水稻组氨酸磷酸转运蛋白OsAHP2的表达及纯化  

Protein Expression and Purification of OsAHP2 in Rice

作　　者：孙丽静[1] 李倩影[1] 王培楠 孙颖 SUN Lijing;LI Qianying;WANG Peinan;SUN Ying(Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Hebei Provincial Laboratory of Crop Genetics and Breeding, Shijiazhuang 050035, China;College of Life Sciences, Hebei Normal University, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Shijiazhuang 050024, China)

机构地区：[1]河北省农林科学院粮油作物研究所,河北省作物遗传育种实验室,河北石家庄050035 [2]河北师范大学生命科学学院,分子细胞生物学教育部重点实验室,河北石家庄050024

出　　处：《华北农学报》2020年第4期46-51,共6页Acta Agriculturae Boreali-Sinica

基　　金：河北省自然科学基金面上项目(2019301089);河北省农林科学院科学技术研究与发展计划项目(2018060304)。

摘　　要：细胞分裂素在植物生长发育和抵御环境胁迫过程中均扮演着重要的角色,其信号是由细胞分裂素受体组氨酸激酶、组氨酸磷酸转运蛋白(HPs)以及反应调节因子组成的复杂的双元组分系统进行传递的。为了获得高纯度的OsAHP2蛋白,从水稻中扩增了OsAHP2全长cDNA,通过酶切连接的方法构建了2个OsAHP2原核表达载体,命名为pET-32a-OsAHP2和pGEX-4T-1-OsAHP2,测序正确后分别转化大肠杆菌表达菌株Rosseta和XA90。SDS-PAGE检测结果显示,0.5 mmol/L IPTG诱导1,3,5 h条件下,pET-32a-OsAHP2重组菌株表达出分子量约为35 ku的融合蛋白,pGEX-4T-1-OsAHP2重组菌株表达出分子量约为42 ku的融合蛋白,筛选出融合蛋白表达量较高的pET-32a-OsAHP2进行后续试验。在0.5 mmol/L IPTG诱导3 h条件下,重组大肠杆菌pET-32a-OsAHP2以可溶性蛋白的形式表达OsAHP2融合蛋白,通过His镍离子亲和层析柱纯化后,获得了大量纯度较好的OsAHP2融合蛋白。Cytokinin plays an important role in plant development and resistance to environmental stresses.Cytokinin signal transduction involves a multistep two-component system composed of a sensor histidine kinase(HK),a histidine phosphotransfer protein(HP),and a response regulator(RR).In order to obtain high purity OsAHP2 protein,the full-length cDNA of OsAHP2 was amplified from rice,and two prokaryotic expression vectors pET-32a-OsAHP2 and pGEX-4T-1-OsAHP2 were constructed.After sequencing,they were transformed into E.coli expression strains Rosseta and XA90,respectively.SDS-PAGE analysis showed that with 0.5 mmol/L IPTG induction for 1,3,5 h,the recombinant strain pET-32a-OsAHP2 expressed a 35 ku fusion protein and the recombinant strain pGEX-4T-1-OsAHP2 expressed a 42 ku fusion protein.The pET-32a-OsAHP2 expressed much more fusion protein than the pGEX-4T-1-OsAHP2 and was used for subsequent experiment.The pET-32a-OsAHP2 expressed fusion protein in the form of soluble protein by 0.5 mmol/L IPTG induction for 3 h.After purification by HIS-Select Nickel Affinity Gel,a large amount of OsAHP2 fusion protein with good purity was obtained.

关 键 词：水稻 组氨酸磷酸转运蛋白 OsAHP2 原核表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学] S511.03[农业科学—作物学]