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目的:克隆金荞麦苯丙氨酸解氨酶(FdPAL)基因DNA和全长cDNA序列,对该基因进行序列分析,原核表达及其活性相关研究。方法:采用同源克隆和RT-PCR技术扩增苯丙氨酸解氨酶基因的DNA序列和全长cDNA序列并对其进行生物信息学分析;构建PAL原核表达载体pET30b(+)-FdPAL,在大肠杆菌Escherich coli BL21(DE3)中进行诱导表达,使用分光光度计法定量测定其酶活,使用薄层色谱法鉴定其催化活性。结果:金荞麦PAL基因DNA序列全长2 583 bp,由2个外显子,1个内含子构成(命名为FdPAL,GenBank登录号为HM628904);cDNA序列包含1个2 169 bp的开放阅读框(ORF),编码722个氨基酸,理论相对标准分子质量78.31 kDa,等电点5.94。SDS-PAGE分析表明,诱导后的新生蛋白质相对标准分子质量为75.37 kDa;诱导4 h后,表达产物的苯丙氨酸解氨酶比活力最高,达到4 386 nmol.g-1.min-1;薄层色谱结果表明,诱导表达产物具有催化苯丙氨酸转化为肉桂酸的活性。结论:首次从金荞麦中克隆到PAL基因,该基因具有植物PAL同源基因的典型特征;重组的金荞麦PAL基因表达载体[pET30b(+)-FdPAL]能有效地在大肠杆菌E.coli BL21(DE3)中表达,并形成具有一定催化功能的酶。
OBJECTIVE: To clone the FdPAL gene DNA and the full-length cDNA sequence of F buckberry, and to study the sequence, prokaryotic expression and activity of FdPAL gene. Methods: The DNA sequence and full-length cDNA sequence of phenylalanine ammonia-lyase gene were amplified by homology cloning and RT-PCR, and their bioinformatics analysis was performed. The prokaryotic expression vector pET30b (+) - FdPAL was constructed, The expression was induced in Escherichia coli BL21 (DE3), the enzyme activity was measured quantitatively using a spectrophotometer, and the catalytic activity was identified by thin layer chromatography. Results: The DNA sequence of PAL gene was 2 583 bp in length, which consisted of two exons and one intron (named FdPAL, GenBank accession number HM628904). The cDNA sequence contained a 2 169 bp open reading (ORF), encoding 722 amino acids, the theoretical relative standard mass of 78.31 kDa, isoelectric point 5.94. SDS-PAGE analysis showed that the relative standard molecular weight of the induced newborn protein was 75.37 kDa. After 4 h of induction, the specific activity of the phenylalanine ammonia-lyase of the expressed product was the highest, reaching 4 386 nmol.g-1 min -1 The results of TLC showed that the induced product had the activity of catalyzing the conversion of phenylalanine to cinnamic acid. CONCLUSION: PAL gene was cloned for the first time from Fagopyrum tataricum, which has the typical characteristics of PAL homologous genes. The recombinant Fagopyrum californica PAL gene expression vector [pET30b (+) - FdPAL] can effectively express in E.coli BL21 (DE3) expression, and the formation of a certain catalytic function of the enzyme.