Identification of the AP2 Subfamily Transcription Factors in Chimonanthus praecox and the Functional Study of CpAP2-L11

doi:10.16420/j.issn.0513-353x.2021-1114

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (2): 382-396.doi: 10.16420/j.issn.0513-353x.2021-1114

• Research Papers • Previous Articles Next Articles

Identification of the AP2 Subfamily Transcription Factors in Chimonanthus praecox and the Functional Study of CpAP2-L11

TIAN Mingkang¹, XU Zhixiang¹, LIU Xiuqun², SUI Shunzhao¹, LI Mingyang¹, LI Zhineng¹^,^*()

1. Key Laboratory of Horticulture Science for Southern Mountains Region（Ministry of Education），Flower Engineering Technology Research Institute of Chongqing，College of Horticulture and Landscape Architecture，Southwest University，Chongqing 400715，China
2. Key Laboratory of Horticultural Plant Biology（Ministry of Education），College of Horticulture and Forestry Science，Huazhong Agricultural University，Wuhan 430070，China

Received:2022-05-19 Revised:2022-11-05 Online:2023-02-25 Published:2023-03-06
Contact: *（E-mail：znli@swu.edu.cn）

Abstract

Abstract:

Members of the AP2 subfamily were identified at the whole genome level in Chimonanthus praecox，and their expression patterns during flower development were analyzed. The expression profile of CpAP2-L11 was investigated，and 35S::CpAP2-L11 was introduced into Arabidopsis thaliana Columbia-0（Col-0）to analyze its function. A total of 20 AP2 subfamily transcription factors were identified，including five，six，and nine members from the euAP2，basalANT，and euANT groups，respectively，and all members of the euAP2 group have no miR172 binding sites. Synteny analysis revealed that 12 members form 16 pairs of duplicated genes，which were subject to purifying selection during the evolutionary process. Most AP2 subfamily members are highly expressed in April and May flower buds（FBs），with low expression during the entire or later stage of the chilling requirement（CR）accumulation；the expression level of CpAP2-L11 increased significantly in FBs initiating blooming when CR accumulation reached 570 CU. qRT-PCR analysis indicated CpAP2-L11 was highly expressed in the young fruit，outer tepals，and stamens，but a low expression level was detected in stems and leaves in C. praecox. The expression level of CpAP2-L11 was downregulated by high- or low-temperature. Ectopic expression of CpAP2-L11 in Arabidopsis showed earlier bolting time and the relative expression of FT，SOC1，LFY and AP1 genes increased significantly. CpAP2-L11 probably participates in chilling-induced dormancy breaking and blooming in winter and FBs differentiation in spring in C. praecox，also promoting flowering in ectopic overexpression Arabidopsis.

Key words: Chimonanthus praecox, AP2 subfamily, transcription factor, flower development

CLC Number:

S685.99

TIAN Mingkang, XU Zhixiang, LIU Xiuqun, SUI Shunzhao, LI Mingyang, LI Zhineng. Identification of the AP2 Subfamily Transcription Factors in Chimonanthus praecox and the Functional Study of CpAP2-L11 [J]. Acta Horticulturae Sinica, 2023, 50(2): 382-396.

Figures/Tables 12

References 54

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用途 Usage	引物名称 Primer name	序列（5′-3′） Sequence
CpAP2-L11基因克隆引物 CpAP2-L11 gene cloning primer	CpAP2-L11-F	AACAAATCGGACGGTTCAAACCACT
CpAP2-L11基因克隆引物 CpAP2-L11 gene cloning primer	CpAP2-L11-R	GAAAATCAAAAACCGATCTCAGCCGG
表达载体构建引物 Expression vector construction primer	eCpAP2-L11-F	CGGGGTACCATGGGGAAACCATCACAGAAG
表达载体构建引物 Expression vector construction primer	eCpAP2-L11-R	ACGCGTCGACTTACTGTCCATCCCAGTCGGC
实时荧光定量PCR qRT-PCR primer	qCpAP2-L11-F	TCCCGACCTTAACGTCTCGTCAGAG
实时荧光定量PCR qRT-PCR primer	qCpAP2-L11-R	GCAGCGGGGAGTTTTGTCGGATTCT
蜡梅内参基因实时荧光定量PCR qRT-PCR primer of reference gene in	CpActin-F	GTTATGGTTGGGATGGGACAGAAAG
蜡梅内参基因实时荧光定量PCR qRT-PCR primer of reference gene in	CpActin-R	GGGCTTCAGTAAGGAAACAGGA
Chimonanthus praecox	CpTublin-F	TAGTGACAAGACAGTAGGTGGAGGT
	CpTublin-R	GTAGGTTCCAGTCCTCACTTCATC
拟南芥内参基因及内源基因实时荧光定量PCR qRT-PCR primer of reference and endogenous genes in Arabidopsis thaliana	AtActin-F	CTTCGTCTTCCACTTCAG
	AtActin-R	ATCATACCAGTCTCAACAC
	AtLFY-F	AGACGGCTGCTTTTGGGATG
	AtLFY-R	TATTCCCCGCCGCATCAGTC
	AtFT -F	GATTGGTGGAGAAGACCTCAGGAAC
	AtFT -R	GCAGCCACTCTCCCTCTGACAAT
	AtAP1 -F	TAGGGCTCAACAGGAGCAGT
	AtAP1 -R	CAGCCAAGGTTGCAGTTGTA
	AtSOC1-F	AATTCGCCAGCTCCAATATG
	AtSOC1-R	CCTCGATTGAGCATGTTCCT

