水葫芦胞质型谷氨酰胺合成酶EcGS1全长cDNA的克隆及序列分析

摘要/Abstract

摘要： 以水葫芦根部总RNA逆转录得到的cDNA为模板, 参照其他植物的胞质型谷氨酰胺合成酶（GS1）氨基酸保守序列设计简并引物, 进行PCR扩增, 以得到的产物为基础, 采用RACE技术获得水葫芦胞质型谷氨酰胺合成酶EcGS1全长cDNA。全长为1434 bp, 开放阅读框为1071 bp, 编码356个氨基酸, 分子量为39.3 kD, 等电点pI为5.52。序列相似性分析显示, 该序列与其他植物的GS1氨基酸序列具有较高的相似性。通过亚细胞定位预测, 确定EcGS1为胞质型谷氨酰胺合成酶。

关键词: 水葫芦, 谷氨酰胺合成酶基因, RACE, 克隆

Abstract: In this study, the template cDNA which was reversely transcribed from the total RNA of Eichharnia crassipes, was subjected to polymerase chain reaction（PCR）with the degenerate primers which was designed according to sequences of the Cytosolic Glutamine Synthetase（GS1）from other plants. The full-length cDNA sequence was achieved with Rapid Amplification of cDNA End method（RACE）from the amplification product. It includes 1434 bp with the open reading frame of 1071 bp which encoded 356 coding amino acids with a predicted size of 39.3 kD and a calculated pI of 5.52. The result of sequence homology analysis showed that the deduced protein had relatively high amino acid identity with GS1s from other plants. The prediction of sub-cellular localization showed that EcGS1 is a cytosolic glutamine synthetase.

Key words: Eichharnia crassipes, Glutamine synthetase, RACE, Cloning

蒋丽花, 傅明辉, 李园枚, 严国花, 郑李军, 陈肖丽, 彭进平. 水葫芦胞质型谷氨酰胺合成酶EcGS1全长cDNA的克隆及序列分析[J]. 生物技术通报, 2014, 0(7): 93-99.

Jiang Lihua, Fu Minghui, Li Yuanmei, Yan Guohua, Zheng Lijun, Cheng Xiaoli, Peng Jinping. Cloning and Analysis of Cytosolic Glutamine Synthetase cDNA from Eichharnia crassipes[J]. Biotechnology Bulletin, 2014, 0(7): 93-99.

