Construction Of A T-DNA Insertional Library Of Colletotrichum Gloeosporioides ES026

Huperzine A (HupA) is a secondary metabolite of Chinese traditional medicine Huperzia serrata (H.serrata), which is a promising drug for Alzheimer’s disease due to its highly efficient acetylcholinesterase inhibitor. Compared with chemical synthetic drugs, it has many characteristics such as complex synthetic technology, high price, poor curative effect etc, so the HupA has been mainly from the wild H.serrata blonging to Huperziaceae plants. Because of the wild Huperziaceae plants’long growth periods, complex ecological environment, excessive resources exploitation, the natural production of HupA has been greatly limited. Based on the endophytic fungi and their symbiotic plants can exchange the genetic material theoretically, separation of endophytic fungi containing HupA had become a new research focus. So far, many endophytic fungi which can produce HupA have been gained by many scholars’ efforts. A HupA-producing endophytic C.gloeosporioides ES026had been previously isolated from H. serrata. However, the yield of HupA in HupA-producing endophytic is very low and the whole HupA biosynthesis pathway is still not yet perfect, what’s more the relevant genes’ researches of HupA biosynthesis pathway is very few, therefore production of this chemical in large quantity from endophytes has not yet been put into practice. In this study, a T-DNA insertion mutant library of C.gloeosporioides ES026was established using Agrobacterium tumefaciens-mediated transformation (ATMT). Intend to lay the foundation for gaining HupA high-yielding strains and cloning the relevant genes of HupA biosynthesis, so as to achieve the purpose of using microorganism to produce HupA. At the same time, in the hope of providing related reference for the further research.The main work of this study as follows:1. A high effieient Agrobacterium tumefacies mediated transformation system for C. gloeosporioides ES026were established. The results showed that:under the condition of25°C,1x106spores/ml, A.tumefaciens OD600=0.3-0.5,400μM acetosyringone and48h co-cultivation in the presence of induction medium, the highest transformation efficiency could be obtained (about95transformants per dish). So far, under this optimized transformed system, a T-DNA insertional mutant library of about4000transformants was constructed.2. Evaluation of genetic stability of the agrobacterium mediated C.gloeosporioides ES026mutant library. To examine the genetic stablility of transformants,13transformants were randomly selected. After five generations in the absence of hygromycinB and cephalosporin, the transformants were transfered on PDA plates containing hygromycinB (200μg/ml) and cephalosporin (150μg/ml), all the tested transformants could grow on the selection PDA plate (containing hygromyeinB and cephalosporin), but the the original strain C.gloeosporioides ES026could not. What’s more, using the specific primers were to amplify the hph gene fragement, the result showed that the DNA fragments with an expected size (1101bp) could be amplified from all tested candidates and binary vector pTFCM, but not from the wild strain C.gloeosporioides ES026. The results suggested the T-DNA insertional transformants with good genetic stability.3.100transformants were randomly selected from the C.gloeosporioides ES026T-DNA insertional mutant library to screen some visual phenotypes. Results showed that only five transformants (EST010005, EST010010, EST010021, EST010043, EST010044) had significant changes compared to that of wild strian C.gloeosporioides ES026on phenotypes. And subsequently, the yield of HupA of these five mutants with morphology change was analyzed by HPLC. The results showed that the yield of HupA produced by transformant EST010044(7.73μg.g-1CDW) increased, about1.83times as high as that of original strian C.gloeosporioides ES026(4.23μg.g-1CDW). However, the yield of HupA produced by transformant EST010005(2.75μg.g-1CDW), EST010043(2.78μg.g-1CDW) decreased, about0.65,0.66times respectively of wild strian C.gloeosporioides ES026(4.23μg.g-11CDW)4. To examine the copy number of phenotypic mutant transformants, genomic Southern blot analysis were used. Among several mutants with morphological change the Southern blot analysis showed that, four mutant strains (EST010005, EST010021, EST010010and EST010044) contained a single T-DNA copy, the percent of single-copy insertion transformants reached80%. While, only one mutant strains EST010043 contained two T-DNA copies. This result also indicated indirectly that the agrobacterium mediated C.gloeosporioides ES026mutant library had a high single-copy insertion proportion.5. Using TAIL-PCR and Inverse-PCR technology, the flanking genomic DNA fragments of EST010044was amplified, and subsequently the targeted gene was analyzed. After NCBI blast, the targeted gene showed the highest sequence similarity to a methyltransferase domain protein gene of C.gloeosporioides. It was inferred that the methyltransferase was correlated with the synthesis of HupA. However, to certify the function of methyltransferase in the synthesis of HupA, more detailed studies remain to be investigated.