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MIAO Yunfeng, ZHOU Dan, DONG Bin, ZHAO Hongbo. Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage[J]. Journal of Zhejiang A&F University, 2021, 38(3): 433-444. doi: 10.11833/j.issn.2095-0756.20200474
Citation: MIAO Yunfeng, ZHOU Dan, DONG Bin, ZHAO Hongbo. Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage[J]. Journal of Zhejiang A&F University, 2021, 38(3): 433-444. doi: 10.11833/j.issn.2095-0756.20200474
shu

Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage

doi: 10.11833/j.issn.2095-0756.20200474
  • 1.

    Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • 2.

    Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • 3.

    College of Landscape Architecture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • Received Date: 2020-07-21
  • Rev Recd Date: 2020-12-30
    • Available Online: 2021-06-09
  • Publish Date: 2021-06-09
  •   Objective  This study aims to investigate the regulation of OfNAC genes on flower opening of Osmanthus fragrans.  Method  From the transcriptome data of O. fragrans ‘Yanhonggui’, the related OfNAC g enes were screened and analyzed to predict the physicochemical properties and structure. Real-time fluorescence quantitative PCR was used to analyze the expression characteristics of the flower opening process.  Result  22 OfNAC sequences were screened from transcriptome. Bioinformatics analysis showed that all of the 22 OfNAC transcription factors contained NAM domains, and the amino acid sequence contained 5 conserved subdomains (A−E). The sequence of conservatism ranging from strong to weak was C, A, D, B, and E. The proportion of different structures in the secondary structure from large to small was random coils, α-helices, extended strands, and β-sheets. Subcellular localization and transmembrane structure prediction showed that OfNAC17, OfNAC17-X2, OfNAC53, OfNAC91, OfNTM1-9 were membrane-bound transcription factors, and most OfNACs were located in the nucleus. During the O. fragrans flowering process, the relative expression levels of OfNAC100-2, OfNAC43 and OfNAC73 reached the peak at the boll stem stage (S4), and then decreased. The relative expression of OfNAC43 was the highest at the boll stem stage (S4). The relative expression of OfNAC71, OfNAC29-1, and OfNAC21/22 increased slowly from the initial stage (S1), reached the highest at the apical shell stage, and then decreased as a whole. The relative expression of OfNAC29-2 increased sharply at the bead stage (S2) and reached the lowest at the boll stem stage (S4).  Conclusion  It is speculated that OfNAC100-2, OfNAC43, OfNAC73, OfNAC71, OfNAC29-1, OfNAC21/22, OfNAC29-2 may be involved in the regulation of O. fragrans flower opening. [Ch, 6 fig. 3 tab. 33 ref.]
  • [1] ZHOU Junjie, WANG Yiguang, DONG Bin, ZHAO Hongbo.  Cloning and expression characterization of OfPSY, OfPDS and OfHYB gene promoters in Osmanthus fragrans . Journal of Zhejiang A&F University, 2023, 40(1): 64-71. doi: 10.11833/j.issn.2095-0756.20220110
    [2] ZHANG Yao, WANG Jiaxuan, CAI Xuan, ZENG Xiangling, YANG Jie, CHEN Hongguo, ZOU Jingjing.  Identification and expression of OfACOs gene family in Osmanthus fragrans . Journal of Zhejiang A&F University, 2023, 40(3): 492-501. doi: 10.11833/j.issn.2095-0756.20220783
    [3] YUE Yuanzheng, HU Hongmin, LIU Jiawei, SHEN Huimin, SHI Tingting, YANG Xiulian, WANG Lianggui.  Functional analysis of OfMYB1R47 transcription factor in Osmanthus fragrans during the formation of aromatic volatiles . Journal of Zhejiang A&F University, 2023, 40(3): 465-474. doi: 10.11833/j.issn.2095-0756.20220456
    [4] HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo.  Cloning and expression analysis of OfABFs gene in Osmanthus fragrans . Journal of Zhejiang A&F University, 2023, 40(3): 481-491. doi: 10.11833/j.issn.2095-0756.20220264
    [5] PANG Tianhong, QIAN Jieyu, FU Jianxin, GU Cuihua, ZHANG Chao.  Sequence and expression analysis of hexokinase gene family members in Osmanthus fragrans . Journal of Zhejiang A&F University, 2021, 38(2): 225-234. doi: 10.11833/j.issn.2095-0756.20200370
