Changes Of Persimmon Fruit Quality And Functional Analysis Of Carotenoid Synthesis Genes DkPSY And DkLCYb

As a common pigment in plants,carotenoids have many important functions in plants and human body.Persimmon is one of the most popular fruits in the fruit market in China,which is rich in carotenoids.However,there are few studies on the gene function analysis of carotenoid synthesis pathway in persimmon.In this experiment,the ‘Heishi' and ‘Huoguan' fruits of different growth and postharvest storage periods were used as research materials to analyze the changes of fruit hardness,soluble solid content,tannin content and pulp color during the growth,development,ripening and softening of persimmon fruits.The changes of carotenoid content in the pulp were analyzed by HPLC,and the changes of expression of carotenoid synthesis related genes were analyzed by q RT-PCR.DkPSY and DkLCYb genes were cloned using ‘Heishi' flesh c DNA 165 days after flowering as template.The function of DkPSY was verified by transient transformation of tomato.The function of DkLCYb was verified by prokaryotic expression and transient transformation of persimmon fruit.The main results are as follows:1.Changes in the quality of persimmon fruit during growth,development,ripening and softening.The hardness of the fruits decreased continuously,especially in the middle and later stages of postharvest storage.During the growth and development of fruit,the content of soluble solids in the pulp first decreased and then increased.During the postharvest ripening and softening periods,the content of soluble solids in ‘Heishi' continued to decrease,while that in ‘Huoguan' decreased and then slightly increased.During the period of fruit growth and ripening,the content of soluble tannin in the flesh decreased gradually,the content of insoluble tannin decreased in the growing period,and increased continuously in the period of postharvest ripening and softening.During the last period of fruit ripening and softening,the content of soluble tannin in the flesh decreased to below 0.5%,and the content of insoluble tannin increased sharply,and the fruit reached the edible standard.During the growth and development of ‘Heishi' and ‘Huoguan' fruits,the flesh turns from green to yellow,and the color richness increases.During the post harvest ripening and softening periods,the flesh turns to orange gradually.The flesh of ‘Heishi' is the most colorful on the 20 th day of postharvest storage,and that of ‘Huoguan' is on the 0 day of postharvest storage.At the end of fruit ripening and softening,the flesh of ‘Heishi' is orange yellow,and the flesh of ‘Huoguan' is orange red.2.Changes in the content of carotenoids during the development,ripening and softening of persimmon fruits.The characteristics of carotenoids accumulation in persimmon were extracted by non saponification method and analyzed.The results showed that the carotenoids in the flesh of ‘Heishi' and ‘Huoguan' gradually accumulated with the development of persimmon fruit and the occurrence of ripening and softening process.The main carotenoids in persimmon flesh are lycopene and ?-carotene.The main carotenoids content in ‘Heishi' is ?-carotene,and that in ‘Huoguan' is lycopene.3.Gene expression analysis of carotenoid synthesis during persimmon fruit growth,development and ripening.q RT-PCR analysis showed that the expression level of carotenoid synthesis related genes DkPSY,DkPDS,DkZDS,DkLCYb,DkLCYe and DkBCH in persimmon fruits increased continuously in the early stages of growth,development and ripening and softening,and decreased significantly in the late stages of ripening and softening.The content of carotenoids in persimmon fruit is greatly affected by DkPSY and DkLCYb.Lycopene in persimmon fruits are mainly transformed through ?,?-branch.4.Functional analysis of DkPSY and DkLCYb genes of persimmon fruit.The total length of the cloned c DNA of DkPSY gene is 1308 bp,encoding 435 amino acids.The transformation of DkPSY in tomato proved that it can effectively regulate the synthesis and accumulation of carotenoids.Overexpression of DkPSY can increase the content of lycopene,zeaxanthin and ?-carotene in tomato,and silencing DkPSY gene can reduce the content of lycopene,?-carotene,zeaxanthin and ?-carotene in tomato.The c DNA sequence of DkLCYb gene was 1515 bp,encoding 503 amino acids.The transformation of DkLCYb in E.coli and persimmon fruits showed that it could promote the transformation of lycopene to ?-carotene.The expression of DkLCYb in engineering bacteria can reduce the content of lycopene in engineering bacteria and generate ?-carotene.The overexpression of DkLCYb can reduce the content of lycopene in persimmon fruit and increase the content of ?-carotene.