Volume 40 Issue 3
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HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo. Cloning and expression analysis of OfABFs gene in Osmanthus fragrans[J]. Journal of Zhejiang A&F University, 2023, 40(3): 481-491. doi: 10.11833/j.issn.2095-0756.20220264
Citation: HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo. Cloning and expression analysis of OfABFs gene in Osmanthus fragrans[J]. Journal of Zhejiang A&F University, 2023, 40(3): 481-491. doi: 10.11833/j.issn.2095-0756.20220264
shu

Cloning and expression analysis of OfABFs gene in Osmanthus fragrans

doi: 10.11833/j.issn.2095-0756.20220264
  • 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 and Architecture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

  • Received Date: 2022-03-31
  • Accepted Date: 2022-07-27
  • Rev Recd Date: 2022-07-27
  • Publish Date: 2023-05-20
  •   Objective  The aim of this study is to explore the expression patterns of OfABF gene family members in different tissues and flower opening process of Osmanthus fragrans ‘Yanhonggui’, and screen the key members involved in regulating flower opening.   Method  The related OfABFs gene sequence was screened from the O. fragrans genome database for the full-length sequence and bioinformatics analysis. The spatio-temporal expression mode of OfABFs gene in different tissues and flower opening process of O. fragrans ‘Yanhonggui’ were analyzed by fluorescence quantitative PCR.   Result  5 OfABFs sequences were screened. Bioinformatics analysis showed that O. fragrans OfABFs gene family protein was hydrophilic and unstable, and had no signal peptide. It was predicted that the protein secondary and tertiary structure had similar characteristics and irregular curl, and α-spiral was the main component of its structure. The 5 OfABFs transcription factors all contained the conserved domain of bZIP transcription factor family. Subcellular localization prediction showed that 5 OfABFs coding proteins were located in the nucleus. The results of fluorescence quantitative PCR showed that the expressions of OfABF1, OfABF2, OfABF3, OfABF4 and OfABF6 were tissue-specific, among which OfABF1, OfABF4 and OfABF6 were highly expressed in flowers. OfABF1 was transcriptionally activated in the apical shell stage, and its expression increased significantly. Although the expression level decreased after boll stem stage, it still maintained a high level. The expression of OfABF2 peaked in the apical shell stage, and the relative expression level was significantly higher than that in other stages. The relative expression levels of OfABF3, OfABF4 and OfABF6 were the highest in flower senescence stage.  Conclusion  OfABF1 and OfABF2 may be related to the blooming process of O. fragrans, while OfABF3, OfABF4 and OfABF6 are more likely to participate in the regulation of O. fragrans flower senescence. [Ch, 10 fig. 4 tab. 32 ref.]
  • [1] LI Li, PANG Tianhong, FU Jianxin, ZHANG Chao.  Screening and identification of ERF transcription factors of B2 subgroup involved in regulating lycopene β-cyclase gene LCYB in Osmanthus fragrans . Journal of Zhejiang A&F University, 2024, 41(): 1-8. doi: 10.11833/j.issn.2095-0756.20240316
    [2] ZHUO Juan, HOU Dan, LIN Xinchun.  Cloning and expression analysis of PhebHLH6 gene from Phyllostachys edulis . Journal of Zhejiang A&F University, 2023, 40(4): 731-737. doi: 10.11833/j.issn.2095-0756.20220553
    [3] LAN Zhixin, HOU Dan, WU Aimin, LIN Xinchun.  Cloning and expression analysis of PeCIGRs gene from Phyllostachys edulis . Journal of Zhejiang A&F University, 2023, 40(5): 982-990. doi: 10.11833/j.issn.2095-0756.20220761
    [4] 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
    [5] 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
    [6] 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
    [7] 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
    [8] MIAO Yunfeng, ZHOU Dan, DONG Bin, ZHAO Hongbo.  Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage . Journal of Zhejiang A&F University, 2021, 38(3): 433-444. doi: 10.11833/j.issn.2095-0756.20200474
    [9] 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
    [10] 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
    [11] 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
    [12] 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
    [13] 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
    [14] 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
    [15] 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
    [16] 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
    [17] 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
    [18] 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.
    [19] ZHOU Yuan, YAO Chong-huai, WANG Cai-yun.  Study on selecting cut-flower cultivars of Osmanthus fragrans . Journal of Zhejiang A&F University, 2006, 23(6): 660-663.
    [20] WU Guang-hong, HU Shao-qing, XUAN Zi-can, XIANG Qi-bai.  Standard of classification and application of sweet Osmanthus . Journal of Zhejiang A&F University, 2004, 21(3): 281-284.
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Cloning and expression analysis of OfABFs gene in Osmanthus fragrans

doi: 10.11833/j.issn.2095-0756.20220264
  • 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 and Architecture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • Received Date: 2022-03-31
  • Accepted Date: 2022-07-27
  • Rev Recd Date: 2022-07-27
  • Publish Date: 2023-05-20

