The Resistant Mechanisms Of CBL To Race 4 Of Soybean Cyst Nematode

Soybean cyst nematode?SCN??Heterodera glycines?is seriously harmful to soybean production.There are currently 16 races of soybean cyst nematode,among which the race 4 is the strongest pathogenicity,mainly distributed in Huanghuaihai of main soybean producing regions.It is the most economical and effective measures to find new sources of resistance and new resistance genes.Based on 3 years test,CBL highly resistance to race 4 of soybean cyst nematode,has been selected.This study explored the resistant mechanisms of CBL to race 4 of soybean cyst nematode to provide a theoretical basis for soybean breeding.The research used acid fuchsin staining to determine the number and life stage of the juveniles in roots,for confirm the type of CBL resistance to race 4 of soybean cyst nematode.The changes of activities of the defensive enzymes were analyzed in roots of CBL after inoculation of soybean cyst nematode,for explore the biochemical mechanism of the resistance of CBL.Using transcriptome sequencing screened the differentially expressed genes,to analysis the molecular mechanism of CBL.At the same time,the CBL-HSPRO₂ gene was obtained by homology cloning,and its related biological information was analyzed,which laid a foundation for further gene identification.The main results are follows:1.CBL resistance to race 4 of soybean cyst nematode showed that the development effect of SCN.CBL cannot resist the penetration of soybean cyst nematode,but inhibit parts of second-stage juveniles developed into third-stage juveniles,and almost all third-stage juveniles developed into fourth-stage juveniles.2.Polyphenol oxidase?PPO?,peroxidase?POD?,phenylalanine ammonia-lyase?PAL?and superoxide dismutase?SOD?participated in the process of CBL resistance to SCN.After inoculated SCN,PPO,POD,SOD and PAL activity compared with uninoculated group showed increased in CBL root,which indicated that the changes of defensive enzymes of POD,PPO,SOD and PAL activity are closely related to CBL resistance to SCN.3.Plant hormone signal transduction,phenylalanine metabolism and energy metabolism et al pathway involved in CBL resistance to race 4 of SCN.In this paper,Illumina HiSeq^TM 2500 technique was used sequence the genes expression of the soybean,which was resistance to race 4 of SCN,after SCN infection 9 day and 17 day.These time points were chosen to contrast with plant gene expression without inoculated cyst nematode.Finally,a total of 1.96×10¹⁰bp bases were obtained.There were 2180 and 4210 genes with alterd expression in roots at 9 day and 17 day after SCN infection,respectively,and a total of 840 common genes expressed between two periods.Among them,the up-regulated genes were 1604 and 1414,and the down-regulated genes were 1116 and 2796,respectively.Using Gene Ontology?GO?,Cluster of Orthologous Group of Proteins?COG?annotation and the Kyoto Encyclopedia of Genes and Genomes?KEGG?annotated the functional of differentially expressed genes.According to GO annotation,there were relatively more differentially expressed genes classified to extracellular region,antioxidant activity and cell killing.According to KEGG,a large number of genes encoding enzymes involved in phenylalanine metabolism,energy metabolism and plant hormone signal transduction were altered.4.The gene of CBL-HSPRO₂ was obtained by homology cloning,which was 1356bp size.Analyzed the physical-chemical properties,domain prediction,and secondary structure prediction of protein coned by CBL-HSPRO₂ gene,and the expression vector pGPFGUS-CBL-HSPRO₂ of CBL-HSPRO₂ gene was constructed and transformed into Agrobacterium.