    [6] WU Qi, WU Hongfei, ZHOU Minshu, XU Qianxia, YANG Liyuan, ZHAO Hongbo, DONG Bin.  Cloning and expression analysis of OfFCA gene at flower bud differentiation stages in Osmanthus fragrans . Journal of Zhejiang A&F University, 2020, 37(2): 195-200. doi: 10.11833/j.issn.2095-0756.2020.02.001
    [7] WANG Qianqian, JIANG Qini, FU Jianxin, DONG Bin, ZHAO Hongbo.  Screening reference genes of Osmanthus fragrans with differing photoperiod and temperature treatments . Journal of Zhejiang A&F University, 2019, 36(5): 928-934. doi: 10.11833/j.issn.2095-0756.2019.05.011
    [8] JIANG Qini, FU Jianxin, ZHANG Chao, DONG Bin, ZHAO Hongbo.  cDNA cloning and expression analysis of OfAP1 in Osmanthus fragrans . Journal of Zhejiang A&F University, 2019, 36(4): 664-669. doi: 10.11833/j.issn.2095-0756.2019.04.005
    [9] LIU Yucheng, WANG Yiguang, ZHANG Chao, DONG Bin, FU Jianxin, HU Shaoqing, ZHAO Hongbo.  Cloning and transient expression assay of OfCCD1 gene promoters from Osmanthus fragrans . Journal of Zhejiang A&F University, 2018, 35(4): 596-603. doi: 10.11833/j.issn.2095-0756.2018.04.003
    [10] LI Jun, DONG Bin, ZHANG Chao, FU Jianxin, HU Shaoqing, ZHAO Hongbo.  EST-SSR primers and their application in cultivar identification of Osmanthus fragrans . Journal of Zhejiang A&F University, 2018, 35(2): 306-313. doi: 10.11833/j.issn.2095-0756.2018.02.015
    [11] LI Bingbing, LIU Guofeng, WEI Shu, HUANG Longquan, ZHANG Jianyun.  Cloning and expression analysis of the tobacco NtPLR1 gene . Journal of Zhejiang A&F University, 2017, 34(4): 581-588. doi: 10.11833/j.issn.2095-0756.2017.04.003
    [12] CAI Zhoufei, CHEN Yaqi, XU Xinlu, WANG Xiaodong, WANG Junyu, ZHANG Rumin, GAO Yan.  Changes of volatile organic compounds released during flowering in four Osmanthus fragrans cultivar groups . Journal of Zhejiang A&F University, 2017, 34(4): 608-619. doi: 10.11833/j.issn.2095-0756.2017.04.006
    [13] FU Jianxin, ZHANG Chao, WANG Yiguang, ZHAO Hongbo.  Reference gene selection for quantitative real-time polymerase chain reaction (qRT-PCR) normalization in the gene expression of sweet osmanthus tissues . Journal of Zhejiang A&F University, 2016, 33(5): 727-733. doi: 10.11833/j.issn.2095-0756.2016.05.001
    [14] WANG Ying, ZHANG Chao, FU Jianxin, ZHAO Hongbo.  Progresses on flower bud differentiation and flower opening in Osmanthus fragrans . Journal of Zhejiang A&F University, 2016, 33(2): 340-347. doi: 10.11833/j.issn.2095-0756.2016.02.021
    [15] XU Yichun, HU Shaoqing, ZHAO Hongbo.  Genetic structure of different natural Osmanthus fragrans populations based on AFLP method . Journal of Zhejiang A&F University, 2014, 31(2): 217-223. doi: 10.11833/j.issn.2095-0756.2014.02.009
    [16] LIN Fuping, ZHOU Shuai, MA Nan, ZHANG Rumin, GAO Yan.  Volatile organic compounds in leaves of Osmanthus fragrans and their effect on airborne microorganisms . Journal of Zhejiang A&F University, 2013, 30(1): 15-21. doi: 10.11833/j.issn.2095-0756.2013.01.003
    [17] YANG Xihong, HUANG Youjun, CHEN Fangfang, HUANG Jianqin.  Identification and expression of FLOWERING LOCUS C homologous genes in Carya cathayensis . Journal of Zhejiang A&F University, 2013, 30(1): 1-8. doi: 10.11833/j.issn.2095-0756.2013.01.001
    [18] YANG Xiu-lian, HAO QI-mei.  Dormancy and germination of Osmanthus fragrans seeds . Journal of Zhejiang A&F University, 2010, 27(2): 272-276. doi: 10.11833/j.issn.2095-0756.2010.02.018
    [19] CHANG Bing-hua, HUYong-hong, XUYe-gen, ZHANG Qiu-xing, ZHANG Wan-li.  Ultrastructures of petal surface of Osmanthus fragrans cultivars . Journal of Zhejiang A&F University, 2007, 24(5): 533-537.
    [20] HU Shao-qing, XUAN Zi-can, ZHOU Xu-lang, WU Guang-hong.  Taxon and clear of Osmanthus fragrans cultivars in Hangzhou . Journal of Zhejiang A&F University, 2006, 23(2): 179-187.
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Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage

doi: 10.11833/j.issn.2095-0756.20200474
  • 1. Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • 2. Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • 3. College of Landscape Architecture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • Received Date: 2020-07-21
  • Rev Recd Date: 2020-12-30
  • Available Online: 2021-06-09
  • Publish Date: 2021-06-09

