沙漠植物羽毛针禾（ Stipagrostis pennata ）糖转运蛋白基因 SpSWEET3的克隆与表达

doi:10.7522/j.issn.1000-694X.2023.00037

摘要/Abstract

摘要：

糖转运蛋白（SWEET）在植物生长发育和逆境胁迫响应中发挥着重要作用。本研究旨在克隆和分析羽毛针禾（Stipagrostis pennata）糖转运蛋白SpSWEET3基因的表达特征，为进一步探究其影响羽毛针禾根部沙套发育的机制及功能奠定基础。利用分子克隆技术克隆获得羽毛针禾SpSWEET3基因的全长开放读码框序列；利用生物信息学方法分析SpSWEET3基因的理化性质；利用qRT-PCR分析基因的表达特征；利用激光共聚焦显微镜观察分析其亚细胞定位。从羽毛针禾中克隆获得576 bp的SpSWEET3基因的全长开放阅读框，该基因编码一个含有191个氨基酸的碱性强疏水性蛋白，相对分子质量为21.619 kD。SpSWEET3属于PQ-loop超家族并含有典型的MtN3_slv结构域，发挥催化胞内糖外排的功能。亚细胞定位分析显示该基因定位于质膜，暗示其可能作为跨膜蛋白发挥功能。qRT-PCR结果显示SpSWEET3基因在羽毛针禾根组织中具有较高的累积表达，表明其对于沙套发育的重要作用；SpSWEET3基因的表达显著受到PEG干旱胁迫的诱导表达，表明其对于干旱适应的重要作用。蛋白质相互作用分析显示SpSWEET3可能通过与参与维持核酸结构稳定、转录因子、跨膜转运等蛋白质相互作用进而发挥转运糖的作用。本研究结果为深入研究SWEET基因的功能及其调控植物组织发育和适应逆境的机制奠定了基础。

关键词: 羽毛针禾（Stipagrostis pennata）, SpSWEET3, 糖转运蛋白, 生物信息学分析, 亚细胞定位, 根鞘, 干旱响应

Abstract:

Sugar transporter （SWEET） plays an important role in plant growth and development and stress response. The purpose of this study is to clone and analyze the expression characteristics of Stipagrosis pennata sugar transporter SpSWEET3 gene， and lay a foundation for further exploring the mechanism and function of its influence on the development of S. pennata root sand trap. The full-length open reading frame sequence of S. pennata SpSWEET3 gene was cloned by molecular cloning technology； The physical and chemical properties of SpSWEET3 gene were analyzed by bioinformatics； The gene expression characteristics were analyzed by qRT-PCR； The subcellular localization was observed and analyzed by laser confocal microscope. SpSWEET3was cloned from the desert plant S. pennata， which includes a 576-bp full-length open reading frame and encodes a basic strongly hydrophobic protein containing 191 amino acids with a relative molecular mass of 21.619 kD. SpSWEET3 belongs to the PQ-loop superfamily and contains the typical MtN3_slv transmembrane domain that catalyzes intracellular sugar efflux. Subcellular localization analysis revealed localization of the gene to the plasma membrane， implying that it may function as a transmembrane protein. qRT-PCR results showed that the SpSWEET3 gene had a higher cumulative expression in S. pennata root tissues， indicating its important role for sand rhizosheath development. Expression of the gene SpSWEET3 was significantly induced by drought stress with PEG， indicating its importance for drought adaptation. Protein interaction analysis showed that SpSWEET3 may transport sugar by interacting with proteins involved in the maintenance of nucleic acid structural stability， transcription factors， and transmembrane transport. The results of this study provide a foundation for in-depth investigation of the functions of SWEET genes and the mechanisms by which they regulate plant tissue development and adaptation to stress.

Key words: Stipagrostis pennata, SpSWEET3, sugar transporter gene, bioinformatics analysis, subcellular localization, rhizosheath, drought response

中图分类号:

Q943

孙博瀚, 杨丹, 王斐, 李榕, 李鸿彬. 沙漠植物羽毛针禾（ Stipagrostis pennata ）糖转运蛋白基因 SpSWEET3的克隆与表达[J]. 中国沙漠, 2023, 43(5): 129-138.

Bohan Sun, Dan Yang, Fei Wang, Rong Li, Hongbin Li. Cloning and expression analysis of sugar transporter gene SpSWEET3 from the desert plant Stipagrostis pennata[J]. Journal of Desert Research, 2023, 43(5): 129-138.

图/表 10

图1 羽毛针禾及其野外生长环境

Fig.1 Stipagrostis pennata plants and the wild growth environment.

表1 本研究使用的引物序列

Table 1 Primers used in this study

引物名称	引物序列(5'-3')	引物功能
SpSWEET3-F	ATGATCACTGGCATACGC	基因克隆
SpSWEET3-R	AGATAACATAGCTGTGTATTTACTACT
SpSWEET3-EF	GCTCTAGAATGATCACTGGCATACGC
SpSWEET3-ER	TCCCCCGGGAGATAACATAGCTGTGTATTTACTACT
q-ELF-F	ACTACCATCCCTATGAGCCAACT	基因表达分析
q-ELF-R	ATCACTGCCAGCCTGAAGACA
q-GAPDH-F	AGTCCGTCGCCATCGTCA
q-GAPDH-R	CGTGCCCATGCCTTCTGT
q-APR6-F	TCAATTCCAAGAAATGGCTCG
q-APR6-R	TGGGTTCCACCAGTAACAAGG
q-SpSWEET3-F	TGACATTTAAAAGAGTGATA
q-SpSWEET3-R	AGAACTCACAACTGGAAGGC

图2 SpSWEET3基因克隆凝胶电泳图

Fig.2 Gel electrophoresis diagram of SpSWEET3 gene cloning

图3 SpSWEET3的序列分析不同颜色字体代表相同或相似氨基酸残基。MtN3_slv保守结构域用实线标出

Fig.3 Sequence analysis of SpSWEET3 protein

图4 羽毛针禾糖转运蛋白基因SpSWEET3的生物信息学分析

Fig.4 Bioinformatic analysis of SpSWEET3

图5 SpSWEET3 的基因结构和保守结构域分析

Fig.5 Gene structure and conserved motif analysis of SpSWEET3

图6 SpSWEET3的表达特征分析

Fig.6 Expression pattern analysis of SpSWEET3

图7 SpSWEET3质粒双酶切鉴定

Fig.7 Double enzymatic identification of SpSWEET3recombinant plasmid

图8 糖转运蛋白SpSWEET3亚细胞定位分析（质壁分离条件下的观察）A：GFP通道下的空载体； B：在明场视野下的空载体； C：复合场下的空载体； D：GFP通道下的SpSWEET3； E：在明场视野下的SpSWEET3； F：复合场下的SpSWEET3

Fig.8 Subcellular localization analysis of SpSWEET3

图9 SpSWEET3蛋白质相互作用分析

Fig.9 Protein interaction analysis of SpSWEET3

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