Excavation Of Key Genes For Meloidogyne Incognita Hatching And Enhancing The Effectiveness Of Aconetin Against Nematodes

Plant-parasitic nematodes(PPNs)are a group of plant pathogenic organisms that cause serious damage to plant growth and agricultural production,led by root-knot nematodes(Meloidogyne spp.,RKNs).Due to the small size of root-knot nematodes,which is difficult to observe,the early symptoms of infestation of plants are not significant,and the knowledge of root-knot nematodes is not sufficient,which are the reasons for the ineffectiveness of current control methods.The development and hatching of eggs is an important part of root-knot nematode growth process,and the mining and research of key genes for hatching will improve the biological knowledge of root-knot nematode and thus carry out green control of root-knot nematode.In our laboratory,we found that aconetin secreted by Bacillus thuringiensis(Bt)HAN055 has sensational virulence activity against J2 stage of Meloidogyne incognita,and the high concentration of aconetin can inhibit the egg hatching of the Meloidogyne incognita.In order to further improve the egg hatching inhibition effect and reduce the cost of using aconetin.In this study,using Meloidogyne incognita as a model,the following research results were obtained:(1)Transcriptome data were obtained for ten different embryonic developmental periods of the Meloidogyne incognita.Based on the Smart-seq2 sampling method for single eggs constructed in our laboratory,the embryonic developmental stages were more precisely classified into single-cell stage(S1),two-cell stage(S2),four-cell stage(S3),eight-cell stage(S4),multi-cell stage(S5),crumpled stage(S6),1.5-fold stage(S7),2-fold stage(S8),Move stage(S9)and L1 stage(S10).Fifty-four egg samples spanning ten embryonic developmental periods were obtained,and 1,249.6 M of data were obtained after transcriptome sequencing.(2)Meloidogyne incognita hatching-related genes are highly expressed at 2-fold stage(S8).that the hatching inhibition by aconetin was increased approximately two fold after interfering with the Mi＿44476 gene of Meloidogyne incognita.Based on the transcriptome information of different embryonic developmental stages of Meloidogyne incognita,20 different gene expression patterns(Clusters)were obtained,and some of them showed high expression at specific periods.The results of GO enrichment analysis showed that 1889 genes(Cluster8),which were highly expressed at the 2-fold stage(S8)of the Meloidogyne incognita,were involved in regulating the cuticle development and molting cycle of Meloidogyne incognita,indicating that the genes highly expressed in the S8 period might affect the hatching process of the Meloidogyne incognita.(3)Construction of the weighted gene co-expression network of Meloidogyne incognita genes and mining of two key genes for hatching inhibition.In this study,a weighted gene co-expression network(WGCNA)of Meloidogyne incognita was constructed based on the WGCNA method,and 15 different gene coexpression modules were obtained during the embryonic development of the Meloidogyne incognita.Based on the correlation between module membership(MM)and gene significance(GS)in the co-expression network,45 hatching key genes were obtained after further mining of the highly correlated green module genes at the 2-fold stage(S8)of the Meloidogyne incognita.Using RNA interference technology,10 of the45 key genes were validated,and the results showed that the hatching rate of Meloidogyne incognita was significantly reduced after interfering with two genes,Mi＿44476 and Mi＿03792.(4)Interference of gene Mi＿44476 enhances the inhibition of hatching of Meloidogyne incognita by aconetin.In this study,using ten concentration gradients(100 μg/m L-1000 μg/m L)of ouabain co-incubated with eggs of Meloidogyne incognita,it was found that the hatching rate of eggs decreased as the concentration of aconetin increased,where the concentration of aconetin for half of the hatching rate was 326.60 μg/m L.Aconetin(300μg/m L)have been used to incubate with Meloidogyne incognita which have been interfered by the two genes Mi＿44476 and Mi＿03792.The experimental results showedBased on the transcriptomic data of Meloidogyne incognita embryonic development,this project uncovered the key genes that inhibit its hatching through the analysis of gene expression patterns and the construction of a weighted gene coexpression network,and deepened the knowledge of the hatching behavior of Meloidogyne incognita.The combined application of aconetin also showed that the interference of the target gene Mi＿44476 could inhibit egg hatching with aconetin.This study deepens the knowledge of hatching behavior of Meloidogyne incognita and provides data resources for nematode embryonic developmental behavior patterns.The strategy of combined application of hatching key genes and aconetin can also be one of the novel tools for green control,which provides new ideas for green control of Meloidogyne incognita.