Identification Of Cryl-Type Genes From Bacillus Thuringiensis Strains By PCR-RFLP

With the aim of identifying the cry1-type genes from Bacillus thuringiensis isolates collected by our laboratory, we developped a PCR-RFLP (Restriction Fragment Length Polymorphism, RFLP) identification system of cry1-type genes. With two universal oligonucleotide primers K5un2 /K3un2, K5un3/K3un3 based on the most conserved regions of all known cry1-type genes, we used DNA templates from eight stardand B. thuringiensis strains and two genetically engineered bacteria containing known cry1-type genes as our references in the PCR system. The PCR amplified products, with size of 1.6-1.7kb and 1.4-1.5 kb, were digested with PstⅠ/XbaⅠ,PstⅠ/EcoRⅠ respectively. And the cry-1 type genes in the ten reference strains were identified on the basis of the restriction fragment length polymorphism. It can be concluded that this method is practical for further identification of cry1-type genes from B.thuringiensis.Furthermore, the PCR-RFLP system was performed to detecting the cry1-type genes from 70 local B. thuringiensis isolates. Ten distinct cry-1 type genes, including cry1Aa, cry1Ab, cry1Ac, cry1Ad, cry1Ae, cry1B, cry1Ca, cry1Cb, cry1D, cry1Ea, were found from the 54 isolates containing cry-1 type genes. No novel cry-1 type gene was identified. Different cry-1 type gene combinations were found from these 54 Bt isolates except isolate WY－290 and isolate 9441 which contained only cry1Aa. Among them, 28 isolates contained two kinds of cry-1 type genes, 18 isolates contained three kinds of cry-1 type genes, and only 6 isolates had four kinds of cry-1 type genes.In addition, the relativity of parasporal crystal morphology, flagella serotype,cry1-type genes, toxin proteins and insecticidal activity to Beet Armyworm (Spodoptera exigua) and Cotton Bollworm (Helicoverpa armigera) of the 70 Bt isolates was also investigated. There was an indirect correlation between the parasporal crystal morphology and insecticidal activity of B. thuringiensis. Isolates having high toxicity to target insects had large bipyramidal , cuboidal crystals.No correlation between flagella serotype and insecticidal activity. Different isolates of the same serotype (H3a3b) had different insecticidal activity while some isolates of different serotype (H7 and H3a3b) had same insecticidal activity.The ICPs genes, toxin proteins and insecticidal activity were highly correlative. Among 54 isolates horboring cry1-type genes, 32 isolates having high toxicity to both two insects and 16 isolates only having high toxicity to Beet Armyworm while low or no toxicity to Cotton Bollworm. All isolates have been proved to have different cry-1 type gene , to express 130-140kDa insecticidal protein. The isolates having no cry1-type genes didn't express the corresponding proteins and had no toxicity to the target insects.