羽毛针禾（ Stipagrostis pennata ）葡萄糖-6-磷酸-1-差向异构酶基因 SpG6P1E 的克隆与表达分析

doi:10.7522/j.issn.1000-694X.2024.00011

摘要/Abstract

摘要：

葡萄糖-6-磷酸-1-差向异构酶（G6P1E）在植物生长发育和逆境胁迫响应中发挥着重要作用。本研究旨在通过克隆和分析沙漠植物羽毛针禾（Stipagrostis pennata）葡萄糖-6-磷酸-1-差向异构酶基因（SpG6P1E），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。利用分子克隆技术从羽毛针禾中克隆获得一个SpG6P1E基因，该基因编码一个含有325个氨基酸的蛋白质，定位于细胞质，为亲水性稳定蛋白。序列及进化分析表明该基因含有一个保守性很高的醛糖异构酶（Aldose_epim）结构域，且与单子叶植物的直系同源基因亲缘性更高。亚细胞定位分析显示该基因定位于细胞质，参与胞质糖代谢过程。qRT-PCR结果显示SpG6P1E基因的表达与羽毛针禾沙套发育过程及可溶性糖含量具有较高的相关性，表明其对于沙套发育的重要作用；SpG6P1E基因的表达显著受到干旱、高温、盐等多种非生物胁迫的诱导，表明其对于胁迫响应的重要作用。蛋白互作网络及注释分析显示SpG6P1E可能通过参与糖合成和糖代谢等一系列过程进而影响羽毛针禾可溶性糖的含量。本研究结果为深入研究SpG6P1E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）, 葡萄糖-6-磷酸-1-差向异构酶, SpG6P1E, 生物信息学分析, 亚细胞定位, 沙套发育, 非生物胁迫

Abstract:

Glucose-6-phospho 1 epimerase （G6P1E） 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 penataSpG6P1E 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. pennataSpG6P1E gene was cloned by molecular cloning technology. SpG6P1E was cloned from the desert plant S. pennata， which includes a 325 amino acids. SpG6P1E was localized in the cytoplasm. Sequence and evolutionary analysis showed that this gene contains a aldose_epim domain， which is closely related to orthologous gene of monocotyledon plants. Subcellular localization analysis showed that the gene was located in cytoplasm and involved in cytoplasmic glucose metabolism. The results of qRT-PCR showed that SpG6P1E expression had a close link with rhizosheath development and soluble sugar content. The expression of SpG6P1E gene was significantly induced by abiotic stress. Protein interaction and annotation analysis indicated that SpG6P1E might affect the content of soluble sugar by participating in a series of processes such as sugar synthesis and sugar metabolism. The results of this study provide a foundation for in-depth investigation of the functions of G6P1E genes and the mechanisms by which they regulate plant tissue development and adaptation to stress.

Key words: Stipagrostis pennata, Glucose-6-phospho 1 epimerase, SpG6P1E, bioinformatics analysis, subcellular localization, rhizosheath, abiotic stress

中图分类号:

Q943

唐蕊, 尹珊, 陈凯露, 李悦涛, 程淋渊, 王景儒, 王斐, 李榕, 李鸿彬. 羽毛针禾（ Stipagrostis pennata ）葡萄糖-6-磷酸-1-差向异构酶基因 SpG6P1E 的克隆与表达分析[J]. 中国沙漠, 2024, 44(4): 126-136.

Rui Tang, Shan Yin, Kailu Chen, Yuetao Li, Linyuan Cheng, Jingru Wang, Fei Wang, Rong Li, Hongbin Li. Cloning and expression analysis of Glucose-6-phospho 1 epimerase SpG6P1E in Stipagrostis pennata[J]. Journal of Desert Research, 2024, 44(4): 126-136.

图/表 9

图1 羽毛针禾及其根部沙套结构

Fig.1 Plant and rhizosheaths of Stipagrostis pennata

表1 本实验所用到的引物及其序列

Table 1 Primers sequences

引物名称	引物序列	引物功能
SpG6P1E-F	ATGGCCGCCGCGACGTCGTCGT	基因克隆
SpG6P1E-R	ACCCTGAAGAAGAACCTTTTCTGGA	基因克隆
SpG6P1E-1300-F	GAGAGGACAGGGTACCATGGCCGCCGCGACGTCGTCGT	亚细胞定位
SpG6P1E-1300-R	GTACTAGTGTCGACTCTAGAACCCTGAAGAAGAACCTTTTCTGGA	亚细胞定位
SpG6P1E-qRT-F	AAGCCTCCTAAAGCAATCCG	荧光定量
SpG6P1E-qRT-R	GTTTACTGGGAAAGGTGGGG	荧光定量
Actin-F	GAAGATCACTGCCTTGCTCC	内参基因
Actin-R	CGATAACAGCTCCTCTTGGC	内参基因

图2 SpG6P1E序列特征分析

Fig.2 Sequence feature analysis of SpG6P1E

图3 SpG6P1E序列保守性分析注：SvG6P1E：XP_034604257.1；MlG6P1E：CAD6259717.1；SiG6P1E：XP_004976707.1；SbG6P1E：XP_021318977.1；ZpG6P1E：KAG8043371.1；OmG6P1E：KAF0892331.1；PhG6P1E：XP_025825614.1；PmG6P1E：RLM64691.1；OgG6P1E：XP_052151522.1；ObG6P1E：XP_006652766.1；OsiG6P1E：EEC77935.1；BdG6P1E：XP_003580451.1；LrG6P1E：XP_047070035.1；TdG6P1E：XP_037479152.1；AetG6P1E：XP_020196260.1；HvG6P1E：XP_044967751.1

