| Soybean[Glycine max(L.)Merr.]is an important food and economic crop in China with an abundance of oil and high-quality protein;however,it is often attacked by a variety of insects during its life cycle.The common cutworm[CCW,Spodoptera litura(Fabricius)]is a major herbivorous pest in southern China.Their larvae are omnivorous,have a large amount of food with many generations,which seriously reduces soybean growth and development and agricultural output.Although the application of chemical pesticides can control insects,the long-term use of chemical pesticides also pollutes the environment and contributes to the development of pesticide resistance.Under millions of years of selection pressure generated by insects,plants have evolved complex defense systems to protect themselves from insect attack.Understanding the mechanism of plant insect resistance is most important for insect-resistant plant breeding.In order to explore the genetic basis and related molecular markers of soybean resistance to CCW,the QTL/SNPs of soybean CCW resistance related traits were identified by using linkage analysis in recombination inbred lines(RILs)(184 families)and association analysis in a natural population(219 accessions).By comparing the transcriptome data of soybean responses to CCW,it was found that the gene GmVQ58,encoding a FxxxVQxxTG(VQ)motif-containing protein,showed markedly higher expression levels in the CCW-susceptible soybean accession than in the CCW-resistant soybean accession.Through a series of experiments,including the transformation of Arabidopsis jav1 mutants and soybean hairy roots,identification of interaction protein,promoter activity analysis and sequence diversity analysis,the role of GmVQ58 in soybean resistance to CCW was preliminarily verified.In addition,CDPKs are best-characterized calcium sensors in plants,which are involved in the regulation of phytohormone signaling and the plant responses to biotic and abiotic stresses.Constitutive expression of GmCDPK38 rescued the susceptibility of Arabidopsis cpk28 mutants to CCW.To better investigate the role of GmCDPK38 in soybean resistance,the endogenous GmCDPK38 was knockout in soybean using the CRISPR/Cas9 genome editing technology.By combining with transcriptome and metabolome analysis,the mechanism of GmCDPK38 was further explored.The main results of this study are as follows:1.Three insect resistance related traits,larval weight,pupal weight and larval period,were investigated in the RILs,which derived from Kefeng No.1 and Nannong1138-2.The linkage analysis was performed in these traits by using the bin-map genetic map.The results showed that all three traits had wide genetic variation and were significantly affected by environmental factors.The highest heritability was 75.17%in larval period,followed by 59.69%in larval weight and 62.1%in pupal weight,respectively.There was a significant positive correlation between larval weight and pupal weight,while there was a significant negative correlation between larval period-larval weight and larval period-pupal weight.Six QTLs were detected by using the composite interval mapping(CIM)method.Of which,four QTLs for larval weight were detected on chromosome 7,10 and 16,respectively.qLW7-1 located on chromosome 7 was observed in El environment,while qLW7-2 could be detected in E2,E3 and E4 environments.Both of the two QTLs were close to the previously reported resistance related QTLs.qLW10-1 located on chromosome 10 was detected in E2 environment,and qLW10-1 located on chromosome 10 was detected in E2 environment.They were both novel QTLs.Two QTLs were detected for pupal weight,which were located on chromosome 2 and 10 respectively.They were all observed in only one environment and were novel QTLs which discovered in this study.2.Three insect resistance related traits,larval weight,pupal weight and larval period,were investigated in a natural population which contains 219 cultivated soybean accessions.The association analysis was carried out based on the high-density soybean array.The results showed that three traits had significant differences among the materials and years.Significant correlations were found among these three traits.The heritability of pupal weight was the highest,followed by larval weight,and the heritability of larval period was the lowest.Using the multi-locus mixed model(MLMM),50,31,and 32 SNPs were identified as associated with larval weight,pupal weight and larval period,respectively.The 50 SNPs associated with larval weight were distributed on chromosomes 1,2,7,8,9,13,16,18,and 20,and they were all uncovered in one environment.The confidence interval of a SNP cluster on chromosome 7 was consinsted with the QTL qLW7-2 that identified by linkage analysis.A total of 31 SNPs were detected for pupal weight.The marker AX-94035553 located on chromosome 7 in both e3 and e4 environments were also consinsted with qLW7-2.Totally,32 SNPs were associated with larval period.The four adjacent markers,AX-93774173,AX-93636682,AX93636687,and AX-93636688,were located on chromosome 10 and could be observed in both e3 and e4 environments.These four SNPs were all the novel SNPs.3.Based on the transcriptome data in our laboratory,the gene GmVQ58,encoding a FxxxVQxxTG(VQ)motif-containing protein,showed differentially expression between the CCW-susceptible soybean accession and the CCW-resistant soybean accession.The expression level of GmVQ58 was higher in the CCW-susceptible soybean accession.The results of qRT-PCR and GUS staining showed that the gene expressed the highest in the leaves and roots and was upregulated significantly after CC W attack.Subcellular localization showed that GmVQ58 was a nuclear localization protein.Constitutive expression of Gm VQ58 rescued the susceptibility of Arabidopsis jav1 mutants to CCW.Overexpression of the gene did not significantly affect soybean hairy roots resistance to CCW,whereas resistance was significantly enhanced in Gm VQ58-RNAi lines.These results indicated that Gm VQ58 may play a role in plant defense against CCW.The interaction between GmVQ58 and GmWRKY32 transcription factor was identified by yeast two-hybrid assay and bimolecular fluorescence complementation assay.The expression of two defense related genes,GmN:IFR and GmVSPβ,downstream of GmWRKY32 was upregulated in Gm VQ58RNAi lines.Sequence diversity analysis showed that the promoter region of GmVQ58 was likely selected during domestication,resulting in different expression patterns in cultivated soybeans relative to wild soybeans.4.Ectopic expression of GmCDPK38 complemented Arabidopsis cpk28 mutants to restore the wild type phenotype.The resistance of complemented Arabidopsis to CCW was similar to that of the wild type.To further investigate the function of GmCDPK38 in soybean resistance to CCW,the endogenous GmCDPK38 was knockout in soybean using the CRISPR/Cas9 genome editing technology.The results of the evaluation of resistance to CCW showed that the gmcdpk38 mutants exhibited obviously antifeedant effect to CCW and affected their growth and development compared with the wild type,suggesting that knockout of GmCDPK38 enhanced the resistance of soybean to CCW.KEGG enrichment analysis of transcriptome and metabonome indicated that differentially expressed genes and metabolites were mainly enriched in the biosynthesis of secondary metabolites related to insect resistance,such as flavonoids,phenylpropane,and terpenoids,followed by signal transduction of plant hormones such as jasmonic acid and plant-pathogen interactions.These results showed that the mutation of GmCDPK38 could alter the expression of jasmonic acidrelated genes and the synthesis of some secondary metabolites associated with insect resistance. |
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