作物学报2024，Vol.50Issue(1)：110-125,16.DOI:10.3724/SP.J.1006.2024.34037
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
摘要
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
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):110-125,16.基金项目
本研究由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)和国家级大学生创新创业训练计划项目(202310389001). This study was supported by the National Key Research and Development Program(2021YFF1000101-5),the National Natural Science Foundation of China(32272156),the Natural Science Foundation of Fujian Province,China(2022J01160),and the National Undergraduate Innovation and Entrepreneurship Training Program Project(202310389001). （2021YFF1000101-5）
