Clone And Preminary Analysis Of The Flanking Pathogenic Related Genes Of T-DNA Insertional Ustilaginoidea Virens Mutants B1178 And B3277

Rice false smut was a fungal disease in rice, which produced by U.virens. This research usedthe technology ATMT, which to construct a U.virens mutant library, the starting strain was P1. The aim of this experiment is to supply a new insight for resolving the pathogenic molecular mechanism of Ustilagionidea virens, which depending on analysis of enhanced virulence mutant strain B3277 of the T-DNA insertion mutant library in U. virens. The wild type strain P1 as a control, statistical analysis mutant strains B3277, such as the pathogenicity, growth rate, sporulation, and other phenotype in this strain. Then found that the growth rate in PSA、TB3、MM and sporulation in PS were all decreased compared with P1, but as for the pathogenicity,its had significantly enhancedin the field. Amplified the T-DNA gene GFP and HPH by using PCR, verified the T-DNA was inserted in DNA of B3277 genome. While southern blot was to know the copy number.And the test displayed thatthe only one foreign gene inserted in B3277. Usedtechnique of hiTAIL-PCR. The aim was to clone and acquire the sequence, which was adjacentto the T-DNA flanking, the length was 258bp (U.VLB) and 347bp (U.VRB), The sequence of LB-terminal of T-DNA only lost 66bp,on the opposite the RB lost the most,302bp in existed only 55bp. The pathogenic of B3277 is increased, and the T-DNA RB and LB were all lost partly, presumably the reason owing to the T-DNA insertion leaded.According to the flanking sequenceswhich got by the hiTAIL-PCR,we could design primers. And combined with the RACE-PCR cloning full-length T-DNA flanking the gene, get a flanking gene Uvt3277, the T-DNA insert in the middle of the gene. Uvt3277 analyzed for bioinformatics and found it has a high homology with some of the low affinity of fungal iron transport proteins. Determination the growth of B3277 on PSA, and medium which added FeSO4.And to measure the capacity of ferrous absorption in strain. Resulted showed, within a certain range of concentrations the B3277 was take advantage of FeSO4 in a normal growth, but the growth forP1 was inhibitedand that slowed down, suggesting that the mutant gene deletion related to ferrous ions. Analysis of gene expressionwhich used by Semi-quantitative RT-PCR. And the results we could conclusion that, Uvt3277 was not expressed comparised with that in P1. The research was obtained a T-DNA flanking gene Uvt3277 (GeneBank Accession No.:KJ158162), and associated with some low affinity transporter protein in fungi, that inferred the gene plays an important role of pathogenic process in U. virens.This experiment is to provide a new insight for resolving the pathogenic molecular mechanism of Ustilagionidea virens, which depending on analysis of mutant strain B1 178 of the T-DNA insertion library in U. virens. The control strain was wild type P1, statistical analysis mutant strains B1178, such as growth rate, sporulation, pathogenicity, the resulted showed that the growth ratein PSA、TB3、MM and sporulation in PS were all decreased compared with P1, the pathogenicity in the field was also decreased. Amplified the T-DNA gene GFP and HPH by using PCR, verified the T-DNA was do inserted in genome DNA of B1178. While Southern blot was to know the inserted copy number of foreign gene, the result showed that only a single copy. The utilizedtechnique of hiTAIL-PCR to clone the sequence of T-DNA flanking, the length was 291bp (U.VLB) and 311bp (U.VRB), The LB-terminal sequence of T-DNA all did not lost,on the opposite the RB lost the most,302bp in only 17bp. The pathogenic of B1178 is decreased, and the T-DNA RB and LB were changed partly, presumably,the reason due to the T-DNA insertion leaded.