理时间,有先升高后降低的趋势,9h基因表达量最高,脯氨酸含量变化与之一致。结论:LmP5CS基因在盐胁迫下脯氨酸含量的变化中起关键作用。Proline is the important osmotic regulation substances in plants and plays a critical role in improving the stress tolerance of plants.The materials is Lycium chinense Miller,which proline content changes significantly after salt stress.After 1.5% NaCl stress,full length cDNA sequence of a putative Δ1-pyrroline-5-carboxylate synthase gene（P5CS）was cloned from Lycium chinense Miller leaves using RT-PCR and 3＇rapid amplification of cDNA ends（RACE）,named LmP5CS,and construct expression vector pH7m24GW,3rc-LmP5CS.Sequence analysis showed that the complete open reading frame（ORF） of this gene is 2154 bp,encoding for a protein of 717 amino acids with an isoelectric point of 6.07 and a molecular weight of 77.5 kDa.After 200 mmol / L NaCl stress,protein expression level increased at first and decreased subsequently,proline content changed in accordance with that.The semi-quantitative PCR result suggests that LmP5CS plays an important role in proline content change responses to salt stress.

关 键 词：枸杞 LmP5CS 基因克隆 脯氨酸 

分 类 号：Q781[生物学—分子生物学]