| Panax notoginseng(Burk)F.H.Chen is a rare traditional Chinese medicine in China,P.notoginsengtotal saponins(PNS)with many kinds of biological activity in anti-tumor,antiviral,reduce cholesterol and improve immunity,is an important drug in the prevention of cardiovascular disease.The planting areas of P.notoginsengare mainly distributed in Yunnan province of China,its unique growth environmentis prone to induce plant diseases and insect pests,especially fungal diseases,including root rot disease.Root rot disease mainly caused by Fusarium solani(Mart.)Saccis the main disease of P.notoginseng.The annual incidence rate of root rot disease was 5%-20%,which was as high as 70%,which seriously affected the yield and quality of the raw materials of P.notoginseng.The preliminary study found that MeJA pretreatment P.notoginseng can significantly improve the resistance to F.solani.Therefore,this study using high-throughput sequencing technology tocarry out transcriptome sequencingand gene expression profile analysis of P.notoginseng roots pretreated by methyl jasmonate(MeJA)and sterile water respectively after F.solani infection.Moreover,Cloninga P.notoginsengresistance related gene PnMAPKK1 induced by exogenous MeJA,and analyzing the expression characteristics and function of PnMAPKK1.Which will help understand deeply the molecular interactions between P.notoginseng and F.solani,and molecular mechanisms of jasmonic acid signal pathway mediated P.notoginseng resistance to F.solani.The results of the present research were as follows:1.Transcriptome sequencing produced a total of 179.007 M high-quality reads(9.13 G bases,Q30%>90),then 198358 transcripts(length ? 200 bp,N50 Length of 1766 bp)were generated through clustering analysis,and finally,a unigene dataset with 80834 sequences(N50 of 1055 bp)was formed through sequencing analysis.Using COG,GO,KEGG,Swissprot,nr and NCBIdatabases toannotateall the unigene,36771 unigenes found corresponding homologous sequences in one or more databases.Then the integrated unigene library was used as the reference for mapping analysis of the 12 groups transcriptome reads,more than 90%reads of each library only mapped to unique transcript,and the percentage of multiple mapped reads was lower than 10%.2.The differentially expressed genes(DEGs)in MeJA pretreated P.notoginseng roots at 24 h were compared with that of sterile water pretreated P.notoginseng roots as control,a total of 1108 DEGs were recieved,including 692 up-regulated genes and 416 downregulated genes.In addition,with P.notoginseng pretreatedby MeJA and sterile water respectively as control,analyzing the DEGs of two groups of P.notoginseng after F.solani infection.At 4 h,12 h,24 h and 48 h after F.solani inoculation,the number of up-regulated DEGs is about 500 in MeJA pretreated P.notoginseng roots.At 72 h after inoculation,3680 DEGs were generatedin MeJA pretreatment group,the expression of 3042 DEGs was up-regulated.On the contrary,the number of sterile water pretreatment group less than 1/3 of MeJA pretreatment group,and the expression of most of genes was inhibited.Making venn diagrams for the different comparison combinations of DEGs,founding a large number of MeJA regulated DEGs also response to F.solani infection.3.Conducting annotation and enrichment of KEGG pathway for DEGs,the results showed that the related genes of terpenoid biosynthetic pathway and plant pathogen interaction pathway were regulated by exogenous MeJA.At 4 h after F.solani inoculation,moreplant hormone signal transduction genes were modulated in sterile water pretreated roots.Similarly,at 12 h after inoculation,more plant hormone signal transduction and plant pathogen interaction genes were differentially expressed in sterile water pretreated roots.At 24 h after inoculation,much more genes related with ribosomes,proteins processing in endoplasmic reticulum(ER)and oxidative phosphorylationand pathway were differentially expressed in sterile water pretreated roots.However,at 48 h and 72 h after inoculation,those genes involved in ribosome,phenylpropanoid biosynthesis,plant hormone signal transduction,plant pathogen interaction and other pathway were regulated in MeJA pretreated roots.4.According to the sequencing results,18 DEGs were selected for qRT-PCR verification.The results showed that the result of qRT-PCR were consistent with that of transcriptome sequencing and gene expression profile analysis,except catalase and peroxidase,the other genes were responsive to exogenous MeJA pretreatment,and their transcriptional levels were induced by F.solani.However,in sterile water pretreated P.notoginseng group,the expression levels of these genes were significantly lower than those in MeJA pretreated P.notoginseng group,or the time of induced expression was significantly delayed when compared with MeJA pretreated P.notoginseng group.The result of qRT-PCR was no doubt that transcriptome sequencing and gene expression analysis can reveal the molecular mechanism of JA signaling pathway mediated P.notoginseng defense response to F.solani and excavate efficiently related disease resistance genes response to JA.5.Based on a differentially expressed gene sequence encoding the MAPKK through transcriptome sequencing,using RACE(rapid amplification of cDNA ends)technology cloned the full-length cDNA of PnMAPKK1,and analyzing its expression characteristics and bioinformatics analysis.The full-length cDNA of PnMAPKK1 was 960 bp and encoded a polypeptide with 319 amino acid residues.The results of quantitative reverse transcriptase PCR(qRT-PCR)showed that the expression of PnMAPKK1 in P.notoginseng is induced during F.solani infection as well as after treatment by MeJA,salicylic acid(SA),ethylene(ETH)and hydrogen peroxide(H2O2).The fusion gene expression vector pBIN m-gfp5-ER-PnMAPKK1 was constructed,and then was transformed into Onion epidermal cells by Agrobacterium tumefaciens-mediaied.Under the laser scanning confocal microscope,target proteins PnMAPKK1 was observed to distributed in the cytoplasm.The over-expression vector PCAMBIA2300S-PnMAPKK1 was constructed and transformed into tobacco(Nicotiana tabacum L.cv Xanthi)plants,nurturing the PnMAPKK1 transgenic plantsuntill T2 generation of transgenic tobacco plants were obtained.The results of qRT-PCR analysis showed that PnMAPKK1 was expressed in all the transgenic lines steadily.Compared with wild type(WT),the PnMAPKKl T2 generation of transgenic tobacco plants improved tolerance to F.solani infection.Induring E solani infection,the transcription level of some defense related genes in PnMAPKK1 transgenic lines were significantly improved.Moreover,the content of MeJA,ETH and SA in PnMAPKK1 transgenic lines were significantly also inereased during F.solani infection.In summary,using high-throughput sequencing technology to do transcriptome sequencing for the 12 samples of MeJA and sterile water pretreated P.notoginseng after F.solani infection,then the gene expression profiles of affinity interaction process and incompatibility interaction process between P.notoginsengand F.solani were obtained.Which is helpful to elucidate the molecular mechanism underlying defense response of P.notoginseng regulated by exogenous MeJA to resist F.solani.PnMAPKK1 as a new MAPKK gene in P.notoginseng,the expression level of PnMAPKK1 was induced by F.solani infection and exogenoussignal molecules treatment.The overexpression of PnMAPKK1 in transgenic tobacco leaf could regulate the expression of some defense related genes,improve the content of MeJA,ETH and SA,and increase resistance to F.solani infection.Therefore,the PnMAPKK1 is a disease-resistant candidate gene that can be used for the genetic engineering technology to improve the resistance of plants. |
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