Identification And Functional Analysis Of Genes Involved In Jasmonate Biosynthetic And Signaling Pathways In Mulberry (Morus Notabilis)

Phytohormones are a class of metabolites produced by plants,which have the characteristics of high effect in low concentrations.Phytohormones have a broad spectrum of physiological effects and they play important roles in growth and development processes(cell division,tropic growth,organ formation,flowering,seed development,senescence),as well as environmental responses(such as high temperature,salinity,pests and diseases).Jasmonate is a kind of cyclopentanone phytohormone which was first separated from fungal fitrate.It is widely distributed in various organs of plants.Extensive research have already shown that jasmonate plays prominent role in plants defense responses against herbivore insects.So far,the mechanism of jasmonate biosynthesis and signal transduction has already been elucidated in model plants.Mulberry is a perennial woody plant,in addition to the traditional sericulture,the role of mulberry in ecology,health care and medicine is also very prominent.However,in natural conditions,mulberry pests are very serious,the traditional pesticide spraying is not only easy to cause pest resistance,but also harmful to human health.Therefore,it is of great significance for mulberry industry to improve the ability of pest resistance in mulberry.In this study,we identified the genes involved in the jasmonate biosynthetic and signal transduction pathways in mulberry(Morus notabilis)genome by bioinformatic method,then conducted bioinformatic analysis.Subsequently,the seedlings of M.notabilis were treated with the silkworm feeding and mechanical wounding,which were then used as materials for transcriptome sequencing.We analysed the expression level of genes and the dynamic changes of phytohormone-related genes in M.notabilis under two kinds of treatment.Afterwards,we screened the interacting proteins of MnNINJAl and MnNINJAs through yeast two hybrid.Finally,we analysed the functions of MnNINJAl and MnNINJAs through transgenic Arabidopsis.The results of this study were as follows: 1.Identification and bioinformatic analysis of genes involved in the biosyntheticand signaling pathways in M.notabilisWe identified 74 genes involved in jasmonate biosynthetic and signaling pathways in the genome of M.notabilis by bioinformatic methods.Among them,39 genes participate in the jasmonate biosynthesis,while the remaining 35 genes invloved in the jasmonate singaling pathway.Domain analysis demonstrated that these genes were conserved in M.notabilis,hierarchical and medians clusterings indicated that these genes showed tissue-biased expresson pattern.OPR is a key enzyme in jasmonate biosynthetic pathway.In the genome of M.notabilis,we identified 7 OPR genes,which were tandem arranged in three scaffolds.Phylogenetic analysis showed that two OPR genes may catalyze the biosynthesis of jasmonate in M.notabilis.Meanwhile,we identified a class of OPR proteins belonging to Group III which was not indentified before,the genes in Group III were all came from monocotyledon plants.Cis-acting elements analysis showed that the MnOPR genes could respond to various phytohormone treatments and environmental stresses.JAZ is the repressor in jasmonate signal transduction pathway.We identified six MnJAZ genes in the genome of M.notabilis.Compared with other reported plant species,mulberry owned the minimum number of JAZ genes with lower redundancy.All six MnJAZs were cloned,compared with that in Arabidopsis,the structures of MnJAZs were found to be very conserved.Sequence alignment revealed that the truncated form of JAZ also exist in mulberry.Multiple sequence alignment and phylogenetic analysis showed that JAZ proteins contained conserved TIFY and Jas domains except for MnJAZ4,both of them can be separated by a conserved intron in mulberry,respectively.Quantitative PCR analysis showed that six MnJAZ genes had different expression patterns.In the genome of M.notabilis,we identified the homologous gene of AtNINJA,but the length of this gene was much shorter than AtNINJA,and the protein encoded by this gene did not contain the conserved EAR motif.Based on these,we conducted molecular cloning by means of designing degenerate primers and finally cloned the full length MnNINJA.Multiple sequence alignment showed that this gene was highly conserved in plants.At the same time,we cloned the predicted MnNINJA gene by using cDNA template of M.notabilis.Sequence alignment revealed that the existance of alternative splicing in MnNINJA,therefore we named the the two transcript forms as MnNINJAl and MnNINJAs,respectively.2.Transcriptome analysis of M.notabilis under silkworm feeding and mechanicalwoundingThe seedlings of M.notabilis were treated with the silkworm feeding and the mechanical wounding.After 1 h,8 h,and 16 h(respectively indicated the early,middle and late stages),the leaves of seedings were collected for transcriptome sequencing.The fold change value of expression level after treatment above 1.5 was used to selected the differentially expressed genes.The sequencing results showed that the number of differentially expressed genes reached maximum at 8 h time piont.Clustering analysis indicated that the expression patterns of differentially expressed genes were similar at 1 h and 8 h time points,but at 16 h time point,there was an obvious difference between feeding and wounding treatment.KEGG enrichment analysis showed that at 1 h time point after treatment,alpha-linolenic acid pathway was significantly enriched,while at 8 h and 16 h time point,photosynthesis-related pathways were mostly enriched.We analysed the differentially expressed genes invloved in the jasmonate biosynthesis and signal transduction pathways in transcriptome data.We found that the expression level of MnAOS1、MnAOC1、MnOPR6、MnLOX2C、MnOPCL1 were strongly induced at 1 h time point after