Cloning And Functional Analysis Of CmLOX08 In Drought And Salt Stresses From Oriental Melon(Cucumis Melo Var. Makuwa Makino)

The oriental melon(Cucumis melo var.makuwa Makino)is an annual herb of Cucurbitaceae melo.In protected cultivation,its yield and quality are seriously affected by abiotic stress.Previous studies have shown that Lipoxygenases(Lipoxygenase,LOXs)play significant roles in abiotic stress responses.The study of LOX genes can provide theoretical basis to improve the stress resistance of the oriental melon by gene engineering.A lot of LOXs have been found in plants in recent decades,but there is relatively little known about oriental melon LOXs.In this study,the gene coding sequence of CmLOX08 was cloned by PCR and analyzed by bioinformatics,using the oriental melon cultivar Yumeiren as material.The subcellular localization of CmLOX08 was identified by transient expression of tobacco.CmLOX08 promoter were analyzed by using the online software PlantCARE and PLACE.The function of CmLOX08 promoter was characterized by tobacco transient expression method.The function of CmLOX08 in drought and salt stress was explored by using virus-induced gene silencing(Virus-induced gene silencing,VIGS)CmLOX08,and CmLOX08 was overexpressed in Arabidopsis thaliana to further verify its function.The main results are described as followings:1.The full-length cDNAs of the oriental melon were cloned from melon leaves.The CmLOX08 harbours a 2790-bp open reading frame(ORF),which consists of 929 amino acid residues with a calculated molecular mass of 104.93 kDa and a theoretical isoelectric point(pI)of 8.44.CmLOX08 protein has the conserved LOX domain of the lipoxygenase family and domain of PLAT LH2 and LH2 related to lipoxygenase.So,CmLOX08 is predicted that is 13-LOXs.We constructed vector by using Infusion method and transferred into Agrobacterium tumefaciens GV3101.CmLOX08 protein was found to be located on cell membrane through transient expression in tobacco leaves.2.CmLOX08 promoter fragments which is 2054 bp were achieved by PCR strategy.The cis-acting elements on the promoter sequence were predicted by bioinformatics software.The CmLOX08-pro sequences achieved above and GeLOX08-pro from the melon genome database share 99.61%identity with each other.The putative cis-regulatory elements of CmLOX08 promoter was analyzed by PlantCARE and PLACE databases and thirty types of potential cis-acting elements responding to signalling molecule and abiotic stress were found,such as ABRE responding to ABA,MYB binding sites related to drought response and GT1GMSCAM4 responding to salt stress,etc.3.Five 5 deletion expression vectors of the CmLOX08 promoter was constructed based on position of cis-acting elements.After the 5 deletion expression vectors of the CmLOX08 promoter transiently expressed in tobacco leaves,the leaves were treated by hormone and environmental stress.The results show that signal substances abscisic acid(ABA),salicylic acid(SA)and hydrogen peroxide(H2O2)can regulate the activity of CmLOX08 promoter.However,no deletion fragment GUS activity was induced by methyl jasmonate.In response to salt,drought,and wounding treatments,-1047 to-1 bp showed the highest GUS expression,indicating that-1047 to-1 bp is the major region regulating promoter activity Similarly,-1284-to-1-bp and-1047-to-1-bp region is a core sequence responding to heat and cold,respectively.The above results show that activity of the CmLOX08 promoter is regulated by various signalling molecules and abiotic stresses,and that the promoter generally functions as a signalling molecule/stress-inducible promoter.4.CmLOX08 was silenced using VIGS technology and melon cotyledon injection method;We constructed overexpression vector of CmLOX08 and transformed into Arabidopsis thaliana by inflorescence infiltration method.The drought treatment of silent plants and CmLOX08 transgenic Arabidopsis showed that compared with the control plants,CmLOX08 silent plants had lower LOX activity and expression level of stress genes,increased the accumulation of hydrogen peroxide,and were more sensitive to drought stress.On the contrary,overexpression of CmLOX08 enhanced the resistance of Arabidopsis to drought stress,reduced oxidative damage and increased the expression of LOX activity and stress response genes.Therefore,CmLOX08 may enhance drought resistance by regulating LOX activity,hydrogen peroxide accumulation and stress gene expression level5.Salt stress treatments on silent plants and CmLOX08 transgenic Arabidopsis showed that compared with the control plants,the LOX activity and expression of stress response genes in silent plants were inhibited,the accumulation of hydrogen peroxide increased,and the resistance to salt stress was reduced However,the LOX activity and expression level of stress genes in transgenic CmLOX08 Arabidopsis were always higher than that in WT,and the accumulation of hydrogen peroxide was lower.In addition,silence of CmLOX08 significantly reduced JA content in leaves,while overexpression of CmLOX08 significantly increased JA content in leaves.These results suggest that CmLOX08 may enhance the resistance to salt stress by regulating LOX activity,hydrogen peroxide accumulation,stress gene expression and JA levelAbove all,The CmLOX08 harbours a 2790-bp open reading frame(ORF)and locates on cell membrane.Activity of the CmLOX08 promoter is regulated by signalling molecules(ABA,SA and H2O2)and abiotic stresses(drought,salt,wounding,heat and cold).CmLOX08 may enhance drought and salt resistance by promoting LOX activity,stress gene expression level and the ability of scavenging reactive oxygen species.In addition,the improvement of salt resistance is related to JA level.