Cloning And Identification Of Herbivore Resistance-related Genes GhJMTs In Cotton(Gossypium Hirsutum)

Methyl jasmonate(Me JA)and jasmonic acid(JA)are naturally occurring compounds known to be produced by many plants,they are major phytohormones that contribute to plant growth and development as well as plant defense.Me JA is biosynthesized from JA by the action of Sadenosyl-L-methionine: jasmonic acid carboxyl methyltransferase(JMT),which belongs to the methyltransferases of SABATH family.JMT has been shown to play important roles in plant defenses.JMT genes have been isolated and characterized from many plant species including rice(Oryza sativa),arabidopsis(Arabidopsis thaliana)and tomato(Solanum lycopersicum).However,up to now JMT gene in cotton has not been identified.In this study,we cloned two jasmonic acid carboxyl methyltransferase genes from Gossypium hirsutum,performed in vitro enzyme assays,and studied their roles of Gh JMTs in the interactions between cotton(G.hirsutum),cotton bollworm(Helicoverpa armigera),and parasitoid(Microplitis mediator).The main research contents and results are as follows:1.Two novel JMTs gene was identified and cloned from leaves of G.hirsutum cv.Zhongmian 12,named as Gh JMT1 and Gh JMT2.Gh JMT1 encoded a protein comprised of 371 amino acids with a predicted molecular masses of 41.5 and an isoelectric points were 5.83,Gh JMT2 encoded a protein comprised of 372 amino acids with a predicted molecular masses of 41.7 k Da and an isoelectric points were 5.96.No signal peptide and transmembrane helices could be predicted for Gh JMT1 and Gh JMT2.Multiple sequence alignment results revealed that Gh JMT1 and Gh JMT2 had conserved motifs and domains typical for JMTs and shared high identity with the JMTs from other plant species.The deduced amino acid sequence of Gh JMT1 shared 57%-65% identity with those of JMT from strawberry,arabidopsis and black cottonwood.The amino acid sequence of Gh JMT2 shared 54%-64% identity.Meanwhile,Gh JMT1 and Gh JMT2 shared 72.7% identity.2.Using headspace collection coupled with GC-MS analysis,quality and relative quantity of cotton volitailes induced by plant hormones Me JA and Methyl salicylate(Me SA)were measured.A total of 21 volatile compounds were detected from Me JA-induced cotton plants.Among these compounds,12 compounds were only detected from Me JA-induced cotton plants while two compounds showed elevated emission levels.When induced by Me SA,volatile emission was less than for plants treated with Me JA.A total of 12 volatile compounds were detected from Me SA-induced cotton plants,and three compounds were de novo synthesized.3.To investigate the catalytic activity of Gh JMT1 and Gh JMT2,the genes were heterologously expressed in Escherichia coli and the proteins were assayed with the potential substrates.Ethyl acetate extracts were analyzed by GC-MS.The results showed that the recombinant Gh JMT1 and Gh JMT2 converted JA into Me JA with the SAM as methyl donor,but they did not catalyze other substrates including salicylic acid(SA),4-hydroxy-benzoic acid and cinnamic acid.4.We used real-time q PCR to further analyze the expression levels of Gh JMT1 and Gh JMT2 after H.armigera feeding.The results showed that the expression of Gh JMT1 and Gh JMT2 was significantly up-regulated after 6h upon onset of herbivory,indicating that Gh JMT1 and Gh JMT2 might play roles in cotton defense against H.armigera.The expression levels of Gh JMT1 were higher than that of Gh JMT2 after herbivory,which indicated that Gh JMT1 and Gh JMT2 were differently regulated in cotton.Furthermore,the transcript levels of Gh JMTs in different tissues of cotton plants treated with plant hormones Me JA and Me SA were analyzed.While Gh JMT1 was up-regulated in both leaves and stems after treated with Me JA,Gh JMT2 was up-regulated only in leaves.However,Gh JMT1 and Gh JMT2 transcript abundance was not influenced by Me SA treatmeat.5.The electroantennogram(EAG)responses of H.armigera moths and M.mediator to standard Me JA and three important volatile compounds,which were realeased by cotton plants induced by Me JA,were also detected.The results showed that H.armigera and its natural enemy effectively identified were responsive to Me JA,β-ocimene,(Z)-3-hexenyl acetate and linalool.Excitingly,M.mediator females were more responsive to Me JA than males,suggesting that Me JA might play a role in host habitat location of female M.mediator.