Analysis Of The Expression Characteristics And Functions Of CpWRKY61 Gene In Hawthorn

In recent years,research on the WRKY transcription factors has been intensively studied,and it has been found that under some biotic or abiotic stress,WRKY protein is highly induced.The transcriptome sequencing of the leaves of Hawthorn plants infected and uninfected with apple chlorotic leaf spot virus(ACLSV)showed that the expression level of CpWRKY61 gene was significantly up-regulated in infected virus plants.In this study,the coding region of CpWRKY61 gene was cloned from Hawthorn and its overexpression vector was constructed.And using agrobacterium-mediated genetic transformation to pour it into the apples,and analyze the role of CpWRKY61 gene in improving abiotic stress tolerance.The apple transgenic plants with ectopic expression of CpWRKY61 and the control plants were subjected to transcriptome sequencing analysis.The main findings are as follows:(1)The CpWRKY61 gene sequence was cloned from Hawthorn by quantitative RT-PCR analysis.The coding region was 1842 bp in length and encoded 613 amino acids.Transcriptional activity assays and subcellular localization experiments demonstrate that CpWRKY61 is a nuclear localization protein with transcriptional activation activity.(2)The expression of CpWRKY61 gene in different organs of Hawthorn was detected by quantitative RT-PCR,which indicated that CpWRKY61 was the mainly expressed in fruits and increased significantly with fruit ripening.The transcription level of the red fruit stage reached the highest,and its expression level was about twice that of the green fruit stage,while the expression level in the young leaves,old leaves,stems and flowers were relatively low.(3)The plant overexpression vector p RI101-CpWRKY61 was constructed.It was transformed into apple 'GL-3' by agrobacterium-mediated genetic transformation,and three CpWRKY61 gene ectopic expression lines were obtained.(4)The phenotypes of OE#2 and OE#4 seedlings in non-transgenic control apples and transgenic apple lines under the 100 m M Na Cl and the 300 m M mannitol abiotic stress and the plant expression at different time were analyzed.The results showed that the damage degree of non-transgenic plants was significantly higher than that of transgenic plants,indicating that CpWRKY61 gene plays an important regulatory role in plant salt stress and drought abiotic stress.(5)High-throughput sequencing analysis was performed on leaves of OE#2,OE#4 and non-transgenic control apple plants in transgenic lines ectopically expressing the CpWRKY61 gene.The downstream genes that may be regulated by the CpWRKY61 gene were detected by quantitative RT-PCR.Three abiotic stress-related downstream genes Md WRKY7,Md WRKY26 and Md WRKY71,which may be regulated by WRKY61 gene,were detected by quantitative RT-PCR.Transcriptome data analysis indicated that CpWRKY61 gene may be involved in the expression of ABA-mediated abiotic stress-related genes(Md WRKY7,Md WRKY26,Md WRKY71,Md NCED5,Md LEA14,Md DREB1b).The downstream genes that may be regulated by the CpWRKY61 gene were detected by quantitative RT-PCR,the results show that the quantitative RT-PCR data is consistent with the transcriptome data.