葡萄果实成熟相关NAC转录因子的筛选、克隆及表达分析  

Screening,cloning and expression analysis of NAC transcription factors related to grape fruit ripening

作　　者：牛早柱 赵艳卓[1] 陈展[1] 宣立锋[1] 牛帅科[1] 褚凤杰[1] 杨丽丽[1] NIU Zaozhu;ZHAO Yanzhuo;CHEN Zhan;XUAN Lifeng;NIU Shuaike;CHU Fengjie;YANG Lili(Shijiazhuang Pomology Institute,Hebei Academy of Agriculture and Forestry,Shijiazhuang 050061,Hebei,China)

机构地区：[1]河北省农林科学院石家庄果树研究所,石家庄050061

出　　处：《果树学报》2022年第7期1137-1146,共10页Journal of Fruit Science

基　　金：国家现代农业产业技术体系建设专项(CARS-29-3);河北省农林科学院创新工程项目。

摘　　要：【目的】NAC转录因子在植物生长发育中起重要调控作用。旨在筛选参与葡萄果实成熟的NAC转录因子,揭示NAC转录家族在葡萄果实成熟过程中的生物学功能。【方法】利用荧光定量PCR技术分析了NAC基因在葡萄果实不同时期的表达水平及其组织特异性,对候选基因的核苷酸序列、蛋白质性质进行分析。同时,构建pGBKT7重组质粒检测转录因子的自激活能力。【结果】红地球葡萄不同发育阶段的NAC基因表达分析中,筛选出11个差异表达的NAC基因。结合其在红巴拉多葡萄中的表达情况,确定4个NAC转录因子为候选基因。VvNAC5、VvNAC11、VvNAC13、VvNAC18基因的开放阅读框长度分别为1083、1092、1098、1062 bp,分别编码360、363、365、353个氨基酸,各蛋白分子质量与等电点不同。蛋白序列对比结果显示4个NAC基因在N端都包含高度保守的NAM结构域,但C端序列高度变异。系统进化树分析显示VvNAC5、VvNAC11、VvNAC13与众多参与组织形成和器官发育相关的NAC蛋白聚为一类,推测同时在组织形成发育过程中发挥作用;VvNAC18与众多调控果实成熟衰老及叶片脱落相关蛋白聚为一类,说明VvNAC18参与葡萄果实的成熟调控过程。分析4个NAC基因的组织特异性,发现VvNAC5、VvNAC18主要在果实中表达,且显著高于其他部位,VvNAC11、VvNAC13在果实中也有较高的表达量。酵母转化试验证明4个NAC蛋白均具有转录激活活性。【结论】筛选出4个NAC转录因子在葡萄果实的成熟过程中发挥重要的调控作用,为探索NAC转录家族调控葡萄果实成熟的作用机制奠定理论基础。【Objective】NAC transcription factor is a type of terrestrial plant-specific transcriptional regulatory factor,which plays an important regulatory role in plant growth and development.However,up to now,the roles of the grape NAC gene family participating in regulating fruit ripening has not been systematically studied.Our present study screened the NAC transcription factors involved in grape ripening and revealed the biological functions of the grape NAC transcription family during grape fruit ripening.【Methods】The fruit of Red Globe in different periods were collected and divided into two parts.One part was used to test the physical and chemical properties,and the other part was used for RNA extraction.Then the RNAs were reverse transcribed into c DNAs for NAC genes expression detection.The expression primers of 74 NAC genes were designed according to the full-length sequence obtained from PlantTFDB.Some NAC genes were screened out by comparing their expression level at different stages of Red Globe fruit with fluorescent quantitative PCR technology.The expression of the genes was detected in different periods of the fruit of Red Balad.Based on the gene expression in the two varieties,four candidate NAC genes were identified and cloned.Nucleic acid sequence of the four NAC genes were analyzed using DNAMAN software.The molecular weight,isoelectric point,instability index,average hydrophilicity and other physical and chemical properties of predicted proteins were analyzed by Protparam software.The transmembrane regions and signal peptide were also analyzed using TMHMM Server v.2.0 and SignalP 4.1 Server.Plant-mPLoc v1.0 was used to predict their subcellular localization.In addition,in order to analyze the protein properties of the candidate NAC genes,a phylogenetic tree was constructed using MEGA X depending on other related studies and 18 NAC proteins of other species obtained through NCBI.At the same time,different tissue expression of the candidate NAC genes was detected by fluorescent quantitative PCR.B

关 键 词：葡萄 果实成熟 转录因子 进化分析 组织表达 

分 类 号：S663.1[农业科学—果树学]