pH指示剂法检测火龙果糖转运蛋白HpSWEET1a和HpSWEET4a的转运活性

doi:10.16420/j.issn.0513-353x.2023-0849

摘要/Abstract

摘要：

针对酵母生长互补方法的缺点，利用pH指示剂指示酵母胞外酸化，建立一种检测候选SWEET（sugars will eventually be exported transporter）基因转运活性的新方法。使用0.006%溴甲酚紫（BCP）作为pH指示剂，获得不同pH值下波长589 nm和432 nm的光吸收值A₅₈₉和A₄₃₂，建立pH值与A₅₈₉/A₄₃₂比值（R_589/432）的相关性函数方程。从火龙果（Hylocereus polyrhizus）果实中分离HpSWEET1a和HpSWEET4a基因，两者编码蛋白序列与拟南芥AtSWEET1和AtSWEET4分别具有66.67%和55.36%的一致性，分别属于SWEET Ⅰ和Ⅱ类成员。利用酵母糖吸收缺陷株系EBY.VW4000表达HpSWEET1a和HpSWEET4a基因，糖饥饿处理后添加葡萄糖或果糖，HpSWEET1a、HpSWEET4a和AtSWEET1（At1g21460，阳性对照）均促使酵母胞外BCP颜色在3 ~ 16 h由紫色变为黄色，说明发生明显的胞外酸化。进一步的试验表明HpSWEET1a、HpSWEET4a和AtSWEET1促进酵母胞外pH值在3 ~ 16 h下降约1.00，载体对照仅下降约0.10。酵母胞外酸化和pH值的明显下降，证明了HpSWEET1a、HpSWEET4a与AtSWEET1功能类似，均具有葡萄糖和果糖转运活性。使用基于超微量分光光度的比色法检测R_589/432值，进一步简化检测步骤和缩短时间。添加葡萄糖后培养0.5 h，HpSWEET1a的R_589/432值的下降水平远大于载体对照，证明该方法有效。本研究中建立的方法从耗时、准确性、操作和通量等方面均优于目前常用的酵母生长互补检测，可广泛应用于定位细胞膜的SWEET转运蛋白研究。

关键词: 火龙果, 糖转运蛋白, pH指示剂, 酵母表达, 比色法

Abstract:

Aiming at the defects of yeast growth complementation method，a new method for detecting the transport activity of candidate SWEET（sugars will eventually be exported transporter）genes was established by using pH indicator to indicate yeast extracellular acidification. In this study，using 0.006% bromocresol violet（BCP）as the pH indicator，the photoabsorption values A₅₈₉ and A₄₃₂under the wavelength of 589 nm and 432 nm at different pH values were measured，and the correlation function equation between pH value and A₅₈₉/A₄₃₂ ratio（R_589/432）was established. HpSWEET1a and HpSWEET4a genes were isolated from pitaya（Hylocereus polyrhizus）fruit，which encoded amino acid sequences sharing identities of 66.67% and 55.36% with Arabidopsis thaliana AtSWEET1 and AtSWEET4，and belonged to the SWEET Ⅰ and Ⅱ clade，respectively. When expressed in the EBY.VW4000 yeast strain with impaired sugar uptake activity，and subjected to sugar starvation treatment followed by addition of glucose or fructose，the overexpression of HpSWEET1a，HpSWEET4a and AtSWEET1（At1g21460，positive control）caused in the extracellular BCP color from purple to yellow in 3-16 h. This change indicated the occurrence of extracellular acidification. Further calculation revealed that HpSWEET1a，HpSWEET4a and AtSWEET1 caused a decline of 1.00 in the extracellular pH value of yeast cells within 3-16 h，while the vector control only showed a decrease of 0.10. The observed extracellular acidification of yeast cells and the significant decrease in pH value demonstrated that HpSWEET1a and HpSWEET4a，similar as AtSWEET1，exhibited the glucose and fructose transport activity. To streamline the procedure and reduce time，the colorimetric method were employed based on ultra-micro spectrophotometer to measure the R_589/432 value. Following the addition of glucose for 0.5 h，the R_589/432 value reduction with HpSWEET1a was significantly higher compared to that of the vector control，indicating the method’s efficacy. The method developed in this study is superior to the commonly used yeast growth complementary detection in terms of time-consuming，accuracy，operation and flux. It also has a wide range of applications for studying the cell membrane-localized SWEET transporters.

Key words: Hylocereus polyrhizus, sugar transporter, pH indicator, yeast expression, colorimetric method

郑乾明, 王小柯, 王红林, 周嘉, 解璞, 马玉华. pH指示剂法检测火龙果糖转运蛋白HpSWEET1a和HpSWEET4a的转运活性[J]. 园艺学报, 2024, 51(9): 2195-2206.

