R2R3-MYB转录因子CitMYB21对柑橘类黄酮生物合成的影响

doi:10.16420/j.issn.0513-353x.2021-1188

摘要/Abstract

摘要：

以‘巴西酸橙’（Citrus aurantium L.）、‘本地早橘’（C. reticulata Blanco）、‘桂花蒂南丰蜜橘’（C. reticulata Blanco）、‘北碚447锦橙’[C. sinensis（L.）Osbeck]等30份柑橘种质为试材，挖掘与柑橘类黄酮生物合成相关的R2R3-MYB转录因子。通过比较转录组分析发现ERF、MYB以及Dof转录因子家族与果实发育过程中类黄酮的代谢密切相关，其中两个R2R3-MYB转录因子基因CitMYB21（Ciclev10021699m）和CitMYB33（Ciclev10012152m）的表达水平与类黄酮生物合成途径中的关键结构基因CitCHS2的表达量明显负相关。系统发育分析显示，这两个基因分别属于R2R3-MYB中的第2和第1亚族。从‘北碚447锦橙’中克隆得到了CitMYB21编码区的全长序列，共804 bp，编码267个氨基酸。CitMYB21的表达有显著的组织特异性及明显的种质特异性，并与类黄酮的含量呈显著负相关。CitMYB21沉默的柑橘植株叶片中类黄酮含量显著增加，而瞬时过表达的果皮中类黄酮的含量明显降低。研究结果表明CitMYB21显著负影响柑橘类黄酮的含量，可能是负调控类黄酮生物合成的重要转录因子。

关键词: 柑橘, 类黄酮, R2R3-MYB转录因子, 负调控, 组织特异性

Abstract:

Brazil Sour Orange（Citrus aurantium L.），Bendizao Mandarin（C. reticulata Blanco），Guihuadinanfeng mandarin（C. reticulata Blanco），Jincheng Beibei 447[C. sinensis（L.）Osbeck] and other 30 germplasms were used for mining the transcription factors related to flavonoid biosynthesis. Comparative transcriptomic analysis revealed that ERF，MYB and Dof transcription factor families are significantly involved in metabolism of flavonoids during fruit development. Correlation analysis of the expressions of CitCHS2，a key gene of flavonoids biosynthesis with the differentially expressed genes from transcriptomic analysis indicated that two R2R3-MYB family member CitMYB21（Ciclev10021699m）and CitMYB33（Ciclev10012152m）were negatively correlated to expression of CitCHS2. Phylogenic analysis revealed that these two genes belong to SG2 and SG1 subgroup of R2R3-MYBs family，respectively. The full length of CitMYB21 coding region is 804 bp and encodes 267 amino acids. The expressions of CitMYB21 were highly tissue and genotype specific，and significantly negatively correlated with the flavonoids contents. Silence of CitMYB21 resulted a significant increase of flavonoids in the leaf of transgenic plants，while transient overexpression of CitMYB21 in the peel of citrus fruit led to a significant decrease of flavonoids. Results of this study indicated that expression of CitMYB21 negatively affect the flavonoid biosynthesis in citrus，suggesting CitMYB21 may be an important transcription factor negatively regulating the biosynthesis of flavonoid.

Key words: citrus, flavonoids, R2R3-MYB transcription factor, negative regulation, tissue specific

中图分类号:

S666

叶子茂, 申晚霞, 刘梦雨, 王彤, 张晓楠, 余歆, 刘小丰, 赵晓春. R2R3-MYB转录因子CitMYB21对柑橘类黄酮生物合成的影响[J]. 园艺学报, 2023, 50(2): 250-264.

YE Zimao, SHEN Wanxia, LIU Mengyu, WANG Tong, ZHANG Xiaonan, YU Xin, LIU Xiaofeng, ZHAO Xiaochun. Effect of R2R3-MYB Transcription Factor CitMYB21 on Flavonoids Biosynthesis in Citrus[J]. Acta Horticulturae Sinica, 2023, 50(2): 250-264.