用途 Usage	引物名称 Primer name	序列（5′-3′） Sequence
CpAP2-L11基因克隆引物 CpAP2-L11 gene cloning primer	CpAP2-L11-F	AACAAATCGGACGGTTCAAACCACT
CpAP2-L11基因克隆引物 CpAP2-L11 gene cloning primer	CpAP2-L11-R	GAAAATCAAAAACCGATCTCAGCCGG
表达载体构建引物 Expression vector construction primer	eCpAP2-L11-F	CGGGGTACCATGGGGAAACCATCACAGAAG
表达载体构建引物 Expression vector construction primer	eCpAP2-L11-R	ACGCGTCGACTTACTGTCCATCCCAGTCGGC
实时荧光定量PCR qRT-PCR primer	qCpAP2-L11-F	TCCCGACCTTAACGTCTCGTCAGAG
实时荧光定量PCR qRT-PCR primer	qCpAP2-L11-R	GCAGCGGGGAGTTTTGTCGGATTCT
蜡梅内参基因实时荧光定量PCR qRT-PCR primer of reference gene in	CpActin-F	GTTATGGTTGGGATGGGACAGAAAG
蜡梅内参基因实时荧光定量PCR qRT-PCR primer of reference gene in	CpActin-R	GGGCTTCAGTAAGGAAACAGGA
Chimonanthus praecox	CpTublin-F	TAGTGACAAGACAGTAGGTGGAGGT
	CpTublin-R	GTAGGTTCCAGTCCTCACTTCATC
拟南芥内参基因及内源基因实时荧光定量PCR qRT-PCR primer of reference and endogenous genes in Arabidopsis thaliana	AtActin-F	CTTCGTCTTCCACTTCAG
	AtActin-R	ATCATACCAGTCTCAACAC
	AtLFY-F	AGACGGCTGCTTTTGGGATG
	AtLFY-R	TATTCCCCGCCGCATCAGTC
	AtFT -F	GATTGGTGGAGAAGACCTCAGGAAC
	AtFT -R	GCAGCCACTCTCCCTCTGACAAT
	AtAP1 -F	TAGGGCTCAACAGGAGCAGT
	AtAP1 -R	CAGCCAAGGTTGCAGTTGTA
	AtSOC1-F	AATTCGCCAGCTCCAATATG
	AtSOC1-R	CCTCGATTGAGCATGTTCCT

名称Name	框Scaffold	位置Position	氨基酸数Amino acids	分子质量/kD Molecular weigh	理论等电点pI
CpAP2-L1	8	633 532 ~ 638 961	593	65.68	6.27
CpAP2-L2	12	6 666 416 ~ 6 670 043	501	55.82	6.29
CpAP2-L3	18	2 386 489 ~ 2 389 495	516	57.15	6.06
CpAP2-L4	18	4 068 635 ~ 4 073 270	650	71.47	7.14
CpAP2-L5	22	197 714 ~ 200 051	389	43.87	7.12
CpAP2-L6	26	6 547 145 ~ 6 551 504	447	49.63	5.88
CpAP2-L7	32	2 029 444 ~ 2 034 037	643	71.22	6.22
CpAP2-L8	70	2 623 638 ~ 2 627 212	479	54.38	6.17
CpAP2-L9	72	5 070 756 ~ 5 074 288	460	50.50	7.63
CpAP2-L10	86	4 575 244 ~ 4 577 540	394	44.47	6.62
CpAP2-L11	92	926 637 ~ 932 737	345	38.67	6.11
CpAP2-L12	120	4 203 257 ~ 4 209 225	650	71.21	6.32
CpAP2-L13	122	2 412 620 ~ 2 417 460	721	78.19	6.06
CpAP2-L14	140	668 231 ~ 673 284	403	45.23	8.38
CpAP2-L15	153	849 837 ~ 855 009	686	75.31	6.02
CpAP2-L16	156	1 808 018 ~ 1 817 462	472	52.60	6.61
CpAP2-L17	243	654 864 ~ 688 642	356	40.11	7.67
CpAP2-L18	463	3 517 149 ~ 3 524 013	496	54.80	6.03
CpAP2-L19	742	586 727 ~ 591 191	396	43.87	5.51
CpAP2-L20	752	2 116 052 ~ 2 120 180	477	51.37	8.07

