Bacillus cereus B-02拮抗作用相关基因的克隆与检测
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摘要
蜡样芽孢杆菌(Bacillus cereus)B-02分离自淄博市感染灰霉病的大棚蔬菜土壤,是一株具有很强的抗灰霉病菌(Botrytis cinerea)活性的拮抗细菌。为探讨菌株对Bo.cinerea的防病机理,利用转座子标签法构建B-02抑菌活性消失的突变体,并通过TAIL-PCR方法研究转座因子的插入位点及其侧翼基因,旨在克隆发现新的抑菌功能相关基因,进而分析该位点所在基因或其侧翼基因对该表型的调控机制。
提取枯草芽孢杆菌(Bacillus subtilis)PY143中携带转座子Tn917的非穿梭性温度敏感型质粒pTV1,电击转化pTV1质粒至野生Ba.cereus B-02菌株,在电压9.0-12.5kv/cm,电阻200Ω,电容25μF的电击转化条件下,得到了具有氯霉素抗性且可以稳定、持续复制的转化子17个,并经质粒电泳及PCR检测证实。将得到的转化子在44.5℃持续地高温诱变,pTV1质粒消除,Tn917插入到Ba.cereus B-02基因组中,筛选得到具有红霉素和林可霉素抗性但失去氯霉素抗性的转座突变子1670个,在本实验中平均转座效率为4.29×10~(-4),通过继代培养,证明突变菌株具有遗传稳定性,表明Tn917插入片段在大多数突变菌株中可以稳定遗传,经PCR检测,可证明pTV1中的Tn917片段插入到Ba.cereus B-02基因组中。进一步测定这些突变子对Bo.cinerea的抑制作用,最终筛选得到了1个对病原菌抑制能力丧失的突变子,命名为B-02-T。随机选择6株突变菌株,以含部分Tn917片段的DIG标记探针和EcoRⅠ消化其基因组进行southern杂交,结果证实突变菌株基因组中有且只有单一的Tn917插入,且可以随机诱变Ba.cereus野生株,产生在不同位点突变的突变株。
采用TAIL-PCR法从突变菌株B-02-T中克隆Tn917插入位点侧翼的序列,分别得到Tn917插入位点上下游两条基因片段,经去除载体污染、上下游基因拼接后,得到一段2705bp的基因序列,将其进行blast比对后,发现其与芽孢杆菌质粒序列同源性较高,同源序列表达的蛋白为假定蛋白,推测其可能为与抑菌活性相关的新基因。
The bacterial strain B-02,isolated from the tomato infected by Botrytis cinerea in Zibo,was a Bacillus cereus strain effective to control Bo.cinerea.To research the antagonizing mechanism of Ba.cereus B-02 against Bo.cinerea at molecular gene level, the antibiosis-free mutant was constructed using transposon tagging technique,and then integration sites and flanking sequences were identified by TAIL-PCR(thermal asymmetric interlaced PCR).It was aimed at to find new antibacterial functional genes and then analyze the regulation mechanism of this phenotype.
In this study,pTV1,a temperature-sensitive suicide vector with Tn917,was introduced by electroporation into Ba.cereus B-02 in the condition of 9.0-12.5kv/cm, 200Ω,25μF.17 transformants with chloramphenicol resistance were obtained and this property was highly stable and inheritable,which was proved by PCR and plasmid electrophoresis.By constant high temperature(44.5℃) mutation,the transposon Tn917 was successfully inserted into the genome of Ba.cereus B-02 and 1670 mutants were obtained resistant to erythromycin and lincomycin but susceptible to chloromycetin.In this study,the average efficiency of transposition was 4.29×10~(-4),no replication fusant was appeared.By successive transfer culture,Tn917 was still inherited in the mutant strains proved by PCR and southern blotting.Among 1670 mutants,1 antibiosis-free mutant was obtained no more antagonizing Bo.cinerea,named as B-02-T.Southern blotting proved that transposon Tn917 mutagenesis resulted in random genome insertions in.Southern blotting analysis showed that Tn917 was randomly integrated into the genome of Ba. cereus B-02 strain,with single insertion copy.
The flanking gene fragment of Tn917 insertion site was cloned by TAIL-PCR amplification using the genome of mutant strain B-02-T.After sequencing and gene splicing of the upper and downstream fragment by removing Tn917 vector sequence,a 2705 bp gene fragment was obtained.Homology alignment revealed that this sequence showed high homology to the plasmid gene of Bacillus which expressed hypothetical protein.From the above,it could be infered that the target gene was a new antibacterial functional gene.
