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Study on Mariner Transposon Random Mutagenesis of Bacillus Thuringiensis in Vivo

Author: LiMingLei
Tutor: YuZiNiu；LiMingShun
School: Huazhong Agricultural University
Course: Microbiology
Keywords: mariner Bacillus thuringiensis Transposable Mutant
CLC: S476.1
Type: Master's thesis
Year: 2009
Downloads: 75
Quote: 1
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Abstract


Bacillus thuringiensis (Bacillus thuringiensis) is a Gram-positive bacteria, widely distributed in the soil can be highly expressed in spore formation insecticidal crystal proteins and the formation of the crystals. Researchers explored by means of various molecules insecticidal crystal protein is highly expressed its mechanism of formation of crystals and achieved certain results, but the specific mode of action of this mechanism, in particular the formation of crystals of the molecular mechanism remains unclear. Mariner transposons in Bacillus thuringiensis transposable efficiency, the preference of the presence or absence of the insertion site and whether to insert hot mariner transposon in Bacillus thuringiensis transposable characteristics for Bacillus thuringiensis bacilli mutant library building and crystal formation mechanism provides a powerful tool. Determination the mariner the integration vector pMarA and pMarB Bacillus thuringiensis crystal the mutant BMB171 and the Bacillus thuringiensis the transposition characteristics in YBT2346. mariner transposon in Bacillus thuringiensis crystal mutations in the strains BMB171 and wild strains YBT2346 of transposition efficiency in 10% or so. BMB171 in the detection efficiency of the insertion site is very low, so that the subsequent insertion site analysis difficult. In YBT2346 in the detection of the insertion sites is also very low, but also detects the same one point. This may be due the pMarA and pMarB two inverted repeat sequence between the E. coli origin of replication, and the detection efficiency of the insertion site by inverse PCR or digested DNA methods with linear pUC19 connected, equally very low . This defect makes the insertion site analysis quite difficult. The 2. Rebuild the mariner integration the carrier pMarA333 and pMarB333. With than pMarA and pMarB, pMarA333 pMarB333 was added between the two inverted repeat sequences of the origin of replication of E. coli, can be cut with the appropriate restriction enzyme treatment of DNA and self-ligated, transformed into E.coli DH5α would can detect the insertion sites. Determination pMarA333 and pMarB333 Bacillus thuringiensis crystal the mutant BMB171 and the Bacillus thuringiensis the transposition characteristics in YBT881. pMarA333 and pMarB333 Bacillus thuringiensis the aphakia mutation the strains BMB171 transposition efficiency of 7.6% and 11.6%, respectively. Transposition in Bacillus thuringiensis YBT881 (CCAM 020673) efficiency of 4.2% and 15.7%, respectively. pMarA333, and pMarB333 displayed than the transposition efficiency of another mariner integration vector pAW068 higher, this may be due to the arising from the difference of the promoter of the transposase enzyme. Restriction analysis found pMarA333 and pMarB333 in Bacillus thuringiensis crystal the mutant strains BMB171 no obvious insertion preference. Insertion sites analysis of Bacillus thuringiensis YBT881 most of the insertion sites of DNA sequence and the two have been completely sequenced Bacillus thuringiensis 97-27 (GI: 49.32824 million) and Al Hakarn (GI: 118 415 003) BLAST analysis . Mariner transposon insertion sites were scattered, and no preference for the non-coding region. In addition, some of the insertion site of the sequence, and it has been determined thuringiensis Bacillus plasmid sequences have relatively high similarity described mariner transposon may be inserted into the plasmid. Thus, mariner transposon mutagens is a relatively good transposition no preference sexual may be randomly inserted into the chromosome and on the plasmid, the coding region and the non-coding region. This we studied the functions of the different sites of the genome is very advantageous. contained pMarA333 and pMarB333 temperature sensitivity the replication origin pe194ts, spectinomycin resistance gene, erythromycin resistance gene and the transposase enzyme promoter P A and CTC has good versatility. In Therefore, pMarA333 and pMarB333 insertion mutant library building of Bacillus thuringiensis and other Gram-positive bacteria, has a relatively high value of potential applications.

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CLC: > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens
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