Volume 40 Issue 3
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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[J]. Journal of Zhejiang A&F University, 2023, 40(3): 465-474. doi: 10.11833/j.issn.2095-0756.20220456
Citation: 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[J]. Journal of Zhejiang A&F University, 2023, 40(3): 465-474. doi: 10.11833/j.issn.2095-0756.20220456
shu

Functional analysis of OfMYB1R47 transcription factor in Osmanthus fragrans during the formation of aromatic volatiles

doi: 10.11833/j.issn.2095-0756.20220456
  • 1.

    College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

  • 2.

    Jiangsu Key Laboratory of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China

  • Received Date: 2022-07-08
  • Accepted Date: 2023-02-13
  • Rev Recd Date: 2023-01-09
  • Publish Date: 2023-05-20
  •   Objective  Fragrance is one of the most important ornamental traits of Osmanthus fragrans. The purpose of this study is to identify the function of OfMYB1R47, a member of the MYB-related gene family of O. fragrans in the formation of aromatic volatiles, so as to provide a new gene node for the study of transcriptional regulation mechanism of aroma synthesis in O. fragrans.   Method  The MYB-related family gene OfMYB1R47 screened from the previous floral transcriptome data was used as the target gene, and O. fragrans ‘Rixianggui’ and Nicotiana benthamiana were used as materials. The characteristics and functions of OfMYB1R47 gene were analyzed by gene sequence and phylogenetic tree, RT-qPCR, subcellular localization, yeast self-activation, as well as the transient overexpression and GC-MS volatile metabolites test in N. benthamiana.   Result  The length of the open reading frame of OfMYB1R47 gene was 1 485 bp, encoding 494 amino acids. Phylogenetic tree analysis showed that OfMYB1R47 had the highest homology gene in Olea europaea subsp. europaea. RT-qPCR analysis showed that the expression pattern of OfMYB1R47 gene increased first, and then decreased with the release of O. fragrans aroma, and the highest expression level was found at the early flowering stage of O. fragrans. Subcellular localization and yeast self-activation tests showed that OfMYB1R47 was mainly distributed in the nucleus and had self-activation activity. Compared with plants transformed into empty vectors, the contents of octanal, β-ionone and other aroma volatiles in the leaves of N. benthamiana with transient over-expression of this gene were significantly changed.   Conclusion  OfMYB1R47 has typical transcription factor characteristics, and its expression pattern is related to the release of O. fragrans, which is involved in the regulation of the synthesis of floral substances such as β-ionone, and can be used as a gene resource for molecular breeding of O. fragrans. [Ch, 7 fig. 2 tab. 39 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] 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
    [4] 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
    [5] 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
    [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] 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
    [12] 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
    [13] 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
    [14] 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
    [15] 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
    [16] 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
    [17] 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.
    [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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Functional analysis of OfMYB1R47 transcription factor in Osmanthus fragrans during the formation of aromatic volatiles

doi: 10.11833/j.issn.2095-0756.20220456
  • 1. College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
  • 2. Jiangsu Key Laboratory of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
  • Received Date: 2022-07-08
  • Accepted Date: 2023-02-13
  • Rev Recd Date: 2023-01-09
  • Publish Date: 2023-05-20

Abstract:   Objective  Fragrance is one of the most important ornamental traits of Osmanthus fragrans. The purpose of this study is to identify the function of OfMYB1R47, a member of the MYB-related gene family of O. fragrans in the formation of aromatic volatiles, so as to provide a new gene node for the study of transcriptional regulation mechanism of aroma synthesis in O. fragrans.   Method  The MYB-related family gene OfMYB1R47 screened from the previous floral transcriptome data was used as the target gene, and O. fragrans ‘Rixianggui’ and Nicotiana benthamiana were used as materials. The characteristics and functions of OfMYB1R47 gene were analyzed by gene sequence and phylogenetic tree, RT-qPCR, subcellular localization, yeast self-activation, as well as the transient overexpression and GC-MS volatile metabolites test in N. benthamiana.   Result  The length of the open reading frame of OfMYB1R47 gene was 1 485 bp, encoding 494 amino acids. Phylogenetic tree analysis showed that OfMYB1R47 had the highest homology gene in Olea europaea subsp. europaea. RT-qPCR analysis showed that the expression pattern of OfMYB1R47 gene increased first, and then decreased with the release of O. fragrans aroma, and the highest expression level was found at the early flowering stage of O. fragrans. Subcellular localization and yeast self-activation tests showed that OfMYB1R47 was mainly distributed in the nucleus and had self-activation activity. Compared with plants transformed into empty vectors, the contents of octanal, β-ionone and other aroma volatiles in the leaves of N. benthamiana with transient over-expression of this gene were significantly changed.   Conclusion  OfMYB1R47 has typical transcription factor characteristics, and its expression pattern is related to the release of O. fragrans, which is involved in the regulation of the synthesis of floral substances such as β-ionone, and can be used as a gene resource for molecular breeding of O. fragrans. [Ch, 7 fig. 2 tab. 39 ref.]

