花生膜联蛋白基因AhAnn1的克隆与表达分析

为挖掘花生抗逆境胁迫相关基因,克隆抗逆相关膜联蛋白Annexin基因,以花生品种花育25号为试验材料,从已知抑制消减杂交文库中获得花生膜联蛋白Annexin类似基因片段,通过RACE技术获得Annexin基因5'-RACE片段。对5'-RACE序列和抑制消减杂交文库中已知基因片段进行拼接,设计特异引物通过逆转录-聚合酶链式反应(RT-PCR)扩增得到该基因,命名为AhAnn1。结果表明,AhAnn1全长为1 277 bp,开放阅读框为951 bp。根据编码区预测AhAnn1编码一条316个氨基酸组成的多肽,预测分子量为36.10 k Da,等电点为7.07。预测该基因编码的蛋白含有膜联蛋白Annexin保守结构域,定位于细胞质中。蛋白序列多重比对和系统发育分析表明,花生与大豆、苜蓿、鹰嘴豆等豆科植物中的膜联蛋白相似性最高,亲缘关系最近。荧光实时定量PCR结果显示,AhAnn1在根系和叶片中的表达量随干旱胁迫程度的增加而增加。由此推测AhAnn1是一种干旱胁迫应答基因,在花生逆境调控中发挥作用。结果为进一步研究花生AhAnn1基因的功能和花生抗逆境胁迫相关分子机理提供了理论基础。

Cloning and Expression of AhAnn1 Gene in Peanut

The aim of this study was to screen Annexin genes related to stress resistance in peanut.A Annexin gene was cloned from the peanut (Huayu 25),named AhAnn1 using RACE technology and RT-PCR.The peanut Annexin like gene was obtained from drought related suppression subtractive hybridization cDNA library in peanut root.The whole sequence of AhAnn1 was 1 277 bp and its open reading frame was 951 bp,encoding a polypeptide of 316 amino acids,with a theoretical molecular weight of 36.10 kDa and an isoelectric point(pI) of 7.07.The protein was predicted to be located in cytoplasm,containing the conserved Annexin domain.Multiple sequence alignments and phylogenetic analysis of Annexin proteins indicated AhAnn1 was most similar with Annexin from Glycine max,Medicago truncatula and Cicer arietinum.The results of Real-time RT-PCR showed that the expression of AhAnn1 in roots and leaves increased with the increase of drought stress.This suggested that AhAnn1 was a drought stress response gene,which played an important role in the regulation of peanut stress.This study provides a theoretical basis for further studying the peanut AhAnn1 gene function and elucidating the molecular mechanism of stress resistance in peanut.