花生类受体蛋白激酶AhAlSRK的原核表达与体外活性分析  

Prokaryotic expression and in vitro functional analysis of peanut receptor like protein kinase AhAlSRK

作　　者：黄舒婷 李霞 苏桂军 詹洁[1,2,3] 王爱勤 何龙飞[1,2,3] 肖冬 HUANG Shu-ting;LI Xia;SU Gui-jun;ZHAN Jie;WANG Ai-qin;HE Long-fei;XIAO Dong(College of Agriculture,Guangxi University,Nanning 530004,China;Key Laboratory of Crop Cultivation and Tillage,Guangxi Colleges and Universities,Nanning 530004,China;National Demonstration Center for Experi-mental Plant Science Education,College of Agriculture,Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学农学院,广西南宁530004 [2]广西高校作物栽培学与耕作学重点实验室,广西南宁530004 [3]广西大学农学院植物科学国家级实验教学示范中心,广西南宁530004

出　　处：《中国油料作物学报》2020年第5期787-795,共9页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(31701356);广西自然科学基金(2016GXNSFBA380223)。

摘　　要：基于对已有转录组数据的分析,发现花生AhAlSRK(LOC107458489)基因在耐铝花生品种99-1507和铝敏感型花生品种中花2号(ZH 2号)受铝毒害后基因表达量出现明显差异,暗示其可能参与花生铝胁迫下信号传递铝胁迫响应机制。花生AhAlSRK基因与拟南芥富含亮氨酸类受体蛋白激酶(leucine-rich repeat receptor-like kinases,LRR-RLKs)VIII_2亚家族中的AT1G56140基因具有相似的结构,为同源基因。为验证花生AhAlSRK蛋白激酶活性,克隆了AhAlSRK的全长编码序列,并基于对AhAlSRK蛋白结构预测的基础上,克隆了AhAlSRK的胞内域,构建其原核表达载体pGEX-6p-1-AhAlSRK-CD,转入大肠杆菌菌株Rosetta 2(DE3)plysS。在1 mmol/L IPTG条件下,16℃低温诱导过夜,成功诱导可溶性GST-AhAlSRK-CD蛋白。重组蛋白GST-AhAlSRK-CD表达效率较高,经过自磷酸化检测验证其具有丝/苏氨酸和酪氨酸激酶活性。实验结果为后续在蛋白质水平上探究AhAlSRK基因响应铝胁迫机理打下基础。Analysis of transcriptome data which was obtained in our previous study revealed that AhAlSRK(LOC107458489)was an Al responsive gene.It was shown a different transcript profile of AhAlSRK under Al stress in Al-tolerant cultivar(99-1507)and Al-sensitive cultivar(ZH 2),suggesting a possible role of AhAlSRK in regulating Al stress signaling transduction in peanut.In this study,we cloned the coding sequence of AhAlSRK.It was predicted that AhAlSRK has similar protein structure with AT1G56140,a member of leucine-rich repeat receptor-like kinases(LRR-RLKs)VIII_2 subfamily.To verify the activity of AhAlSRK,the coding region of intracellular kinase domain was amplified and cloned into the prokaryotic expression vector pGEX-6p-1.The fusion protein was expressed in Escherichia coli Rosetta 2(DE3)plysS.GST-AhAlSRK-CD was induced successfully with 1mmol/L IPTG at 16℃overnight.A kinase assay revealed that AhAlSRK autophosphorylated on both serine/threonine and tyrosine residues.The findings provided some important information of AhAlSRK for future research on AhAlSRK-mediated Al response pathway.

关 键 词：花生 类受体蛋白激酶 原核表达与纯化 自磷酸化 

分 类 号：S565.2[农业科学—作物学] Q946.2[生物学—植物学]