Aluminum Responsive Transcriptome Analysis And Functional Study Of ZjOMT In Zoysia Japonica

With the change of global environment and climate and the improvement of industrialization and urbanization,soil acidification in southern China has become a serious problem,which has caused great harm to plant growth and has a great impact on human living environment and life quality.It is a direct method to cultivate aluminum-resistant plant varieties and apply them in production and life,and it is beneficial for ecology,environmental protection and economy.Zoysia japonica is an excellent warm-season turf grass,widely grown in the vast areas of southern China.It can grow well in acidified soil with high aluminum content.It's an aluminum tolerant plant with large biomass.In order to reveal the molecular mechanism of aluminum tolerance in Z.japonica,the aluminum responsive transcriptome of Z.japonica was analyzed.In addition,the sequence and functional characteristics of ZjOMT,an O-methyltransferase gene in Z.japonica,were studied.The gene was also expressed in prokaryotes and Arabidopsis thaliana.The main results are as follows;(1)analysis of aluminum response transcriptome in Z.japonicaAbout 34743 Unigenes were obtained by sequencing the transcriptomes of Z.japonica treated with aluminum(T1)and control(CK),where 14555 were differentially expressed genes including 4845 down-regulated genes and 9710 up-regulated genes.Among the differentially expressed genes,93.17%are known genes and 314 of the known genes have not been annotated yet.The annotated genes contain a large number of transporter genes,transcription factors,heat shock protein genes,ion channel protein genes,genes involved in redox reaction,pectin synthesis,citric acid synthesis,and other aluminum resistance related genes.The expression levels of XLOC 007305,XLOC 010540 and XLOC 037486 related to ion binding in the unannotated genes were significantly increased under aluminum stress.KEGG enrichment showed that the glycolysis pathway,ascorbic acid metabolism,oxidative phosphorylation,photosynthesis and fatty acid biosynthesis in Z.japonica under aluminum stress were closely related to aluminum stress.(2)cloning and expression analysis of ZjOMT gene from Z.japonicaAn O-methyltransferase gene ZjOMT was cloned from Z.japonica.The ZjOMT coding sequence is 1080 bp,and the encoded protein sequence contains 359 amino acids.Under aluminum stress,the expression of ZjOMT in leaves and roots increased by 20.86 times and 31.18 folds.(3)function of ZjOMT gene from Z.japonicaProkaryotic expression of ZjOMT protein gene in E.coli resulted in an eight-fold increase in melatonin synthesis and a significant increase in aluminum resistance of the recombinant vector.ZjOMT gene was further transformed into Arabidopsis thaliana.The melatonin content of transgenic plants increased significantly.The highest melatonin content of three transgenic A.thaliana lines overexpressing ZjOMT gene was 72 ng/g,which was 4.0 folds higher than that of wild type.The other two strains were 3.2 and 2.7 times higher melatonin content than wild type,respectively.Under 200 ?M aluminum chloride stress,the root lengths of transgenic plants were 20%,35%and 37%less than those of the control,while those of wild-type plants under stress were 60%less than that of wild-type plants.The root lengths of wild type A.thaliana plants exposed to AlCl3 were only 57-78%of those with over-expressed ZjOMT genes.The MDA contents of transgenic plants under 200 ?M AlCl3 stress were 81.1%,73.5%and 64.4%of those in wild-type plants,respectively.The Al tolerance of A.thaliana expressing ZjOMT gene was significantly higher than that of wild type Arabidopsis.The expression of ZjOMT gene can increase the melatonin content in plants,and improve the ability of tolerance to aluminum toxicity.ZjOMT can be used as an effective gene for improving the melatonin content and quality of crops,and also can be regarded as a genetic resource for plant genetic engineering of resistance to aluminum toxicity.