Cloning, Sequencing And Expression Of The Mosquitocidal Toxin Genes From Bacillus Sphaericus

Bacillus sphaericus strains have different toxicity and active spectrum against mosquito larvae. Among them, 1AB872 strain has high activity to susceptible Culex quinquefasciatus colony and medium activity to the B. sphaericus-resistant colony, while LP I -G strain shows same medium toxicity to both susceptible and resistant C. quinquefasciatus colonies. With the aim of underlining the molecular basis of toxin genes for these differences, we undertook the cloning, sequence analysis and expression of two toxin genes from B. sphaericus strainsThe binary toxin gene from B. sphaericus 1AB872 was located on a 3.5Kb HindIII chromosomal DNA fragment. After the total DNA was totally digested by HindlII, approximate 3.5Kb fragments were extracted and ligated into a shuttle vector pBU4. Two recombinant plasmids pBS 1 and pBS2, with their inserted fragment in the opposite orientation, were screened by HindIII restriction analysis and Southern blot. Sequence analysis revealed that the foreign fragment was composed of 3479bp and the sequence of the binary toxin gene was totally identical to that of the reference strain 2362. B. thuringiensis transformants Bt-B SI and Bt-B S2, which were obtained by electroporating the recombinant plasmids pBS 1 and pBS2 into an acrystalliferous B.t.i strain 4Q-7, expressed the binary toxin in a high level and then formed rhombus crystal and other irregular crystals during their sporulation. Toxicity bioassays showed that Bt-BS 1 and Bt-BS2 performed high toxicity only to susceptible mosquito larvae, with LC50 values of 2.32x lO~ and 5.89x 1O~ respectively, but no toxicity to resistant mosquito larvae. Therefore, we deduced that the activity of 1AB872 against resistant mosquito larvae was perhaps contributed to Mtx toxin or other unknown toxins, whose modes of action might be different from that of the binary toxin. Meanwhile, it was noticed that toxicity of Bt-BS2 against susceptible mosquito larvae was higher3than that ofBt-BSI and this might be explained by the inserted direction of the binary toxin gene perhaps influencing its expression in the recipient strain.In addition, cloning of the mosquitocidal toxin gene from Bacillus sphaericus LPI-G strain in E.coli was also studied in this paper. The total DNA of LP1-G was digested partially and randomly. The purifed fragments were ligated into the vector pUC 18 and the part of the genome library was contructed. Through toxicity bioassays, a strain E-UL68 was screened out, which performed midium toxicity against susceptible colony and some colonies of resistant mosquito larvae. Its LC50 values against susceptible and C3-41-resistant mosquito larvae were 2.58x1O~ and 3.66x1O~ respectively, equally to that of the wild strain LPI-G. Sequence analysis showed that the foreign fragment from LPI-G was composed of 3876bp and contained a whole gene, whose sequence was identical to that of mtxi gene from B. sphaericus 5511-1. Because the binary toxin of LP1-G has no toxicity against susceptible and resistant mosquito, while final whole culture of LP I -G has medium toxicity against susceptible and resistant mosquito, therefore, it was deduced that either the mosquitocidal toxin expressed by mtxl gene or other mosquitocidal active factors produced during the sporulation was perhaps responsible for the activity of LP1-G strain against susceptible and resistant mosquito.