摘要： 为验证羊草脂氧合酶同源蛋白基因 LcLHP 与盐胁迫的关系,利用RACE(Rapid amplification of cDNA ends)克隆技术从羊草中克隆得到抗盐碱相关基因脂氧合酶同源蛋白基因(LHP,Lipoxygenase homology protein)的cDNA全长序列,其GenBank登录号为KJ472894。 LcLHP 基因开放阅读框为537 bp,编码178个氨基酸。LcLHP中含有PLAT/LH2结构域,该结构域蛋白家族的许多成员受胁迫诱导。同源性分析显示,LcLHP与互花米草PLAT/LH2家族蛋白脂氧合酶、PLAT植物胁迫结构域包含蛋白等同源性较高,蛋白功能预测显示其N 端可能存在信号肽并可能具有酶功能。RT-PCR分析显示,在一定盐碱胁迫条件下,随着处理试剂Na₂CO₃溶液处理时间的延长, LHP 基因在羊草中的表达量呈单峰趋势,在12 h时达到峰值;构建重组表达载体pGEX-LcLHP 并进行IPTG原核诱导表达培养,获得了GST-LcLHP融合蛋白并用GST抗体进行Western Blot试验得到验证。

关键词: 羊草, 盐碱胁迫, LcLHP, RACE克隆, 表达分析

Abstract: In order to know the relationships between Lipoxygenase and salt stress,a full length cDNA named LcLHP related to saline-alkali tolerance was cloned from Leymus chinensis by RT-PCR and RACE(Rapid amplification of cDNA ends). LcLHP gene (GenBank:KJ472894) with 537 bp open reading frame encoded a protein with a predicted length of 178 amino acids.LcLHP has PLAT/LH2 structural domain,which is induced by stress.Homology analysis showed that the deduced amino acid sequence of LcLHP shared high homology with PLAT/LH2 protein family of Spartina alterniflora and PLAT stress protein.Protein function prediction showed N terminal of LcLHP may have signal peptide,act as enzyme.The expression of LcLHP was analyzed by RT-PCR under salinity-alkalinity stressed condition.With the extension of the salinity-alkalinity stressed time,the expression of LcLHP increased from 0 to 12 h in the leaf,and then decreased,the peak of expression level arrived at 12 h.Construction of pGEX-LcLHP expression vector,IPTG induced prokaryotic expression,we got GST-LcLHP fusion protein and verified by Western Blot.

Key words: Leymus chinensis, Saline-alkali stress, LcLHP, RACE cloning, Expression analysis

中图分类号: 

• Q78