油桐ACPase基因克隆及生物信息学分析
Cloning and bioinformatics analysis of ACPase from tung tree
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摘要: 【目的】克隆油桐酸性磷酸酶(ACPase)基因的全长序列,为进一步分析油桐ACPase的功能奠定基础。【方法】根据油桐转录组酸性磷酸酶蛋白全长序列设计引物,利用RT-PCR从油桐种子中克隆ACPase基因,对其cDNA序列、核苷酸序列的相似性、理化性质、疏水性、跨膜结构、二级结构及三级结构进行分析预测,同时进行多序列比对并构建系统树。【结果】克隆获得油桐ACPase基因,其开放阅读框为1152 bp,编码383个氨基酸残基,相对分子质量为40.94 kDa,理论pI为5.30,属可溶性不稳定蛋白。同源性和进化树的预测分析结果显示,油桐ACPase与毛果杨的ACPase蛋白同源性达84%。油桐ACPase存在磷酸酶基因家族的保守区域,ACPase编码蛋白的大部分氨基酸属于亲水性氨基酸。其蛋白二级结构主要由不规则卷曲、α-螺旋和β-折叠构成。ACPase蛋白包含1个跨膜螺旋区域,其相应氨基酸位置在136~156;包含1个疑似跨膜域,其相应氨基酸位置在255~275;每个跨膜结构域为由20个氨基酸残基组成的螺旋,同时跨膜蛋白的N端和C端位于细胞膜胞质的一侧。【结论】油桐ACPase基因的表达可能与其体内磷营养的分解利用等过程有关。Abstract: ObjectiveThe full-length sequence of acid phosphatase(ACPase)gene in tung tree was cloned to provide ref-erences for further studies on the functions of ACPase. MethodThe primers were designed according to the full-length se-quence of acid phosphatase gene in transcriptome of tung tree. ACPase gene from tung tree seeds was cloned by RT-PCR. Bioinformatic analysis was used to predict cDNA sequence and its deduced nucleotide sequence similarity, physicochemical properties, hydrophobicity, transmembrane structure, secondary structure, and tertiary structure of ACPase. Multiple se-quences alignment and phylogenetic trees were also constructed. ResultACPase in tung tree was cloned. The largest open reading frame of ACPase had 1152 bp in length and encoded a polypeptide of 383 amino acid residues. The estimated molecu-lar weight and isoelectric point of the putative protein was 40.94 kDa and 5.30, respectively. The ACPase was a soluble unsta-ble protein. BLASTs search result and phylogenetic tree analysis showed that peptide encoded by ACPase of tung tree had 84%similarity with ACPase of Populus trichocarpa. ACPase of tung tree had conservative regions of the phosphatase gene family. Most amino acid encoded by ACPase belonged to hydrophilic amino acid, and its protein secondary structure was mainly con-sisted of irregular coli,α-helix andβ-sheet. ACPase protein contained one transmembrane spiral region with its amino acid in 136-156 and one suspected transmembrane region with its amino acid in 255-275. Every transmembrane region was spirals consisted of 20 amino acid residues. N-terminal domain and C-terminal domain of the transmembrane protein were on one side of cytomembrane cytoplasm.ConclusionThe cloned ACPase of tung tree may be involved in the process of phosphorus nutri-tion in plants and the decomposition of soil organic phosphorus.