鹅掌楸属GST家族基因的克隆与表达分析

Cloning and Expression Analysis of GST Family Genes in Liriodendron

该研究以鹅掌楸属植物为试验材料,在北美鹅掌楸和鹅掌楸转录组数据库中筛选出54条GST基因家族的EST序列,通过比对将其归为Tau、Zeta、Theta和Phi等4个亚类。在这4个亚类中各选取1条EST序列设计引物,通过反转录PCR(RT-PCR)与cDNA末端快速扩增(RACE)技术,从鹅掌楸属植物中克隆出4条全长分别为1 150、932、1 022和1 067bp的基因,并分别命名为LtGSTparCb、LtGSTZ-like、LtGSTF13a和LcGSTT1。对LtGSTparCb、LtGSTZ-like、LtGSTF13a和LcGSTT1这4个基因编码蛋白质的结构预测发现,其蛋白质的分子质量分别为25.17、25.34、24.49和28.09kD;理论等电点分别是5.60、6.53、6.66和9.20,即LtGSTparCb、LtGSTZlike、LtGSTF13a蛋白质属于酸性蛋白,LcGSTT1属于碱性蛋白;不稳定系数分别是33.01、36.97、43.19和47.96,即LtGSTparCb、LtGSTZ-like为稳定蛋白,LtGSTF13a、LcGSTT1为不稳定蛋白;α-螺旋(alpha helix)是这4个蛋白质二级结构的主要成分。利用在线软件TargetP并结合GST蛋白质在其他植物中亚细胞定位的结果,推测这4种蛋白质极有可能位于鹅掌楸属植物的细胞质内。实时荧光定量PCR分析结果表明,这4个基因在北美鹅掌楸各个组织中均有表达,其中LtGSTparCb和LtGSTF13a在叶中的表达量最高,在其他组织中的表达量均较低;LtGSTZ-like在花器官中的表达量最高,在叶中的表达量最低,在其他组织中的表达量居于两者之间;LcGSTT1在叶中表达量最高,花器官中表达量其次,在茎和叶芽中的表达量偏低。该研究结果可为鹅掌楸属GST家族基因的功能研究奠定基础。

54 EST sequences,belonging to Tau,Zeta,Theta and Phi classes,were mined from the transcriptome databases of Liriodendron.4 EST sequences were selected for designing primers and corresponding genes,named LtGSTparCb,LtGSTZ-like,LtGSTF13a,LcGSTT1,were cloned by reverse transcription PCR and rapid amplification of cDNA ends technology.The full cDNA length of LtGSTparCb,LtGSTZ-like,LtGSTF13a and LcGSTT1 were 1 150,932,1 022 and 1 067 bp respectively.The prediction results of protein structure showed that the four proteins molecular weights were about 25.17,25.34,24.49 and 28.09 kD,respectively;the theoretical isoelectric points(pI)were 5.60,6.53,6.66,9.20,which mean that LtGSTparCb,LtGSTZ-like,LtGSTF13a were acidic protein and LcGSTT1 was basic protein;the instability indexes were 33.01,36.97,43.19,47.96,so that LtGSTparCb and LtGSTZ-like were classified as stable proteins,whereas,LtGSTF13a and LcGSTT1 proteins were unstable;Alpha helix was the mainly secondary structure component of the four proteins.The four proteins were predicted located at cytoplasm in Liriodendron.Expression profile by real-time PCR showed that they were expressed in all eight tissues studied,while the expression levels varied significantly among eight tissues.The expression of LtGSTparCb and LtGSTF13a in leaves was the highest while lower in other tissues.LtGSTZ-like had the highest expression level in floral organs and lowest expression in leaves.LcGSTT1 had the highest expression level in leaves and lower in floral organs,which was least in stem and leaf buds.The results could lay a foundation for the further studies of function about GST family genes in Liriodendron.