吡咯伯克霍尔德氏菌JK-SH007高GC基因PCR体系的扩增研究

Exploration of GC-rich Gene PCR System from Burkholderia pyrrocinia JK-SH007

对生防菌吡咯伯克霍尔德氏菌JK-SH007基因组上高GC基因PCR扩增体系进行探讨。通过对该菌株基因组进行100℃处理5 min;PCR反应体系为10μL,在体系中使用高保真LA Taq(Mix)酶,并分别加入不同体积分数的DMSO、2×GC bufferⅠ、2×GC bufferⅡ,然后选用适当的PCR程序对产几丁质酶基因(bcc1,GC含量71.1%)进行扩增,在此基础上,选择最适体系对产ACC脱氨酶基因(acd S,GC含量66.86%)、内切葡聚糖酶基因(GC含量74.71%)、色氨酸单加氧酶基因(iaa M,GC含量6.62%)、嗜铁素合成相关基因(cepR,GC含量64.44%)进行PCR扩增,将扩增出的片段与PMD19-T vector连接后转化至大肠杆菌JM-109中进行测序。结果表明:JK-SH007菌株基因组经高温处理后,在PCR反应体系中加入10%DMSO可成功扩增出该菌株bcc1基因(2 484 bp)。该体系同样适用于acd S、内切葡聚糖酶、iaa M、cepR等基因,而且测序结果与预期的序列一致。因此,该体系具有广泛性,而且简单可靠,成本低,扩增效率高,具有很强的实用性,为洋葱伯克霍尔德氏菌群菌株及其他菌株高GC含量基因的克隆提供了参考依据。

GC-rich gene PCR system from biocontrol strain Burkholderia pyrrocinia JK-SH007 was established. The genome of strain JK-SH007 was processed for 5 min at 100 ℃. The total volumes of PCR were 10 μL. The PCR reactions added high-fidelity LA Taq （Mix） polymerase and different DMSO, 2 × GC bufferⅠ, 2 × GC bufferⅡ, respectively. Then the producing chitinase gene （bcc1, GC content： 71.1%） was amplified by the appropriate PCR procedures. And on this basis, the best PCR system was adopted for producing ACC deaminase gene （acdS, GC content： 66.86%）, endoglucanase gene （GC content： 74.71%）, tryptophan monooxygenase gene （iaaM, GC content： 68.62%） and siderophore biosynthesis-related gene （cepR, GC content： 64.44%）, respectively. The amplified product was cloned into the PMD19-T vector and then transformed into E.coli JM-109 for sequencing. The result showed that the PCR system added 10% DMSO could be successfully amplify bcc1 gene （2 484 bp） and be equally applicable to acdS gene, endoglucanase gene, iaaM gene and cepR gene. Moreover, the sequencing results were in complete accord with the expected sequences. So this PCR system is a widerange, simple, high quality, low-cost and effective way for amplification the GC-rich genes. All of these provide an important reference for the cloning of GC-rich genes of Burkholderia cepacia complex strains and others.