黄瓜细胞质型谷氨酰胺合成酶基因(GS1)的克隆及其在低氮条件下的表达

doi:10.3864/j.issn.0578-1752.2012.15.011

摘要/Abstract

摘要： 【目的】分离和克隆黄瓜谷氨酰胺合成酶GS1基因，分析其序列特征，了解其在低氮条件下的表达情况。【方法】依据黄瓜基因组数据库中Csa015274基因编码区全序列，应用引物设计软件Primer Premier 5.0设计引物，从黄瓜叶片cDNA中克隆该基因，用生物信息学方法对获得的cDNA序列及推定氨基酸序列进行分析鉴定，并用实时荧光定量PCR法研究GS1基因在不同氮素浓度下的表达变化。【结果】分离到GS1基因，GenBank登录号为JQ277263。该基因长1 071 bp，编码356个氨基酸，与甜瓜(Cucumis melo L.)GS1基因同源性高达97%。该基因编码的蛋白是1个不稳定的疏水蛋白，无跨膜结构，无信号肽，存在蛋白激酶C磷酸化位点，酪蛋白激酶Ⅱ磷酸化位点，N-十四酰化位点，酪氨酸激酶磷酸化位点等活性位点。GS1基因表达模式分析显示，在低氮条件下，该基因下调表达，随着氮素浓度的增高GS1基因的表达量增加。在高浓度的氮素水平下，该基因的表达同样受到抑制。【结论】成功从黄瓜叶片中分离克隆到GS1基因，该基因具有已知物种GS1基因的特征,可用于该基因的功能研究。

关键词: 黄瓜, GS1基因, 克隆, 序列分析, 低氮

Abstract: 【Objective】The purpose of this study is to clone full-length cDNA of key enzyme gene GS1 in plant nitrogen metabolism and to investigate its sequence characteristics and to analyze its expression under low nitrogen conditions.【Method】Sequences of primers were designed based on the Csa015274 gene coding region in the cucumber genome database using Primer Premier 5.0. The gene cDNA sequence was cloned from cucumber leaves using RT-PCR techniques; bioinformatics methods were used to analyze cDNA sequence obtained and putative amino acid sequence, qRT- PCR method were used to study the expression of GS1 gene under different nitrogen conditions. 【Result】 Cucumber GS1 gene (JQ277263) was got from cDNA by PCR. The gene is 1 071 bp, encoding 356 amino acids. Analysis of the nucleotide sequence by BLAST on line indicated that the homology of GS1 was up to 97% between Cucumis sativus L. and Cucumis melo L. The protein encoded by this gene is an unstable and hydrophobic protein with protein kinase C phosphorylation site, casein kinase II phosphorylation site, N-myristoylation site, tyrosine kinase phosphorylation site, and other active sites. Expression patterns analysis showed that the gene are down-regulated under low nitrogen conditions. And as the nitrogen concentration increased GS1 gene expression was increased. However, the expression of this gene is also inhibited at the high nitrogen level.【Conclusion】Gene GS1 was firstly isolated and characterized from cucumber. This gene had genetic characteristics similar with all species and it could provide reference for functional research.

Key words: Cucumis sativus L., GS1 gene, clone, sequence analysis, low nitrogen conditions

冯卓, 秦智伟, 武涛, 何红梅. 黄瓜细胞质型谷氨酰胺合成酶基因(GS1)的克隆及其在低氮条件下的表达[J]. 中国农业科学, 2012, 45(15): 3100-3107.

FENG Zhuo, QIN Zhi-Wei, WU Tao, HE Hong-Mei. Cloning and Expression of Cytosolic Glutamine Synthetase (GS1) in Cucumis sativus L. Under Low Nitrogen Conditions[J]. Scientia Agricultura Sinica, 2012, 45(15): 3100-3107.

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