扩展青霉PF898碱性脂肪酶基因组DNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有重要工业生产价值的碱性脂肪酶(PEL) .在通过 3′RACE和 5′RACE获得PEL完整的cDNA序列的基础上 ,通过PCR方法首次克隆了该脂肪酶的完整的基因组DNA序列 (GenBank登录号为AF330 6 35 ) .该脂肪酶DNA全长 14 0 4bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区DNA由 1135个碱基组成 ,含有 5个内含子 ,大小分别为 5 8bp、4 7bp、5 0bp、5 6bp和 6 9bp .在已报道的丝状真菌脂肪酶中 ,PEL基因的内含子数量最多 ,而其大小与其它丝状真菌脂肪酶基因的内含子一样 ,均为只有几十个碱基的小内含子 .PCR扩增获得的PLEDNA序列还包括由 195个碱基组成的 3′端非编码区序列 ,74个碱基的部分 5′端非编码区序列 .PELDNA全长序列中的 - 2 4至 - 2 7nt为TATAbox ,终止码TGA下游15 6nt出现AATAAA序列 ,TGA下游 182位出现poly(A)尾 ,为典型的真核基因结构 .同源性序列分析表明 ,PEL与其它真菌来源脂肪酶的基因组DNA序列同源性约为 39%～ 4 9% ,PEL内含子之间或PEL内含子与其它丝状真菌脂肪酶基因的内含子之间的序列同源性约 4 2 %～ 5 7% .

Cloning and Sequencing of Genomic DNA Encoding Alkaline Lipase from Penicillium expansum PF898

Penicillium expansum PF898 produces an alkaline lipase (PEL) with industrial value. Based on the cloning of PEL cDNA by the methods of 3′RACE and 5′RACE,two primers were designed and the full\|length genomic DNA was amplified from total DNA of the fungus by the method of PCR.The amplified DNA sequence of 1404 bp includess PEL coding area,3′and 5′non\|coding region.Analysis of the nucleotide sequence indicated that the genomic DNA of PEL (GenBank accession number AF3306635)was composed of 1135 bp and had six exons and five shorth introns (58 bp,47 bp,50 bp, 56 bp and 69 bp).The number of introns of PEL is more than that of other fungi lipases that have been sequenced and they are all short introns.The 3′non coding region is composed of 195 bp with an AATAAA sequence appeared at position 156 nt and the poly(A) tail is at the position 182 nt downstream of stop coden TGA. 5′non\|coding region of 74 bp was sequenced and the TATA box was found at -24 to-27 nt of the gene.The homology is about 39%～49% between the genomic DNA sequence of PEL and that of other lipases from fungi.The homology is about 42%～57% between the introns of lipases from PEL and other several fungus.