Abstract:Aiming at enriching novel lepidoptera insecticidal genes, Bacillus thuringiensis strains BN23-5 was analyzed and identified by the PCR-restricted fragment length polymorphisms (PCR-RFLP) method, which indicated that crystal(cry) gene of cry2Ab-type gene was contained. According to the NCBI open cry2Ab-type gene encoding area, a pair of degenerate primer was designed. A full-length of 1 902 bp cry2Ab-type gene fragment, which was amplified by PCR with the degenerate primers using strain BN23-5 genome plasmid DNA as template, was inserted into the Escherichia coli expression vector pET-28a to obtain the recombinant plasmid pET-2Ab, and then transformed into E. coli BL21(DE3) cell. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) verified that the target gene could express as 65 kD protein in E. coli BL21(DE3) strain by isopropyl-β-d-thiogalactoside (IPTG) induced at low temperature. What's more, expressed product of cry2Ab32 existed in bacteria precipitation after ultrasonic broken but not in the supernatant expressed product. The encoded protein composed 634 amino acid residues and shared a 96.5% sequence homology with Cry2Ab1 with 21 amino acids difference, which was designated as cry2Ab32 (GenBank No. KJ710647) by International Nomenclature Commitee of B. thuringiensis endotoxin. Bioactivity of cry2Ab32 gene expressed product was tested against Plutella xylostella, Ostrinia fumacalis, Laphygma exigua and Helicoverpa armigera by feeding method. The bioassay results indicated that expressed product of cry2Ab32 had a high insecticidal activity against P. xylostella, O. fumacalis and H. armigera, with median lethal concentration (LC50) of 8.99 μg/mL (95% confidence: 5.82~14.91 μg/mL), 19.34 μg/mL (95% confidence: 9.64~28.31 μg/mL) and 21.24 μg/mL (95% confidence: 13.35~30.32 μg/mL), respectively. However, Cry2Ab32 protein had no insecticidal activity against L. exigua, but could obviously inhibit the growth of L. exigua. In view of few previous reports about the high insecticidal activity of cry2Ab-type gene against O. fumacalis pest, the result was a major breakthrough for its prevention and control. The discovery of new gene cry2Ab32 will provide more choices for important agricultural pests prevention, and supply materials for further study of Cry2Ab-type protein structure and insecticidal mechanism.
[1]Akhurst RJ,James W, Bird LJ,Beard C.Resistance to the CrylAc δ-endotoxin of Bacillus thuringiensis in the Cotton Bollworm,Helicoverpa armigera (Lepidoptom: Nocmidae)[J].Journal of Economic Entomology, 2003, 96(4):1290-1299
[2]Ali M I, and Luttrell R G.Susceptibility of bollworm and tobacco budworm (Lepidoptera: Noctuidae) to Cry2Ab2 insecticidal protein[J].Journal of economic entomology, 2007, 100(3):921-931
[3]Chitkowski RL, Turnipseed SG, Sullivan MJ, Bridges WC.Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var[J].kurstaki Berliner proteins for management of noctuid (Lepidoptom) pests. Journal of Economic Entomology, 2003, 96(3):755-762
[4]Chen ZY, Li CY, Liu JB, Zhang J, Huang DF.Expression and insecticidal activities analysis of silent gene cry2Ab3 from Bacillus thuringiensis C002 Strain[J].Acta Microbiologica Sinica, 2002, 42(5):561-566
[5]Escudero IR, Estela A,Porcar M,Martinez C,Oguiza JA,Escriche B,Ferre J, Caballerol P.Molecular and insecticidal characterization of a Cry lI protein toxic to insects of the families Noctuidae,Tortricidae,Plutellidae and Chrysomelidae[J].Applied and Environmental Microbiology, 2006, 72(7):4796-4804
[6]Jain D, Udayasuriyan V, Arulselvi PI, Dev SS, Sangeetha P.Cloning,characterization,and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1[J].Applied Biochemistry Biotechnology, 2006, 128(3):185-194
[7]Kuo WS, Chak KF.Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA[J].Applied and Environmental Microbiology, 1996, 62(4):1369-1377
[8]Li CY, Zhang J, Song FP, Han LL, Li GX, Huang DF.Expression and insecticidal activity of a novel gene cry2ab4 from Bacillus thuringiensis strain B-Pr-88[J].Shengwu Gongcheng Xuebao, 2007, 23(4):634-638
[9]Rasapirose S, Itsares T, Watanalai P.Coexpression of the silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis subsp. aizawai SP41 leads to formation of amorphous crystal toxin and enhanced toxicity against Helicoverpa armigera[J].Journal of Invertebrate Pathology, 2014, 116:48-55
[10]Reyes RA, Ibarra JE.Plasmid patterns of Bacillus thuringiensis type strains[J].Applied and Environmental Microbiology, 2008, 74(1):125-129
[11]Sauka DH, Cozzi JG, Benintende GB.Screening of cry2 genes in Bacillus thuringiensis isolates from Argentina[J].Antonie Van Leeuwenhoek, 2005, 88:163-165
[12]Sambrook J, Fritsch EF, Maniatis T.A Laboratory of Molecular Cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2002
[13]Schagger H, Von JG.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa[J].Anal Biochem, 1987, 166(2):368-379
[14]Sevim A, Emine E, Zihni D, Demir S.A Novel cry2Ab gene from the indigenous isolate Bacillus thuringiensis subsp[J].Kurstaki. Journal of Microbiology Biotechnology, 2012, 22(1):133-140
[15]Song FP, Zhang J, Huang DF, Xie TJ, Yang ZW, Dai LY, Li GX.Establishment of PCR-RFLP identification system of cry genes from Bacillus thuringiensis[J].Sci Agric Sin, 1998, 31:19-24(宋福平,张杰,谢天健,杨自文,戴莲韵,李国勋. 苏云金芽抱杆菌cry基因PCR-RLFP鉴定体系的建立. 中国农业科学,1998,31(3):13-18
[16]Song FP,Zhang J,Gu AX,Wu Y,Han LL,He KL,Chen ZY,Yao J,Hu YQ,Li GX, Huang DF.Identification of cry1I-type genes genefrom Bacillus thuringiensis strains and characterization of a novel crylI-type gene[J].Appliedand Environmental Microbiology, 2003, 69(9):5207-5211
[17]Swiecicka I, Bideshi DK, Federici BA.Novel isolate of Bacillus thuringiensis subsp thuringiensis that produces a quasicuboidal crystal of Cry1Ab21 toxic to larvae of Trichoplusia ni[J].Applied and Environmental Microbiology, 2008, 4(4):923-930
[18]Tabashnik BE,Dennehy TJ,Sims MA,Larkin K, Head GP, Moar, WJ, Carrière Y.Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab[J].Applied and Environmental Microbiology, 2002, 68(8):3790-3794
[19]Wang YB, Lang ZH, Zhang J, He KL, Song FP, Huang DF.Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize ( Zea mays L[J].Chinese Science Bulletin, 2008, 53(20):3185-3190