甜瓜果实发育相关SWEET糖转运蛋白基因的鉴定与功能初步分析

doi:3969/j.issn.1004-1524.2023.02.08

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (2): 308-318.DOI: 3969/j.issn.1004-1524.2023.02.08

甜瓜果实发育相关SWEET糖转运蛋白基因的鉴定与功能初步分析

李苹芳¹^,²(), 姚协丰²^,^*(), 徐锦华², 朱凌丽², 羊杏平²^,^*()

1.浙江开放大学教学中心农学教研部, 浙江杭州 310012
2.江苏省高效园艺作物遗传改良重点实验室,江苏省农业科学院蔬菜研究所, 江苏南京 210014

收稿日期:2022-05-20 出版日期:2023-02-25 发布日期:2023-03-14
通讯作者: 姚协丰,羊杏平
作者简介:羊杏平,E-mail: 1394654153@qq.com
*姚协丰,E-mail:yaoxiefeng@jaas.ac.cn;
李苹芳(1982—),女,江苏苏州人,博士,副研究员,研究方向为瓜类蔬菜栽培和育种,农民大学生培养。E-mail: 621856792@qq.com
基金资助:
国家自然科学基金(31872130);2021年度高校国内访问学者教师专业发展项目(FX2021220);国家西甜瓜产业体系(CARS-25)

Identification and preliminary functional characterization of SWEET sugar transporters involved in fruit development of melon (Cucumis melo L.)

LI Pingfang¹^,²(), YAO Xiefeng²^,^*(), XU Jinhua², ZHU Lingli², YANG Xingping²^,^*()

1. Department of Agriculture, Teaching and Researching Center, Zhejiang Open University, Hangzhou 310012, China
2. Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Received:2022-05-20 Online:2023-02-25 Published:2023-03-14
Contact: YAO Xiefeng,YANG Xingping

摘要/Abstract

摘要：

甜瓜是我国,也是世界上最重要的夏令水果之一。甜瓜果实内所含碳水化合物的种类和数量很大程度上决定其品质和产量。SWEET(sugars will eventually be exported transporters)糖转运蛋白具有运输葡萄糖和其他寡糖的功能,最近研究表明,SWEET糖转运蛋白在果实发育中可能起调控作用。本研究从甜瓜基因组中鉴定获得18个SWEETs糖转运蛋白基因,进一步通过RT-PCR并结合实时荧光定量PCR(quantitative real-time PCR,qPCR)方法,筛选到3个SWEETs基因在整个果实发育期内或某个发育时期表达量较高。亚细胞定位显示,两个SWEETs基因(CmSWEET3,CmSWEET7a)定位在细胞膜上。进一步通过酵母表达发现,甜瓜CmSWEET7a在体外具有转运葡萄糖和果糖的功能。本研究为揭示SWEET糖转运蛋白在甜瓜果实发育过程的调控作用奠定了基础。

关键词: 甜瓜, 果实, 糖转运, SWEETs

Abstract:

Melon (Cucumis melo L.) is one of the most important summer fruits in China and worldwide. The quality and yield of melon fruits are largely determined by the types and amounts of carbohydrates contained in the fruits. SWEET(sugars will eventually be exported transporters) are a new class of proteins with the ability to transport glucose and other oligosaccharides, and recent studies have shown that SWEET sugar transporters may play a role in regulating the fruit development. In this study, 18 SWEETs genes were identified from the melon genome, and further through RT-PCR combined with quantitative real-time PCR (qPCR) methods, three SWEETs genes with high expression level were identified during the whole or certain period of fruit development stages. Subcellular localization showed that two SWEETs genes(CmSWEET3,CmSWEET7a) were localized on the cell membrane. Further cloned into a yeast vector system, it was found that CmSWEET7a has the function of transporting glucose and fructose in vitro. This study laid the foundation for revealing the regulatory role of SWEET in the development process of melon fruit.

Key words: melon, fruit, sugar transporter, SWEET

中图分类号:

S652

李苹芳, 姚协丰, 徐锦华, 朱凌丽, 羊杏平. 甜瓜果实发育相关SWEET糖转运蛋白基因的鉴定与功能初步分析[J]. 浙江农业学报, 2023, 35(2): 308-318.

LI Pingfang, YAO Xiefeng, XU Jinhua, ZHU Lingli, YANG Xingping. Identification and preliminary functional characterization of SWEET sugar transporters involved in fruit development of melon (Cucumis melo L.)[J]. Acta Agriculturae Zhejiangensis, 2023, 35(2): 308-318.

