Functional Analysis Of (E)-β-ocimene Synthase Gene GhTPS16 From Gossypium Hirsutum

Cotton is an important economic crop in China.The damage of insects such as Helicoverpa armigera and Lygocoris lucorum is one of the crux factors that restricts development of the cotton industry.Insect-induced plant volatiles play an important role in both direct and indirect defense of plants,and the use of plant volatile functional components to regulate insect behavior can effectively reduce the population of pests and has a wide range of potential applications.In this study,we firstly isolated a terpene synthase gene GhTPS16 from G.hirsutum cv.CCRI12,and identify the gene function by the prokaryotic expression in vitro and enzymatic reaction.Then,transgenic technology was used to get the transgenic tobacco,and the ecological function of(E)-β-ocimene in the regulation of insect behavior in living plants was further clarified in the model of the tobacco-H.armigera/A.lucorμm-Microplitis mediator/Peristenus spretus.The results are as follows:1.(E)-β-ocimene is the most common terpene component in cotton volatiles induced by H.armigera and A.lucorμm,with the average release rate of 891 ± 2.56 ng/μL.EAG results showed that both H.armigera and A.lucorμm,as well as their natural enemies,M.mediator and P.spretus had done response to the(E)-β-ocimene standard(P < 0.05).The EAG response of female of M.mediator was significantly higher than that of male(0.01 mg/mL,0.1 mg/mL,P < 0.05),and the EAG response of female of P.spretus was significantly higher than that of male(0.01 mg/mL,0.1 mg/mL,1 mg/mL,100mg/mL,P < 0.05),The EAG response of male of H.armigera was higher than that of female(10mg/mL,P < 0.05),and there was no significant difference in EAG responses between females and males.Behavioral bioassays showed that the three insects,A.lucorμm,M.mediator and P.spretus had preferred to(E)-β-ocimene standard.(P < 0.05).and M.mediator and P.spretus can be strongly attracted by(E)-β-ocimene.(P < 0.01)。2.Based on the genome and transcriptome data of G.hirsutum,the cotton(E)-β-ocimene synthase gene GhTPS16 was identified,and the full-length is 1815 bp,belonging to the TPSb subfamily.Then,soluble recombinant protein was obtained by prokaryotic expression and identified in vitro enzymatic reaction.The results shows that the recombinant protein could catalyze the production of(E)-β-ocimene by the substrate GPP,with the average release rate of 243 ± 10.28 ng/μL and couldn’t catalyze the substrates NPP、FPP and GGPP.3.Transgenic Nicotiana tabacum plants overexpressing GhTPS16 were generated.Real-time PCR showed that the expression level of GhTPS16 in the transgenic tobacco was about 1000 to 2500 times higher than that in the wild type tobacco plants.Moreover,transgenic mutant plants release large amounts of(E)-β-ocimene,with the average release rate of 1000 ± 170 ng/μL.Behavioral bioassays showed that the transgenic mutants not only significantly attracted the natural enemies-M.mediator and P.spretus,but also attracted A.lucorμm and the larvae of H.armigera,as well as the oviposition of H.armigera(P < 0.05).