Screening and verification of JAZ9 gene interacting proteins in Vitis vinifera

【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 in-teractions 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 subgroup Ⅰ, VvJAZ4 and VvJAZ9 are closest to AtJAZ1 and AtJAZ2 in Arabidopsis. In subgroup Ⅱ, VvJAZ1 and VvJAZ11 are closest to AtJAZ9 in Arabidopsis. In subgroup Ⅲ, 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.