Studies On The Specific Cry Genes From Bacillus Thuringiensis Strains

1.Silent cry genes from Bacillus thuringiensis (Bt)The CAPS (cleaved amplified polymorphic sequences, CAPS) method of cryll genes from Bt strains was established. By using this system, cry] I-type genes of 142 Bt strains were determined. Among them, 98 strains contained cry]I-type genes, including cry] Ia-type gene in 68 strains, cry]Ib-type gene in 29 strains, cry] Ic-type gene in 8 strains, but no strain contained cry]Id-type gene. However, PCR digested fragments of 6 strains were different from the known cry] I genes, so they maybe contain a novel-type cry] I gene. According to the conserved regions of both downstream sequences of cryl-type genes and upstream sequences of cryll-type genes, a pair of specifically universal primers, S5una and S3una, was designed for identification of linkages between cry] -cry]! genes. 71 strains detected had positive products by PCR amplification. The results indicated that both cry] I-type genes and linkages of cryll-cryl gene were widespread in Bt strains.The linkage sequences of cryl-cryll genes from Bt strains BtcOO8 and J8 were cloned and sequenced. They were registered in GenBank database with accession number AF373208 and AF373209, respectively. The sequence analysis showed that a number of transcriptional terminations and no promoter were found in interval sequences between cry] and cry]! genes. It was proved that cry]! genes were silent in BtcOO7 and J8..Based on the results of identification for cry]! gene from Bt strains, a novel cry] I-type gene from Bt strain BtcOO7 had been cloned, and it encoded a crystal protein that was composed of 719 amino acids with MW 8lkDa and isoelectric point pH 5.91. Because the identities of amino acid sequences between this Cry 11 protein and known Cry 11 proteins were all less than 95%, the novel cryll gene was designated as cry] Ie] by the international Bt 6-endotoxin nomenclature committee (BENC). It was a first holotype cry gene identified and cloned in China.A novel cry]Ja gene from Bt strain BtcOO 1 had been cloned, and it encoded a crystal protein composed of 719 amino acids with MW 8lkDa and isoelectric point pH 5.995. By BENC, this gene was designated as cry]!a8 with accession number AF373207 in GenBank database.Both cry]!e] and crylla8 gene were inserted into expression vector pET-2 I b, and recombinant plasmids pETB-1IE and pBO8IIA were obtained and transformed into E. coil4L2I (DE3), respectively. The cry] lel and cry] 1a8 genes were highly expressed in 8L2 1(DE3). Cryl tel toxin was highly active against both Ostriniafurnacalis (LC50 =16.2 lppm) andPiutelIa xylostella (LC50 1 97.88ng/mL) larvae. Cry 1 1a8 protein was highly toxic to bothOstriniafurnacalis and Plutella xylostella larvae. LC50 against Ostriniafurnacalis neonate waslower than 23ppm, and LC50 against PluteIla xylostella larvae was 2227.38ng/mL02.The Cry toxins against Spodopera e.xiguaA novel cry] Ca gene from Bt strain J8 had been cloned, and it encoded a crystal protein that was composed of 1189 amino acids with MW 134.7kDa and isoelectric point pH 4.745. This gene was designated as cry] Ca7 by BENC. Accession number was AYO 15492 in GenBank database. Additionally, another crylCa gene from BtcOO8 was cloned, whose sequence were 100% identity with that of cry] Ca7.A novel cry] Cb gene from Bt strain BtcOO8 had been cloned, and it encoded a crystal protein that was composed of 1176 amino acids with MW l33kDa and isoelectric point pH 4.32. By Bt b-endotoxin nomenclature committee was this gene designated as cry] Cb2 with accession number AY007686 in GenBank database.Both cry] Ca7 and cry] Cb2 gene were inserted into vector pET-21b and transformed intoBL2 I (DE3), respectively. Cry 1 Ca7 toxin was active to Spodoptera exigua with LC502.74ng/mg (diet dry weight) , and not active to Ostrinia furnacalis and Plutella xylostella.Ciyl Cb protein was moderately toxic to Plutella xylostella.3.Site-mu tagenesis and domain swapping of Cry toxinBy PCR method, two different fragments of 5?terminal c...