红麻亚硝酸还原酶基因<i>HcNiR</i>的克隆与表达分析

张超; 邓勇; 黄思齐; 伍应保; 张高阳; 满百膺; 李德芳

doi:10.19303/j.issn.1008-0384.2022.005.007

红麻亚硝酸还原酶基因HcNiR的克隆与表达分析

doi: 10.19303/j.issn.1008-0384.2022.005.007

张超^{1, 2,},
邓勇²,
黄思齐²,
伍应保¹,
张高阳¹,
满百膺¹,
李德芳^2, ,

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

基金项目: 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）

详细信息

作者简介:
张超（1985−），男，博士，讲师，主要从事麻类遗传育种研究（E-mail：zhangchao_20180604@163.com）

通讯作者:
李德芳（1962−），男，博士，研究员，主要从事一年生麻类遗传改良研究（E-mail：chinakenaf@126.com）

中图分类号: S 563.5
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出版历程
- 收稿日期: 2021-10-29
- 修回日期: 2022-01-08
- 网络出版日期: 2022-06-19
- 刊出日期: 2022-05-28

Cloning and Expression of Nitrite Reductase Gene HcNiR in Kenaf

1.
College of Life Science, Shangrao Normal University, Shangrao, Jiangxi　334001, China
2.
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, Hunan　410205, China

摘要

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。
- 红麻 /
- 亚硝酸还原酶 /
- HcNiR基因 /
- 基因克隆 /
- 表达分析
Abstract: 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 1395 bp 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 26 potential 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.
- kenaf /
- nitrate reductase /
- HcNiR /
- gene cloning /
- expression analysis

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图 1 HcNiR基因克隆的琼脂糖凝胶电泳检测结果

M-DL2000 DNA Marker; 1为HcNiR-1, 2为HcNiR-2, 3为HcNiR-3。

Figure 1. Gel electrophoresis of HcNiR cloning result

M: DL2000 DNA marker; 1: HcNiR-1; 2: HcNiR-2; 3: HcNiR-3.

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图 2 HcNiR蛋白保守结构域的预测

Figure 2. Predicted conserved domain of HcNiR protein

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图 3 HcNiR蛋白质亲水和疏水性分析

Figure 3. Hydrophilicity and hydrophobicity of HcNiR protein

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图 4 HcNiR蛋白潜在磷酸化位点预测结果

A为潜在磷酸化位点；B为磷酸化势。

Figure 4. Predicted phosphorylation sites in HcNiR protein

A: Potential phosphorylation site; B: Phosphorylation potential.

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图 5 红麻HcNiR基因编码的蛋白产物二级结构

Figure 5. Secondary structure of HcNiR encoded protein

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图 6 HcNiR基因编码蛋白质产物的三级结构

Figure 6. Tertiary structure of HcNiR encoded protein

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图 7 HcNiR跨膜结构预测

Figure 7. Predicted transmembrane structure of HcNiR

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图 8 HcNiR信号肽预测

Figure 8. Predicted signal peptide of HcNiR

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图 9 HcNiR的氨基酸序列对比

HcNiR：红麻；HsNiR：木槿（XP_039006864.1）；TcNiR：可可树（XP_007042430.2）；GhNiR：陆地棉（ADJ68001.1）；GrNiR：雷蒙德氏棉（XP_012463711.1）。

Figure 9. Amino acid sequences of HcNiR

HcNiR: H. cannabinus; HsNiR: H. syriacus (XP_039006864.1); TcNiR: Theobroma cacao (XP_007042430.2); GhNiR: Gossypium hirsutum (ADJ68001.1); GrNiR: Gossypium raimondii (XP_012463711.1).

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图 10 HcNiR基因同源进化树比对

Figure 10. Homologous evolutionary trees of HcNiR

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图 11 HcNiR在不同组织中的表达情况

不同小写字母表示不同组织间差异显著（P＜0.05）。

Figure 11. Expressions of HcNiR in various tissues

Data with different lowercase letters indicate significant difference at P＜0.05.

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表 1 引物序列

Table 1. Primer sequence

引物名称 Primer names	引物序列（5′-3′） Primer sequence(5′-3′)
HcNiR-F	ATGACAGATGGGAGATTTATGATG
HcNiR-R	GCATTTTCCACTTCTTCTTCCC
HcNiR-qPCR-F	TCTTGGTTACAGGGGCAATAGAC
HcNiR-qPCR-R	TGGACACCAAGATAGTCTCTCCT
beta-actin-F	ATCCTCCGTCTTGACCTTG
beta-actin-R	TGTCCGTCAGGCAACTCAT

下载: 导出CSV

表 2 HcNiR基因编码蛋白质产物一级结构预测分析

Table 2. Primary structure of HcNiR encoded protein

一级结构特征 Characteristics of primary structure	氨基酸数量 Number of amino acids	等电点 pI	相对分子质量 Relative molecular mass/Da	分子式 Molecular formula	正电荷残基 Arg+Lys	负电荷残基 Asp+Glu	平均疏水性 Average hydrophobicity	脂肪系数（AI） Fatty coefficient	不稳定系数（Ⅱ） Instability coefficient (Ⅱ)	半衰期 Estimated half-life/h
预测结果 Prediction result	464	5.490	51683.220	C₂₂₇₆H₃₆₃₂N₆₄₄O₆₈₁S₂₄	54	64	−0.289	89.480	37.930	30

下载: 导出CSV

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