大蒜蔗糖:蔗糖1-果糖基转移酶基因As-1-SST的克隆与表达分析

Cloning and Expression Analysis of Sucrose:Sucrose 1-Fructosyltransferase As-1-SST Gene in Garlic

植物果聚糖是一类重要的可溶性碳水化合物,其在植物中的积累可提高植物的抗逆性。为了解大蒜蔗糖:蔗糖1-果糖基转移酶的序列特征和功能,本研究采用TA克隆方法(Original TA Cloning Kit)得到乐都紫皮大蒜As-1-SST基因全长序列,利用BLAST、DNAMAN、ProtParam、SWISS-MODEL、MEGA等生物信息工具分析其序列特征,通过荧光定量PCR(qRT-PCR)分析As-1-SST基因在大蒜根、假茎、叶片和鳞芽中的表达差异及其对低温和干旱胁迫的响应情况。结果表明,大蒜As-1-SST基因全长1872 bp,编码623个氨基酸,推测蛋白质分子质量为69.76 kDa,理论等电点为5.19,为不稳定亲水性蛋白;亚细胞定位预测结果显示,As-1-SST蛋白主要定位于液泡,该蛋白无信号肽,包含2个特异位点,属于糖苷水解酶32(GH32)家族。在进化关系上,大蒜As-1-SST与百合科的洋葱1-SST亲缘关系最为接近。qRT-PCR分析表明,As-1-SST基因在根中的表达量最高,其次是假茎,在鳞芽和叶片中表达水平较低,具有明显的组织特异性;不同组织As-1-SST对于低温及干旱胁迫的响应差异显著,低温胁迫显著诱导了根、假茎、叶片中As-1-SST的表达,而干旱胁迫只显著提高了鳞芽中As-1-SST的表达量,说明大蒜各组织As-1-SST对逆境信号的响应机制不同。本研究为进一步鉴定大蒜果聚糖合成酶基因的生物信息学功能和表达调控机制提供了一定的理论依据。

Plant fructan, one of the most important soluble carbohydrate for plant defense against abiotic stress. In order to investigate the function and expression patterns of As-1-SST gene which encodes sucrose: sucrose 1-fructosyltransferase(1-SST) in garlic, the full length of As-1-SST gene was cloned from garlic cultivar Ledu purple skin garlic by original TA cloning kit. Bioinformatics tools including BLAST, DNAMAN, ProtParam, SWISS-MODEL, and MEGA were used to analyze the sequence information, and quantitative real-time PCR(qRT-PCR) was performed to detect the expression levels of As-1-SST gene in garlic roots, pseudostems, leaves, and scale buds and its response to low temperature and drought stress. The length of As-1-SST gene was 1 872 bp, which encoded 623 amino acids. The predicted molecular mass was 69.76 kDa, and the pI was 5.19. The subcellular localization of predicted As-1-SST protein was within the vacuoles. As-1-SST is an unstable hydrophilic protein, which can be classified into glycoside hydrolase 32(GH32) family. Phylogenetic analysis indicated that garlic As-1-SST is close to onion 1-SST in Liliaceae. qRT-PCR analysis demonstrated that the expression of As-1-SST in roots was the highest, followed by pseudostems, the lower was in scale buds and leaves, showed obvious tissue specificity. The response of As-1-SST in different tissues to low temperature and drought stress was significantly different. Low temperature stress significantly induced the expression of As-1-SST in roots, pseudostems, and leaves, while drought stress only significantly increased the expression of As-1-SST in scale buds, which indicated that the response mechanism of As-1-SST in garlic tissues to stress signals was different. The results of this study provide a theoretical basis for further studies on the biology function and expression mechanism of garlic defensins.