Analysis Of GhCOI1 And GhMYC2 By VIGS In The Cotton Resistance Against Verticillium Wilt

In order to deal with biotic or abiotic stress,plants have obtained the corresponding physiological functions in the long evolution to carry out the response or defense function.As a kind of metabolic substance,plant hormones play an important role in the process of growth and development.Jasmonic acid(JA)and its methyl jasmonate(MeJA)is an important signal molecule in plants.It can activate the activity of many related transcription factors and regulate the plant defense response.At present,there are many reports on the upstream regulatory proteins,transcription factors and downstream effector genes in the process of JA signal transduction.However,the study of JA signal transduction in crops,especially in cotton and other economic crops,needs to be further studied.Coronatine-insensitive 1(COI1)is a key regulator of jasmonic acid signaling pathway.In Arabidopsis thaliana,the COI1 deletion mutant showed no susceptible or response to jasmonic acid,and could not initiate the corresponding response when infected by the pathogen.The results show that MYCs(Myelocytomatosis proteins,MYCs)is a key transcription factor in the JA response pathway,and is widely found in animals and plants.MYC2 transcription factor is a member of the plant bHLH transcription factor family,which plays an important role in JA induced response and JA signal transduction.Virus-induced gene silencing(VIGS)is a kind of technology that can be used to study the function of the gene,which is able to induce the endogenous gene silencing.We use bioinformatics cloned the COI1 and MYC2 in cotton,and through VIGS technology to silence the expression of these two genes to analyze the resistance reaction of Verticillium dahliae V991.We determined their disease resistance in cotton,and provided a theoretical basis for the breeding of cotton and molecular mechanism of plant response to stress.The results were shown as following:1.Through the NCBI database(http://www.ncbi.nlm.nih.gov/),we cloned the full-length sequence of COI1 and MYC2 genes in cotton.The full length of the target gene was amplified by using cotton genomic cDNA as template,and two genes of GhCOI1 and GhMYC2 were cloned by enzyme digestion and sequencing analysis,the specific primers designed by DNAMAN program.2.We analyzed the tissue specificity of GhCOI1 and GhMYC2 gene expression,and the results showed that the two genes had the highest expression in roots.Theresults showed that GhCOI1 and GhMYC2 gene expression induced by Verticillium dahliae infection.The highest expression level of GhCOI1 was observed after inoculation with 12 h,and the highest expression of GhMYC2 was observed after inoculation with 3h.The expression of these two genes decreased with the increase of time.3.Using DNAMAN program to design specific primers,we amplified the target gene sequences.The target sequence was inserted into the virus vector pYL-156.After restriction enzyme digestion and sequencing,we obtained recombinant vector pYL-156-GhCOI1 and pYL-156-GhMYC2.The vector of pYL-156-GhCOI1 and pYL-156-GhMYC2 were introduced into Agrobacterium tumefaciens GV3101 to obtain the engineering bacteria of VIGS.Inoculating engineering bacteria of VIGS into the cotyledons of cotton seedling without true leaves.We obtained 20 plants with target gene silencing.4.When the control leaves of plants with PDS gene silence appeared white spot,we extracted the total RNA from the control plants and the target gene silencing plants,then obtained cDNA by reverse transcription,and finally detected by RT-PCR and qPCR.The results showed that the expression of target gene was silenced and the gene silencing effect was more than 85%.5.The silencing plants were inoculated with V991,showed decreased resistance to V991 of these two gene silencing plants.Silencing plants showed more susceptible to the disease,while the control plants of Verticillium dahliae have obvious disease tolerance.These results indicated that the target gene involved in cotton resistance to V991,and act as a positive regulator of regulation of cotton disease resistance.