盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析

doi:10.11983/CBB24015

摘要/Abstract

摘要： AP3/DEF (APETALA3/DEFICIENS)基因为MADS-box基因家族的B类基因, 在花发育过程中主要参与调控花瓣和雄蕊发育。对盐肤木(Rhus chinensis) AP3/DEF同源基因进行克隆及功能分析, 有助于探究其在盐肤木雄蕊发育过程中的作用。采用RT-PCR技术获得盐肤木AP3/DEF同源基因CDS; 利用NCBI CD Search对其序列和结构域进行比较分析; 利用酵母双杂交系统, 对AP3/DEF同源蛋白与盐肤木中其它MADS-box转录因子进行蛋白互作验证; 通过实时荧光定量PCR分析盐肤木AP3/DEF同源基因的时空表达模式; 用过表达拟南芥(Arabidopsis thaliana)验证盐肤木AP3/DEF同源基因在花器官发育中的功能。结果表明, 克隆得到2个盐肤木AP3/DEF同源基因分别命名为RcAP3 (GenBank: OR962160)和RcTM6 (GenBank: OR962159), 根据其氨基酸保守结构域比对及系统进化分析, 发现这2个蛋白序列与漆树科的芒果(Mangifera indica)和阿月浑子(Pistacia vera) AP3/DEF同源蛋白亲缘关系最近。酵母双杂交结果表明, RcAP3和RcTM6与盐肤木B类蛋白RcPI、C类蛋白RcAG和Rcag存在互作关系, 但与A类和E类蛋白不存在互作关系。实时荧光定量PCR分析结果显示, RcAP3和RcTM6基因在不同性别盐肤木花芽快速发育期高表达, 在花芽发育早期和开花后表达水平较低; RcAP3在雌花、雄花和两性花的花芽分化过程中均维持较高的表达水平, 而RcTM6在两性花中显著表达, 在雄花和雌花中表达量很低。两性花快速生长期, RcAP3在花瓣和雄蕊中高表达且差异很小, 而RcTM6在雄蕊中的表达量显著高于其它花器官。RcAP3基因能恢复拟南芥ap3-3突变体花瓣和雄蕊的缺陷表型, RcTM6过表达则导致拟南芥花瓣、雄蕊和子房缩短, 花药败育, 表明盐肤木中同属AP3/DEF亚家族的同源基因RcAP3和RcTM6存在功能分化。RcAP3促进花瓣和雄蕊发育, 而RcTM6抑制雄蕊发育。研究结果为进一步研究盐肤木性别分化的分子机制奠定了基础。

关键词: 盐肤木, RcAP3, RcTM6, 表达模式, 功能分析

Abstract: The APETALA3/DEFICIENS (AP3/DEF) gene is a B-class gene of the MADS-box gene family, which is mainly involved in the regulation of petal and stamen development during flower development. Cloning and analysis of the AP3/DEF homologous genes from Rhus chinensis can help exploring their role in the development of stamen. The CDS of AP3/DEF homologous gene was obtained using RT-PCR; its sequence and structural domains were compared and analyzed by NCBI CD Search; interactions between AP3/DEF homologous proteins and other MADS-box transcription factors in R. chinensis were verified by using yeast two-hybrid system; real-time fluorescence PCR was used to analyze the spatial and temporal expression patterns of the AP3/DEF homologous genes; overexpression in Arabidopsis was conducted to verify the function of the AP3/DEF homologous genes in flower organ development. Two coding sequences of AP3/ DEF homologous genes in R. chinensis were cloned and named RcAP3 (GenBank: OR962160) and RcTM6 (GenBank: OR962159). Based on the alignment of their conserved amino acid domains and phylogenetic analysis, these two protein sequences showed the closest genetic relationship to the AP3/DEF homologous gene-encoded proteins in the Anacardiaceae family, including Mangifera indica and Pistacia vera. The yeast two-hybrid results showed that RcAP3 and RcTM6 interacted with the B-class protein RcPI, the C-class proteins RcAG and Rcag in R. chinensis, but not with the A-class and E-class proteins. Real-time fluorescence quantification results demonstrated that RcAP3 and RcTM6 were highly expressed during the rapid growth stage of flower buds in different sexes of R. chinensis, with lower expression levels during the early bud development and after flowering. RcAP3 exhibited high expression levels during buds differentiation in female, male, and hermaphrodite flowers, while RcTM6 showed significant expression in hermaphrodite flowers and very low expression in male and female flowers. During the rapid growth stage of hermaphrodite flowers, RcAP3 was highly expressed in both petals and stamens with minimal differences, whereas RcTM6 showed significantly higher expression in stamens compared to other floral organs. RcAP3 could restore the petal and stamen phenotypes in the ap3-3 mutant of Arabidopsis, while overexpression of RcTM6 resulted in shortened petals, stamens, and aborted ovaries in Arabidopsis. Functional differentiation exists between the AP3/DEF subfamily homologous genes, RcAP3 and RcTM6. RcAP3 promotes petal and stamen development, while RcTM6 inhibits stamen development. These findings provide a foundation for further research on the molecular mechanisms underlying sex differentiation in R. chinensis.

