甘蔗割手密种转录因子NAP亚家族的鉴定及SsNAP2a参与叶片衰老的功能分析

doi:10.3724/SP.J.1006.2024.34037

Abstract

Abstract:

NAP (NAC-like, Activated by APETALA3/PISTILLATA) is a subfamily of the transcription factor NAC gene family, which is widely involved in regulating plant growth and development, leaf senescence, and response to hormones and abiotic stress. Firstly, the NAP subfamily members were identified from the genomic database of Saccharum spontaneum, and phylogenetic analysis, conserved domains, and cis-regulatory elements were predicted using comparative genomics and various bioinformatics methods. Secondly, the allele SsNAP2a of the SsNAP2 member was isolated from the cDNA library of a wide accession SES208. The relative expression characteristics of the SsNAP2a were detected by qRT-PCR under hormone and abiotic stresses at different growth and development stages. Finally, transient overexpression and subcellular localization performed the function of SsNAP2a gene. The results showed that five NAP subfamily members were identified in the genome of S. spontaneum. The subcellular localization predicted that the encoded proteins of all members were localized in the nucleus. The K_a/K_s ratio of five gene pairs was less than 1, indicating that purifying selection was crucial in the evolution. Phylogenetic analysis revealed that 46 NAP members, including five representative angiosperms (Arabidopsis thaliana, Ananas comosus, Oryza sativa, Zea mays, and Sorghum bicolor), 12 reported species, and the S. spontaneum, were classified into four Clades. The evolution order was Clade I > Clade II > Clade III > Clade IV. In addition, the promoter regions of SsNAP members predicted many cis-acting elements in response to abscisic acid, jasmonic acid, low temperature, and other stresses. We speculated that they were involved in various hormone and abiotic stress-related response pathways. Furthermore, the full-length cDNA sequence of the SsNAP2a gene (GenBank accession number: OQ335094) was isolated from the wild accession SES208, with an open reading frame of 1173 bp and encoding 390 amino acid residues. The amino acid sequence similarity between SsNAP2 and SsNAP2a proteins was 97.70%. There was a difference of 10 amino acid residues, indicating that the autopolyploid allelic sequences of Saccharum species were significant difference. The qRT-PCR demonstrated that the SsNAP2a gene was constitutively expressed in various tissues of S. spontaneum, especially in the senescent bark and root, and its expression level was significantly induced under the treatment of ethylene, ET, abscisic acid (ABA), low temperature at 4℃, and high temperature at 40℃. However, the relative expression level of the SsNAP2a gene was significantly down-regulated under 8% polyethylene glycol (PEG) stress. Subcellular localization revealed that the SsNAP2a-GFP fusion protein was located in the cell nucleus of Nicotiana benthamiana leaves. After transient overexpression of the SsNAP2a gene for seven days, the leaves of N. benthamiana displayed obvious curling and shriveling phenotype. The relative expression level of ET synthesis-related genes (NtEFE26, NtAccdeaminase) was significantly up-regulated, while salicylic acid, jasmonic acid, and ABA synthesis-related genes (NtPR-1a/c, NtPR3, and NtAREB1) were significantly down-regulated, indicating that the SsNAP2a gene was involved in multiple hormone signaling pathways to induce leaf senescence. These results lay a foundation for exploring the biological functions of NAP subfamily gene members involved in sugarcane leaf senescence and provide candidate gene resources for breeding anti-senescence new cultivars.

Key words: Saccharum spontaneum, NAP gene, leaf senescence, hormone stress, functional analysis, sugarcane

WANG Heng-Bo, FENG Chun-Yan, ZHANG Yi-Xing, XIE Wan-Jie, DU Cui-Cui, WU Ming-Xing, ZHANG Ji-Sen. Genome-wide identification of NAP transcription factors subfamily in Saccharum spontaneum and functional analysis of SsNAP2a involvement in leaf senescence[J].Acta Agronomica Sinica, 2024, 50(1): 110-125.

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名称 Primer name	引物序列 Primer sequences (5′-3′)	备注 Annotation
SsNAP2a-F	ATGACGACGATGATGTCGGCG	基因克隆 Gene cloning
SsNAP2a-R	CTAGAAATGAGGAAGCATGTGATGG	基因克隆 Gene cloning
qRT-PCR-SsNAP2a-F	CCTCCAAGCTCAGGAACGTC	定量PCR引物 qRT-PCR primers
qRT-PCR-SsNAP2a-R	CTCGTAGTCGGCGAGCG	定量PCR引物 qRT-PCR primers
qRT-PCR-SsUBC-F	GATTCTACTGCGGATGGATTGA	甘蔗内参基因 Reference gene in sugarcane
qRT-PCR-SsUBC-R	CAACTTTGGGATGCTTGATACAC	甘蔗内参基因 Reference gene in sugarcane
pMDC202-SsNAP2a-F	CTCGACTCTAGAACTAGTATGACGACGATGATGTCGGCG	过表达载体构建 Construction of overexpression vector
pMDC202-SsNAP2a-R	ATTTTTTCTACCGGTACCCTAGAAATGAGGAAGCATGTGATGG	过表达载体构建 Construction of overexpression vector
pSuper-1300-SsNAP2a-F	GGGGCCCGGGGTCGACATGACGACGATGATGTCGGCG	亚细胞定位载体构建 Construction of subcellular localization vector
pSuper-1300-SsNAP2a-R	CCCTTGCTCACCATGGTACCGAAATGAGGAAGCATGTGATGG	亚细胞定位载体构建 Construction of subcellular localization vector
qPCR-AREB1-ABA-F	ATCCAGAAAAACAGAAAAGAGTGAT	脱落酸通路相关基因 Abscisic acid pathway related gene
qPCR-AREB1-ABA-R	CAACACTACTTCCACCCTCCC	脱落酸通路相关基因 Abscisic acid pathway related gene

名称 Name	基因编号 Sequence ID	氨基酸残基数 Number of amino acid	相对分子量 Molecular weight (kD)	理论等电点 Theoretical pI	不稳定系数 Instability index	平均疏水性 Grand average of hydropathicity	高粱直系同源基因 Orthologous gene from sorghum	非同义和同义替换率 K_a/K_s
SsNAP1	Npp.01C032290.1	371	40.285	9.04	41.98	-0.50	Sobic.001G385800.1	0.46
SsNAP2	Npp.02D037500.1	392	41.876	7.29	44.69	-0.26	Sobic.002G420700.1	0.65
SsNAP3	Npp.03B008200.1	340	36.933	6.33	55.52	-0.45	Sobic.003G105800.1	0.48
SsNAP4	Npp.05B003240.1	396	43.899	6.22	49.34	-0.65	Sobic.005G018500.1	0.34
SsNAP5	Npp.08D002510.1	409	44.912	6.06	51.39	-0.61	Sobic.008G021800.1	0.30

Genome-wide identification of NAP transcription factors subfamily in Saccharum spontaneum and functional analysis of SsNAP2a involvement in leaf senescence

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