Abstract:   Objective  The aim of this study is to explore the expression patterns of OfABF gene family members in different tissues and flower opening process of Osmanthus fragrans ‘Yanhonggui’, and screen the key members involved in regulating flower opening.   Method  The related OfABFs gene sequence was screened from the O. fragrans genome database for the full-length sequence and bioinformatics analysis. The spatio-temporal expression mode of OfABFs gene in different tissues and flower opening process of O. fragrans ‘Yanhonggui’ were analyzed by fluorescence quantitative PCR.   Result  5 OfABFs sequences were screened. Bioinformatics analysis showed that O. fragrans OfABFs gene family protein was hydrophilic and unstable, and had no signal peptide. It was predicted that the protein secondary and tertiary structure had similar characteristics and irregular curl, and α-spiral was the main component of its structure. The 5 OfABFs transcription factors all contained the conserved domain of bZIP transcription factor family. Subcellular localization prediction showed that 5 OfABFs coding proteins were located in the nucleus. The results of fluorescence quantitative PCR showed that the expressions of OfABF1, OfABF2, OfABF3, OfABF4 and OfABF6 were tissue-specific, among which OfABF1, OfABF4 and OfABF6 were highly expressed in flowers. OfABF1 was transcriptionally activated in the apical shell stage, and its expression increased significantly. Although the expression level decreased after boll stem stage, it still maintained a high level. The expression of OfABF2 peaked in the apical shell stage, and the relative expression level was significantly higher than that in other stages. The relative expression levels of OfABF3, OfABF4 and OfABF6 were the highest in flower senescence stage.  Conclusion  OfABF1 and OfABF2 may be related to the blooming process of O. fragrans, while OfABF3, OfABF4 and OfABF6 are more likely to participate in the regulation of O. fragrans flower senescence. [Ch, 10 fig. 4 tab. 32 ref.]

HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo. Cloning and expression analysis of OfABFs gene in Osmanthus fragrans[J]. Journal of Zhejiang A&F University, 2023, 40(3): 481-491. doi: 10.11833/j.issn.2095-0756.20220264
Citation: HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo. Cloning and expression analysis of OfABFs gene in Osmanthus fragrans[J]. Journal of Zhejiang A&F University, 2023, 40(3): 481-491. doi: 10.11833/j.issn.2095-0756.20220264
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  • bZIP转录因子家族是植物中最大、最保守且功能最多样的基因家族之一，广泛参与调节多种生物学过程[1-2]。ABF (ABRE binding factor)转录因子属于bZIP基因家族的A亚族[3]，特异存在于植物中，ABFs是ABA信号转导途径的关键基因，能够特异识别ABA响应元件(ABA-responsive element，AREB)，并通过与AREB结合参与下游响应ABA信号靶基因的调控[4]。月季Rosa chinensis中RhABF2基因能够与含有ABRE元件的RhFer1启动子结合，共同调节铁平衡参与调控花瓣衰老过程[5]；睡莲Nymphaea colorata bZIP家族A亚组的11个成员的启动子区域有大量ABA响应元件，在ABA信号转导中具有重要作用[6]；菊花Chrysanthemum×morifolium中锌指蛋白BBX19和ABF3互作，通过诱导ABA响应基因表达来调控菊花的耐旱性[7]。桂花Osmanthus fragrans属于木犀科Oleaceae木犀属Osmanthus常绿乔木，是中国传统十大名花之一。温度、湿度是影响桂花花开放的重要因素。为了解ABFs转录因子在桂花中的作用，本研究以秋桂品种‘堰虹桂’O. fragrans‘Yanhonggui’为材料，在桂花基因组中筛选出相关OfABFs基因序列，通过实时荧光定量PCR(RT-qPCR)分析OfABFs基因在‘堰虹桂’不同组织和花开放进程中的时空表达模式，为后续阐明ABFs基因家族各成员的生物学功能提供理论支持。