Key words: Rhus chinensis, RcAP3, RcTM6, expression pattern, functional analysis

顾磊, 张棋, 张霞, 杨冰冰, 王芳岚, 刘文, 陈发菊. 盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析. 植物学报, 2024, 59(4): 533-543.

Lei Gu, Qi Zhang, Xia Zhang, Bingbing Yang, Fanglan Wang, Wen Liu, Faju Chen. Cloning and Functional Analysis of APETALA3/DEFICIENS Homologous Gene from Rhus chinensis. Chinese Bulletin of Botany, 2024, 59(4): 533-543.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24015

https://www.chinbullbotany.com/CN/Y2024/V59/I4/533

图/表 10

表1 引物序列

Table 1 Primer sequences used in this study

Primers	Primer sequence (5'-3')	Primer usage
RcAP3-CDS-F	ATGGCTCGAGGAAAGATCCAG	CDS cloning and positive plant identification
RcAP3-CDS-R	CTAGTCAAGCAAGGGGGAGG
RcTM6-CDS-F	ATGGGTCGCGGAAAGATTG
RcTM6-CDS-R	TCAACCAAGGCTGAGATTGTTG
qRcAP3-CDS-F	GAAGAAGGTAAGGAGTGTGACAG	Real-time fluorescent quantitative PCR
qRcAP3-CDS-R	AGGTGGTGAGATCTGAGCCTG
qRcTM6-CDS-F	CGTCCGTGAAAGAAAGTACCATG
qRcTM6-CDS-R	GCGTACAGGTTAGATGCTCC
qPP2A-F	TCCACCGTCCGATCATCAGAAC	Reference gene
qPP2A-R	GCACGTTCCATTCCTCCACC	Reference gene
ap3-jianceF1	ATGGCGAGAGGGAAGATCC	Detection of ap3 gene-pure plants in Arabidopsis thaliana
ap3-jianceR1	GATCAAGAGGATAGAGAACCAGACAAACAGA
ap3-jianceF2	ACAGTTTCCTCTTGGTTTCTTGC
ap3-jianceR2	CGCATCAAGAATTTAACCAACCAGCG

表2 构建系统进化树蛋白序列登录号

Table 2 GenBank number of protein sequences used for constructing the phylogenetic tree

Species	Protein	GenBank ID
Rhus chinensis	RcAP3	OR962159
R. chinensis	RcTM6	OR962160
Nicotiana tabacum	NtDEF	CAA65288
Petunia × hybrida	PhDEF	AAQ72510
Solanum lycopersicum	SolyDEF	CAJ53871
Antirrhinum majus	AmDEF	P23706
Torenia fournieri	TofoDEF	BAG24492
Gerbera hybrid	GDEF2	CAA08803
Arabidopsis thaliana	AtAP3	NP_191002
Pistacia vera	PvAP3	OR962159
Mangifera indica	MinAP3	XP_044510378
Lotus japonicus	LojaAP3	AAX13301
Gongora galeata	GogaDEF	ACR16038
Oncidium hybrid	OMADS9	ADJ67235
Dendrobium crumenatum	DecrAP3	AAZ95249
Spiranthes odorata	SpodDEF	ACR16049
Vanilla planifolia	VaplDEF	ACR16055
Asparagus officinalis	AODEF	BAC75969
Muscari armeniacum	MaDEF	BAE48147
Tulipa gesneriana	TGDEFA	BAC75970
T. gesneriana	TGDEFB	BAC75971
Lilium longiflorum	LMADS1	AAM27456
Monotropa hypopitys	MhTM6	AQM52303
Tradescantia ohiensis	TrohDEF	BAD80745
Commelina communis	CocoAP3	BAD80747
Oryza sativa	OsMADS16	Q944S9
Chimonanthus praecox	ChprAP3	ABK34952
Akebia trifoliata	AktAP3-1	AAT46097
Hydrangea macrophylla	HmTM6	AAF73932
Petunia × hybrida	PhTM6	AAS46017
Helianthus annuus	HAM91	AAO18231
Rosa rugosa	MASAKO B3	BAB63261
Prunus avium	PaTM6	BAT57494
Malus domestica	MdTM6	BAC11907
Ma. domestica	MdMADS13	CAC80856
Pistacia vera	PvTM6	XP_031264419
Mangifera indica	MinTM6	XP_044469431
Philadelphus pubescens	PhpTM6	ACY08886
Vitis vinifera	VvTM6	ABI98021
Saurauia zahlbruckneri	SzTM6	ACY08897
Actinidia chinensis	AcAP3	ADU15473
Ar. thaliana	AtPI	NP_197524

