甘蔗磷酸丙糖/磷酸转运蛋白基因ShTPT的克隆与表达分析

Cloning and Expression Analysis of the Phosphate Triose/Phosphate Transporter Gene ShTPT in Sugarcane

磷酸丙糖/磷酸转运蛋白(triose-phosphate/phosphate translocator,TPT)是一种磷酸丙糖转运蛋白,在植物碳代谢途径以及非生物胁迫中发挥重要作用。蔗糖具有广泛的食用价值和重要的经济价值,甘蔗是制糖的主要原料,蔗糖占中国产糖量的80%以上,因此实现甘蔗高产高抗目标至关重要。本研究以甘蔗品种新台糖22号(ROC22)为材料,克隆获得甘蔗磷酸丙糖/磷酸转运蛋白基因ShTPT,利用生物信息学方法对ShTPT进行蛋白理化性质、保守结构域、跨膜结构预测和蛋白序列比对。结果显示:ShTPT基因CDS全长1221 bp,编码406个氨基酸,蛋白分子量为43.63 kDa,等电点(pI)为9.80,富含丙氨酸、亮氨酸,不稳定系数为42.51,亲水系数为0.583,是一种不稳定的疏水性蛋白;ShTPT蛋白不含信号肽并具有9个跨膜结构域;保守结构域预测显示ShTPT含有1个TPT结构域,符合TPT家族特征;进化分析结果显示,ShTPT与高粱SbTPT(XP_002454867.1)、玉米ZmTPT(NP_001105497.1)聚在一起,同源性分别为97.04%和94.13%。进一步对ShTPT进行亚细胞定位分析发现ShTPT定位在叶绿体。甘蔗组织表达模式分析表明,ShTPT主要在叶片中表达,在根部和茎中的表达量极低。在PEG模拟的干旱胁迫条件下ShTPT的表达量表现出升-降-升的趋势,表明其响应干旱胁迫。该研究结果表明ShTPT是一个定位于叶绿体的跨膜转运蛋白,可能参与甘蔗叶片中的原初碳代谢化合物的转运,响应干旱胁迫。本研究初步确定甘蔗ShTPT基因在叶片中碳同化物的转运和非生物胁迫方面发挥重要作用,为进一步研究其功能提供理论依据。

The triose-phosphate/phosphate translocator(triose-phosphate/phosphate translocator,TPT)is a triose phos-phate transporter that plays an important role in plant carbon metabolism pathways as well as abiotic stresses.Sucrose has a wide range of edible value and important economic value.Sugarcane is the main raw material for sugar production,and sucrose from sugarcane accounts for more than 80%of China's sugar production,so it is very important to achieve the goal of high yield and high resistance of sugarcane.In this study,the sugarcane variety ROC22 was used as the ma-terial to clone the sugarcane triose/phosphate transporter gene ShTPT,and the physicochemical properties,conserved domains,transmembrane structure prediction and protein sequence comparison of ShTPT were carried out by bioinfor-matics methods.The results showed that the CDS of ShTPT was 1221 bp,encoding 406 amino acids,the molecular weight of the protein was 43.63 kDa,and the isoelectric point(pI)was 9.80.It was rich in alanine and leucine,its insta-bility coefficient was 42.51,and its hydrophilic coefficient was 0.583,which was an unstable hydrophobic protein.ShTPT did not contain signal peptides but had 9 transmembrane domains.Conserved domain prediction showed that ShTPT contained a tpt domain,which was consistent with the characteristics of the TPT family.The results of phyloge-netic analysis showed that ShTPT was clustered with Sorghum bicolor SbTPT(XP_002454867.1)and Zea mays ZmTPT(NP_001105497.1)with homology of 97.04%and 94.13%,respectively.Further subcellular localization analysis of ShTPT showed that ShTPT was localized in chloroplasts.Tissue expression pattern analysis of sugarcane showed that ShTPT was mainly expressed in leaves,and the expression level in roots and stems was very low.Under the drought stress conditions simulated by PEG,the expression of ShTPT showed an upward trend,indicating that it responded to drought stress.The results of this study suggest that ShTPT is a chloroplast-localized transmembrane transporter,which may be involved in the transport of primary carbon metabolic compounds in sugarcane leaves and responsive to drought stress.This study preliminarily determined that ShTPT gene plays an important role in the transport of carbon assimi-lates in leaves and abiotic stress,which provides a theoretical basis for further study of its function.