小麦ArfGAP基因的克隆、半定量RT-PCR分析及原核表达  

Cloning,Semi-quantitative RT-PCR Analysis and Prokaryotic Expression of ArfGAP Gene in Triticum aestvum L.

作　　者：许峰[1] 闫素辉[1] 张从宇[1] 时侠清[1] 李文阳[1] 张子学[1] 

机构地区：[1]安徽科技学院农学院,安徽凤阳233100

出　　处：《麦类作物学报》2015年第12期1631-1638,共8页Journal of Triticeae Crops

基　　金：安徽省教育厅自然科学重点项目(KJ2015A221);安徽科技学院重点学科项目(AKZDXK2015A03)

摘　　要：为明确小麦ArfGAP基因在非生物胁迫中所行使的功能,采用电子克隆技术分离普通小麦ArfGAP基因后对其在不同非生物胁迫下的表达特性进行了分析,并在大肠杆菌中表达了该基因。结果表明,从扬麦18号中克隆得到的TaArfGAP基因ORF全长为2 121bp。生物信息学分析表明,TaArfGAP蛋白氨基端方向存在一个典型的ArfGAP蛋白结构域,在亲缘关系上与山羊草、乌拉尔图小麦和短柄草较近,而与甘蓝等作物关系较远。半定量RT-PCR分析表明,TaArfGAP基因在干旱、PEG和NaCl胁迫下显著上调表达,但在高温条件下则显著下调表达。SDS-PAGE检测结果表明,IPTG终浓度为0.8~1.0mmol·L-1、诱导温度为24℃及诱导时间为10h时,在分子量大小为81kD左右可以得到较大的可溶性表达蛋白量,且与预期结果一致,说明TaArfGAP基因在原核表达体系中成功表达。To verify the role of ArfGAP gene to abiotic stress in wheat,ArfGAP was cloned from common wheat by using silicon cloning technology,and expression characteristics of ArfGAP was analyzed under different stress conditions,and prokaryotic expression of ArfGAPin E.coli was conducted.The results showed that the full length of TaArfGAP was 2 121 bp in Yangmai 18.Bioinformatics analysis indicated that TaArfGAP protein has a classic ArfGAP protein domain at N-terminal,and it has a close relationship to the homologous genes in Aegilops tauschii,Triticum urartu,and Brachypodium distachyon,whereas it had a distant relationship with Brassica rapaand other dicotyledon.Semi-quantitative RT-PCR analysis showed that the expression of TaArfGAPgene was significantly up-regulated by dehydration,PEG,and sodium chloride in contrast to heat treatment.The results of SDS-PAGE indicated that the prokaryotic expression of TaArfGAP gene was successfully completed.A huge amount of soluble protein about 81 kD could be obtained under the conditions of the induction temperature up to 24 ℃ and 0.8-1.0mmol·L-1 IPTG for 10 h.

关 键 词：小麦 ADP核糖基化因子GTP水解酶激活蛋白 半定量RT-PCR 融合蛋白 

分 类 号：S512.1[农业科学—作物学] S330