Expression And Functional Analysis Of Of Pomegranate Seed Lignin Biosynthesis Key Genes

Pomegranate?Punica granatum L.?is one of the most important fruit trees in China,and it has a wide economic value.Pomegranate fruit has high medical and edible value.Its grain hardness directly affects the taste and edible rate of fruit,so grain hardness is one of the most important components of fruit quality.In existing pomegranate germplasm resources,some soft seed pomegranate cultivars are popular among the market because of their low lignin content,soft kernel,and no need to spit seeds.In order to study the influence factors of lignin content in soft seed pomegranate and hard seed pomegranate development process,and analyze the function of related genes.In this paper,the key genes of pomegranate lignin biosynthesis pathway?PgHCT,PgF5H,PgC3H?were cloned in the development process of hard seed variety‘Hongyu'and soft seed variety‘Tunisian soft seed',the expression pattern of key genes at these different developmental stages was also analyzed.At the same time,the PgHCT overexpression vector was constructed and infected with Arabidopsis to obtain the transgenic Arabidopsis thaliana plant with PgHCT gene.At the same time,lignin content and gene expression characteristics in different tissues of T₂ generation plants were analyzed.The specific results are as follows:1.Obtain the complete cDNA sequence of PgHCT,PgF5H and PgC3H genes of lignin biosynthesis.Biological information showed that the amino acid sequence of PgHCT?GenBank:MH104887?was 80%,81%,and 82%respectively high consistency with the LlHCT of Leucaena glauca,CaHCT of Coffea atraica and HcHCT of Hibiscus cannabinus.The amino acid sequence of PgF5H?GenBank:MH104888?was 77%,78%and 78%respectively high consistency with the EgF5H of Eucalyptus globulus,MsF5H of Medicago sativa,and AkF5H of Acacia koa.The amino acid sequence of PgC3H?GenBank:MH104886?was 88%,89%and 91%,respectively high consistency with the HcC3H of Hibiscus cannabinus,PtC3H3 of Populus tomentosa and EgC3H of E.globulus respectively.2.Fluorescence quantitative analysis of the expression of lignin biosynthesis pathway in‘Hongyu'pomegranate seed coat at different growth stages.The expression of PgHCT and PgC3H showed a tendency to go down and then go down.The expression of PgF5H showed a wave trend with the growth of pomegranate seed coat.Compared with‘Tunisian soft seed',the expression tendency of PgHCT,PgF5H and PgC3H was the same,but the expression of gene was significantly different:the expression of PgHCT genes in the pomegranate of‘Tunisia soft seed'was higher than that of‘Hongyu'before the mid-term development,but no significant difference in late stage;The expression of PgC3H was higher than that of‘Hongyu'before the mid-term development,lower than that of‘Hongyu'in late stage;The expression of PgF5H gene was lower than that of‘Hongyu'in early stage,lower than that of‘Hongyu'in middle stage,significantly lower than that of‘Hongyu'in late stage.Among the three genes,only PgHCT showed the most consistent expression tendency in two pomegranate seed pellicle.and3.Lignin content of Arabidopsis thaliana strains was determined by T₂ transgenic PgHCT gene.The content of lignin in A.thaliana was affected by the transfer of PgHCT gene.Compared with the wild type,the content of lignin in the pod of the transgenic plants was different from that of the wild type plants,and the differences in other tissues were not obvious.4.PgHCT gene expression in transgenic expression in different tissues were detected. The results showed that PgHCT gene was expressed in all tissues and significant difference in the root,the highest expression level in leaves,the lowest expression level was positively correlated with lignin content in tissue.