CAJ | 학술논문

苏云金芽胞杆菌(Bacillus thuringiensis, Bt)是一种已被广泛用于农业害虫生物防治的昆虫病原细菌。本研究从永春天湖山矿区采集的159份样品中分离出1株Bt，光学显微镜检测发现菱形伴孢晶体。以cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11和cry1I的通用引物对该Bt分离菌进行Cry毒素基因分析，利用SDS-PAGE和双向电泳-质谱法(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)对Bt分离菌进行蛋白鉴定。结果表明，分离菌含有cry1基因型，经克隆、测序后可知，菌株中的cry1亚基因型与cry1Ac有极高的同源性。分离株表达的晶体蛋白约为130 kD，通过蛋白质谱分析表明，杀虫蛋白为Cry1Ac，与PCR-RFLP所得出的结果一致；通过质谱分析还鉴定出分子伴侣GroEL、F0F1型ATP-β亚基合成酶、延伸因子Tu和丙酮酸脱氢酶。生物信息学分析结果表明，这些蛋白主要参与帮助新生肽的正确折叠、能源生产和转换，以及提高细胞的应激能力以抵御外界不良刺激。本研究从煤矿中分离鉴定出Bt菌，为发现新资源、新型杀虫蛋白基因以及利用微生物治理重金属污染提供基础资料。
소운금아포간균(Bacillus thuringiensis, Bt)시일충이피엄범용우농업해충생물방치적곤충병원세균。본연구종영춘천호산광구채집적159빈양품중분리출1주Bt，광학현미경검측발현릉형반포정체。이cry1/7/9、cry2、cry3、cry4/10、cry5、cry6、cry8、cry11화cry1I적통용인물대해Bt분리균진행Cry독소기인분석，이용SDS-PAGE화쌍향전영-질보법(2-dimensional electrophoresis/mass spectrometry,2-DE/MS)대Bt분리균진행단백감정。결과표명，분리균함유cry1기인형，경극륭、측서후가지，균주중적cry1아기인형여cry1Ac유겁고적동원성。분리주표체적정체단백약위130 kD，통과단백질보분석표명，살충단백위Cry1Ac，여PCR-RFLP소득출적결과일치；통과질보분석환감정출분자반려GroEL、F0F1형ATP-β아기합성매、연신인자Tu화병동산탈경매。생물신식학분석결과표명，저사단백주요삼여방조신생태적정학절첩、능원생산화전환，이급제고세포적응격능력이저어외계불량자격。본연구종매광중분리감정출Bt균，위발현신자원、신형살충단백기인이급이용미생물치리중금속오염제공기출자료。
Bacillus thuringiensis (Bt) is an insect pathogenic bacteria that has been widely used in biological control of agricultural insect pests. In this study, one Bt strain was isolated from 159 samples collected from coalfields in Tianhu Mountain, Yongchun County, Fujian Province. Bipyramidal parasporal crystal of isolated Bt strain was observed under microscope. PCR results showed that isolated Bt strain carried cry1 type gene using general primers of cry1/7/9, cry2, cry3, cry4/10, cry5, cry6, cry8, cry11 and cry1I genes, then cry1 gene was cloned and sequenced. Homology analysis of nucleotide sequence of cry1 showed that it was of high similarity with the reported cry1Ac genes. Results of SDS-PAGE and 2-dimensional electrophoresis/mass spectrometry (2-DE/MS) showed that isolated Bt strain encoded approximately 130 kD crystal protein, which was then identified as insecticidal protein Cry1Ac by mass spectrometry (MALDI-TOF/TOF-MS). Molecular chaperone GroEL, ATP F0F1 synthase subunit beta, elongation factor Tu and pyruvate dehydrogenase were identified by MALDI-TOF/TOF-MS, respectively. Further understanding of protein functions were conducted through bioinformatics analysis, leading to correct folding of nascent peptides, energy production and conversion, as well as improving the ability of cells to withstand adverse external stimuli. This work provides basic data for discovering new Bt resources, new insecticidal protein and using microbial to control heavy metal pollution.