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[J]. Journal of Zhejiang A&F University, 2023, 40(3): 465-474. doi: 10.11833/j.issn.2095-0756.20220456
Citation: 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[J]. Journal of Zhejiang A&F University, 2023, 40(3): 465-474. doi: 10.11833/j.issn.2095-0756.20220456
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  • 桂花Osmanthus fragrans是木犀科Oleaceae木犀属Osmanthus常绿木本植物，具有花朵形小繁多、花香迷人的特点，是一种应用广泛的优良园林树种[1−2]，并在食品和香水等商品中具有较高的应用价值[3]。有研究表明：桂花花香挥发物依据化学结构可划分为6个类别，即萜烯类、酯类、烷烃类、酮类、醇类和醛类[4−6]。也有研究通过气相色谱-嗅觉测量法(GC-Olfactometry)分析，共鉴定出11种对桂花香气形成具有贡献的萜类活性物质[7]；同时，在‘日香桂’O. fragrans‘Rixianggui’中也发现芳樟醇的含量与花香强度的变化高度相关[8]；此外，β-紫罗兰酮、芳樟醇及其衍生物等关键花香物质在不同桂花品种中相对含量的改变还是导致桂花品种间花香感官品质差异的重要原因[9]。

    花香物质合成的转录调控是一个复杂的过程，其中转录因子对植物次生代谢物合成途径中结构基因的转录调控被认为是植物挥发性芳香物质生物合成的重要环节[10−11]。在桂花中，转录因子OfWRKY3和OfERF61可以直接调控催化产生挥发性芳香物紫罗兰酮的类胡萝卜素裂解双加氧酶4基因(OfCCD4)的表达，并认为它们可能参与了调节酮类花香物质紫罗兰酮的合成[12−13]。此外，有研究通过气相色谱-质谱联用法(GC-MS)分析桂花花香挥发物，并结合桂花全基因组WRKY和MYB转录因子的鉴定，获得了一批与桂花萜烯类花香物质合成相关的候选基因[3, 14]。

    根据MYB结构域的数量，MYB转录因子分为4个亚家族：1R-MYB、R2R3-MYB、3R(R1R2R3)-MYB和4R-MYB蛋白[15]。其中，R2R3-MYB已被证实与多种生物过程有关，其在调控挥发性香气物质的合成与释放以及增强非生物胁迫能力等方面均具有重要的作用[15−18]。1R-MYB蛋白统称为MYB-related蛋白，是最大的MYB基因亚家族，在某些物种中MYB-related成员的数量甚至是R2R3-MYB的2倍[18−19]。目前，在R2R3-MYB亚家族中已鉴定出多个与花香挥发物合成有关的成员，如在花香模式植物矮牵牛Petunia hybrida中已鉴定出4个通过调控苯基/苯丙烷代谢途径中结构基因的表达，来参与花香物质合成的R2R3型MYB转录因子[20−21]；此外，在玫瑰Rosa rugosa[22]、留兰香Mentha spicata[23−24]、姜花Hedychium coronarium[25]、百合Lilium brownii var. viridulum[26]等观赏植物中，R2R3-MYB转录因子也会参与调控植物花香物质的合成，但关于MYB-related亚家族成员在调节植物花香合成的研究仍鲜有报道。

