Study On The Mechanism Of Anthocyanin And Apigenin Metabolization In Celery

Celery(Apium graveolens L.),a biennial plant of Apiaceae,is one of the most important leaf vegetables worldwide.Celery leaves and petioles are the main edible parts.Nowadays,there are five basic types of celery:white,green,red,yellow and purple celery based on their petiole color.Rich anthocyanin agricultural products have good food value and economic value.Thus,it is significant to study and cultivate purple celery.In this thesis,the purple celery(named 'Nanxuan liuhe purple celery')was obtained from 'Liuhe yellow celery',local cultivar celery of Nanjing city,China.The purple celery was purified and the law of inheritance was studied by artificial selfing and method of hybridization,respectively.The genes involved in anthocyanin and apigenin biosynthesis was assembed,cloned and identificatied in purple celery.Furthermore,the function of Ag3GT and AgFNS genes were studied by transgene.The research results are as follows:1.The purple celery obtained from 'Liuhe yellow celery',which is widely cultivated in Nanjing city.The celery purified by selfing,and named 'Nanxuan liuhe purple celery'.The F1 and F2 hybrid seeds obtained from hybrization of purple celery and non-purple celery('Liuhe yellow celery' and 'Jinnan shiqin').The results showed that the purple trait of celery is a dominant trait and control by a single gene.The main components of anthocyanins in the petiole of 'Nanxuan liuhe purple celery' identified to be cyanidin-based anthocyanin by U-V spectrophotometer and HPLC.2.'Liuhe yellow celery' and 'Baiqin' were used as the materials of celery tissue culture.The results showed that the rate of bacterial contamination from the celery seeds disinfected was only 0.1%by 20%sodium hypochlorite for 30 min.The time required for celery seed germination decreased after disinfection by sodium hypochlorite.Compared 1/2MS,MS,1/2 B5(Gamborg's B5),and B5 medium,the most appropriate medium for celery seedling growth was MS medium without any hormone.Hypocotyls were used as explants of celery tissue culture,the appropriate medium for callus induction and callus conservation was B5 medium supplemented with 1.5 mg·L-1 IAA and 1.0 mg·L-1 6-BA,the maximum callus inductivity was 88.2%in this medium.Each celery callus produces 3-7 well-differentiated buds,the differentiation rate reach to 93.5%on callus induction medium under 50?mol×(m2s)-1 of light intensity.No-vitrificated celery tissue culture seedling with a number of rooting obtained under 300 ?mol×(m2s)-1 of light intensity.It is needed 45-60 d to obtain tissue culture seedling of celery.3.The anthocyanin and apigenin biosynthesis related genes were identified from the transcriptome database of celery,and clone from purple clery 'Nanxuan liuhe purple celery'.The results showed that AgPAL,AgC4H,AgCHS,AgCHI,AgFNS,AgF3H,AgF3'H,AgDFR,AgANS,and Ag3GT genes were successfully clone and highly similarity with others species of Apiaceae family.The transcript levels of the AgPAL,AgC4H,AgCHS,AgCHI,AgF3H,AgF3'H,AgDFR,AgANS,and Ag3GT were higher in the petioles of'Nanxuan liuhe purple celery' than in the petioles of 'Liuhe yellow celery'.However,AgFNS gene showed higher expression in the petioles of 'Liuhe yellow celery' than in the petioles of 'Nanxuan liuhe purple'.4.The Ag3GT gene was clone from 'Nanxuan liuhe purple celery' and transformed into the plant 'Liuhe yellow celery' by transgenic method.The results showed that overexpressing Ag3GT gene high anthocyanin accumulated in the petioles of transgenic celery.Accordingly,the transcript levels of AgPAL,AgC4H,AgCHS,AgCHI,AgF3H,AgF3 'H,AgDFR,AgANS and Ag3GT genes were upregulated in the petioles of transgenic plants.However,apigenin content and the expression levels of AgFNS gene were higher in the petioles of transgenic celerys than in the petioles of the control celery plants.5.The AgFNS gene was clone from 'Nanxuan liuhe purple celery' and transformed into 'Nanxuan liuhe purple celery'.The results showed that the anthocyanin content in the petioles of transgenic AgFNS gene celery was lower than that in the wild-type celery plants,and apigenin content increased in the petioles of transgenic celery.The transcript levels of the AgPAL,AgC4H,AgCHS,and AgCHI genes were upregulated,whereas those of the AgF3H,AgF3 'H,AgDFR,AgANS,and Ag3GT genes were downregulated in the petioles of transgenic plants compared with wild-type celery plants.