Expression and Enzyme Activity Identification of Cell Wall Invertase Gene SmCWIN6 from Red Pitaya

doi:10.13560/j.cnki.biotech.bull.1985.2025-0388

Abstract

Abstract:

Objective Cell wall invertase (CWIN) degrades sucrose into glucose and fructose, and acts as the key enzyme for sugar metabolism in higher plants. The physiological functions of CWINs in soluble sugar metabolism of red pitaya (Selenicereusmonacanthus) fruit were investigated, which provides a basis for regulating and improving fruit flavor and quality. Method Total protein from adult stem tissues and fruit pulp at various developmental stages was extracted to detect the CWIN enzyme activity; the sequence domains of SmCWIN family were analyzed; the expression patterns of SmCWINs in flower, stem, fruit peel and pulp tissues were detected by gene digital expression profiles and real-time fluorescent quantitative PCR. The subcellular localization of SmCWIN6 was detected by transiently transformed in tobacco mesophyll cells. By expressed in the baker’s yeast (Saccharomyces cerevisiae) and then total protein was extracted, the enzyme activity of SmCWIN6 was analyzed in vitro. Result The CWIN enzyme activity in adult stems of red pitaya was low, reached highest at 23 d after pollination in fruit, and then decreased significantly with fruit ripening. Of the red pitaya SmCWIN family, only SmCWIN6 contained domains and conserved amino acid residues related to sucrose degradation. SmCWIN6 was the only CWIN member that expressed in fruits and mainly expressed at 20-25 d after pollination; after then gradually down-regulated with fruit development, and weakly expressed at fruit ripening (30 d after pollination). Subcellular localization assay together with the plasmolysis experiment showed that SmCWIN6 protein was located into the apoplast space, possibly in the cell wall. After been expressed by yeast cells and detected in vitro, the SmCWIN6 protein degraded sucrose into glucose and fructose with the maximum enzyme activity at pH=3.0. Conclusion Red pitaya SmCWIN6 degrades sucrose to produce glucose and fructose in the apoplast, is expressed at 20-25 d after pollination in fruits, and involved in the extracellular soluble sugars metabolism during fruit development.

Key words: pitaya, cell wall invertase, apoplast, yeast expression, sucrose degradation

ZHENG Qian-ming, YAN Shuang, XIE Pu, WANG Hong-lin. Expression and Enzyme Activity Identification of Cell Wall Invertase Gene SmCWIN6 from Red Pitaya[J]. Biotechnology Bulletin, 2025, 41(8): 267-275.

Figures/Tables 4

References 38

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名称 Name	序列 Sequence （5′-3′）
CWIN6-qRT-F	GGAATGGGTCTTGATACCTCAG
CWIN6-qRT-R	GCAGTGGTTGGATCCCTAAA
β-ACT-F	CTTCCATACCAATGAATGAGG
β-ACT-R	AACCGCCAAGAGTAGTTCTG
CWIN6-GFP-F	ACTAGGGTCTCGCACCATGGGGTTTTATTCTTTGATGATGAT
CWIN6-GFP-R	ACTAGGGTCTCTCGCCAGCTATGCTAGCTTTCTTCATGCTCC
CWIN6-Y-F	TGGATCCCCCGGGCTGCAGGAATTCATGGGGTTTTATTCTTTGA
CWIN6-Y-R	TTGGGTACCGGGCCCCCCCTCGAGCTAAGCTATGCTAGCTTTCT

名称 Name	序列 Sequence （5′-3′）
CWIN6-qRT-F	GGAATGGGTCTTGATACCTCAG
CWIN6-qRT-R	GCAGTGGTTGGATCCCTAAA
β-ACT-F	CTTCCATACCAATGAATGAGG
β-ACT-R	AACCGCCAAGAGTAGTTCTG
CWIN6-GFP-F	ACTAGGGTCTCGCACCATGGGGTTTTATTCTTTGATGATGAT
CWIN6-GFP-R	ACTAGGGTCTCTCGCCAGCTATGCTAGCTTTCTTCATGCTCC
CWIN6-Y-F	TGGATCCCCCGGGCTGCAGGAATTCATGGGGTTTTATTCTTTGA
CWIN6-Y-R	TTGGGTACCGGGCCCCCCCTCGAGCTAAGCTATGCTAGCTTTCT

名称 Name	基因ID Gene ID	定位 Location	位置 Position （aa）
名称 Name	基因ID Gene ID	定位 Location	信号肽 Signal peptide	GH32N结构域 GH32N domain	GH32C结构域 GH32C domain
SmCWIN1	HU02G02988.1	细胞壁Cell wall	1-19	48-363	366-559
SmCWIN2	HU02G03468.1	细胞壁Cell wall	1-19	48-342	345-538
SmCWIN3	HU07G01984.1	细胞壁Cell wall	1-27	46-366	369-562
SmCWIN4	HU07G01985.1	细胞质Cytoplasm	-	10-327	330-523
SmCWIN5	HU07G01986.1	细胞壁Cell wall	1-34	61-378	381-575
SmCWIN6	HU09G00322.1	细胞壁Cell wall	1-31	61-380	383-576
SmCWIN7	HU09G01009.1	细胞质Cytoplasm	-	7-104	107-287

名称 Name	基因ID Gene ID	定位 Location	位置 Position （aa）
名称 Name	基因ID Gene ID	定位 Location	信号肽 Signal peptide	GH32N结构域 GH32N domain	GH32C结构域 GH32C domain
SmCWIN1	HU02G02988.1	细胞壁Cell wall	1-19	48-363	366-559
SmCWIN2	HU02G03468.1	细胞壁Cell wall	1-19	48-342	345-538
SmCWIN3	HU07G01984.1	细胞壁Cell wall	1-27	46-366	369-562
SmCWIN4	HU07G01985.1	细胞质Cytoplasm	-	10-327	330-523
SmCWIN5	HU07G01986.1	细胞壁Cell wall	1-34	61-378	381-575
SmCWIN6	HU09G00322.1	细胞壁Cell wall	1-31	61-380	383-576
SmCWIN7	HU09G01009.1	细胞质Cytoplasm	-	7-104	107-287

蛋白 Protein	位点/结构域位置 Site or domain position （aa）
蛋白 Protein	63	65-69	90	125	191-193	245-249	282	285
AtcwINV1	W	NDPNG	W	W	RDP	WECPD	D	K
SmCWIN1	W	N - - - G	W	Y	RDP	WECPD	V	-
SmCWIN2	W	N - - - G	W	Y	RDP	WECPD	V	-
SmCWIN3	W	NDPNG	W	W	RDP	WECPD	F	S
SmCWIN4	W	N - - - G	W	W	RDP	WECPD	F	L
SmCWIN5	W	N - - - A	W	W	RDP	WECPD	D	K
SmCWIN6	W	NDPNG	W	W	RDP	WECPD	D	K
SmCWIN7	-	- - - - -	-	-	- - -	- - - - -	M	D