图/表 8

参考文献 38

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引物名称	上游引物序列	下游引物序列
Primer name	Forward primer sequence(5'-3')	Reverse primer sequence(5'-3')
CmSWEET1	TCTTGGAAATGCAACTGCTC	GGCAATTCAACATCGTCATC
CmSWEET2	AAAGTGTGGAGTTTATGCCATTT	CCCAACATAGTTCCTATTCCATT
CmSWEET3	CTCCGCAAGTTCCTTATGGT	CAATGGGCTTCCCACTAGAT
CmSWEET5d	GGCCTCTTTCTGTCACCAGT	GCATTCCATAGAACACCCAA
CmSWEET7a	GAGGGAAGGTAAAGGGAAGG	TGTTGCGAGTCCTATCAAGC
CmSWEET7c	ATGGAAGAAGGGATCAGTGG	CCGTACAATGTCCACACCAT
CmSWEET10	TTCTTCAGCCAAACATGGAC	CGGGTGCAATAGATCAGATG
CmSWEET12a	ACGCGGATGAGTTCTTTCTT	GAGCCTTCTTGGGAGCATAG
CmSWEET12c	TCGATACTCATCACCCTGGA	AAGTGCCACCACATATGGAA
CmSWEET12d	TCGAAAGAGAGTTTCTTCCTTACA	GCAGCAGGGTTATGGGTATT
CmSWEET15	CTCCTTTGGTTGTGTCATCG	AATGAAGTGAATGGCCACAA
CmSWEET17c	AACGGAGATGATGGAGGAAG	TTGGCTTTGGAAGGCTTAAC
Actin7	TGCCCAGAAGTTCTATTCCAGC	CATAGTTGAACCACCACTGAGGAC
TOPO-SWEET7a	CAC CAT GGT TTC TTT GGT GC	TCAAGCTCCATGGACCTCAG
TOPO-SWEET3	CACCATGAGATCTCTCTACACCAT	CTAGGAGTTATTTTGATGTGGA
TOPO-SWEET17c	CACCATGGCTGCTAGTTTGAGCT	TCAAAATTTTCCATCTTCCACC
NEV-SWEET7a:	GCGGCCGC AAGCTTGTAAAAGAA AT GGT TTC TTT GGT GC	GCGGCCGCTCAAGCTCCATGGACCTC
NEV-SWEET3:	GCGGCCGC AAGCTTGTAAAAGAA ATGAGATCTCTCTACACC	GCGGCCGCCTAGGAGTTATTTTGATG

引物名称	上游引物序列	下游引物序列
Primer name	Forward primer sequence(5'-3')	Reverse primer sequence(5'-3')
CmSWEET1	TCTTGGAAATGCAACTGCTC	GGCAATTCAACATCGTCATC
CmSWEET2	AAAGTGTGGAGTTTATGCCATTT	CCCAACATAGTTCCTATTCCATT
CmSWEET3	CTCCGCAAGTTCCTTATGGT	CAATGGGCTTCCCACTAGAT
CmSWEET5d	GGCCTCTTTCTGTCACCAGT	GCATTCCATAGAACACCCAA
CmSWEET7a	GAGGGAAGGTAAAGGGAAGG	TGTTGCGAGTCCTATCAAGC
CmSWEET7c	ATGGAAGAAGGGATCAGTGG	CCGTACAATGTCCACACCAT
CmSWEET10	TTCTTCAGCCAAACATGGAC	CGGGTGCAATAGATCAGATG
CmSWEET12a	ACGCGGATGAGTTCTTTCTT	GAGCCTTCTTGGGAGCATAG
CmSWEET12c	TCGATACTCATCACCCTGGA	AAGTGCCACCACATATGGAA
CmSWEET12d	TCGAAAGAGAGTTTCTTCCTTACA	GCAGCAGGGTTATGGGTATT
CmSWEET15	CTCCTTTGGTTGTGTCATCG	AATGAAGTGAATGGCCACAA
CmSWEET17c	AACGGAGATGATGGAGGAAG	TTGGCTTTGGAAGGCTTAAC
Actin7	TGCCCAGAAGTTCTATTCCAGC	CATAGTTGAACCACCACTGAGGAC
TOPO-SWEET7a	CAC CAT GGT TTC TTT GGT GC	TCAAGCTCCATGGACCTCAG
TOPO-SWEET3	CACCATGAGATCTCTCTACACCAT	CTAGGAGTTATTTTGATGTGGA
TOPO-SWEET17c	CACCATGGCTGCTAGTTTGAGCT	TCAAAATTTTCCATCTTCCACC
NEV-SWEET7a:	GCGGCCGC AAGCTTGTAAAAGAA AT GGT TTC TTT GGT GC	GCGGCCGCTCAAGCTCCATGGACCTC
NEV-SWEET3:	GCGGCCGC AAGCTTGTAAAAGAA ATGAGATCTCTCTACACC	GCGGCCGCCTAGGAGTTATTTTGATG

