Preliminary Research Of Anthocyanidins In Lonicera Japonica Var. Chinensis And Its Formation Mechanism

Honeysuckle is one of the most commonly used herbal medicines in clinical, and derived frombuds or open flowers of Lonicera japonica Thunb. Lonicera japonica var. chinensis is a variantion ofLonicera japonica Thunb that generated by natural mutagenesis. Compared with the phenotype of L.japonica, L. japonica var. chinensis have purple young branches, young leaves and Corolla outside withpurple, inside with white. L. japonica var. chinensis have stronger flavor and stronger resistance than L.japonica. In the research, through the transcriptome sequencing data of L. japonica and L. japonica var.chinensis, we find that gene LRCrtH of L. japonica var. chinensis in flower buds has several SNPmutations, and predict it may be the main cause of L. japonica var. chinensis is red. In order to depthlystudy the coloring process and mechanism of L. japonica var. chinensis, using bioinformatics analysis,gene cloning and expression, chemical composition analysis and other methods, we initiatory explore tothe coloring mechanism of L. japonica var. chinensis and the type of anthocyanin.Research results areas follow:(1) By sequence analysis and prediction of protein structure and function, we got the key enzymegenes of carotenoids dehydrogenation--LYCrtH, LYPDS, LYZDS and LYZ-ISO from L. japonicatranscriptome sequencing data. We cloned these cDNA sequences from flower buds of L. japonica var.chinensis and L. japonica. The results show that, compared to L. japonica, the gene LRCrtH of L.japonica var. chinensis has two non-synonymous mutations in amino acid sites, and other genes haveno mutation. By constructing a prokaryotic expression vector, we proved LRCrtH gene normal express.(2) By HPLC-MS anaylsing the component of cyanidin, total flavonoids in petal of L. japonica var.chinensis and using the quantitative and semi-quantitative method to anaylse the content of cyanidin,total flavonoids and carotenoids, the results show that cyanidin is the main reason for flower buds ispurple.(3) Throught using bioinformatics analysis, we gained the relevant gene sequences--LDOX, DFR,BZ1and GT1)from cyanidin biosynthetic pathway. To analyse transcripted level of these genes in L.japonica var. chinensis and L. japonica by Real-time PCR analysis method, we found that transcriptedlevel of these genes in L. japonica var. chinensis is higher than L. japonica.(4) Taking bisulfite method and site-specific DNA methylation detection technology, we analysed thepromoter CpG island methylation sites of same gene family Phosphoethanolamine N-methyltransferase(PNMT) in L. japonica var. chinensis and L. japonica. The results show the gene promoter sequences ofLYPNMT has a small amount of CpG island methylation phenomenon, but LRPNMT have not.