Cloning Of Upstream Region Of Cyp51 Gene From Ustilago Maydis And Analysis By Bioinformatics

Corn smut is a common disease of agriculture mainly caused by Ustilago maydis destroying the ear of maize. When the fungal disease happened, it would be harmful to agricultural production and bring about enormous crop losses. Cytochrome P450 (CYP51) is the only member of the superfamily with mixed-function of monooxygenase which can be widely found in higher plants and animals, fungi and yeast cells. The enzyme is named by its maximum absorption in the 450nm wavelength. CYP51 of Fungi, known as sterol 14-a-demethylase (P450 14DM) is a key enzyme in the biosynthesis of membrane ergosterol. It will lead to the destruction and loss of function of membrane structure and ultimately to fungal death without CYP51. At present, about one third of the fungicide market is represented by demethylation inhibitors (DMIs) fungicides which act as potent inhibitors for the fungal sterol 14a-demethylase. However, as such widespread use of fungicides, it was gradually increased that the fungi become more and more resistant to these fungicides. It was reported that the fungal resistance was related to the over-expression of cyp51 gene which can lead to the fungal resistance by the ability of fungicide degradation. The enhancement effect of promoter was another main factor to gene over-expression. The core promoter structure contains TATA-box and CAAT-box, and also the AP-4, GATA-1, CdxA, Dfd, Oct-1 and some other specific response elements and transcription factor binding sites with the specific function of regulating gene transcription. As Ustilago maydis is one of the important model organism to study the mechanism of resistance owned by filamentous fungi, it is very significant to master the upstream regulatory sequence of its cyp51 gene and the mechanism of resistance.In this study, the upstream promoter was successfully obtained by using the chromosome walking technique, based on 5’-end sequence of cyp51 gene from Ustilago maydis. Then some bioinformatic softwares were utilized to deeply study the structure and function of the upstream promoter of cyp51 gene from Ustilago maydis. The results mainly include the following two parts.1. Based on the 5’-end sequence of cyp51 gene, three specific reverse primers GSP1, 2,3 were designed and utilized the chromosome walking technique to clone the promoter via three thermal asymmetric PCR reactions and then analysised by Bioinformatics. The transcription start site of cyp51 gene promoter was predicted by the NNPP on-line bioinformatics software of (http://www.fruitfly.org/seq.tools/promoter.html). The TFSEARCH (http://www.cbrc. jp/research/db/TFSEARCH.html) and MATCH (http://www.gene-regulation.com/pub/ programs.html#matc) were used to find the transcriptional factor binding sites by searching the database (http://transfac.gbf.de/TRANSFAC/). It is benefit to deeply study the expression and regulation mechanism in future.2. Constructed the recombinant plasmid pET-Um-GFP and pGL-Um to study the promoter structure and function. The promoter regulating green fluorescence protein(GFP) expressive plasmid pET-Um-GFP transformed Ecoli BL21 strains and high expressed at 37℃by 1.0mmol/L IPTG for 36 hours. The results showed that pET-Um-GFP transforming bacteria liquid sent out higher intensity GFP than the pET-GFP control group respectively observed under visible and ultraviolet light. In order to detect the transcription activity of the promoter, recombinant plasmid pGL-Um transfected the HeLa cell lines. Dual luciferase reporter gene expression detection system displays that the luciferase activities of transfected groups contained the recombinant plasmid pGL-Um was 2.2 times higher than control group, indicating promoter of cyp51 gene from Ustilago maydis activated the expression of luciferase gene in cells.This thesis mainly analyzed and studied the structure of upstream regulatory promoter of cyp 51 gene from Ustilago maydis, which will be benefit to deeply interpret the function by mutation and truncated and also laid a theoretical foundation for the development and screening of the new and highly efficient anti-fungal drug.