斜卧青霉114-2 cbh1基因的TAIL-PCR克隆及与抗阻遏突变株JU-A10的比较

Cloning CellobiohydrolaseⅠfrom Penicillium decumbens 114-2 with TAIL-PCR and comparing with its derepressed mutant JU-A10

【目的】研究斜卧青霉(Penicillium decumbens)114-2与其抗阻遏突变株JU-A10外切酶基因序列的差异。【方法】用热不对称交错PCR(TAIL-PCR)和RT-PCR扩增得到斜卧青霉114-2外切葡聚糖酶Ⅰ(cbh1)基因全长和cDNA全长。【结果】cbh1基因全长为1500bp,含有两个内含子,编码453个氨基酸(GenBank,EF397602)。克隆并分析了1.9kb的cbh1基因上游序列,分别发现了葡萄糖代谢抑制因子CREⅠ与纤维素酶转录调控蛋白ACEΙ的两个的潜在结合位点。【结论】在相同的培养条件下,其抗阻遏突变株JU-A10的外切酶活明显高于野生株114-2。两菌株的cbh1基因序列完全一致,说明外切酶活明显提高不是由于cbh1基因发生突变引起的。

[Objective] We studied the differences in gene sequence of cellobiohydrolase Ⅰ gene （cbhl） from PeniciUium decumbens 114-2 and its derepressed mutant JU-A10. [Methods] We cloned cbhl and its full-length cDNA from Penicillium decumbens 114-2 by the modified thermal asymmetric interlaced polymerase chain reaction （TAIL-PCR） and RT-PCR. [Results] The total length of cbhl was 1500 bp. It contained 2 introns and encoded 453 amino acids （GenBank Accession No.EF397602）. The upstream sequence （1.9 kb） of cbhl gene was also cloned and sequenced. It contained two putative binding sites for the carbon catabolite repressor CRE Ⅰ and two putative binding sites for cellulases transcriptional regulator ACE Ⅰ. [Conclusion] The derepressed strain JU-A10 was a multiple mutant of the wild strain114-2. The mutant produced several times more cellobiohydrolase activity per ml of culture medium when compared with 114-2. The cbhl gene sequence of the mutant was the same with the wild strain. While four single base mutations were detected on the upstream sequences （1.9 kb） of cbhl gene.The result suggests that the evidently enhanced cellobiohydrolase activity of the mutant is not due to cbhl protein-coded sequence. The true reason maybe refer to single base mutations of the upstream sequence that effect the transcription regulation of mutant JU-A10. As a result, the secretion of CBH Ⅰ increased.