    • 1.   材料与方法

      1.1.   材料

    • 以浙江农林大学桂花资源圃的桂花品种‘堰虹桂’盆栽植物为材料，选取株龄相同、长势一致的健康植株，分别于起始1期(圆珠期前3 d，B1)，起始2期(圆珠期前2 d，B2)，起始3期(圆珠期前1 d，B3)，圆珠期(S1)，顶壳期(S2)，铃梗期(S3)，初开期(S4)，盛开期(S5)，盛开末期(S6)，衰老期(S7)共10个时期进行取样(图1)[8]。同样，取桂花不同组织的样品，包括根、新叶、新枝、老叶、老枝、盛开期的花朵，每个时期样品采集3份，液氮速冻后储藏于−80 ℃冰箱。

      Figure 1.  Phenotypic of O. fragrans in different periods

      • 1.2.   方法

      1.2.1.   桂花OfABFs转录因子鉴定及简单克隆

    • 桂花核苷酸序列来源于桂花基因组数据库[9]，从拟南芥Arabidopsis thaliana信息资源(TAIR)数据库(http://www.arabidopsis.org/)和PlantTFDB (http://planttfdb.cbi.pku.edu.cn/blast.php)数据库中获得拟南芥的ABFs氨基酸序列，以此作为查询对象，使用NCBI-Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi)在桂花基因组蛋白数据库中进行同源比对，确定桂花中的ABFs基因序列。利用NCBI-CDD (https://www.ncbi. nlm.nih.gov/cdd/)对OfABFs进行保守功能域预测。使用SMART (http://smart.embl-heidelberg.de/)分析OfABFs蛋白结构域。

      RNA的提取使用RNA prep Pure Plant Plus Kit提取试剂盒[天根生化科技(北京)有限公司]，使用核酸分析仪(赛默飞世尔科技公司)对RNA的纯度与浓度进行检测，并用质量分数为1%的琼脂糖凝胶电泳检测RNA的完整性。使用HiScriptⅡ Q Select RT SuperMix for qPCR(+gDNA wiper)试剂盒(南京诺维赞生物科技股份公司)，对花开放不同时期的cDNA进行合成。

      运用Primer 5.0进行简单克隆引物设计(表1)，以花开放时期的花芽cDNA为模板进行PCR扩增。PCR反应体系(20 μL)：上下游引物(10 μmol·L−1)各1 μL、cDNA模板1 μL、GreenTaqMix10 μL和ddH2O 7 μL。PCR扩增程序：预变性95 ℃，3 min；变性95 ℃，15 s，退火温度以各自引物最佳温度为准，15 s，延伸72 ℃，3 min，35个循环；72 ℃延伸5 min。将PCR反应产物进行1%琼脂糖凝胶电泳检测，目的片段回收纯化后与pMD-18T载体连接，连接产物转化大肠埃希菌Escherichia coli DH5α感受态细胞，PCR初步鉴定阳性克隆后送杭州有康生物科技有限公司测序，经比对后确定序列。

      基因名称引物序列(5′→3′)产物长度/bp引物温度/℃
      OfABF1 F: ATGGGGAGTAATTTGAACTTCAAGAATCCG
      R: TTACCATGGACCTGTCTGAGTTCGTCTCA      		 1 557 54
      OfABF2 F: ATGGGGTCATACATGAACTTCAAG
      R: CTACCAAGGTCCCGTCAGCA      		 1 353 58
      OfABF3 F: ATGGGGAGTAATTTTAACTTCAAGAATTTTGG
      R: TTACCATGGCCCTGTCTGTGTACGTC      		 1 455 56
      OfABF4 F: ATGTGGAGTAATTTTAACTTCAAGAATGT
      R: TCACCATGGCCCCGTTTG      		 1 572 56
      OfABF6 F: ATGAACTTTGGGCCGGACACATCA
      R: CTACCAAGGTCCTGTCAGCGTCCTTCT      		 1 329 45

