The Function Identification And Regulation Mechanism Study Of ClWRKY20 In Watermelon Under Low Temperature And High Salt Stresses

Watermelon,as a widely cultivated fruit and economic crop in the world,plays an important role in agricultural production.Originated in tropical desert area,watermelon needs enough light and relatively high temperature during its growth.Low temperature and low light are the key factors limiting watermelon cultivation in early spring in northern China.At the same time,with the increased area of protected cultivation,the secondary salinization of soil has becomes a difficult problem in protected cultivation,which seriously restricts the development of watermelon industry.Therefore,the research on low temperature and salt tolerance mechanism of watermelon is of great significance in production.The WRKY transcription factor plays an important role in plant responses to various stresses.In our previous studies,57 WRKY genes were identifiedin watermelon plants.Expression profiles of these 57 WRKY genes in response to cold stress were analyzed,and CIWRKY20 was selected as the candidate gene for further study since it showed a significant response to low temperature.Its function and regulation mechanism in watermelon stress responses was not clear.So in this study,ClWRKY20 was cloned from watermelon leaves by the RT-PCR method,and transformed into Arabidopsis by constructing overexpression vectors PMDC32-C1WRKY20.The salt and low temperature tolerance of CIWRKY20 transgenic arabidopsis thalianawas identified.At the same time,transcriptome sequencing technology was used to preliminarily analyze the regulation pathway of CIWRKY20 under low temperature.The main results of this paper were as follows:(1)ClWRKY20 was cloned from watermelon leaves by the RT-PCR.Sequence analysis showed that the full length of the open reading frame of ClWRKY20 was 1023 bp,encoding 340 amino acids.The molecular weight of the encoded protein was about 37.94 KD and the isoelectric point was 4.94.NCBI blast found that the ClWRKY20 showed a similarity of over 75%with WRKY46 of Cucumis melo、Cucumis sativus、Cucumis melo var.makuwa.(2)ClWRKY20 was expressed in different degrees in root,stem and leaf of watermelon seedlings,with the highest expression in leaf.Real-time PCR showed that C1WRKY20could be induced by salicylic acid,mannitol,ethephon,hydrogen peroxide,sodium chloride and mechanical damage.The gene may be involved in various signal transduction pathwaysand plants stress responses.(3)ClWRKY20 was localized in nucleus.The fusion expression vector of pH7LIC5.0-N-eGFP-ClWRKY20 was successfully constructed,and its expression in tobacco lower epidermal cells was detected using the confocal microscopy.It was found that the green fluorescences was distributed in the nucleus.The results conformed to the functional properties of transcription factors.(4)Acquisition of transgenic arabidopsis thaliana plants.Over-expression vectors of ClWRKY20 was constructed and transformed into wild Arabidopsis thaliana by agrobacterium-mediated transformation.Finally,three pure lines OE1,OE2 and OE5 were obtained.(5)Overexpression of ClWRKY20 in arabidopsis thaliana increasedlow temperature resistance.Under normal culture conditions,there was no significant difference between the growth states of wild type and transgenic plants.After treatment at 4℃ for 7 days,the root length of transgenic plants was significantly longer than that of wild-type plants.After treatment at-20℃ for 1h,thawing at 4℃ for 12h,and then placed under normal conditions for 7d.Compared with transgenic plants,the death rate of wild-type plants increased more significantly and its wilting degree was also more serious.The several physiological indexes related to stress resistance were also measured It was showed that the conductivities and MDA contents of transgenic plants were significantly lower than that of wild-type plants,indicating that the degree of cell damage in transgenic plants was significantly reduced.Compared with wild type plants,the F0 value of transgenic plants increased less,the FV/FM and proline contents was higher.All these values fully showed that the introduction of CIWRKY20 into Arabidopsis plants could significantly improve their tolerance to low temperature.(6)Overexpression of ClWRKY20 improved salt tolerance in arabidopsis thaliana.After salt treatment,the germination rate and root length of transgenic plants were significantly higher than that of wild-type plants.The salt tolerance phenotype of transgenic plants was obvious,The conductivities and MDA contents of transgenic plants were significantly lower than that of wild-type plants.At the same time,more proline was accumulated in wild-type plants,indicating that wild type plants were sensitive to high salt.All these indicated that over-expression of ClWRKY20 also significantly improved the salt stress resistance of Arabidopsis plants.(7)Transcriptome results showed that the differentially expressed genes of wild type and transgenic arabidopsis thaliana were mainly concentrated on the plant hormone signaling pathway.Especially,the genes participated in the uxin and jasmonic acid signaling pathways were significantly upregulated.Such as AUX1 and Aux/IAA,which were all up-regulated after low temperature treatment in transgenic plants,and down-regulated or not significantly changed after low temperature treatment in wild-type plants.In the jasmonate signaling pathway,AT1632630 was highly expressed in transgenic plants without low temperature treatment.and was significantly up regulated in both wild type and transgenic plants after low temperature treatment,It was possible that CIWRKY20 regulated the cold tolerance of transgenic plants through the signaling pathways of auxin and jasmonic acid..However,its regulatory mechanism needs further experimental verification.