红麻亚硝酸还原酶基因HcNiR的克隆与表达分析  

Cloning and Expression of Nitrite Reductase Gene HcNiR in Kenaf

作　　者：张超 邓勇[2] 黄思齐[2] 伍应保 张高阳[1] 满百膺 李德芳[2] ZHANG Chao;DENG Yong;HUANG Siqi;WU Yingbao;ZHANG Gaoyang;MAN Baiying;LI Defang(College of Life Science,Shangrao Normal University,Shangrao,Jiangxi 334001,China;Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha,Hunan 410205,China)

机构地区：[1]上饶师范学院生命科学学院,江西上饶334001 [2]中国农业科学院麻类研究所,湖南长沙410205

出　　处：《福建农业学报》2022年第5期600-608,共9页Fujian Journal of Agricultural Sciences

基　　金：江西省教育厅科学技术项目(GJJ180890);国家麻类产业技术体系红麻品种改良项目(CARS-16-E05);中国农业科学院科技创新工程一年生麻类育种项目(ASTIP-IBFC03)。

摘　　要：【目的】了解红麻亚硝酸还原酶基因HcNiR生物信息学特性及组织表达特异性,为培育红麻氮高效利用品种提供理论依据。【方法】以红麻材料349叶片的cDNA为模板,利用PCR扩增HcNiR基因的CDS序列,采用生物信息学方法分析HcNiR的氨基酸组成、蛋白质跨膜结构、信号肽、高级结构以及蛋白的同源进化树;采用实时荧光定量PCR检测HcNiR基因在红麻不同组织的表达情况。【结果】HcNiR基因cDNA全长1395 bp,编码蛋白含有464个氨基酸,包含2个保守的亚硝酸和亚硫酸还原酶4Fe-4S结构域及铁氧蛋白部分结构域。HcNiR蛋白是一个不含跨膜转运结构与信号肽的亲水稳定性蛋白质,该蛋白质等电点是5.49,分子量51.68 kDa;具有26处潜在磷酸化位点。在其蛋白二级结构中,α-螺旋和无规则卷曲所占比例超过70%。通过氨基酸序列同源性分析发现,红麻HcNiR氨基酸序列与木槿HsNiR氨基酸序列相似性较高,达到97.37%,都含有铁-硫/铁血红素结合位点。进化树分析结果表明,红麻HcNiR基因与木槿HsNiR基因亲缘关系较近。组织特异性表达结果显示,红麻HcNiR基因在叶中的表达量高于根。【结论】HcNiR基因编码蛋白含亚硝酸和亚硫酸还原酶4Fe-4S结构域及铁氧蛋白部分结构域;HcNiR基因具有组织表达特异性,在红麻叶片中表达较高,推测其主要在初级氮的同化过程中发挥重要调控作用。【Objective】Bioinformatics and expressions of the nitrate reductase gene of kenaf(Hibiscus cannabinus)were studied for breeding varieties highly efficient in nitrogen utilization.【Method】From the leaf of kenaf 349,the coding sequences(CDS)of HcNiR was amplified by PCR.Bioinformatics method was applied to analyze the amino acid sequences,protein transmembrane structure,protein signal peptide,high-level structures,and homologous evolutionary tree associated with the gene,while the expression in various tissues detected by qRT-PCR.【Result】The full length of HcNiR cDNA was 1395bp encoded 464 amino acids.The amino acid sequence contained two conserved nitrite and sulfite reductase 4Fe-4S domains and two conserved nitrite/sulfite reductase ferredoxin-like half domains.The predicted stable,hydrophilic HcNiR protein with an isoelectric point of 5.49 and molecular weight of 51.68 kDa had no transmembrane domain or signal peptide.It contained 26potential phosphorylation sites in a secondary structure that consisted of more than 70%in the forms of alpha helix and irregular coils.The amino acid sequence of HcNiR was 97.37%homologous with that of H.syriacus,and both included nitrite and sulfite reductases iron-sulfur/siroheme-binding sites.The phylogenetic tree on HcNiR showed it closely related to HsNiR.The HcNiR expression was higher in the leaves than in the roots of a kenaf plant.【Conclusion】HcNiR contained two conserved nitrite and sulfite reductase 4Fe-4S domains and two conserved nitrite/sulfite reductase ferredoxin-like half domains.The gene was abundantly expressed in the kenaf leaves and speculated to be mainly involved in the process of primary nitrogen assimilation.

关 键 词：红麻 亚硝酸还原酶 HcNiR基因 基因克隆 表达分析 

分 类 号：S563.5[农业科学—作物学]