基因名 Gene name	甜瓜基因组ID Melon genome IDs	拟南芥同源基因 Arabidopsis hits	E值 E-value	开放阅读框 ORF length/bp	多肽Deduced polypeptide
					长度	分子量	等电点	跨膜区域
					Length/aa	MW/ku	pI	TM
CmSWEET1	MELO3C008417.2.1	AtSWEET1	6.00×10^-94	759	252	27.7	9.25	7
CmSWEET3	MELO3C005869.2.1	AtSWEET3	3.00×10^-43	627	208	23.5	9.06	5
CmSWEET5a	MELO3C002256.2.1	AtSWEET5	2.00×10^-72	552	183	21	9.42	5
CmSWEET5b	MELO3C009951.2.1	AtSWEET5	9.00×10^-67	747	248	28.2	8.91	7
CmSWEET5c	MELO3C009950.2.1	AtSWEET5	5.00×10^-63	705	234	26	8.72	7
CmSWEET7a	MELO3C016259.2.1	AtSWEET7	3.00×10^-74	789	262	29.1	9.1	7
CmSWEET7b	MELO3C005758.2.1	AtSWEET7	3.00×10^-39	504	265	29.1	9.66	7
CmSWEET9a	MELO3C008674.2.1	AtSWEET9	4.00×10^-76	798	262	30.1	9.07	7
CmSWEET9b	MELO3C031232.2.1	AtSWEET9	4.00×10^-59	639	212	24.3	9.8	6
CmSWEET10	MELO3C026184.2.1	AtSWEET10	5.00×10^-82	891	296	33	9.23	7
CmSWEET12a	MELO3C026183.2.1	AtSWEET12	1.00×10^-79	894	297	33.3	8.85	7
CmSWEET12b	MELO3C002381.2.1	AtSWEET12	2.00×10^-72	876	291	32.9	6.44	7
CmSWEET12c	MELO3C002380.2.1	AtSWEET12	3.00×10^-38	729	242	27.4	8.97	5
CmSWEET15	MELO3C022341.2.1	AtSWEET15	2.00×10^-74	822	273	30.9	9.19	6
CmSWEET17a	MELO3C000200.2.1	AtSWEET17	2.00×10^-42	582	193	21.6	7.66	4
CmSWEET17b	MELO3C004222.2.1	AtSWEET17	1.00×10^-31	456	151	16.6	8.67	5
CmSWEET17c	MELO3C027076.2.1	AtSWEET17	2.00×10^-27	615	204	22.5	7.8	4
CmSWEET17d	MELO3C031734.2.1	AtSWEET17	4.00×10^-16	501	166	19.5	9.86	2

基因名 Gene name	甜瓜基因组ID Melon genome IDs	拟南芥同源基因 Arabidopsis hits	E值 E-value	开放阅读框 ORF length/bp	多肽Deduced polypeptide
					长度	分子量	等电点	跨膜区域
					Length/aa	MW/ku	pI	TM
CmSWEET1	MELO3C008417.2.1	AtSWEET1	6.00×10^-94	759	252	27.7	9.25	7
CmSWEET3	MELO3C005869.2.1	AtSWEET3	3.00×10^-43	627	208	23.5	9.06	5
CmSWEET5a	MELO3C002256.2.1	AtSWEET5	2.00×10^-72	552	183	21	9.42	5
CmSWEET5b	MELO3C009951.2.1	AtSWEET5	9.00×10^-67	747	248	28.2	8.91	7
CmSWEET5c	MELO3C009950.2.1	AtSWEET5	5.00×10^-63	705	234	26	8.72	7
CmSWEET7a	MELO3C016259.2.1	AtSWEET7	3.00×10^-74	789	262	29.1	9.1	7
CmSWEET7b	MELO3C005758.2.1	AtSWEET7	3.00×10^-39	504	265	29.1	9.66	7
CmSWEET9a	MELO3C008674.2.1	AtSWEET9	4.00×10^-76	798	262	30.1	9.07	7
CmSWEET9b	MELO3C031232.2.1	AtSWEET9	4.00×10^-59	639	212	24.3	9.8	6
CmSWEET10	MELO3C026184.2.1	AtSWEET10	5.00×10^-82	891	296	33	9.23	7
CmSWEET12a	MELO3C026183.2.1	AtSWEET12	1.00×10^-79	894	297	33.3	8.85	7
CmSWEET12b	MELO3C002381.2.1	AtSWEET12	2.00×10^-72	876	291	32.9	6.44	7
CmSWEET12c	MELO3C002380.2.1	AtSWEET12	3.00×10^-38	729	242	27.4	8.97	5
CmSWEET15	MELO3C022341.2.1	AtSWEET15	2.00×10^-74	822	273	30.9	9.19	6
CmSWEET17a	MELO3C000200.2.1	AtSWEET17	2.00×10^-42	582	193	21.6	7.66	4
CmSWEET17b	MELO3C004222.2.1	AtSWEET17	1.00×10^-31	456	151	16.6	8.67	5
CmSWEET17c	MELO3C027076.2.1	AtSWEET17	2.00×10^-27	615	204	22.5	7.8	4
CmSWEET17d	MELO3C031734.2.1	AtSWEET17	4.00×10^-16	501	166	19.5	9.86	2

甜瓜果实发育相关SWEET糖转运蛋白基因的鉴定与功能初步分析

Identification and preliminary functional characterization of SWEET sugar transporters involved in fruit development of melon (Cucumis melo L.)

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