小麦棉子糖合成酶基因(TaRS)的克隆及特性分析  

Cloning and Characterization Analysis of Raffinose Synthase Gene(TaRS) in Wheat(Triticum aestivum)

作　　者：乔梦[1] 田原[1] 杨瑞[1] 付金梅[2] 康妮[1] 闵东红[2] 张小红[1] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]西北农林科技大学农学院,杨凌712100

出　　处：《农业生物技术学报》2017年第8期1245-1254,共10页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2014ZX0800203B和No.2016ZX08002002-010);陕西省科技攻关项目(No.2014K02-04-05);陕西省科技统筹创新计划(No.2014KTZB02-01-01)

摘　　要：棉子糖(raffinose)作为一种重要的可溶性碳水化合物广泛存在于高等植物中,并且参与植物抵抗各种非生物逆境胁迫。棉子糖合成酶(raffinose synthase,RS,EC2.4.1.82)是催化肌醇半乳糖苷和蔗糖产生棉子糖的关键酶。本研究通过同源比对克隆到了一个小麦(Triticum aestivum)的棉子糖合成酶基因(triticum aestivum raffinose synthse,TaRS),该基因定位于小麦3B染色体上。序列分析显示,TaRS基因具有一个完整的开放阅读框(2 349 bp),包含两个保守模体,即Kx D和Rxxx D,属于糖苷水解酶超家族GHD(glycoside hydrolases,clan D)。在进化关系上,小麦TaRS蛋白与山羊草(Aegilops tauschii)的棉子糖合成酶XM020298559.1亲缘关系最近(相似度为98.47%)。Southern杂交结果表明,TaRS基因在中国春小麦基因组中存在至少4个拷贝。亚细胞定位结果显示,TaRS蛋白定位在小麦叶肉细胞的细胞膜上。对该基因进行组织表达特异性分析发现,TaRS在小麦的根、茎、叶和种子中都有表达,但在叶片中的表达量最高。将TaRS在大肠杆菌(Escherichia coli)中进行异源表达,通过高效液相色谱(high performance liquid chromatography,HPLC)法检测发现,TaRS能够以蔗糖和肌醇半乳糖苷为底物在体外合成棉子糖,并且反应的最适pH在8.0左右。此外,TaRS的表达受到脱水、42℃高温、高盐和4℃低温4种胁迫的诱导,分别于胁迫12、1、1和48 h时表达量达到最大。本研究为进一步利用TaRS基因进行小麦抗逆育种提供了理论依据。Raffinose is ubiquitously occurred in higher plant as an important soluble carbohydrate, which is known to be involved in various abiotic stresses. Raffinose synthase （RS, EC2.4.1.82） is the key enzyme that catalyses the reversible galactosylation, yielding raffinose and myo-inositol. In this study, the raffinose synthase gene TaRS of wheat （Triticum aest＆um） was cloned by homologous alignment. The TaRS was assigned to wheat chromosome 3B. Sequence analysis revealed that TaRS had a whole open reading frame （ORF, 2 349 bp） and belonged to the glycoside hydrolase super family （GH-D） which contained 2 conservative motifs, KxD and RxxxD. In evolutionary relationship, TaRS shared the highest homology with the indicated RS from Aegilops tauschii （XM020298559.1）. Southern blot assay showed that there existed at least 4 allelic copies of TaRS in Chinese spring wheat genome. Subcellular localization analysis revealed that TaRS protein was localized at the cell membrane in wheat protoplasts. Tissue specific analysis showed that TaRS expressed in root, stem, leaf and seed, but had the highest expression level in leaves. Heterologous expression was conducted in Escherichia coli, and the crude extract was able to utilize sucrose and galactinol as substrates to synthesis raffinose ir~ vitro based on the result of high performance liquid chromatography （HPLC）. Furthermore, TaRS displayed an optimum activity at about pH 8.0. Expression pattern analysis under multiple abiotic stresses indicated that the expression of TaRS was induced by dehydration, high temperature, salinity, and low temperatur, and reached the highest expression level at 12, 1, 1, 48 h, respectively. These results indicated that TaRS may play critical roles in wheat tolerance of abiotic stresses and provides theoretical basis for further studying in wheat abiotic stress tolerance breeding.

关 键 词：中国春小麦 基因克隆 棉子糖合成酶 酶活 非生物逆境胁迫 

分 类 号：S42[农业科学—植物保护] Q78[生物学—分子生物学]