ZHENG Qianming, WANG Xiaoke, WANG Honglin, ZHOU Jia, XIE Pu, MA Yuhua. The Transport Activity Detection of Pitaya Sugar Transporters HpSWEET1a and HpSWEET4a by pH Indicator Method[J]. Acta Horticulturae Sinica, 2024, 51(9): 2195-2206.

图/表 9

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引物 Primer	序列（5′-3′） Sequence
AtSWEET1-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAACATCGCTCACAC
AtSWEET1-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGTTAAACTTGAAGGTCTT
HpSWEET1a-ORF-F	TAACGAACAAGAAATATAAG
HpSWEET1a-ORF-R	ATCATACATTGACATACAGA
HpSWEET1a-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAATATTCCCCATTT
HpSWEET1a-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGCTAGGCATGCCCAGTT
HpSWEET4a-ORF-F	CTGCCTCTACGGTGCTAAGG
HpSWEET4a-ORF-R	ACTGTGATTATTCTCGCGCA
HpSWEET4a-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAAACACTCCCTGTT
HpSWEET4a-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGTCATGCTGGTGGTGGCT

引物 Primer	序列（5′-3′） Sequence
AtSWEET1-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAACATCGCTCACAC
AtSWEET1-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGTTAAACTTGAAGGTCTT
HpSWEET1a-ORF-F	TAACGAACAAGAAATATAAG
HpSWEET1a-ORF-R	ATCATACATTGACATACAGA
HpSWEET1a-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAATATTCCCCATTT
HpSWEET1a-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGCTAGGCATGCCCAGTT
HpSWEET4a-ORF-F	CTGCCTCTACGGTGCTAAGG
HpSWEET4a-ORF-R	ACTGTGATTATTCTCGCGCA
HpSWEET4a-Y-F	GTGGATCCCCCGGGCTGCAGGAATTCATGAAACACTCCCTGTT
HpSWEET4a-Y-R	AATTGGGTACCGGGCCCCCCCTCGAGTCATGCTGGTGGTGGCT

时间/h Time	基因Gene	麦芽糖Maltose	葡萄糖Glucose	果糖Fructose
1	pDR196（对照Control）	-0.7646 ± 0.0059 b	-0.0141 ± 0.0024 a	—
	AtSWEET1	-0.6568 ± 0.0126 a	-0.6159 ± 0.0126 e	—
2	pDR196（对照Control）	-0.9148 ± 0.0265 c	-0.0396 ± 0.0041 b	-0.0270 ± 0.0038 a
	AtSWEET1	-0.9339 ± 0.0040 c	-0.9204 ± 0.0017 f	-0.1811 ± 0.0053 b
3	pDR196（对照Control）	-0.9749 ± 0.0060 d	-0.0574 ± 0.0042 c	—
	AtSWEET1	-1.0190 ± 0.0005 e	-1.0977 ± 0.0037 g	—
9	pDR196（对照Control）	—	—	-0.0221 ± 0.0056 a
	AtSWEET1	—	—	-0.2676 ± 0.0105 c
16	pDR196（对照Control）	-0.9781 ± 0.0017 d	-0.1490 ± 0.0107 d	-0.0229 ± 0.0156 a
	AtSWEET1	-1.0242 ± 0.0007 f	-1.1392 ± 0.0017 h	-0.9844 ± 0.0363 d

时间/h Time	基因Gene	麦芽糖Maltose	葡萄糖Glucose	果糖Fructose
1	pDR196（对照Control）	-0.7646 ± 0.0059 b	-0.0141 ± 0.0024 a	—
	AtSWEET1	-0.6568 ± 0.0126 a	-0.6159 ± 0.0126 e	—
2	pDR196（对照Control）	-0.9148 ± 0.0265 c	-0.0396 ± 0.0041 b	-0.0270 ± 0.0038 a
	AtSWEET1	-0.9339 ± 0.0040 c	-0.9204 ± 0.0017 f	-0.1811 ± 0.0053 b
3	pDR196（对照Control）	-0.9749 ± 0.0060 d	-0.0574 ± 0.0042 c	—
	AtSWEET1	-1.0190 ± 0.0005 e	-1.0977 ± 0.0037 g	—
9	pDR196（对照Control）	—	—	-0.0221 ± 0.0056 a
	AtSWEET1	—	—	-0.2676 ± 0.0105 c
16	pDR196（对照Control）	-0.9781 ± 0.0017 d	-0.1490 ± 0.0107 d	-0.0229 ± 0.0156 a
	AtSWEET1	-1.0242 ± 0.0007 f	-1.1392 ± 0.0017 h	-0.9844 ± 0.0363 d

基因 Gene	转录本 Transcript	ORF/bp	跨膜结构域数量 TMD number	FPKM
基因 Gene	转录本 Transcript	ORF/bp	跨膜结构域数量 TMD number	果实 Fruit	茎 Stem
HpSWEET1a	c31195_g1	789	7	4.66	9.69
HpSWEET4a	c58868_g1	729	7	0.87	0.10