The analysis of Bacillus thuringiensis vegetative insecticical protein gene cloning and expression / 生物工程学报

Three kinds of Bacillus thuringiensis serotype-subsp. Leesis(H33) strain YBT-833, subsp. Aizawai(H7) strain YBT-1416 and subsp. Kurstaki(H3ab) strain YBT-1535, which were isolated by our lab, are chosen as original strain to clone vegetative insecticidal protein gene. Southern hybridization showed that vip genes are all localized at roughly 4-5 kb size-fractionated XbaI fragments of total DNA from YBT-833, YBT-1416 and YBT-1535. Three subgenomic libraries containing the vip gene fragment, were constructed with pUC19 as vector. Then, three vegetative insecticidal protein gene vip83, vip14 and vip15 are obtained from the libraries through the methods of colony-blot-in-situ screening and enzyme-cut detection. Comparision of DNA sequence made out that only vip83 gene exist five different base pairs with known vip genes. Because the sequences of vip14 and vip15 are the same, two of the three genes, vip83 and vip14, were subcloned to shuttle vehicle pHT315 to get recombinant plasmids pBMB8901 and pBMB8902 in turn. The plasmids were separately transformed into vip Bt. receptors BMB171 and 4Q7 to obtain four engineered strains BMB8901-171, BMB8902-171, BMB8901-4Q7 and BMB8902-4Q7. SDS-PAGE results indicated that all recombinant strains express 88 kD vegetative insecticidal protein. Bioassay also showed that the proteins of genes vip83 and vip14 both have certain toxicity to Lepidopteran insect larvae such as Heliochis armigera, Spodotera exigua and Plutella xylostella. While the toxicity of vip protein from four engineered strains to Plutella xylostellas are highest, whose LC50 value is 28.6, 31.6, 45.4 and 37.6 microL/mL respectively. This study will contributed to construct high efficacy and wide spectrum engineered strains on theory and reality.

THE SYNERGISM OF VEGETATIVE INSECTICIDAL PROTEIN VIP AND CRY PROTEIN FROM BACILLUS THURINGIENSIS / 微生物学通报

In this paper, vegetative( vip83 ) and crystal(cry1Ac10 and cry1Ca) insecticidal protein genes from Bacillus thuri ngiensis were simultaneously electrospored into the plasmid-free strain BMB17 1. By the means of the specific P CR detection, the recombinant strains BMB2830-171 contained cry 1Ac10 and vip83, and BMB2 882-171 had cry1Ca and vip83 , were obtained respectively. Under the control of r ecombinant strains with one gene, bioassay of the synergism between vegetative V ip83 and crystal Cry1Ac10( or Cry1Ca )insecticidal proteins to three important Lepidopteran pests were done. The results, by analysis of statistic bio-so ft, showed that the synergia relation of vegetative Vip83 and crystal Cry1Ac10 i nsecticidal protein toxic to Heliothis armigera wascounteracted, while Plu tella xylostella and Spodotera exigua unobservable. There was no synergis tic action between Vip83 and Cry1Ca insecticidal proteins with Spodotera exigu a as tested insect. Bu t their cooperation to Heliothis armigera was minus, and the counterpart to Plutella xylostella plus, whose cotoxicity factor is 32.6. The experiment of bi-g ene genetic stability also suggested that the synergia effection had certain molecu lar genetic stability in the same cell. This performance can be contributed to construct high-effect and wide-spectrum engineered strain.