细胞学结合转录组分析发掘绿豆花瓣开放及衰老关键基因  

Identification of critical genes for petal opening and senescence in mungbean by cytology and transcriptome analysis

作　　者：林云 周琰琰 刘金洋 闫强 陈景斌 吴然然 薛晨晨 陈新[1,2] 袁星星 LIN Yun;ZHOU Yanyan;LIU Jinyang;YAN Qiang;CHEN Jingbin;WU Ranran;XUE Chenchen;CHEN Xin;YUAN Xingxing(Institute of Cash Crops,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Life Sciences,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]江苏省农业科学院经济作物研究所,江苏南京210014 [2]南京农业大学生命科学学院,江苏南京210095

出　　处：《南京农业大学学报》2025年第2期348-359,共12页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(32301894);国家食用豆产业技术体系生物防治与综合防控岗位科学家项目(CARS-08-G15);江苏省种业“揭榜挂帅”项目(JBGS[2021]004);江苏省“一带一路”国际合作项目(BZ2022005)。

摘　　要：[目的]本研究目的在于分析绿豆花开放过程中的细胞学变化,结合转录组学发掘调控绿豆花开放过程中的关键基因,并探究其分子机制。[方法]以‘苏绿1号’为研究对象,对花开放过程中各时期的花瓣进行细胞学观察,根据结果锁定花瓣衰老的关键时期,对该时期的花瓣进行转录组测序,根据前后时期表达量变化以及功能注释挖掘关键基因,最终通过烟草及拟南芥的遗传转化试验验证功能。[结果]细胞学试验显示在B2至B4期,绿豆表皮细胞膨大,细胞壁变薄,叶绿体降解,迅速进入程序性死亡,细胞结构崩坏。转录组分析结果检测到细胞壁合成和代谢以及细胞壁松弛和细胞扩张相关基,并鉴定到10个NAC(NAM/ATAF1/2/CUC2)家族基因表达量的强烈变化,qPCR检测发现其中VrNAC72b是一个在花瓣中特异表达的基因,并且表达量在B3和B4期迅速升高。在烟草中瞬时表达VrNAC72a/b导致叶片黄化,在拟南芥中过表达VrNAC72b也会导致阳性植株的叶片提前衰老,NBT和DAB染色显示阳性植株各组织中的活性氧含量升高。[结论]绿豆开花过程中细胞膨大并迅速死亡,导致花瓣张开并随后凋落,此过程中大量细胞壁相关基因参与,并且NAC家族基因也起到重要作用,其中VrNAC72b表达量发生显著变化,并引起花瓣细胞的程序性死亡。[Objectives]This study was conducted to analyze the cytological changes in the process of mungbean flower blooming,explore the key genes with transcriptomic,and reveal the underlying molecular mechanism.[Methods]‘Sulyu 1’was used in this study,cytological observations were conducted on the petals of each stage during the blooming process.Based on the cytological results,the critical stages of petal senescence were identified,and transcriptome sequencing was performed on the petals before and after these stages.Key genes were identified based on changes in expression levels and functional annotation,and their functions were ultimately verified through genetic transformation experiments in tabacco and Arabidopsis.[Results]Cytological experiments showed that from B2 to B4 stage,the epidermal cells of petals expanded,with cell wall thinning and chloroplasts degradation,subsequently enter programmed cell death rapidly,resulting in cell structure collapse.The transcriptome analysis detected cell wall synthesis and metabolism,as well as cell wall relaxation and cell expansion related genes,and identified 10 NAC family genes with significant changes in the expression levels,qPCR detected that VrNAC72b was a gene specifically expressed in petals,and its expression level rapidly increased at B3 and B4 stages.Expression of VrNAC72a/b in tobacco leaded to leaf yellowing,while overexpression of VrNAC72b in Arabidopsis leaded to early leaf senescence.NBT and DAB staining showed an increase in reactive oxygen species content in various tissues.[Conclusions]During the flowering process of mungbean,the cells expanded and died rapidly,leading to petal opening and subsequent littering.A large number of cell wall related genes were involved in this process,and the NAC family genes also played an important role.Among them,the expression level of VrNAC72b changed significantly,causing programmed cell death in petal cells.

关 键 词：绿豆 花瓣 转录组 衰老 细胞学 

分 类 号：S431.12[农业科学—农业昆虫与害虫防治]