      Table 1.  Primers for Cloning of OfABFs in O. fragrans

      • 1.2.2.   桂花OfABFs基因染色体定位分析

    • 利用TBtools[10]从桂花基因组Gff文件中提取桂花OfABFs基因的染色体位置信息，通过在线分析工具TBtools进行绘图可视化。

      • 1.2.3.   桂花OfABFs转录因子结构分析

    • 用DNAMAN对已筛选的桂花OfABFs转录因子进行氨基酸序列比对，分析其保守结构域和氨基酸序列同源性；通过在线分析软件MEME 5.1.0分析ABFs的保守基序(http://meme-suite.org/tools/meme)，并用TBtools对其进行绘图和可视化。对桂花OfABFs二级结构特征采用在线软件SOPMA (https://npsaprabi.ibcp.fr/cgi-bin/npsa-sopma.html)进行预测分析；使用在线网站Swiss-Model (https://swissmodel.expasy.org/)构建OfABFs蛋白的三级结构模型。

      • 1.2.4.   桂花OfABFs转录因子蛋白质理化性质分析及亚细胞定位预测

    • 运用ExPASy在线软件(http://web.expasy.org/protparam/)对OfABFs进行蛋白质、分子量、理论电点、酸碱性等预测分析和编码；利用SignalP 4.1 Server分析桂花OfABFs蛋白有无信号肽。亚细胞定位预测通过在线工具wolfpsort (https:// wolfpsort.hgc.jp/)完成。

      • 1.2.5.   桂花OfABFs转录因子系统进化树构建

    • 从美国国家生物信息中心(NCBI) (www.ncbi.nlm.nih.gov)中获取拟南芥、烟草Nicotiana tabacum、水稻Oryza sativa、大麦Hordeum vulgare、小麦Triticum aestivum、番茄Solanum lycopersicum、油橄榄Olea europaea的ABFs氨基酸序列，使用MEGA 11.0软件进行同源聚类，建立系统发育树并选择Bootstrap (1 000次)评估检测系统进化树。

      • 1.2.6.   桂花OfABFs转录因子的表达分析

    • 利用Primer Premier 5.0对5个OfABFs基因进行荧光定量特异性引物设计(表2)，桂花OfACT作为内参基因[11]，以‘堰虹桂’不同组织及不同花开放时期的花芽cDNA为模板进行RT-qPCR扩增。反应体系为20 μL：cDNA模板2 μL，2×Taq Pro Universal SYBR qPCR Master Mix 10 μL，上下游引物(10 μmol·L−1)各0.4 μL，ddH2O 7.2 μL。RT-qPCR程序：95 ℃ 30 s；95 ℃ 5 s，60 ℃ 30 s，共40个循环；然后以95 ℃持续15 s，60 ℃持续1 min，95 ℃持续15 s作为熔解曲线分析程序。每个样品设置3次重复。

      基因名称用于荧光定量的引物序列(5′→3′)
      OfABF1F: GGAAGGATGGGAGTGGTAGTGR: TTAGGCTTTCCAGCCAACTGA
      OfABF2F: TGGTGCAAAAGATGGAAGTGCR: ATTTGCTGTCCAAGAGGGCTG
      OfABF3F: GAGTATGGTGGCGGGAAGAATR: GGTAGGCTTTCCAGCAAACTG
      OfABF4F: TCTGGAGAGAGAGGTGGAATGAR: CAAACTGGGCATCTTCCCTTAC
      OfABF6F: GAAGGGGTAGTGGAGGATCAAR: ACCACCATAAATTCCACCAGC

      Table 2.  Specific primers for RT-qPCR of OfABFs in O. fragrans

    2.   结果与分析

      2.1.   桂花OfABFs转录因子鉴定及克隆

    • 通过对桂花基因组数据分析，得到5条OfABFs基因序列，依次命名为OfABF1、OfABF2、OfABF3、OfABF4、OfABF6。用‘堰虹桂’花开放时期的花芽(混样) cDNA为模板进行PCR扩增(图2)。结果显示：OfABFs的开放阅读框(ORF)长度为1329~1572 bp，编码442~523个氨基酸；其中基因OfABF4氨基酸序列最长，编码523个氨基酸；基因OfABF6的氨基酸序列最短，编码442个氨基酸。

      Figure 2.  PCR amplification of OfABFs of O. fragrans

      • 2.2.   桂花OfABFs基因家族染色体定位分析

    • 根据‘堰虹桂’基因组Gff文件定位5个OfABFs基因。结果显示：5个桂花OfABFs基因分布在5条染色体上，且各个基因在染色体上分布的数量相等，1号、6号、11号、12号和22号染色体上各分布1个OfABF基因(图3)。

