Genome-based Identification And Expression Of AsA Metabolic Genes In Rosa Roxburghii Tratt

The fruit of Rosa roxburghii Tratt.,also known as Chinese rose,is renowned for its high content of vitamin C（L-ascorbic acid,AsA）and is referred to as the‘king of vitamin C’.Although our research group has previously identified the main pathway and some regulatory factors involved in AsA synthesis in R.roxburghii,the AsA metabolic pathway and gene family have not been systematically identified at the genomic level.In this study,we used the main cultivar‘Guinong 5’as material and combined R.roxburghii genome data completed by our research group with online databases such as NCBI to comprehensively identify members of the AsA metabolic pathway gene family and their bioinformatics characteristics.Based on this,we analyzed their expression characteristics through qRT-PCR analysis and screened key candidate genes related to AsA accumulation for functional verification.The main results obtained are as follows:1.Identification of AsA metabolism-related genes based on the R.roxburghii genome.Based on the AsA metabolic pathway in higher plants,73 candidate genes were identified from the R.roxburghii genome,covering all reported genes and families in the AsA metabolic pathway,including 35 genes from11 gene families（RrPMIs,RrPMMs,RrGMPs,RrGMEs,RrGGPs,RrGPPs,RrGDHs,RrGLDHs,RrGal URs,RrGLOases and RrMIOXs）in four AsA biosynthesis pathways;27 genes from two gene families（RrAOs and RrAPXs）in oxidation pathways;and 11 genes from two gene families（RrMDHARsand RrDHAR）in regeneration pathways.The specific gene family names were assigned sequentially according to their location order on the R.roxburghii genome chromosome.2.Spatial and temporal expression characteristics of AsA metabolic pathway-related genes and their correlation with AsA accumulation in R.roxburghii.There are significant differences in the content of AsA in R.roxburghii leaves,flowers,stems,and fruits.The content of AsA in fruits increases significantly with development time and is significantly higher than that in other organs.qRT-PCR analysis showed that most genes have spatial-temporal specificity in expression.Among the 46 genes involved in the synthesis pathway and regeneration pathway of AsA,the expression of 15 genes was basically consistent with the accumulation of AsA and showed a significant positive correlation,namely RrPMI1,RrPMI4,RrGMP1,RrGMP2,RrGME1,RrGGP2,RrGPP2,RrGDH1,RrGDH2,RrGLDH,RrGal UR4,RrGal UR4,RrMDHAR4,RrMDHAR8,and RrDHAR.Combining their transcription levels during AsA accumulation process and their correlation coefficients with AsA content preliminarily determined that RrGME1,RrGGP2,RrGDH2,and RrMDHAR8 are key candidate genes for regulating high accumulation of fruit ascorbic acid（AsA）in R.roxburghii.3.The effects of key candidate gene silencing and overexpression on AsA accumulation.Since our research group has previously conducted transient expression verification of RrGGP2 in R.roxburghii fruit,this study only verified the functions of RrGME1,RrGDH2,and RrMDHAR8 in regulating AsA accumulation in jujube fruit through transient overexpression and VIGS silencing.The results showed that the expression levels of corresponding genes were upregulated by 1.9-2.7 times in overexpressed fruits compared to controls,and the content of AsA increased by 15.5-37.6%.In silenced fruits,the expression levels of genes decreased by 48.2-64.2%,and the content of AsA decreased by 24.8-45.1%.In addition,stable overexpression of RrGGP2 in tomato plants significantly promoted AsA synthesis,with an increase in AsA content by 1.78-2.27 times in transgenic plant leaves and 3.14-4.11 times in fruits respectively.These results once again confirm the positive role played by these four key structural genes in promoting AsA accumulation,with RrGGP2 and RrGDH2 having the most significant effect.4.Basic bioinformatics analysis of the gene families where the key candidate genes are located and their expression response to exogenous hormones.The four key candidate genes,RrGMEs（2members）,RrGGPs（3 members）,RrGDHs（4 members）,and RrMDHARs（10 members）belong to NADB＿Rossmann superfamily,DUF4922 superfamily,AKR＿SF superfamily,and Pyr-redox-2superfamily respectively.Analysis of gene promoter elements found that among the 19 members in these4 gene families,except for RrMDHAR1,all other members have a certain number of hormone response elements with more ABA response elements,GA₃ response elements and MeJA response elements.Correlation analysis between AsA content and gene expression under exogenous hormone feeding treatment on detached fruits showed that all genes have different degrees of expression responses to different exogenous hormones.Among them,ABA can mainly promote AsA synthesis by up-regulating the expression of RrGME1 while MeJA mainly has an opposite effect on AsA accumulation by down-regulating the expression of RrGME2;GA3’s regulatory role is not obvious.