粗毛栓菌atp9基因的克隆、表达及序列分析

Cloning,Expression and Sequence Analysis of the Subunit Gene Atp9 Unit in Trametes Gallica

ATP合酶是生物体内能量代谢的关键酶,参与多种氧化磷酸化和光合磷酸化反应.atp9基因是ATP合酶的重要组成部分,其编码了ATP合酶A亚基上第9亚单位,与呼吸作用和光合作用密切相关.本研究利用atp9基因在进化过程中高度保守的特点,据已知近缘真菌基因序列,设计并合成了一对引物,以粗毛栓菌mRNA反转录得到的cDNA第一链为模板,PCR扩增得到atp9基因完整序列,并连接于原核表达载体pET32a(+)上.测序与序列分析表明:该克隆片段全长222 bp,共编码73个氨基酸,翻译的蛋白质分子量是7.35 kDa.转化大肠杆菌后经IPTG诱导,可高效表达外源融合蛋白,分子量大小与预测结果一致.经过同源比对和进化树分析,该克隆基因编码的氨基酸序列与可可丛枝病菌(Crinipellis perniciosa)和瓣环栓菌(Trametes cingulata strain ATCC 26747)中相对应的氨基酸序列相似度最高.本实验为未来进一步研究粗毛栓菌atp9基因和其蛋白功能,阐明其调控和作用机制奠定了基础.

ATP synthase is a key enzyme of energy metabolism, which was involved in a variety of oxidative phosphorylation and photophosphorylation in vivo. The atp9 gene encoding the ninth part of ATP synthase A sub-unit is an important component of ATP synthase. It is closely related with respiration and photosYnthesis. For its highly conserved sequence in evolution, one pair of primers were designed and synthesized according to the known gene sequences fi＇om closely related fungi. The first strand of Trametes gallica eDNA was amplified by reverse transcription and then the complete atp9 gene sequences were obtained by PCR. And then the product of PCR was ligated to the prokaryotie expression vector pET32a （ ＋ ） and transformed into E. coli for the expression of fusion protein. The sequenced and bioinfonnatics analysis showed that： the complete length of atp9 gene was 222 hp, and the peptide it encoded had 73 amino acids with a molecular mass of 7.35 kDa as predicted. Homology comparison and phylogenetic analysis indicated that the amino acid sequence of Trametes gallica atp9 gene possessed the highest homology with Crinipellis perniciosa and Trametes cingulata. This research laid the foundation for probing the function and metabolic pathways of atpA gene in the future.