      Figure 3.  Chromosome location analysis of OfABFs genes in O. fragrans　　　　　　　

      • 2.3.   桂花OfABFs转录因子结构分析

    • 利用SMART在线分析软件对桂花OfABFs进行功能结构域预测。结果显示：5个桂花ABFs均含有BRLZ结构域(图4)。BRLZ结构域是bZIP家族特有的保守域，由碱性区和亮氨酸拉链区组成，能够识别特定的DNA序列，以二聚体形式发挥功能[12]。通过DNAMAN软件，将OfABFs氨基酸序列进行比对(图5)。结果显示：OfABF3和OfABF4同源性最高，OfABF1和OfABF6同源性最低；5个C端均含有能被激酶识别的保守序列RXXS/T。

      Figure 4.  Functional domains of OfABFs in O. fragrans

      Figure 5.  Sequence alignment of OfABFs transcription factor in O. fragrans

      对桂花ABFs氨基酸序列进行保守基序分析发现(图6)：OfABFs蛋白结构域中含有10个蛋白基序(Motif 1~Motif 10)，其中Motif 7在除OfABF4外的4个蛋白中均被鉴定到，Motif 8在除OfABF6外的4个蛋白中均被鉴定到，其余8个Motif高度保守，是OfABFs核心结构域的组成部分。

      Figure 6.  Conserved motifs OfABFs proteins in O. fragrans

      桂花OfABFs蛋白的二级结构预测表明：桂花OfABFs蛋白的二级结构均含有α-螺旋、β-折叠、延伸链、无规则卷曲，不同结构占比从大到小依次为无规则卷曲、α-螺旋、延伸链、β-折叠。其中，β-折叠占比最小，均在5%以下；延伸链占比为10%~13%；α-螺旋和无规则卷曲占二级结构总量的83%，为OfABFs蛋白二级结构的主要构成元件(表3)。

      基因名称α-螺旋/％β-折叠/％延伸/％无规则卷曲/％基因名称α-螺旋/％β-折叠/％延伸/％无规则卷曲/％
      OfABF127.804.0513.3254.83OfABF428.494.7811.8554.88
      OfABF228.671.7812.2257.33OfABF631.673.1710.8654.30
      OfABF330.993.1011.1654.75

      Table 3.  Secondary structure analysis of protein OfABFs in O. fragrans

      桂花OfABFs蛋白三级结构分析显示(图7)：OfABFs蛋白三级结构均含丰富的α-螺旋和无规则卷曲，这与其二级结构预测结果一致。含无规则卷曲较多的蛋白稳定性要低于含α-螺旋、β-折叠多的蛋白，在OfABFs蛋白中无规则卷曲比例均高于50%，为不稳定蛋白，这与其理化性质分析预测结果一致(表3)。

      Figure 7.  Prediction of tertiary structure of the OfABFs protein in O. fragrans

      • 2.4.   桂花OfABFs转录因子蛋白理化性质分析及亚细胞定位预测

    • 对桂花ABFs家族蛋白理化性质的分析结果表明：桂花氨基酸长度为450~523 个，相对分子质量为25.7~56.2 kDa，理论等电点为4.77~10.03，编码氨基酸序列最长的的是OfABF4，其相对分子质量最大，为56.2 kDa；编码氨基酸序列最短的是OfABF2，其相对分子质量也最小，为25.6 kDa。5个OfABFs基因成员的不稳定系数为46.76~54.43。如果将不稳定系数＞40的蛋白判断为不稳定蛋白，那么5个基因的蛋白不稳定系数均高于40，均为不稳定蛋白。桂花ABFs蛋白脂溶性系数为65.29~78.07，亲水性总平均系数为−0.659~−0.482，均小于0，为亲水性蛋白。信号肽分析结果显示：桂花ABFs编码蛋白均不含有信号肽，表明桂花ABFs家族蛋白均非分泌蛋白。亚细胞定位预测发现(表4)：5个编码蛋白均定位在细胞核，这与其作为转录因子的生物学功能相吻合。

      基因名称序列名称氨基酸
      长度/个      		相对分
      子量/kDa      		等电点酸性氨
      基酸/个      		碱性氨
      基酸/个      		不稳定
      系数      		脂溶性
      系数      		亲水性平
      均系数      		亚细胞定
      位预测      		信号肽
      OfABF1gui0017720.151855.69.24495646.7678.07−0.482细胞核无
      OfABF2gui0167370.145025.74.77251646.9571.04−0.536细胞核无
      OfABF3gui0180650.148451.99.35435052.8067.77−0.615细胞核无
      OfABF4gui0305040.152356.25.38605250.6168.66−0.659细胞核无
      OfABF6gui0100630.144246.810.03355054.4365.29−0.593细胞核无

