中国农业科学 ›› 2024, Vol. 57 ›› Issue (21): 4376-4390.doi: 10.3864/j.issn.0578-1752.2024.21.016

• 研究简报 • 上一篇    

利用酵母双杂交系统筛选花生AhSAP1的互作蛋白

朱艳婷(), 党浩, 牛思杰, 林婧怡, 杨华, 杨强, 张冲, 蔡铁城, 庄伟建, 陈华()   

  1. 福建农林大学农学院/豆科油料植物遗传与系统生物学研究中心/福建农林大学闽台作物生物育种农业农村部重点实验室/作物遗传育种与综合利用教育部重点实验室,福州 350002
  • 收稿日期:2024-07-18 接受日期:2024-08-29 出版日期:2024-11-01 发布日期:2024-11-10
  • 通信作者:
    陈华,E-mail:
  • 联系方式: 朱艳婷,E-mail:yantingzhu1215@163.com。
  • 基金资助:
    国家自然科学基金(32272155); 福建省科技厅高校产学合作项目(2021N5007)

Screening of Interaction Proteins with AhSAP1 in Peanut Using the Yeast Two-Hybrid System

ZHU YanTing(), DANG Hao, NIU SiJie, LIN JingYi, YANG Hua, YANG Qiang, ZHANG Chong, CAI TieCheng, ZHUANG WeiJian, CHEN Hua()   

  1. College of Agriculture, Fujian Agriculture and Forestry University/Research Center of Leguminous Oil Plant Genetics and Systems Biology/Key Laboratory of Fujian-Taiwan Crop Biological Breeding and Agriculture, Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University/Key Laboratory of Crop Genetics and Comprehensive Utilization, Ministry of Education, Fuzhou 350002
  • Received:2024-07-18 Accepted:2024-08-29 Published:2024-11-01 Online:2024-11-10

摘要:

【目的】种子大小直接影响花生的产量。前期通过QTL定位获得调控花生种仁大小的关键转录因子AhSAP1,构建花生胚酵母双杂交cDNA文库,并以AhSAP1为诱饵进行酵母双杂交筛选互作蛋白,分析候选互作蛋白基因的时空表达特征,为深入研究AhSAP1调控花生种仁发育的分子机制奠定基础。【方法】用SMART(switching mechanism at 5′ end of the RNA transcript)法构建花生胚大肠杆菌cDNA文库并进行鉴定;构建诱饵载体pGBKT7-AhSAP1,并鉴定其对酵母细胞的毒性以及自激活性。将花生胚cDNA文库质粒与诱饵质粒pGBKT7-AhSAP1共转Y2H Gold酵母菌株,筛选长势较好且呈蓝色的阳性菌落,测序比对,获得候选互作蛋白基因序列,预测生物学功能。利用RNA-seq明确候选互作蛋白基因在花生不同组织器官、受外源植物激素诱导及低钙胁迫诱导下的表达特征。根据功能注释结果,选取可能参与植物种子发育的候选因子,扩增其CDS全长序列,构建到靶标载体pGADT7后,分别与pGBKT7-AhSAP1共转化酵母细胞进行点对点酵母双杂交互作验证。【结果】花生胚大肠杆菌次级cDNA文库滴度为1.05×108 cfu/mL,重组率为98%,平均插入片段大小约1 000 bp,文库质量较高;成功构建酵母双杂交诱饵载体pGBKT7-AhSAP1,在酵母细胞中没有自激活性且对酵母菌没有毒性;筛选得到68个酵母阳性克隆,经序列相似性比对,去除重复,共获得60个候选互作蛋白,主要参与能量产生与代谢、翻译、核糖体结构和生物发育、转录、信号转导机制、翻译后修饰、无机离子运输和代谢、染色质结构等。选取12个候选互作蛋白与AhSAP1进行一对一酵母双杂交验证,有8个候选互作蛋白与AhSAP1发生互作。【结论】成功构建了花生胚发育不同时期的混合cDNA文库,筛选出60个与AhSAP1互作的候选互作蛋白,主要涉及能量产生与代谢、翻译、核糖体结构和生物发生、转录、信号转导机制、翻译后修饰、无机离子运输和代谢、染色质结构等,这些候选互作蛋白基因在花生根、茎、叶、花序、果针、果皮、种皮及胚中均有表达,明确了8个候选互作蛋白与AhSAP1的互作关系。

关键词: 花生, 种子大小, AhSAP1, 酵母双杂交, 互作蛋白

Abstract:

【Objective】Seed size directly affects the yield of peanut. The key transcription factor AhSAP1, which regulates peanut seed size, was obtained by QTL mapping in the early stage, but the molecular mechanism of AHSAP1 regulating peanut seed size remains unclear. In this paper, a peanut embryo yeast two-hybrid cDNA library was constructed and AhSAP1 was used as bait to screen interacting proteins, and the spatial and temporal expression characteristics of candidate interacting protein genes were analyzed. It laid the foundation for further study on the molecular mechanism of AhSAP1 regulating peanut kernel development. 【Method】The peanut embryo Escherichia coli cDNA library was constructed and identified by SMART (switching mechanism at 5′ end of the RNA transcript) method. The decoy vector pGBKT7-AhSAP1 was constructed and its toxicity and self-activation to yeast cells were evaluated. The peanut embryo cDNA library plasmid and the bait plasmid pGBKT7-AhSAP1 were co-transformed into Y2H Gold yeast strains, and the positive colonies with good growth and blue color were screened and sequenced to obtain the candidate interacting protein gene sequences and predict the biological functions. The expression characteristics of candidate interacting protein genes in different tissues and organs of peanut, induced by exogenous plant hormones and induced by low calcium stress were determined by RNA-seq. According to the functional annotation results, the candidate factors that may be involved in plant seed development were selected, their CDS full-length sequences were amplified, and the target vector pGADT7 was constructed, and point-to-point yeast two-hybrid interaction was verified with pGBKT7-AhSAP1 co-transformed yeast cells. 【Result】The titer of peanut embryo Escherichia coli secondary cDNA library was 1.05×108 cfu/mL, the recombination rate was 98%, the average insert fragment size was more than 1 000 bp, and the library quality was high. The yeast two-hybrid decoy vector pGBKT7-AhSAP1 was successfully constructed, which had no self-activation in yeast cells and no toxicity to yeast. Sixty-eight yeast-positive clones were screened, and 60 candidate interacting proteins were obtained by sequence similarity comparison and removal of duplicating, which were mainly involved in energy production and metabolism, translation, ribosome structure and biological development, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. Twelve candidate interacting proteins were selected for one-to-one yeast two-hybrid verification with AhSAP1, and 8 candidate interacting proteins were found to interact with AhSAP1. 【Conclusion】The mixed cDNA library of peanut embryo development at different stages was successfully constructed, and 60 candidate interacting proteins with AhSAP1 were screened. The candidate interacting proteins were mainly involved in energy production and metabolism, translation, ribosome structure and biogenesis, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. These candidates interacting protein genes were expressed in root, stem, leaf, inflorescence, fruit needle, pericarp, seed coat and embryo, and the interaction between 8 candidate interacting proteins and AhSAP1 was confirmed.

Key words: peanuts, seed size, AhSAP1, yeast two-hybrid, interacting protein