中国中药杂志

2018, v.43(03) 484-492

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丹参均一化酵母双杂交文库的构建及与SmJAZ8互作蛋白的筛选
Construction of yeast two-hybrid library of Salvia miltiorrhiza and screening of Sm JAZ8 interaction protein

杨梦丹;赵英;麻鹏达;
YANG Meng-dan;ZHAO Ying;MA Peng-da;College of Life Science,Northwest A & F University;College of Science,Northwest A & F University;

摘要(Abstract):

为构建高质量的丹参c DNA文库以及通过酵母双杂交获得丹参Sm JAZ8互作蛋白基因。研究以丹参的根、茎、叶、花、毛状根为材料,利用SMART(switching mechanism at 5'end of RNA transcript)方式合成全长c DNA,并结合基于双链的特异性核酸酶(duplex-specific nuclease,DSN)均一化技术,构建丹参的均一化全长c DNA文库。通过文库鉴定,检测到c DNA文库库容为1.45×106,重组率为100%,插入片段平均大小为500~2 000 bp。构建p DEST-p GADT7-Sm JAZ8重组载体,将该载体转化Y2HGold菌种,通过酵母双杂交进行互作蛋白的筛选,最终筛选到了丹参Dna J蛋白和UBQ蛋白。该研究既成功构建了丹参均一化全长c DNA文库,又为后续丹参功能基因筛选和基因功能的研究奠定了基础。
The study is aimed to construct high quality Salvia miltiorrhiza c DNA library and obtain the Sm JAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study,full-length cDNA was synthesized from roots,stems,leaves,flowers and hairy roots of S. miltiorrhiza. The full-length c DNA library was synthesized by SMART method and constructed with DSN homogenization technique.The results showed that the library capacity was 1. 45 × 10~6,the recombination rate was 100%,and the average size of the insert was500-2 000 bp. The recombinant vector of p DEST-p GADT7-Sm JAZ8 was constructed and transformed into Y2 HGold strain. The interaction protein was screened by yeast two-hybrid system. The Dna J protein and UBQ protein were screened by yeast two-hybrid system.This study has successfully constructed a full-length c DNA library of S. miltiorrhiza,and laid the foundation for the follow-up study on functional gene screening and gene function of S. miltiorrhiza.

关键词(KeyWords): 丹参;均一化;酵母双杂交;序列分析
Salvia miltiorrhiza;homogenization;yeast two-hybrid;sequence analysis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31670295);; 中央高校基本科研业务费专项基金项目(2452017159)

作者(Author): 杨梦丹;赵英;麻鹏达;
YANG Meng-dan;ZHAO Ying;MA Peng-da;College of Life Science,Northwest A & F University;College of Science,Northwest A & F University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20180105.018

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