图/表 14

表1 种质名单

Table 1 List of citrus germplasms

种类 Specific name	种质编号 Code	名称 Name	种类 Specific name	种质编号 Code	名称 Name
宽皮橘 Citrus reticulate Blanco	ZY004	桂花蒂南丰蜜橘 Guihuadinanfeng Mandarin	宽皮橘 C. reticulate Blanco	ZY198	半野生橘杂种No.3 Hybrid of Semi-wild No.3
	ZY013	槾橘 Manju		ZY208	无核早橘 Seedless Zaoju
	ZY014	野橘 2 Yeju 2		ZY215	丙中洛橘 Bingzhongluoju
	ZY026	酸橘 Suanju		ZY222	红橘 Hongju
	ZY035	鹅蛋香柑 E Dan Xianggan		ZY007	津优 Jinyou
	ZY036	汉源黄果柑 Hanyuan Huangguogan		ZY008	扁柑 Biangan
	ZY037	德昌金钱橘 Dechang Jinqianju		ZY011	久贺早生温州 Kuga Wase
	ZY049	冰糖香柑 Bingtang Xianggan		ZY012	北口温州 Kitaguchi Wase
	ZY084	青红橘 Qinghongju	温州蜜柑 C. unshiu Macf.	ZY067	久能温州 Kunou Wase
	ZY085	红橙 Hongcheng		ZY070	繁田温州 Shigeta Wase
	ZY090	元阳酸橘 Yuanyang Suanju		ZY124	大分1号温州 Dafen No.1 Wase
	ZY108	沅江南橘 Yuanjiang Nanju		ZY145	湘慈43号 Xiangci No.43
	ZY119	马塔里橘 Matari Tangerine		ZY168	谷口系日南1号温州
	ZY151	新生系3号椪柑 No.3 Ponkan			Taniguchi Line Nichinan No.1 Wase
	ZY174	春见 Harumi		ZY231	桥本温州 Hashimoto Wase
				ZY002	平阳橘 Duong Mandarin

表2 所用引物

Table 2 Sequences of primers

引物名称 Primers name	序列（5′-3′） Sequence	酶切位点 Restriction site
OE-CitMYB21F	CCATTTAAATATGGTGAGAGCTCCATGCTG	SwaⅠ
OE-CitMYB21R	CGGGATCCCGACTTCTCATCAAAAAGGAAGGA	BamHⅠ
VIGS-CitMYB21F	TCCCCCGGGACAACAAGAGCCCGTGATTA	SmaⅠ
VIGS-CitMYB21R	CGGGATCCAACTATAGACAGTGGGTCGGC	BamHⅠ
q-CitMYB21F	CATGCTGCGAGAAGATGGGA
q-CitMYB21R	CTGCTTGTTTGGGAAGTGCC
Actin-F	CATCCCTCAGCACCTTCC
Actin-R	CCAACCTTAGCACTTCTCC
pTRV1-F	TTGGGTTGCTACTGATTCGACT
pTRV1-R	CTGTAAGGACCATCATACTTCGC
pTRV2_GFP-F	CTGCCCGACAACCACTACCT
pTRV2_GFP-R	CTTGTACAGCTCGTCCATGCC

图1 ‘巴西酸橙’和‘本地早橘’花后160 d vs. 100 d果实间差异表达基因数量（A）及维恩图（B）

Fig. 1 The number of differentially expressed genes（A）and Venn diagram（B）between the fruits of Brazil Sour Orange and Bendizao Mandarin at 160 d vs. 100 d after flowering

图2 ‘巴西酸橙’和‘本地早橘’花后160 d vs. 100 d果实共有的差异表达基因KEGG功能注释

Fig. 2 KEGG functional annotation of co-shared differently expression genes between Brazil Sour Orange and Bendizao Mandarin at 160 d vs. 100 d after flowering

图3 与CitCHS2表达相关的基因功能注释

Fig. 3 Functional annotation of genes related to CitCHS2 gene expression

图4 类黄酮生物合成途径中结构基因启动子的顺式作用元件

Fig. 4 Analysis of Cis-regulatory elements in the promoters of structural genes in flavonoids biosynthesis pathway

图5 与类黄酮生物合成相关的R2R3-MYB的鉴定 A：与结构基因CitCHS2相关的R2R3-MYB转录因子；B：预测的R2R3-MYB与差异表达基因中筛选的R2R3-MYB的交集。

Fig. 5 Identification of candidate R2R3-MYB transcription factors related to flavonoids biosynthesis A：Correlation analysis of R2R3-MYB transcription factors with flavonoids biosynthesis related structural genes CitCHS2. B：The Venn diagram of predicted R2R3-MYB and identified R2R3-MYB from DEGs.

