Cloning And Functional Analysis Of The Key Enzyme Genes Involved In The Flavonoids Biosynthesis In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge(Danshen),which has purple flowers,is a traditional medicinal model plant in the Labiatae family.It has a white-flowered variant,named Salvia miltiorrhiza Bge f.alba.Flavonoids,which are the chemical basis for determining the color of plant tissues,have strongly antioxidant effects,delay aging,treat and prevent various disease,and protect plants defensing biotic and abiotic stresses,and so on.However,the genes involved in flavonoids biosynthesis in danshen plants have been identificated and analyzed,very little information is available to illuminate their functional roles.Therefore,in this study,basing on the comparative analysis of the spatial and temporal expression profiles of the genes involved in flavonoids biosynthesis in danshen and white-flowered danshen,we researched and analyzed the molecular mechanisms of the flavonoids genes in color phenotype difference of the two kinds of danshen,and discussed the crosstalk of metabolic flow between flavonoids and non-flavonoids pathways.The main details are as follows:1.On the basis of the homology cloning and transcriptome database,5 key genes involved in flavonoids biosynthesis were cloned from the petals in S.miltiorrhiza,were named SmCHI,SmANS,SmF3 H,SmF3’H,and SmUF3 GT,respectively.Bioinformatics analysis showed that these genes kept highly conservative in the evolution,and owned highly homology with the Labiatae plants.2.qRT-PCR was performed to analyze the emporal and spatial expression patterns of the genes(SmCHI、SmANS、SmF3H、SmF3’H、SmUF3GT、SmCHS、SmFNSⅡ、SmDFR、SmF3’5’H、SmDFR、SmWD1 和 SmWD2)involved in the flavonoid biosynthesis in different tissues and flowers of different flowering stages in S.miltiorrhiza and S.miltiorrhiza Bge f.alba.The results showed that they expressed differently in the two kinds of danshen.SmANS was expressed at the higher levels in the purple tissues such as purple petals,calyxes and pistils,but showed almost no expression in stamens of danshen.SmANS barely unexpressed in the white petals,green calyxes and calyxes and pistils,but expressed at the highest levels in the stamens(with yellow and purple blue pollen)of white-flowered danshen.And these results indicated that SmANS could be the most crucial factor for the formation the color phenotypic difference of the two kinds of danshen.In addition,the three gene(SmANS,SmF3’5’H,and SmFNS Ⅱ)showed highly expression levels in the stamens of white-flowered danshen,whereas had extremely low expression levels in the stamens of purple-flowered danshen,suggested the coordinate expression of the three genes mainly controlled the formation of yellow-purple blue pollen of white floweres.SmF3’H expressed highly in the acrial part including leaves,stems,calyxes and petals both in danshen and white-flowered danshen,that may be relative to the abundant flavonoids in these tissues.Besides,SmF3’H also expressed highly in the stamens of white-flowered danshen,but showed the low expression levels in the pistils and stamens of danshen,which showed that this gene also participated in the pollen development.The superposition of expression effects of all these genes together with the other flavonoids genes determines the coloration of various tissues of the two kinds of danshen.3.In the purple flower development process of Danshen,the expression levels of SmCHS,SmCHI,SmF3 H,SmF3’H,SmF3’5’H,SmPAP1,SmWD2,SmANS,SmFNSⅡ,and SmUF3 GT were extremely low in the unpigmented young bud(F1),all of them showed a trend of slow growth and then rapid growth when the purple pigment began to precipitate,the expression levels of the top 7 genes reached a peak in purple petals in full-bloom flower(F4),and the expression of SmANS、SmFNSⅡ and SmUF3 GT increased rapidly in the pre-bloom flower(F3)but declined sharply in full-bloom purple flower(F4).The expression levels of SmDFR showed a gradual increasing trend.The expression of SmWD1 was highest in F1,and despalyed a sharply decreasing,then recovering and then decreasing again trend.In the white flower development process of white-flowered danshen,the expression patten as follows: the expression of SmCHS,SmCHI,SmDFR,SmUF3 GT,SmF3’H,and SmANS showed the highest levels in the young yellow bud,yet decreased