Study On Drought Resistance Function Of VyMYB24 Gene In Vitis Yeshanensis

The MYB gene is one of the largest families of transcription factors in plants,and plays an important role in the growth and development of plants,in cell morphology,organ differentiation,secondary metabolism,and resistance to stress.Chinese wild ’Yanshan’ grapes are extremely drought resistant,but the role of the MYB gene in the stress resistance process of Yanshan grapes is unclear.In this study,Chinese wild ’Yanshan’ grape（Vitis yeshanensis ’Yan Shan’）was used as material to clone and obtain VyMYB24 gene.Nicotiana benthamiana was genetically transformed by Agrobacterium tumefaciens mediated leaf disk method to obtain stable genetic transgenic tobacco grass（OE1 and OE3）.Its functions in plant growth and development and drought stress were studied.The main results are as follows:（1）VyMYB24 gene（Uniprot database registration number:VIT₁4s0066g01090）cDNA total length 570 bp,encoding 190 amino acids,molecular weight of 21.569 kD,localized in the genome Chr14 chromosome.VyMYB24 contains a highly conserved R2-R3 DNA domain,which is a typical R2R3 MYB transcription factor;Subcellular localization showed that VyMYB24 was a nuclear protein;The expression analysis of VyMYB24 gene under stress showed that the gene was induced by drought,high temperature and salt stress,among which drought stress was the most intense.（2）Agrobacterium agrobacterium-mediated leaf disc method was used to transfer VyMYB24 to Ben’s tobacco and obtain stable genetic transgenic tobacco plants（OE1,OE3）.The relevant growth and development indexes of transgenic plants were measured,and it was found that the overexpression of VyMYB24 gene caused dwarfing of transgenic tobacco plants,and the phenotype at all growth periods was smaller than that of wild type,which was specifically manifested as plant dwarfing,internodal shortening,smaller shape of reproductive organs,smaller leaf area,and darker leaf color,etc.These results show that VyMYB24 gene is involved in regulating the morphological development of tobacco plants.（3）The content of endogenous gibberellin GA1+3 in tobacco was determined,and it was found that the GA1+3 content of transgenic tobacco plants was significantly lower than that of wild-type tobacco plants（P<0.05）,which were 47.72%and 46.17%of wild-type tobacco plants,respectively.;The expression analysis of gibberellin synthesis and metabolism-related genes found that the expression levels of GA20ox and GA3ox genes related to gibberellin synthesis in transgenic tobacco plants（OE1,OE3）were lower than those in wildtype tobacco plants,while gibberellin In addition to GA2oxl and GA2ox3 genes,the expression levels of other genes in transgenic tobacco plants（OE1,OE3）were higher than those in wild-type tobacco plants.It indicated that VyMYB24 may reduce the endogenous gibberellin content of transgenic plants by downregulating gibberellin synthesis-related genes and up-regulating gibberellin degradation genes.In addition,after spraying exogenous gibberellin GA3,it was found that the dwarf phenotype of VyMYB24 transgenic tobacco plants（OE1,OE3）was restored to a certain extent,and the difference in plant height was reduced.It indicated that VyMYB24 gene regulates the gibberellin pathway and affects the plant height of tobacco plants.（4）Under drought stress,the wilting rates of VyMYB24 transgenic tobacco plants（OE1,OE3）were 26.67%and 30.00%,respectively,while the wilting rate of wild-type tobacco plants reached 80.00%,and the wilting rate of transgenic tobacco plants was significantly lower in wild-type tobacco plants（P<0.05）;meanwhile,the root fresh weight was significantly higher than that in wild-type tobacco plants（p<0.05）.These results indicated that the VyMYB24 gene could enhance the drought resistance of tobacco plants under drought stress.（5）Under drought stress,both the hydrogen peroxide（H2O2）content and the superoxide anion（O2·-）production rate in transgenic and wild-type tobacco leaves increased,but the accumulated H2O2 in transgenic tobacco was higher than that in wild-type tobacco.content and O2·-production rate were significantly lower（p<0.05）,H2O2 content was only 64.93%and 72.57%of wild-type tobacco,and O2·-production rate was only 67.38%and 66.20%of wild-type tobacco;Benzidine（DAB）and nitro blue tetrazolium（NBT）staining gave the same results.These results indicated that under drought stress,VyMYB24 gene could enhance the drought resistance of tobacco plants by reducing the accumulation of reactive oxygen species in leaf tissues of tobacco plants.（6）Under drought stress,VyMYB24 gene can significantly increase the SOD,POD,CAT and other antioxidant enzyme activities and proline content of transgenic tobacco plants（OE1,OE3）（p<0.05）,and significantly reduce transgenic tobacco plants（OE1,OE3）plant MDA content and relative conductivity（p<0.05）,SOD enzyme activity reached 1.91 times and 1.70 times that of wild type tobacco plants;POD enzyme activity reached 1.40 times and 1.32 times that of wild type tobacco plants;CAT enzyme activity.The activity reached 1.84 times and 1.68 times that of wild type tobacco plants;the proline content reached 176.09%and 213.95%of wild type tobacco plants;the malondialdehyde content was 49.07%and 51.07%of wild type tobacco plants,respectively;the relative conductance The rates were 0.38%and 0.42%,respectively,while the relative conductivity of wild-type tobacco plants reached 0.73%.These results indicated that under drought stress,VyMYB24 gene could enhance the drought resistance of tobacco plants by up-regulating antioxidant enzyme activity and proline content,and down-regulating malondialdehyde content and relative conductivity.（7）Under drought stress,the expression levels of NtERD10C,NtERD10D,NtLEA,NtSOD,NtPOD,NtCAT,NtP5CS,NtP5CR genes related to drought resistance in transgenic tobacco plants（OE1,OE3）were all up-regulated,and were significantly higher than those in wild-type tobacco（p<0.05）.The results showed that VyMYB24 gene could increase the expression of tobacco drought resistance-related genes under drought stress and enhance the drought resistance of tobacco plants.