Cloning And Fuctional Analysis Of Two Key Genes In The Procyanidins Synthesis In Gansu Sainfoin

Procyanidins(Condensed tannins)in organisms play an important physiological function in Uv-resistance,disease resistance,insect resistance and scavenging free radicals,adjusting the dormancy and germination of seed,and influence the palatability and digestibility of forage,and also have a medicinal and health value.Two key enzyme genes in the path of procyanidins synthesis in O.viciaefolia Scop.cv.Gansu.were cloned by the method of molecular biology,namely the anthocyanidin reductase(ANR)and leucoanthocyanidin reductase(LAR).We also forecast the structure and function of the protein encoded by the ANR gene and LAR gene by using bioinformatics software,and construct two the plant expression vectors containing the bar and GFP for double marking.Based on transgenic technique,their functions in procyanidins biosynthesis were studied.The relationship between procyanidins content and these gene expression levels were discussed by the semi-quantitative analysis and procyanidins content analysis.The main results of this study are as follows:1)The content of total procyanidins and insoluble procyanidins were low in leaves,their content were high in stem and reproductive organs.Interestingly,the contents of soluble procyanidins were high in the leaves,and low in the stem,flower and fruit.total anthocyanins,insoluble procyanidins and soluble procyanidins content were high at vegetative stage,and low at reproductive stage,which is contrary trend in the stem.Therefore,leave is a key synthesis organ of procyanidins,but not the main storage organs of procyanidins.Anthocyanins content in flower was the highest levels in reproductive organs.The anthocyanins content in stem and leaf in general was higher in reproductive growth phase than the vegetative growth stage.It shows that anthocyanins play an important physiological function in the form of flower color and reproductive growth.2)BAN gene was cloned by RT-PCR from O.viciaefolia Scop.cv.Gansu.The gene sequence shared a high level of similarity with the BAN gene in GenBank(accession No: HM152980)and its homology was 99.41% by sequencing analysis.Their open reading frame(ORF)was 1020 bp,encoding 339 amino acid residues.The amino acid sequence was analyzed and predicted,it contained many important functional sites of anthocyanin reductase,so it was inferred that BAN gene expression has a function of synthesizing the monomer of condensed tannins.The sequence of BAN gene was submitted to Gen Bank and was given the accession number KM924437.The BAN gene expression vector with the double-marker green fluoresce protein(GFP)gene and herbicide resistance bar gene was constructed and named CPB-BAN-GFP.3)LAR gene was cloned by RT-PCR from O.viciaefolia Scop.cv.Gansu.the LAR gene sequence from O.viciaefolia Scop.cv.Gansu.shared a high level of similarity with the LAR gene in GenBank(accession No: HM152980)and its homology was 98.34% by sequencing analysis.Their open reading frame(ORF)was 1089 bp,encoding 362 amino acid residues.The coding sequence of amino acids is obviously different from those in other genus plants of Leguminosae.The sequence of LAR gene was submitted to GenBank and was given the accession number KP013623.It is referred that LAR gene expression is related to difference of procyanidins accumulation in O.viciaefolia Scop.cv.Gansu.The LAR gene expression vector with double-marker green fluoresce protein(GFP)gene and herbicide resistance bar gene was constructed and named CPB-LAR-GFP.4)The expression level of BAN and LAR in O.viciaefolia Scop.cv.Gansu.were measured by Real-time PCR.The expression levels were different in different organs.The expression level of BAN gene was the highest in fruit,followed by leaf,bud,flower and stem,The expression level of LAR gene was the highest in bud,followed by fruit,flower,stem and leaf.5)Plant expression vector CPB-BAN-GFP and CPB-LAR-GFP were imported into agrobacterium LBA4404 respectively.Alfalfa hypocotyls were infected by two bacterium solutions respectively.We get callus by co-cultivation and differentiation cultivation,then complete fluorescent identification,herbicide-resistance identification and PCR identification.It is verified that BAN and LAR gene has been integrated into the alfalfa callus.The callus integrated with the two gene have higher procyanidins than the control,it is confirmed that heterologous expression BAN and LAR genes in alfalfa can raise the content of procyanidins in tissues.In this study,the gene BAN and LAR in procyanidins biosynthesis path were cloned from O.viciaefolia Scop.cv.Gansu.Bioinformatics analysis showed that the sequence,structure,structural domain,catalytic activity sites of the encoding protein from the genes have high conservative.It is inferred that their function is similar to other plants.Transgenic experiments show that the heterologous expression of BAN and LAR gene can raise procyanidins accumulation in alfalfa callus.The relationship between procyanidins and their biosynthesis genes are explained at the level of molecular biology.These studies enrich the basic research in procyanidins biosynthesis,and lay the foundation for the variety improvement of sainfoin and alfalfa.