Molecular Biological Characterization of Mosquitocidal Strain, Bacillus thuringiensis subspecies mogi

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농업생명과학대학 농생물학과(곤충학전공)
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서울대학교 대학원
B.thuringiensisovoidal-shaped crystalsmosquitocidalfull genome sequencethree-domain cry geneover-expression
학위논문 (박사)-- 서울대학교 대학원 : 농생물학과(곤충학전공), 2014. 2. 제연호.
Bacillus thuringiensis subspecies mogi was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. Plasmids from this species have been implicated in pathogenicity as they carry genes responsible for a variety of entomo-pathogenic diseases. The purpose of this study was to characterize the B. thuringiensis subsp. mogi strian, determine the full genome sequence, and investigate the molecular genetics of expression of novel toxin-related cry genes which located on the plasmid in B. thuringiensis subsp. mogi.
As a primary study, the flagellated vegetative cells of B. thuringiensis subsp. mogi were agglutinated with the H3 reference antiserum and further agglutinated with 3b and 3d monospecific antisera but non-reactive to 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. B. thuringiensis subsp. mogi showed activity against dipteran larvae, Culex pipiens molestus and Culex pipiens pallens while no lepidopteran toxicity. It produced three small ovoidal-shaped parasporal crystals combined together and whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the identification of the protein by nano-LC-ESI-IT MS analysis, the putative peptides of Cry27Aa, Cry39ORF2, and Cry20-like were detected. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis subsp. mogi contained only megaplasmids (> 30 MDa) on which the toxin genes were occasionally located.
Second, full genome sequence of the novel B. thuringiensis subsp. mogi strain was determined. The 6.0 Mb genome of B. thuringiensis subsp. mogi contains three replicons: a circular chromosome (5.40 Mb) encoding 5,652 predicted open reading frames (ORFs) and two mega-plasmids, pMOGI364 (364,564 bp) and pMOGI222 (222,348 bp). The G+C contents of these replicons ranged from 31.3% to 34.2% for pMOGI364 and pMOGI222, respectively, and did not significantly deviated from that of the chromosome (35.2%). There were seventeen toxin-related genes existed in these two mega-plasmids, and six of them (cry19Bb1, cry73Aa with cry40orf2, cry20Bb1, cry27Ab1, cry4Aa and cry56Ba1 with cry39orf2) belonged to the group of three-domain cry toxins.
Finally, to investigate the role of six novel three-domain cry genes in crystal production of B. thuringiensis subsp. mogi, the transcription level of these toxin genes were analyzed by quantitative PCR (qPCR). The results clearly indicated that all of these cry genes were successfully transcribed in wild type B. thuringiensis subsp. mogi strain in different transcription time with different maximum levels. Then, these cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, under the control of its own promoter, and introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. Another vector p1KSD, which containing a strong chimeric cyt1Aa promoter combined with the STAB-SD sequence was constructed and used to over-express the cry genes. To determine the function of the cry39orf2 and over-express the cry56Ba1 in cry56Ba1 operon, different combinations of Cry56Ba1 and Cry39ORF2 were synthesized in strain Cry-B. The stable inclusion in recombinant cells suggests that Cry39ORF2 assists in synthesis and crystallization of Cry56Ba1 by functioning like the C-terminal domain characteristic of Cry protein in the 130 kDa mass range. In addition, the increased Cry56Ba1 yield under the cyt1A-p/STAB-SD promoter has broadened the possibility of application in other toxins.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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