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

作物学报 ›› 2024, Vol. 50 ›› Issue (1): 110-125.doi: 10.3724/SP.J.1006.2024.34037

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

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

王恒波¹(), 冯春燕¹(), 张以星¹, 谢婉婕¹, 杜翠翠¹, 吴明星¹, 张积森²^,^*()

¹福建农林大学农业农村部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗工程技术研究中心 / 福建农林大学生命科学学院, 福建福州 3500022
²广西大学亚热带农业生物资源保护与利用国家重点实验室，广西南宁，530004

收稿日期:2023-02-23 接受日期:2023-06-29 出版日期:2024-01-12 网络出版日期:2023-07-20
通讯作者: *张积森, E-mail: zjisen@126.com
作者简介:王恒波, E-mail: wanghengbo_0354@126.com; 冯春燕, E-mail: 1151137075@qq6.com
**同等贡献
基金资助:
国家重点研发计划项目(2021YFF1000101-5);国家自然科学基金项目(32272156);福建省自然科学基金项目(2022J01160);国家级大学生创新创业训练计划项目(202310389001)

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

WANG Heng-Bo¹(), FENG Chun-Yan¹(), ZHANG Yi-Xing¹, XIE Wan-Jie¹, DU Cui-Cui¹, WU Ming-Xing¹, ZHANG Ji-Sen²^,^*()

¹Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / National Sugarcane Engineering Technology Research / College of Life Sciences Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
²State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, China

Received:2023-02-23 Accepted:2023-06-29 Published:2024-01-12 Published online:2023-07-20
Contact: *E-mail: zjisen@126.com
About author:**Contributed equally to this study
Supported by:
National Key Research and Development Program(2021YFF1000101-5);National Natural Science Foundation of China(32272156);Natural Science Foundation of Fujian Province, China(2022J01160);National Undergraduate Innovation and Entrepreneurship Training Program Project(202310389001)

摘要/Abstract

摘要：

NAP (NAC-like, activated by apetala3/pistillata)是转录因子NAC基因家族中一类参与调控植物生长发育、叶片衰老及响应激素和非生物胁迫应答的亚家族。本研究利用甘蔗割手密种基因组数据和生物信息学方法, 首先, 基于比较基因组学对NAP亚家族成员进行鉴定、系统进化分析、保守结构域及顺式调控元件预测; 其次, 克隆获得割手密种SsNAP2的等位基因SsNAP2a, 分析该基因在不同生长发育阶段的表达及其在激素和非生物胁迫下的表达特征; 最后, 利用瞬时表达和亚细胞定位分析SsNAP2a基因的功能。结果表明, 在割手密种基因组中共鉴定5个NAP亚家族成员, 亚细胞定位预测所有成员编码蛋白均定位于细胞核上, 这些基因的K_a/K_s比值均小于1, 表明纯化选择在演化中起到关键作用。系统聚类分析表明, 5个代表性的被子植物(拟南芥、菠萝、水稻、玉米和高粱)与已报道的12个物种及甘蔗的NAP亚家族成员, 共计46个, 分为4个Clade, 其演化的顺序Clade I > Clade II> Clade III > Clade IV。此外, 在SsNAP亚家族成员的启动子区域预测到较多响应脱落酸和茉莉酸、低温等逆境胁迫的顺式作用元件, 推测其参与多种激素和非生物胁迫相关应答通路。进一步, 从割手密种SES208中克隆到SsNAP2a基因, 该cDNA全长序列1173 bp (GenBank登录号为OQ335094), 编码390个氨基酸残基, 其与等位基因SsNAP2编码蛋白的序列相似性为97.70%, 有10个氨基酸残基的差异, 表明同源8倍体的割手密种等位基因序列差异较大。qRT-PCR分析表明, SsNAP2a基因在割手密种不同生长发育阶段中组成型表达, 尤其在衰老的蔗皮和根中的表达量最高; 在乙烯利(ethylene, ET)、脱落酸(abscisic acid, ABA)、4℃低温和40℃高温处理下, 其表达量呈现显著诱导表达; 而在8%聚乙二醇(polyethylene glycol, PEG)胁迫下显著下调表达。亚细胞定位表明, SsNAP2a融合蛋白定位在细胞核上。瞬时过表达SsNAP2a基因7 d后, 本氏烟(Nicotiana benthamiana)叶片有明显的卷曲、皱缩等衰老现象, ET合成相关基因(NtEFE26, NtAccdeaminase)显著上调表达, 而水杨酸、茉莉酸和ABA合成相关基因(NtPR-1a/c、NtPR3和NtAREB1)显著下调表达, 表明SsNAP2a基因参与多种激素信号传导途径诱发叶片衰老。以上研究结果为挖掘甘蔗NAP亚家族基因成员参与叶片衰老的生物学功能奠定了研究基础, 也为培育甘蔗抗衰老分子育种提供候选的基因资源。

关键词: 割手密种, NAP基因, 叶片衰老, 激素胁迫, 基因功能, 甘蔗

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

王恒波, 冯春燕, 张以星, 谢婉婕, 杜翠翠, 吴明星, 张积森. 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[J]. 作物学报, 2024, 50(1): 110-125.

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

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

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

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