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

【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.Besides,the transcriptional activation activity were detected using yeast one-hybrid technology.【Results】In this study,11 differentially expressed NAC genes were screened out through the expression in Red Globe.They were detected in another grape variety(Red Balad).Based on the expression change in the NAC genes in the two grape varieties,4 differentially expressed NAC genes-VvNAC5,VvNAC11,VvNAC13,VvNAC18 were identified as candidate genes.They were cloned and sequenced using the cDNA of Red Globe as the template.It was found that the ORF lengths of VvNAC5,VvNAC11,VvNAC13 and VvNAC18 genes were 1083,1092,1098,1062 bp,encoding 360,363,365 and 353 amino acids,respectively.The molecular weights and isoelectric points of these proteins were different.All the proteins were hydrophilic proteins.The comparison of the protein sequences showed that the 4 NAC genes contained highly conserved NAM domains at the N-terminus,but the C-terminus sequences were highly variable.The results of phylogenetic tree analysis divided these NACs into three branches.Branch one included a number of NAC transcription factors involved in stress response.The second branch contained VvNAC18 and the aggregation of many proteins that regulate fruit maturation and senescence and leaf shedding,indicating that VvNAC18 might participate in the ripening process of grape fruit.The third branch mainly consisted of the NAC proteins related to the formation of meristems and organ development.VvNAC5,VvNAC11and VvNAC13 all belongs to the last branch,which might play a role in the process of tissue formation and development.The expression of the 4 NAC genes in 6 different tissues and organs of grapes displayed different patterns.VvNAC5 and VvNAC18 were mainly expressed in fruit,and significantly more active than in other parts.VvNAC11 and VvNAC13 had the highest expression in roots and leaves,respectively,but also had a higher expression level in fruit,indicating that they may be involved in the development and maturation of fruit.The four candidate NAC genes were recombined with the pGBKT7vector,and the recombinant plasmids and the empty PGBKT7 plasmid were transformed into yeast AH109.It was found that the yeasts transformed with the recombinant plasmid and the pGBKT7 empty plasmid grew normally on SD-Trp single-deficient medium.Yeasts transformed with p GBKT7 empty plasmids could not grow on SD-Trp-His-Ade deficient medium plates.Yeasts transformed with PGBKT7-NACs recombinant plasmids grew on SD-Trp-His-Ade(X-α-gal)deficient medium plates,and decomposed X-α-gal to produce a blue substrate,indicating that all the four NAC transcription factors had transcriptional activation activity.【Conclusion】NAC transcription factors play an important regulatory role in the development and maturation of fruits.Based on the gene expression in the two grape varieties,4 NAC transcription factors(VvNAC5,VvNAC11,VvNAC13,and VvNAC18)were screened out and cloned.The results showed that the 4 NAC transcription factors may be involved in fruit development and maturation.