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【目的】克隆柑橘青霉菌(意大利青霉菌,Penicillium italicum)CYP51的同源基因,并对基因序列进行生物信息学分析。【方法】通过PCR及染色体步移技术得到基因的完整序列及上下游调控序列。通过生物信息学手段对基因的结构进行分析:使用NNPP分析软件预测转录起始位点,并利用TFSEARCH1.3软件分析转录因子结合位点。利用SWISS-MODEL在线软件对蛋白进行同源模建。【结果】克隆得到了意大利青霉菌CYP51的同源基因,命名为Pi CYP51B。获得的Pi CYP51B及上下游调控区序列总长为3 496 bp,包括上游910 bp和下游834 bp的序列。Pi CYP51B基因的开放阅读框全长1 575 bp,编码524个氨基酸。该基因含有3个分别为74、51、52 bp的内含子,分别位于247 bp与320 bp之间,519 bp与569 bp之间,1 635 bp与1 686 bp之间。Pi CYP51B 5′上游调控区序列的总长为579 bp。生物信息学分析结果显示:转录起始位点位于上游458 bp处;上游调控区不仅包含启动子的核心结构序列TATA盒(位于-25 bp处),也包含多个转录因子结合位点,如Abd-B、ADR1、AP-4、GATA-1、Cdx A、Clox和Oct-1等。在上游调控序列中嘌呤含量高,而且从+387 bp处开始存在4个连续高嘌呤含量的热激转录因子特异性结合位点(HSF);在+106 bp处开始存在3个连续的Cdx A转录因子结合位点。选用人CYP51晶体结构(PDB ID:3LD6)为模板,利用SWISS-MODEL在线软件构建了意大利青霉菌CYP51B蛋白的结构模型。【结论】克隆了意大利青霉Pi CYP51B基因,其上游含有多个热激应答转录因子特异性结合位点(HSF)表明其参与逆境应答。该基因作为意大利青霉菌CYP51的同源基因,可能与青霉菌对真菌药物的抗药性密切相关。
【Objective】 The objective of this study was to clone the homologous genes of CYP51 from Penicillium italicum and to analyze the gene sequence by bioinformatics. 【Method】 The complete sequence of the gene and upstream and downstream regulatory sequences were obtained by PCR and chromosome walking techniques. The structure of the gene was analyzed by bioinformatics: the transcriptional start site was predicted using the NNPP analysis software and the transcription factor binding site was analyzed using TFSEARCH 1.3 software. Homology modeling of proteins using SWISS-MODEL online software. 【Result】 The homologous gene of Penicillium italicum CYP51 was cloned and named as Pi CYP51B. The total length of Pi CYP51B and upstream and downstream regulatory regions was 3 496 bp, including the sequence of 910 bp upstream and 834 bp downstream. The full-length open reading frame of Pi CYP51B gene is 1 575 bp, encoding 524 amino acids. The gene contains three introns of 74,51,52 bp respectively located between 247 bp and 320 bp, between 519 bp and 569 bp, between 1 635 bp and 1 686 bp. The total length of the Pi CYP51B 5 ’upstream regulatory region sequence is 579 bp. Bioinformatics analysis showed that the transcriptional start site was located at 458 bp upstream. The upstream regulatory region contained not only the TATA box (located at -25 bp) of the core sequence of the promoter, but also a number of transcription factor binding sites, such as Abd-B, ADR1, AP-4, GATA-1, CdxA, Clox, Oct-1 and the like. Purine content was high in the upstream regulatory sequence, and four continuous high purine content heat-sensitive transcription factor-specific binding sites (HSF) existed at +387 bp. Three consecutive Cdx A Transcription factor binding site. CYP51 crystal structure (PDB ID: 3LD6) was selected as a template, and the structural model of CYP51B protein of Penicillium chinense was constructed by online software SWISS-MODEL. 【Conclusion】 Clone of the CYP51B gene of Penicillium italia, which contains multiple heat shock-responsive transcription factor-specific binding sites (HSFs) upstream, indicates its involvement in stress response. This gene, as a homologous gene of Penicillium sp. CYP51 in Italy, may be closely related to the resistance of fungi to penicillium.