甜高粱蔗糖转运蛋白SUT5的表达及功能分析

Expression and Functional Analysis of the Sweet Sorghum Sucrose Transporter SUT5

为揭示甜高粱蔗糖转运蛋白SdSUT5基因的功能及在不同非生物胁迫下的表达模式,本研究利用同源克隆获得一个甜高粱SUT5基因,并进行生物信息学及亚细胞定位分析。采用实时荧光定量PCR法研究该基因在低温、高温、盐和干旱4种非生物逆境下不同组织的响应特性,同时通过转化己糖转运缺陷型酵母EBY.VW4000验证SdSUT5基因的功能。生物信息学分析结果表明,该基因全长1 602 bp,共编码534个氨基酸,理论等电点为8.63,属于稳定亲水性蛋白;存在12个跨膜区,43个磷酸化位点;二级结构以α-螺旋和无规则卷曲为主。亚细胞定位试验显示,SdSUT5蛋白定位在质膜。利用己糖转运缺陷型酵母EBY.VW4000证明SdSUT5基因编码蛋白具有转运蔗糖的功能。系统进化树表明甜高粱SUT5蛋白与甘蔗和玉米的亲缘关系较近。实时荧光定量PCR分析表明,低温、高温、干旱和盐胁迫后,甜高粱不同部位SUT5基因均有不同程度的响应。甜高粱SdSUT5基因的克隆和功能分析为甜高粱的抗逆育种提供了理论参考。

To reveal the functions of sweet sorghum sucrose transporter SdSUT5 gene and its expression patterns under different abiotic stresses, a sweet sorghum SUT5 gene was obtained by homologous cloning and bioinformatics and subcellular localization analysis was performed in this study. The real-time fluorescence quantitative PCR method was used to study the response characteristics of SdSUT5 gene in different tissues under four abiotic stresses: low temperature, high temperature, salt and drought, and at the same time the function of the SdSUT5gene was verified by transforming the hexose transporter-deficient yeast EBY.VW4000. Results of bioinformatics analysis showed that the gene was 1 602 bp in length, encoding 534 amino acids, the theoretical isoelectric point was 8.63, and belongs to a stable hydrophilic protein. There were 12 transmembrane regions and 43 phosphorylation sites;the secondary structure was dominated by α-helix and random coil. The experiment of subcellular localization showed that SdSUT5 protein was localized at the plasma membrane. The hexose transporter-deficient yeast EBY.VW4000 was used to demonstrate that theprotein encoded by SdSUT5 gene has the function of transferring sucrose. The phylogenetic tree showed that the sweet sorghum SUT5 protein was more closely related to sugarcane and maize. Real-time fluorescence quantitative PCR analysis showed that the SUT5 gene responded with different degrees in different parts of sweet sorghum after suffering stresses of low temperature, high temperature,drought and salt. The analysis of cloning and function of the SdSUT5 gene in sweet sorghum has provided theoretical references for breeding sweet sorghum for stress resistance.