白菜亚硝酸还原酶基因BcNiR的克隆及表达分析  

Molecular Cloning and Characterization of Nitrite Reductase Gene BcNiR from Non-heading Chinese Cabbage

作　　者：孙菲菲[1,2] 蒋芳玲[1,2] 侯喜林[1,2] 李英[1,2] 杨学东[1,2] 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室,南京210095 [2]农业部南方蔬菜遗传改良重点开放实验室,南京210095

出　　处：《园艺学报》2009年第10期1511-1518,共8页Acta Horticulturae Sinica

基　　金：国家‘863’计划项目(2006AA10211C9)

摘　　要：以白菜品种‘苏州青’自交系叶片cDNA为模板,采用RT-PCR、3′RACE和5′RACE技术,获得了编码亚硝酸还原酶基因(NiR)的cDNA全序列1852bp,包含有1749bp的开放阅读框,编码583个氨基酸,命名为BcNiR。所推导的氨基酸序列与拟南芥NiR1、烟草nii2编码的氨基酸序列具有较高同源性,分别为83%和76%。生物信息学分析表明,BcNiR具有完整的NiR蛋白结构,含血红素蛋白β-化合物区域,一个明显的西罗血红素siroheme结合位点和4Fe-4S区域,可以在SWiSS-MODEL数据库中搜索到与之相近的三维结构。RT-PCR结果显示,该基因在叶片中的表达量远远高于根系,在以0、10、20、30、40mmol.L-1硝态氮各分别处理0、2、4、6、8、12h的试验中,30mmol.L-1处理4h可使BcNiR的表达量达到最大;5和10mmol.L-1铵态氮处理试验表明,高浓度的铵抑制该基因的表达。The BcNiR gene was cloned using RT-PCR and 3＇/5＇RACE techniques with cDNA isolated from non-heading Chinese cabbage [Brassica campestris L. ssp. chinensis （L.） Makino], which was nitrate- induced for 4 hours. The cDNA of BcNiR was 1 852 bp containing a 1 749 bp opening-reading frame （ORF） encoding 583 amino acids. Further comparison showed that BcNiR had high homology to Arabidopsis thaliana NiRI gene and Nicotiana tabacum nii2 gene, were 83% and 76%, respectively. The predicted BcNiR protein was found to have a hemoprotein beta-compnent （ferrodoxin-like）, a siroheme-binding site and 4Fe- 4S region. A similar 3D structural were obtained from the SWiSS-MODEL database. Real-time PCR analysis showed that, the expressions of BcNiR were awfully higher in leaves than that in roots. Furthermore, in nitrate treatment experiments, both the maximum expressions of BcNiR in roots and leaves were induced by 30 mmol·L^-1 NO^3- - N at 4 h. 5 and 10 mmol·L^-1 NH4^＋ - N treatments indicated that high level of NH4^＋ - N treatment restrained the expression of BcNiR.

关 键 词：白菜 亚硝酸还原酶 克隆 基因表达 

分 类 号：S634.3[农业科学—蔬菜学]