图1 RcAP3和RcTM6基因的PCR扩增 M: DL 2000 DNA分子量标准

Figure 1 PCR amplification of RcAP3 and RcTM6 M: DL 2000 DNA marker

图2 RcAP3和RcTM6的相对表达模式 (A), (B) RcAP3 (A)和RcTM6 (B)在花芽不同发育时期的表达(单因素方差分析, 沃勒-邓肯检测, 不同小写字母表示在P<0.05水平差异显著); (C) RcAP3和RcTM6在两性花花芽中期不同器官中的表达水平(* P<0.05, *** P<0.001)。

Figure 2 The relative expression patterns of RcAP3 and RcTM6 (A), (B) Expression of RcAP3 and RcTM6 at different stages of flower bud development (One-way analysis of ANOVA by Waller-Duncan, different lowercase letters indicate significant differences at P<0.05); (C) Expression levels of RcAP3 and RcTM6 in different organs in the middle stage of flower buds of hermaphroditic flower (* P<0.05, *** P<0.001).

图3 RcAP3 (A)和RcTM6 (B)的蛋白质保守结构域以及与其它植物的氨基酸序列多重比对(C)

Figure 3 The protein conserved domain of RcAP3 (A), RcTM6 (B), and the alignment of amino acid sequences of RcAP3 and RcTM6 with other plants (C)

图4 基于盐肤木RcAP3和RcTM6蛋白的系统进化树红色星号表示本研究中的蛋白。

Figure 4 Phylogenetic tree based on RcAP3 and RcTM6 proteins The red asterisk indicates the protein in this study.

表3 RcAP3和RcTM6蛋白理化性质分析

Table 3 Analysis of physicochemical properties of RcAP3 and RcTM6 proteins

Parameter	RcAP3	RcTM6
Formula	C₁₁₁₈H₁₈₂₄N₃₂₈O₃₄₈S₅	C₁₁₂₅H₁₇₈₉N₃₂₅O₃₄₃S₆
Molecular mass (kDa)	25.59	25.55
Total number of atoms	3623	3588
Theoretical pI	9.23	9.33
Total number of negatively char- ged residues (Asp+Glu)	30	29
Total number of positively char- ged residues (Arg+Lys)	35	36
Instability index	48.17	40.95
Aliphatic index	86.82	73.50
Grand average of hydropathicity	-0.754	-0.749
Signal peptide	None	None
Transmembrane domain	None	None

图5 RcAP3 (A)和RcTM6 (B)蛋白质二级结构分析

Figure 5 Secondary structure analysis of RcAP3 (A) and RcTM6 (B) proteins

图6 酵母双杂交分析RcAP3与RcTM6蛋白的相互作用模式 AD: 激活结构域; BD: 结合结构域; NC: 阴性对照; PC: 阳性对照

Figure 6 Yeast two-hybrid analysis of RcAP3 and RcTM6 protein interaction patterns AD: Activation domain; BD: Binding domain; NC: Negative control; PC: Positive control

图7 转基因拟南芥表型比较 (A) 野生型(WT)拟南芥(Col-0); (B) 空载体pBI121转Col-0拟南芥; (C), (D) 35S::RcAP3转Col-0拟南芥; (E)-(I) 35S::RcTM6转Col-0拟南芥; (J) ap3-3突变体拟南芥; (K), (L) 35S::RcAP3转ap3-3突变体拟南芥。(A)-(H), (J)-(L) Bars=1 mm; (I) Bar=1 cm

Figure 7 Comparison of phenotypes of transgenic Arabidopsis thaliana (A) Wild-type (WT) of A. thaliana (Col-0); (B) pBI121 empty vector to Arabidopsis Col-0; (C), (D) 35S::RcAP3 transgenic Arabidopsis in a Col-0 background; (E)-(I) 35S::RcTM6 transgenic Arabidopsis in a Col-0 background; (J) ap3-3 homozygous mutants; (K), (L) 35S::RcAP3 transgenic Arabidopsis in a ap3-3 homozygous background. (A)-(H), (J)-(L) Bars=1 mm; (I) Bar=1 cm

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