Differential Expression Analysis Of Ginseng Gene Under Benzoic Acid Stress And Cloning And Transformation Of Response Gene WRKY7

Panax ginseng C.A.Meyer is a traditional Chinese herbal medicine which belongs to the Araliaceae family,and continuous cropping obstacle made the yield and quality of which decreased seriously,sometimes even causes crop failure.It has been confirmed that autotoxicity was one of the most important causes of continuous cropping obstacles,and allelochemicals released by Panax ginseng may lead to the change of physical and chemical properties,and the diversity of microbial community of ginseng cultivating soil.So far,there are few studies on the differential gene expression of Panax ginseng responsive to autotoxin stress.In the present research,based on annotation information,the differential expression of functional genes of Panax ginseng under benzoic acid stress was analyzed.And then,the full length sequence of a WRKY transcription factor gene,tentatively named as WRKY7,was successfully cloned,and its functional domain,steric configuration and spatial conformation,amino acid sequences,etc.,were predicted by bioinformatic analysis method.Furthermore,the full-length gene was transferred into Arabidopsis thaliana successfully,and the seeds of the third generation pure lines were harvested.The more detail results were shown as follows:(1)In total,the differential expression of 7 physiological related genes,including 2 reactive oxygen species(ROS)-related genes,2 proline-related genes,1 malondialdehyde(MDA)-related gene and 2 peroxidase(POD)-related genes,under benzoic acid stress was confirmed by real-time quantitative PCR.Results showed that,except for 1 proline-related gene,the other genes were up-regulated.(2)A total of 13 differentially expressed transcription factor genes,including 8 WRKY genes,4 MYB genes and 1 AP2/ERF gene,under benzoic acid stress were researched.Among of them,except for 1 MYB gene was down-regulated,the other transcription factor genes were up-regulated.(3)According to the transcriptome sequencing data of ginseng under benzoic acid stress,a WRKY transcription factor gene,temporarily named as WRKY7,was cloned by reverse transcription-PCR.Based on the bioinformatic analysis results,the length of the cDNA was 1216 bp.The open reading frame(ORF)of which was 1014 bp,encoded 337 amino acids,containing 1 WRKY domain,and the zinc finger domain was C2HC.Which indicated that the protein product of WRKY7 belongs to the third WRKY transcription factor family.Alignment results showed that WRKY7 has the highest homology(87%)to WRKY6(JF927162.1)and WRKY9(JF927165.1)in Panax quinquefolius.(4)Over-expression vector pCAMBIA2300-WRKY7 was constructed and transformed into Agrobacterium GV3101.Positive clone was identified by colony-PCR and transformed into Arabidopsis thaliana through floral-dip method and screened by Kan-MS culture.After three generations of cultivation,the transgenic Arabidopsis pure lines were obtained,which laid a solid foundation for further functional characterization.