QTL Analysis Of Soybean Resistance To Common Cutworm (Spodoptera Litura Fabricius) And Functional Study Of GmCDPK1

Soybean (Glycine max (L.) Merr.) is one of the most important economic crops which provide quality protein and oil for human. However, various insect pests significantly affect the yield and quality of soybean. The common cutworm (CCW; Spodoptera litura Fabricius)is a serious herbivorous insect pest because of its polyphagous appetite for many crops,especially for soybean, and its rapid rate of multiplication. Although the application of chemical insecticides can kill CCW, this management technique also damages the environment and increases the cost to farmers. The use of host plant resistance to pest would contribute to integrated pest management in soybean. Cultivating resistant germplasms may be a sustainable and economical approach for soybean production. This strategy would reduce both crop losses and the cost of insecticide application; therefore, elite soybean cultivars with resistance to CCW would have both environmental and economic benefits.In order to explore the genetic machanism of soybean resistance to CCW and MAS application, the QTL of soybean resistance related traits, LW (larva weight), PW (pupa weight) and PD (pupa days), were widely investigated using RIL-based linkage analysis and germplasm-based association mapping. Meanwhile, we conducted a genome-wide analysis of soybean CDPK and identified 39 CDPK genes. Moreover, the expression pattern were analysis by qPCR. At last, the gene GmCDPK1 was cloned and overexpressed in Arabidopsis mutant and wild type for functional verification. The main results of this study were as follows:1. QTL analysis of soybean resistance to CCW relatid triats (LW, PW and PD) were conducted using recombination inbred line (RIL) accessions. The results showed that there were significant differences aomong recombination inbred lines for each trart and between years. The phenotype correlaion analysis showed that three traits were highly correlated and significant. Five significant QTL spred in five chromosomes were detected using CIM(Composite interval mapping) methods under two years or BLUP. Among them, three QTLs detected under two years and BLUP; one QTL detected under one year and BLUP.Three QTLs were consisted with the previous reports; the other two QTLs were identified in this research first time.2. Genome-wide association analysis with 1142 SNPs and three CCW resistance related traits (LW, PW and PD) were proformed under two years or BLUP. The results showed that were broad diversity existed in the association population either phenotype or genotype.There were 63 significane SNPs were detected with three traits. Most of these genome-wide association studies (GWAS) loci were located within genomic regions previously identified for these traits. The region on chromosome 6, which co-associated with LW and PD, included several QTLs for resistance to reactions to biotic stress. On chromosome 7, two SNPs mapped a loci that have been reported resistance to CCW, corn earworms, soybean aphid and bean pyralid infestations..3. We conducted a genome-wide analysis of soybean CDPK and identified 39 CDPK genes, which the phylogenetic analysis indicated could be divided into four groups.Quantitative real-time RT-PCR was performed on eight selected CDPK genes to confirm their expression patterns in response to wound and CCW attack treatments; the expression of two genes (CPK3 and CPK31) was rapidly induced by both treatments, especially for CPK31 expression significantly increased after wound or CCW attack, which suggested CPK31 genes might be response to CCW stresses in soybean.4. We cloned and constructed GmCDPK1/CPK31 plant expression vector overexpressed in Arabidopsis mutants and wild type. CCW feeding experiment was performed to evaluation the insecticidal activity of transgenic plants. We found the GmCDPKl restored the Arabidopsis mutants which growth phenotype partially resembling with reduced rosette growth,leaf deformation and anthocyanin accumulation as well as elevated transcript levels of JA-regulated genes, and negative regulation of resistance to insects. The expression patterns of JA markerd gene Thi2.1, VSP2 and PDF1.2 were significantly expression in Arabidopsis mutants comparison of overexpression GmCDPKl plants. While no significant changes in gene expression observed for JA biosynthesis genes AOS and OPR3 in mutants or transgenic lines. This implies that the GmCDPKl probably did not result from impaired JA metabolism but from the regulation JA biosynthesis activity.5. Previous studies indicated that Arabidopsis mutants shown function as negative modulate ROS generation. We found the transgenic lines restored ROS leves comparison of Arabidopsis mutants after induced by CCW. To further investigate function of GmCDPKl,subcellular localization and fluorescence double molecular complementation (BiFC) was conduct to function analysis of GmCDPKl, then we found GmCDPK1 interacts with GmRBOHD on the plasma. Taken together, we found GmCDPKl regulating developmental JA-dependent growth processes and dependent of JA-mediated defense responses for plant resistance to insects; on the other hands GmCDPK1 interacts with GmRBOHD on the plasma and participate in ROS burst by phosphorylate the ROHD proteins for defense responses in plants.