Licorice Sqs1 Gene Polymorphism And Its Accumulation Products,

Glycyrrhiza is commonly used in Chinese medicine with, its active component glycyrrhizie acid (GA). However, the content of GA varies greatly among different plants, and the reason for which is still unknown. This study, intend to elucidate the different GA content by the investigation of the gene mechanism of GA biosynthesis. Many studies have indicated that changes in amino acid sequence resulted from gene mutation lead to changes in enzyme activity resulting in varieties in accumulation of target products. Therefore, the internal genetic factors for the difference of GA content can be explored by view of gene polymorphism of key enzymes in GA biosynthesis route. GA is a secondary metabolite synthesized by mevalonate pathway in which squalene synthase (SQS) andβ-amyrin syntheses (PAS) play important roles.β-amyrin is formed by the catalysis which form glycyrrhizie acid after further oxidation. Therefore, in the present study, the above mentioned two key enzymes were used as study objects to investigate their influences on the biosynthesis of GA in plants.In addition, biosynthetic pathways of a natural product often involve 15 to 20 enzymes forming a metabolic network with, complex regulation and feedβAck mechanism. In many current studies on bioengineering, a great number of target products were obtained through constructing a complete metabolic pathway in which co-expression of multiple genes is definitely involved. Therefore, co-expression of multiple genes has become a research focus. In this study, we expect to obtain more P-amyrin through co-expressing the above mentioned two genes.As a result, in our study, artificial cultivated Glycyrrhiza uralensis Fisch under the same condition was used. On theβAsis of systematic analysis of the relationship between functional gene sequence and GA content, the genotypes with polymorphism were screened and the engineering P Acteria for co-expression of the two genes was constructed. The influence of gene polymorphism on accumulation of target products was studied and the presumption that co-expression of multiple genes increase the synthesis of target products was testified. This work lays aβAsis for subsequent artificial molecular breeding of Glycyrrhiza and mass production of GA by fermentation technology.The main conclusions in this study are as follows:1. Significant difference on GA content was observed in Glycyrrhiza plants even grown under the same condition with a scope from 0.9087% to 4.9164%.2. A gene polymorphism of Glycyrrhiza SQS1 existswith at least 3 genotypes. However, through cluster analysis, no direct relationship was observed between gene polymorphism and GA content. It is proβAbly the GA level is co-regulated by multiple enzymes, while individual enzyme plays a limited role. Therefore, further study on the mechanism of combined effect of multiple enzymes in the whole metabolic pathway is needed.3. The polymorphism of SQS1 gene influences the accumulation of SQ andβA. Through introduction of different genotypes of SQS1 genes into yeast cells, it was noted that SQSs had great influence on the accumulation of squalene and downstream productβAS-β-amyrin. The net increases of squalene were-0.0009mg/ml (-1.8%),0.0043mg/ml (8.6%), and 0.004mg/ml (8.0%), respectively; and that ofβ-amyrin were 0,0.0025(50%), and 0.0007 mg/ml (14%), respectively.4. Gene co-expression can increase the synthesis of target products. Co-expressionof desirable genes increases downstream products significantly, especially the highest level ofβA was found in bSd2P strain with co-expression of genes SQS1 and PAS, which was 1.5 fold of that in bP strain, and 1.5 fold of that in bSd1P strain with lowest level ofβA, indicating that co-expression of desirable genes can obtain more target products.5. There is a mRNA alternative splicing (AS) on Glycyrrhiza SQS1 gene, but the influence of AS on glycyrrhizie acid biosysthesis and related mechanism requires further study.