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

doi:10.3969/j.issn.1004-1524.2022.08.14

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (8): 1703-1712.DOI: 10.3969/j.issn.1004-1524.2022.08.14

• Horticultural Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S682.31

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.

Figures/Tables 8

Fig.1 Floral structure of Phalaenopsis

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.

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

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.

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

Fig.5 Phylogenetic analysis of PhaSEP3 protein in Phalaenopsis

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

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

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