Abstract:   Objective  This study aims to investigate the regulation of OfNAC genes on flower opening of Osmanthus fragrans.  Method  From the transcriptome data of O. fragrans ‘Yanhonggui’, the related OfNAC g enes were screened and analyzed to predict the physicochemical properties and structure. Real-time fluorescence quantitative PCR was used to analyze the expression characteristics of the flower opening process.  Result  22 OfNAC sequences were screened from transcriptome. Bioinformatics analysis showed that all of the 22 OfNAC transcription factors contained NAM domains, and the amino acid sequence contained 5 conserved subdomains (A−E). The sequence of conservatism ranging from strong to weak was C, A, D, B, and E. The proportion of different structures in the secondary structure from large to small was random coils, α-helices, extended strands, and β-sheets. Subcellular localization and transmembrane structure prediction showed that OfNAC17, OfNAC17-X2, OfNAC53, OfNAC91, OfNTM1-9 were membrane-bound transcription factors, and most OfNACs were located in the nucleus. During the O. fragrans flowering process, the relative expression levels of OfNAC100-2, OfNAC43 and OfNAC73 reached the peak at the boll stem stage (S4), and then decreased. The relative expression of OfNAC43 was the highest at the boll stem stage (S4). The relative expression of OfNAC71, OfNAC29-1, and OfNAC21/22 increased slowly from the initial stage (S1), reached the highest at the apical shell stage, and then decreased as a whole. The relative expression of OfNAC29-2 increased sharply at the bead stage (S2) and reached the lowest at the boll stem stage (S4).  Conclusion  It is speculated that OfNAC100-2, OfNAC43, OfNAC73, OfNAC71, OfNAC29-1, OfNAC21/22, OfNAC29-2 may be involved in the regulation of O. fragrans flower opening. [Ch, 6 fig. 3 tab. 33 ref.]

MIAO Yunfeng, ZHOU Dan, DONG Bin, ZHAO Hongbo. Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage[J]. Journal of Zhejiang A&F University, 2021, 38(3): 433-444. doi: 10.11833/j.issn.2095-0756.20200474
Citation: MIAO Yunfeng, ZHOU Dan, DONG Bin, ZHAO Hongbo. Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage[J]. Journal of Zhejiang A&F University, 2021, 38(3): 433-444. doi: 10.11833/j.issn.2095-0756.20200474
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  • NAC(NAM-ATAF1/2-CUC2)是植物中最大的转录因子家族之一，成员众多，功能多样[1]。NAC转录因子参与植物多个生物学过程，如植物茎尖分生组织的形成[2]、植物次生生长[3]、组织愈合过程中的细胞增殖[4]、生物和非生物胁迫响应[5-6]、植物开花时间调节[7-8]、叶片和花瓣衰老[9]和果实成熟时颜色变化等[10]。花瓣细胞的伸长和扩展也受到NAC转录因子的调控。花瓣的扩展是引起植物花开放的重要原因[11-13]，主要受花瓣细胞的膨压变化、细胞壁代谢变化等影响[14]。研究发现：NAC转录因子NAP可能通过抑制拟南芥Arabidopsis thaliana花瓣细胞扩张而影响花器官生长，且在花发育细胞分裂向细胞扩展过渡阶段发挥重要作用[15]。RhNAC3可以作为上游调控因子结合具有调节渗透压功能的基因从而影响月季Rosa hybrida花瓣细胞扩张[16]。桂花Osmanthus fragrans是中国传统的十大名花之一，花是其最重要的观赏器官。桂花花的开放受到温度、湿度等环境因子的影响，其具体的分子机制至今尚未阐明。本研究通过桂花NAC家族基因鉴定和筛选，预测它们的理化性质，运用荧光定量PCR分析花开放进程中的表达模式，筛选出参与桂花花开放调控的关键NAC转录因子成员，为明确其在花开放中的功能作用奠定基础。