treatment,which was consistent with KEGG enrichment analysis.In the jasmonate signal transduction pathway,the expression level of MnMYC2 only showed a significant increase at 1 h time point,while MnJAZ1、MnJAZ3、MnJAZ4 were upregulated significantly at all time points.The transcript level of MnJAZs under silkworm feeding indicated the reliability of transcriptome data.We then conducted clustering analysis for the genes invloved in other phytohormone pathways under feeding and wounding treatment.The results showed that genes involved in the biosynthesis and signal transduction of gibberellin,ethylene,salicylic acid,abscisic acid were strongly induced in response to insect feeding and mechanical wounding.Among them,the majority of genes involved in gibberellin and Auxin signal transduction pathways showed decreased expression levels.Because we did not identifed MnNINJAl in transcriptome data,we detected the expression level of MnNINJAl in response to feeding treatment at 0 h、1 h、0.5 h、1 h、2 h、4 h、8 h、12 h time points.The results of quantitative PCR revealed that transcript level of MnNINJAl decreased firstly and then increased,and the expression pattern were similar between the damaged and undamaged sites.These results indicated MnNINJAl played an inhibitory role in the jasmonate signaling pathway.Phytohormones treatment showed that besides methyl jasmonate,MnNINJAl could respond to salicylic acid,gibberellin and ethylene.Western Blot analysis showed that MnNINJAl expressed in eight tissues of M.notabilis with the highest expression level in young leaves,which was not consistent with its transcript level.However,we could not detected the expression of MnNINJAs protein in all tissues of M.notabilis.3、Functional analysis of Mn NINJAl and MnNINJAsWe identified the proteins interacting with MnNINJAl and MnNINJAs by yeast two hybrid.Finally,through yeast mating method,we screened 6 clones(including eight genes)and 3 clones(including two genes)interact with MnNINJAl and MnNINJAs,respectively.After validation,only the number 14、52、99、100 were positive clones for MnNINJAl,others were self-activated or fase positive,while three clones interacted with MnNINJAs were both positive.In yeast two hybrid screening,MnJAZ4 and MnJAZ5 interacted with MnNINJAl,while MnJAZ1 and MnJAZ3 interacted with MnNINJAs,so we constructed all six MnJAZs prey plasmids and conducted yeast two hybrid experiment.The results indicated that all six MnJAZs proteins interacted with MnNINJAl,for MnNINJAs,it had a weak association with MnJAZ4,but could interact with the remaining five MnJAZs.We analysed two positive clones numbered 99 and 100,they encoded SUMO conjugating enzyme E2(SCE)and 4-coumarate-Co A ligase-like 5(OPCL),respectively,therefore we named them as MnSCE1 and MnOPCL1.OPCL1 participates in jasmonate biosynthesis,the expression level of MnOPCL1 was significantly increased when mulberry was subjected to silkworm feeding and mechanical wounding.SCE1 involved in sumoylation process.Studies showed that AtSCE1 played important roles in abscisic acid responses,embryo development ending in seed dormancy,temperature responses and the process of virus infecting plants.By means of plant transgenic technology,we overexpressed MnNINJAl and MnNINJAs in model plants Arabidopsis thaliana.Quantitative PCR and Western Blot analysis indicated that MnNINJAl and MnNINJAs were successfully overexpressed in Arabidopsis.Compared with wide type,transgenic Arabidopsis did not show significant difference in phenotype.Therefore,we selected jasmonate-responsive genes AtPDF1.2 and AtVSP2 for quantitative PCR experiment.The transcript level of AtVSP2 was significantly downregulated in all transgenic Arabidopsis lines overexpressed MnNINJAs.For AtPDF1.2,the expression level was not affcted by MnNINJAs.MnNINJAl could upregulated the transcript level of AtPDF1.2.For AtVSP2,MnNINJAl could upregulate its expression level in a certain concentration,but it will repress its expression when MnNINJAl accumulated to a amount above the threshold.Meanwhile,overexpression MnNINJAl and MnNINJAs in Arabidopsis affected the transcript levels of other phytohormones responsive gene.The expression levels of salicylic acid and abscisic acid responsive gene AtPR-1 and AtERD10 showed an upregulation in transgenic Arabidopsis,while that of auxin downstream genes AtIAA11 and AtARF8 showed a downward trend in all transgenic lines.The expression levels of gibberellin-responsive genes AtGASA1 and AtGASA6 were also significantly affected by MnNINJAl and MnNINJAs in Arabidopsis.In our study,we identified 74 genes involved in the jasmonate biosynthesis and signal transduction in the genome of M.notabilis via bioinformatic methods.Domain analysis,phylogenetic analysis,multiple sequence alignments and gene structures demonstrated that these genes in M.notabilis have highly homology to that in reported plant species,implying they play a similar role in M.notabilis.Transcriptome analysis of M.notabilis under feeding and mechanical wounding treatments indicated that differentailly expressed genes shared similar expression pattern both in early and middle stages,while in late stage,they showed greater difference.Meanwhle,these two kinds of treatments activated jasmonate biosynthesis and signal pathways with large amount of genes strongly been induced.Expression levels of genes in other phytohormones pathways showed up-or down-regulation to different extent.The expression pattern in response to feeding and hormone treatment as well as the results of yeast two hybrid screen showed that MnNINJAl was able to respond to other hormones and physiological processes in addition to the jasmonate signaling pathway.Transgenic experiments demonstrated that heterologous expression of MnNINJAl and MnNINJAs had a significant effect on the expression levels of genes downstream of phytohormones like jasmonic acid,salicylic acid,abscisic acid,auxin and gibberellin in Arabidopsis.