Function Identification Of CcCCR Gene,cloning Of CcCAD Gene And Construction Of VIGS Vector In Capsicum Chinense

Pepper is popular all over the world due to its pungent and strong aroma,spicy taste is a unique characteristic of pepper,and the main component of spicy taste is capsaicin.In recent years,capsaicin has been widely used in food industry,biological control,medical care and other aspects.Therefore,the demand of the world is increasing,but due to the origin environment and extraction process,the yield of capsaicin is relatively low.Varieties with high capsaicin content,such as Millet pepper,Ghost pepper,Chaotian pepper and Capsicum chinense,are important materials for making sauces or extracting capsaicin.However,there are some problems in breeding methods such as introduction and hybridization,such as natural conditions limitation and long domestication time,conventional breeding methods are time-consuming and laborious for increasing capsaicin content.Therefore,a more comprehensive understanding of the pathways of capsaicin biosynthesis and hhe function of related genes,so the regulation of capsaicin synthesis by means of genetic engineering has become a hot spot in the quality research of capsicum.The basic framework of capsaicin biosynthesis and the genes directly involved have been clarified,secondary metabolites such as capsaicin and lignin are products of the phenylpropane metabolic pathway of capsicum.Their synthetic pathways share common precursors such as Cinnamic acid,Coumaric acid,Feroyl-CoA,etc.Among them,the downstream regulatory genes of the specific pathway of lignin synthesis are mainly regulated by cinnamoyl-CoA reductase（CCR）and cinnamyl alcohol dehydrogenase（CAD）,so the expression regulation of CCR and CAD genes may affect the synthesis and accumulation of capsaicin.In this study,the CAD1 gene was cloned from Hainan Capsicum chinense,and heterologous expression vectors of CcCCR1,CcCCR2 and vectors of CcCCR1,CcCCR2,CcCAD1 VIGS were constructed.The functions of CcCCR1 and CcCCR2 in the regulation of lignin and capsaicin metabolism in Capsicum chinense were studied,and the expression characteristics of CcCAD1 in different tissues of capsicum were analyzed.The main results are as follows:1.The two genes CcCCR1 and CcCCR2 cloned from Capsicum chinense were designed with BamHI and SacI restriction sites to amplify the full length of the target gene ORF,and the over-expression vector of pBI121-CcCCRs was successfully constructed to heterogenously express CcCCR1 and CcCCR2 in Arabidopsis thaliana.The results showed that the lignin content of transgenic CcCCR1/2 plants was 13.02%and 24.46%higher than that of wild-type Arabidopsis,respectively,and the lignin content of transgenic CcCCR2plants was more significant.It was inferred that CcCCR1/2 genes might be involved in regulating the specific synthesis of lignin in yellow lantern capsicum,in which CcCCR2played a major role.2.According to the specific primer amplification target fragments of CcCCR1 and CcCCR2 gene sequences,the VIGS vector pTRV2-CcCCRs was constructed,induce culture after transferring to Agrobacterium respectively in the 5d after flowering young fruit period injection infection,infection after two weeks to candidate genes of the normal growth of fruit quantitative qPCR detection,in the fruit and stem CcCCRs expression was significantly decreased,and the CcCCR1 and CcCCR2 silent for the rest of the five candidate genes expressed in pulp and the placenta,the expression of CAD gene was down-regulated,the expression levels of PAL,4CL,pAMT and CS genes involved in the synthesis of capsaicin phenylpropane were all increased in the placenta,CS gene was reduced to no expression in pulp,and the remaining three genes were generally induced to increase.40d after flowering CcCCR1 silenced the fruit,the content of lignin in pulp and placenta decreased by 10.2%and 13.5%,and the content of capsaicin in pulp and placenta increased by 5%and 9.6%,respectively.In CcCCR2 silencing fruits,the contents of lignin in pulp and placenta decreased by 18.8%and 22.8%respectively,while the contents of capsaicin in pulp and placenta increased by 16.2%and 19.1%respectively.After CcCCR2gene silencing,the lignin content was decreased more significantly than that of CcCCR1,and the content of capsaicin was increased more significantly,so it is speculated that CcCCRs gene positively regulates the biosynthesis of lignin and CcCCR2 plays a more important role in the biosynthesis of lignin and capsaicin content,there is a clear competitive relationship between lignin and capsaicin content,which is increased by inhibiting the synthesis of lignin in pepper fruit.3.The full-length cDNA of CAD1 gene was cloned from the young leaves of Capsicum chinense by RT-PCR and named CcCAD1.The cDNA sequence of CcCAD1was 1074 bp long and encoded 357 amino acids.CcCAD1 has no transmembrane structure and is a hydrophilic protein located in cytoplasm.Domain analysis showed that CcCAD1contained two Zn²⁺binding sites and one NADP⁺coenzyme binding site characteristic of CAD,belonging to the MDR superfamily.Homologous comparison showed that its consistency with tomato CAD1 protein was as high as 96.09%.The results of real-time PCR showed that the expression of CcCAD1 gene in pepper roots,stems,leaves,placenta and pulp was different,and the magnitude of its relative expressions is,in turn leaf>pulp>stem>root>placenta.The pTRV2-CcCAD1 silencing vector was successfully constructed by BamHI and SacI double enzyme digestion,which laid a foundation for the subsequent function identification of CcCAD1 gene.This study shows that CcCCRs gene promotes lignin synthesis and CcCCR2 gene plays a major role.It is preliminarily demonstrated that reducing CcCCRs gene expression can inhibit lignin synthesis to improve capsaicin synthesis.Therefore,the whole study can provide a theoretical basis for the subsequent improvement of the quality of chilli through biotechnology and the improvement of the molecular mechanism of chilli pepper regulation.