提取枯草芽孢杆菌(Bacillus subtilis)PY143中携带转座子Tn917的非穿梭性温度敏感型质粒pTV1,电击转化pTV1质粒至野生Ba.cereus B-02菌株,在电压9.0-12.5kv/cm,电阻200Ω,电容25μF的电击转化条件下,得到了具有氯霉素抗性且可以稳定、持续复制的转化子17个,并经质粒电泳及PCR检测证实。将得到的转化子在44.5℃持续地高温诱变,pTV1质粒消除,Tn917插入到Ba.cereus B-02基因组中,筛选得到具有红霉素和林可霉素抗性但失去氯霉素抗性的转座突变子1670个,在本实验中平均转座效率为4.29×10~(-4),通过继代培养,证明突变菌株具有遗传稳定性,表明Tn917插入片段在大多数突变菌株中可以稳定遗传,经PCR检测,可证明pTV1中的Tn917片段插入到Ba.cereus B-02基因组中。进一步测定这些突变子对Bo.cinerea的抑制作用,最终筛选得到了1个对病原菌抑制能力丧失的突变子,命名为B-02-T。随机选择6株突变菌株,以含部分Tn917片段的DIG标记探针和EcoRⅠ消化其基因组进行southern杂交,结果证实突变菌株基因组中有且只有单一的Tn917插入,且可以随机诱变Ba.cereus野生株,产生在不同位点突变的突变株。
采用TAIL-PCR法从突变菌株B-02-T中克隆Tn917插入位点侧翼的序列,分别得到Tn917插入位点上下游两条基因片段,经去除载体污染、上下游基因拼接后,得到一段2705bp的基因序列,将其进行blast比对后,发现其与芽孢杆菌质粒序列同源性较高,同源序列表达的蛋白为假定蛋白,推测其可能为与抑菌活性相关的新基因。
The bacterial strain B-02,isolated from the tomato infected by Botrytis cinerea in Zibo,was a Bacillus cereus strain effective to control Bo.cinerea.To research the antagonizing mechanism of Ba.cereus B-02 against Bo.cinerea at molecular gene level, the antibiosis-free mutant was constructed using transposon tagging technique,and then integration sites and flanking sequences were identified by TAIL-PCR(thermal asymmetric interlaced PCR).It was aimed at to find new antibacterial functional genes and then analyze the regulation mechanism of this phenotype.
In this study,pTV1,a temperature-sensitive suicide vector with Tn917,was introduced by electroporation into Ba.cereus B-02 in the condition of 9.0-12.5kv/cm, 200Ω,25μF.17 transformants with chloramphenicol resistance were obtained and this property was highly stable and inheritable,which was proved by PCR and plasmid electrophoresis.By constant high temperature(44.5℃) mutation,the transposon Tn917 was successfully inserted into the genome of Ba.cereus B-02 and 1670 mutants were obtained resistant to erythromycin and lincomycin but susceptible to chloromycetin.In this study,the average efficiency of transposition was 4.29×10~(-4),no replication fusant was appeared.By successive transfer culture,Tn917 was still inherited in the mutant strains proved by PCR and southern blotting.Among 1670 mutants,1 antibiosis-free mutant was obtained no more antagonizing Bo.cinerea,named as B-02-T.Southern blotting proved that transposon Tn917 mutagenesis resulted in random genome insertions in.Southern blotting analysis showed that Tn917 was randomly integrated into the genome of Ba. cereus B-02 strain,with single insertion copy.
The flanking gene fragment of Tn917 insertion site was cloned by TAIL-PCR amplification using the genome of mutant strain B-02-T.After sequencing and gene splicing of the upper and downstream fragment by removing Tn917 vector sequence,a 2705 bp gene fragment was obtained.Homology alignment revealed that this sequence showed high homology to the plasmid gene of Bacillus which expressed hypothetical protein.From the above,it could be infered that the target gene was a new antibacterial functional gene.
引文
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[2]童蕴慧,郭桂萍,徐敬友,等.拮抗细菌对番茄植株抗灰霉病的诱导[J].中国生物防治,2004,20(3):187-189.
[3]童蕴慧,徐敬友,陈夕军,等.番茄灰霉病菌拮抗菌的筛选和应用[J].江苏农业研究,2001,22(4):25-28.
[4]李桂霞,马汇泉,刘婧,等.番茄灰霉病高效拮抗菌株的鉴定及通过导入β-1,3-葡聚糖酶基因提高其生防效果[J].中国生物工程杂志,2007,27(4):44-49.
[5]李桂霞,马汇泉,刘婧,等.番茄灰霉病高效拮抗菌株的鉴定及抗菌活性研究[J].山东农业科学,2007,2:69-72.
[6]杜立新,冯书亮,王容燕,等.拮抗BS-208菌株对番茄灰霉病诱导抗性的初步研究[J].华北农学报,2005,20(6):84-87.
[7]林东,徐庆,刘忆周,等.枯草芽孢杆菌SO113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001,9(1):77-80.
[8]汪澈,何月秋,张永庆.枯草芽孢杆菌B9601-Y2抑菌蛋白活性及产生条件的研究[J].植物病理学报,2005,35(1):30-36.
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