皮状丝孢酵母脂肪酸CoA连接酶(Facl)基因的克隆与分析

Cloning and Characterization of a Gene Encoding Fatty Acid Co A Ligase( Facl)from Trichosporon Cutaneum

从皮状丝孢酵母(Trichosporon cutaneum)中克隆了Facl基因及其5'-上游序列,并对Facl基因及其5'-上游序列进行了分析。根据前期的基因组文库测序结果,获得了898 bp DNA序列。以此为基础,2次使用染色体步移(Genome walking)方法,分别获得了854 bp和1 236 bp 5'-DNA序列。根据获得的5'和3'端序列,合成引物用于扩增Facl基因的全长DNA和c DNA序列。分析发现,皮状丝孢酵母Facl基因c DNA包含一个1 662 bp的开放读码框,DNA不含内含子序列。同时获得了482 bp的5'-上游序列。在5'-上游序列区域存在一系列预测的顺式作用元件。相似性分析的结果表明T.cutaneum和Rhodococcus erythropolis的Facl氨基酸序列相似性最高。该研究旨在为后继的Facl基因的功能和表达的研究、油脂代谢的遗传改造奠定基础。

The Facl gene and its 5 ＇-flanking sequence has cloned from Trichosporon cutaneum and amalysis.Based on the former result of genome library sequencing, 898 bp DNA DNA sequence, the 5＇ genomic DNA was cloned by genome walking m were amplified respectively in two genome walking experiments. On the sequence was obtained. Based on the 898 bp ethod; 854 bp and 1 236 bp DNA sequences basis of the 5＇and 3＇termini sequences, prim- ers were synthesized to amplify the full - length genomic DNA and eDNA sequence. The full - length Facl eDNA con- tained 1 662 bp open - reading frame （ ORF）, while the full - length DNA did＇ t contain intron sequence. In addi- tion, 482 bp 5＇- flanking sequence was obtained. There were a number of predicted cis - acting elements in 5＇- flan- king sequence. The results of similarity analysis revealed that the highest identity of Facl amino acid sequence could be found between T. cutaneum and Rhodococcus erythropolis. The study has laid foundation for further research on the function and overexpression of Facl gene, genetic manipulation of lipid metabolism as a success.