蜡梅AP2亚家族转录因子鉴定及CpAP2-L11功能研究

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

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

蜡梅AP2亚家族转录因子鉴定及CpAP2-L11功能研究

田明康¹, 徐智祥¹, 刘秀群², 眭顺照¹, 李名扬¹, 李志能¹^,^*()

1.西南大学园艺园林学院，南方山地园艺学教育部重点实验室，重庆市花卉工程技术研究中心，重庆 400715
2.华中农业大学园艺林学学院，园艺植物生物学教育部重点实验室，武汉 430070

收稿日期:2022-05-19 修回日期:2022-11-05 出版日期:2023-02-25 发布日期:2023-03-06
通讯作者: *（E-mail：znli@swu.edu.cn）
基金资助:
重庆市自然科学基金项目(cstc2020jcyj-msxmX1014);中央高校基本科研业务费项目(XDJK2020B059)

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）

摘要/Abstract

摘要：

对蜡梅（Chimonanthus praecox）AP2（Apetala2，AP2）亚家族成员进行全基因组鉴定，并分析其在花发育过程中的表达模式；研究CpAP2-L11的表达特异性，过表达拟南芥（Arabidopsis thaliana）并观察其表型。共鉴定出20个蜡梅AP2亚家族转录因子，其中euAP2、basalANT和euANT组分别有5、6和9个成员，且euAP2组全部成员无miR172结合位点。共线性分析发现有12个成员形成了16对复制基因，并在进化过程中受纯化选择。多数AP2亚家族成员在4、5月花芽中高表达，在花芽进行需冷量积累的过程或后期低表达，仅CpAP2-L11在花芽需冷量积累到570 CU（Chill units，CU）的始花期高表达，同时CpAP2-L11在蜡梅幼果、外轮花被片和雄蕊中高表达，在茎、叶中低表达，并受高温和低温诱导表达量降低。拟南芥异源表达CpAP2-L11，抽薹时间显著提前，且FT、SOC1、LFY和AP1基因表达量显著升高。蜡梅CpAP2-L11可能参与低温诱导打破蜡梅休眠导致寒冬开花及春季花芽分化，且促进过表达拟南芥早开花。

关键词: 蜡梅, AP2亚家族, 转录因子, 花发育

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

中图分类号:

S685.99

田明康, 徐智祥, 刘秀群, 眭顺照, 李名扬, 李志能. 蜡梅AP2亚家族转录因子鉴定及CpAP2-L11功能研究[J]. 园艺学报, 2023, 50(2): 382-396.

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.

图/表 12

参考文献 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

蜡梅AP2亚家族转录因子鉴定及CpAP2-L11功能研究

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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蜡梅AP2亚家族转录因子鉴定及CpAP2-L11功能研究

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

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