Identification Of GA2ox,GA3ox And GA20ox Families Of Gibberellin Oxidase Genes And Salt Tolerance Functional Analysis Of GA2ox7 In Vitis Vinifera

Salt stress has a significant impact on crop cultivation and production in northwest China.The research on the mechanism of plant salt tolerance is deepening,especially the identification and mining of salt tolerance related genes has been one of the focuses of biological research.Gibberellin participates in various abiotic stress responses of plants,and exogenous application of GA₃ can improve plant tolerance to salt stress.The gibberellin oxidase genes GA2ox,GA3ox,and GA20ox have been studied in some model plants to improve the salt stress tolerance of plants.Therefore,identification through bioinformatics methods and q RT-PCR analysis of Vv GA2ox,Vv GA3ox and Vv GA20ox in this paper.The salt-tolerant gene GA2ox7 obtained from the screening was transformed into Arabidopsis thaliana,and the salt-tolerant function of the gene was analyzed.The main findings are as following:1.In this paper,24 members of the grape gibberellin oxidase families were identified by bioinformatics method.The gene identification numbers（ID）were utilized to rename for GA2ox,GA3ox,GA20ox family,which was named Vv GA2ox1-Vv GA2ox11,Vv GA3ox1-Vv GA3ox6 and Vv GA20ox1-Vv GA20ox7.Physical and chemical properties analysis showed that there were significant differences in molecular weight,isoelectric point and CDS sequence size of GA2ox,GA3ox and GA20ox genes.The nucleus,cytoplasm and chloroplast were the principal localized organelles of the family genes.It was based on the analysis of phylogenetic tree,gene structure and motif of this gene family that can be divided into 7 subgroups.2.It was based on the results of promoter molecules of Vv GA2ox,Vv GA3ox and Vv GA20ox gene families,which the promoter elements mainly with various hormone response elements,and contains abiotic stress elements,such as ABA,GA₃,Me JA,low temperature,drought,salt and other stress response elements.The analysis of the expression of this gene family in grape transcription data under abiotic stress showed that most of the GA2ox,GA3ox and GA20ox genes were highly expressed under ABA,Na Cl,PEG and 5℃treatment.Grape tissue expression analysis showed that the expression levels of Vv GA2ox and Vv GA20ox in different tissues of grape at different developmental stages were higher than those of Vv GA3ox.Finally,the relative expression of Vv GA2ox,Vv GA3ox and Vv GA20ox gene families treated with GA₃ and uniconazole was detected by q RT-PCR,and it was found that some Vv GA2oxs genes were up-regulated under GA₃ treatment.Simultaneously,uniconazole upregulated the expression of Vv GA3ox and Vv GA20ox.Among them,under salt stress and exogenous GA₃ treatment,the expression of Vv GA2ox7 was the highest.3.The GA2ox7 gene of grape was cloned.The length of the CDS was 100 2 bp,and the corresponding amino acid number was 334.It was a hydrophilic protein.The subcellular localization vector p BI221-GA2ox7-EGFP was constructed to verify the subcellular prediction results.The results showed that GA2ox7 protein belonged to nuclear and cytoplasmic localization protein.The construction of over-expression vector p CAM-GA2ox7-FLAG was accomplished,and successfully transformed into Arabidopsis thaliana of this gene.Subsequently,various physiological indexes and antioxidant enzyme activities were detected in transgenic Arabidopsis thaliana leaves after Na Cl or Na Cl+GA₃ treatment.It was found that the electrical conductivity,MDA and H₂O₂ in transgenic Arabidopsis thaliana leaves remained unchanged at first or decreased then increased,chlorophyll and proline content increased at first and then decreased,POD,SOD and CAT activity increased at first and then decreased,and 8 h treatments time was an important turning point.And the changes of these physiological indexes were more noticeable under the co-treatment of Na Cl+GA₃.The expression of salt tolerance related genes and flowering regulatory genes were determined at 8 h.The results showed that salt tolerance related genes KAT1,APX1,LEA,P5CS1,AVP1 and CBF1 were up-regulated in transgenic Arabidopsis thaliana,flower inhibition gene FLC was up-regulated and flower-promoting gene FT was down-regulated in transgenic Arabidopsis thaliana.In addition,after8 h treatments,the contents of GA₃ and ABA in transgenic Arabidopsis thaliana were determined.The results showed that the content of GA₃ in transgenic Arabidopsis thaliana decreased significantly,while the content of ABA increased significantly,and the changes of the two hormones were more obvious under Na Cl+GA₃ co-treatment.These results show that the heterologous expression of GA2ox7 could enhances the salt tolerance of transgenic Arabidopsis.