红肉火龙果细胞壁转化酶基因SmCWIN6的表达和酶活性鉴定

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

摘要/Abstract

摘要：

目的细胞壁转化酶（cell wall invertase, CWIN）分解蔗糖生成葡萄糖和果糖，是高等植物糖代谢关键酶。探讨CWINs在红肉火龙果（Selenicereus monacanthus）果实可溶性糖代谢中的生理功能，为调控和改良果实风味和品质提供基础。方法提取成年态茎和各发育时期果肉的总蛋白，检测CWIN酶活性，分析SmCWIN家族序列结构域，利用基因数字表达谱和实时荧光定量PCR检测SmCWINs在花、茎、果皮和果肉中的表达模式，通过瞬时转化烟草叶肉细胞进行SmCWIN6的亚细胞定位，利用酿酒酵母表达并提取总蛋白，离体检测SmCWIN6的酶活性。结果红肉火龙果成年态茎的CWIN酶活性较低，在果实授粉后23 d时最高，此后，随果实成熟明显下降。红肉火龙果SmCWIN家族仅SmCWIN6含有蔗糖分解相关的结构域和保守的氨基酸残基。SmCWIN6在果实中表达，主要在授粉后20-25 d表达，此后，随果实发育逐渐下调表达，果实成熟时（授粉后30 d）表达微弱。亚细胞定位结合质壁分离试验表明，SmCWIN6蛋白定位于质外体，可能位于细胞壁。经酵母表达获得重组蛋白并离体检测，SmCWIN6可分解蔗糖产生葡萄糖和果糖，在pH = 3.0具有最大酶活性。结论 SmCWIN6在质外体分解蔗糖产生葡萄糖和果糖，主要在果实授粉后20-25 d表达，参与果实发育期间胞外可溶性糖代谢。

关键词: 火龙果, 细胞壁转化酶, 质外体, 酵母表达, 蔗糖分解

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

郑乾明, 晏霜, 解璞, 王红林. 红肉火龙果细胞壁转化酶基因SmCWIN6的表达和酶活性鉴定[J]. 生物技术通报, 2025, 41(8): 267-275.

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.

图/表 4

参考文献 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