名称Name	框Scaffold	位置Position	氨基酸数Amino acids	分子质量/kD Molecular weigh	理论等电点pI
CpAP2-L1	8	633 532 ~ 638 961	593	65.68	6.27
CpAP2-L2	12	6 666 416 ~ 6 670 043	501	55.82	6.29
CpAP2-L3	18	2 386 489 ~ 2 389 495	516	57.15	6.06
CpAP2-L4	18	4 068 635 ~ 4 073 270	650	71.47	7.14
CpAP2-L5	22	197 714 ~ 200 051	389	43.87	7.12
CpAP2-L6	26	6 547 145 ~ 6 551 504	447	49.63	5.88
CpAP2-L7	32	2 029 444 ~ 2 034 037	643	71.22	6.22
CpAP2-L8	70	2 623 638 ~ 2 627 212	479	54.38	6.17
CpAP2-L9	72	5 070 756 ~ 5 074 288	460	50.50	7.63
CpAP2-L10	86	4 575 244 ~ 4 577 540	394	44.47	6.62
CpAP2-L11	92	926 637 ~ 932 737	345	38.67	6.11
CpAP2-L12	120	4 203 257 ~ 4 209 225	650	71.21	6.32
CpAP2-L13	122	2 412 620 ~ 2 417 460	721	78.19	6.06
CpAP2-L14	140	668 231 ~ 673 284	403	45.23	8.38
CpAP2-L15	153	849 837 ~ 855 009	686	75.31	6.02
CpAP2-L16	156	1 808 018 ~ 1 817 462	472	52.60	6.61
CpAP2-L17	243	654 864 ~ 688 642	356	40.11	7.67
CpAP2-L18	463	3 517 149 ~ 3 524 013	496	54.80	6.03
CpAP2-L19	742	586 727 ~ 591 191	396	43.87	5.51
CpAP2-L20	752	2 116 052 ~ 2 120 180	477	51.37	8.07

基因1 Gene 1	基因2 Gene 2	非同义替换率Ka	同义替换率Ks	Ka/Ks	选择类型 Selection type	复制类型 Duplication type
CpAP2-L1	CpAP2-L11	0.59	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L3	CpAP2-L4	0.78	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L3	CpAP2-L8	0.20	0.66	0.30	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L3	CpAP2-L11	0.66	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L4	CpAP2-L7	0.19	0.82	0.24	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L4	CpAP2-L1	0.18	1.02	0.17	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L6	CpAP2-L11	0.67	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L8	CpAP2-L11	0.66	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L12	CpAP2-L3	0.72	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L12	CpAP2-L4	0.51	2.37	0.22	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L12	CpAP2-L8	0.66	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L12	CpAP2-L11	0.51	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L14	CpAP2-L10	0.16	0.73	0.22	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L14	CpAP2-L11	0.49	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L15	CpAP2-L11	0.55	2.48	0.22	纯化选择Purify selection	全基因组复制或片段复制WGD or SD
CpAP2-L16	CpAP2-L10	0.58	NaN	NaN	纯化选择Purify selection	全基因组复制或片段复制WGD or SD

Identification of the AP2 Subfamily Transcription Factors in Chimonanthus praecox and the Functional Study of CpAP2-L11

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株系Line	莲座叶数 Number of rosette leaves	时间/d Time			株高/cm Plant height
株系Line	莲座叶数 Number of rosette leaves	抽薹1 cm Bolting 1 cm	第1朵花开放 The first flower oping	第1个果荚形成 The first pod formation	株高/cm Plant height
野生型Wild type	10.3 ± 0.9 a	27.4 ± 0.9 a	30.6 ± 1.2 a	33.8 ± 1.1 a	25.04 ± 0.81 a
OE10-12	8.3 ± 0.9 b	25.5 ± 1.2 b	28.4 ± 1.4 b	30.9 ± 1.6 b	18.88 ± 0.88 b
OE9-5	7.9 ± 0.7 bc	25.0 ± 1.0 b	27.8 ± 1.0 b	30.3 ± 0.9 b	18.48 ± 0.47 b
OE7-6	7.4 ± 0.6 c	22.6 ± 1.1 c	25.6 ± 1.2 c	28.3 ± 1.4 c	17.06 ± 0.55 c

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