Cloning And Functional Analisis Of Monocarboxylate Transporter Gene(BcMCT1) In Botrytis Cinerea

Botrytis cinerea is an important plant pathogenic fungus,which can infect more than 200 kinds of plants,such as tomato,grape,strawberry and so on.In order to effectively combat Botrytis cinerea,our research builted Botrytis cinerea T-DNA insertion mutant library by Agrobacterium mediated transformation technology.In this study,the technique of the Agrobacterium tumefaciens-mediated transformation(ATMT)by T-DNA insertion of B.cinerea was established.A library of 107 transformants was constructed.Genetic stability test of the marker hygromycin B-resistant gene(hph)was performed and stable transformants were obtained and subcultured through single-spore isolates.In order to screen the cell wall-defective mutant,mutants were screened by using Calcofluor White and 27 mutants were obtained.By testing the hygromycin resistace gene in T-DNA with Tail-PCR,it was confirmed that T-DNA has inserted into genome of above mutants but only five mutants have value to be further studied(Q-74,Q-20,Q-25,Q-33,Q-103).The Q-74 mutant is monocarboxylate transporter(MCT)gene of the T-DNA insertion mutants.In the RT-PCR and Southern blotting analysis,the gene of BcMCTl was detected because of T-DNA insertion and there was one copy insertion in mutant Q-74.In order to study the function of BcMCT1 gene,the gene complementary strain was constructed(BcMCT1-C).By comparing and analyzing the phenotypes of wild type,mutant Q-74 and complementation strain,it was showed that BcMCT1 was associated with growth rate,cell wall integrity,infection.In addition,we arranged MCT in B.cinerea by BLAST search and found that there are six MCT-related genes.But so far,the function of these genes is still unknown.The results of this experiment can provide some theoretical and experimental basis for further study of the six MCTs gene and their function in B.cinerea.