羊草脂氧合酶同源蛋白基因LcLHP的克隆与表达分析

Cloning and Expression of Lipoxygenase Homology Protein from Leymus chinensis

为验证羊草脂氧合酶同源蛋白基因Lc LHP与盐胁迫的关系,利用RACE(Rapid amplification of c DNA 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c DNA全长序列,其Gen Bank登录号为KJ472894。Lc LHP基因开放阅读框为537 bp,编码178个氨基酸。Lc LHP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,Lc LHP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p GEX-Lc LHP并进行IPTG原核诱导表达培养,获得了GSTLc LHP融合蛋白并用GST抗体进行Western Blot试验得到验证。

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 amplifica-tion of cDNA ends）.LcLHP gene （GenBank：KJ472894）with 537 bp open reading frame encoded a protein with a predicted length of 1 78 amino acids.LcLHP has PLAT /LH2 structural domain,which is induced by stress.Homolo-gy 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 1 2 h in the leaf,and then decreased,the peak of expression level arrived at 1 2 h.Construction of pGEX-LcLHP ex-pression vector,IPTG induced prokaryotic expression,we got GST-LcLHP fusion protein and verified by Western Blot.