一个家蝇未知功能基因的克隆及其生物信息学分析
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摘要
家蝇(Musca domestica L.)属于双翅目昆虫,在世界范围内分布极其广泛。作为一种重要的资源昆虫,家蝇可广泛的应用于化工、农业、医药、食品、化妆品、环保、印染、造纸以及酶固定化载体等领域。目前,国内外学者在家蝇的形态学、生物学、生态学特性、免疫机理和人工饲养应用等方面做了大量的研究,而在分子生物学领域的基础研究则较少。检测发现一个家蝇EST序列(NCBI登录号为：ES608747)在数据库中不存在同源序列,初步判断其基因可能是家蝇特有的未知功能基因,因此对此家蝇未知功能基因(unknown functional gene of Musca domestica,简称UFGMD)的cDNA全长序列进行克隆和分析显得很有必要,可为进一步研究此基因的功能和作用机理打下基础。
本文以一已知家蝇EST序列为基础,通过RACE技术克隆得到其cDNA全长序列,采用在线生物信息学软件对其编码蛋白的性质、结构和功能进行预测分析,主要研究结果如下：
1.根据已知的ES608747序列设计特异引物GSP1进行5'-RACE反应,克隆得到的UFGMD基因5'端为726bp；再根据已知的5'端序列设计引物GSP2进行3'-RACE反应,得到UFGMD基因3'端为308bp,将UFGMD基因5'端和UFGMD基因3'端进行拼接得到的UFGMD基因cDNA全长序列为824bp。克隆得到的UFGMD基因包含一个588bp的开放阅读框(49bp-636bp),编码的蛋白具有195个氨基酸。
2. UFGMD编码的蛋白不具有信号肽序列,不属于分泌蛋白,去除N端甲硫氨酸(M)的氨基酸序列即为成熟的UFGMD蛋白。利用在线分析软件对UFGMD蛋白理论分子量、理论等电点、分子式、氨基酸组成、二硫键、摩尔消光系数、脂溶指数、不稳定指数、亲水性指数和磷酸化位点进行了预测。
3.综合二级结构和疏水簇的预测结果,确定UFGMD蛋白含有5个α螺旋和11个p折叠；利用SAM-T08对UFGMD蛋白的三级结构进行预测,在二级结构水平上,UFGMD蛋白具有6个α螺旋和11个β折叠,与UFGMD蛋白二级结构的预测及其相近,仅多一个α螺旋。
4.经blastn检测显示：不存在与UFGMD基因同源性较高的序列,但部分区域与S6k和S6k耳的同源性非常高；FASTA检测显示：不存在与UFGMD蛋白同源性在40%以上的序列,结果中仅拟嗜凤梨果蝇(Drosophila pseudoananassae)着丝粒组蛋白(NCBI登录号为：AAL78894.1)属于昆虫蛋白序列,其同源性为27.6%。结合亚细胞定位分析结果,UFGMD蛋白可能在细胞核和高尔基体中发挥作用,其具体功能有待进一步的研究。
Housefly (Musca domestica L.) belongs to Diptera, extremely broad distribution in the worldwide. As an important resource insect, housefly was widely used in chemical industry, agriculture, medicine, food, cosmetics, environmental protection, printing and dyeing, paper making, and carrier of enzyme immobilization and other fields. At present, a lot of research on the housefly has done in the fields of morphology, biology, ecological characteristics, immune mechanism and breeding, however, basic research in the field of molecular biology is less. An EST sequences does not exist homologous sequences in the database, it may be an unknown functional gene that unique to the housefly, so cloning and analysis the cDNA sequences of the unknown functional gene of Musca domestica is necessary, that can prepare for further study of the mechanism and function to the gene.
In this paper, based on an EST sequences from housefly, its cDNA sequence was cloned by RACE; using online bioinformatics software, the character, structure and function of protein was predicted, the main findings are as follows:
According to the specific primer GSP1 for 5'-RACE reaction, the 5'-end of UFGMD (726bp) was cloned; then design specific primer GSP2 to clone the 3'-end of UFGMD (308bp) through 3'-RACE reaction. Obtain an 824bp full-length cDNA sequence through splice the 5'-end and 3'-end of UFGMD. It contains an open reading frame with 588bp (49bp-636bp), encodes a protein with 195 amino acids.
UFGMD pro-protein does not have a signal peptide, so not belong to secreted protein; remove the N-terminal methionine (M), the amino acid sequence is the mature UFGMD protein. Using the online analysis software, its theoretical molecular weight and isoelectric point, molecular formula, amino acid composition, disulfide bonds, molar extinction coefficient, aliphatic index, instability index, grand average of hydropathicity, phosphorylation sites were predicted.
Comprehend secondary structure prediction and hydrophobic cluster analysis, the UFGMD protein contains 5 a-helices and 11β-strands; using the SAM-T08 to predict tertiary structure of the UFGMD, that has 6 a-helixs and 11 P-strands on the secondary structure level, and similar to the result of secondary structure prediction, increased by only one a-helix.
