Cloning and Functional Analysis of APETALA3/DEFICIENS Homologous Gene from Rhus chinensis

doi:10.11983/CBB24015

Abstract

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

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[J]. Chinese Bulletin of Botany, 2024, 59(4): 533-543.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24015

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

Figures/Tables 10

References 33

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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

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

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

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

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