参考文献

[1] 韩娜, 葛荣朝, 赵宝存, 等.植物谷氨酰胺合成酶研究进展[J].河北师范大学学报, 2004, 28（4）：407-410.
[2] Sun H, Huang QM, Su J. Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance [J]. Journal of Plant Physiology and Molecular Biology, 2005, 31（5）：492-498.
[3] Man HM, Boriel R, El-Khatib R, Kirby EG. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability [J]. New Phytologist, 2005, 167（1）：31-39.
[4] Fuentes SI, Allen DJ, Ortiz-Lopez A, et al. Overexpression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations [J]. Exp Bot, 2001, 52：1071-1081.
[5] Rana NK, Mohanpuria P, Yadav SK. Expression of tea cytosolic glutamine synthetase is tissue-specific and induced by cadmium and salt stress [J]. Biologia Plantarum, 2008, 52（2）：361-364.
[6] Lee HJ, Abdula SE, Jang DW, et al. Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress [J]. Plant Cell Rep, 2013, 32：1521-1529.
[7] Pascual MB, Jing ZP, Kirby EG, et al. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin [J]. Phytochemistry, 2008, 69：382-389.
[8] Tabuchi M, Sugiyama K, Ishiyama K, et al. Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1；1, a cytosolic glutamine synthetase1；1 [J]. The Plant Jounal, 2005, 42：641-651.
[9] Ishiyama K, Inoue E, Tkahashi AW, et al. Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutam-ine synthetase in Arabidopsis [J]. Biol Chem, 2004, 279（16）：16598-16605.
[10] Martin A, Lee J, Kichey T, et al. Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production [J]. The Plant Cell, 2006, 18：3252-3274.
[11] Teixeira J, Pereira S, Cánovas F, et al. Glutamine synthetase of potato（Solanum tuberosum L. cv. Désirée）plants：cell- and organ-specific expression and differential developmental regulation reveal specific roles in nitrogen assimilation and mobilization [J]. Experimental Botany, 2005, 56（412）：663-671.
[12] Castro-Rodríguez V, García-Gutiérrez A, Canales J, et al. The glutamine synthetase gene family in Populus [J]. BMC Plant Biology, 2011, 11：119.
[13] Seabra AR, Vieira CP, Cullimore JV, et al. Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds [J]. BMC Plant Biology, 2010, 10：183-198.
[14] 陈胜勇, 李观康, 汪云, 等.谷氨酰胺合成酶的研究进展[J].中国农学通报, 2010, 26（22）：45-49.
[15] 濮培民.改善太湖马山水厂水源区水质的物理-生态工程实验研究[J].湖泊科学, 1993, 5（2）：171-180.
[16] 窦鸿身, 蹼培民, 张圣照, 等.太湖开阔水域凤眼莲的放养实验[J]. 植物资源与环境, 1995, 4（1）：54-60.
[17] 孙文浩, 俞子文, 余叔文.城市富营养化水域的生物治理和风眼莲抑制藻类生长的机理[J].环境科学学报, 1989, 9（2）：187-195.
[18] 蒋艾青.凤眼莲对城郊污水鱼塘的净化试验[J].淡水渔业, 2003, 33（5）：43-44.
[19] 袁桂良, 刘鹰.凤眼莲对集约化甲鱼养殖污水的静态净化研究[J].农业环境保护, 2001, 20（5）：322-325.
[20] Casabianca LM. Large production of Eichhornia crassipes of paper industry effluent [J]. Bioresoure Technol, 1995, 54：35-38.
[21] 袁蓉, 刘建武, 成旦红, 等.凤眼莲对多环芳烃（萘）有机废水的净化[J].上海大学学报：自然科学版, 2004, 10（3）：272-276.
[22] 贺丽虹, 沈颂东.水葫芦对水体中氮磷的清除作用[J].淡水渔业, 2005, 35（3）：7-9.
[23] 宋伟, 韩士群, 刘海琴, 等.水葫芦去除污水中氮磷的效果[J].安徽农业科学, 2008, 36（25）：1107-11079.
[24] Paula MM, Lígia ML, Isabel MS, et al. Expression of the plastid-located glutamine synthetase of Medicago truncatula accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids [J]. Plant Physiol, 2003, 132（1）：390-399.
[25] Llorca O, Betti M, González JM, et al. The three-dimensional structure of an eukaryotic glutamine synthetase：Functional implications of its oligomeric structure [J]. J Struct Biol, 2006, 156：469-479.
[26] Sakamoto A, Ogawa M, Masumura T, et al. Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L. [J]. Plant Molecular Biology, 1989, 13：611-614.
[27] Ochs G, Schock G, Trischler M, et al. Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus [J]. Plant Molecular Biology, 1999, 39：395-405.
[28] Rana NK, Mohanpuria P, Yadav SK. Cloning and characterization of a cytosolic glutamine synthetase from Camellia sinensis（L.）O. Kuntze that is upregulated by ABA, SA, and H²2O² [J]. Mol Biotechnol, 2008, 39：49-56.
[29] 王小纯, 张同勋, 李高飞, 等.小麦谷氨酰胺合成酶基因克隆与其表达特性分析[J].河南农业大学学报, 2012, 46（5）：487-492.
[30] 王江, 张景六.稻属谷氨酰胺合成酶家族的系统分析[J].植物生理学通讯, 2007, 43（2）：277-282.