1.   材料与方法

    1.1.   材料

  • 以桂花品种‘堰虹桂’O. fragrans‘Yanhonggui’盆栽植株为材料，种植于浙江农林大学桂花资源圃。选取株龄相同、生长一致的健康植株，分别于起始期(S1)以及花开放不同阶段：圆珠期(S2)、顶壳期(S3)、铃梗期(S4)、初开期(S5)、盛花期(S6)进行取样[17]。每个时期样品采集3份，取样时间均为10:00，液氮速冻后储藏于−80 ℃冰箱备用。

    • 1.2.   方法

    1.2.1.   桂花OfNAC转录因子的鉴定及理化性质分析

  • 本研究从已构建的桂花‘堰虹桂’转录组数据库[18]中对NAC转录因子进一步进行NCBI-Blast(http://www.ncbi.nLm.nih.gov/BLAST/)比对分析。通过PlantTFDB(http://planttfdb.cbi.pku.edu.cn/blast.php)以及Pfam(https://pfam.xfam.org/)查询候选基因中的NAC的结构域，并通过NCBI(https://www.ncbi.nlm.nih.gov/cdd)进行保守功能域预测，利用TBtools工具进行桂花NAC保守结构域可视化分析，TBtools-ORF finder分析开放阅读框[19]，并通过NCBI-Blast获得开放阅读框完整的桂花OfNAC序列。运用ExPASy在线软件(http://web.expasy.org/protparam/)预测编码蛋白质分子量、理论电点、酸碱性等。

    • 1.2.2.   桂花OfNAC转录因子结构和亚细胞定位预测分析

  • 用DNAMAN对已筛选的桂花OfNAC转录因子氨基酸序列进行比对，分析其保守亚结构域；在线分析软件MEME 5.1.0分析NAC的保守基序(http://meme-suite.org/tools/meme)，对桂花OfNAC蛋白质二级结构以及跨膜结构分别采用在线软件SOPMA(https://npsaprabi.ibcp.fr/cgi-bin/npsa-sopma.html)和TMHMM Server V.2.0(http://www.cbs.dtu.dk/services/TMHMM/)进行分析。用在线分析软件Wolfp sort Genscript(https://www.genscript.com/wolf-psort.html?src=leftbar)进行亚细胞定位预测。

    • 1.2.3.   桂花OfNAC转录因子系统进化树构建

  • 从PlantTFDB(http://planttfdb.cbi.pku.edu.cn/blast.php)中获取拟南芥、水稻Oryza sativa的NAC序列，使用MEGA 6.0软件构建系统发育树，采用邻近法(neighbor-joining)，1 000 次Bootstrap抽样。

    • 1.2.4.   桂花OfNAC转录因子表达分析

  • RNA提取采用UltraClean Polysaccharide and Phenol Plant RNA Purification Kit试剂盒(诺禾致源公司，中国天津)，方法参照说明书。使用核酸分析仪(IMPLEN公司，德国)对RNA的纯度与浓度进行检测，并用质量分数1%的琼脂糖凝胶电泳检测RNA的完整性。依据说明书使用PrimeScript™RT Master Mix Perfect Real Time(TaKaRa，中国大连)试剂盒对桂花不同花开放时期的cDNA进行合成。利用Primer Premier 5.0对22个OfNAC基因进行定量特异性引物设计，桂花OfACT作为内参基因[20]，引物序列如表1所示。荧光定量反应体系如下：SYBR Premix Ex Taq 10 μL，cDNA 2 μL，上游引物和下游引物(10 μmol·L−1)各8 μL，双蒸水(ddH2O)补足至20 μL，每个样品设置3次生物学重复。反应程序为：95 ℃预变性30 s，95 ℃变性5 s，60 ℃复性30 s，共40个循环；然后以95 ℃持续5 s，60 ℃持续1 min，95 ℃持续15 s作为溶解曲线分析程序。循环阈值由Light Cycler 480软件自动计算。最后根据${2^{{\rm{ - }}\Delta \Delta {C_{\rm{t}}}}} $法计算目的基因的相对表达量。

