Cloning And Functional Analysis Of Lysozyme Gene Of Soybean Cyst Nematode(Heterodera Glycines)

Soybean cyst nematode(Heterodera glycines)is one of the most important soil-borne pathogens in soybean.Nematodes are often threatened by pathogens during development.In order to prevent pathogen infection,nematodes usually express antibacterial proteins,such as lysozyme.But the specific defense mechanisms of nematodes on pathogens are still unclear.Lysozyme is an important defense enzyme in nematodes.The functional study of the plant nematode lysozyme gene helps to explain the specific defense mechanism of nematodes and provides a theoretical basis for the prevention and control of plant nematodes.In this study,three lysozyme genes were verified in soybean cyst nematode by screening transcriptome library.In situ hybridization technique was used to determine the expression of lysozyme gene in soybean cyst nematode.The qPCR method was used to detect the transcriptional properties of lysozyme gene in the defense of nematodes,and the expression pattern of three lysozyme genes in nematodes after bacterial infection of nematodes was analyzed.The defensive function of lysozyme was preliminarily studied by measuring the 1-7 day survival rate of three strains of soybean cyst nematode placed after lysozyme gene RNA interference.The inhibitory effect of the lysozyme protein expressed in vitro on bacteria was determined by counting the number of colonies and the turbidity assay.In situ hybridization results indicated that the three lysozyme genes Hg23324,Hg21698 and Hg17337 were expressed in intestinal cells.The results of gene expression change analysis showed that Hg23324 gene expression was significantly up-regulated under the induction of Bacillus subtilis and Bacillus thuringiensis.The expression of Hg21698 and Hg17337 genes was significantly up-regulated under the induction of Staphylococcus aureus.It is speculated that lysozyme is involved in the defense of nematodes.The function of lysozyme was analyzed by RNA interference technique.After dsRNA soaking,the transcriptase gene transcription level was significantly decreased by qPCR.The detection experiment of nematode survival rate showed that the survival rate of nematodes in Bacillus subtilis and Bacillus thuringiensis decreased after the nematode lysozyme gene Hg23324 interfered.After the lysozyme gene Hg17337 interfered,the survival rate of nematodes in Staphylococcus aureus and Bacillus thuringiensis decreased.After the lysozyme gene Hg21698 interfered,the survival rate of nematodes in Staphylococcus aureus and Bacillus thuringiensis decreased.It is speculated that the interference of the lysozyme gene causes the nematode immune system to be affected,becomes more sensitive to bacterial infection,and the survival rate decreases.The above indicates that lysozyme can play a specific role to help the nematode resist bacteria.The tobacco protein is extracted after transient expression of the lysozyme gene tobacco.The inhibitory effect of the crude protein on the bacteria was determined by counting the number of colonies and the turbidity measurement method.The results showed that Hg23324 recombinant protein can inhibit Bacillus subtilis.Hg17337 recombinant protein can inhibit Staphylococcus aureus.Hg23324 and Hg21698 recombinant proteins can inhibit Bacillus thuringiensis.From these results,it was revealed that lysozyme can function in vitro.In this study,the lysozyme gene of soybean cyst nematode was isolated for the first time.The expression level and antibacterial function of the nematode resistance bacteria were studied in detail,which provided a reference for the research of nematode defense mechanism and provided a better direction for the study of the mechanism of nematode biological control.