    基于前期桂花全基因组和转录组的测序工作[27]，本研究筛选出了1个与花香释放规律较为一致的MYB-related转录因子，并根据其在染色体上的位置命名为OfMYB1R47。本研究拟先通过实时荧光定量PCR (RT-qPCR)技术，分析OfMYB1R47基因在‘日香桂’不同发育时期中的表达特性；并结合亚细胞定位和酵母自激活实验进行基因特性分析；然后将该基因在本氏烟草Nicotiana benthamiana中瞬时表达；最后通过GC-MS分析检测瞬时转化植株与瞬时转化空载植株中主要挥发性有机物质质量分数的变化，进而探讨该基因在桂花花香合成过程中的功能，旨在揭示桂花花香合成的转录调控机制。

    • 1.   材料与方法

      1.1.   材料

    • 2021年10月，采集南京林业大学校园内‘日香桂’5个不同发育阶段的花朵，分别为铃梗期(S1，聚伞花序开始分散延伸)，香眼期(S2，花被纹理明显，花顶端出现孔隙)，初花期(S3，大部分花处于半闭合状态，花梗挺立)，盛花期(S4，花大部分或近全部呈开放状态)和末花期(S5，花全部开放，花瓣有所褶皱)[28]。所有样品采集后立即放入液氮中速冻处理，并置于−80 ℃超低温冰箱保存。每个花期具有3个生物学重复。此外，还采集了瞬时转化OfMYB1R47基因和空载体的本氏烟草叶片，用于后续目的基因半定量表达分析。本氏烟草种子由南京林业大学风景园林学院保存。

      • 1.2.   桂花OfMYB1R47转录因子的克隆及进化分析

    • 使用RNAprep Pure Plant试剂盒[天根生化科技(北京)有限公司]从桂花5个花期提取桂花的总RNA。随后从总RNA中去除gDNA后，用SuperMix反转录试剂盒[天根生化科技(北京)有限公司]合成cDNA，条件如下：65 ℃ 5 min，冰浴2 min，42 ℃ 30 min，最后85 ℃ 5 s[29]。将cDNA用去离子水稀释10倍，用于后续的基因克隆。使用 Primer Premier 5.0软件设计克隆OfMYB1R47转录因子编码区的引物(表1)，基因克隆的反应体系为：cDNA 1 μL、上下游引物各1 μL、PrimeSTAR mix 10 μL，ddH2O补至20 μL。PCR扩增程序为：98 ℃ 10 s；58 ℃ 30 s，72 ℃ 90 s，35个循环；72 ℃ 10 min[30]。通过凝胶电泳检测后，使用北京全式金生物技术公司的DNA切胶回收试剂盒对扩增产物进行收集，再将目标片段连接至用SmaI和KpnI 酶切过的Super1300载体上，并通过热激法转化大肠埃希菌Escherichia coli，随后挑选阳性菌落送至测序公司测序，最后通过冻融法将测序正确的质粒转化根癌农杆菌Agrobacterium tumefaciens。此外，使用美国生物技术信息中心(NCBI)在线网址(https://blast.ncbi.nlm.nih.gov/Blast.cgi)得到了与OfMYB1R47序列同源性较高的物种中的基因序列，并用MEGA 7.0软件，采用邻接法(Neighbor-joining)分析1 000次来构建系统进化树。

      引物名称引物作用上游引物(5′→3′)下游引物(5′→3′)
      1300-MYB1R47 基因克隆　　 aagcttctgcaggggcccgggATGGAATCCAAAGTT TATAGGAATCC gcccttgctcaccatggtaccGACAAGGCAACTCTCA TGTTGTGA
      pGBKT7-MYB1R47 酵母自激活　 atggccatggaggccgaattcATGGAATCCAAAGTTT ATAGGAATCC gcaggtcgacggatccccgggGACAAGGCAACTCTC ATGTTGTGA
      OfACTIN 荧光定量分析 CCCAAGGCAAACAGAGAAAAAAT ACCCCATCACCAGAATCAAGAA
      OfMYB1R47 ATCGCCTGGAGTGAATGCTAC CACCAAGTAATGCGTTCACAGC
      NbCCD4.1 TACCACCAAACAAACAGTAGAGC TCAATGAAAGCGTTCACGAAA
      NbCCD4.2 ACAAGAAAAGCCAACCCCATC TGGAAATGATGGCCCTACTGT
      NbCCD4.3 ACGGTTTCCACGGGCTTTT GATGACACCCATGCCCTCTT
      NbCCD1 TGGAGAGGCGAGAATAGAGGG ACGGGGAGGTTGGTAAGAGG
      NbL25 半定量分析　 GCTAAGGTTGCCAAGGCTGTC TAAGGTATTGACTTTCTTTGTCTGA

