Bt Transgenic And RNA Interference Against Soybean Cyst Nematode(Heterodera Glycines)

In recent years,plants have suffered from the damage of parasitic nematodes during their growth.Soybean cyst nematode(SCN)disease is one of the most harmful diseases in soybean production,which causes great loss to soybean production in the world every year.Soybean cyst nematodes mainly live in the soil or parasitic in plants,so it is difficult to control the damage,and the existing control methods have some limitations.Although the selection of resistant species is one of the most economical and effective measures,due to the numerous and evolving SCN physiological species,a single resistant species is no longer good enough to prevent nematode damage.Therefore,it is very important to establish a better control system of soybean cyst nematode.A large amount of soybean cysts nematode material is the premise of nematode control research,the field environment is complex,sampling needs a lot of manpower and material resources,so it is of great significance to culture nematode in the laboratory.In this study,the artificial passage of soybean cyst nematode was successfully realized by exploring the growth and incubation conditions of soybean cyst nematode.This study also explored the detection method of soybean resistance to cyst nematode.The soybean root tissue was stained at different stages after nematode infection,and the results showed that the best staining results were obtained 12 days after nematode infection.We also found that comparing the number of plant cysts in soybean roots is also a way to measure the insect resistance of soybean.The establishment of soybean cyst nematode laboratory system will provide a good foundation for the research of soybean insect resistance in the future.Bacillus thudngiensis(Bt)is the most widely used biological insecticide at present.It is a grampositive bacterium that widely exists in the natural environment such as soil.During the spore formation process,it produces semisporal crystal protein with insecticidal activity,which is mainly encoded by cry gene.At present,few nematocidal genes have been discovered,and some studies have found that Cry6Aa2 and Cry5B have insecticidal effects on root-node nematode.In this study,we optimized Cry6Aa2 and Cry5B genes according to soybean codon preference to study their toxic effects on soybean cyst nematode.At the same time,this study designed the Bt gene and plant tissue specific promoter binding insect resistance strategy.Because plant tissue-specific promoters can specifically express foreign genes at specific sites,they can avoid the bottleneck of transgene,and previous work in the laboratory has excavated the promoter p4176 that can specifically express in soybean root.After obtaining the vector for specific expression of Bt gene in soybean roots,we transformed soybeans by Agrobacterium rhizogenes K599.No transformed roots with successful expression were identified,which may be due to the weak promoter affecting the expression of cry gene.However,this experiment provided a new idea for the control of soybean cyst nematode.RNA interference(RNAi)is another method to control plant pests,which can achieve the effect of preventing and controlling insects by silencing the expression of specific genes in insects.In this study,we compared soybean cyst nematode with Caenorhabditis elegans and screened out 9 genes related to the growth of second instar larvae of C.elegans as target genes.By directly feeding artificially hatched second instar larvae with transcriptional target genes in vitro,qRT-PCR was used to detect the relative expression levels of target genes in nematodes,and five target genes that could effectively inhibit the growth of soybean cystis nematodes were obtained.We also designed and constructed RNAi vectors with silencing target genes,and transferred these RNAi vectors into Agrobacterium rhizogenes K599.Through Agrobacterium tumefaciens mediated transformation of soybean roots,a series of soybean root transformation lines expressing nematode RNAi were obtained.This method is a new strategy for green control of soybean cyst nematode.In summary,this study had an in-depth understanding of the characteristics of soybean cyst nematode and the existing problems in its control,and tried to establish the technical system of soybean anticyst nematode.