Cloning, Function Identification And Expression Analysis Of The Important Functional Genes Invloved In The Flavonoids Biosynthesis Pathway Of Golden-buckwheat[Fagopyrum Dibotrys (D. Don) Hara]

Golden-buckwheat[Fagopyrum dibotrys (D. Don) Hara)]is one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of China.It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese medicinal materials, the active extract of rhizome(flavonoids secondary metabolites) of Fagopyrum dibotrys is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum dibotrys is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum dibotrys largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of functional genes research relating to active components of medicine were barely reported at home and abroad.This thesis aims to elucidate biosynthetic pathway of flavonoids through cloning and characterizing key genes,thus provides theory foundation for laying the groundwork for future producing medicinal second metabolic products on a large scale by genetic engineering.The main results in this rasearch are as follows:1.Cloning and functional analysis of leucoanthcyanidin4-reductase(FdLAR)gene from Fagopyrum dibotrysBased on the methods of degemerate PCR and RACE technique,the full length cDNA of leucoanthcyanidin4-reductase named as FdLAR was cloned from Fagopyrum dibotrys(GenBank accession:JN793953).The sequence analysis revealed that the full-length cDNA of FdLAR is1158 bp long fragment containing a1176bp open reading frame(ORF) encoding a polypeptide of391amino acids.Blastn and blastp analysis on NCBI showed that the amino acid sequence of the protein encoding by FdLAR gene shares a high similarity with other reported LAR proteins,especially with LAR from strawberry (Fragaria chiloensis,ADP37950.1)(75%identity).The phylogenetic analysis indicated that the genetic diatance between FdLAR and LAR from castor (Ricinus communi) is the nearest.The bioinformatics analysis showed that the FdLAR protein contains highly conserved NADPH and substrate specific binding site at N-terminal.There is no signal peptide in the deduced amino acid sequence and the FdLAR protein which is the hydrophilic protein with the typical tertiary tructure of enzyme is localized in motochondrial matrix predicted by the bioinformatics software.The prokarytic expression vector of FdLAR and FdDFR genes were constructed and transformed into Escherichia coli Transetta(DE3),also the FdDFR and FdLAR proteins expressed and purified.The FdDFR/FdLAR-NADPH enzymatic reaction system was recongnized as a whole to determine the enzyme activity.The results showed that the recombinant protein of FdDFR/FdLAR could catalyze (+)-DHQ(Dihydroquercetin)as an initial sunbstrate to change into downstream end-product (+)-catechin which demonstrated that both of the recombinant proteins FdDFR and FdLAR had enzymatic activity.The plant over-expression vector of FdLAR named FdLAR-2301G was consrructed and transformed into tobacco using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101(including FdLAR-2301G recombinant plasmid).15transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In15lines of transgenic plants, it was variable of total flavonoids contents in different lines and all of them were improved in varied degree. The content of total flavonoids in leaves of the number T2transgenic tobacco plant increased significantly, which is the highest and being up to7folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdLAR in transgenic line number T2proved that improved flavonoids content was positively co-related with the expression of FdLAR.2. Cloning and functional analysis of chalcone synthase (FdCHS)gene from Fagopyrum dibotrysBased on the nuclectide sequence submitted on the NCBI(GenBank accession number:GU169470),the FdCHS gene was cloned by reverse transcription PCR(RT-PCR)from the first strand of cDNA of the Fagopyrum dibotrys. The prokarytic expression vector and plant over-expression vector were constructed respectively in order to reveal the preliminary charecterization of the FdCHS gene. the FdCHS protein was expressed in Escherichia coli Transetta(DE3) and then was purified by the His Bind Purification Kit..Took the4-Coumaroyl-CoA and Malonyl-CoA as substrates to determine the enzyme activity,and the enzyme activity determination of the recombinant protein showed that the prokaryotic expressed reconmbinant protein of FdCHS could catalyze4-Coumaroyl-CoA and Malonyl-CoA to change into downstream substances,which demonstrated that the recombinant protein had enzymatic activity. The plant over-expression vector FdCHS-2301G was constructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens GV3101.11transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS(β-glucuronidase)histochemical staining and PCR and real-time quantitative PCR(qPCR) analysis.In11lines of transgenic plants, it was variable of total flavonoids contents in different lines and10of them were improved in varied degree. The content of total flavonoids in leaves of the number T3transgenic tobacco plant increased significantly, which is the highest and being up to3.4folds of the wild type tobacco.In subsequent molecular detection by qPCR,the high expression level of FdCHS in transgenic line number T3proved that improved flavonoids contene was positively co-related with the expression of FdCHS.However,positive transgenic line number T10did not show FdCHS function,which may be resulted from the gene silence of introduced FdCHS by co-suppression.The q-PCR also did not detect the expression the FdCHS in the number T10tobacco plant.3The biosynthesis of flavonoids in Fagopyrum dibotrys and expression analysis of FdCHS, FdDFR,FdLAR and transcription regulator FdMYBPl genes.The paper analysed the relationship between the flavonoids contents in different tissues of Fagopyrum dibotrys at different stages and the three important structural genes FdCHS,FdDFR, FdLAR and one transcription regulator FdMYBPl gene.The results showed that the the flavonoids contents display the virous patterns during different development stages.During the vegetatibe multiplication,the overall condition of the flavonoids contents was leaf>root>item and rhizome>flower>leaf>leaf during reproductive growth.The expression levels of the three structural genes in the root or rhizome approximately kept consistency with each other which was high at both ends and low in the middle during the whole development stages.Although the four genes had the high expression in item and low expression in rhizome,the flavonoids contents were high in rhizome and low in item. We sepculated that the flavonoids synthesis and accumulation occurred at different parts of Fagopyrum dibotrys plant.The item may be the main synthesis site of flavonoids synthesised and transport to the rhizome which may be the main accumulation site of flavonoids.In order to verify the assumption furthmore,we detemined the enzyme activity of the key enzyme system DFR/LAR which was closely related to the flavonoids biosynthesis in Fagopyrum dibotrys.The results showed that the enzyme activity of DFR/LAR in item are higher than other tissues during reproductive growth which suggested that there were massive enzyme reaction occurring in item. Summarization above results,the flavonoids synthesis and accumulation had the tissue specificity.