吡咯伯克霍尔德氏菌JK-SH007高GC基因P CR体系的扩增研究

摘要

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( acdS, 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( iaaM, 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(2484 bp)。该体系同样适用于acdS、内切葡聚糖酶、 iaaM、 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 10μL. 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 PMD ® 19-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 re-sults were in complete accord with the expected sequences. So this PCR system is a wide-range, simple, high qual-ity, 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.