      Table 1.  Primers used in this study

      • 1.3.   桂花OfMYB1R47转录因子的RT-qPCR分析以及瞬时转化植株的半定量表达分析

    • 按照上述方法提取了桂花5个花期的总RNA，并将其反转录为cDNA，用于基因表达分析。使用Primer Premier 5.0设计OfMYB1R47转录因子的RT-qPCR引物。并且选择桂花OfACTIN作为内参基因[31]。每个RT-qPCR的表达量均来源于3个生物学重复和3个技术重复。原始数据使用SPSS 26.0中的Turkey检验进行差异显著性分析，利用2−ΔΔCt方法进行表达量分析。同时，提取瞬时转化OfMYB1R47基因和空载体植株叶片中的总RNA，并反转录成cDNA进行目的基因OfMYB1R47的半定量表达分析[14]。烟草NbL25基因被选为内参基因。此外，还选取了与桂花OfCCD4和OfCCD1同源的基因NbCCD4和NbCCD1来探究OfMYB1R47在烟草挥发性香气物质合成过程中潜在的调控关系。

      • 1.4.   桂花OfMYB1R47转录因子的亚细胞定位及酵母自激活

    • 使用不含终止密码子，但包含了OfMYB1R47转录因子蛋白质编码区(CDS)的Super 1300载体以及SmaI和KpnI限制位点构建了35S∷OfMYB1R47∷GFP。然后将含有35S∷OfMYB1R47∷GFP的根癌农杆菌(GV3101)和阴性对照载体(EV)注射到生长了35 d的本氏烟草叶片中进行亚细胞定位分析。注射所用的烟草植株生长在温度为(26±2) ℃、光强为144 µmol·m−2·s−1、光照为15 h光/9 h暗的生长室中。注射完载体的烟草浇透水后放置在生长室中生长2 d后，滴入稀释100倍后的4,6-二脒基-2-苯基吲哚(DAPI)染液，再放置在LSM710激光共聚焦显微镜下观测绿色荧光信号。

      此外，利用EcoRI和SmaI限制性酶切位点，将缺失终止密码子的OfMYB1R47基因的CDS区域连接到pGBKT7载体上。然后使用酵母菌株AH109 (上海唯地)获得pGBKT7-OfMMB1R47和pGBKT7载体。最后经PCR阳性检测后，挑取阳性克隆的单菌落在选择性培养基SD/-Trp、SD/-Trp-Ade和SD/-Trp-Ade+X-α-gal中黑暗培养3 d，在恒温培养箱(30 ℃)中观察目的基因是否具有自激活活性。

      • 1.5.   桂花OfMYB1R47转录因子在本氏烟草叶片中的瞬时表达及芳香成分分析

    • 联合顶空固相微萃取(SPME)和GC-MS方法检测瞬时转化植株与瞬时转化空载体植株中主要挥发性有机物质量分数的变化。目的基因瞬时转化方法同1.4。测定叶片中芳香性挥发物的方法如下：每30 mL SPME瓶底部加入1.5 g新鲜叶片样品，中间加入1 µL稀释1万倍的癸酸乙酯[29]。在65 ℃条件下将盖瓶中间暴露30 min后，将萃取头放到加热的注射器端口3 min，并在250 ℃下进行解吸附作用。随后，通过Trace DSQ GC-MS装置鉴定解吸后叶片中的芳香挥发物[32]，并用正构烷烃混标DLM-1342-5计算挥发性成分的保留指数(IR)，在美国国家标准技术研究所(NIST)数据库中(https://webbook.nist.gov/chemistry/cas-ser/)，与含有DB-5MS (30.00 m×0.25 mm×0.25 μm)的值进行比较，从而确认花香成分[32−33]。此外，采用以下公式计算芳香挥发物质量分数：各组分质量分数=(各组分峰面积×内标质量/内标峰面积/样品质量)。在SIMCA 14.1软件中，采用主成分分析法(PCA)对数据组内的重复性和组间的差异性进行分析，借助正交偏最小二乘判别分析法(OPLS-DA)和变量重要性投影值(VIP)对转基因组和对照组间关键的有机挥发物进行鉴定。

