黄瓜亚硝酸还原酶基因(CsNiR)的克隆及对外植体分化的影响

Cloning of nitrite reductase gene(CsNiR)in cucumber and its effect on explant differentiation

[目的]本文旨在揭示黄瓜亚硝酸还原酶基因(CsNiR)的表达特征及其在黄瓜再生体系中的功能。[方法]以黄瓜自交系CCMC为试验材料,克隆CsNiR的编码区序列全长,对其进行生物信息学分析;利用本氏烟草表皮细胞瞬时表达系统对CsNiR蛋白进行亚细胞定位,并采用RT-qPCR技术分析CsNiR基因在不同组织中的表达量。对不同基因型黄瓜子叶节外植体的NiR活性与外植体分化率进行相关性分析,测定在不同培养条件下不同基因型黄瓜子叶节外植体CsNiR基因表达量,并统计在不同NH+4/NO-3比例(1∶5、1∶2、1∶1、2∶1)培养条件下外植体分化率情况。[结果]CsNiR基因ORF全长为1752 bp,编码583个氨基酸,具有含血红素蛋白β-化合物区域和4Fe-4S区域的完整NiR蛋白结构。系统进化树分析发现植物中CsNiR高度保守,黄瓜与甜瓜氨基酸序列相似性高达96%。RT-qPCR结果表明,CsNiR基因在黄瓜成熟叶表达量最高,雄花中次之,在茎尖和侧芽等分化组织中表达量最低,具有组织特异性。亚细胞定位结果表明CsNiR主要在细胞膜和叶绿体中表达。对不同基因型黄瓜子叶节的NiR活性与再生率的测定发现,NiR活性与黄瓜外植体分化率显著负相关。在不同基因型黄瓜外植体诱导分化过程中,CsNiR基因的表达在含有诱导激素培养基上的表达量显著低于无诱导激素的培养基,当NH+4/NO-3为1∶5时,黄瓜子叶节外植体分化率最高。[结论]植物中NiR高度保守,CsNiR基因的表达具有特异性,在茎尖等分生组织表达含量最低,NiR活性与外植体分化率呈负相关关系,降低NH+4/NO-3比例有助于提高黄瓜外植体分化率。

[Objectives]This paper aimed to reveal the expression characteristics of nitrite reductase gene(CsNiR)and its function in cucumber regeneration system.[Methods]The coding region sequence full-length of CsNiR was cloned from cucumber inbred line CCMC,of which the bioinformatics analysis was carried out.The subcellular localization of CsNiR protein was identified by the transient expression system of Nicotiana benthamiana epidermal cells.The expression pattern of CsNiR in different tissues of CCMC was detected by RT-qPCR.Correlation between NiR activity and explant differentiation rate in different genotypes cucumber cotyledonary-nodes were analyzed.Transcriptional changes of CsNiR in different genotypes of cucumber cotyledonary-nodes under different culture conditions were also analyzed by RT-qPCR,and the differentiation rates of explants under different NH+4/NO-3 ratios(1∶5,1∶2,1∶1,2∶1)were also counted.[Results]The ORF of CsNiR gene was 1752 bp in length and encoded 583 amino acids.It had a complete NiR protein structure containing the heme proteinβ-compound region and the 4Fe-4S region.Phylogenetic tree analysis showed that CsNiR was highly conserved in plants,and the amino acid sequence similarity between cucumber and melon reached to 96%.RT-qPCR suggested that CsNiR had tissue-specific expression characteristics where showed the highest expression in mature leaves of cucumber,followed by male flowers,but the lowest expression in the meristem containing organs such as shoot tips and lateral buds.Subcellular localization results indicated that CsNiR was mainly expressed in cell membrane and chloroplast.The endogenous NiR activity and regeneration rate of cotyledonary-nodes from different cucumber genotypes showed significant negative correlation.The expression of CsNiR in cotyledonary-nodes under induce-medium was significantly lower than that in the inducer-free medium.When NH+4/NO-3 ratio was 1∶5,the explant differentiation rate of cucumber cotyledonary-nodes was highest.[Conclusions]NiR homologs were highly conserved in plant.The cucumber CsNiR gene showed tissue specific expression and the lowest in the shoot meristem.The NiR activity was negatively correlated with the explant differentiation.Decreasing the NH+4/NO-3 ratio would help to increase differentiation rate of cucumber explants.