Genetic Diversity And Gene Expression Of Flower Pattern And Color In Camellia Japonica L.

Camellia japonica L.is one of the ten famous traditional flowers in China,commonly used in indoor landscape and garden landscaping.At the same time,C.japonica polyphenols and flavonoids have pharmacological functions,and is a plant with high ornamental and economic value.There are a large number of C.japonica species which are widely introduced and cultivated all over the world,but there are taxonomic differences in C.japonica species,and coupled with the long-term introduction and cultivation,there are a large number of species with the same name or different names.To address this phenomenon,this study analyzed the genetic diversity of 53 C.japonica species and combined with phenotypic trait clustering analysis to provide a basis for the classification of C.japonica species.Flower type and flower color are two important ornamental traits of C.japonica.Flower type is mainly regulated by the ABCDE type genes in flower organ development,and the regulatory mechanism of ABC type genes in C.japonica has been relatively clear,but the function of E-type genes in it is relatively shallowly studied.In this study,we carried out cloning and expression analysis of E-type genes,and also genetic transformation of E-type genes to provide technical support for the transformation of flower type by genetic engineering.In this study,we analyzed the expression of key genes for anthocyanin synthesis in C.japonica,and also studied the content of secondary metabolites such as anthocyanins in different varieties and healing tissues,in order to provide a theoretical basis for the innovative use of C.japonica germplasm resources.The main findings of the study are as follows:1.Genetic diversity analysis of 53 C.japonica species.The genetic diversity and population structure of 53 C.japonica species(lines)were analyzed using phenotypic traits combined with CDDP molecular marker technology.The results showed that two traits,petal number and inner secondary color shape,had higher coefficients of variation than other traits,48.71% and 65.05%,respectively;the polymorphism of 28 primers in CDDP molecular marker results was98.68%,PIC was 0.905,and effective alleles,Nei’s genetic similarity coefficient and Shanno information index were 1.380,0.245 and 0.386,respectively.Based on the population structure analysis,the 53 C.japonica species(lines)were divided into four taxa,and the phenotypic trait observations were correlated with the CDDP marker results.65 loci out of 529 loci were significantly associated with 11 phenotypes(p<0.01),and both flower diameter and flower type were associated with 11 loci.Both traits were associated with 11 loci.2.Cloning and expression analysis of C.japonica E-type gene.Using RACE cloning technique,three E-type genes,Cj SEP1a(1235 bp),Cj SEP3(967 bp)and Cj SEP4a(1725 bp),were isolated from the fully retrorse variety’Cochineal peony’ with NCBI accession numbers OP172861,OP172863 and OP172864,respectively.,OP172863,OP172864.All three genes do not have transmembrane structural domains,have a low probability of being signal peptides,are hydrophilic nuclear proteins,and have K-boxes,which are MADS-box specific protein structural domains,with an α-helical protein structural backbone.After homology analysis,the Cj SEP1 a,Cj SEP3 and Cj SEP4 a genes were more homologous with the E-type genes of the homologous species C.japonica,with a clustering value of 100.q PCR analysis showed that the Cj SEP1 a gene was more expressed in the semi-and fully heavy petal varieties than in ’Beauty Tea Cj SEP3 gene was not much different from Cj SEP1 a gene,but the overall expression was higher than Cj SEP1 a in six different types of C.japonica varieties;Cj SEP4 a gene was more highly expressed in complete heavy petal and peony type heavy petal than single petal type,especially in heavy petal varieties almost The expression of the gene was higher in the fully heavy petal and peony heavy petal than in the monopetalous type,especially in the heavy petal variety,which was almost three times that of the monopetalous type,but lower in the rest of the varieties,and extremely low in the torqueflower type.3.Construction of plant expression vector of C.japonica E-type gene and genetic transformation of healing tissue.The p BI121-SEP-GFP plant expression vector was constructed with three E-type genes as target genes.The single-petaled variety ’Beauty Tea’ was used as the material to induce healing tissue culture and to obtain healing tissues in good condition.The genetic transformation system of C.japonica healing tissue was established,and the Etype genes were successfully and stably genetically transformed into ’Beauty Tea’healing tissues.Ten positive transgenic materials were obtained after PCR verification.The transgenic positive materials p BI121-Cj SEP1a-GFP and p BI121-Cj SEP4a-GFP were used as templates for real-time fluorescence quantification reaction,and the Cj SEP1 a gene in p BI121-Cj SEP1a-GFP and Cj SEP4 a in p BI121-Cj SEP4a-GFP were found to be overexpressed,while Cj SEP4 a in Cj SEP1a-GFP and Cj SEP1 a expression in p BI121-Cj SEP4a-GFP were extremely low compared with the CK group,while the Cj SEP3 gene was repressed expression in both materials,a phenomenon indicating a synergistic expression situation between the two E-type genes.4.Analysis of expression patterns of key genes of the anthocyanin synthesis pathway in healing tissues of C.japonica petals and ’Beauty Tea’ of different colors.The expression of the CHS gene was higher in the yellow variety ’ Ki-no-senritsu’,while the expression of both F3’H and DFR genes were not as high as in the white control variety ’Masterpiece’ on the pigment biosynthesis pathway of different colors of C.japonica.In the healing tissues,except for CHI and UFGT genes,the expression of the remaining five genes(CHS,F3 H,F3’H,DFR,and ANS)was higher in the healing tissue at 30 d of culture than in the healing tissue at 35 d.This indicates that the expression of genes related to anthocyanin synthesis in C.japonica differs depending on the variety,and also that the expression of genes related to anthocyanin synthesis in the healing tissues of C.japonica at different culture stages provides clues for further research on the targeted synthesis of anthocyanin in C.japonica.5.Determination of secondary metabolite content of petals and healing tissues.This study showed that the contents of total phenols,flavonoids,proanthocyanidins and anthocyanins in C.japonica petals were higher than those in the healing tissues,with the highest total phenol content of 143.53 mg/g in the petals of the red variety ’Chian’ and the highest of 50.353 mg/g in the dried samples of the healing tissues at 40 d;the highest flavonoids,proanthocyanidins and anthocyanidins in the purple-red variety The correlation analysis between the expression of key genes in the pigment synthesis pathway and the content of each secondary metabolite showed that total phenols were highly and significantly correlated with F3H(P<0.05).(P<0.01),flavonoids and anthocyanins were significantly positively correlated with F3 H gene(P<0.05),ANS and UFGT genes(P<0.01),anthocyanins were highly significantly negatively correlated with CHI and UFGT genes(P<0.01),and F3’H gene(P<0.05).