The Function Research Of Three Kinds Of Rice Anthocyanin Biosynthesis Regulated Trans-factors

Plants are flexible chemical factory that can produce tens of thousands of chemicals.Some of these chemicals are known as secondary metabolites.Anthocyanins and beta-carotene are important secondary metabolites in plants,which play an important role in human nutrition and health.Anthocyanin is a kind of water-soluble flavonoid pigments and it is a strong scavenger of free radicals.Beta-carotene is one of the most important carotenoids as the provitamin A.Beta-carotene can be converted into vitamin A in the human body and then help body eliminate or reduce the symptoms caused by vitamin A deficiency.Rice is the staple food for more than half the world's population.At present,the main rice cultivars are white rice that lacks anthocyanin.Rice endosperm is also lacks of provitamin A.People who take rice as the staple diet and are not supplied by enough additional foods,would face serious health problems.Therefore,breeding high quality rice with high content of anthocyanin and beta-carotene is important.Three rice endogenous transcription factors OsCl,OsB2 and OsPAC1,which may regulate the biological sysnthesis of anthocyanidin,were isolated from the black rice variety Heishuai(HS),and were overexpressed in white rice varieties Chao2-10 and ZH11 in this study.The expressional levels of structural genes in the anthocyanin biosynthetic pathway were detected in OsC1,OsB2 and OsPAC1 overexpressing rice,which lays the foundation for the cultivation of rice varieties with abundant anthocyanins in the endosperm.Secondly,we hope to develop rice containing both anthocyanins and beta-carotene by hybridizating GRH(Golden Rice of Huazhong Agricultural University)containing beta-carotene with HS containing anthocyanins.The main results obtained in this study are listed as follows:1.Three rice endogenous transcription factors OsC1,OsB2,and OsPAC1,which may regulate the biological sysnthesis of anthocyanidin,were isolated from black rice HS.The overexpression vectors for each of these three genes were constructed respectively.Two dual-gene overexpression vectors OsC1 + OsB2 and OsC1 +OsPACl were also constructed successfully.2.The overexpression vectors of three single genes were transformed into japonica rice cultivars Chao2-10 and ZH11 respectively.Comfirmed by using PCR screening and Southern blotting analysis,OsC1-overexpressed Chao2-10(referred to as CC)and ZH11(referred to as CZ),3 single-copy families and 18 single-copy families were obtained;for OsC1-overexpressed Chao2-10(referred to as PC)and ZH11(referred to as PZ),11 single-copy families and 9 single-copy families were obtained;for OsB2-overexpressed Chao2-10(referred to as BC)and ZH11(referred to as BZ),both of them,28 strains of To generation plant were obtained.3.Expressional analysis of structural genes involved in the anthocyanin biosynthetic pathway demonstrated that all of the detected genes were up-regulated in CC or CZ except for OsDFR.OsCHS,OsCH1,OsANS,OsUFGT were up-regulated in PC,but the expressional levels of OsF3H and OsF3 1H kept unchanged;OsCHS,OsANS,OsUFGT were up-regulated in PZ,but the expressional levels of OsCH1,OsF3H and OsF3'H kept unchanged.OsCHS,OsCHI,OsF3'H,OsANS,OsUFGT were up-regulated in BC or BZ,but the expressional levels of OsF3H kept unchanged.4.Phenotypic observation showed that anthocyanin accumulation was found in resistant calli,leaves,leaf sheath,apiculus of CC.High Performance Liquid Chromatography(HPLC)analysis showed that two kinds of anthocyanin cyanidin-3-O-glucoside and peonidin-3-O-glucoside were accumualted in CC.In other transgenic rice,such as CZ,PC,PZ,BC and BZ,none anthocyanin accumulation was observed.5.OsCl and OsB2 were co-transformed into Chao2-10 and ZH11.Phenotypic observation showed that anthocyanin accumulation was observed in the resistant calli of Chao2-10 overexpressing OsC1 and OsB2,but there are no anthocyanin accumulation was observed in ZH11 overexpressing OsCl and OsB2.6.Rice GRHA(GRH with Anthocyanin)containing anthocyanins and beta-carotene were acquired by hybridizating GRH containing beta-carotene with HS containing anthocyanins.HPLC analysis showed that both cyanidin-3-O-glucoside and peonidin-3-O-glucoside were accumualted in HS and GRHA.The content of cyanidin-3-O-glucoside and peonidin-3-O-glucoside in GRHA were 130.12 ?g/g and 0.42 ?g/g respectively,and were 73.69 ?g/g and 0.62 ?g/g respectively in HS.The anthocyanin content of GRHA was higher than HS about 75.67%,and the content of beta-carotene in GRHA(13.31 ?g/g)was also higher than GRH(10.22 ?g/g)about 30.23%.