玉米岩藻糖基转移酶基因SPINDLY的克隆及表达分析

Cloning and expression analysis of maize fucosyltransferase gene SPINDLY

关键词:  玉米, ZmSPY, 基因克隆, 亚细胞定位, 激素应答

Key words:  maize, ZmSPY, gene cloning, subcellular localization, hormone response

摘要:

SPINDLY(SPY)是植物中特有的岩藻糖基转移酶,其介导的O-岩藻糖基化修饰作用在维持细胞内稳态和调控植物生长发育等过程中发挥了重要作用。为探究玉米(Zea mays)岩藻糖基转移酶基因(ZmSPY)的功能,该研究采用生物信息学方法对ZmSPY蛋白的保守结构域、氨基酸序列以及理化性质等进行分析,并从玉米根系组织中克隆该基因,构建GFP融合蛋白表达载体,分析其蛋白质亚细胞定位,同时通过外源激素处理分析ZmSPY对不同激素[赤霉素(GA)、吲哚乙酸(IAA)、6-氨苄基嘌呤(6BA)、脱落酸(ABA)]的应答。结果表明:(1)ZmSPY蛋白归属于TPR和SPY超家族,主要由TPR结构域以及催化结构域构成。(2)SPYs高度保守,ZmSPY与高粱(Sorghum bicolor)SbSPY的序列同源性最高。(3)ZmSPY 编码序列(coding sequence,CDS)区为2 736 bp,编码911个氨基酸,所编码蛋白为亲水蛋白,无跨膜结构域,二级、三级结构以α-螺旋和无规卷曲为主,并含有33个糖基化位点。(4)亚细胞定位研究显示,ZmSPY主要定位于细胞核。(5)ZmSPY表达水平受GA、IAA、6BA、ABA等外源激素信号调控,并呈现出不同程度的响应。该研究结果为玉米SPY的功能探究提供了数据支撑,也为植物中O-岩藻糖基化修饰的相关研究提供了理论参考。

Abstract:

SPINDLY(SPY)is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation. Previous studies have indicated that the SPY protein regulates plant growth and development by modulating various intracellular processes in Arabidopsis thaliana. Its mediated O-fucosylation plays an important role in maintaining cell homeostasis and regulating plant growth and development, however protein O-fucosylation regulated by SPY in other plants largely remain unknown. Maize(Zea mays )is one of the most important cereals crops for supplying foods, fibers, and fuels to humans. In order to explore the function of maize fucosyltransferase gene(ZmSPY), this study first analyzed the conserved domain, amino acid sequence and physicochemical properties of ZmSPY protein by bioinformatics means, and cloned the gene from maize root tissue to construct the GFP fusion protein expression vector. The subcellular localization of ZmSPY was analyzed, and its response to different hormone treatments(GA, IAA, 6BA, ABA)was determined by exogenous hormone application. And the results were as follows:(1)ZmSPY proteins belong to the TPR and SPY superfamilies, and structural analysis demonstrated that ZmSPY had TPR(Tetratricopeptide repeat)and catalytic domains.(2)Phylogenetic analysis shows that SPYs are highly conserved, and ZmSPY exhibits strong homology to SPY in Sorghum bicolor.(3)Sequence analysis shows that the CDS region of ZmSPY is 2736 bp. Physicochemical analysis indicates that ZmSPY, which contains 911 amino acids and 33 glycosylation sites, is hydrophilic and non-secretory. Its secondary and tertiary structure is largely composed of alpha helix and random coil.(4)The subcellular localization of ZmSPY is predominantly observed in the nucleus.(5)The expression of ZmSPY is induced by phytohormones including GA, IAA, 6BA and ABA, and exhibits various expression patterns. This study provides foundational information on SPY in maize, which could contribute to further investigation of SPY and its effect on O-fucosylation in cereal plants.