番茄花柄脱落过程的酵母双杂交cDNA文库构建  

The Construction of a Yeast Two-hybrid cDNA Library of Tomato Pedicel during Abscission

作　　者：姜赟 史自航[1] 付欣 成丽娜 张璐 葛思琪 许涛[1] 李天来[1] JIANG Yun;SHI Zi-hang;FU Xin;CHENG Li-na;ZHANG Lu;GE Si-qi;XU Tao;LI Tian-lai(College of Horticulture/Key Laboratory of Ministry of Education/Collaborative Innovation Center of Protected Vegetable Surrounds Bohai Gulf Region,Shenyang Agricultural University,Shenyang 110161,China)

机构地区：[1]沈阳农业大学园艺学院/设施园艺省部共建教育部重点实验室/环渤海湾地区设施蔬菜优质高效生产协同创新中心,沈阳110161

出　　处：《沈阳农业大学学报》2018年第2期129-135,共7页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(31672197;31572167);国家重点研发计划支持项目(2016YFD0201004);国家现代农业产业技术体系建设专项资金项目(CARS-25)

摘　　要：为进一步阐明脱落的分子机理,采用SMART(switching mechanism at 5'end of the RNA transcript)技术,在酵母菌株Y187中构建番茄花柄脱落过程的酵母双杂交cDNA文库。以中蔬6号番茄花柄为试材,选取其不同脱落阶段的离区,提取总RNA,利用SMART技术合成双链cDNA,产物经CHROMA SPINTM+TE-400柱纯化后,与载体pGADT7-Rec共转化入酵母菌Y187感受态中,在缺少亮氨酸的培养基(SD-Leu with agar)上培养收集所有克隆。经鉴定,cDNA文库的转化效率为5.6×10~6cfu·μg^(-1),文库容量为2.4×10~7cfu,文库滴度为4.9×10~8cfu·mL^(-1),平均插入片段长度大于1000bp,重组率为100%。随机挑取20个单克隆测序,比对NCBI数据库分析获得了19个已知蛋白序列和1个未知蛋白序列。已知功能的序列编码的蛋白分为4类,包括酶类蛋白、转录因子、载体蛋白和逆境响应蛋白,参与蛋白质磷酸化、水分运输和氧化还原反应等生物过程,其中7个序列之前被报道参与脱落。结果表明获得该文库质量较高,为筛选番茄花柄脱落过程中的互作蛋白奠定了基础。In order to illustrate the molecule mechanism of abscission,a yeast two-hybrid cDNA library of tomato pedicel abscission zone(AZ)was constructed by SMART cDNA synthesis technology.Total RNA was extracted from flower pedicel AZ which is in different abscission points.Using SMART technology,cDNA was synthesized and purified by CHROMASPINTM+TE-400 column.Finally the purified PCR product was co-transformed with plasmid pGADT7-Rec into competent yeast Y187.The transformed yeast cells grew on the SD-Leu medium plates.All the growing clones were harvested and then established the cDNA library.The cDNA library transformation efficiency was 5.6×106 cfu·μg-1.Harvested library had 2.4×107 cfu independent clones.The library titer was 3.2×108 cfu·mL-1,average length of the insertions was more than 1000 bp and recombination rate was 100%.Twenty single clone sequences of the cDNA library were blasted by NCBI database,containing nineteen sequences with known functions and one unkown sequence.The proteins coding by the sequences with known functions are divided into four parts including enzyme,transcription factor,carrier and stress-responding protein.These proteins are involved in many biological processes including protein phosphorylation,water transportation,oxidation-reduction process,etc.Seven sequences involves in abscission.The results indicate that our library can be used to screen the interaction proteins for participating in tomato pedicel abscission.

关 键 词：番茄 SMART技术 酵母双杂交 CDNA文库 脱落 花柄离区 

分 类 号：S625.2[农业科学—园艺学]