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 Saccha-rum spontaneum and functional analysis of SsNAP2a involvement in leaf senes-cence
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
作者信息
- 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
- 折叠
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 Ka/Ks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.
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 phyloge-netic analysis,conserved domains,and cis-regulatory elements were predicted using comparative genomics and various bioinfor-matics 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 sub-cellular localization predicted that the encoded proteins of all members were localized in the nucleus.The Ka/Ks 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 Sor-ghum 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 re-sponse to abscisic acid,jasmonic acid,low temperature,and other stresses.We speculated that they were involved in various hor-mone 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 encod-ing 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 overex-pression 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 senes-cence.These results lay a foundation for exploring the biological functions of NAP subfamily gene members involved in sugar-cane leaf senescence and provide candidate gene resources for breeding anti-senescence new cultivars.
关键词
割手密种/NAP基因/叶片衰老/激素胁迫/基因功能/甘蔗Key words
Saccharum spontaneum/NAP gene/leaf senescence/hormone stress/functional analysis/sugarcane引用本文复制引用
基金项目
国家重点研发计划项目(2021YFF1000101-5)
国家自然科学基金(32272156)
福建省自然科学基金(2022J01160)
国家级大学生创新创业训练计划项目(202310389001)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据