    基因名称用于荧光定量的引物序列(5′→3′)基因名称用于荧光定量的引物序列(5′→3′)
    OfNAC100-1F: TGAACAAGATTGAGCCTTGGGOfNAC104F: TGCATTTTACATTGGTGAAGATGTC
    R: CCTTTCCTGTGGCTTTCCAGR: GCTCGTACACTTGACACACCA
    OfNAC53F: AGATTGTGGGGATGAAGAAAAOfNAC92F: TCCTAGTCGGAATGAAGAAAACTC
    R: CAACTCCATATCAGTAAGCCGR: ATGGCTTTCTAATCTGTATTCGTGC
    OfNTM1-9F: GGTTGCTCTAATGCCCACTTCOfNAC72-1F: GGAAAAGCCCCCAAAGGAAC
    R: CTGGTTCCGTAGCACGATACTR: CCCAATCATCCAACCTTGAGC
    OfNAC73F: AGGCAAGGATGGCCAAATTCOfNAC72-2F: ACGTAGGAAAAGCACCAAAAGG
    R: TTGTGCCATCTTGTTTCTCCR: AGCATCCAACCTTGCGCTTC
    OfNAC43F: AGGCTACTGGTCGTGATAAAGOfNAC29-1F: TTACAAGGGAAGGCCTCCAAAG
    R: GGGGTCATGAGTGTCGTCCR: TTGAGCCATTTTGCGTGTTAGG
    OfNAC91F: TCTACAAAGGTCGTGCTCCGOfNAC29-2F: CCCAAAGGGCGTCAAAACTG
    R: CCCTGACCAGGATAAGTGCCR: GCACACAATCATCCAACCTCA
    OfNAC50-X2F: TGGCAAAGGGTATTGGAAAGCOfNAC71F: CTATCGTGGAAGAGCACCACT
    R: TCGCCCACTATGGAAAACAAGR: TCCCTGAAATCTTGGGGTGTC
    OfNAC50F: AAGCAACTGGAAAGGATCGCOfNAC2F: TTGGGAATAAAGAAGGCTCTGGTG
    R: AATTCTGCATCGCAAAGCCTGR: ACACAACACCCAATCATCAAGCCTC
    OfNAC21/22F: GAAGGGAAGCCTGGTTGGAATOfNAC100-2F: TCAGAGGAAAAATCCTCGTCGG
    R: CCCAATCCTCCTTGACAGATGR: TTTGGGAGGTTGTGGATCGAG
    OfNAC56F: TCTATGGTGGAAAGCCTCCTOfNAC32F: AAGCCTTGGTTTTCTATGCCG
    R: CATCAAGCCTTAAAGAGCCCR: AAGCTGTTGTTCTTGTTTCGA
    OfNAC17-X2F: TCCTGTTGGGGTGAAGAAGAOfACTF: CCCAAGGCAAACAGAGAAAAAAT
    R: ATAGTCATCCTGTGCATCCTGCR: ACCCCATCACCAGAATCAAGAA
    OfNAC17F: TGGTCTTCCATAAAGGTCGTGC
    R: TTGTACAGAGCATAGCAATCCCGTG

    Table 1.  Specific primers for RT-PCR of OfNAC in O. fragrans

2.   结果分析

    2.1.   桂花OfNAC转录因子鉴定及理化性质分析

  • 通过对桂花‘堰虹桂’转录组数据分析，并结合NCBI-Blast筛选，对其中22个桂花OfNAC基因进行分析。结果显示：它们均含有NAC家族蛋白特有的NAM保守功能域(图1)。通过ExPASy对22条桂花OfNAC转录因子进行理化性质分析(表2)，结果表明：22条桂花OfNAC基因的氨基酸(OfNAC104~OfNAC91)长度为186~609个；蛋白质(OfNAC104~OfNAC17)相对分子量为21 498.77~68 408.12；等电点为4.59~9.33，其中OfNAC100-1、OfNAC73、OfNAC56、OfNAC72-1、OfNAC72-2、OfNAC29-1、OfNAC29-2、OfNAC32等8个蛋白质等电点大于7，偏碱性，其余14个OfNAC等电点小于7，偏酸性。OfNAC的不稳定系数为29.27~52.83，OfNAC53、OfNAC73、OfNAC56、OfNAC92、OfNAC29-2等5个蛋白质不稳定系数低于40，为稳定蛋白质，其余17个不稳定系数均高于40，为不稳定蛋白质。脂溶性指数为53.37(OfNAC71)~77.70(OfNAC17-X2)，总平均疏水值为−0.816~−0.530，蛋白质的平均疏水指数均小于0，表明OfNAC均为亲水性蛋白质(表2)。

