人参质体ATP/ADP转运蛋白基因PgAATP1的克隆及表达分析
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摘要
目的克隆人参Panax ginseng中质体ATP/ADP转运蛋白基因(ATP/ADP transporter protein,AATP),为深入研究其在人参中的生物学功能奠定基础。方法利用人参14个组织部位转录组测序的转录组数据库,通过与NCBI下载的其他植物中AATP的m RNA序列进行本地Blast比对,从中筛选出质体ATP/ADP转运蛋白基因。利用PCR技术克隆获得该基因的全长,通过生物信息学软件分析该基因编码蛋白的信息。利用人参14个组织部位的转录组数据库分析该基因在人参不同组织中的表达模式,并利用实时荧光定量PCR检测茉莉酸甲酯处理条件下该基因的表达模式。结果获得人参AATP1基因全长c DNA,命名为Pg AATP1,该基因长度为1 866 bp,编码621个氨基酸,蛋白质相对分子质量为67 897.23,等电点为9.58。该蛋白与其他物种中的质体ATP/ADP转运蛋白比较类似,分析发现Pg AATP1在人参中所有组织中表达量均比较高,在果肉和叶片中表达量最高。实时荧光定量PCR检测发现Pg AATP1基因在茉莉酸甲酯处理条件下表达持续上调。结论获得Pg AATP1基因全长cDNA序列,该基因在淀粉合成旺盛的组织中表达量较高,且响应茉莉酸甲酯处理。
Objective To clone the full-length cDNA of the ATP/ADP transporter protein(AATP) genes in Panax ginseng to provide resources and some knowledge necessary for the further gene function study. Methods The mRNA sequence of the AATP genes in other plants were downloaded on the website of NCBI and used to perform local Blast alignment in the transcriptome of Jilin ginseng from 14 tissues. The AATP gene in Panax ginseng was cloned by PCR, and analyzed using bioinformatics software and online resources. The expression pattern of PgAATP1 gene in 14 tissues of Panax ginseng was analyzed using the expression profile of transcriptome and its expression level under methyl jasmonate was deceted by quantitative real-time PCR. Results A full-length cDNA sequence was successfully cloned from Panax ginseng and named as PgAATP1, which was 1866 bp in length and encoded 621 amino acids. The relative molecular mass of PgAATP1 protein calculated was 67 897.23 Da, and the isoelecric point calculated was 9.58. It was found that the protein was similar to the plastid AATP in other species. The expression of this gene was high in all tissues but higherin fruit flesh and leaf blade, and the expression of PgAATP1 gene was up-regulated by methyl jasmonate. Conclusion We have obtained the full-length of PgAATP1 gene. This gene expressed higher in tissues of vigorous starch synthesis and responsing to methyl jasmonate.
Objective To clone the full-length cDNA of the ATP/ADP transporter protein(AATP) genes in Panax ginseng to provide resources and some knowledge necessary for the further gene function study. Methods The mRNA sequence of the AATP genes in other plants were downloaded on the website of NCBI and used to perform local Blast alignment in the transcriptome of Jilin ginseng from 14 tissues. The AATP gene in Panax ginseng was cloned by PCR, and analyzed using bioinformatics software and online resources. The expression pattern of PgAATP1 gene in 14 tissues of Panax ginseng was analyzed using the expression profile of transcriptome and its expression level under methyl jasmonate was deceted by quantitative real-time PCR. Results A full-length cDNA sequence was successfully cloned from Panax ginseng and named as PgAATP1, which was 1866 bp in length and encoded 621 amino acids. The relative molecular mass of PgAATP1 protein calculated was 67 897.23 Da, and the isoelecric point calculated was 9.58. It was found that the protein was similar to the plastid AATP in other species. The expression of this gene was high in all tissues but higherin fruit flesh and leaf blade, and the expression of PgAATP1 gene was up-regulated by methyl jasmonate. Conclusion We have obtained the full-length of PgAATP1 gene. This gene expressed higher in tissues of vigorous starch synthesis and responsing to methyl jasmonate.
引文
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[14] Wang K, Jiang S, Sun C, et al. The spatial and temporal transcriptomic landscapes of Ginseng, Panax ginseng C.A. Meyer[J]. Sci Rep, 2015, 5:18283,doi:10.1038/srep18283.
[15]岳跃冲,范燕萍.植物萜类合成酶及其代谢调控的研究进展[J].园艺学报, 2011, 38(2):379-388.
[16] Trentmann O, Horn M, Scheltinga A C T, et al.Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae[J]. PLo S Biol,2007, 5(9):e231.
