An Oriental Melon Gene CmLOX09 Responded To Stress,Hormones And Signal Substances And Transformated Into Tomato

Lipoxygenase(LOX)is a key enzyme in the pathway of plant eighteen carbonic acid metabolisms which catalyzes lipids and polyunsaturated fatty acids into certain active compounds and extensively involved in plant growth and development,ripening and senescence,biosynthesis of aroma compounds,stress responses and defense reaction.LOXs can catalyze the oxygenation of polyunsaturated fatty acids(PUFA)with a(Z,Z)-1,4-pentadiene structure to form fatty acid hydroperoxides.These hydroperoxides were act as substrates for seven downstream branches of the LOX pathway to form a wide variety of active compounds,collectively called oxylipins.Currently,the hydroperoxide lyase(HPL)and allene oxide synthase(AOS)branches of LOX pathway whose involvement in plant defense has been established.In our previous study,five 13-LOX genes responded to abiotic stress and signal substances,but it is unclear whether the 9-LOX is involved in biotic and abiotic stress.Promoter analysis revealed that motifs of CmLOX09 might participate in gene expression regulated by stresses,hormones and signal substances.We analyzed the expressions of CmLOX09,CmHPL and CmAOS in leaves of four-leaf stage seedlings of oriental melon cultivar 'Yumeiren' under stress,hormons and signal substances,in addition,the contents of Green leaf volatiles(GLVs)and jasmonic acid(JA)were measured after MeJA and inoculated with Podosphaera xanthii treatment,respectively.The results were described as follow:1.Promoter analysis by Plant CARE revealed that CmLOX09 promoter has various cis-regulatory elements,such as light responsive elements(3-AF1 binding site,Box 4,ACE,AT1-motif and so on),hormone elements(p-box element),signal substance elements(ABRE element,TCA-element),fungal elicitor responsive elements(Box-W1 element),and cis-acting element involved in defense and stress responsiveness(TC-rich Repeats).The results indicated that CmLOX09 might response to hormones,signal substances,biotic stress and other stresses.2.We analyzed the expressions of CmLOX09,CmHPL and CmAOS under stress,hormones and signal substances.Real time qPCR revealed that CmLOX09,CmHPL and CmAOS were all induced by wounding.CmLOX09 was induced by auxin(IAA)and gibberellins(GA3),however,CmHPL and CmAOS showed differential responded to IAA and GA3.CmLOX09,CmHPL and CmAOS were all induced by hydrogen peroxide(H2O2)and methyl jasmonate(MeJA),while inhibited by abscisic acid(ABA)and salicylic acid(SA).CmLOX09,CmHPL and CmAOS were all induced by Podosphaera xanthii.3.The emission of 2-hexynol and 2-hexenal at 6h,72h and 168h after MeJA treatment were significantly higher than the control,the results showed that CmLOX09 responded to MeJA might through the HPL pathway to produce C6 alcohols and aldehydes.4.After infection with Podosphaera xanthii,the levels of JA in level 1 and level 3 were significantly higher than the level 0,the results showed that CmLOX09 responded to Podosphaera xanthii might through the AOS pathway to form JA.5.The overexpression vector of CmLOX09 was obtained by Gateway technology;CmLOX09 was transfered into tomato genome using the Agrobacterium-mediated method;T0 transgenic plants were identification by PCR,qRT-PCR,Western blot,vivo fluorescence imager and spraying with 0.000750%Basta.In summary,the results suggested that CmLOX09 might play positive role in plant defense response.CmLOX09 responded to MeJA might through the HPL pathway to produce C6 alcohols and aldehydes.CmLOX09 responded to Podosphaera xanthii might through the AOS pathway to form JA.Two tomato transgenic plants of CmLOX09 overexpression were acquired using the agrobacterium-mediated method.The data generated will strive to lay the foundation to understand the precise roles of CmLOX09 in transgenic plant in defense response.