sharply in the yellow middle bud(F2).The expression of SmCHS,SmCHI and SmDFR rised a little again after a fall in F3 stage.The expression of SmUF3 GT and SmF3’H always kept the downward trend.The expression of SmANS rised after a fall in F3 stage and then decreased in F4 stage.However,SmF3 H,SmF3’5’H and SmFNSⅡ expressed lowest in F1 stage,and increased to the peak in F3 stage,then decreased in F4 stage,which suggested they took part in the coloration of pollen in white floweres.SmWD1 and SmWD2 expressed lowerly in F1 stage,and then increased all the time.These suggested the coordinate expression of the most key genes involved in flavonoids biosynthesis to regulate the color of flowers in danshen and white-flowered danshen.The lower expression levels of the most flavonoids genes decided that the petals did not appear purple pigment in white petals.4.GFP fusion proteins were build and expressed transiently in tobacco leaves to investigate the subcellular localization,the results showed that SmCHI,SmANS,SmUF3 GT,and SmF3‘H protein localizated in the Golgi and cell membrane and nucleus of the epidermal cell in tobacco leaves.In addition,SmCHI and SmF3‘H also localized in ER,peroxisome and plastid.Beyond that SmANS were localized in the ER and plastid.SmUF3 GT were also localized in the peroxidase.SmF3‘H was predicted to localize in the cytoplasm.These poly-subcellular localization of the five flavonoid genes indicated that the SmF3‘H and other flavonoids key enzymes may formate the multienzyme complex and anchor to ER,then produce and transport the anthocyanin and other flavonoids in and out the cells.5.The transgenic plants of SmANS-overexpressed in danshen and white-flowered danshen,as well as SmANS-RNAi in danshen were obtained by the Agrobacterium tumefaciens mediated genetic transformation system.SmANS overexpression enhanced the accumulation of delphinium chloride,and added the ingredient of cyanidin-3,5-di-O-glucoside,which were positive correlated with the up-regulating of the expression levels of most flavonoids genes including SmCHS、SmCHI、SmF3’H and SmDFR.Moreover,the overexpression of SmANS reduced the concentration of salvianolic acid B and romarinic acid,and down-regulated the expression levels of the SmPAL,SmC4H1,Sm4CL2,SmRAS and SmCYP98A14,also altered the synthesis of proanthocyanidin monomers in the transgenic danshen and white-flowered danshen plantlets.SmANS-RNAi decreased the concentration of total anthocyanin,down-regulated the expression levels of the most flavonoids genes but SmDFR and SmF3 H.Meanwhile,the danshen transgenic plantlets of SmANS-overexpression mainly displayed the purple-red coloration in the leaves.The SmANS-overexpressed white-flowered danshen transgenic plantlets restored the purple-red phenotype in the leaf margins and stem segments.These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in danshen,but also acts as a ―switch‖ for the coloration of white-flowered danshen.SmANS not only controlled the acumulation of anthocyanin,but also regulated the salvianolic acid sub-pathway and the proanthocyanidin sub-pathway.7.The danshen plantlets of SmF3’H-overexpressed and RNAi were also obtained for the functional analysis.The overexpression of SmF3’H,altered the ratio of the dihydroquercetin,dihydrokaempherol and dihydromyricetin,improved the contents of delphindin chloride and total anthocyanin,up-regulated the expression levels of SmCHS,SmCHI,SmFNSⅡ,SmF3’5’H,SmDFR,and SmANS.However,the silence of SmF3’H decreased the production of delphindin chloride and total anthocyanin,reduced the contents of the three kinds of dihydroflavonols in most danshen transgenic plantlets,and down-regulated the expression of SmF3’5’H.These indicate that SmF3’H gene altered the flavonoids‘s composition and accumulation,and it was coordinated expression with other genes in the flavonoids pathway.SmANS-overexpression decreased the concentration of total phenolics,but SmANS-RNAi increased the the concentration of total phenolics.However the accumulation of salvianolic acid B and romarinic acid were all enhanced in parts of SmANS-overexpression and RNAi lines.The crosstalk between the flavonoids and salvianolic acid pathways was induced by the flavonoids genes.These revealed that the downstream genes of flavonoids pathway also had regulatory effects on the salvianolic acid sub-pathway.