      Table 4.  Physicochemical properties of OfABFs proteins of O. fragrans

      • 2.5.   桂花OfABFs转录因子多序列比对和进化分析

    • 构建系统进化树进一步研究OfABFs的进化关系，结果表明(图8)：8个物种的25条氨基酸序列可分为5个分支(分支Ⅰ~Ⅴ)，其中OfABFs分布在2个分支中。OfABF1、OfABF3和OfABF4均分布在分支Ⅰ，该分支包括油橄榄OeTRAB1、烟草NtABF2、马铃薯Solanum tuberosum StABF、马铃薯StABF1、番茄SlAREB1和拟南芥AtABF2，说明OfABF1、OfABF3和OfABF4在进化过程中与这些物种亲缘关系较近；OfABF2和OfABF6与番茄SlABF4聚为分支Ⅱ。

      Figure 8.  Studies on the relationship between O. fragrans OfABFs and the evolution of ABFs transcription factors in different species

      • 2.6.   桂花OfABFs转录因子在不同组织和花开放时期的表达分析

    • 利用荧光定量分析5个基因在根、嫩枝、老枝、新叶、老叶等不同组织中的表达情况。定量结果(图9)显示：OfABF1在花中表达量最高，在老叶和新叶中表达没有显著差异，在新枝和老枝中相对表达量最低；OfABF2在老叶中表达最高，其次为新叶，在花中表达略高于枝；OfABF3在叶中表达水平相对其他组织高，在花和枝中表达较低。OfABF4在花中相对表达量最高，其次是新叶和老叶，老枝中表达量最低，OfABF6的相对表达量在花中最高，在其他组织中的表达量均较低。OfABF1、OfABF4、OfABF6在所有组织中均有表达，但在花中的相对表达水平最高，尤其是OfABF6。

      Figure 9.  Expression results of 5 OfABFs genes in different tissues

      由图10可见：起始1期(B1)到起始3期(B3)花芽芽体肥大并延伸。圆珠期(S1)到盛开末期(S6)为花开放时期，OfABF1在顶壳期(S2)被转录激活，表达显著升高，铃梗期(S3)后表达水平虽呈下降趋势，但仍保持较高水平；OfABF2在起始1期(B1)到圆珠期(S1)时期几乎不表达，在顶壳期(S2)的表达量到达峰值，且相对表达水平显著高于其他时期；OfABF3和OfABF4在花朵衰老期(S7)的相对表达量最高，在盛开末期(S6)的表达量其次，圆珠期(S1)至盛开期(S5)表达趋势变化不显著；OfABF6的相对表达量在花朵衰老期(S7)最高。由此说明：OfABF1和OfABF2可能与桂花花开放进程有关，OfABF3、OfABF4和OfABF6可能参与桂花花衰老的调控。

      Figure 10.  Express an results of 5 OfABFs genes in different flower opening periods

    3.   讨论

    • ABF转录因子在模式植物拟南芥、烟草以及园艺作物中的研究较多。ABFs已在拟南芥、烟草、杨树Populus中被鉴定[12-15]。LI等[16]对29种陆地植物中的95种ABFs蛋白长度、分子量进行分析，发现ABF的氨基酸长度为254~485 个；分子量为27.81~52.95 kDa。目前，关于桂花OfABFs的研究较少，通过鉴定与分析桂花OfABF转录因子，可进一步了解桂花OfABFs的生物学功能及其表达分析。

      OfABFs蛋白的二级结构组成与烟草、睡莲、野菊Chrysanthemum indicum、蓝莓Vaccinium corymbosum、大豆Glycine max[6, 17-20]等其他物种中的结构相类似，均以α-螺旋和无规则卷曲为主要构成元件，少量β-折叠和延伸链散布于整个多肽链中。说明在基因变异和进化时，虽然基因外显子的数量上发生变化，但仍具有其保守性，这与杨颖等[2]的研究结果基本一致。基于蛋白行使特定功能依赖其三维结构，本研究使用蛋白三级结构预测软件SWISS-MODEL同源模拟桂花OfABFs蛋白三级结构，结果显示：OfABF2与烟草[17]中的同源蛋白质NtABF1、NtABF2三级结构类似，除OfABF2蛋白外，其余成员蛋白三级结构在空间结构上相似度较高，进一步说明ABF蛋白在进化上较保守，具有对植物生长发育的重要功能。序列比对结果显示：5个OfABFs的C端均具有bZIP转录因子特有的BRLZ结构域，且含有保守序列RXXS/T，推测OfABFs转录因子可通过被磷酸化激活，从而结合下游基因启动子区域的ABRE元件，调控相关下游胁迫响应基因[20-21]。