    Figure 1.  NAM conserved functional domain about NAC

    序列名称氨基酸长度/个相对分子量等电点碱性氨基酸酸性氨基酸不稳定系数脂溶性指数总平均疏水值亚细胞定位
    位置预测值/%
    OfNAC100-134538 986.118.69403640.0661.01−0.574细胞核61.54
    OfNAC5355862 807.474.60518838.8769.37−0.564内质网42.42
    OfNTM1-960668 053.705.63718651.4962.43−0.725细胞核76.92
    OfNAC7330033 768.958.79393438.2169.47−0.754细胞核76.92
    OfNAC4339945 513.665.77435546.3163.26−0.752细胞核69.23
    OfNAC9160967 741.524.98638652.8371.07−0.593细胞核76.92
    OfNAC50-X238943 765.295.29465847.8369.43−0.629叶绿体58.33
    OfNAC5039944 952.525.35466150.4567.69−0.652叶绿体46.15
    OfNAC21/2229633 613.156.54333451.2666.52−0.570细胞核71.43
    OfNAC5632235 946.518.62373439.4864.81−0.725细胞核92.30
    OfNAC17-X257364 828.714.87639140.6077.70−0.530细胞核53.85
    OfNAC1760668 408.124.85609746.7974.62−0.571细胞核30.77
    OfNAC10418621 498.774.59193146.9668.60−0.695细胞核84.61
    OfNAC9232536 813.716.47404229.2771.35−0.579细胞核38.46
    OfNAC72-133938 289.938.64403740.1464.96−0.671细胞核84.61
    OfNAC72-234238 848.468.64423940.0461.64−0.762细胞核100
    OfNAC29-128032 538.567.71363542.9661.96−0.816细胞核52.50
    OfNAC29-227531 330.369.33352633.8161.35−0.741细胞核100
    OfNAC7130334 864.615.42334351.8953.37−0.795细胞核85.71
    OfNAC229634 249.556.09353850.0166.22−0.735细胞核69.23
    OfNAC100-233437 778.806.51384043.1067.96−0.540细胞核85.71
    OfNAC3226330 253.458.45383543.9169.32−0.635细胞核61.54
    　　说明：不稳定系数大于40为不稳定序列，小于40为稳定序列

    Table 2.  Physicochemical properties and secondary structure analysis of OfNAC of O. fragrans

    • 2.2.   桂花OfNAC转录因子结构及亚细胞定位分析

  • 使用DNAMAN软件对桂花OfNAC转录因子的氨基酸序列进行比对，结果显示(图2)：22个OfNAC的氨基酸序列均具有保守的亚结构域(A~E)。对这5个亚结构域进行分析发现：A区较为保守的氨基酸是甘氨酸(G)、苯丙氨酸(F)、脯氨酸(P)、谷氨酸(E)；B区较为保守的氨基酸是脯氨酸(P)；C区较为保守的氨基酸是苯丙氨酸(F)、精氨酸(R)、色氨酸(W)、甘氨酸(G)；D区较为保守的氨基酸是甘氨酸(G)、赖氨酸(K)、色氨酸(W)；E区没有完全稳定的氨基酸。各保守亚域的保守性由强到弱依次为C、A、D、B、E。对所有OfNAC的氨基酸序列进行保守基序分析发现(图3)：大多数OfNAC均含有较保守的元件，除OfNAC73只有2个保守元件(元件1、元件2)外，其余OfNAC均含有4个保守元件(元件1、元件3、元件4、元件5)。另外，22条OfNAC转录因子中元件7、元件9和元件10出现在一些OfNAC蛋白质的C端，且对应的序列亲缘关系较近。桂花OfNAC蛋白质二级结构分析表明：OfNAC蛋白质的二级结构包括α-螺旋、β-折叠、延伸链和无规则卷曲4种结构，且不同结构占比从大到小依次表现为无规则卷曲、α-螺旋、延伸链、β-折叠(表3)。OfNAC32的无规则卷曲占比最高，为67.30%。此外，OfNAC73、OfNAC92、OfNAC29-2结构中α-螺旋占比均小于延伸链。跨膜结构预测分析表明(图4)：OfNAC17、OfNAC17-X2、OfNAC53、OfNAC91、OfNTM1-9均在C端有1个跨膜结构。亚细胞定位预测分析表明：OfNAC53定位于内质网，OfNAC50-X2和OfNAC50定位于叶绿体，其余OfNAC蛋白质均定位在细胞核中。

    Figure 2.  Sequence alignment of OfNAC transcription factor

    Figure 3.  Evolution analysis of 22 OfNAC transcription factors and analysis of conserved motif

    序列名称二级结构占比序列名称二级结构占比
    α-螺旋/%β-螺旋/%延伸/%无规则卷曲/%α-螺旋/%β-螺旋/%延伸/%无规则卷曲/%
    OfNAC100-115.073.1914.7866.96 OfNAC1724.592.8112.7159.90
    OfNAC5321.333.7614.5260.39OfNAC10418.823.7615.5961.83
    OfNTM1-923.273.149.7463.86OfNAC9214.153.6915.6966.46
    OfNAC7313.003.6720.0063.33OfNAC72-117.993.5416.2262.24
    OfNAC4321.554.2614.2959.90OfNAC72-216.963.5116.0863.45
    OfNAC9120.362.6310.5166.50OfNAC29-13.213.5717.8655.36
    OfNAC50-X231.115.9112.3450.64OfNAC29-215.644.3616.7363.27
    OfNAC5027.825.0110.5356.64OfNAC7116.174.9514.5264.36
    OfNAC21/2217.233.0415.2064.53OfNAC221.624.0511.8262.50
    OfNAC5616.153.1115.8464.91OfNAC100-216.473.5914.0765.87
    OfNAC17-X225.832.9712.9158.29OfNAC3217.112.6612.9367.30

