Preliminary Study On Genetic Transformation System Of Verbena Bonariensis And Cloning And Functionalidentification Of VBWRKY32

Verbena bonariensis has the advantages of soft color,strong drought tolerance,moderate water demand,easy survival and so on.It is widely distributed in areas with strong light and high temperature,and has the high ornamental value.But in the planting of V.bonariensis,the yield and appreciation of V.bonariensis are seriously affected due to the cold environment in winter.In this study,by analyzing the transcriptomic data of Verbena bonariensis under low temperature stress,we preliminarily explored the cold-responsed mechanism of V.bonariensis and screened the VbWRKY32 gene;the VbWRKY32 gene was transformed into V.bonariensis by using the genetic transformation system constructed in this study and the cold-resistant function of VbWRKY32 gene was identified.1.Six cDNA libraries were established by using illumina sequencing technology for transcriptome analysis of V.bonariensis leaves under low temperature stress.A total of271,920 unigenes were obtained.Of these,205,539 were annotated and 19,003 were differentially expressed genes?DEGs?.This study analysed the enrichment of metabolic pathways of V.bonariensis DEGs and the expression level changes of DEGs related to photosynthetical system,membrane system,antioxidant enzyme system,phytohormone and transcription factors under low temperature stress.2.In the process of establishing the regeneration system of V.bonariensis,the leaves of aseptic plantlets were used to induce adventitious bud on the MS medium supplemented with 1.0 mg/L 6-BA and 0.1 mg/L NAA.The average shooting rate was 62.67%and the multiplication coefficient was 1.03.On this basis,we explored that the optimum inoculation mode of leaves was that the back of leaves contacted the medium and the optimum temperature of inducing adventitious buds was 35?,and found the bud point of adventitious buds was mainly located at the leaves bases and leaves tips.In the Agrobacterium tumefaciens mediated transformation system,the OD₆₀₀ value and the time of infection were 0.4 and 10 min,respectively.the concentration of Carbenicillin?Carb?and kanamycin?Km?supplemented in the medium were 450 mg/L and 1 mg/L,respectively.The probability of getting positive seedling was 60.00%.3.The VbWRKY32 gene was screened from transcriptomic data of V.bonariensis under cold stress.The full-length sequence of VbWRKY32 gene was cloned and its characteristics were analyzed by bioinformatics.The plant cloning vector pEASY-VbWRKY32 was constructed.The plasmids containing pEASY-WRKY32 and pCAMBIA 2300 expression vectors were digested by BamH and Kpn I.VbWRKY32 gene was transferred into V.bonariensis by constructing genetic transformation system and VbWRKY32 transgenic lines were obtained.By Real time Quantitative PCR?qPCR?experiments,it was proved that the expression level of VbWRKY32 gene in wild type?WT?of V.bonariensis was significantly higher than that of stem and root.4.Under low temperature stress,the transgenic V.bonariensis had lower the relative electrolyte conductivity,the content of malondialdehyde?MDA?,hydrogen peroxide?H₂O₂?and superoxide anion?O₂^-?than WT.The activities of antioxidant enzymes and the content of osmotic adjustment substances were higher than WT.The qPCR technique was used to detect the expression of related cold resistance genes.Under low temperature stress,the expression levels of VbPOD,VbSOD,VbCAT and VbAPX6,VbCor413im1 and VbCor413pm2,VbAMY3,VbBAM1 and VbP5CS were significantly higher in transgenic lines compared with WT.Under normal conditions,there was no significant apparent difference between transgenic lines and WT;under cold stress,the transgenic lines showed better cold resistance,and the recovery rate was higher than that of WT.Above all,this study provided and analyzed the transcriptomic data of V.bonariensis under low temperature stress,and successfully transferred VbWRKY32 gene by using the established genetic transformation system.It was proved that VbWRKY32 was a positive regulated gene for V.bonariensis under low temperature stress,which can improve the cold resistant ability of transgenic V.bonariensis,and was an excellent breeding gene for plants to increase low temperature tolerance.