Screening Of Protein From Bacillus Thuringiensis With Insecticidal Activity Against Chrysomelidae Pests And Cloning Of Novel Genes

The most destructive insects of coleopteran are mainly the Scarabaeoidea and Chrysomeloidea,Scarabaeoidea of grubs are recognized as the most difficult subterranean pests to control,Chrysomeloidea of Colaphellus bowringi Baly is an important pest to cruciferous crops,The Cry8proteins from Bacillus thuringiensis have good insecticidal activity against Scarabaeoidea pests,however,there are few reports on the activities of Chrysomeloidea pests.The studies have shown that some Bt full-length proteins,activated by protease,produce new insecticidal activities against some pests,and become an important way to discover new genes,new proteins and new insecticidal activities.Previous studies in our laboratory found that Cry8Ha1 protein digested by chymotrypsin had insecticidal activity on C.bowringi.For the study of protease digestion on insecticidal activities of Cry8Ea1,Cry8Ga1 and Cry8X proteins.In this paper,the trypsin and chymotrypsin were used to digest three full-length proteins,the insecticidal activity of the digestibility proteins to the C.bowringi was measured respectively,Cry8Ea1 digestion protein was found to have good insecticidal activity against C.bowringi,the peptide retained insecticidal activity against grubs.At the same time,a new sip gene with insecticidal activity against C.bowringi was cloned from Bt strains preserved in laboratory.The main results are as follows:（1）.Cry8Ea1,Cry8Ga1,Cry8Ha1 and Cry8X proteins from Bt were digested by trypsin and chymotrypsin,respectively.Cry8 full-length proteins and two enzyme digested proteins were used to determine the bioactivity of coleopteran pests of the first hatched larvae of C.bowringi.The results of 48-hour bioassay showed that:the four full-length Cry8 proteins had no significant activity on the C.bowringi;the four Cry8 proteins were digested by trypsin,only the Cry8Ea1 protein show strongly insecticidal activity,the LC₅₀ of Cry8Ea1 protein digested by trypsin was 34.01μg/m L（95%confidence interval was 26.95-40.27μg/m L）;the four Cry8 proteins were digested by chymotrypsin,only the Cry8Ha1 protein show strongly insecticidal activity,the LC₅₀ of Cry8Ha1 protein digested by chymotrypsin was 35.67μg/m L（95 confidence interval was 29.99-41.38μg/m L）.It is consistent with the previous results.These results indicated that the full-length proteins Cry8Ea1 and Cry8Ha1,which had no activity against Chrysomelidae pests,showed a good toxic and killing effect on C.bowringi after digestion by protease.（2）The cleavage sites of Cry8Ea1 digestible protein were analyzed,it was speculated that the digestion sites of trypsin on Cry8Ea1 protein were Met⁴⁸ and Arg⁶⁴⁵,and the digestion sites of chymotrypsin were Ser⁵⁷ and Ile⁶⁴⁶.The former digestion product has insecticidal activity,while the latter loses the insecticidal activity,the results showed the different cutting sites of protease would affect the insecticidal activity of the protein.（3）Four Cry8 proteins are digested by trypsin and chymotrypsin and it were used to determine the bioactivity of the 2-day of H.parallela and 2-day H.obeita of coleopteran pests,and the results of the 14-day corrected mortality of 2-day-old larva of H.obeita of Cry8 proteins that were digested by trypsin was generally low,the corrected mortality rates of Cry8Ga1,Cry8Ha1 and Cry8X digested by trypsin on 2-day-old H.parallela larvae of the species were 53.3%,56.7%and 43.3%,respectively.The corrected mortality rates of Cry8Ea1,Cry8Ha1 and Cry8X proteins activated by chymotrypsin in 2-day-old larva of H.obeita were 41.4%,62.0%and 82.7,respectively,among which the digested Cry8X proteins showed better.The corrected mortality rates of Cry8Ea1,Cry8Ha1 and Cry8X proteins activated by chymotrypsin in 2-day-old larvae of H.parallela were50.0%,63.3%and 26.7%,respectively,and the digested Cry8Ha1 proteins were more virulent to 2-day-old larvae of H.parallela.（4）Using 14 standard strains containing sip genes in the laboratory as experimental materials,14 sip genes were cloned and compared with the amino acid sequences of the 14 sip genes.Six sip genes with significant differences were selected for expression:4A9-sip,4BZ1-sip,6A7-sip,4D18-sip,Bt185（Bt185-60）-sip and Bt185（JJX-sip02）-sip.The results of 48-hour bioactivity assay showed that the corrected mortality of 4BZ1-sip,Bt185（Bt185-60）-sip and Bt185（JJX-sip 02）-sip proteins for the first hatched larvae of C.bowringi were 25.1%,30.7%and 55.7%,respectively.The results of 48-hour bioassay showed that the soluble and insoluble components of 4A9-sip,6A7-sip and 4D18-sip proteins had no insecticidal activity against the hatching larva of C.bowringi.In this study,we successfully screened the Cry8Ea1 protein digested by trypsin with high virulence to the incubated larva of C.bowringi,and the Bt185（JJX-sip02）-sip gene was expressed,it also has a certain insecticidal activity to the incubated larva of C.bowringi.The results of this study will provide ideas for the discovery of new insecticidal activities of known Bt proteins and the screening of new Bt insecticidal proteins.It provided new resources for the control of coleopteran pests.