Identification Of Grape Cation Peroxidase Gene Family And Its Involvement In Scirpusin A Synthesis

Grape was deeply loved by consumers,because of its high water content,sweet and sour taste,high nutritional value and medicinal value.However,stilbenes which were common plant polyphenols,which had biological activity,were the secondary metabolites of grape,which were closely related to the quality and disease resistance of grape in the process of growth and development.In addition,they also had biological activities such as antioxidant,antibacterial and anti-tumor.Scirpusin A was a stilbene compound with less research but high attention.As a quality function factor of grape,its pharmacological activity has attracted people's attention.However,the pathway of biosynthesis was still unclear.In our laboratory,it was found that the precursor of Scirpusin A was ?-viniferins.Therefore,based on the previous research,the project intends to establish a linear marker system from precursor to product synthesis with"postharvest grapes" as the material.The key genes involved in Scirpusin A synthesis were excavated and verified by yeast protein expression.Grape cationic peroxidase(VvCP)is an important catalytic enzyme that plays an important role in plant defense mechanism.Meanwhile,through transcriptome sequencing analysis,this gene may be involved in biosynthesis of Scirpusin A.Therefore,by studying the VvCP gene family and verifying its biological functions,the regulatory mechanism of grape fruit Scirpusin A synthesis was elucidated.The research results of this project will help to clarify the specific pathway of synthesis of polyphenols in postharvest fruits.The results of this study are mainly composed of the following two parts:(1)In this study,the members of CP gene family in the genomes of six plant species were identified using bioinformatics methods and the gene structures,the Cis-elements on their promoters,the physiochemical characteristics of the encoded proteins and the phylogenetic trees were also studied.Tissue expression of VvCP genes in wine grapes and its expression pattern under UV-B stress were analyzed by RT-qPCR.9,9,7,4,18 and 11 proteins were obtained from Cabernet Sauvignon,Cicer arietinum,Jatropha curcas,Sesamum indicum,Quercus suber and Arachis duranensis,respectively.The phylogenetic tree showed that the family was clustered into 4 subfamilies,with 22 genes in the first subfamily and 11,4 and 21 genes in the second,third and fourth subfamilies respectively.The introns of the VvCP gene are distributed between 2 and 4.Phylogenetic analysis showed that the VvCP gene on the same chromosome had a distinct evolutionary relationship.The results showed that 9 VvCP genes were distributed on 4 chromosomes.Cis-acting elements analysis showed that there were many different response elements in these gene promoters and the a light responsive element was common to the VvCP gene.RT-qPCR analysis of the expression profiles revealed that they were significantly different in expression in different tissues.At the same time,the expression of VvCPs was different after UV-B treatment at different time.The plant expression vector pCAMBIA2300-GFP-VvCP11 of green fluorescent protein of VvCP 11 gene was constructed for subcellular analysis and localization,and the plant expression vector was introduced into onion epidermal cells by Agrobacterium mediated method.The recombinant plant expression vector was expressed in onion epidermis.After dark culture for 24?28 h,the subcellular localization of VvCP11 was observed by laser confocal microscopy.The results showed that VvCP11 protein was located in the cell membrane of onion epidermal cells,and there was green fluorescence in the whole cell membrane.(2)By means of molecular biology,it was verified whether VvCP11 gene of Vitis vinifera grape had the function of catalyzing ?-viniferins reaction to produce Scirpusin A.Using grape as material,RNA was extracted and reverse transcribed into cDNA,VvCP11 gene was cloned homologously,and the eukaryotic expression vector pPic9KVvCPll was constructed.The expression vector was transferred into Pichia pastoris GS115 by electric shock transformation method for in vitro verification.The recombinant yeast containing the target gene VvCP11 was induced and expressed in medium containling methanol.After 48 hours of culture,the secretory protein was extracted from the culture medium,and then SDS-PAGE and Western Blot were used to verify the protein,which was about 33.8 kDa.After that,the substrate was fed to the BMGY medium containing recombinant yeast,Scirpusin A was detected by UHPLC-QTOF-MS2,which proved that VvCP11 had catalytic function to produce Scirpusin A in vitro.