The Synergism Between Bacillus Sphaericus 100 Kilo-dalton Mosquitocidal Toxin And Cyt1Aa Toxin In Bacillus Thuringiensis

Mtxl (100-kilodalton mosquitocidal toxin) is a kind of toxin synthesized in vegetative growth stage of Bacillus sphaericus (B.s). The purified Mtxl has the same toxicity as the binary toxin from B.s. However it is susceptible to protease(s) and cannot express in a large amount in the sporulation phase of natural occurring strain of B.s.With the aim of increasing the expression and stability of Mtxl, the mtxl gene originated from B. sphaericus SSII1 was cloned to a shuttle vector pBU4. Two recombinant plasmids pMT9 and pMT4 were obtained, with the inserted fragments in the opposite orientations. Both of the E. coli transformants could express the Mtxl toxin and had the medium toxicities against both the resistant and susceptible Culex.qninqiiefasciatus larvae. The two B. thuringiensis subsp. israelensis recombinants, which contained recombinants plasmid pMT4 and pMT9 respectively, were obtained by electroporation. The bioassay results showed that the recombinants B-pMT9 and B-pMT4 had toxicities both to resistant and susceptible C.quinqnefasciatus larvae during vegetative growth stage, having the LC?o values similar to that of .fi.sSSII-1. However, the toxic levels of the final sporulated cultures of recombinants B-pMT4 and B-pMT9 differed, with a LCso value of 2 49mg/ml for B-pMT9 and little toxicity for B-pMT4 By using the plasmid pMT9, mtxl gene from B.sphaericus was ligated with p20 and cytJAa gene, giving recombinant plasmid pMPX2. The recombinant B.thuringiensis subsp. israelensis B-pMPX2 can produce a rhombus crystalline inclusion body during its sporulation SDS-PAGE analysis proved that CytlAa had overexpressed during the sporulation of the recombinant. It was found that B-pMPX2 strain remained stable toxicity, whatever during vegetative phase or sporulation phase, which was similar to the expression of Mtxl gene in thestrain of protease(s)-deficient B. sphaericus. Meanwhile, a synergism between CytlAa and Mtxl toxin has been observed during the bioassay of different recombinants. The bioassay result that the LTsoof the FWC of B-pMT9, B-pMPX2 and B-pBA30 against 3rd-4rd instar larvae of C.quinquefasciatus were 28.53 h, 17.22 h and 7.61 h respectively, indicates that the CytlAa play a key role in the synergism with Mtx 1 for faster mosquitocidal activity.