米黑根毛霉脂肪酶基因密码子优化及重叠延伸PCR合成

目的]对米黑根毛霉脂肪酶基因的密码子进行优化,并采用重叠延伸PCR合成设计的基因序列。方法]根据毕赤酵母(Pichiapastoris)密码子使用偏好性,对米黑根毛霉脂肪酶成熟肽基因进行全面密码子优化,根据优化后的氨基酸编码序列设计合成30条长度约为48 bp的寡核苷酸片段,并采用重叠延伸PCR法扩增合成全长基因序列。结果]经凝胶电泳和酶切鉴定、测序分析表明,合成的目的基因与设计的序列相一致。结论]该研究为脂肪酶基因在毕赤酵母中的构建及后续表达奠定了基础。

Codon Optimization of Rhizomucor miehei Lipase Gene and Its Synthesis by Overlapping PCR

Objective ] The aim was to optimize the codon of Rhizomucor miehei lipase （RML） gene and synthesize its sequence by overlap- ping PCR. Method] According to the bias in codon choice of the high expression gene in Pichia pastoris, the gene encoding the mature por- tion of Rhizomucor miehei lipase with high specific activity was optimized. And according to the coding sequence of optimized amino acids, 30 oligonucleotide fragments at length of 48 bp were synthesized, respectively, and the full-length gene was amplified by overlap extension PCR method. Result] The synthetic gene sequences were aligned with the designed sequence by gel electrophoresis, restriction enzyme digestion and sequencing analysis. Conclusion] This study provided foundation for the construction and subsequent expression of the lipase gene in Pichia pastoris.