| Persimmon anthracnose is caused by the infection of Colletotrichum gloeosporioides Penz. Which occurred widely in any country cultivating persimmon all over the world. For recent years, Wuheshi (Diospyros kaki cv.Wuheshi) was grown with the large scale in Chunan area, Zhejiang province and anthracnose disease tend to be more severe. Some of mechanisms of pathogenesis had been studied systemically in our lab, such as pathogen identification, disease occurrence conditions, pathogenesis of the pathogen, as well as mechanisms of interaction between pathogen and host and so on. However, many works are limited on the cellular level. It is a key means to understand mechanisms of pathogen pathogenesis by making the use of molecular biological technology. These are of great importance not only in plant pathology but also in practice of produce, providing the clue for probing the pathway of disease control. Therefore, in this thesis, the pathogenesis-related genes of C. gloeosporioides were cloned by genomic library construct with SuperCosl vector and Agrobacterium tumefaciens-mediated transformation (ATMT) technique of C. gloeosporioides.1 Genomic library constructionThe genomic library was constructed by SuperCosl vector. A method extracting high quality genomic DNA of C. gloeosporioides was set up by utilizing SDS and proteinase K and about 40 kbp DNA fragments were produced by partial digestion of enzyme. After ligation and packaging of the DNA fragments and vector, XL1-Blue MR bacteria was transfected by them and At last 4.0×10~4 cosmid clones were obtained, being equivalent to 10~5 clones per microgram genomic DNA. The clones were stored with 15% glycerol at -70℃. Analysis of 50 recombinants selected randomly showed that 98% recombinants contained the exogenous DNA fragment with about 40.5 kb of average size. If the size of genomic DNA was calculated as 50 Mb, the library capacity reached up to 31.75 and the exact probability of having any given DNA sequence in the library is as high as 99%. Colony titration in stock was around 4.6×10~8 cfu/ml, even after three times thawing, it still reached to 4.0×10~8 cfu/ml. From the results above, A high quality genomic library had been constructed, which accorded with the requirement for the screening target gene.2 Agrobacterium tumefaciens-mediated transformation (ATMT) of C. gloeosporioides and screening of mutantsIn this experiment, a binary vector pATMTl was transformed into Agrobacterium AGL-1 by electroporation and then them and 800ul conidial suspension (1×10~6 cfu/ml) of Colletotrichum gloeosporioides TSG001 was co-cultivated in cultures containing acetosyringone,. After two rounds of screening on cultures containing hygromycin B, total 787 transformants were obtained. PCR analysis showed that 14 transformants selected randomly, containing T-DNA, reached up to 92.8 % .The phenotype analysis displayed that number of transformants variety is 22 in growth rate, 30 in colony color, 32 in conidium-produced ability and 18 in conidium morphology. Meanwhile, 5 weak pathogenicity and 6 non-pathogenicity transformants were obtained by pathogenicity test as well.3 Analysis of Non-pathogenicity mutantsBiological characteristics analysis Six non-pathogenicity mutants was analyzed further. Mutants Cg-10, Cg-32, Cg-305, Cg-573, Cg-608 and Cg-613 displayed a significant difference on the growth rate compared with the wild type TSG001 strain and also there was the differences among them in colony patterns and colors. It was found that all the six mutants produced less conidia than TSG001 strain(187.5×10~6 conidium/plate). Among them, the conidial production of Cg-10,Cg-32,Cg-305 and Cg-608 was less than 1/100 that of TSG001 strain. The conidia germination and appressoria formation rate of five transformants were lower than that of TSG001 strain except for Cg-608, and especially conidial germination rate of both Cg-32 and Cg-613 was 2% and appressoria formation rate of Cg-613 was 1%, while there was no appressoria in transformant Cg-32 experiment.Identification of copy number of T-DNA insertion Analysis of Southern hybrization for six non-pathogenicity mutants showed that 4 mutants Cg-10,Cg-32,Cg-573 and Cg-613 were single copy insertion except three-copy mutant Cg-305 and two-copy mutant Cg-608.Analysis of flanking sequences of single copy non-pathogenic mutants According to insert sequences, the primers were designed, and flanking sequences were amplified by inverse PCR, TAIL-PCR and hiTAIL-PCR. These sequences were obtained from right and left flanking sequences of T-DNA insert sites of mutants Cg-10 and Cg-32 as well as right of Cg-613. The Blastn search results showed that sequences obtained from mutants Cg-10, Cg-32 and Cg-613 were homologous to sterol 24-C-methyltransferase gene, 40S ribosomal protein S11 gene and eukaryotic translation initiation factor 3 gene, respectively. |
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