Molecular Mechanism Of Induced Resistance In Populus Simonii ×p.Nigra By Lymantria Dispar Herbivory Based On Transcriptome And Metabolome Analysis

Populus simonii×P.nigra is an important greening tree species in the Three North region,and it is also the main tree species in the northern windbreak forest,which has good wind and sand fixation effect.Lymantria dispar is the worldwide forest pest,which is widely distributed and omnivorous,causing huge economic losses in forestry production.Therefore,to explore the interaction mechanism between P.simonii × P.nigra and L.dispar,and dig out the functional genes of P.simonii × P.nigra resistance to L.dispar herbivory,provide a basis for the effective control of L.dispar and breeding of insect resistant varieties.This study will be imporant to both in theory and practice.In this paper,transcriptome and metabolome were used to analyze the differentially expressed genes and metabolites in P.simonii× P.nigra responding to L.dispar herbivory and mechanical wounding,and then the expression of key differential genes and enzymatic activities were further determined.The specific research results are as follows:1.BGISEQ-500 high-throughput sequencing platform was used to construct the transcriptome library of P.simonii × P.nigra leaves treated by herbivory and mechanical wounding.The 62.43Gb data were obtained by sequencing.After filtering,70116 Unigenes were assembled with an average length of 1567bp.Finally.62326(NR:88.89%),66225(NT:94.45%),47332(Swissprot:67.51%),49093(KOG:70.02%),48222(KEGG:68.77%),47802(go:68.18%)and 48414(Pfam:69.05%)Unigenes were functionally annotated according to the functional database.By comparing and analyzing the differentially expressed genes,it was found that the number of differentially expressed genes between the L.dispar herbivory group and the control group were the highest(12152),while the number of differentially expressed genes between L.dispar herbivory group vs the mechanical wounding group and between the mechanical wounding group vs the control group were only 7935 and 6440,respectively.Among these differential genes annotated according to GO functions.The 138372 Unigenes annotated in molecular functions were the most,followed by biological process(105094),the least is cellular component(82586).The 3666 differential genes related to herbivory and 1171 differential genes related to mechanical wounding were screened out.2.Based on LC-MS non-targeted metabolomics method,the metabolites and its contents were identified in leaves of P.simonii × P.nigra by herbivory and mechanical wounding.The XCMS software was used to extract the metabolite ion peaks.The detected material peaks had 9108 ion peaks in positive mode and 7656 ion peaks in negative mode.Principal component analysis(PCA)combined with t-test statistical method was used to classify,discriminate and screen the metabolites.By sorting the contribution of metabolites to the second principal component(PC2),we screened out the different metabolites related to the specific L.dispar herbivory group,which are ubiquinone(37.62-flod)and cimicifugoside(53.89-fold),and the different metabolites related to the specific mechanical wounding were arbutin(59.9-fold),strictosamide(2.17-fold).3.According to the analysis of KEGG metabolic pathway,these differential genes are mainly enriched in phenylpropanoid biosynthesis pathway(ko00940)and flavonoid biosynthesis pathway(ko00941)based on transcriptome and metabolome.Among them,38 differentially expressed genes were enriched in phenylpropanoid biosynthesis pathway(ko00940)while 30 differentially expressed genes were enriched in flavonoid biosynthesis pathway pathway(ko00941).A total of 52 and 45 differentially expressed genes were upregulated in L.dispar herbivory group and mechanical wounding group,respectively.And the expressions of cinnamoyl-CoA reductase gene(CCR-1,CCR-2),dihydroflavonol 4-reductase gene(DFR),flavanone 3-hydroxylase gene(F3H),flavonol synthetase gene(FLS),leucoanthocyanidin reductase gene(LAR),chalcone synthetase gene(CHS-1,CHS-2),peroxidase gene(POD)and cinnamyl-alcohol dehydrogenase gene(CAD)were induced up-regulation by L.dispar herbivory,while those of LAR and CAD were inhibited down-regulation.The activities of CCR,DFR,FLS,CHS,POD and CAD in P.simonii ×P.nigra were induced by L.dispar herbivory and mechanical wounding.The activity of CHS and DFR in L.dispar herbivory group was the most significant,which was 6.91?5.56 fold of those in the control group,while DFR and FLS in the mechanical wounding group were the most significant,which were 2.30-2.31 fold of those in the control group.L.dispar herbivory and mechanical wounding can induce CCR,DFR,FLS,CHS,POD,and CAD enzyme activities.However,FLS and LAR enzymatic activities did not change significantly after L.dispar herbivory and mechanical wounding treatments.These results showed that the related synthetases and genes in phenylpropanoid biosynthesis(ko00940)and flavonoid biosynthesis(ko00941)pathways were involved in the defensive response to the herbivory and mechanical wounding.The research results have enriched the research of P.simonii × P.nigra anti-insect function,and provided theoretical basis and new ways for the control of L.dispar and the selection of insect-resistant varieties in forestry engineering.