番茄叶绿体外膜转运蛋白SlToc159-2的多克隆抗体制备以及果实中蛋白表达分析  

Polyclonal antibody preparation and protein expression analysis of tomato chloroplast outer membrane transporter SiToc159-2 in fruit

作　　者：王琪[1,2] 闫见敏 岳江[1,2] 王馨敦 陈精艺 WANG Qi;YAN Jianmin;YUE Jiang;WANG Xindun;CHEN Jingyi(College of Agriculture,Guizhou University,Guiyang 550025,China;Vegetable Research Institute,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州大学蔬菜研究院,贵阳550025

出　　处：《植物生理学报》2024年第11期1707-1716,共10页Plant Physiology Journal

基　　金：国家自然科学基金(31960604);贵州省自然科学基金项目(黔科合基础[2021]重点040号);贵州省自然科学基金项目(黔科合基础[2019]1107号);贵州省生物学国内一流学科建设学科开放基金项目(GNYL[2017]009FX4KT43);贵州大学人才引进科研项目(贵大人基合字[2014]18号)。

摘　　要：核编码叶绿体蛋白通过叶绿体被膜上的TOC-TIC装置靶向导入叶绿体,对于叶绿体生物发生以及植物生长发育至关重要。Toc159家族蛋白是前体蛋白导入叶绿体这一过程中最重要的受体。以前期克隆到的番茄SlToc159-2基因为模版,选取SlToc159-2基因N端409~745个氨基酸之间包括5个保守GTPase基序的cDNA序列,亚克隆至原核表达载体pGEX-6P-1,融合N端GST标签,并转化大肠杆菌BL21(DE3),探索不同诱导条件下目的蛋白表达。GST-SlToc159-2409-745重组蛋白在1 mmol·L^(-1) IPTG,16℃ 4 h诱导表达效果最好,融合蛋白主要以包涵体形式存在。融合蛋白经过亲合纯化,凝胶过滤层析纯化,离子柱纯化后作为抗原,进行3次免疫注射兔子,1周后采心脏血作为多克隆抗血清,通过抗原亲和纯化,得到多克隆抗体anti-SlToc159-2409-745,将纯化后的抗体通过间接酶联免疫(ELISA)和蛋白质印迹(Western blot)分析,表明所制备的抗体具有很好的效价,效价比为1:128000,同时具有良好的灵敏度和特异性。通过免疫印迹检测SlToc159-2在番茄不同成熟度果实中的表达情况,结果显示,SlToc159-2在绿熟期表达量较低,在果实成熟过程中表达量升高。SlToc159-2抗体的成功制备为深入探究番茄中复杂的叶绿体蛋白导入机制奠定了基础。Nuclear-encoded chloroplast proteins are targeted into the chloroplast through the Toc-TiC apparatus on the chloroplast envelope,which is essential for chloroplast biogenesis and plant growth and development.Toc159 family proteins are the most important receptors in the process of importing precursor proteins into chloroplasts.The tomato S/Toc159-2 gene cloned in the previous stage was used as the template,and the cDNA sequence including 5 conserved GTPase motifs between the 409-745 amino acids of the N-terminal of the S/Toc159-2 gene was selected and subcloned into the prokaryotic expression vector pGEX-6P-1,The N-terminal GST tag was fused and transformed into Escherichia coli BL21(DE3)to explore the expression of the target protein under different induction conditions.The GST-SIToc159-2409-745 recombinant protein induced the best expression at 1 mmol·L^(-1) IPTG,16℃ for 4 h,and the fusion protein mainly existed in the form of inclusion bodies.The fusion protein was purified by affinity purification,purified by gel filtration chromatography,purified by ion column and used as an antigen.After three times of immunization and injection of rabbits,heart blood was collected as polyclonal antiserum after one week,and the polyclonal antibody anti-sIToc159-240974,the purified antibody was analyzed by indirect enzyme-linked immunosorbent assay(ELISA)and Western blot,indicating that the prepared antibody had a good titer,the titer ratio was 1:128000,and at the same time Has good sensitivity and specificity.The expression of sIToc159-2 in tomato fruits of different maturity was detected by Western blotting.The results showed that the expression level of sIToc159-2 was lower at the mature green stage and increased during the fruit ripening process.The successful preparation of SIToc159-2 antibody laid a foundation for further investigation of the complex chloroplast protein transport mechanism in tomato.

关 键 词：番茄 叶绿体外膜转运蛋白 Toc159 原核表达 抗体制备 蛋白表达分析 

分 类 号：S641.2[农业科学—蔬菜学]