欧洲葡萄VvJAZ9基因互作蛋白的筛选与验证

Screening and verification of JAZ9 gene interacting proteins in Vitis vinifera

【目的】探究葡萄JAZ家族基因特点及在低温胁迫下的表达情况,以期筛选出与低温胁迫相关的家族成员,并进一步筛选其互作蛋白,为后续葡萄抗寒机制研究提供理论依据。【方法】以拟南芥和葡萄基因组数据为基础,获得葡萄JAZ家族成员基因并进行同源克隆、染色体定位、理化性质以及蛋白家族聚类等分析,利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术分析VvJAZ家族成员基因在低温胁迫下的表达情况,应用酵母双杂交技术进行互作蛋白筛选和验证,并通过双分子荧光互补试验验证候选互作蛋白与VvJAZ9蛋白的互作关系。【结果】葡萄中有11个JAZ家族基因,随机分布于7条染色体上,都具有JAZ家族蛋白中特有且高度保守的TIFY和Jas结构域,均属于碱性不稳定亲水蛋白;聚类分析表明,葡萄JAZ家族蛋白可分为3个亚族,其中VvJAZ9与拟南芥AtJAZ1和AtJAZ2的亲缘关系最近,VvJAZ1与VvJAZ11、VvJAZ5与VvJAZ6同源关系最近。qRT-PCR结果表明,VvJAZ9是葡萄JAZ家族成员基因中受低温诱导表达最为明显的1个基因。通过酵母双杂交试验筛选获得26个可能与VvJAZ9存在互作关系的候选蛋白;通过q RT-PCR分析候选蛋白的相关基因在低温胁迫下的表达模式,结果表明HAP5A基因受低温诱导表达趋势与筛选出的VvJAZ9基因类似,推测HAP5A可能是JAZ9候选互作蛋白中响应低温最为显著的1个蛋白;通过同源克隆获得了VvHAP5A基因序列,全长636 bp,编码211个氨基酸,进一步分析发现该基因是NF-YC类型的转录因子,通过酵母双杂交和双分子荧光互补试验也证实了JAZ9与HAP5A蛋白全长有互作关系。【结论】鉴定并克隆到了葡萄的11个VvJAZs基因,VvJAZ9和VvHAP5A基因均响应葡萄低温诱导表达,并且二者还具有蛋白互作关系,可为进一步研究葡萄JAZ基因参与低温胁迫的机制提供参考。

【Objective】The aims of this experiment were to investigate the characteristics of grapevine JAZs gene family and its expression pattern in response to low temperature stress,to screen out the JAZs family members which related to low temperature stress,to screen the candidate substrates of the low temperature-induced JAZ proteins by the yeast two-hybrid assay,and to provide a theoretical basis for the study on cold resistance mechanism in grapevines.【Methods】Based on the genomic analysis of Arabidopsis thaliana and grapevine,the grapevine JAZs gene family members were obtained and analyzed by homologous cloning,and their chromosomal localization,physicochemical properties and protein family clustering were also investigated.We used the qRT-PCR experiment to analyze the expression of the JAZs gene family members under low temperature treatment conditions,and the yeast twohybrid was used to screen the candidate substrates of low temperature-induced JAZ protein,and the yeast two-hybrid assay and bimolecular fluorescence complementation assay were used to verify the interactions between the candidate protein and the target JAZ protein.【Results】Eleven JAZs gene family members were cloned by homologous sequences in grapevine.These genes were randomly distributed on seven chromosomes,all of which have TIFY and Jas structural domains that are unique and strongly conserved in JAZs family proteins,and all of them belong to basic hydrophilic proteins.Cluster analysis showed that grapevine JAZs family proteins can be divided into three subfamilies.In subgroupⅠ,VvJAZ4 and VvJAZ9 are closest to AtJAZ1 and AtJAZ2 in Arabidopsis.In subgroupⅡ,VvJAZ1 and VvJAZ11 are closest to AtJAZ9 in Arabidopsis.In subgroupⅢ,VvJAZ3,VvJAZ5,VvJAZ6,VvJAZ7 and VvJAZ8 are most closely related to AtJAZ7 and AtJAZ8 in Arabidopsis.The following qRT-PCR results showed that among the JAZs gene family members,the transcripts of VvJAZ9 were significantly induced by low temperature,with the lowest level at 0 h and the highest level at 24 h.The expression of VvJAZ2 and VvJAZ10 genes showed an increasing trend at first,followed by a decreasing expression,and then gradually increasing again.The expression of VvJAZ3,VvJAZ5/6 and VvJAZ8 was at a low level before 12 h and peaked at 24 h.The expression of VvJAZ1 showed a decreasing trend firstly,and then showed an increasing expression pattern,while the expression of VvJAZ11 showed a decreasing expression trend in response to low temperature treatment,with the lowest expression level at 24 h.According to the qRT-PCR assay,we found that VvJAZ9 was one of the most significantly low temperature-induced gene among the grapevine JAZs gene family members.By the yeast two-hybrid assay,we obtained 26 candidate proteins to be the candidate substrates of the VvJAZ9 protein,including bZIP,HAP5A,CONSTANS-LIKE 2,STOP1,CSN5B,GAI1,NINJA and so on.Based on the conserved structural domains of 26 VvJAZ9 candidate reciprocal proteins,the homologous sequences of some proteins were obtained,and the mRNA sequences corresponding to the related genes were obtained by the blast analysis in the NCBI database.The qRT-PCR primers for these candidate protein-related genes were designed by Primer 6.0 software.The results indicated that the bZIP gene showed a rising trend at first,and then showed a decreasing expression trend after treatment with low temperature,and the maximum level was presented at 3 h.The transcripts of CONSTANS-LIKE 2,STOP1 and GAI2 showed a down regulation toward at first,and then presented the increasing expression trend,and the lowest expression point was observed at 6 h after cold treatment.The expression of CSN5B showed a doublepeaked trend,and the maximum expressions were at 6 h and 24 h,respectively.The expression of NINJA showed a gradually decreasing trend.The transcripts of HAP5B and GAI1 increased at first and then decreased,and both of them reached to the maximum expression at 3 h.However,the expression level of HAP5A showed a gradually increasing expression trend,which was similar to the low temperature-induced expression trend of VvJAZ9 we screened.By the qRT-PCR analysis of the candidate genes,we considered that the expression of HAP5A gene was similar to the VvJAZ9 gene in terms of low temperature.Therefore,the HAP5A might be one of the most significant expression of the JAZ9 candidate interacting protein among the JAZ9 candidate substrates in response to low temperature.Furthermore,VvHAP5A was cloned by homologous sequences,and its ORF includes 636 nucleotides,encoding 211 amino acids.Further analysis revealed that the VvHAP5A gene is a NF-YC type transcription factor gene,and the interacting relationship between JAZ9 and the full length of HAP5A protein was also confirmed by the yeast two-hybrid and bimolecular BiFC experiments.【Conclusion】The 11 VvJAZs genes from grapes were identified and cloned,and both VvJAZ9 and VvHAP5A responded to low temperature in grapevines,and they also had protein interaction relationship.These results can provide reference for further study on the mechanism of grape JAZ genes involved in low temperature stress.