基于苎麻疫霉菌诱导的棉花组学分析克隆硝酸还原酶基因<i>GhNIA1</i>

doi:10.7668/hbnxb.20196564

摘要/Abstract

摘要：

为了探究棉花响应苎麻疫霉菌侵染的关键基因并初步解析其参与互作的分子机制,利用Illumina Hiseq^TM平台对感棉铃疫病棉花种质885系进行了转录组测序和TMT标记定量蛋白组检测,通过差异基因(蛋白)筛选和GO/KEGG富集分析获得响应病原侵染的关键基因,并对关键基因进行生物信息学分析,利用实时荧光定量技术(qPCR)明确关键基因在胁迫环境下的表达模式。结果表明,转录组和蛋白组联合分析共获得50个在基因与蛋白层面均存在显著差异的候选序列;GO和KEGG富集分析显示,差异基因主要富集于植物激素信号转导、植物-病原菌互作、谷胱甘肽代谢以及次级代谢产物生物合成等通路;进一步通过序列比对获得7个候选抗病相关基因,利用qPCR技术对7个候选抗病相关基因检测后发现,Gh_D02G091500、Gh_D06G194600、Gh_A01G241100、Gh_A06G192400和Gh_D09G161000的表达受苎麻疫霉菌诱导,其中棉花硝酸还原酶基因的表达差异倍数最高,差异显著性最为明显,并获得了棉花硝酸还原酶基因全长,命名为GhNIA1。综上,GhNIA1可能是参与棉花-苎麻疫霉菌互作的关键基因,并初步明确了GhNIA1的基因特征,为解析GhNIA1参与棉花抗棉铃疫病分子机制及抗病品种选育奠定了基础。

关键词: 棉花, 苎麻疫霉菌, 转录组测序, 基因克隆, 表达分析

Abstract:

To investigate the key genes in cotton in response to Phytophthora boehmeriae infection and preliminarily elucidate the molecular mechanisms underlying their interaction,it performed transcriptome sequencing and TMT-based quantitative proteomic analysis on the boll rot-susceptible cotton germplasm line 885 using the Illumina Hiseq^TM platform.By screening for differentially expressed genes(proteins)and conducting GO/KEGG enrichment analyses,key genes involved in the stress response were identified.Bioinformatic analysis was further carried out on these candidate genes,and their expression patterns under pathogen stress were validated via Real-time Quantitative PCR(qPCR).The results showed that combined transcriptomic and proteomic screening identified 50 candidate sequences with significant differential abundance at both the transcriptional and translational levels.GO and KEGG enrichment analyses revealed that the differentially expressed genes were primarily enriched in pathways such as plant hormone signal transduction,plant-pathogen interaction,glutathione metabolism,and the biosynthesis of secondary metabolites.Further sequence alignment screened seven candidate genes related to disease resistance.qPCR verification demonstrated that the expression of Gh_D02G091500,Gh_D06G194600,Gh_A01G241100, Gh_A06G192400,and Gh_D09G161000 was significantly induced by P.boehmeriae.Among these,the cotton nitrate reductase gene exhibited the highest fold change and the most significant differential expression.The full-length sequence of this nitrate reductase gene was cloned and designated GhNIA1.In conclusion,it identified GhNIA1 as a key gene involved in the cotton-P.boehmeriae interaction and provided preliminary characterization of GhNIA1.These findings lay a theoretical foundation for further analysis of the molecular mechanism by which GhNIA1 participates in cotton resistance to boll rot and for breeding resistant varieties.

Key words: Gossypium spp., Phytophthora boehmeriae, RNA-seq, Gene cloning, Expression analysis

中图分类号:

S435.62

赵存鹏, 秦臻, 李丹, 刘素恩, 郭宝生, 王凯辉, 张海娜, 高鑫隆, 王海燕, 耿军义. 基于苎麻疫霉菌诱导的棉花组学分析克隆硝酸还原酶基因GhNIA1[J]. 华北农学报, 2026, 41(4): 34-43. doi: 10.7668/hbnxb.20196564.

ZHAO Cunpeng, QIN Zhen, LI Dan, LIU Suen, GUO Baosheng, WANG Kaihui, ZHANG Haina, GAO Xinlong, WANG Haiyan, GENG Junyi. Cloning of the Nitrate Reductase Gene GhNIA1 Based on Omics Analysis of Cotton Induced by Phytophthora boehmeriae[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 34-43. doi: 10.7668/hbnxb.20196564.

http://www.hbnxb.net/CN/Y2026/V41/I4/34

图/表 8

表1 试验所用引物

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	用途 Use
q-Gh_D11G285900-F	CCACCAACCCTCTTATCC	实时荧光定量
q-Gh_D11G285900-R	GAAGTTGGGATACCTACGAT	PCR引物
q-Gh_D06G194600-F	GGAGCCAACAAGTTCACA
q-Gh_D06G194600-R	GCAAAGATCATACCACCAA
q-Gh_D09G161000-F	CCAAGCCAAGGACTACCA
q-Gh_D09G161000-R	AAGATCATCCCACCAAAA
q-Gh_A06G192400-F	AAGCAACGAAGAAGGGAA
q-Gh_ A06G192400-R	AAGGAAAGTCGCCACAAA
q-Gh_A01G241100-F	GACTACCATGATCCTCCAC
q-Gh_ A01G241100-R	TACCAAGAATGCCAACAC
q-Gh_D02G091500-F	GTTGTTGACGTCGGGTTATATT
q-Gh_D02G091500-R	CCAACTTCACCGATGCTACT
q-Gh_A13G043200-F	TTTAGTTGTTAGCCAGATTT
q-Gh_A13G043200-R	GATGAAATCAGCAGTCGC
Histone H3-F	TCAAGACTGATTTGCGTTTCCA
Histone H3-R	GCGCAAAGGTTGGTGTCTTC
GhNIA1-F	ATGATGAACAACTGCTGG	PCR 扩增引物
GhNIA1-R	AAATACCAACAAAGAATCCT

图1 棉铃接菌后表型鉴定结果每组棉铃左侧皆为对照组,右侧皆为试验组。

Fig.1 Phenotypic identification results of cotton bolls after inoculation The cotton bolls on the left side served as the control group, while the cotton bolls on the right side were the experimental group.

