Based On The Engineering Bacteria Of Licorice Hmgr, Sqs And Beta As Gene Cnvs On Its Expression

Nowadays researches about the molecular mechanism of glycyrrhizin production process are of great importance for the following reasons. As widely used herbal medicine, Licorice is of great demand in both China and other countries. As the key therapeutic compound of licorice, Glycyrrhizin is very effective in the treatment of inflammatory, aging, high-blood pressure and so on. However, nowadays the amount of glycyrrhizin in planted Licorice is too low to reach the standard level. For that matter, it is necessary to improve the amount, while knowing the production process of glycyrrhizin would definitely be the key problem for finding the method.According to the biological principles, Copy number variation(CNV) is highly possible to influence the production of the key enzyme in production process and then takes effect on the biosynthesis. CNV is very popular in recent years and it has been found to be related to many diseases. In plant researches, it has even been created to improve the amount of production. Therefore, CNV is of high possibility to take effect on the Glycyrrhizin production.In CNV research, engineering bacteria are better than transgenic plants as the expression system. Unlike transgenic plants which take a long time to grow up and are complicated, engineering bacteria grow fast and are easy to be operated. What’s more, they are similar to the plants system at the translation process modification.As a result, in order to find out how CNV influences the expression of the key genes, construction of expression vectors and Real time PCR are needed to screen the exact inserted copy number of genes-HMGR, SQS, β-AS. Finally RT-PCR technique is used to figure out the expression difference between Pichia Pastoris strains with CNVs.The results of this paper are as follows:1. Engineering bacteria with CNVs of Licorice gene-HMGR:1,2,4,8,13,44copies; engineering bacteria with CNVs of Licorice gene-SQS:1,2,4,10,16,47; engineering bacteria with Licorice gene-β-AS:lcopy.2. Real time PCR method for copy number screening. 3. In engineering bacteria with Licorice gene-HMGR,4copies show the highest gene expression while44copies show the lowest, and the highest is3times of the lowest, which indicates that CNVs of gene HMGR could take effect on gene expression. In engineering bacteria with Licorice gene-SQS, there is no great difference among them, which indicates that CNVs of gene SQS are not effective.-on gene expression. Engineering bacteria with lcopy of Licorice gene-β-AS are not detected for expression difference.