剑麻AhKH1775基因克隆及其酵母双杂交诱饵载体构建  

Molecular Clone and Construction of Yeast Two-Hybrid Bait Vector for KH1775 Gene in Agave Hybrid No.11648

作　　者：汪询 杨倩 柯智 周文钊[2] 杨子平[2] 吕玲玲 Wang Xun;Yang Qian;Ke Zhi;Zhou Wenzhao;Yang Ziping;LüLingling(College of Horticulture,Hainan University,Haikou,570228;Zhanjiang City Key Laboratory for Tropical Crops Genetic Improvement,South Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Sciences,Zhanjiang,524091;College of Landscape Architecture,Huaihua University,Huaihua,418099)

机构地区：[1]海南大学园艺学院,海口570228 [2]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,湛江524091 [3]怀化学院风景园林学院,怀化418099

出　　处：《分子植物育种》2023年第15期4935-4940,共6页Molecular Plant Breeding

基　　金：海南省自然科学基金项目(320MS088,321QN300);现代农业产业技术体系建设专项(CARS-16);广东省自然科学基金项目(2022A1515011841,2021A1515012421)共同资助。

摘　　要：KH结构域蛋白参与调控植物开花时间、花器官形成、叶发育、miRNA产生和对生物非生物胁迫的响应。前期实验发现AhKH1775可能参与调控剑麻株芽的发育,但是其调控机制仍不清楚。KH结构域蛋白可以通过复合体的方式参与转录后基因表达调控。为获得与AhKH1775相互作用的蛋白,本研究克隆了AhKH1775的完整编码框,构建了酵母双杂交诱饵载体,并检测了诱饵载体的毒性和自激活性。结果显示,克隆获得一个918 bp的编码框,成功构建了诱饵载体,诱饵载体无毒性,但存在自激活性,25 mmol/L的3-AT可以抑制诱饵载体的自激活性。本研究结果为进一步筛选与AhKH1775相互作用的蛋白和深入研究AhKH1775在剑麻中的功能提供依据。KH domain proteins have been found that involved in the regulation of flowering time,flower organ formation,leaf development,miRNA biogenesis,abiotic and biotic stress signal.Based on our previous results,AhKH1775 may involve in regulating the development of bulbil in Agaves.However,the molecular mechanism of the regulation development of bulbil is still unknown.KH domain proteins participated in the regulation of gene expression at post transcriptional level through interact complexes.To obtain the target proteins,the open read frame of AhKH1775 was first cloned and the pGBKT7-AhKH1775 bait expression vector was constructed,and we tested the toxicity and self-activation of bait vectors.The results showed that the AhKH1775 gene was amplified and the bait plasmid pGBKT7-AhKH1775 was successfully constructed.Bait plasmid has no toxicity to yeast cells,but has autoactivation,which was inhibited by 25 mmol/L 3-AT.The results can provide scientific basis for screening proteins interacting with AhKH1775 and further functional study of AhKH1775 in Agave Hybrid No.11648.

关 键 词：剑麻 KH结构域 基因克隆 酵母双杂交 诱饵载体 

分 类 号：S563.8[农业科学—作物学]