柑橘青霉菌CYP51B基因和上游调控区克隆及生物信息学分析

Cloning and bioinformatic analysis of CYP51B gene and its upstream regulatory region in Penicillium italicum

摘要:

【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PiCYP51B。获得的PiCYP51B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。PiCYP51B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。PiCYP51B 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、CdxA、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CdxA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PiCYP51B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。

Abstract:

[Objective] This study aimed to clone and characterize a CYP51 homologous gene in Penicillium italicum. [Methods] PCR and Genome walking strategies were used to obtain the gene sequence and its flanking region. Gene structure was analyzed by bioinformatic strategy: Gene transcriptional start site was predicted by NNPP software and TFSEARCH1.3 software was used to predict transcriptional factor binding sites. Homology modeling was performed using human CYP51 protein as the template through the online software SWISS-MODEL. [Results] A CYP51 homologous gene was successfully cloned from Penicillium italicum and named as PiCYP51B. A 3 496 bp sequence including 910 bp 5′ flanking sequence and 834 bp 3′ flanking sequence was obtained. The ORF of PiCYP51B is predicted to encode a protein of 525 amino acids. PiCYP51B contains three introns length 74, 51 and 52 bp, located between 247 bp and 320 bp, 519 bp and 569 bp, 1 635 bp and 1 686 bp respectively. The transcriptional start site is located 458 bp upstream of the initiation codon; the upstream regulatory region not only contain the core structure TATA box (located at 25 bp and 105 bp upstream of the initiation codon), but also contain several transcriptional factor binding sites such as Abd-B, ADR1, AP-4, GATA-1, CdxA, Clox and Oct-1. The proportion of purine is relatively high in the upstream regulatory region. From 387 bp upstream, there are four consecutive heat shot protein transcriptional factor binding sites (HSF); there are three consecutive CdxA transcriptional factor binding sites from 106 bp upstream. To further analysis the structure of PiCYP51B, homology modeling was performed using the online software SWISS-MODEL. [Conclusion] PiCYP51B, being the homologous gene of CYP51 may connect with the resistance to pesticides of P. italicum.