图2 不同时间点差异表达基因统计

Fig.2 Statistical analysis of differentially expressed genes at different time points

图3 差异表达基因富集分析及差异蛋白对应的生物过程 A:1. 转硫途径,2. 二萜生物合成过程,3. 细胞壁大分子分解代谢过程,4. 几丁质分解代谢过程,5. 翻译,6. 胞质核糖体大亚基,7. 胞质核糖体小亚基,8. 液泡,9. 核糖体,10. 胞质,11. (+)-δ-杜松烯合酶活性,12. UDP-糖基转移酶活性,13. 镁离子结合,14. 核糖体结构组分,15. 血红素结合;B:1. 毒素分解代谢过程,2. 几丁质分解代谢过程,3. 谷胱甘肽代谢过程,4. 蛋白质丝氨酸/苏氨酸磷酸酶活性的调控,5. 脱落酸激活的信号通路,6. RISC 复合体,7. 氨甲酰磷酸合成酶复合物,8. 过氧化物酶体,9. 胞质溶胶,10. 细胞质,11. 几丁质酶活性,12. 谷胱甘肽转移酶活性,13. 脱落酸结合,14. 蛋白磷酸酶抑制活性,15. FAD 结合。

Fig.3 Enrichment analysis of DEGs and the biological processes corresponding to differentially expressed proteins A:1. Transsulfuration,2. Diterpenoid biosynthetic process,3. Cell wall macromolecule catabolic process,4. Chitin catabolic process,5. Translation,6. Cytosolic large ribosomal subunit,7. Cytosolic small ribosomal subunit,8. Vacuole,9. Ribosome,10. Cytosol,11. (+)-delta-cadinene synthase activity,12. UDP-glycosyltransferase activity,13. Magnesium ion binding,14. Structural constituent of ribosome,15. Heme binding; B:1. Toxin catabolic process, 2. Chitin catabolic process, 3. Glatayhione metabolic process, 4. Regulation of protein serine/threonine phosphatase activity, 5. Abscisic acid-activated signaling pathway, 6. RISC complex, 7. Carbamoyl-phosphate synthase complex, 8. Peroxisome, 9. Cytosol, 10. Cytoplasm, 11. Chitinase activity, 12. Glutathione transferase activity, 13. Abscisic acid binding, 14. Protein phosphatase inhibitor activity, 15. FAD binding.

表2 候选基因特征分析

Tab.2 Analysis characteristics of the candidate genes

基因名 Gene ID	通路编号 Pathway_ID	序列比对 Blast	信号肽 Signal peptide	核酸/bp Nucleotide	亚细胞定位 Subcellular localization
Gh_D11G285900	ko00030	磷酸糖异构酶	无	846	细胞核
Gh_D06G194600	ko04962	主要内在蛋白	无	1 613	细胞膜
Gh_D09G161000	ko04924	主要内在蛋白	无	1 606	细胞膜
Gh_A06G192400	ko04962	主要内在蛋白	无	1 791	细胞膜
Gh_A01G241100	ko04924	主要内在蛋白	无	1 455	细胞膜
Gh_A13G043200	ko00680	磷酸甘油酸脱氢酶	无	2 623	细胞核
Gh_D02G091500	ko00520	硝酸还原酶	无	1 314	细胞核

图4 棉花GhNIA1基因PCR扩增结果与序列分析 M.DL 2000 Marker; A.GhNIA1扩增结果;B.GhNIA1序列分析;C.GhNIA1聚类分析。

Fig.4 PCR results and multiple alignments of GhNIA1 gene M.DL2000 Marker; A.Amplification results of GhNIA1; B.Sequence analysis of GhNIA1;C.Cluster analysis of GhNIA1.

图5 GhNIA1基因特征分析 A.疏水性分析;B.磷酸化位点预测;C.保守结构域分析;D.信号肽预测;E.跨膜结构域预测;F.二级结构预测;G.三级结构预测。

Fig.5 Analysis of gene characteristics of GhNIA1 gene A.Hydrophobic analysis; B.Phosphorylation site prediction; C.Conserved domain analysis; D.Signal peptide prediction; E.Transmembrane region prediction; F.Secondary structure prediction; G.Tertiary structure prediction.

图6 差异表达基因在侵染棉铃不同时间点的表达模式分析不同字母表示不同组间差异显著(P<0.05);内参基因.Histone H。

Fig.6 Expression pattern analyses of DEGs during the different points in infection times Different letters indicate significant differences between groups(P<0.05); Reference gene.Histone H.

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基于苎麻疫霉菌诱导的棉花组学分析克隆硝酸还原酶基因GhNIA1

Cloning of the Nitrate Reductase Gene GhNIA1 Based on Omics Analysis of Cotton Induced by Phytophthora boehmeriae

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Cloning of the Nitrate Reductase Gene GhNIA1 Based on Omics Analysis of Cotton Induced by Phytophthora boehmeriae

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