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

【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 was obtained induced and purified.Western Blot was used to detect FaCSN5-His target protein by SDS-PAGE and Anti-His antibody.【Results】Evolutionary tree analysis showed that FaCSN5 was highly homologous to FvCSN5 and rosa CSN5b,with similarity rates of 100%and 94.24%,respectively.The base and amino acid sequence similarity between the cloned FaCSN5 and the eight gene sequences of Yanli strawberry reached 98.97%and 99.35%,respectively.Bioinformatics analysis showed that the coding sequence of FaCSN5 was 1080 bp,encoding 359 amino acids.The analysis of physicochemical properties of amino acids showed that the molecular formula of FaCSN5 was C1797H2773N471O558S14,the molecular weight of the protein was 40.35 ku,and the isoelectric point(pI)was 4.93,which was an acidic protein.The protein contained 48 negatively charged amino acid residues(Asp+Glu)and 31 positively charged amino acid residues(Arg+Lys).The instability coefficient was 41.41,and the average hydrophilicity is-0.421.It was inferred that the protein should be an unstable hydrophobic protein.Conserved domain analysis showed that FaCSN5 had a conserved MPN domain.Phylogenetic tree analysis showed that FaCSN5 had high homology with FvCSN5 and rose CSN5b,and the similarity rates were 100%and 94.24%,respectively.The pET30a-FaCSN5 fusion expression vector was constructed for prokaryotic expression in Escherichia coli.The results of SDS-PAGE and Western Blot showed that the size of FaCSN5-His target protein was about 66 ku.The transient expression of Nicotiana benthamiana showed that the FaCSN5-GFP fusion protein was localized in the nucleus and cytoplasm.RT-qPCR analysis showed that the expression level of FaCSN5 was the highest in the root,followed by FR,PR,stem,leaf,and flower.The expression level of FaCSN5 was the lowest in the seed,and the expression level in the root was 8 times higher than that in the seed,indicating that FaCSN5 had tissue specificity.During fruit development,the expression level was the highest at the Full red stage and the lowest at the De-greening stage.From the Degreening green stage,the expression level increased with fruit development,indicating that FaCSN5 might be involved in the development of strawberry fruit.Agrobacterium-mediated transient infestation of strawberry fruit with FaCSN5 overexpression could promote strawberry fruit ripening;silencing FaCSN5 expression inhibited strawberry fruit ripening.The FaCSN5 promoter contained cis-acting elements in response to methyl jasmonate and gibberellin.After treatment with MeJA and GA,the expression level of FaCSN5 gene was slightly lower than that of the control group after 1 h of MeJA treatment.After 2-5 h of treatment,the expression level of FaCSN5 gene was higher than that of the control group.The expression level of FaCSN5 gene was higher than that of the control group at 1-5 h after GA treatment,and the expression level was the highest at 1 h after treatment.After ABA treatment,the expression level of FaCSN5 gene was higher than that of the control group,and the expression level was the highest after 3 h of treatment.The results showed that the expression level of FaCSN5 gene was induced by MeJA,GA,and ABA,to various degrees.【Conclusion】The protein height of FaCSN5 was about 66 ku,which was localized in the nucleus and cytoplasm.The expression of FaCSN5 was induced by abscisic acid,methyl jasmonate,and gibberellin.FaCSN5 might promote strawberry fruit ripening through multiple hormonal regulations.