为初步探究黄曲霉McrA的生物学功能,采用反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)克隆黄曲霉转录因子AfMcrA基因,并对其编码蛋白的理化性质、保守结构域、亚细胞定位、二级结构等进行预测。实时荧光定量聚合酶链式反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)对不同培养时间AfMcrA的表达情况进行分析。结果表明,AfMcrA基因全长1 448 bp,由3个内含子和4个外显子组成。cDNA为1 101 bp,编码366个氨基酸。生物信息学预测,AfMcrA是一个分子质量为39.82 kDa,理论等电点为8.97,不稳定的亲水蛋白,且不含信号肽和跨膜结构域;AfMcrA具有一个高度保守的Zn(Ⅱ)2Cys6锌指结构域,属于C6转录转因子;PredictNLS预测AfMcrA亚细胞定位于细胞核中;AfMcrA二级结构主要由无规则卷曲组成,延伸链、α-螺旋和β-转角含量较低。qRT-PCR结果显示,AfMcrA的相对表达量在培养6 d时达到峰值,分别为培养2 d和8 d的15.79倍和14.76倍。结合qRT-PCR结果及已报道McrA的功能,推测AfMcrA可能参与黄曲霉次级代谢产物合成的调控。
To preliminary explore the biological functions of Aspergillus flavus McrA, the transcription factor gene AfMcrA was cloned by reverse transcription-polymerase chain reaction(RT-PCR). The physicochemical properties, conserved domain, secondary structure, and subcellular localization of AfMcrA were predicted by online tools. The relative expression of the transcript levels of AfMcrA in different cultural periods was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that the genomic DNA of AfMcrA was 1 448 bp, containing 4 extrons and 3 introns. The nucleotide sequence of the cDNA of AfMcrA contained an open reading frame of 1 101 bp, encoding a protein of 366 amino acids. Bioinformatics analysis predicted that AfMcrA was an unstable hydrophilic protein with a relative molecular mass of 39.82 kDa. The theoretical isoelectric point of AfMcrA was 8.79,without transmembrane domain nor signal peptide. AfMcrA had a conserved Zn(II)2Cys6 zinc finger domain, termed as a C6 transcription factor. PredictNLS analysis predicted that AfMcrA was located in the nucleus. The secondary structure of AfMcrA primarily consisted of random coils, and the proportion reached 84.43%. qRT-qPCR results revealed that the relative expression level of AfMcrA reached its peak at 6 days after inoculation, which was 15.79 and 14.76 times of that at 2 and 8 days, respectively. Combined with our results and related reports on McrA, it was speculated that AfMcrA may be involved in the regulation of secondary metabolites of A. flavus.
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