Isolation And Fuctional Analysis Of A Glutathione-S-Transferase Gene Family From Lilium Regale Wilson

Plants often suffered from diverse abiotic and biotic stresses,such as phytopathogens,drought,chilling,high salt,and heavy metals.Biotic and abiotic stresses cause a large amount of reactive oxygen species(ROS)in plant cells.However,the excessive ROS are particularly harmful to plant cells,and they rapidly inactivate enzymes,cause peroxidation of lipids,damage nucleic acids,inhibit DNA and protein sythesis,destroy membranes,and ultimately lead to cell death.Glutathione S-transferases(GSTs)are ubiquitous enzymes in animals and plants with a variety of functions encoded by a large gene family.GSTs catalyze the conjugation of GSH to electrophilic molecules in cells,thereby reducing the level of ROS,and maintain the balance of the intracellular redox state.In this study,the full-length cDNA sequences of 9 GST genes in Lilium regale Wilson were obtained with the method of rapid amplification the cDNA end(RACE),based on the 9 expression sequence tags(ESTs)from a suppression subtractive hybridization cDNA library of L.regale infected by Fusarium oxysporum.The bioinformatics analysis was performed to understand the gene family of GSTs and the properties of the putative GST proteins.The quantitative reverse transcription-PCR(QRT-PCR)analysis was used to detect the expression characteristics of the L regale GST gene family.We constructed a plant expression vector of LrGSTU5 and transferred it into tobacco(Nicotiana tabacum L.cv Xanthi)by agrobacterium tumefaciens-mediated transformation.Finally,we studied the functional of LrGSTU5 by analysis of the LrGSTU5 overepressed T1 tobacco.The results of the present research are as follows:1.Nine full-length cDNA sequences of GST gene were isolated by RACE,and made fully bioinformatics analysis for understanding the gene and its coded protein properties.The full-length cDNAs of L.regale GST genes ranged from 828 bp to 1076 bp,and the predicted polypeptides of LrGSTs were from 216 amino acid residues to 239 amino acid residues.Additionally,a phylogenetic analysis grouped the L.regale GST genes into 4 classes including 5 tau GSTs,1 lambda GST,1 theta GST,and 2 phi GSTs.2.QRT-PCR analysis revealed the expression patterns of the L regale GST genes family,and they showed different expression profiles in the vegetative organs.The expression of LrGSTF2,LrGSTL1,LrGSTU2 and LrGSTU3 was up-regulated after treatment with SA,JA,ET,and H2O2,but LrGSTFl and LrGSTT1 were down-regulated.The ET slightly induced the the L.regale GST genes,meanwhile,the SA evidently repressed the expression of the L.regale GST genes.The expression patterns of the 9 novel L.regale GST genes in resistant L.regale and susceptible Lilium Oriental Hybrid ’Siberia’ were rather different after inoculation with Fusarium oxysporum.Three genes LrGSTF1,LrGSTF2 and LrGSTU2,showed relatively high expression levels in L.regale-F.oxysporum interaction comparison to ’Siberia’-F.oxysporum interaction.Moreover,they showed similar expression patterns in L.regale-F.oxysporum interaction,and all of them were evidently induced by F.oxysporum in L.regale with the highest expression levels at 12 hpi.However,LrGSTU1 and LrGSTU3 had relatively high expression levels in ’Siberia’-F.oxysporum interaction.3.pCAMBIA2300S-LrGSTU5 was constructed for stable tansformation of tobacco.Then LrGSTU5 transgenic plants were generated through A.tumefaciens-mediated transformation and 30 positive transgenic plants were obtained by PCR.QRT-PCR analysis demonstrated that the LrGSTU5 gene was steadily expressed in the T1 transgenic tobacco lines.Antifungal activity of the crude protein extracts of the LrGSTU5 transgenic tobacco lines showed different levels of resistance against Botrosphaeria dothidea,F.oxysporum,Phomopsis sp,and Alternaria sp.Subsequently,the 3 selected LrGSTU5 transgenic tobacco lines displayed enhanced resistance to F.oxysporum and drought stress in vivo assay.In addition,overexpression of LrGSTU5 in the transgenic lines significantly increased the activities of superoxide dismutase(SOD),GST,and ascorbate peroxidase(APX),but reduced the rate of superoxidate anion production and degree of membrane lipid oxidation.Therefore,the LrGSTU5 transgenic tobacco enhanced resistance to stresses.In conclusion,the members of L.regale GST genes family not only were induced by several signaling molecules,but also participated in defense reaction against F.oxysporum infection.The LrGSTU5 was induced by ET and SA,and the overexpression of LrGSTU5 in transgenic tobacco increased activities of several antioxidant enzymes and enhanced F.oxysporum and drought stress resistance.Therefore,the cloning and function analysis of L.regale GST gene family helped to further understand the molecular mechanism of resistance in L.regale additionally,this study supplied several genes for plant stress-tolerance gene engineering.