Study On Cry Insecticidal Genes And Function Proteome Of Crystal Proteins From Bacillus Thuringiensis 4.0718

Biotechnology of modern plant protect_is very important for insuring food safety and humanhealth. In the past few years, it has been a world-wide trend to use Bt to replace chemicalinsecticide for pest control in plant. However, further studies are needed to increase itsinsecticidal toxicity, seek new cry functional gene and deepen its proteome analysis.This paper employs microbial technology, molecular biotech and methodology ofProteomics. An insecticidal strain 4.0718 of Ballilus thuringiensis was bred, its cry gene wasstudied systematically and a new insecticidal gene was obtained. And for the first time, theinsecticidal crystal proteins from Balillus thruingiensis were researched using proteome analysis.Also, high toxin insecticidal recombinant strain was constructed by combining B. thuringiensiscry gene and tchiB gene from tobacco.The wide application of Bt as a microbial insecticide in agricultural production depends to alarge degree on the breeding of a high toxin strain. In the research, 30 strains of Bacillusthuringiensis were isolated from 858 samples of dry land, vegetable garden and rice soils indifferent ecological regions of Hunan province, and selected as an original from the 30 strainswas strain 701^2c typical of B. thuringiensis.By means of multiple mutagenesis of UV and double NTG+ LiCl and through microscopicexamination of smear dyeing, it was first found that the insecticidal property of B.thuringiensisis closely related with the shape, size and number of the crystal protein and the proportion ofspore and crystal protein. After screenings, one highly toxic mutant strain 4.0718 was obtained.Tests showed that its toxicity to the larval of Helicoverpa armigera was 4.527 times higher thanthat of the control strain and the lethal rate reached 20%, 97% and 100% respectively 6, 12 and24h after treatment. The virulence of 4.0718 did not change after ten-generation transmission.This method to a simple and efficient way of breeding high toxin insecticidal stains. Still, thefermentation conditions of a highly toxic mutant strain 410718 of Bacillus thuringiensis werediscovered by using orthogonal experiment and bioassays of insecticidal activity.Using a PCR strategy, this study identified rapidly Bacillus thuringiensis strain 4.0718 thatharbored the known cry1 and cry2 type genes. These results suggested the novel Bacillusthuringiensis 4. 0718 had plenty of cry -type genes. This was evidence that Bt 4. 0718 hasconsiderable application and research value. To construct a new fusion protein to enhance thetoxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 wascombined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process,the Enterokinase site sequence was inserted into the midst of the two genes. Then the fusion gene fragment carrying the upstream promoter region and the downstream terminator region ofcry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzymedigestions and subclonings a new expression vector pHUAccB6and pHUAccB7 was obtained.The two vectors was transformed into B. thuringiensis acrystalliferous strain XBU001 withelectroporation to obtain the recombinant strain HAccB7 and HAccB6.Under AFM and SEM,there were some bipy ramidal crystals with a size of 1.5×3.0μm. Bioassay showed that the fusioncrystals from recombinant strain HAccB7 and HAccB6 were high toxic against third-instarlarvae of Plutella xylostella with the LC₅₀ (after 72 h) value of 9.10μg/ml. The fusion proteinswhich have constructed had more toxicity than Cry1Ac crystal proteins. The study will enhancethe toxicity of B. thuringiensis Cry toxins protein and make a ground for construction the fusiongenes of B.thuringiensis cry gene and other foreign toxin genes and the recombinant strain withhightoxicity.Bt insecticidal protein has been separated, using two modified methods that two-phaseliquid partition and isoelectric sediment point. The extracted insecticidal crystal proteins werespearateed by 2DE, and submitted to MS identified.We systemly studied the ICPs proteome.Comparatively from three Bt kurstaki 4.0718,wuhanensis 140, israelensis H14 and twoengnerring strains Although pH3-10 and pH4-7gels were used by us, most of the spots were inthe gel pH range 4 to 7.The ICPs are usually high molecular weight, hydrophobic proteins. Thereare 129 matched spots between HAc5and HAccB5 maps (the matching rate was 10%), usingisoelectric point methods 90±9 spots in HAc5, 193±11spots in HAccB5 strain.There are 80matched spots between gels of HAc5and HAccB5 (the matching rate was 10%), Thecharacteristics of the 2-DE maps of ICPs from these three strains and two engineering strainpartially explain such differences.After digestion in gel with TPCK-trypsin, Cry1Ac amid the differentially expressedproteins were identified by MALDI-TOF-MS and searched in the SWISS-PROT database withMASCOT in two strains, it is found that the function of some of the different proteins, such astoxins protein (Cry1Ac,Cry2Aa,CryIG, CryH2, Cry1Ab16, Cry2Ac,), proteinsynthesis,transporter (ATPsynthase F1, gamma subunit, ATP synthase F1, beta subunit,translation elongation factor Ts, translation elongation factor G), metabolism (branched-chainamino acid aminotransferase, Enolase, pyruvate dehydrogenase complex E3 component,pyruvate dehydrogenase complex e1 component, beta subunit, fructose-bisphosphate aldolase,classâ…±, putative respiratory nitrate reductase delta chain, Negative regulator of geneticcompetence clpC/mecB), protein folding(chaperone protein dnak), hypothetical protein tlr2278,synthase beta chain, TEM-7 beta-lactamase, unknown hypothetical. we found two commonproteins. The heat shock proteins (Hsp60) and Translation Elongation Factor Tu, which help with protein refolding and prevent protein degradation, an exogenous insecticidal toxity gene hada significance effect on the expression sperectra of ICPs and on the virulence of the Bt strains aswell.In summary, it is a very usefull reference thought for us to screen, optimize fermentableconditions, fuse between cry gene and other gene, and analyse function protein for hight toxicitystrains from B.thuringiensis. Thus, the characteristic proteins of Cry, as identified in theirinsecticidal crystal proteomes, could also be used as new screening markers of the high toxicity.Cry and function proteomic has been identified the first systemic investigation B. thuringiensisstrain 4.0718, this bring forth new ideas that domain of different protoxin is close relationshipbetween insecticidal toxicity, recombinant mechanism and insect recepter.