| Stylosanthes spp. is the most important and commercial forage legume in tropical area. Stylosanthes anthracnose, is a global severe disease in stylo’s production and extension, mainly caused by Colletotrichum gloeosporioides. A better understanding of molececular basic of this disease is not only beneficial for Stylosanthes anthracnose control, but also can serve as a model for understanding other plant-pathogenic fungus interaction.In this study, we identified T-DNA mutants from the ATMT mutant library, which displayed reduced pathogenicity. The flaking sequences of T-DNA were identified by using Tail-PCR. Gene function was analyzed by using biological character analysis、 bioinformatics analysis and gene knockout technology. These resultts were showed as follows:（1） Two pathogenicity-weaked mutants from the ATMT mutant library was identified, which named after H621and H800.（2） Biological character analysis showed that:the two mutants’ growth rate, germination rate, toxin activities, were significantly decline, moreover, polygalacturonase activities of H800mutant was declined.（3） The right and left T-DNA flaking sequence of H621were non-adjacent in the wild type CH008, and the right and left T-DNA flaking sequence H800were adjacent in the CH008. Sequence alignment analysis showed that the T-DNA inserted into the fourth exon of StCg-ALS gene and the first exon of StCg-ssu gene in the H621. And the T-DNA inserted into the the promoter region of StCg-800gene in the H800.（4）The results of bioinformatics analysis:StCg-ssu nucleotide sequence showed99%similarity to XM007279438.1from strawberry Colletotrichumgloeosporioides. And the coding sequence was5172bp long and encoded a1723amino acid protein,which was hydrophilic protein and belong to ssu processome component. StCg-ssu gene had a U3snoRNA structural domain, which was involved in the processing of pre-rRNA. StCg-ALS nucleotide sequence showed98%similarity to XM007284118.1from strawberry Colletotrichum gloeosporioides. And the coding sequence was2079bp long and encoded a692amino acid protein, which was acetolactate synthetase and belong to Thiamine pyrophosphate （TPP） family. StCg-ALS gene had three domains:N-terminal TPP binding domain,TPP central domain,C-terminal TPP binding domain. StCg-800gene was774bp long, included only one exon, no intron, encoded a257amino acid protein, which was a hypothetical and unstable water-soluble protein, located in cytoplasm, no signal peptide, belonged to growth factor and enzyme.（5）Using gene knockout technology to seek out StCg-ALS,StCg-800gene biological function. The delection of the StCg-ALS gene made the Colletotrichum gloeosporioides change a lot:no aerial hyphae, stronger hyphae, lost ability of forming Conidium,declined polygalacturonase activities and toxins activities,lost pathogenicity. These changes indicated that StCg-ALS gene played an important role in the Colletotrichum gloeosporioides’s vital movement and pathogenicity. The delection of the StCg-800gene didn’t change Colletotrichum gloeosporioides any,which indicated that the StCg-800gene had nothing to do with Colletotrichum gloeosporioides’s pathogenicity. |