      系统发育结果显示：桂花ABFs基因家族成员全部分布在以双子叶植物油橄榄、番茄、马铃薯、烟草为主的分支Ⅰ和分支Ⅱ中，无家族成员分布在以单子叶植物小麦、大麦、水稻等为主的分支Ⅲ、分支Ⅳ和分支Ⅴ中。由此推测，桂花ABFs与双子叶植物亲缘关系较近，与单子叶植物的亲缘关系较远。表明在进化过程中，ABF在种属间是高度保守的。

      根据亲缘关系推测桂花OfABFs基因功能，如在分支Ⅰ中，OfABF1、OfABF3、OfABF4与马铃薯StABF、番茄SlAREB1、烟草NtABF2、拟南芥AtAREB1亲缘性较高。在对环境的胁迫应答过程中，番茄SlAREB1的过表达可增加物种干旱和盐胁迫耐受性[22]，诱导有机酸积累的变化并参与其合成的基因编码酶的表达[23]；烟草NtABF2与拟南芥AtAREB1响应非生物胁迫，前者通过与胁迫响应基因启动子区的ABRE元件结合相关基因的转录[16, 23-24]，后者受ABA含量等渗透胁迫诱导，作为关键基因参与ABA信号调控通路以增强物种抗旱性[25]，显著提高ABA依赖性逆境响应基因的表达水平[25-28]；在分支Ⅱ中，OfABF2、OfABF6与番茄SlABF4同源性较高，且SlABF4作为ABA响应因子，ABA通过其对SICOL4产生负调控作用，使其与下游乙烯通路相关的基因互作[29]，调控乙烯的合成和信号转导进而调控番茄果实的成熟进程[29-30]。由此推测：桂花OfABFs可以作为ABA响应因子，参与ABA信号转导，影响植物整个生长发育过程[31]。牡丹Paeonia suffruticosa切花保鲜的研究发现：ABA以诱导乙烯大量合成的方式间接加速牡丹切花的衰老[32]。还有研究发现：在月季花瓣衰老过程中内源ABA含量迅速上升，促进RhABF2的表达量上调，从而推测RhABF2在月季花瓣衰老过程中发挥调节作用[7]。在桂花中OfABF3、OfABF4和OfABF5的相对表达量在衰老期急剧上升，推测在桂花花瓣衰老过程中内源ABA含量上升，OfABF3、OfABF4和OfABF5可能参与调节桂花花瓣的衰老。推测OfABFs基因响应内源ABA，参与桂花衰老的可能性较大。5个OfABFs基因在桂花中的功能值得进一步探讨。

    4.   结论

    • 以桂花品种‘堰虹桂’为材料，在基因组数据库中筛选出相关5条OfABFs基因序列。生物信息学分析得出：桂花ABFs蛋白具有亲水性，较不稳定，无信号肽段。其蛋白二、三级结构预测具有相似特性，无规则卷曲和α-螺旋为其结构组成的主要元件；5条OfABFs基因序列均含有bZIP转录因子家族保守结构域；亚细胞定位预测表明：5条OfABFs编码蛋白均定位在细胞核。荧光定量PCR结果表明：OfABF1、OfABF2、OfABF3、OfABF4和OfABF6在桂花中的表达具有组织特异性，其中OfABF1、OfABF4和OfABF6在花中表达较高。OfABF1在顶壳期(S2)被转录激活，表达显著升高，铃梗期(S3)后表达水平虽呈下降趋势，但仍保持较高水平；OfABF2在顶壳期(S2)表达量达到峰值，且相对表达水平著显高于其他时期；OfABF3、OfABF4和OfABF6在花朵衰老期(S7)的相对表达量最高。推测OfABF1、OfABF2可能与桂花花开放进程有关，而OfABF3、OfABF4和OfABF6响应ABA参与调控花朵衰老的可能性较大。

Reference (32)

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