图6 候选R2R3-MYB成员的系统发育分析 Cit：柑橘；At：拟南芥；Ptr：杨树；Vv：葡萄；Md：苹果；Pp：桃；Pb：梨；Ft：荞麦；Gm：大豆；Fa：草莓；Fc：智利草莓；Ph：矮牵牛；Cs：茶树；Nt：水仙。

Fig. 5 Phylogenetic analysis of candidate R2R3-MYB members Cit：Citrus clementina；At：Arabidopsis thaliana；Ptr：Populus trichocarpa；Vv：Vitis vinifera；Md：Malus × domestica；Pp：Prunus persica；Pb：Pyrus bretschneideri；Ft：Fagopyrum tataricum；Gm：Glycine max；Fa：Fragaria × ananassa；Fc：Fragaria chiloensis；Ph：Petunia × hybrida；Cs：Camellia sinensis；Nt：Narcissus tazetta.

图7 CitMYB21克隆及其表达量与类黄酮含量的相关性分析 A：CitMYB21克隆产物电泳图；B：CitMYB21基因结构图及与Cs5g29830序列相似性比对；C：30份种质（详见表1）中CitMYB21表达量与类黄酮含量。

Fig. 7 CitMYB21 gene cloning and correlation analysis of its expression and flavonoid content A：Gel electrophoresis of cloned production of CitMYB21. B：The structure and sequence similarity of CitMYB21 and Cs5g29830. C：Relative expression level of CitMYB21 and flavonoids content in 30 citrus germplasms（See Table 1）.

图8 ‘桂花蒂南丰蜜橘’CitMYB21表达的组织特异性

Fig. 8 Analysis of tissue specific expression of CitMYB21 in Guihuadi Nanfeng Mandarin

图9 ‘桂花蒂南丰蜜橘’叶片发育过程中CitMYB21表达水平和类黄酮含量的变化

Fig. 9 The change pattern of CitMYB21 expression and flavonoids content during leaf developing of Guihuadi Nanfeng Mandarin

图10 ‘北碚447锦橙’基因沉默植株（A、B）和过表达果皮（C、D）中CitMYB21表达水平和类黄酮含量 ** 表示处理间差异极显著（P < 0.01）。

Fig. 10 CitMYB21 expression level and flavonoid content in Beibei 447 Jincheng gene-silenced plants（A，B）and overexpressed peels（C，D） ** indicate extremely significant differences between different treatments at 0.01 level.

表3 ‘北碚447锦橙’转基因植株中CitMYB21表达与类黄酮含量的相关性

Table 3 The correlation of CitMYB21 expression and flavonoids content in transgenic lines of Beibei 447 Jincheng

转化方式 Transformation method	r 相关性Correlation	P 显著性Significance
病毒诱导的基因沉默Virus induced gene slicing	-0.8	< 0.01
瞬时表达Transient expression	-0.8	> 0.05

表4 CitMYB21表达与类黄酮合成相关基因表达水平的相关性

Table 4 The correlation analysis of expression level of CitMYB21 and structural genes related to flavonoids biosynthesis

基因 Gene	CitPAL	CitC4H	Cit4CL1	Cit4CL2	CitCHS2	CitCHI	条件 Condition
CitMYB21	-0.7^**	0.3	-0.5^*	0.3	-0.4	-0.5	叶片发育早期Leaf early developmental stage
CitMYB21	0.1	-0.4	0.5^*	-0.5	0.6^*	0.4	叶片发育后期Leaf late developmental stage
CitMYB21	0.1	0.9^**	-0.6	0.7	-0.6	0.8^**	组织部位Tissue
CitMYB21	0.3	0.6^*	-0.4	-0.5	-0.7^**	-0.6^**	基因沉默Gene silencing
CitMYB21	0.2	0.8	-0.3^**	0.7	-0.9^**	-0.9^**	瞬时表达Transient expression

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