Study On Gene Cloning And Activity Of The Novel Bt Insecticidal Protein With High Toxicity Against Laodelphax Striatellus

Laodelphax striatellus is a very important hemipteran pest that preys on rice and substantially affects the safety of rice production.It can not only suck the juice of rice and other crops through the piercing-sucking mouthparts,plunder its nutrients,directly harm the crops,but also spread plant viruses between crops such as rice and corn.It has seriously affected the safe production of rice,corn and other crops.Such pests are mainly controlled by chemical pesticides,but the long-term prevention and control of these pests with chemical pesticides has led to an increase in the resistance of the rice planthopper,as well as serious environmental pollution and food safety problems.New and efficient methods for the safe control of rice planthoppers are urgently needed.Bacillus thuringiensis?Bt?has been widely used for the green control of a variety of rice lepidopteran pests.In recent years,based on a large number of Bt strain resources isolated and screened in our laboratory,two wild strains that are activity against L.striatellus have been found,and three new genes,cry64Ba,cry64Ca,and cry78Aa,have been cloned from them.These gene expression products has high toxicity against L.striatellus.This shows that it is feasible to select new insecticidal protein with high toxicity against rice planthopper from Bt strain resources.This study based on the high-throughput sequencing of the Bt strain genome,using the cry64Ba,cry64Ca and cry78Aa genes that exhibit high activity against L.striatellus as reference.We used bioinformatics technology to analyze and mine new Bt insecticidal genes,and used L.striatellus as the target pest,combined with traditional bioactivity assay,to clone new genes with significant insecticidal activity against L.striatellus,provide important strains and genetic resources for the biological control of L.striatellus.The main results are as follows:?1?Using bioinformatics technology and BLAST comparison,one hundred and sixty-two homologous nucleotide sequences to the cry78Aa1 gene were obtained from the 1368 Bt strains draft database completed in the early stage of the laboratory.And Seventeen candidate strains containing cry78-like genes were obtained.?2?Bioassay results showed that total crystal proteins of of P14E12,B4F11,C8D6,T5D8,and B15C9 strain showed certain insecticidal activity against the third instar nymph of L.striatellus through bioassays,with a fatality rate of 40-80%.The collection locations of these strains were consistent,all from Beijing Baiwang Mountain forest park.?3?The Bt insecticidal gene annotation of 5 strains of P14E12,B4F11,C8D6,T5D8,and B15C9strains was completed.The protein structure prediction found that the proteins encoded by the cry78-like genes contained in these strains all contain the conserved domains of RicinB?lectin and Toxin?10 superfamily.?4?The protein samples of inactive strains Bt P14C1,P14F6,C14F11,P10C11,A4A4,and B9B3were digested with trypsin,and the lethality rate of L.striatellus was increased.The activity of total crystal protein of C14F11 and P10C11 strains before and after digestion by trypsin was significantly different.?5?A total of 8 cry78-like genes were cloned from B4F11,A3F10,P10C11,P14F6,T5D8,A4A4,C14F11,and P14E12 strains.The amino acid sequence similarity between the proteins encoded by these genes and Cry78Aa1 was 31.8-54.3%;Seven new genes have been successfully expressed.The molecular weight of these proteins is between 41.6-43.7 kDa,which is consistent with the predicted protein molecular weight.Among them,Protein 6,Protein 10,and Protein 11 are soluble in expression,and the protein molecular weights are 42.5,43.7,and 42.4 kDa,respectively.The results of preliminary screening of bioassay show that these three Cry78-like proteins had a certain degree of insecticidal activity against third-instar nymphs of L.striatellus,and the corrected mortality rate at 30?g/mL is96.73%,40.99%,and 26.23%,respectively.?6?Select Protein 6 with the best initial screening activity for in-depth research.gene6 is derived from Bt strain B4F11.Protein 6 encoded by this gene is composed of 380 amino acid residues,with a predicted molecular weight of approximately 42.5 kDa,and contains the conserved domains of Ricin?B?Lectin and Toxin?10 superfamily.It has high insecticidal activity against third-instar nymphs of L.striatellus,with a lethal LC₅₀0 of 9.72?g/mL.the amino acid sequence similarity with Cry78Aa1 is54.3%,and it is named cry78Ba1 by the Bt?-Endotoxin International Nomenclature Committee,which is a second-level new gene.?7?The Cry78Ba1 protein does not display sequence homology to currently known allergen,and can be degraded and inactivated rapidly when heated at 90°C for 10 min.The Cry78Ba1 protein was completely degraded and inactivated within 15 s of simulated gastric juice treatment,but it still had insecticidal activity after digestion in simulated intestinal fluid for 1 h.It is speculated that the Cry78Ba1 protein can be rapidly degraded in gastric juice after being ingested by humans or higher animals,and should have lost its toxicity when entering the intestine.Within the test range of this study,no safety risk to humans or mammals has been identified for this protein in terms of digestion and absorption.To sum up,based on the draft genome data of Bt strains obtained by high-throughput sequencing technology and the bioinformatics method to compare the homologous nucleotide sequences of active genes,this study started from the candidate strain activity screening and identification of homologous gene activity,and excavated Bt strains and genes that are highly toxic against L.striatellus.This shows that there is a great possibility that Bt strains that are active against piercing-sucking mouthparts such as L.striatellus among wild Bt strains are found.The research results provide new ideas for discovering Bt strains and genes that are highly insecticidal against Hemiptera piercing-sucking mouthparts pests.At the same time,the Cry78Ba1 protein obtained in this study has created favorable conditions for the efficient and green control of the rice planthoppers.