The Development Of Agrobacterium Tumefaciens-Mediated Transformation And Its Application In The Study Of Triterpenes Biosysthesis Of The Medicinal Fungus Ganoderma Lucidum

Ganoderma lucidum is one of the traditional medicinal fungus which has many biological and pharmacological activities. As a kind of Basidiomycetes, G. lucidum has a high value for nutrition and health care, but the molecular biological study of G. lucidum is very limited. Triterpenes, which is one kind of the secondary metabolites of G. lucidum, is considered as the main active ingredient.Nowadays, studies attempting to enhance the triterpenes production in G. lucidum are divided into two branches. One has focused on changing fermentation parameters and adding inducer to improve the production of triterpenes. The other has been directed to regulating the expression levels of specific genes involved in the biosynthesis of the triterpenes. But the research on the gene expression and regulation mechanism of the triterpenes biosynthetic pathway is very less, which mainly due to the lack of transgenic methods.In this paper, we initially established the Agrobacterium tumefaciens-mediated transformation (ATMT) method and the reporter gene expressing system in G. lucidum. First, we obtain a bunch of transformants by co-cultured of A. tumefaciens LBA4404strain and fungus protoplasts. We then confirmed that the foreign gene has been successfully integrated into the genome of the fungus by PCR amplification and Southern Blot analysis. We optimized this transformation method by adjusting the parameters of the co-culture. We obtained200transformants per105protoplasts by adding0.2mM acetosyringone, cultured at25℃for36hours based on the bacterium protoplast ratio of1000:1. The transformation efficiency is significantly higher than electric shock method and REMI method in preliminary studies. Besides, our research applied four fungal promoters and compared the influence to transformation efficiency. The result showed that the plasmid contain a cassette in which hph is under the control of promoter of Gl-gpd get the highest transformation efficiency, while the promoter of Le-gpd and Le-ras gets similar, the promoter of L. edodes gets the lowest Our result demonstrated that endogenesis promoter can significantly improve the transformation efficiency. We created the reporter system based on GUS, since GUS got higher expression efficiency than EGFP.We applied over-expression technology in our research in order to study the function of mevalonate pyrophosphate decarboxylase. As far as we know, it is the first time to apply the over-expression technology in G. lucidum research. We created Gl-mvd over-expression vector, and transformed it into G. lucidum by ATMT. We examined the expression of Gl-mvd gene at the mRNA level in randomly picked four transformants, by using Real-time PCR analysis. The result showed that, the expression level of Gl-mvd gene in GMOE9was over4.5times than that in wild-type strain G20. And the other three GMOEs, the expression levels of Gl-mvd were over2.5times than G20. We also examined the expression of Gl-mvd at the protein level in the above GMOEs using Western Blot analysis. The results showed that the expression of Gl-mvd at protein level in the four GMOEs was not significantly increased. To better understand the influence to phenotypic character by over-expression of Gl-mvd, we measured the content of triterpenes in the GMOEs. The result shows that, GMOE10, compared with G20, the triterpenes content was increased101%. The triterpenes content of the other three over-expression transformants were also significantly increased. In order to explore the mechanism of the increasing of GMOEs triterpenes content, we further examined the expression of key enzyme gene involved in triterpenes biosysthesis pathway at mRNA levels in GMOEs. The result showed that expression level of Gl-hmgs, Gl-hmgr, Gl-fps, Gl-sqs and Gl-osc gene were increased also.Transient transfection analysis has been widely used in the study of promoter and other regulatory elements analysis. In this study, we initially applied transient transfection technology into G. lucidum gene promoter analysis and analyzed the promoter of squalene synthase. We transformed the5’deletion vectors into G lucidum by using ATMT and analyzed its activity by examining the GUS activity. The result is consistent with the bioimformatics analysis. Especially the deletion of CAAT-box and other cis-acting element has significant reduction of promoter activity. However, one in the region of-868to-673deletion, we found that the phenomenon of promoter activity rose. But in this region about bioinformatics analysis did not predict the existence of the element inhibited promoter activity. The deletion of50bp was used for further analysis. The deletion from-868to-821regions was no significant effect on promoter activity. With the deletion from-822to-776and-775to-721regions, the promoter activity increased significantly. This result indicated that there was a structure which can significantly reduce the promoter activity within these two regions. In preliminary studies in the laboratory, we have found that MeJA has significant induction effect for the biosynthesis of triterpenoids and the expression of Gl-sqs gene was significantly increased. In the bioinformatics analysis of Gl-sqs promoter, we found that there were three potential MeJA response elements in Gl-sqs promoter. We analyzed the three potential MeJA response elements using the transient transfection, and the result showed that all of the elements have the ability to respond to the Gl-sqs expression in the induction of MeJA. This result is a foundation for researchers in greater detail to improve the accumulation of triterpenes in the future research and also shed new light on G. lucidum triterpenes biosynthesis.