‘南通小方柿’cDNA文库构建及DkGA2ox2调控因子的筛选  

Construction of cDNA Library from’Nantongxiaofangshi’and Screening the Regulatory Factor DkGA2ox2

作　　者：叶夏林 曹丽芳 董雨菡 侯应军 渠慎春[1] Ye Xialin;Cao Lifang;Dong Yuhan;Hou Yingjun;Qu Shenchun(Laboratory of Tree Biotechnology,College of Horticulture,Nanjing Agricultural University,Nanjing,210095)

机构地区：[1]南京农业大学园艺学院,果树生物技术实验室,南京210095

出　　处：《分子植物育种》2022年第21期7082-7090,共9页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2019YFD1000600);中央高校基本科研业务费专项(KYZZ2021002)共同资助。

摘　　要：本研究利用酵母单杂技术筛选DkGA2ox2基因上游转录因子,为进一步研究其表达调控机制提供理论基础。利用HiTAIL-PCR技术获得启动子序列,连入诱饵载体,转化进诱饵菌株。利用Gateway技术构建‘南通小方柿’cDNA文库,再共转化诱饵菌株,筛选得到转录因子。通过HiTAIL-PCR共获得1205 bp启动子序列。构建的文库库容为1.4×107,插入片段大小在750~2000 bp间,重组率100%。初步筛选出HB蛋白、FLL3蛋白和EIN3/EIL1蛋白3个候选蛋白,为探索DkGA2ox2基因上游调控机制提供了理论支撑。In this study,the potential upstream transcription factors of DkGA2ox2 gene were screened by yeast one-hybrid technique,which provide a theoretical basis for further study of its expression regulation mechanism.The promoter sequence was obtained by Hi TAIL-PCR,then inserted into bait vector and transformed into bait strain.The cDNA library of’Nantongxiaofangshi’was constructed by Gateway technology,and then co transformed into bait strains to obtain transcription factors.A total of 1205 bp promoter sequence was obtained by HiTAIL-PCR.The estimated cDNA library storage capacity is almost 1.4×107,the size of inserted fragments ranged from 750 to2000 bp and the recombination rate was 100%.Three candidate proteins,HB protein,FLL3 protein and EIN3/EIL1 protein,were preliminarily screened,which provided theoretical support for exploring the upstream regulation mechanism of DkGA2ox2 gene.

关 键 词：DkGA2ox2 HiTAIL-PCR GATEWAY技术 CDNA文库 酵母单杂 

分 类 号：S665.2[农业科学—果树学]