【作者】 石晓艳；

【导师】 马凤鸣；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2011， 博士

【摘要】 甜菜是我国北方特有的糖料作物,在我国北方农业生产中占有重要地位。其产量仅次于甘蔗,甜菜糖占世界食糖总量的30%左右。在世界产糖大国中,我国是少数既产甜菜糖又产甘蔗糖的国家之一,我国甜菜的种植面积最高年份达67万公顷,居世界第3位,总产量居世界第6位。然而,目前黑龙江省乃至全国甜菜单产不高、总产不稳,特别是含糖率呈下降趋势,已成为限制我国甜菜糖业发展的障碍性因子。究其原因,除品种选育滞后外,氮素供应不合理是主要原因之一。由于氮源和氮素代谢在作物产量中发挥着中心作用,确定氮素状态调控的基因网络,鉴定重要基因的功能,阐明该基因表达蛋白生化特性,将对农业发展有着广泛的影响。在甜菜所需的全部营养中,氮素是影响产量的最关键、最活跃因素,氮素过量会造成甜菜产糖量的降低。硝态氮(NO3-)是旱田作物可利用氮素的主要形式, NR是NO3-无机同化的限速酶,NiR是NO3-无机同化的控制酶。与NR相比较,有关NiR研究尚少。亚硝酸还原酶(NiR)是氮素同化途径的关键酶,由于NiR活力一般要比NR活力高得多,所以NiR作为硝酸盐还原的限制步骤的可能性就被排除了,致使国内外以往的初级氮素同化研究忽略了NiR的控制酶作用,造成氮的无机同化代谢研究偏重于NR的不平衡局面。本研究借助现代分子生物学手段,首次克隆甜菜亚硝酸还原酶编码基因的cDNA全长序列以及基因组gDNA全长序列;首次确定了甜菜亚硝酸还原酶基因编码蛋白的多肽序列和功能区域;首次推测出甜菜亚硝酸还原酶蛋白的3D构象;并通过实时荧光定量PCR的方法(RT-PCR),在转录水平上研究该基因在NO3--N和NH4+-N两种不同形态氮素条件下以及NO3--N和NH4+-N分别处理不同浓度下的表达变化,揭示氮素条件和甜菜NiR基因表达的关系;另外在氮素诱导的基础上,设置不同浓度蛋白抑制剂放线菌酮处理,以及NO2-处理和NaCl处理,研究甜菜NiR基因的表达;初步研究了环境因子对于亚硝酸还原酶基因表达的影响。具体的研究内容和结论如下:1.根据GenBank已知甜菜部分DNA序列(AF173663)和菠菜mRNA(X07568)全长序列,进行同源比对,在保守区设计巢式PCR引物,利用RACE方法扩增得到甜菜亚硝酸还原酶基因的5′和3′未知区域。2.依据得到的5′和3′序列设计引物,扩增得到中间片段P0。3.将5′端和3′端以及中间片段P0进行拼接,以拼接序列设计引物,成功地从甜菜叶片中首次分离了NiR的全长基因,登录号为HQ224499,长度为2014bp。该序列含有一个长度1830bp的开放读码框(ORF),编码599个氨基酸,经BLAST分析,核苷酸序列及其推导的氨基酸序列与其它植物NiR基因具有较高的同源性。4.运用PCR扩增技术首次克隆了甜菜NiR基因组序列,登录号为HQ419065,测序结果表明,基因组DNA长度为2815bp,含有4个外显子和3个内含子,内含子长度分别为531bp,135bp和269bp;4个外显子长度分别为437bp,355bp,289bp和799bp。5.采用TopPred在线工具分析了NiR的理化特性,并构建了NiR的系统发育树,结果表明,甜菜NiR为易溶、亲水性强的蛋白,理论等电点pI为7.21,相对分子量为66.88kDa,该蛋白C端存在一个跨膜区。甜菜NiR与菠菜(Spinacia oleracea)NiR的亲缘关系最近。二级结构预测结果显示,NiR包含29.05%的螺旋,21.04%的延伸链和49.92%的自由卷曲。卷曲结构和螺旋结构是甜菜NiR的二级结构的主体。另外,应用同源建模法进行NiR三维结构的预测,3D结构包括352个H-bonds;21个Helices;30处Strands;66个Turns。6.分析甜菜NiR基因编码蛋白的结构域,所推导的氨基酸序列具有完整NiR结构,含血红素蛋白β-化合物区域(hemoprotein beta-component, ferrodoxin-like, Domain:氨基酸133~203,氨基酸392~461),4Fe-4S区域(4Fe-4S region, Domain:氨基酸211~371,氨基酸517~580)。甜菜NiR没有信号肽,是非分泌型蛋白。甜菜NiR氨基酸序列的517~533区域的TGCpnsCgqvqvaDIGF为NiR的活性位点。7.实时荧光定量PCR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;不同浓度NaCl处理下NiR的基因表达无明显的规律。更多还原

