甜菜亚硝酸还原酶基因(NiR)的克隆与表达分析

doi:10.3724/SP.J.1006.2011.01406

摘要/Abstract

摘要： 以甜菜品种甜研7号叶片的cDNA为模板，采用RT-PCR和3′/5′RACE技术，获得了编码亚硝酸还原酶基因(NiR)的cDNA全序列2 014 bp, 包含有1 830 bp的开放阅读框，编码599个氨基酸。所推导的氨基酸序列与菠菜及拟南芥NiR编码的氨基酸序列均具93%的同源性。生物信息学分析表明，甜菜NiR具有完整的NiR蛋白结构，含血红素蛋白β-化合物区域和4Fe-4S区域, 并利用分析软件预测其三维结构。实时荧光定量结果显示，在以0、10、20、30、40、50、80和160 mmol L^–1 NO₃^–-N处理72 h的试验中，50 mmol L^–1处理可使甜菜NiR的表达量达到最大；以0、2、4、8、16、32、64和128 mmol L^–1 NH₄⁺-N处理48 h的试验表明，8 mmol L^–1和64 mmol L^–1处理条件下甜菜NiR表达量相对较高。硝态氮和铵态氮不同配比处理48 h的试验中，NO₃^–-N和NH₄⁺-N比例为80:20可使甜菜NiR的表达量达到最大；在氮素诱导的基础上，蛋白抑制剂放线菌酮处理9 h，随着处理浓度的增大，NiR的表达量逐渐下降；不同浓度NO₂^–处理的试验中，40 mmol L^–1处理下NiR的表达量最大。

关键词: 甜菜, 亚硝酸还原酶, cDNA, 克隆, 基因表达

Abstract: The NiR gene was cloned using RT-PCR and 3′/5′RACE techniques. The cDNA of NiR gene isolated from sugarbeet was 2 014 bp containing a 1 830 bp opening-reading frame (ORF), encoding 599 amino acids. Further comparison showed that NiR gene had high homology to both of Spinacia oleracea NiR gene and Arabidopsis thaliana NiR gene, which was 93%. The predicted NiR protein found to have a hemoprotein beta-component (ferrodoxin-like), and 4Fe-4S region, its 3D structure was predicted by analysis software. Real time PCR analysis showed that when using 0, 10, 20, 30, 40, 50, 80, and 160 mmol L^–1 NO₃^–-N treated for 72 hours, the expression of NiR gene was the highest at 50 mmol L^–1; when using 0, 2, 4, 8, 16, 32, 64, and 128 mmol L^–1 NH₄⁺-N treated for 48 hours, the expression of NiR gene showed two peaks at 8 mmol L^–1and 64 mmol L^–1, respectively. Furthermore, in experiments treated with different ratios of NO₃^–-N to NH₄⁺-N for 48h, the expression of NiR gene was the highest when the ratio was 80:20. Cycloheximide 9h treatment experiments showed that NiR gene expression decreased with increasing the treating concentration. NO₂^–treatments indicated that the maximum expression of NiR gene was induced by 40 mmol L^–1 NO₂^–.

Key words: Sugarbeet, Nitrite Reductase, cDNA, Cloning, Gene expression

石晓艳, 曾彦达, 李世龙, 王玉波, 马凤鸣, 刁志伟. 甜菜亚硝酸还原酶基因(NiR)的克隆与表达分析[J]. 作物学报, 2011, 37(08): 1406-1414.

DAN Xiao-Yan, CENG Pan-Da, LI Shi-Long, WANG Yu-Bei, MA Feng-Ming, DIAO Zhi-Wei. Molecular Cloning and Characterization of Nitrite Reductase Gene from Sugarbeet[J]. Acta Agron Sin, 2011, 37(08): 1406-1414.

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