蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达

doi:10.3969/j.issn.1004-1524.2022.08.14

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1703-1712.DOI: 10.3969/j.issn.1004-1524.2022.08.14

蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达

许申平(), 张燕, 梁芳, 蒋素华, 牛苏燕, 崔波, 袁秀云^*()

郑州师范学院生物工程研究中心,河南郑州450044

收稿日期:2021-10-22 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 袁秀云
作者简介:*袁秀云,E-mail: yuanxiuyun@zznu.edu.cn
许申平(1983—),女,河南郑州人,博士,副教授,研究方向为兰花栽培生理与分子生物学。E-mail: shenpingxu@163.com
基金资助:
河南省科技攻关计划(212102110116);河南省科技攻关计划(222102110470)

Cloning of PhaSEP3 gene in Phalaenopsis and its expression in floral organ mutants

XU Shenping(), ZHANG Yan, LIANG Fang, JIANG Suhua, NIU Suyan, CUI Bo, YUAN Xiuyun^*()

Institute of Bioengineering, Zhengzhou Normal University, Zhengzhou 450044, China

Received:2021-10-22 Online:2022-08-25 Published:2022-08-26
Contact: YUAN Xiuyun

摘要/Abstract

摘要：

E类MADS-box是花器官发育分子模型中必不可少的基因,通过突变体研究其表达模式将为深入理解兰科植物花器官的分子机理与完善花发育调控理论提供依据。采用RACE(rapid-amplification of cDNA ends)技术从蝴蝶兰花瓣中克隆了一个E类MADS-box基因PhaSEP3(GenBank登录号为MZ436812),并采用实时荧光定量PCR(qRT-PCR)分析了该基因在蝴蝶兰不同组织和5种突变体中的表达水平。结果表明,该基因cDNA全长为1 236 bp,具有753 bp的开放阅读框(ORF),可编码250个氨基酸,其C端具有SEPⅠ和SEPⅡ基序。系统进化分析显示,该基因编码蛋白质与蝴蝶兰属的PeSEP3和AGL9亲缘关系最近。组织特异表达分析表明,PhaSEP3基因主要在生殖器官和授粉后子房中表达;在不同突变体中,PhaSEP3基因在侧萼唇瓣化突变体的萼片和唇瓣中表达水平显著升高;在退化雄蕊瓣化突变体的侧瓣、唇瓣和子房中表达水平显著降低,在蕊柱中的表达水平显著升高;在侧瓣唇瓣化突变体的侧瓣和唇瓣中表达水平显著升高;在侧瓣退化突变体的侧瓣中表达水平显著降低,而在蕊柱和子房中表达水平显著升高;在侧瓣雄化突变体中,该基因的表达水平在萼片和侧瓣中均显著升高。分析认为,PhaSEP3基因主要调控蝴蝶兰花器官各轮组织与授粉后子房的发育,在突变体花器官中,PhaSEP3类基因可能与其他花发育基因互作参与花器官形态的发育调控。该研究结果为进一步理解兰科植物花器官多样性的调控机理提供了资料。

关键词: 蝴蝶兰, SEPALLATA基因, 花器官, 突变体

Abstract:

The class E MADS-box is one of the essential genes in molecular models of floral organ development. Studying its expression pattern in mutants will provide basis for understanding the molecular mechanism of flower organs in orchids and improving the regulation theory of flower development. In this study, a Class E MADS-box gene PhaSEP3 (GenBank accession number: MZ436812) was cloned from petal in Phalaenopsis using the method of rapid-amplification of cDNA ends (RACE), and the expression level of this gene in different tissues and five mutants of Phalaenopsis was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the cDNA full-length of PhaSEP3 gene was 1 236 bp with open reading frame (ORF) of 753 bp encoding 250 amino acids, and with SEPⅠ and SEPⅡ motifs in its C-terminal. The phylogenetic analysis indicated that the protein was highly homologous to PeSEP3 and AGL9 from Phalaenopsis. The expression analysis showed that PhaSEP3 gene was expressed mainly in the reproductive organs and ovary after pollination. In different floral organ mutants, the expression level of the PhaSEP3 gene was increased significantly in sepal and lip in the lip-like lateral sepal mutant. The expression level of this gene was decreased significantly in petal, lip and ovary, while was increased significantly in column in the mutant of degenerated stamen converting petals. The expression level of this gene was increased significantly in petal and lip in the lip-like lateral petals mutant, and was decreased significantly in petal along with increased significantly in column and ovary in the petal degeneration mutant. However, the expression level of PhaSEP3 gene was increased significantly in the sepal and petal in the mutant of lateral petals with anther. It was concluded that PhaSEP3 gene mainly regulated the development of floral tissues and ovary after pollination in Phalaenopsis. In the floral organ mutants, the class E gene might interact with other floral development genes to regulate the morphological development of floral organ. The results provided further understandings of the molecular mechanism of floral organ diversity in orchid.