The blastn analysis showed that:There is no high homology sequence with UFGMD gene, but partial sequences are high homology to S6k and S6kⅡ. FASTA analysis showed that:there is no sequence that homology more than 40% with UFGMD protein, only the centromeric histone from Drosophila pseudoananassae (NCBI accession number: AAL78894.1) is insect protein, the homology is 27.6%. Combined with subcellular localization analysis, UFGMD protein may play a role in the nucleus and the Golgi apparatus; its specific function needs to be further studied.
本文以一已知家蝇EST序列为基础,通过RACE技术克隆得到其cDNA全长序列,采用在线生物信息学软件对其编码蛋白的性质、结构和功能进行预测分析,主要研究结果如下：
1.根据已知的ES608747序列设计特异引物GSP1进行5'-RACE反应,克隆得到的UFGMD基因5'端为726bp；再根据已知的5'端序列设计引物GSP2进行3'-RACE反应,得到UFGMD基因3'端为308bp,将UFGMD基因5'端和UFGMD基因3'端进行拼接得到的UFGMD基因cDNA全长序列为824bp。克隆得到的UFGMD基因包含一个588bp的开放阅读框(49bp-636bp),编码的蛋白具有195个氨基酸。
2. UFGMD编码的蛋白不具有信号肽序列,不属于分泌蛋白,去除N端甲硫氨酸(M)的氨基酸序列即为成熟的UFGMD蛋白。利用在线分析软件对UFGMD蛋白理论分子量、理论等电点、分子式、氨基酸组成、二硫键、摩尔消光系数、脂溶指数、不稳定指数、亲水性指数和磷酸化位点进行了预测。
3.综合二级结构和疏水簇的预测结果,确定UFGMD蛋白含有5个α螺旋和11个p折叠；利用SAM-T08对UFGMD蛋白的三级结构进行预测,在二级结构水平上,UFGMD蛋白具有6个α螺旋和11个β折叠,与UFGMD蛋白二级结构的预测及其相近,仅多一个α螺旋。
4.经blastn检测显示：不存在与UFGMD基因同源性较高的序列,但部分区域与S6k和S6k耳的同源性非常高；FASTA检测显示：不存在与UFGMD蛋白同源性在40%以上的序列,结果中仅拟嗜凤梨果蝇(Drosophila pseudoananassae)着丝粒组蛋白(NCBI登录号为：AAL78894.1)属于昆虫蛋白序列,其同源性为27.6%。结合亚细胞定位分析结果,UFGMD蛋白可能在细胞核和高尔基体中发挥作用,其具体功能有待进一步的研究。
Housefly (Musca domestica L.) belongs to Diptera, extremely broad distribution in the worldwide. As an important resource insect, housefly was widely used in chemical industry, agriculture, medicine, food, cosmetics, environmental protection, printing and dyeing, paper making, and carrier of enzyme immobilization and other fields. At present, a lot of research on the housefly has done in the fields of morphology, biology, ecological characteristics, immune mechanism and breeding, however, basic research in the field of molecular biology is less. An EST sequences does not exist homologous sequences in the database, it may be an unknown functional gene that unique to the housefly, so cloning and analysis the cDNA sequences of the unknown functional gene of Musca domestica is necessary, that can prepare for further study of the mechanism and function to the gene.
In this paper, based on an EST sequences from housefly, its cDNA sequence was cloned by RACE; using online bioinformatics software, the character, structure and function of protein was predicted, the main findings are as follows:
According to the specific primer GSP1 for 5'-RACE reaction, the 5'-end of UFGMD (726bp) was cloned; then design specific primer GSP2 to clone the 3'-end of UFGMD (308bp) through 3'-RACE reaction. Obtain an 824bp full-length cDNA sequence through splice the 5'-end and 3'-end of UFGMD. It contains an open reading frame with 588bp (49bp-636bp), encodes a protein with 195 amino acids.
UFGMD pro-protein does not have a signal peptide, so not belong to secreted protein; remove the N-terminal methionine (M), the amino acid sequence is the mature UFGMD protein. Using the online analysis software, its theoretical molecular weight and isoelectric point, molecular formula, amino acid composition, disulfide bonds, molar extinction coefficient, aliphatic index, instability index, grand average of hydropathicity, phosphorylation sites were predicted.
Comprehend secondary structure prediction and hydrophobic cluster analysis, the UFGMD protein contains 5 a-helices and 11β-strands; using the SAM-T08 to predict tertiary structure of the UFGMD, that has 6 a-helixs and 11 P-strands on the secondary structure level, and similar to the result of secondary structure prediction, increased by only one a-helix.
The blastn analysis showed that:There is no high homology sequence with UFGMD gene, but partial sequences are high homology to S6k and S6kⅡ. FASTA analysis showed that:there is no sequence that homology more than 40% with UFGMD protein, only the centromeric histone from Drosophila pseudoananassae (NCBI accession number: AAL78894.1) is insect protein, the homology is 27.6%. Combined with subcellular localization analysis, UFGMD protein may play a role in the nucleus and the Golgi apparatus; its specific function needs to be further studied.
引文
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