| Liquorice, as a Chinese traditional medicine, holding the title of "Guolao". The glycyrrhizic acid (GA) as the main active ingredient in liquorice has important pharmacological effects, but also is the measure of the quality of the licorice. With the increasing scarcity of wild licorice, the problem of which the content of GA lower and varies greatly among different artificially cultivated licorice becoming more and more serious and need to be solved.In recent years with the development of understanding the plant secondary metabolism, studying the key genes as one of the hot research areas. And it also attract great attention because of the abroad application of molecular biology. But there is few study from glycyrrhizic acid biosynthetic pathway to solve the problem of GA varies greatly among different plants. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) which catalyzes the conversion of HMG-CoA to mevalonate, played a significant role in GA biosynthesis. So HMGR was chosen as to investigate the effect on the GA biosynthesis.So in this repect, the main intention is cloning the HMGR from Liquorice as the first step. Then on the basis of systematic analysis of the relationship between HMGR sequence and GA content, the genotypes with polymorphism were screened. And the influence of gene polymorphism on accumulation of target products was studied from the different activities.The main conclusions in this study are as follows:1. A full- lenght HMGR cDNA was isolated from Liquorice using cobminatoiral methods of RT-PCR andRACE and named as GuHMGR for the frist time. (Genbank AccessionNo. GQ845405). Sequence analysis revealed that the complete Sequence of GuHMGR gene was 1842bp. The coding sequence of the HMGR gen was 1722bp which deduced 574 amino acids. Bioinformatics softwares were used to predict the coded protein structure, the formula for GuHMGR is C2657H4303N745O815S26, molecular weight is 60.5584kDa, and the isoelectric Point is 7.09. 2. GuHMGR is polymorphism in liquorice. According to the missense mutation of amino acid, the plants were divided into five types:-HSL (D1, D2),-HSV (D3, D4, G2, G4, G5), GALSV (Gl), GALLV (G3), D5 to heterozygosity (--HSL/GALLV). Combined with content analysis of glycyrrhizin, inserted GA genotype was found present in high specific content of glycyrrhizin group; L genotype which is of the V/L gene mutantions was found only in the low group.3. The HMGR gene was subcloned into the Escherichia coli expression pET-32a, and the recibined plasmids were transformed into E.coli BL21 (DE3). Then we successfully isolate and purifie the protein with the catalytic activity from E.coli.5. The polymorphism of GuHMGR gene influences the accumulation of product MVA. In our study, the quantity of the MVA which was catalyticde by the genotype with GA insertion is 2-3 times of the MVA which was catalyticde by the genotype with NO-GA insertion. V/L genotype of two mutations found the products are close to each other. So we consider that insertion mutant genotype GA with higher catalytic efficiency; V/L genotype mutation has little effect on product accumulation. |