【Abstract】 Sugar beet (Beta vulgaris L.) was an endemic sugar crop and played an important role in agricultural production in north China.Its yield was secondary to sugarcane, and beet sugar was about 30% of the world’s sugar yield.China was one of the big country which produced beet sugar and cane sugar.Planted area of sugarbeet in China was reached 67 million ha,which ranked 3rd in the world,and.total output was resides the 6st..But yields of sugar beet were not high and the total output was instable, particularly sugar content showed a downward trend in Heilongjiang province and the national. All had become the obstacle factors to the development of sugar beet. Unreasonable application of nitrogen was one of the main reasons besides the breeding lag .As nitrogen source and nitrogen metabolism played an central role in crop yield,determined the gene network of nitrogen regulation, identified the function of important genes, clarified the biochemical characteristics of gene expression protein, all will have a wide range influence on agricultural development.Nitrogen was the key and active factor in the necessary nutrients, and excess nitrogen can cause the reduction of sugar production in the sugar beet.Nitrate (NO3-) was the the main form of available nitrogen in dry crops, NR was the rate-limiting enzyme of NO3- inorganic assimilation,and NiR was the control enzyme of NO3- inorganic assimilation. Compared with the NR, few studies on NiR.Nitrite reductase (NiR) was a key enzyme of nitrogen assimilation pathway.NiR activity generally much higher than NR activity,so NiR as a limiting step of nitrate reduction was ruled out.Resulting in primary nitrogen assimilation studies neglected to its role of control enzyme at home and abroad in the past, and caused an unbalanced situation that emphasis on the NR in inorganic nitrogen assimilation metabolism.Modern molecular biotechnology methods was used in this study.NiR cDNA complete cds and NiR gDNA full length sequence of sugarbeet was first cloned; The peptide sequence and the functional domain of sugar beet NiR was identified for the first time; The 3D conformation of sugar beet NiR was conjectured for the first time;The expression of NiR gene on the transcription level was studied by RT-PCR under different proportion of NO3-N and NH4+-N, different NO3--N , different NH4+-N, then revealed the relationship of nitrogen and NiR gene expression in sugar beet ; Also on the basis of the nitrogen-induced, different concentrations of cycloheximide treatment , NO2- treatment and NaCl treatment was seted, which in order to study the NiR gene expression.The environmental factors on nitrite reductase gene expression was preliminary studied.The specific study contents and conclusions are as follows:1.According to the partial DNA sequence of beet (AF173663)and the mRNA sequence of Spinach(X07568)on GenBank , primers was designed in the conserved region, nitrite reductase gene 5′and 3′unknown region was amplified by RACE technique..2.Primers was designed according to the nitrite reductase gene 5′and 3′sequence , intermediate part was amplified.3.Taked the splicing sequences of 5′, 3′and P0 as the target sequences to design primers, NiR gene was firstly cloned from sugar beet. The accession number was HQ224499. The full length of NiR was 2014bp.The sequence included an ORF with 1830bp, encoded 599 amino acids,The cDNA and amino acid sequences showed high identity with other plant NiR genes by BLAST analysis.4.Sugar beet NiR genomic DNA was isolated by PCR and its sequence contained 4 exons and 3 introns. The accession number was HQ419065. The full length of genomic DNA was 2815bp, and the lengths of introns were respectively 531bp, 135bp and 269bp; the lengths of exons were respectively 437bp,355bp,289bp and 799bp.5.Physical and chemical properties of NiR was predicted with different methods,and the NiR phylogenetic tree was constructed.The results of primary structure analysis showed that NiR was soluble and hydrophobic, with a pI (isoelectric point) of 7.21 and MW (molecular weight) of 66.88kDa. A transmembrane region was found in C-end . The homology of spinach NiR and sugar beet NiR was the highest.Secondary structure analysis indicated that NiR contained 29.05% alpha helix, 21.04% extended strand and 49.92% random coil. Helix and coil structures were the main body of secondary structure .3D structure was constructed by homology method and contained 352 H-bonds,21 Helices,30 Strands,66 Turns.6.Protein structure was analysed, the amino acid sequence had a complete structure .The predicted NiR protein found to have a hemoprotein beta-component (ferrodoxin-like), and 4Fe-4S region, NiR was non-secreted protein with no signal peptide. The region TGCpnsCgqvqvaDIGF was the active site of NiR.7.Real time PCR analysis showed that, 0, 10, 20, 30, 40, 50, 80, and 160 mmol L–1 NO3–-N processing 72 hours experiments, the expression of NiR was higher at 50 mmol L–1; 0, 2, 4, 8, 16, 32, 64, and 128 mmol L–1 NH4+-N processing 48 hours experiments, the expressions of NiR were higher at 8 mmol L–1and 64 mmol L–1, respectively; Furthermore, in different nitrogen forms and ratios treatment 48 hours experiments, The expression of NiR gene was higher when the ratio of NO3–-N and NH4+-N was 80:20; Cycloheximide treatment nine hours experiments showed that NiR expression decreased with increasing treatment concentration; NO2– treatments indicated that the maximum expressions of NiR was induced by 40 mmol L–1NO2–; The expression of NiR had no significant law under different concentration NaCl treatment.更多还原