Key words: Phalaenopsis, SEPALLATA gene, floral organ, mutant

中图分类号:

S682.31

许申平, 张燕, 梁芳, 蒋素华, 牛苏燕, 崔波, 袁秀云. 蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达[J]. 浙江农业学报, 2022, 34(8): 1703-1712.

XU Shenping, ZHANG Yan, LIANG Fang, JIANG Suhua, NIU Suyan, CUI Bo, YUAN Xiuyun. Cloning of PhaSEP3 gene in Phalaenopsis and its expression in floral organ mutants[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1703-1712.

图/表 8

图1 蝴蝶兰花部结构

Fig.1 Floral structure of Phalaenopsis

图2 蝴蝶兰突变体花器官 A,内山姑娘;B,S1024;C,火鸟;D,富乐夕阳;E,婚宴。

Fig.2 Mutant flower organs of Phalaenopsis A, Ney Shai Gu Niang; B, Red Coral S1024; C, Sogo Beach; D, Fuller's Sunset; E, Wedding Promenade.

表1 引物信息

Table 1 Information of primers

引物类别 Primer category	引物名称 Primer name	引物序列 Primer sequence (5'-3')
保守片段扩增	SEP-F	GMTGAAGAKGATYGAGAACAAGA
Conserved fragment amplification	SEP-R	TAGCTGGTCRAGCATSAACTG
3'RACE	outer	AGCAATTAGAGAGGCAACTGGAC
	inner	ATCAAGGGAGACCCAGAATAGTC
5'RACE	outer	TCTTTGCAAGGCTTCTACTCTGGA
	inner	TACTTGTGTTGCTGCAGAACTCG
ORF扩增	ORF-F	ATGGGAAGAGGGAGAGTGGAG
ORF amplification	ORF-R	CATAATGTAATAAGGGGGAAAGCC
qRT-PCR	qSEP-F	GAGCCCTAAAGATAAGGTTTGAGG
	qSEP-R	ACTGTTGAAGTAGTCGCCGTTG
内参基因	Act-F	GCAGCATGAAGATCAAGGTGG
Reference genes	Act-R	GCCTTAGAAATCCACATCTGTTG

图3 蝴蝶兰PhaSEP3基因扩增 A,保守片段扩增;B,3' RACE;C,5' RACE;D,开放读码框扩增。1,保守片段;2,3' RACE片段;3,5' RACE片段;4,开放阅读框片段;M,DL2000 分子量标记。

Fig.3 Amplification of PhaSEP3 gene in Phalaenopsis A, Conserved region amplification; B, 3' RACE; C, 5' RACE; D, ORF amplification. 1, Conserved region fragment; 2, Fragment of 3' RACE; 3, Fragment of 5' RACE; 4, Fragment of ORF; M, DL2000 marker.

图4 PhaSEP3基因编码的氨基酸序列与其他植物SEP3蛋白的氨基酸序列比对

Fig.4 Multiple sequence alignment of amino acid sequences of PhaSEP3 with other SEP3s from different species

图5 蝴蝶兰PhaSEP3蛋白的系统进化分析

Fig.5 Phylogenetic analysis of PhaSEP3 protein in Phalaenopsis

图6 PhaSEP3基因在不同组织与子房发育中的表达

Fig.6 Expression of PhaSEP3 in different tissues and developing ovary

图7 PhaSEP3基因在蝴蝶兰花器官突变体中的表达 A,S1024;B,内山姑娘;C,火鸟;D,婚宴;E,富乐夕阳;*表示差异显著(P<0.05)。

Fig.7 Expression of PhaSEP3 in floral organ mutants of Phalaenopsis A, Red Coral S1024; B, Ney Shai Gu Niang; C, Sogo Beach; D, Wedding Promenade; E, Fuller's Sunset; * indicated significant difference (P<0.05).

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蝴蝶兰PhaSEP3基因的克隆及其在突变体中的表达

Cloning of PhaSEP3 gene in Phalaenopsis and its expression in floral organ mutants

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