    2.   结果与分析

      2.1.   桂花OfMYB1R47转录因子的克隆及编码蛋白分析

    • 以桂花‘日香桂’盛花期花朵的cDNA为模板，使用特异性引物扩增获得了长度为1 485 bp，可编码494个氨基酸的OfMYB1R47基因序列。进一步分析发现：OfMYB1R47基因具有MYB-related基因家族的保守结构域(图1)。将从NCBI在线网址中获得的13个物种中与桂花同源性较高的目的基因进行系统发育树的构建，结果表明：OfMYB1R47与木犀科木樨榄Olea europaea subsp. europaea中的基因(CAA3020406.1)具有最高的同源性(图2)。

      Figure 1.  Amino acid sequence of OfMYB1R47

      Figure 2.  Phylogenetic tree analysis of OfMYB1R47

      • 2.2.   RT-qPCR分析

    • 为了得到可靠的基因表达结果，选择内参基因ACTIN进行了OfMYB1R47基因在桂花花朵发育5个典型花期的表达分析(图3)。发现OfMYB1R47基因在5个花期中的表达展现出与桂花花朵转录组数据较为相似的表达趋势，即OfMYB1R47的表达在开花期间呈现先上升后下降的模式，在S3时期达到最高值，S3~S5时期逐渐下降。

      Figure 3.  Expression pattern of OfMYB1R47 in the five flower development stages of O. fragrans

      • 2.3.   亚细胞定位及酵母自激活

    • 经过亚细胞定位分析，发现在细胞核和细胞质中均检测到了35S::OfMYB1R47::GFP融合蛋白的绿色荧光蛋白(GFP)荧光信号，经DAPI染色后发现细胞核绿色荧光与DAPI染色结果重合，证实了OfMYB1R47转录因子主要定位于细胞核(图4)。除此之外，将该基因的编码区克隆到酵母表达载体pGBKT7中，然后将阴性对照pGBKT7和含有OfMYB1R47转录因子的pGBKT7载体在连续稀释的SD/-Trp、SD/-Trp-Ade和SD/-Trp-Ade+x-α-gal培养基上培养并进行对比。结果表明：pGBKT7-OfMYB1R47在SD/-Trp和SD/-Trp-Ade的平板上能够正常生长，并在含有x-α-gal的SD/-Trp-Ade培养基上变成蓝色，这表明OfMYB1R47转录因子具有自激活功能(图5)。

      Figure 4.  Subcellular localization analysis of OfMYB1R47 in the N. benthamiana leaves

      Figure 5.  Transcriptional activation analysis of OfMYB1R47

      • 2.4.   瞬时表达植物挥发性香气成分的验证与测定

    • OfMYB1R47基因的半定量表达分析表明：在瞬时转化OfMYB1R47的植株中，都可以检测到OfMYB1R47的表达，而在转化空载体植物中均未检测到OfMYB1R47的表达(图6A)，这说明本氏烟草的瞬时转化体系较为成功，可以用于后续基因功能的分析。从瞬时表达OfMYB1R47本氏烟草叶片中共鉴定出17种芳香性挥发物，包括6种酯类、6种醛类、2种酮类等(表2)。通过对瞬时表达OfMYB1R47基因和空载体植株挥发物的主成分PCA分析，发现瞬时转OfMYB1R47基因的植株可以与瞬时转化空载体的植株很好地区分开来，表现出较显著的代谢差异(图6B)。同时，最小偏二乘法(OPLS-DA)分析的结果也表明瞬时表达OfMYB1R47和空载体的组分可以完全分离(图6C)。综上所述，瞬时超量表达OfMYB1R47可导致本氏烟草叶片芳香性挥发物质量分数的构成发生改变。

