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
OfMYB
1
R
47 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
College of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
Jiangsu Key Laboratory of Landscape Architecture, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
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
OfMYB
1
R
47, 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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.]
Keywords:
Osmanthus fragrans
,
MYB-related transcription factor,
Gas Chromatography and Mass Spectrometry (GC-MS),
aroma
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
ZHANG Yao, WANG Jiaxuan, CAI Xuan, ZENG Xiangling, YANG Jie, CHEN Hongguo, ZOU Jingjing.
Identification and expression of OfACOs gene family in
Osmanthus fragrans
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2023, 40(3): 492-501.
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10.11833/j.issn.2095-0756.20220783
HONG Fanglei, LU Yao, YU Shijiao, HU Zhinuo, MIAO Yunfeng, ZHONG Shiwei, ZHAO Hongbo.
Cloning and expression analysis of OfABFs gene in
Osmanthus fragrans
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2023, 40(3): 481-491.
doi:
10.11833/j.issn.2095-0756.20220264
PANG Tianhong, QIAN Jieyu, FU Jianxin, GU Cuihua, ZHANG Chao.
Sequence and expression analysis of hexokinase gene family members in
Osmanthus fragrans
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2021, 38(2): 225-234.
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10.11833/j.issn.2095-0756.20200370
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.
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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
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2020, 37(2): 195-200.
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WANG Qianqian, JIANG Qini, FU Jianxin, DONG Bin, ZHAO Hongbo.
Screening reference genes of
Osmanthus fragrans
with differing photoperiod and temperature treatments
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2019, 36(5): 928-934.
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JIANG Qini, FU Jianxin, ZHANG Chao, DONG Bin, ZHAO Hongbo.
cDNA cloning and expression analysis of
OfAP
1 in
Osmanthus fragrans
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2019, 36(4): 664-669.
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LI Jun, DONG Bin, ZHANG Chao, FU Jianxin, HU Shaoqing, ZHAO Hongbo.
EST-SSR primers and their application in cultivar identification of
Osmanthus fragrans
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2018, 35(2): 306-313.
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LIU Yucheng, WANG Yiguang, ZHANG Chao, DONG Bin, FU Jianxin, HU Shaoqing, ZHAO Hongbo.
Cloning and transient expression assay of
OfCCD
1 gene promoters from
Osmanthus fragrans
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2018, 35(4): 596-603.
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10.11833/j.issn.2095-0756.2018.04.003
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
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2017, 34(4): 608-619.
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10.11833/j.issn.2095-0756.2017.04.006
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.
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WANG Ying, ZHANG Chao, FU Jianxin, ZHAO Hongbo.
Progresses on flower bud differentiation and flower opening in
Osmanthus fragrans
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2016, 33(2): 340-347.
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10.11833/j.issn.2095-0756.2016.02.021
HOU Dan, FU Jianxin, ZHANG Chao, ZHAO Hongbo.
Flower scent composition of
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‘Yanhong Gui’‘Yu Linglong’ and ‘Hangzhou Huang’, and their emission patterns
. Journal of Zhejiang A&F University,
2015, 32(2): 208-220.
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XU Yichun, HU Shaoqing, ZHAO Hongbo.
Genetic structure of different natural
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YANG Xiu-lian, HAO QI-mei.
Dormancy and germination of
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CHANG Bing-hua, HUYong-hong, XUYe-gen, ZHANG Qiu-xing, ZHANG Wan-li.
Ultrastructures of petal surface of Osmanthus fragrans cultivars
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2007, 24(5): 533-537.
HU Shao-qing, XUAN Zi-can, ZHOU Xu-lang, WU Guang-hong.
Taxon and clear of
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cultivars in Hangzhou
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2006, 23(2): 179-187.
ZHOU Yuan, YAO Chong-huai, WANG Cai-yun.
Study on selecting cut-flower cultivars of
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2006, 23(6): 660-663.
WU Guang-hong, HU Shao-qing, XUAN Zi-can, XIANG Qi-bai.
Standard of classification and application of sweet
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2004, 21(3): 281-284.
HUANG Bin, CHEN Huangqin, SHAO Nongquan. The ethanol extract of
Osmanthus fragrans
attenuates
Porphyromonas gingivalis
lipopolysaccharide-stimulated inflammatory effect through the nuclear factor erythroid 2-related factor-mediated antioxidant signalling pathway [J].