    Table 3.  Secondary structure analysis of OfNAC of O. fragrans

    Figure 4.  Transmembrane structure prediction related to OfNAC protein

    • 2.3.   桂花OfNAC转录因子系统进化树

  • 为了进一步研究OfNAC的进化关系，对桂花、拟南芥和水稻的NAC蛋白构建系统进化树。结果表明：22个桂花OfNAC与拟南芥和水稻的NAC蛋白质共划分为11个亚族(图5)，除a、b、c、e、h亚族外，其他亚族均有OfNAC分布。其中i亚族桂花NAC蛋白质较多，有8个OfNAC蛋白质。

    Figure 5.  Phylogenetic tree of OfNAC, A. thaliana and O. sativa

    • 2.4.   桂花OfNAC转录因子在不同花开放时期的表达分析

  • 为了研究OfNAC在桂花花开放进程中的表达模式，对其进行荧光定量表达分析(图6)。OfNAC100-2、OfNAC43、OfNAC73相对表达量在铃梗期(S4)出现表达高峰，在此之后相对表达量大幅度降低，其中OfNAC43只在铃梗期(S4)相对表达量最高，其余时期相对表达量很低；OfNAC56从铃梗期开始呈现上升趋势，并在盛花期(S6)到达顶峰；OfNAC71、OfNAC29-1、OfNAC21/22从起始期(S1)缓慢上升，在顶壳期 (S3)到达最高，随后急剧下降；OfNAC29-2在圆珠期(S2)相对表达量达到最大，之后在铃梗期(S4)相对表达达到最低。OfNAC在花开放不同阶段的表达情况说明：OfNAC100-2、OfNAC43、OfNAC73、OfNAC71、OfNAC29-1、OfNAC21/22、OfNAC29-2可能参与花开放的调控，其中又以OfNAC43和OfNAC29-2关联最为紧密。

    Figure 6.  Expression results of 22 OfNAC genes in different flower opening periods

    OfNAC50-X2、OfNAC50、OfNAC17-X2整体呈现上升趋势，并在初开期(S5)达到最高；OfNAC100-1、OfNAC92、OfNAC72-1、OfNAC91、OfNTM1-9、OfNAC53在起始期(S1)表达量较高，整体呈下降趋势；OfNAC104、OfNAC32、OfNAC2、OfNAC17、OfNAC72-2均呈现先下调后上升的表达趋势，到盛花期(S6)相对表达量较高。这说明，这些基因成员可能不参与桂花花开放的调控。

3.   讨论

  • NAC转录因子最初在矮牵牛Petunia hybrida中发现[21]。在模式植物拟南芥、水稻以及园艺作物中的研究较多。在拟南芥、水稻、黄瓜Cucumis sativus、紫花苜蓿Medicago sativa中分别有151、117、91、113个NAC基因被鉴定[22-24]。目前关于桂花OfNAC转录因子的报道较少。通过桂花OfNAC转录因子的鉴定与分析，可为进一步了解桂花OfNAC的生物学功能及其在花开放过程中的作用奠定基础。

    本研究通过转录组测序鉴定出22条开放阅读框完整的OfNAC转录因子，对它们进行跨膜结构预测发现：OfNAC17、OfNAC17-X2、OfNAC53、OfNAC91、OfNTM1-9这5个转录因子均在C端有1个跨膜结构，推测它们可能是NAC膜结合转录因子。大多数OfNAC蛋白质预测定位在细胞核中的可能性较高，因此推测，OfNAC转录因子可能大多依旧在细胞核内发挥功能。目前，已经在模式植物中发现NAC膜结合转录因子，如拟南芥和水稻NAC基因组中已发现分别有18和6个膜结合NAC转录因子[25-26]，其中拟南芥膜结合转录因子NTM1调节细胞分裂素信号传导而参与细胞分裂；NTL8调节拟南芥开花时间，过表达转基因植株出现晚花现象；ANAC089被确定为另一个膜结合转录因子，也有调控拟南芥开花时间的功能[27-28]。由此推测，桂花OfNAC17、OfNAC17-X2、OfNAC53、OfNAC91、OfNTM1-9可能也具有调控开花时间的功能。

