COP9亚基FaCSN5在草莓果实发育过程中的功能分析  

Functional analysis of the COP9 subunit FaCSN5 during strawberry fruit development

作　　者：高佳慧 冀桂明 李文静 郭家选[2] 沈元月[1] 高凡[2] GAO Jiahui;JI Guiming;LI Wenjing;GUO Jiaxuan;SHEN Yuanyue;GAO Fan(College of Plant Science and Technology,Beijing University of Agriculture/Beijing Key Laboratory of New Technology in Agricultural Application,Beijing 102206,China;College of Bioscience and Resources Environment,Beijing University of Agriculture/Key Laborato-ry for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs,Beijing 102206,China)

机构地区：[1]北京农学院植物科学技术学院·农业应用新技术北京市重点实验室,北京102206 [2]北京农学院生物与资源环境学院·农业农村部华北都市农业重点实验室,北京102206

出　　处：《果树学报》2023年第12期2536-2547,共12页Journal of Fruit Science

基　　金：国家自然科学基金资助项目(32272648,32072516,32030100)。

摘　　要：【目的】探究草莓COP9信号复合体亚单位5(constitutive photomorphogenic signalosome subunit 5,CSN5)在草莓果实发育过程中的功能。【方法】以草莓品种红颜为材料,根据草莓果实发育过程中的转录组数据,筛选并克隆FaCSN5基因。基于生物信息学对其功能域、理化性质、蛋白结构等进行预测。通过SDS-PAGE和Western Blot检测FaCSN5-His目的蛋白,利用烟草对其进行亚细胞定位分析。利用RT-qPCR检测FaCSN5的时空表达水平,利用农杆菌介导瞬时侵染草莓果实,观察记录表型,检测FaCSN5基因表达水平。利用圆片温育和外源激素处理试验检测外源激素对FaCSN5基因表达的诱导影响。【结果】系统进化树分析表明FaCSN5、FvCSN5和月季CSN5b同源性较高,相似率分别为100%和94.24%。克隆的FaCSN5与艳丽草莓8条基因序列的碱基和氨基酸序列相似率分别达98.97%和99.35%。FaCSN5基因的编码区为1080 bp,编码359个氨基酸,具有一个保守的MPN结构域。p ET30a-FaCSN5融合表达载体的大肠杆菌原核表达表明FaCSN5-His目的蛋白大小约66 ku。亚细胞定位显示FaCSN5-GFP融合蛋白定位为细胞核和细胞质。FaCSN5在根中表达水平最高,在种子中最低,在根中的表达量是种子中表达量的8倍;果实发育过程中在全红期表达量最高,在褪绿期表达量最低,从褪绿期开始随着果实发育表达量升高。农杆菌介导瞬时侵染草莓果实,FaCSN5过表达能够促进草莓果实成熟;沉默FaCSN5表达则会抑制草莓果实成熟。FaCSN5启动子上含有响应茉莉酸甲酯和赤霉素的顺式作用元件,且其表达受到这两种激素及脱落酸的诱导。【结论】FaCSN5可能是通过多种激素调控促进草莓果实成熟。Benihoppe strawberry was used as experimental material.Firstly,based on the transcriptome data of the five developmental stages(SG,LG,Wt,IR and PR)of strawberry fruit,a gene with increased expression level during fruit development from the large green fruit was screened.Because it contained MPN conserved domain and had 100%sequence similarity with diploid strawberry FvCSN5(XM_004291211),the gene was named FaCSN5.Total RNA was extracted from the samples using a total RNA extraction kit(Guangzhou Magen Biotechnology Co.,Ltd.),and the cDNA was synthesized by reverse transcription using Hi fair®Ⅲ1st Strand cDNA Synthesis Super Mix for qPCR(Shanghai Yeasen Biotechnology Co.,Ltd.)kit,and then the full-length CDS sequence of FaCSN5 was obtained by PCR.Secondly,some bioinformatics techniques were used in this study.The molecular formula,molecular weight,isoelectric point,and fat solubility index of encoding protein were analyzed in the Prot Param website.The conserved domain of FaCSN5 was analyzed by the NCBI website and Blast tool.The transmembrane domain of FaCSN5 protein was analyzed by TMHMM 2.0.The secondary and tertiary structures of FaCSN5 were analyzed online using NPS and Swiss Model.The phylogenetic tree of FaCSN5 homologous genes was constructed by MEGA11 software.Thirdly,the spatiotemporal expression levels of FaCSN5 were detected by RT-qPCR using Agrobacterium-mediated transient transformation of strawberry fruits,and the phenotypes were observed and used to detect the expression levels of the FaCSN5 gene.The cis-acting elements of FaCSN5 gene promoter were analyzed by the online software Plant CARE.The induction of FaCSN5 gene expression by exogenous hormones was detected by disc incubation and exogenous hormone treatment experiments.The subcellular localization was observed by Agrobacterium-mediated transient transformation of tobacco mesophyll cells.Finally,the fulllength CDS sequence of FaCSN5 was constructed into pET30a vector by homologous recombination,and the pET30a-FaCSN5 fusion expression vector

关 键 词：草莓 红颜 CSN5 表达分析 亚细胞定位 激素诱导 

分 类 号：S668.4[农业科学—果树学]