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YONG Yubing, ZHANG Yue, LYU Yingmin. A MYB-related transcription factor from
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Functional analysis of
OfMYB
1
R
47 transcription factor in
Osmanthus fragrans
during the formation of aromatic volatiles
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
Osmanthus fragrans
/
MYB-related transcription factor
/
Gas Chromatography and Mass Spectrometry (GC-MS)
/
aroma
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
OfMYB
1
R
47, 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
OfMYB
1
R
47 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
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
]
。
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
OfMYB
1
R
47。本研究拟先通过实时荧光定量PCR (RT-qPCR)技术，分析
OfMYB
1
R
47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。
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
OfMYB
1
R
47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，ddH
2
O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
OfMYB
1
R
47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-
MYB
1
R
47 基因克隆 aagcttctgcaggggcccgggATGGAATCCAAAGTT TATAGGAATCC gcccttgctcaccatggtaccGACAAGGCAACTCTCA TGTTGTGA
pGBKT
7-
MYB
1
R
47 酵母自激活 atggccatggaggccgaattcATGGAATCCAAAGTTT ATAGGAATCC gcaggtcgacggatccccgggGACAAGGCAACTCTC ATGTTGTGA
OfACTIN
荧光定量分析 CCCAAGGCAAACAGAGAAAAAAT ACCCCATCACCAGAATCAAGAA
OfMYB
1
R
47 ATCGCCTGGAGTGAATGCTAC CACCAAGTAATGCGTTCACAGC
NbCCD
4.1 TACCACCAAACAAACAGTAGAGC TCAATGAAAGCGTTCACGAAA
NbCCD
4.2 ACAAGAAAAGCCAACCCCATC TGGAAATGATGGCCCTACTGT
NbCCD
4.3 ACGGTTTCCACGGGCTTTT GATGACACCCATGCCCTCTT
NbCCD
1 TGGAGAGGCGAGAATAGAGGG ACGGGGAGGTTGGTAAGAGG
NbL
25 半定量分析 GCTAAGGTTGCCAAGGCTGTC TAAGGTATTGACTTTCTTTGTCTGA
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
OfMYB
1
R
47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
−ΔΔ
Ct
方法进行表达量分析。同时，提取瞬时转化
OfMYB
1
R
47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
OfMYB
1
R
47的半定量表达分析
[
14
]
。烟草
NbL
25基因被选为内参基因。此外，还选取了与桂花
OfCCD
4和
OfCCD
1同源的基因
NbCCD
4和
NbCCD
1来探究
OfMYB
1
R
47在烟草挥发性香气物质合成过程中潜在的调控关系。
使用不含终止密码子，但包含了
OfMYB
1
R
47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35
S
∷
OfMYB
1
R
47∷
GFP
。然后将含有35
S
∷
OfMYB
1
R
47∷
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。
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(
I
R
)，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。
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35
S
::
OfMYB
1
R
47::
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
OfMYB
1
R
47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
OfMYB
1
R
47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：
pGBKT
7-
OfMYB
1
R
47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
OfMYB
1
R
47转录因子具有自激活功能(
图5
)。
OfMYB
1
R
47基因的半定量表达分析表明：在瞬时转化
OfMYB
1
R
47的植株中，都可以检测到
OfMYB
1
R
47的表达，而在转化空载体植物中均未检测到
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB1R
47基因和空载体植株挥发物的主成分PCA分析，发现瞬时转
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47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。
Cas号出峰时间/min名称物质质量分数/(μg·g
−1
)RILVIP
P
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。
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，
OfMYB
1
R
47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，
OfMYB
1
R
47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
OfMYB
1
R
47
-
2植株中
NbCCD
4.1
、NbCCD
4.2
、NbCCD
4.3以及
NbCCD
1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
)。
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
OfMYB
1
R
47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
OfMYB
1
R
47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：
OfMYB
1
R
47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
OfERF
61可通过调节
OfCCD
4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：
OfMYB
1
R
47转录因子还具有较强的自激活活性。可见，
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47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
OfMYB
1
R
47植株中
NbCCD
4.1
、NbCCD
4.2
、NbCCD
4.3以及
NbCCD
1基因的表达明显上升，表明在本氏烟草中瞬时过表达
OfMYB
1
R
47激活了
NbCCD
4.1
、NbCCD
4.2
、NbCCD
4.3和
NbCCD
1等与β-紫罗兰酮合成相关的酶基因的表达，从而导致
OfMYB
1
R
47瞬时表达植物中β-紫罗兰酮相对含量的变化。
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Figure 1 Amino acid sequence of
OfMYB
1
R
47
Figure 2 Phylogenetic tree analysis of OfMYB1R47
Figure 3 Expression pattern of
OfMYB
1
R
47 in the five flower development stages of
O. fragrans
Figure 4 Subcellular localization analysis of OfMYB1R47
in the
N. benthamiana
leaves
Figure 5 Transcriptional activation analysis of OfMYB1R47
Figure 6 Functional analysis of
OfMYB
1
R
47