      Figure 6.  Functional analysis of OfMYB1R47

      Cas号出峰时间/min名称物质质量分数/(μg·g−1)RILVIPP
      EVOfMYB1R47
      141-43-5 2.50 乙醇胺 ethanolamine 0.017 0±0.008 5 0.026 8±0.020 5 − 0.529 9 0.491
      644-78-0 4.02 2-羟基查酮 2-hydroxychalcone 0.081 5±0.059 5 0.044 2±0.003 4 − 0.961 8 0.391
      3777-69-3 5.86 2-戊基呋喃 furan, 2-pentyl- 0.043 9±0.012 6 0.043 6±0.004 9 1 001 0.194 6 0.975
      124-13-0* 5.99 辛醛 octanal 0.052 9±0.007 1 0.026 2±0.006 5 1 009 1.302 2 0.009
      2548-87-0 6.67 E-2-辛烯醛 2-octenal, (E)- 0.011 2±0.003 2 0.015 8±0.003 4 1 066 1.004 8 0.173
      124-19-6 7.07 正壬醛 nonanal 0.354 0±0.052 0 0.254 6±0.047 3 1 110 1.118 8 0.071
      18829-56-6 7.73 反-2-壬烯醛 2-nonenal, (E)- 0.154 0±0.107 8 0.299 9±0.066 8 1 162 1.141 6 0.117
      112-31-2 8.14 癸醛 decanal 0.144 7±0.038 5 0.088 5±0.019 5 1 209 1.131 7 0.087
      432-25-7 8.42 β-环柠檬醛 1-cyclohexene-1-carboxaldehyde, 2,6,6-trimethyl- 0.021 2±0.002 2 0.017 6±0.004 3 1 219 0.850 0 0.277
      54-11-5 9.82 尼古丁 pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)- 0.043 7±0.040 1 0.010 3±0.006 3 1 360 1.028 4 0.286
      79-77-6* 10.99 β-紫罗兰酮 β-ionone 0.007 4±0.000 3 0.013 6±0.002 0 1 492 1.272 6 0.006
      166273-38-7 11.20 5-羟基戊酸- 2,4-二叔丁基苯基酯 pentanoic acid, 5-hydroxy-,
      2,4-di-t-butylphenyl esters      		 0.024 2±0.006 7 0.023 4±0.013 2 0.354 2 0.934
      6846-50-0 11.83 2,2,4-三甲基-1,3-戊二醇二异丁酸酯 2,2,4-trimethyl-1,
      3-pentanediol diisobutyrate      		 0.018 2±0.005 8 0.020 1±0.009 4 0.515 5 0.780
      84-69-5 14.11 邻苯二甲酸二异丁酯 1,2-benzenedicarboxylic acid, bis
      (2-methylpropyl) ester      		 0.022 3±0.004 7 0.016 1±0.001 0 1.135 7 0.294
      112-39-0 14.43 棕榈酸甲酯 hexadecanoic acid, methyl ester 0.011 3±0.005 3 0.014 4±0.002 9 1 929 0.777 2 0.424
      84-74-2 14.94 邻苯二甲酸二丁酯 dibutyl phthalate 0.025 3±0.003 6 0.017 0±0.001 6 1.291 2 0.023
      5129-61-3 15.94 异硬脂酸甲酯 heptadecanoic acid, 16-methyl-, methyl ester 0.000 0±0.000 0 0.005 3±0.001 1 2 103 1.365 1 0.015
      　　　　说明：EV表示阴性对照载体；VIP表示变量重要性投影值；RIL表示与NIST谱库或网站中文献的保留指数对比鉴定值；*表示相对含量发生显著改变。

      Table 2.  Peak time and content of aromatic volatiles in transient expression plants

      基于OPLS-DA分析，通过筛选VIP＞1和P＜0.05的差异物质，发现与瞬时表达空载体相比，OfMYB1R47瞬时表达植株中辛醛和β-紫罗兰酮的相对含量都发生了显著改变(表2和图6D)。从GC-MS的峰面积来看，OfMYB1R47瞬时表达植株中，辛醛的面积明显比对照瞬时表达植株小，表现为辛醛的相对含量显著下降；而β-紫罗兰酮的面积明显比对照瞬时表达植株大，表现为β-紫罗兰酮的相对含量明显上升(图6E)。此外，RT-qPCR结果显示：与瞬时表达空载体植株相比，在瞬时表达OfMYB1R47-2植株中NbCCD4.1、NbCCD4.2、NbCCD4.3以及NbCCD1等与β-紫罗兰酮合成相关的酶基因的表达量都显著上升(图7)。

