豆梨NADH型硝酸还原酶基因克隆、表达及酶活性分析

Cloning,expression and enzyme activity analysis of Nitrite Reductase Gene from Pyrus calleryana

【目的】克隆豆梨硝酸还原酶基因(PcNR),并对其序列特征、表达特点及酶活性进行分析。【方法】采用电子克隆、RT-PCR和长片段PCR,得到PcNR的cDNA和DNA序列,利用生物信息学方法进行序列分析,半定量RT-PCR检测其表达特点,并测定硝酸还原酶活性。【结果】获得了豆梨硝酸还原酶基因(PcNR)的cDNA编码区,其长度为2 712 bp,编码903个氨基酸,对应的基因组DNA序列长4 115 bp,包括4个外显子和3个内含子。PcNR编码的多肽含有硝酸还原酶特征序列:含钼因子、血红素结合区域和FAD绑定区域,属于NADH型硝酸还原酶。PcNR与湖北海棠MhNR(JN632526)、桃PpNR(AB061670)和野草莓FvNR(XM_004300392)蛋白间的相似性较高,并与蔷薇科植物NR处于系统进化树的同一分枝上。在无氮处理的豆梨叶片中几乎检测不到PcNR的表达;随着培养液中NO3-浓度的提高,其表达量先上升后下降;此外,供氮时间、环境温度及光照均可调控该基因的表达。豆梨叶片中硝酸还原酶的活性变化规律与PcNR表达量变化趋势相一致。【结论】首次从豆梨中克隆获得PcNR基因的cDNA和DNA序列,揭示了其序列特征,并对调控该基因表达和硝酸还原酶活性的相关因素进行分析,为开展梨砧木氮素营养研究提供资料。

【Objective】The aim of this study was to clone the full-length cDNA and DNA of a gene encoding nitrate reductase from Pyrus calleryana Dcne.,investigate its sequence characteristics and analyze itsexpression in different environmental conditions in addition of the activities of nitrate reductase.【Method】The full-length cDNA and DNA sequences of PcNR were isolated by electronic cloning together with PCR techniques from P.calleryana,and the bioinformatics characteristics were analyzed using online software.The expression profiles of PcNR and the nitrate reductase activities of leaves were determined using semiquantitative RT-PCR and sulfanilamide colorimetric method,respectively.【Result】The obtained cDNAof PcNR was 2 712 bp in length with Genbank accession number KC545876,which encoded 903 aminoacid residues.The genomic DNA of PcNR was 4 115 bp in length with 4 exons and 3 introns.The deducedprotein of PcNR belonged to NADH-type NR subfamily and shared common structural features with that of other higher plants,which included three distinguish able sequence regions named the Mo cofactor,thecytochrome b5-like Fe-heme domain,and a flavin adenine nucleotide(FAD) cofactor binding domain,respectively.PcNR was highly similarity to MhNR from Malus hupehensis(JN632526),PpNR fromPrunus persica(AB061670) and FvNR from Fragaria vesca(XM_004300392).Furthermore,PcNR andNRs of other Rosaceae plants,were located in the same branch of the phylogenetic tree.Semi-quantitativ eRT-PCR results demonstrated that the mRNA abundance of PcNR was response to different nitrogen status.PcNR was only slightly expressed in leaves without nitrogen supply,however,its abundance increased sharply and then decreased along with the increase of nitrate concentrations.In addition,PcN Rexpression was also regulated by treatment time,temperature and illumination.In all treatments,nitrater eductase activity exhibited a positive correlation to the expression abundance of PcNR in leaves.【Conclusion】The cDNA and DNA of PcNR gene of P.calleryana were firstly isolated and characterized.Then,the relationships between several regulation factors of PcNR expression and the corresponding nitrate reductase activity were disclosed preliminarily.This study will provide some useful information for the research on nitrogen nutrition of pear rootstocks.