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

doi:10.7668/hbnxb.20196564

Abstract

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

CLC Number:

S435.62

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.

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Figures/Tables 8

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

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.

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

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.

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

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.

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.

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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