| Cotton verticillium wilt is caused by Verticillium dahliae, a kind of soil-borne fungal pathogen, with long latent period. It can cause a harm to growth and development of a variety of plants, especially cotton. V. dahliae renders cotton to decrease the yield and fiber quality, resulting in serious economic loss.Plants have acquied the highly conservative defense mechanism against pathogens in the long process of evolution through the interaction with pathogens.Cotton BOP genes involved in abscission zone differentiation regulation and defense response. GhBOP1/2 have low expression level, and can be significantly induced by verticillium dahliae infection. At present, the BOP genes of Arabidopsis and tobacco have been characterized in many reports, but its function and molecular mechanism in cotton remains to be addressed. Thus, we performed this study, and major resultswere shown as following:1. The GhBOP2 gene was clone from upland cotton by using RT-PCR technique.the full-length cDNA sequence of GhBOP2 contains 1449 bp, encoding 482 amino acids. The protein structure contains a BTB domain and two ANK domains, which has high similarity with other homologous protein, indicted that its evolution is quitely conservative.2. the virus-induced gene silencing vectors, pYL156-GhBOP1’,pYL156-GhBOP2’, and pYL156-GhBOP1’2’, were constructed, and introduced into Agrobacterium tumefaciens GV3101 strain by electric shock. Agrobacterium cultures were then injected in the cotton cotyledon before leaf emerged. Two weeks after inoculation, the positive control plants silenced GhPDS, a marker gene associated with chloroplast biosynthesis, showed albino in leaves. We extracted the RNA of silencing plants and the control to carry out semi-RT-PCR analysis. The results show that the expression level of GhBOP1 and GhBOP2 in all silenced plants were reduced.3. Inoculating V. dahliae V991 to the silenced cotton plants and the control,,compared with the control, the plant resistance to V. dahliae was weakened in plants silencing GhBOP1 or/and GhBOP2 12 days after inoculation. There is similardefense-sensitive phenotype between plants silencing GhBOP1 or GhBOP2, while the plants silencing both genes showed more sensitive to pathengens compared to those silencing single gene. Besides, the silenced plants occasionally grow a little leaves on the petiole, which suggested GhBOP also participates in differentiation and development of leaf.4. We constructed the plant expression vector pBin438-GhBOP1, and introdued it into Agrobacterium LBA4404 strain. The transgenic tobacco plants were developed by leaf discs transformation method. Inoculating pathogen assays showed that transgenic plants conferred high resistance against pathogens.5. We constructed the prokaryotic expression vectors of pET28a-GhBOP1 and pGEX6p-1-GhNPR1. the recombinant GhBOP1 protein with His tag and GhNPR1 protein with GST tag were incuced and purified respectively, whose molecular weight is 53.6 and 65.5 kDa.6. To further investigate the GhBOP function, we emplied the luciferase complementary test(LCI) to analyze the protein interaction. We successfully constructed the vector NLuc-GhBOP1, and the vector CLuc-GhNPR1/TGA1/TGA2/TGA3/TGA4/TGA6, which were then introduced into Agrobacterium GV3101 strain. The tobacco leaves were injected with Agrobaterium culture containing responding vectors with non-needle syringe. 48 hours after inoculation, collecting data from detached leaves through the fluorescence scanning system, the results suggested GhBOP1 protein can interact with GhNPR1 protein, and can also interact with TGA1, TGA2, TGA3, TGA4, TGA6 transcription factors.In conclusion, the GhBOP1 and GhBOP2 can synergetically affect cotton resistance against V. dahliae. Both genes silenced will highly increase desease-sensitive against pathogens. GhBOP1 responses to pathogen investion possibly through interaction with NPR1 protein or TGAs. GhBOP gene not only has a certain disease resistance, but also regulates leaf growth and development. |
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