      Figure 7.  Expression of CCD4 and CCD1 gene in transiently transformed plants

    3.   讨论与结论

    • MYB-related和R2R3-MYB转录因子为植物中MYB基因家族的2个主要成员，它们在调节植物生长发育过程和响应非生物胁迫等方面均具有重要的作用。目前，已有较多的与植物挥发性有机化合物合成相关的R2R3-MYB转录因子成员被鉴定出来[24, 34−35]，然而，MYB-related成员在花香挥发物合成过程中的功能目前仍鲜有报道。植物MYB-related转录因子通常具有一段高度保守的由51或52个氨基酸残基构成的结构域，该结构域可以用于识别不同类型的顺式作用元件并发挥调控作用，这也是MYB-related基因成员的重要特征[16, 36−37]。在OfMYB1R47转录因子的N端也发现了这段保守结构域，表明该基因为典型的MYB-related转录因子成员。通过系统进化树分析，在13个代表性物种中均发现了OfMYB1R47的直系同源基因，然而这些同源基因的功能仍然未知。

      花香调控基因的表达水平往往随着花朵的发育而表现出显著的变化，且往往与不同花期花香释放的强度变化具有较高的相关性[25]。为了获得较为可靠的基因表达分析结果，本研究使用桂花ACTIN这个内参基因进行RT-qPCR分析，结果发现：OfMYB1R47转录因子在以内参基因为参考的分析结果中表现为先上升后下降的表达趋势，这与桂花花香物质释放的规律一致[38]。在姜花、矮牵牛、留兰香、蜡梅Chimonanthus praecox等观赏植物中研究表明：MYB转录因子家族中的成员可与花香合成酶基因的启动子结合，通过调控相应靶基因的表达水平来调节植物挥发性有机化合物的合成[20, 24−25, 39]。在桂花中，也发现核定位转录因子OfERF61可通过调节OfCCD4的表达来影响桂花重要花香物质β-紫罗兰酮的合成[13]。在本研究中，通过亚细胞定位预测发现：OfMYB1R47转录因子蛋白主要位于细胞核内，为典型的核定位转录因子。同时，酵母自激活实验发现：OfMYB1R47转录因子还具有较强的自激活活性。可见，OfMYB1R47转录因子在桂花花香合成过程中可能具有重要的调控作用。

      目前，在本氏烟草中进行基因功能瞬时验证的体系已经在本源物种转基因较为困难的植物中广泛应用[13, 25-26]。本研究将OfMYB1R47转录因子在本氏烟草中进行了瞬时超量表达，发现瞬时转化空载体的植株可与瞬时转化OfMYB1R47载体的植株较好地划分为2个类群，这说明在本氏烟草中，异源表达OfMYB1R47基因后会引起本氏烟草芳香性挥发物构成的改变。OPLS-DA的VIP值分析表明：辛醛和β-紫罗兰酮是导致瞬时转化OfMYB1R47载体植株与瞬时转化空载体植株的芳香性挥发物聚类发生明显区分的重要香味物质，且辛醛和β-紫罗兰酮的含量与转化空载体的植株相比都发生了显著变化，这表明OfMYB1R47转录因子在芳香性挥发物的合成过程中具有重要的调节作用。进一步研究表明：瞬时表达OfMYB1R47植株中NbCCD4.1、NbCCD4.2、NbCCD4.3以及NbCCD1基因的表达明显上升，表明在本氏烟草中瞬时过表达OfMYB1R47激活了NbCCD4.1、NbCCD4.2、NbCCD4.3和NbCCD1等与β-紫罗兰酮合成相关的酶基因的表达，从而导致OfMYB1R47瞬时表达植物中β-紫罗兰酮相对含量的变化。

      本研究鉴定了一个具有转录激活活性，在桂花花朵中的表达模式与花香释放规律一致，且参与调控芳香挥发物合成的核定位转录因子OfMYB1R47，有助于加深对MYB-related转录因子亚家族功能的了解，可为今后桂花的花香分子育种提供可靠的基因资源。

Reference (39)

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