Isolation And Functional Analysis Of Vaccinium Corymbosum WRKY Transcription Factors VcWRKY24 And VcWRKY33

Vaccinium corymbosum is an extremely important cold fruit tree resource and has extremely high nutritional health value.During the growth process,Vaccinium corymbosum often suffers from adversity damage such as salinization and low temperature,which affects its yield and quality.In order to adapt to the stress environment,plants have evolved a series of physiological and molecular stress resistance mechanisms to improve tolerance.WRKY transcription factors are widely involved in the response of plants to abiotic and abioti c stresses during the growth and development of plants.The regulation and expression of WRKY genes help to improve the adaptability of plants to adversity.At present,most researches on Vaccinium corymbosum focus on its fruit quality and physiological characteristics,but there are few studies on the molecular mechanism of Vaccinium corymbosum adapting to adversity stress.Therefore,excavating the resistance gene in Vaccinium corymbosum and exploring its molecular mechanism under adversity stress are of great significance for breeding new varieties of resistance blueberry.In this study,the VcWRKY24 and VcWRKY33 genes were cloned by using Vaccinium corymbosum as the test material.The gene sequence and expression pattern under different tissues and different stress conditions were analyzed;By overexpressing VcWRKY24 and VcWRKY33 in Arabidopsis,respectively,the functions of these two genes in response to salt stress and low temperature stress were studied.The main research results obtained are as follows:1.Using Duchenne Bilberry c DNA as a template,the VcWRKY24 and VcWRKY33 genes of Vaccinium corymbosum are obtained by PCR amplification and cloning.The open reading frames of VcWRKY24 gene and VcWRKY33 gene are 1632 bp and 1167 bp,respectively.Sequence analysis results show that VcWRKY24 and VcWRKY33 proteins contain two complete WRKY domains,and the C-terminal contains two C₂H₂ zinc finger structures.2.The results of semi-quantitative PCR and fluorescent quantitative PCR showed that the expressions of VcWRKY24 and VcWRKY33 genes were tissue-specific.Both VcWRKY24 and VcWRKY33 genes were expressed in the roots,leaves and stems of Vaccinium corymbosum,and the expression levels were higher in leaves.Under the conditions of salt stress（200 m M Na Cl）and low temperature stress（4℃）,the expression levels of VcWRKY24 and VcWRKY33 genes showed a trend of increasing first and then decreasing with increasing stress time.3.Build VcWRKY24 and VcWRKY33 overexpression vectors respectively,and transform Arabidopsis thaliana by Agrobacterium infection method to obtain VcWRKY24 and VcWRKY33gene overexpression plants.The wild type and transgenic Arabidopsis were treated with low temperature and salt stress,respectively.Under stress,the leaves of the wil d-type Arabidopsis thaliana shriveled,while most of the leaves of the over-expressed plants of VcWRKY24 and VcWRKY33 were still green,and the measurement results of physiological indicators also showed that there were significant differences between wild-type and over-expressed plants.Seven days after the treatment was stopped,the survival rate of VcWRKY24 and VcWRKY33overexpressing plants was much higher than that of wild-type plants.In summary,overexpression of VcWRKY24 and VcWRKY33 genes can enhance the tolerance of Arabidopsis to low temperature and salt stress.