中国农业科学 ›› 2022, Vol. 55 ›› Issue (8): 1630-1641.doi: 10.3864/j.issn.0578-1752.2022.08.013

• 园艺 • 上一篇    下一篇

茶树CsHIPP26.1互作蛋白的筛选与验证

范延艮(),王域(),刘富浩,赵秀秀,向勤锃,张丽霞()   

  1. 山东农业大学园艺科学与工程学院/作物生物学国家重点实验室,山东泰安 271018
  • 收稿日期:2021-08-20 接受日期:2021-12-10 出版日期:2022-04-16 发布日期:2022-05-11
  • 联系方式: 范延艮,E-mail: 876562801@qq.com。|王域,E-mail: 1113405407@qq.com。
  • 基金资助:
    山东省现代农业产业技术体系创新团队项目(SDAIT-19-05);山东省“双一流”奖补资金项目(SYL2017YY03);鲁渝科技协作计划项目(2020LYXZ005)

Screening and Verification of CsHIPP26.1 Interaction Protein in Tea Plant

FAN YanGen(),WANG Yu(),LIU FuHao,ZHAO XiuXiu,XIANG QinZeng,ZHANG LiXia()   

  1. College of Horticulture Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong
  • Received:2021-08-20 Accepted:2021-12-10 Published:2022-04-16 Online:2022-05-11

摘要:

【背景】‘黄金芽’属于光照敏感型黄化茶树(Camellia sinensis)品种,叶片色泽呈现强光黄化、弱光复绿的特点,但叶色响应光照的黄化机制并不明确。前期通过对黄化叶片、遮阴复绿叶片以及常绿品种叶片的蛋白组研究发现,重金属相关异戊二烯化植物蛋白CsHIPP26.1(TEA000549)的表达响应光照强度,表明CsHIPP26.1可能参与调控‘黄金芽’叶色黄化的光响应过程。【目的】通过筛选与CsHIPP26.1互作的光信号响应相关的蛋白,为叶片色泽响应光照信号变化提供科学依据。【方法】以‘黄金芽’茶树1芽2叶为材料进行CsHIPP26.1和互作基因的克隆,经酵母双杂交筛库,然后将筛选得到的目的蛋白进行酵母双杂交点对点验证、体内双分子荧光互补(BiFC)和体外pull-down等技术进行蛋白互作的进一步验证。【结果】通过酵母双杂交对茶树cDNA文库进行筛选,共筛选到26个候选互作蛋白,主要集中在细胞组分、结合以及催化活性方面发挥作用,其中生物素合成代谢过程富集程度较高,与光信号通路及叶绿素合成相关的蛋白只有编号为TEA026466.1的bHLH30转录因子。克隆bHLH30转录因子后发现,该转录因子与茶树光信号传导途径蛋白PIF4处于同一进化树分支,且含有与茶树PIF4蛋白相同的HLH和ACT结构域,将该bHLH30转录因子命名为CsPIF4.2,GenBank登记号为MW116834。进一步通过体外Pull-down蛋白互作和体内双分子荧光互补(BiFC)验证发现,CsHIPP26.1和CsPIF4.2蛋白能够发生互作,并且发生互作的部位在细胞核内。【结论】初步筛选出26个与CsHIPP26.1互作的蛋白,并验证发现CsHIPP26.1能够在细胞核内与其中一个光敏色素互作因子CsPIF4.2发生蛋白相互作用。

关键词: HIPP, PIF4, 蛋白互作, 黄化品种, 光响应

Abstract:

【Background】 Huangjinya is a light sensitive chlorotic tea (Camellia sinensis) variety, the leaf color of which presents yellow under strong light and presents green under weak light, but the chlorotic mechanism of leaf color in response to light is not clear. Previous proteomic studies on etiolated leaves, shaded green leaves and evergreen leaves found that the expression of heavy metal-associated isoprenylated plant protein CsHIPP26.1 (TEA000549) responded to light intensity, indicating that CsHIPP26.1 may be involved in regulating the light response process of leaf color etiolation of Huangjinya. 【Objective】The proteins related to the light signal response interacting with CsHIPP26.1 were screened to provide a scientific basis for leaf color response to light signal changes. 【Method】The gene were cloned from one bud and two leaves of Huangjinya. And the screened target protein was further verified by yeast two hybrid point-to-point verification, in vivo bimolecular fluorescence complementarity (BiFC), and in vitro pull-down techniques. 【Result】 The tea cDNA library was screened by yeast two hybrid, and a total of 26 candidate interaction proteins were screened, which mainly played a role in cell components, binding and catalytic activity. Among them, the enrichment degree of biotin anabolism process was high, and the proteins related to light signal pathway and chlorophyll synthesis were only the bHLH30 transcription factor, and its gene ID was TEA026466.1. After cloning the gene of bHLH30 transcription factor, it was found that the transcription factor was in the same evolutionary tree branch as tea light signal transduction pathway protein PIF4, and contained the same HLH and ACT domains as tea PIF4 protein. Therefore, the bHLH30 transcription factor was named CsPIF4.2, GenBank registration number: MW16834. And through the pull-down protein interaction in vitro and bimolecular fluorescence complementarity (BiFC) in vivo, it was found that CsHIPP26.1 and CsPIF4.2 proteins could indeed interact, and the site of interaction was in the nucleus. 【Conclusion】 26 proteins interacting with CsHIPP26.1 were preliminarily screened, and it was found that CsHIPP26.1 could interact with one of the phytochrome interacting factors CsPIF4.2 in the nucleus.

Key words: HIPP, PIF4, protein interaction, yellowing varieties, light response