    桂花22个OfNAC与拟南芥和水稻NAC蛋白质共同划分为11个亚族。根据亲缘关系推测相关OfNAC基因的功能，如OfNAC100-1、OfNAC100-2与拟南芥At5G6140.1、At5G7680.1、At5G39610.1、At3G29035.1亲缘性较高，其中At5G39610.1(ANAC92/ATNAC2)存在于部分或完全开放的花的花器官中，且参与叶和花的衰老过程[29]。在月季中，RhNAC100是At5G39610.1(ANAC92/ATNAC2)的同源基因，乙烯通过微调microRNA164-RhNAC100模块来调节细胞的扩张从而影响月季花瓣的扩展[30]，OfNAC100-1和OfNAC100-2与拟南芥At5G39610.1亲缘关系较近，所以推测OfNAC100-1与OfNAC100-2可能有调节花瓣扩展的相关功能。OfNTM1-9与At1G33060.1同源性较高，且At1G33060.1是膜结合转录因子，在拟南芥各个器官均有表达，影响叶片衰老[25]，推测OfNTM1-9可能作为NAC膜结合转录因子参与细胞衰老。

    花开放的主要原因是花瓣细胞的扩展[11-13]，对OfNAC基因在桂花花开放时期的荧光定量表达分析表明：OfNAC在桂花花开放过程中发挥重要作用，且不同的OfNAC在此过程中表达模式有差异。OfNAC71、OfNAC29-1、OfNAC21/22在顶壳期相对表达到达最大。罗云等[31]发现：OfEXPA2和OfEXLA1在桂花花开放的顶壳期相对表达急剧增加并到达顶峰，推测此时期的高丰度表达是后续花开放的基础。OfNAC71、OfNAC29-1、OfNAC21/22与OfEXPA2和OfEXLA1表达模式相似，推测它们参与花开放进程。OfEXPA4在花芽萌发期至圆珠期表达量较低，而顶壳期开始急剧上升，铃梗期表达量达到最大。罗云等[31]推测：OfEXPA4是参与花瓣扩展的关键基因，OfNAC100-2、OfNAC43、OfNAC73相对表达量也在铃梗期出现峰值，推测它们亦作为关键基因参与桂花花瓣扩展。对月季的研究发现：RhNAC2和RhEXPA4参与月季花瓣细胞扩展，且RhEXPA4的表达可能受到RhNAC2的调控[32]，拟南芥中NAC25和NAC1L被确定是EXPA2(EXPANSIN2)的上游调控因子[33]。以此推测，NAC转录因子可能与EXP的启动子结合，是EXP的上游调控因子，则OfNAC29-2在圆珠期相对表达最大，可能是作为OfEXPA2和OfEXLA1调控因子调节OfEXP影响花瓣细胞扩张从而为后续花瓣展开奠定基础，所以OfNAC100-2、OfNAC43、OfNAC73、OfNAC71、OfNAC29-1、OfNAC21/22、OfNAC29-2可能是参与桂花花开放的关键基因。由于OfNAC43、OfNAC29-2相对表达差异明显，推测它们与花开放关系更为紧密，但这些关键基因在桂花开花中的具体作用还需要进一步研究。

4.   结论

  • 本研究从桂花品种‘堰虹桂’转录组数据中筛选得到22条OfNAC序列，生物信息学分析结果表明：22条OfNAC转录因子氨基酸序列含有5个保守的亚结构域(A~E)，其保守性由强到弱依次为C、A、D、B、E；亚细胞定位及跨膜结构预测表明：OfNAC17、OfNAC17-X2、OfNAC53、OfNAC91、OfNTM1-9是膜结合转录因子，且大多数 OfNAC 定位在细胞核。实时荧光定量PCR分析表明：在桂花花开放进程中，22个OfNAC基因表达模式不同，其中OfNAC100-2、OfNAC43、OfNAC73相对表达量在铃梗期(S4)达到顶峰，在此之后相对表达降低。OfNAC43在铃梗期骤然升高，并且在此时期相对表达最大；OfNAC71、OfNAC29-1、OfNAC21/22从起始期呈缓慢上升趋势，在顶壳期到达最高，随后整体呈现下降趋势；OfNAC29-2在圆珠期相对表达量陡然上升，在铃梗期相对表达最低，推测OfNAC100-2、OfNAC43、OfNAC73、OfNAC71、OfNAC29-1、OfNAC21/22、OfNAC29-2等成员极有可能参与调控桂花的花开放。

Reference (33)

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