摘要
为深入研究小麦TaSnRK2.1在小麦-叶锈菌互作过程中的功能,PCR扩增TaSnRK2.1的开放阅读框(ORF)全长,对其进行生物信息学分析。结果表明,该基因开放阅读框全长为1029 bp,编码342个氨基酸,相对分子质量38.78 kDa,等电点为5.78。构建重组原核表达载体pColdⅡ-TaSnRK2.1,并转化大肠杆菌表达菌株BL21(DE3)。使用终浓度0.5 mmol/L IPTG诱导基因表达,并进行电泳检测。结果显示,诱导蛋白(包涵体)在约39 kDa处有明显条带,与预测蛋白分子量大小一致。利用Ni-NTA亲和层析纯化获得目的蛋白,并将之作为抗原制备兔源多克隆抗体。经酶联免疫吸附试验(ELISA)检测,抗体效价为1∶25600,采用Western-blotting检测分析该抗体的特异性,结果显示该多克隆抗体能够特异结合TaSnRK2.1。本试验为进一步研究TaSnRK2.1在小麦-叶锈菌互作过程中的功能奠定了基础。
In order to further study the function of wheat TaSnRK2.1 in the interaction of wheat and Puccinia triticina, the full-length open reading frame(ORF) of TaSnRK2.1 was amplified by PCR and analyzed by bioinformatics. The results showed that the ORF of the gene was 1 029 bp and encoded 342 amino acids. Its molecular weight was 38.78 k Da with an isoelectric point of 5.78. The recombinant prokaryotic expression vector p Cold II-TaSnRK2.1 was constructed and transformed into E. coli expression strain BL21(DE3). Gene expression was induced using a final concentration of 0.5 mmol/L IPTG, and the expressed protein was detected by electrophoresis. The results showed that the expressed protein had a distinct band at about 39 k Da, which was consistent with the predicted protein molecular weight. The target protein was purified by Ni-NTA affinity chromatography and used as antigen to prepare a rabbit polyclonal antibody. The antibody titer was 1:25 600 measured by Enzyme Linked Immunosorbent Assay(ELISA). The specificity of the antibody was analyzed by Western-blotting assay. The result showed that the polyclonal antibody could specifically bind to TaSnRK2.1. This experiment laid the foundation for further study of the function of TaSnRK2.1 in the interaction of wheat and Puccinia triticina.
作者
乔金柱
麻楠
孙天杰
李姗
王冬梅
QIAO Jinzhu;MA Nan;SUN Tianjie;LI Shan;WANG Dongmei(Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology/College of Life Sciences,Hebei Agriculture University,Baoding 071001,China)
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2019年第5期8-14,共7页
Journal of Hebei Agricultural University
基金
国家自然科学基金项目(31171472
31871548)
高等学校博士学科点专项科研基金资助课题(优先发展领域)(20111302130001)
河北省应用基础研究计划重点基础研究项目(12967149D)
