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作物学报 ›› 2011, Vol. 37 ›› Issue (11): 1949-1955.doi: 10.3724/SP.J.1006.2011.01949

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甜菜nia基因的克隆及不同氮素形态诱导的差异表达

丁广洲1,2，侯静2，陈丽1，马凤鸣2,*，陈连江1,*   

  1. 1 中国农业科学院甜菜研究所, 黑龙江哈尔滨 150080; 2东北农业大学, 黑龙江哈尔滨 150030
  • 收稿日期:2011-04-02 修回日期:2011-07-15 出版日期:2011-11-12 网络出版日期:2011-09-06
  • 通讯作者: 马凤鸣, E-mail: fengming_ma@sohu.com, Tel: 0451-55191947; 陈连江, E-mail: chen4004@163.com, Tel: 0451-86609481
  • 基金资助:

    本研究由国家自然科学基金项目(30571096)资助。

Cloning of nia Gene and Its Differential Expression Induced by Different Nitrogen Forms in Sugar Beet (Beta vulgaris L.)

DING Guang-Zhou1,2, HOU Jing2,CHEN Li1,MA Feng-Ming2,*,CHEN Lian-Jiang1,*   

  1. 1 Sugar Beet Institute of Chinese Academy of Agricultural Sciences, Harbin 150080, China; 2 College of Agriculture, Northeast Agricultural University, Harbin 150030, China
  • Received:2011-04-02 Revised:2011-07-15 Published:2011-11-12 Published online:2011-09-06
  • Contact: 马凤鸣, E-mail: fengming_ma@sohu.com, Tel: 0451-55191947; 陈连江, E-mail: chen4004@163.com, Tel: 0451-86609481

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被引次数

3

摘要： 利用同源序列克隆方法从二倍体甜菜品种Ty7中获得氮素诱导nia基因片段，通过RACE技术克隆nia基因全长序列，该基因ORF长度2 718 bp，编码905个氨基酸，并已在GenBank上登录(EU163265)，基因组中nia以低拷贝数存在。nia编码蛋白的等电点为6.12，推测分子量为102 kD，以NADH为电子供体。为揭示不同氮素形态和处理对甜菜nia基因表达的影响，采用半定量PCR方法检测不同氮素形态诱导nia基因mRNA的表达，同时测定酶活力。结果表明，当铵态氮诱导nia基因时，低浓度的铵离子能促进基因的表达，过高浓度的铵离子抑制基因的表达。当硝态氮诱导nia基因时，随处理浓度的增加，nia的表达加强，呈正相关关系。用30 mmol L–1硝态氮诱导4 h后，nia基因表达达最高值，约在6 h后，表达明显下降。

关键词: nia基因克隆, 氮素形态和处理, 基因表达

Abstract: From diploid species Ty7, a cDNA clone related to nitrate reduction was isolated by Homology-based cloning. According to its sequences information, a novel full-length cDNA termed nia (accession number: EU163265) was obtained by using rapid amplification of cDNA ends (RACE). Nia was 3 247 bp long containing a 2 718 bp ORF, encoded 905 amino acids with a theoretical molecular weight of 102 kD and an isoelectric point of 6.12. Southern bloting proved that nia gene of Ty7 existed in the form of low copies. It was also proved that this clone was the style of NADH-NR. To reveal the effect of nitrate and ammonium nitrogen on nia expression, we determined NR activity in sugar beet under the treatment of nitrate and ammonium. Gene transcripts of nia were detected by semi-quantitative PCR. Efficiency of mRNA synthesis from each sample was estimated by quantitative PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that when nia gene was induced by ammonium, the gene expression was promoted with low concentration of ammonium ion, while inhibited by high concentration of ammonium ion. When nia gene was induced by nitrate, being positively correlated with the concentration, the gene expression was enhanced with the increase of the concentration. By 30 mmol L–1 nitrate induction for four hours, nia gene expression reached the highest value, and at about six hours of induction, the expression decreased significantly.

Key words: Nia gene cloning, Nitrogen form and processing, Gene differential expression

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