Fig.3 Sequence conservative analysis of SpG6P1E

图4 G6P1E家族结构域与motif分析

Fig.4 G6P1E family domain and motif analysis

图5 SpG6P1E的表达分析

Fig.5 The qRT-PCR expression analysis of SpG6P1E

图6 沙套发育过程中可溶性糖含量

Fig.6 Contents of soluble sugar in rhizosheath development

图7 SpG6P1E亚细胞定位

Fig.7 Subcellular localization of SpG6P1E

图8 蛋白互作网络及注释分析

Fig.8 Protein interaction network and annotation analysis

参考文献 26

1	张晓芹.西北旱区典型生态经济树种地理分布与气候适宜性研究［D］.北京：中国科学院大学，2018.
2	Bengough A G.Water dynamics of the root zone：rhizosphere biophysics and its control on soil hydrology［J］.Vadose Zone，2012，11（2）：1-12.
3	Pang J， Ryan M H， Kadambot H M，et al.Unwrapping the rhizosheath［J］.Plant and Soil，2017，418：129-139.
4	龙丽红，王慧，马晓丽，等.古尔班通古特沙漠羽毛针禾（Stipagrostis pennata）种群种子雨特征［J］.干旱区研究，2014，31（3）：516-522.
5	Yamada K， Osakabe Y， Mizoi J，et al.Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides［J］.Biochemistry & Molecular Biology，2010，285（2）：1138-1146.
6	Mahmood T， Mehnaz S， Fleischmann F，et al. Soil sterilization effects on root growth and formation of rhizosheaths in wheat seedlings［J］.Pedobiologia，2014，57（3）：123-130.
7	Nambiar E.Uptake of Zn65from dry soil by plants［J］.Plant & Soil，1976，44（1）：267-271.
8	Liu T Y， Chen M X， Zhang Y，et al.Comparative metabolite profiling of two switchgrass ecotypes reveals differences in drought stress responses and rhizosheath weight［J］.Planta，2019，250（4）：1355-1369.
9	陈尚武.课堂教学随笔：异构酶、变位酶、消旋酶、差向异构酶与互变异构酶［J］.生命的化学，2020，40（3）：433-435.
10	Wurster B， Hess B.Glucose-6-phosphate-1-epimerase from baker's yeast.a new enzyme［J］.FEBS Letters，1972，23（3）：341-344.
11	高亚南.中国石竹中葡萄糖-6-磷酸-1-差向异构酶基因的功能鉴定及表达分析［D］.呼和浩特：内蒙古农业大学，2021.
12	Ison J， Kalas M， Jonassen I，et al.EDAM：an ontology of bioinformatics operations， types of data and identifiers， topics and formats［J］.Bioinformatics，2013，29（10）：1325-1332.
13	Kelley L A， Mezulis S， Yates C M，et al.The Phyre2 web portal for protein modeling， prediction and analysis［J］.Nature Protocols，2015，10（6）：845-858.
14	Jumper J， Evans R， Pritzel A，et al.Highly accurate protein structure prediction with AlphaFold［J］.Nature，2021，596（7873）：583-589.
15	Varadi M， Anyango S， Deshpande M，et al.AlphaFold protein structure database：massively expanding the structural coverage of protein-sequence space with high-accuracy models［J］.Nucleic Acids Research，2022，50（D1）：D439-D444.
16	Chou K C， Shen H B.Cell-PLoc 2.0：an improved package of web-servers for predicting subcellular localization of proteins in various organisms［J］.Natural Science，2010，2（10）：1090.
17	Tamura K， Stecher G， Kumar S.MEGA11：molecular evolutionary genetics analysis version 11［J］.Molecular Biology and Evolution，2021，38 （7）：3022-3027.
18	Bailey T L， Johnson J， Grant C E，et al.The MEME suite［J］.Nucleic Acids Research，2015，43（W1）：W39-W49.
19	Chen C， Wu Y， Li J，et al.TBtools-II：A" One for All， All for One" bioinformatics platform for biological big-data mining［J］.Molecular Plant，2023，16（11）：1733-1742.
20	Li R， Cui K， Xie Q，et al.Selection of the reference genes for quantitative gene expression by RT-qPCR in the desert plant Stipagrostis pennata ［J］.Scientific Reports，2021（1）：21711.
21	张丽霞，张霞，王绍明，等.公路阻碍作用对古尔班通古特沙漠南缘羽毛针禾种群基因流的影响［J］.江苏农业科学，2019，47（9）：104-109.
22	Graille M， Baltaze J P， Leulliot N，et al.Structure-based functional annotation：yeast ymr099c codes for a D-hexose-6-phosphate mutarotase［J］.Journal of Biological Chemistry，2006，281（40）：30175-30185.
23	Lv B， Guo Y， Zhao X，et al.Glucose-6-phosphate 1-Epimerase CrGlu6 contributes to development and biocontrol efficiency in clonostachys chloroleuca［J］.Journal of Fungi，2023，9（7）：764.
24	Samolski I， de Luis A， Vizcaíno J A，et al.Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants，chitin，or glucose using a high-density oligonucleotide microarray［J］.BMC Microbiology，2009，9（1）：1-14.
25	Sheshukova E V， Komarova T V， Pozdyshev D V，et al.The intergenic interplay between aldose 1-epimerase-like protein and pectin methylesterase in abiotic and biotic stress control［J］.Frontiers in Plant Science，2017，8：1646.
26	申龙斌，李瑞梅，段瑞军，等.海马齿 Spmet 基因表达产物的亚细胞定位及其组织表达特异性分析［J］.中国农学通报，2012，28（27）：213-217.

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