Screening Of Arabidopsis Thaliana Mutants For The Resistance To Botrytis Cinerea Toxin And Gene Analysis Of The Mutant

Botrytis cinerea Pers., an important plant pathogenic fungus, often cause gray mold in vegetables, fruits and flowers, which may cause great losses. Toxin is one of the most important virulence factors of Botrytis cinerea, but the plant-toxin interaction mechanism is unclear. Therefore understanding the mechanism of toxin effect can provide the basis for the pathogenic mechanism of the fungi and the breeding resistant varieties to the disease.We measured the toxicity of Botrytis cinerea toxin in Arabidopsis thaliana by the methods of radicle growth inhibition and membrane permeability change. We cultured the toxin of Botrytis cinerea in the modified Fries Ⅲ, and used chloroform to extract the crude toxin. We found that different concentrations of toxin made a different growth inhibition of Arabidopsis thaliana. The higher concentration of toxin we used, the inhibition rate of the radicle growth was greater. When the toxin concentration was0.1%, there was no effect on the seeds germination rate, but significantly inhibited radicle growth with83%inhibition rate; when the toxin concentration was0.2%, the seeds germination rate was97.2%, but the young roots can hardly growth; when used the toxin concentration of0.3%, all the seeds of Arabidopsis thaliana can not germinate.The B.cinerea toxin also could make changes in the permeability of the A.thaliana leaf cell membrane, cause the leakage of cytoplasmic. The damage of different toxin concentrations on cell membrane was variety, and the higher concentration caused the greater damage. When the toxin concentration was0.1%, the injury in cell membrane was slight with8.31%; when the toxin concentration was0.8%, the damage was obvious with53.72%injury rate; when the toxin concentration increased to1%, the injury rate was65.48%.According to the experimental results, we established the methods and standards which used to analyze the sensitivity of A. thaliana to toxin, using the method of radicle growth inhibition with0.1%toxin concentration. We cultured A. thaliana in MS medium plates with upright method, and then we measured the radicle length and calculated the inhibition rate after14d.The mutant library of Arabidopsis thaliana was provided by the Chinese Academy of Sciences Institute of Genetics and Developmental Biology, and there were total of12100lines. We used the toxin to screen for A. thaliana mutants, and the wild-type A. thaliana which grow in non-toxic medium as control. Tow weeks later, the radicle length of each treatment was measured. Library screening showed that most mutants were very sensitive to the toxin, and that is, they couldn’t germinate in the medium containing the toxin. We found that the mutant group AM-96showed a decrease sensitivity of toxin with100%seed germination rate and0.6cm radicle length after two weeks. At the same time, the length of wild-type; plants was1.42cm, and the inhibition rate was58%.We treated AM-96leaves with toxin, and found that it’s symptoms were much lighter than that of the wild type leaves; we used pathogen B.cinerea to inoculate AM-96, and we observed that the leave was only slightly yellow, and wild-type leave was yellow serious and larger. We can detect sequence from the AM-96mutants, nothing in wild-type. The sensitivity of the reverse mutation AMR-96to the toxin was increase, similar to that of wild-type plants, and we also failed to detect the insertion sequence in it. Thus, the exogenous gene sequence in A. thaliana was related to the sensitivity of the toxin.We amplified the flanking sequences of the insert fragments by Tail-PCR, and then compared it by NCBI. We found that it was the A. thaliana kinesin motor protein-related sequences (KMPR).We used bioinformatics to analyze this protein and found that this protein exists in many plants, including some important cultivated plants, such as:Vitis vinifera、Oryza sativa、Hordeum vulgare、Zea mays and Sorghum bicolor. We constructed the phylogenetic tree by the method of MP. We found that the relationship between Arabidopsis KMP and dicots KMP was closer than other species. The degree of variation was positively correlated with the evolution.