甘蔗单糖转运蛋白基因ShSTP7序列分析及组织表达特征测定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1537

生物技术通报 ›› 2022, Vol. 38 ›› Issue (4): 72-78.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1537

• 作物品质遗传与改良专题（专题主编: 刘巧泉教授） • 上一篇下一篇

甘蔗单糖转运蛋白基因ShSTP7序列分析及组织表达特征测定

赵婷婷(), 王俊刚, 王文治, 冯翠莲, 冯小艳, 张树珍()

中国热带农业科学院热带生物技术研究所农业农村部热带作物生物学与遗传资源利用重点实验室海南热带农业资源研究院海南省热带农业生物资源保护与利用重点实验室中国热带农业科学院甘蔗研究中心,海口 571101

收稿日期:2021-12-09 出版日期:2022-04-26 发布日期:2022-05-06
通讯作者: 张树珍,女,博士,研究员,研究方向：甘蔗生物技术;E-mail： zhangsz2007@163.com
作者简介:赵婷婷,女,博士,助理研究员,研究方向：甘蔗基因工程;E-mail： zhaotingting@itbb.org.cn
基金资助:
国家自然科学基金项目(31901593);国家重点研发计划资助(2018YFD1000503);现代农业产业技术体系建设专项资金(CARS-170301)

Sequence and Tissue Expression Analysis of Monosaccharide Transporter Gene ShSTP7 in Sugarcane

ZHAO Ting-ting(), WANG Jun-gang, WANG Wen-zhi, FENG Cui-lian, FENG Xiao-yan, ZHANG Shu-zhen()

Institute of Tropical Bioscience and Biotechnology of Chinese Academy of Tropical Agricultural Sciences,Key Laboratory of Biology and Genetic Resources of Tropical Crops/Key Laboratory for Biology and Genetic Resources of Tropical Crops of Hainan Province,Hainan Institute for Tropical Agricultural Resources/Sugarcane Research Center of Chinese Academy of Tropical Agricultural Sciences,Ministry of Agriculture and Rural Affairs,Haikou 571101

Received:2021-12-09 Published:2022-04-26 Online:2022-05-06

摘要/Abstract

摘要：

糖转运蛋白基因在调控甘蔗产量形成、糖分积累过程中发挥重要作用,对其功能研究有助于揭示甘蔗糖分积累机制及其在甘蔗品种遗传改良上的应用。利用RT-PCR、Real-time PCR、原生质体亚细胞定位等技术,分离甘蔗中单糖转运蛋白基因ShSTP7,并对其编码蛋白生物学特性、亚细胞定位情况及ShSTP7基因表达模式进行分析。结果表明从甘蔗叶中克隆出1 619 bp ShSTP7 cDNA序列,其中包含1 557 bp完整阅读框,编码518个氨基酸;ShSTP7编码蛋白ShSTP7属于易化扩散超家族单糖转运蛋白家族成员,含有12个跨膜结构域及STP糖转运保守结构域,与割手密、高粱、玉米STP7高度同源;ShSTP7-GFP融合蛋白定位于细胞膜;ShSTP7主要在甘蔗叶片中表达,茎秆中表达量低,在高糖甘蔗茎尖未成熟1-3茎节中的表达量高于低糖甘蔗,在高糖甘蔗叶和第4-24茎节中的表达低于低糖甘蔗。这些研究结果表明甘蔗中ShSTP7是一种细胞膜单糖转运蛋白,可能负责质外体中单糖的吸收,在甘蔗叶片糖分运输过程中发挥重要作用,研究结果将为解析甘蔗中单糖转运蛋白基因功能及应用奠定基础。

关键词: 单糖转运蛋白, ShSTP7, 糖吸收, 表达分析, 亚细胞定位, 甘蔗

Abstract:

Sugar transporter protein（ST）genes play important roles on yield formation and sugar accumulation in sugarcane（Saccharum spp. hybrids）. The function analysis of ST genes facilitates to clarify the sugar accumulation mechanism and sugarcane genetic improvement. The ST gene ShSTP7 was isolated from sugarcane,and its biological characteristics,subcellular location and expression pattern of ShSTP7 were analyzed by bioinformatics software and techniques including RT-PCR,real-time PCR and protoplast subcellular location. The results showed that a sugar transporter gene ShSTP7 cDNA was isolated from sugarcane leaves. This gene was 1 619 bp length containing 1 557 bp open reading frame and encoded 518 amino acids. ShSTP7-encoded protein ShSTP7 belonged to facilitated diffusion superfamily and sugar transporter family. It had 12 transmembrane structures and sugar transport conserve domain. ShSTP7 was highly homologous with STP7 from S. spontaneum,Zay mays and Sorghum bicolor. The subcellular location analysis indicated that the green fluorescence signal of ShSTP7-GFP fusion protein appeared in the region of plasma membrane. The expression analysis of ShSTP7 demonstrated that it mainly expressed in sugarcane leaves and less expressed in sugarcane stalks. The expression of ShSTP7 in immature internodes 1-3 of high-sugar-content sugarcane cultivar was higher than that of low-sugar-content sugarcane cultivar. However,the expression of ShSTP7 in the leaves and internodes 4-24 of high-sugar-content sugarcane cultivar was lower than that of low-sugar-content sugarcane cultivar. These results indicate that ShSTP7 is a sugar transporter on plasma membrane uptaking sugars from apoplasmic space and plays an important role on sugar transport in sugarcane leaves,which lays a foundation in clarifying the gene function of monosaccharide transporters and its application.

