| Glycyrrhiza uralensis Fisch. of Leguminous family is the main source of medicinal licorice (Radix Glycyrrhiza). Glycyrrhizin content is one of the most important indexes to estimate the quality of medicinal licorice. There are many key enzymes involved in the regulation of the Glycyrrhizin biosynthetic pathway. The current use of molecular biology and genetic engineering of those enzyme genes has become a hot research. In this study, we selected the first important rate-limiting enzyme—squalene synthase of Glycyrrhiza uralensis (GuSS) as the research subject. Cloning GuSS gene by RT-PCR method, summing up the gene sequence polymorphism law with gene sequencing, were amid to reveal the molecular mechanism on squalene synthase regulation to glycyrrhizic acid metabolism and to provide a theoretical basis of obtaining high-quality medicines. The polymorphism squalene synthase expressed in E. coli expression system, then different enzymes were purified for the catalytic reaction. After reaction the catalysate squalene were measured by GC-MS. Comparison between squalene production is to further verify the squalene synthase gene polymorphism on regulation of enzyme, which will bring potential applications of improving glycyrrhizic acid content by the use of biotechnology. Basing on the purpose and content above, The following conclusions can be drawn in this study:1. This study revealed the gene polymorphism of GuSS on the base of cloning 2 forms of licorice squalene synthase gene—GuSSl and GuSS2. The phenomenon of GuSS gene polymorphism was quite abundant, including Coding scingle nucleotide polymorphism (cSNP), Insertion/deletion length polymorphism (InDel) and aternative splicing(AS).21 cSNPs of nucleotide sites were detected in 15 GuSS1 sequences, accounting for 1.69% of sequence length, and the proportion of transitions and transversions was 2.14:1 with 80.9% missense mutations. InDels also exist with 26.7% occurrence rate. In addition,9 nucleotide sites cSNPs were found in 11 GuSS2 sequences, accounting for 0.73% of sequence length, the proportion of transitions and transversions was 8:1 with 55.55% missense mutations. In the level of amino acid, non-conservative amino acid subsititution of GuSS1 was 53.94% in frequency with 2 mutation in structure domains, whereas 60% GuSS2 were non-conservative amino acid subsititution with 1 mutation in structure domains.2. This study established the methods of squalene synthase gene expression in E. coli and purification. Connecting the selected sequences SS1 A-c,SS1 F-c,SS2A and SS2F with gene polymorphism to expressing vector pET-32a(+), structuring recombinant vector pETSS, molecular weight of 67kD recombinant fusion protein were expressed in the host bacteria E.coli BL21(DE3) with the condition of 30℃0.4mmol·L"'IPTG 4h induced, in which were mainly the soluble form. Proteins using Ni-NTA affinity chromatography purification were obtained as a satisfactory result.3. The polymorphism of GuSS significantly affected on the accumulation of squalene. The four purified squalene synthase used to catalyzed the substrate FPP. Then the catalysate squalene were detected by GC-MS analysis method with the 18.44 retention time (RT). Squalene synthase produced different amount of squalene in different reaction times. In 30min reaction, squalene catalyzed from SS2A was 2.5 times higher than the other same average; in 50min, squalene catalyzed from SS1F-c was 6.1 times higher than the other same average. The catalytic efficiency of enzyme SS1F-c was higher than other samples. "ASNGPL" amino acid type can increase the catalytic ability of GuSS1, "EGSRLS" amino acid type can reduce the catalytic ability of GuSS1; whereas in GuSS2 in 330 non-conservative amino acid mutations on the enzyme catalytic activity was not obvious. The squalene accumulation capacity of GuSSl and GuSS2 enzymes was different, because the catalytic ability of GuSS1 different individuals was quite different and the catalytic ability of GuSS2 different individuals was stable. |
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