Molecular Mechanism Of T1N6₂2 From Arabidopsis Thaliana Against Botrytis Cinerea

Gray mold of vegetables, caused by the pathogen Botrytis cinerea, is a very serious disease in the world. By means of molecular and genetic engineering, exploring resistance-related genes against Botrytis cinerea, researching the mechanism of defence, is one very important and effective way to cultivate varieties of vegetables which resistance is significantly enhanced. A Botrytis susceptible mutant named t1n6₂2 was achieved by previous research. Real-Time PCR, RT-PCR and complementation were used to confirm that T1N6₂2 was a positive regulator against Botrytis cinerea and a negative regulator against Pst DC3000（Pseudomonas syringae pv. tomato DC3000）, T1N6₂2 ragulated the resistance of Arabidopsis by SA and JA signaling pathway. In this research, RT-PCR was used to confirm the expression pattern of T1N6₂2 in different organizations and developmental stages. Double-mutants crossing between t1n6₂2 and single-mutant of SA and JA pathway were inoculated with Botrytis cinerea and Pst DC3000, expression level of ICS1, PR1, JAR1 and PDF1.2 were measured by RT-PCR. Based on these, the relation between T1N6₂2 and SA/JA signaling pathway was clarified. The subcellular localization and tissue localization of gene T1N6₂2 were confirmed by transient expression of GFP and GUS staining respecively. Expression and purification of T1N6₂2 protein obtained by prokaryotic expression. With these work, we can illustrate molecular mechanism of T1N6₂2 in Arabidopsis against Botrytis cinerea and lay a foundation for resistant breeding.1. Using RT-PCR analysis expression of T1N6₂2 in rosette leaf, cauline leaf, flower,silique, stem and root, we found T1N6₂2 in rosette leaf, cauline leaf, flower, stem and root are similar, but flower, stem and root are comparatively high, silique has the lowest expression of all. Expression of T1N6₂2 of different stages（7 d, 14 d, 21 d, 28 d and 35 d）are measured by RT-PCR, the results show that T1N6₂2（the whole plant） in the first three weeks has an increasing tendency, and then has a decreased trend, and shows a significant decrease at 5 weeks.2. Double mutants t1n6₂2/jar1, t1n6₂2/eds5 and t1n6₂2/Nah G are already have, and were inoculated with Botrytis cinerea and Pst DC3000. The results show that WT was immune and double-mutants were highly susceptible to Botrytis cinerea. Expression of Bc ACTIN on the leaves after inoculated was measured by RT-PCR, the results show Bc ACTIN of double-mutants were significant higher than WT, these indicate the increment of Botrytis cinerea in double-mutants were higher than WT and double-mutants are susceptible to Botrytis cinerea. WT and double-mutants t1n6₂2/jar1, t1n6₂2/eds5 and t1n6₂2/Nah G were inoculated with Pst DC3000, and we found all of them showed sensitivity of Pst DC3000. We also tested the cfu of leaves after insulated with Pst DC3000,the result showed double-mutants were more susceptible to Pst DC3000 than WT.3. Wild-type and double-mutants t1n6₂2/jar1, t1n6₂2/eds5 and t1n6₂2/Nah G were inoculated with Botrytis cinerea, expression level of ICS1, PR1, JAR1 and PDF1.2 were measured by RT-PCR. We found that expression level of JAR1 was decreased in t1n6₂2/jar1, expression level of JA marker gene PDF1.2 in the WT was higher than double-mutants, but was not obvious, and it was increased at 48 h. The results indicate that T1N6₂2 gene affects the resistance of Arabidopsis against Botrytis cinerea by suppressing expression of JA synthesizing related gene JAR1. WT and double-mutants t1n6₂2/jar1,t1n6₂2/eds5 and t1n6₂2/Nah G were inoculated with Pst DC3000, expression level of ICS1, PR1, JAR1 and PDF1.2 were measured by RT-PCR. We confirmed that ICS1 and PR1 were increased in t1n6₂2/eds5, but lower than WT level. Expression of ICS1 in t1n6₂2/Nah G was increased at 0.5 h quickly and then decreased gradually. PR1 was increased at 2 h significantly with SA accumulation, and then decreased significantly, and greatly lower than wild-type level. These results indicated that T1N6₂2 regulated resistance of Arabidopsis by affecting SA accumulation as a negative regulator.4. We successfully constructed subcellular localization vector GFP-T1N6₂2. Using agrobacterium transformation to transform GFP-T1N6₂2 vector into tobacco. With the CLSM, we can definite that gene T1N6₂2 is positioned in the cytoplasm and nucleus.5. We successfully constructed tissue localization vector T1N6₂2pro::GUS, which was transformed into Arabidopsis thaliana. We found T1N6₂2 expresses in the hypocotyls,roots, leaves, sepals and anthers by GUS Staining.6. We successfully constructed prokaryotic expression vector GST-T1N6₂2, which was transformed into BL21. By the way mentioned above, we can definite the best conditions（IPTG 0.1 mmol/L, induced in 3 h） of the prokaryotic expression of T1N6₂2 protein.Then we get the purified T1N6₂2 protein with GST affinity purification.7. We successfully constructed expression vector FLAG-T1N6₂2, which carries FLAG-tag. The vector lays a foundation for further Ch IP researching.