剑麻酵母双杂交cDNA表达文库的构建及与AhKNOX2相互作用蛋白的筛选  

Construction of Yeast Two-hybrid Library of Agave hybrid No.11648 and Screening of Proteins Interacting with AhKNOX2

作　　者：杨子平[1] 孙艺桓 杨倩 鹿志伟 张燕梅[1] 李俊峰[1] 周文钊[1] YANG Ziping;SUN Yihuan;YANG Qian;LU Zhiwei;ZHANG Yanmei;LI Junfeng;ZHOU Wenzhao(South Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Sciences/Zhanjiang City Key Laboratory for Tropical Crops Genetic Improvement,Zhanjiang,Guangdong 524091,China;Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国热带农业科学院南亚热带作物研究所/湛江市热带作物遗传改良重点实验室,广东湛江524091 [2]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《热带作物学报》2020年第9期1748-1755,共8页Chinese Journal of Tropical Crops

基　　金：现代农业产业技术体系建设专项资金项目(No.CARS-16);国家自然科学基金青年基金项目(No.31801679);中国热带农业科学院基本科研业务费专项资金项目(No.1630062018010)。

摘　　要：植物KNOX(Knotted1-likehomeobox)可通过蛋白质相互作用参与调控落果、纤维细胞发育、次生细胞壁发育、开花等事件。为筛选剑麻叶中与AhKNOX2相互作用的蛋白,采用SMART同源重组技术构建剑麻叶片酵母双杂交表达文库,利用AhKNOX2为诱饵筛选与之相互作用的蛋白。结果表明:文库总容量为3.9’106CFU,转化效率为9.15’105CFU/μgpGADT7-Rec,插入片段大小主要分布于0.5~3.0kb之间,重组率为92%,保存的文库细胞密度为1.35’108/mL,文库滴度为2.22’107 CFU/mL。构建的pGBKT7-AhKNOX2诱饵载体存在自激活性,5 mmol/L的3-氨基-1,2,4-三唑(3-Amino-1,2,4-triazole,3-AT)能抑制诱饵载体的自激活,通过筛选获得了16个与AhKNOX2相互作用的蛋白。本研究成功构建剑麻叶片酵母表达文库,并筛选获得与AhKNOX2相互作用的蛋白,该结果为进一步研究AhKNOX2在剑麻中的功能奠定前期基础,为培育高纤维产量的剑麻新品种提供基因资源和理论基础。To obtain the proteins interacting with AhKNOX2 in Agave hybrid No.11648,a full length-expression cDNA library was constructed in yeast strain Y187 using homologous recombination-mediated SMART technology.The capacity of the cDNA library was 3.9×106 CFU and the transformation efficiency was about 9.15×105 CFU/μg pGADT7-Rec.The PCR amplification of 24 clones randomly selected from the cDNA library indicated that the length of most inserts was ranged from 0.5 kb to 3.0 kb,with a recombination rate of 92%.The cell density was 1.35×108/mL and the titer was up to 2.22×107 CFU/mL.The bait vector(pGBKT7-AhKNOX2)was transformed into yeast strain Y2HGold,colonies appeared on SD/-Trp/-His media and was inhibited by 5 mmol/L 3-AT.After screening the library using bait plasmids,a total of 16 proteins interacting with AhKNOX2 were obtained by sequencing and homology analysis.In conclusion,a high-quality cDNA library was constructed and 16 proteins interacting with AhKNOX2 were screened from cDNA library.

关 键 词：剑麻 AhKNOX2 酵母双杂交 CDNA文库 蛋白质相互作用 

分 类 号：S563.8[农业科学—作物学] Q78[生物学—分子生物学]