冬瓜均一化酵母双杂交cDNA文库的构建及鉴定  

Construction and Identification of a Homogenized Yeast Two-hybrid cDNA Library in Benincasa hispida

作　　者：陈凤 闫晋强 李梅兰[1] 江彪[2] CHEN Feng;YAN Jinqiang;LI Meilan;JIANG Biao(College of Horticulture,Shanxi Agricultural University,Jinzhong 030801,China;Vegetable Research Institute,Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory for New Technology Research on Vegetables,Guangzhou 510640,China)

机构地区：[1]山西农业大学园艺学院,山西晋中030801 [2]广东省农业科学院蔬菜研究所/广东省蔬菜新技术研究重点实验室,广东广州510640

出　　处：《广东农业科学》2021年第8期58-64,共7页Guangdong Agricultural Sciences

基　　金：广东省重点领域研发计划项目(2020B020220003);广东省基础与应用基础研究基金-粤穗联合基金(2020A1515111138);广东省农业科学院科技创新战略专项资金(高水平农科院建设)-人才项目(R2020PY-JG003);广东省蔬菜新技术研究重点实验室开放基金(201902)。

摘　　要：【目的】通过构建冬瓜酵母双杂交cDNA文库,为开展冬瓜基因功能研究提供技术支撑。【方法】以冬瓜高代自交系B227(冬瓜参考基因组测序材料)的根、茎、叶、雄花、嫩果等不同组织为材料,利用CTAB法提取RNA、分离纯化获得mRNA并进行双链cDNA合成和DSN均一化处理后,与pDONR222载体进行BP重组反应,转化大肠杆菌DH10B感受态细胞,构建初级文库;提取初级文库质粒,与pGADT7-DEST载体通过LR重组反应,转化大肠杆菌DH10B感受态细胞,构建酵母杂交cDNA次级文库。利用倍比稀释法计算文库库容量,并通过菌落PCR计算文库重组率和插入片段的大小。【结果】初级文库库容量为8.24×10^(6) CFU,冬瓜均一化酵母双杂交次级cDNA文库库容量为1.03×10^(7) CFU;次级文库中插入片段主要集中在850~3000 bp,重组率为100%,具有良好的多态性。【结论】成功构建了冬瓜均一化酵母双杂交cDNA文库,文库完整性较高、质量较好,符合酵母双杂交实验要求,可应用于后续相关基因产物互作蛋白的筛选等试验,为冬瓜基因功能研究提供前提条件。【Objective】The study was carried out to provide technical support for the researches on gene function of wax gourd by constructing a wax gourd yeast two-hybrid cDNA library.【Method】The roots,stems,leaves,male flowers and tender fruits of the wax gourd inbred line B227(wax gourd cultivar used for genome sequencing)were used as materials to extract RNA by using the CTAB method.Then,the extracted RNA was used to isolate and purify mRNA,and double-stranded cDNA synthesis and DSN homogenization treatment were carried out.Later,cDNA was ligated with pDONR222 vector through BP recombination and transformed into E.coli DH10B competent cells to construct a primary library.LR recombination reaction was performed with plasmid extracted from primary library and pGADT7-DEST vector,and the products were transformed into E.coli DH10B competent cells to construct a secondary yeast two-hybrid cDNA library.The library capacity was calculated by the multiple dilution method,and the recombination rate and the sizes of the inserted fragments were calculated by colony PCR.【Result】The library quality test showed that the primary library capacity was 8.24×10^(6) CFU,and the homogenized secondary yeast two-hybrid cDNA library capacity of wax gourd was 1.03×10^(7) CFU.The insert fragments mainly concentrated between 850 bp and 3000 bp with good polymorphism and the recombination rate was 100%.【Conclusion】A homogenized yeast two-hybrid cDNA library in wax gourd was successfully constructed.The library is highly integrated and of good quality,which meets the requirements of yeast two-hybrid experiments.The library can be used in related experiments such as screening of interaction proteins,laying a foundation for the study on gene function of wax gourd.

关 键 词：冬瓜 DSN均一化 CDNA文库 酵母文库 

分 类 号：S642.3[农业科学—蔬菜学]