Screening And Identification Of Gene From Bacillus Thuringiensis Isolates Toxic To The Larvae Of Holotrichia Oblita

Holotrichia oblita is one of subterranean insect pest difficult to control which lead to a tremendous loss for corp. Bacillus thuringiensis(Bt) is the most widely used insecticidal microorganism in agricultural pest prevention. During spore-forming different shapes parasporal crystals which is insecticidal crystal proteins encoded by cry or cyt gene were produced. Screening of Bt strain resources is the effective and green approach to control this insect pest. In order to obtain Bt isolates with higher insecticidal activity for Holotrichia oblita, 500 Bt isolates in our laboratory were investigated. The further study involved Bt isolates diversity analysis, microscopy observation of the Bt isolates, analysis of crystals protein, gene identification and the histopathological observation in the midguts of H. oblita larvae infected Bt, and the main results as follow:Screening Bt isolates toxic to the larvae of H. oblita: 500 Bt isolates in our laboratory were investigated and 42 isolates toxic to larvae of H. oblita were obtained. The corrected mortality of 10 isolates was higher than 60%, while corrected mortality of 261-1 strain reached 100% in 7 days. Corrected mortality of the others in 14 days were between 30%~60%.Bt isolates diversity analysis: Through the Bt isolates diversity analysis method set up in our laboratory, the 42 Bt isolates were analyzed with the condition of genomic DNA as template, optimized general primers and PCR amplification system. Then the PCR products were digested by HinfI and 33 RFLP profiles were obtained. These strains were exactly classified into 14 different types by diversity analysis. According to the insecticidal activity, one isolate was selected from each type. 1126-1, P65-1, QDL47-1, 261-1, FTL84, 78-2, 127-2, 78-3, 1198-1, S10A2, B8E12, QDL40-2, FCD114 and B8E11 were selected for further identification of cry genes.Microscopy observation of the Bt isolates and analysis of crystals protein: Results of optical and scanning electronic microscope observation indicated that 78-3, 127-2 and 1198-1 contained crystals in rhombus and 261-1, FCD114, P65-1, FTL84, 78-2 and 1126-1 contained crystals in spherical shape, nevertheless, other strains contained no crystals. SDS-PAGE analysis indicated that these strains of P65-1, 1126-1, 127-2, 78-3, 261-1, FCD114, 1198-1 and 78-2 could express protein with about 130 kDa. QDL47-1 and B8E11 could express protein with about 97 kDa, others expressed protein with about 60 kDa.Gene identification: The result of gene identification with general primer of cry3, cry8, cry18, cry23, cry37 and cry43 showed that four isolates of P65-1, 1126-1, FCD114 and 78-2 contained five novel cry8 gene, including cry8 Ma, cry8 Ca, cry8 Ab, cry8 Ga, cry8 Ea respectively. The rest of 10 strains, including strain 261-1 which was the highest toxic to larvae of H. oblita of 42 strains, didn’t contain cry genes detected in this paper, such as cry3, cry8, cry18, cry23, cry37, cry43. The result of LS-MS/MS indicated that Cry8 protein could be detected from P65-1, 1126-1, FCD114 and 78-2.Histopathological observation in the midguts of H. oblita larvae infected Bt: The histopathological results in the midguts of H. oblita larvae infected by mixture of crystals and spores produced by tested strains were observed and investigated, and the results indicated that 261-1, 1198-1, FCD114, 1126-1 and QDL40-2 could cause the obvious histopathological changes in the midguts of H. oblita.In conclusion, the results of this study provide the foundation for control H. oblita and further cloning of novel cry gene from the candidate strains.