Key words: monosaccharide transporter protein, ShSTP7, sugar uptake, expression analysis, subcellular location, Saccharum spp. hybrids

赵婷婷, 王俊刚, 王文治, 冯翠莲, 冯小艳, 张树珍. 甘蔗单糖转运蛋白基因ShSTP7序列分析及组织表达特征测定[J]. 生物技术通报, 2022, 38(4): 72-78.

ZHAO Ting-ting, WANG Jun-gang, WANG Wen-zhi, FENG Cui-lian, FENG Xiao-yan, ZHANG Shu-zhen. Sequence and Tissue Expression Analysis of Monosaccharide Transporter Gene ShSTP7 in Sugarcane[J]. Biotechnology Bulletin, 2022, 38(4): 72-78.

图/表 5

表1 引物序列设计及用途

Table 1 Design and application of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Application of primers
ShSTP7-F	TGGTAGTGGCTCGTCTTGGAG	克隆引物
ShSTP7-R	TCTTCACTTGTGATTGTTGCTGG	克隆引物
ShSTP7Q-F	GGACCCATTTCTGGAGAA	荧光定量引物
ShSTP7Q-R	TTGCAGTAGTTGTTCTTCC	荧光定量引物
GADPH-F	CACGGCCACTGGAAGCA	荧光定量引物
GADPH-R	TCCTCAGGGTTCCTGATGCC	荧光定量引物
ShSTP7G-F	ACTAGTATGGCGGGCGGCGG- GATG	载体构建引物
ShSTP7G-R	ACTAGTCTTGTGATTGTTGC- TGGCAGGT	载体构建引物

图1 甘蔗ShSTP7 cDNA序列PCR扩增电泳图

Fig.1 PCR amplification electrophoresis of sugarcane ShSTP7 cDNA

图2 甘蔗ShSTP7氨基酸序列同源性（A）、跨膜结构（B）及遗传进化树分析（C） A：物种间ShSTP7氨基酸序列同源性分析;B：甘蔗ShSTP7跨膜结构分析;C：甘蔗ShSTP7遗传进化树分析。SsSTP7：割手密Saccharum spontaneum（Sspon.002D0015220）;SbSTP7：高粱Sorghum bicolor（XP_002460212.1）;ZmSTP7：玉米Zea mays（NP_001141959.1）;PhSTP7：黍Panicum hallii（XP_025801232.1）;SiSTP7：小米Setaria italica（XP_004968460.1）;OsSTP7：水稻Oryza sativa（XP_015612499.1）;AtSTP7：拟南芥Arabidopsis thaliana（NP_192114.1）;BdSTP7：短柄草Brachypodium distachyon（XP_003578119.1）;TuSTP7：小麦Triticum urartu（EMS63503.1）

Fig.2 Amino acid sequence homology（A）,transmembrane structure（B）and phylogenetic tree analysis（C）of sugarcane ShSTP7 A：Amino acid sequence homology of ShSTP7. B：Transmembrane structure analysis of ShSTP7. C：Phylogenetic tree analysis of ShSTP7

图3 不同甘蔗品种中ShSTP7表达模式分析 A：ROC22中ShSTP7的表达;B：28NG251（HS）和MUCK CHE（LS）中ShSTP7的表达。YL：未成熟叶;ML：成熟叶;J1：茎节1-2;J2：茎节4-5;J3：茎节7-8;J4：茎节10-11;J5：茎节12-13;J6：茎节15-16;J7：茎节18-19;J8：茎节23-24

Fig.3 Expression pattern analysis of ShSTP7 in different sugarcane cultivars A：Expression pattern of ShSTP7 in ROC22. B：Expression pattern of ShSTP7 in 28NG251（HS）and MUCK CHE（LS）. YL：Immature leave. ML：Mature leave. J1：Internode 1-2. J2：Internode 4-5. J3：Internode 7-8. J4：Internode 10-11. J5：Internode 12-13. J6：Internode 15-16. J7：Internode 18-19. J8：Internode 23-24

图4 甘蔗ShSTP7-GFP融合蛋白水稻叶肉细胞原生质体亚细胞定位结果 A：ShSTP7-GFP融合表达载体Spe I酶切鉴定;B：ShSTP7-GFP和pCAMBIA1302-GFP空载体在水稻叶肉细胞原生质体中的亚细胞定位,由左至右分别为荧光明场融合、荧光场、明场图像

Fig.4 Subcellular location result of ShSTP7 -GFP fusion protein in protoplast of rice mesophyll cells A：Enzyme digestion by Spe I of ShSTP7-GFP fusion expression construct. B：Subcellular locations of ShSTP7-GFP and pCAMBIA1302-GFP in protoplast of rice mesophyll cells. From left to right, images are fluorescence and bright merged field, fluorescence field and bright field, respectively

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甘蔗单糖转运蛋白基因ShSTP7序列分析及组织表达特征测定

Sequence and Tissue Expression Analysis of Monosaccharide Transporter Gene ShSTP7 in Sugarcane

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