黄金树B类MADS-box基因表达特征分析

doi:10.11983/CBB15053

摘要/Abstract

摘要： 采用同源克隆技术, 从黄金树(Catalpa speciosa)花芽中克隆得到B类MADS-box基因CaspAP3和CaspPI的cDNA序列。序列分析表明, CaspAP3基因cDNA序列的完整开放阅读框(ORF)为696 bp, 编码231个氨基酸残基; CaspPI基因cDNA序列的ORF为639 bp, 编码212个氨基酸残基。蛋白质序列相似性比对和分子系统发生分析表明, CaspAP3属于AP3/DEF进化支, 其C末端包含保守的euAP3基序和PI-derived基序, 而CaspPI聚类于PI/GLO进化支, 其C末端包含保守的PI基序。半定量RT-PCR分析结果表明, CaspAP3和CaspPI基因均仅在花瓣和雄蕊中表达。实时荧光定量PCR分析表明, CaspAP3和CaspPI基因在花瓣和雄蕊原基分化期至成熟期均有表达, 这2个基因在雄蕊中表达高峰出现的时间均早于花瓣; 且花瓣中的CaspAP3和CaspPI基因表达高峰均出现在快速伸长阶段; 这与花瓣和雄蕊的形态发育阶段相吻合。

关键词: 黄金树, 花发育, B类基因, 基因克隆, 基因表达

Abstract: The cDNA sequence of B-class MADS-box genes CaspAP3 and CaspPI involved in flower development were isolated from the flower bud of Catalpa speciosa by homology cloning. The open reading frame (ORF) of CaspAP3 was 696 bp, encoding 231 amino acids, and the ORF of CaspPI was 639 bp, encoding 212 amino acids. Molecular phylogeny analysis and protein sequence alignment suggested that CaspAP3 groups with the AP3/DEF lineages and the C domain of CaspAP3 contained two conserved motifs, euAP3 and PI-derived motifs. However, CaspPI grouped with the PI/GLO lineages, and the C domain of CaspPI contained a conserved PI motif. CaspAP3 and CaspPI mRNA expression was mainly concentrated in petals and stamens but occurred at the primordium to mature stages of petals and stamens during morphological differentiation of organs. The expression of both factors peaked earlier in stamens than in petals, and the peak expression for both in petals appeared at the stage of rapid elongation. Meanwhile, the time of expression was the same with morphological differentiation of petals and stamens.

Key words: Catalpa speciosa, flower development, B-class MADS-box genes, gene cloning, the expression of gene

景丹龙, 夏燕, 张守攻, 王军辉. 黄金树B类MADS-box基因表达特征分析. 植物学报, 2016, 51(2): 210-217.

Danlong Jing, Yan Xia, Shougong Zhang, Junhui Wang. Expression Analysis of B-class MADS-box Genes from Catalpa speciosa. Chinese Bulletin of Botany, 2016, 51(2): 210-217.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15053

https://www.chinbullbotany.com/CN/Y2016/V51/I2/210

图/表 7

表1 引物序列

Table 1 The sequences of primers

Primer name	Primer sequences (5′→ 3′)
FLAP3F	GCAAACTCAAATCTTGAAAATC
FLAP3R	CACATTATGCGACAGAATTCAT
FLPIF	GAGAACCAAATTAAGAGAAAGACCA
FLPIR	GCACCAAGACAACCACATACGTA
RTactinF	ATGATGCTCCAAGAGCTGTG
RTactinR	AGCAAGATCGAGACGTAGGA
RTAP3F	TACATCAGCCCCATTATAACGA
RTAP3R	TTATTCAAGCAAGGCAAACGTG
RTPIF	ACCACAAGTTGTCTGGGAAGAG
RTPIR	AATCTGCAACTCCCTGATGATC
qRTactinF	GATGATGCTCCAAGAGCTGT
qRTactinR	TCCATATCATCCCAGTTGCT
qRTAP3F	CTTGAAGAAGCTGAAGGAGGTT
qRTAP3R	CTTGCTGGTATCAATCTGGTTG
qRTPIF	GAGAATGACAGCATGCAGATTG
qRTPIR	ATTATCGTCCATTGGATCCAGA

表2 黄金树的花芽长度

Table 2 The size of the flower buds of Catalpa speciosa

Length of flower buds (mm)	The stages and morphology of C. speciosa
1−3	In early stage of floral buds formation, the petals and stamens differentiated and elongated rapidly
4−13	At flower buds elongation stage, the size of petals and stamens increased rapidly
30−55	At flowering phase, the stamens reached mature stage, and the petals completely opened

图1 AP3和PI亚家族基因分子系统发生分析

Figure 1 Phylogenetic analysis of AP3 and PI subfamily

图2 CaspAP3和CaspPI基因编码的氨基酸序列与AP3和PI亚家族相关蛋白质序列的比较比对中的第1个下划线区域表示MADS结构域, 第2个下划线表示K区, 在M区和K区之间的是I区, PI-derived、euAP3和PI基序用黑框标出。

Figure 2 Comparison of deduced amino acid sequences encoded by CaspAP3 and CaspPI, and related members of AP3 and PI subfamily The first underlined regions represent the MADS domain and the second K domain, the region between the MADS- and K-domain is I domain. The PI-derived motif, euAP3 motif and PI motif are boxed.

图3 黄金树不同组织中CaspAP3和CaspPI基因的半定量RT-PCR分析 ste: 茎; j-le: 幼叶; m-le: 成熟叶; sep: 萼片; pet: 花瓣; sta: 雄蕊; car: 雌蕊; cap: 蒴果; see: 种子

Figure 3 Semi-quantitative RT-PCR of CaspAP3 and CaspPI gene in different tissues of Catalpa speciosa ste: Stem; j-le: Juvenile-leaf; m-le: Mature leaves; sep: Sepal; pet: Petal; sta: Stamen; car: Carpel; cap: Capsule; see: Seed

图4 黄金树花器官的形态学观察 (A)−(C) 1−3 mm的花芽, 刚分化出的花瓣和雄蕊; (D)−(F) 4−13 mm花芽的快速生长期; (G), (H) 30−55 mm的开花期花芽。pe: 花瓣; sta: 雄蕊。Bar=500 μm

Figure 4 The characterization of the floral organ in Catalpa speciosa (A)−(C) The flower buds of 1−3 mm, the newly differentiated petal and stamen; (D)−(F) The flower buds of 4−13 mm, the sizes of petal and stamen increased rapidly; (G), (H) The flower buds of 30−55 mm, the petal and stamen reached mature stage, and the flowers reached anthesis. pe: Petal; sta: Stamen. Bar=500 μm

图5 黄金树CaspAP3和CaspPI基因在花芽不同发育时期的表达量 (A) CaspAP3和CaspPI基因在黄金树花芽分化时期的相对表达量; (B) CaspAP3和CaspPI基因在花芽延伸和开花时期的相对表达量

Figure 5 Quantitative real-time PCR analysis of CaspAP3 and CaspPI expressed in floral buds of Catalpa speciosa at different developmental stages (A) Relative expression patterns of CaspAP3 and CaspPI at differentiation stage of flowers; (B) Relative expression patterns of CaspAP3 and CaspPI at elongation and flowering stages of flowers

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