[17] Neuhaus H E, Thom E, M?hlmann T, et al. Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.[J]. Plant J,2010, 11(1):73-82.
[18] Schünemann D, Heldt H W. ADP/ATP Translocator from pea root plastids(comparison with translocators from spinach chloroplasts and pea leaf mitochondria)[J]. Plant Physiol, 1993, 103(1):131-137.
[19] Sahintóth M, Karlin A, Kaback H R. Unraveling the mechanism of the lactose permease of Escherichia coli[J]. PNatl Acad Sci USA, 2000, 97(20):10729-10732.
[20]王冲之,丁家宜.不同培养基及外源激素对西洋参毛状根的生长和皂甙含量的影响[J].植物资源与环境学报, 2001, 10(4):1-4.
[2] Heldt H W. Adenine nucleotide translocation in spinach chloroplasts[J]. Febs Lett, 1969, 5(1):11-14.
[3] Emes M J, Neuhaus H E. Metabolism and transport in non-photosynthetic plastids[J]. J Exp Bot, 1997, 48(12):1995-2005.
[4] Kampfenkel K, M?hlmann T, Batz O, et al. Molecular characterization of an Arabidopsis thaliana c DNA encoding a novel putative adenylate translocator of higher plants[J]. Febs Lett, 1995, 374(3):351-355.
[5] Mikami K, Ichimura K, Iuch S. Molecular characterization of a cDNA encoding a novel small GTP-binding protein from Arabidopsis thaliana L.[J].Biochim Biophys Acta, 1997, 1354(2):99-104.
[6] M?hlmann T, Tjaden J, Schw?ppe C, et al. Occurrence of two plastidic ATP/ADP transporters in Arabidopsis thaliana L.[J]. Eur J Biochem, 1998, 252(3):353-359.
[7] Yuen C Y L, Leelapon O, Chanvivattana Y, et al.Molecular characterization of two genes encoding plastidic ATP/ADP transport proteins in cassava[J]. Biol Plantarum, 2009, 53(1):37-44.
[8] Wang F, Ye Y, Niu Y, et al. A tomato plastidic ATP/ADP transporter gene SlAATP increases starch content in transgenic Arabidopsis[J]. Physiol Mol Biol Pla, 2016, 22(4):497-506.
[9] Wang F, Chen X, Zhang F, et al. A soybean plastidic ATP/ADP transporter gene, GmAATP, is involved in carbohydrate metabolism in transgenic Arabidopsis[J].Plant Biotechnol Rep, 2017, 11(3):135-146.
[10] Geigenberger P, Tjaden J S A, Quick P W, et al. Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity[J]. Plant Physiol, 2001, 125(4):1667-1678.
[11]王雁楠.甘薯淀粉合成相关基因IbAATP和Ib SSI的克隆及功能分析[D].北京:中国农业大学, 2015.
[12]于林卉,刘保申,李文兰,等.玉米ZmAATP基因的克隆及遗传转化载体的构建[J].山东农业大学学报:自然科学版, 2012, 43(3):321-327.
[13]黄伟奇.转ATP/ADP转运蛋白基因水稻的获得及其初步研究[D].福州:福建农林大学, 2005.
[14] Wang K, Jiang S, Sun C, et al. The spatial and temporal transcriptomic landscapes of Ginseng, Panax ginseng C.A. Meyer[J]. Sci Rep, 2015, 5:18283,doi:10.1038/srep18283.
[15]岳跃冲,范燕萍.植物萜类合成酶及其代谢调控的研究进展[J].园艺学报, 2011, 38(2):379-388.
[16] Trentmann O, Horn M, Scheltinga A C T, et al.Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae[J]. PLo S Biol,2007, 5(9):e231.
[17] Neuhaus H E, Thom E, M?hlmann T, et al. Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.[J]. Plant J,2010, 11(1):73-82.
[18] Schünemann D, Heldt H W. ADP/ATP Translocator from pea root plastids(comparison with translocators from spinach chloroplasts and pea leaf mitochondria)[J]. Plant Physiol, 1993, 103(1):131-137.
[19] Sahintóth M, Karlin A, Kaback H R. Unraveling the mechanism of the lactose permease of Escherichia coli[J]. PNatl Acad Sci USA, 2000, 97(20):10729-10732.
[20]王冲之,丁家宜.不同培养基及外源激素对西洋参毛状根的生长和皂甙含量的影响[J].植物资源与环境学报, 2001, 10(4):1-4.
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