芋ADP-葡萄糖焦磷酸化酶(AGPase)基因家族的克隆、生物信息学及表达分析

Cloning,Bioinformatics and Expression Analysis of ADP-glucose Pyrophosphorylase Gene Family in Colocasia esculenta

芋(Colocasia esculenta)为天南星科芋属成员,在世界范围内广泛种植,其球茎主要贮藏物质为淀粉。ADP-葡萄糖焦磷酸化酶(AGPase)是淀粉合成的第一个关键酶和限速酶,在植物淀粉合成中发挥关键作用,但目前关于芋AGPase基因家族的分子结构特征和表达模式还不清楚。本研究以芋新品种‘荔浦芋1号’为试材,从全长转录组注释信息中筛选到6个AGPase基因家族成员,利用RT-PCR技术克隆了6个基因的编码区,利用生物信息学对其理化性质、进化关系和保守motif进行分析;同时利用转录组学和荧光定量RT-PCR技术对6个基因在不同组织和球茎不同发育阶段的表达模式进行分析。结果表明:克隆获得6个芋AGPase基因编码区的cDNA序列,序列长度为1398~2028bp,编码的蛋白大小为350~543 aa,其中4个基因(CeAGPL1~CeAGPL4)编码AGPase的大亚基,2个基因(CeAGPS1,CeAGPS2)编码AGPase的小亚基。系统进化分析显示,所有的AGPase分成了大亚基和小亚基2个群组,4个大亚基分在了大亚基组,2个小亚基分在了小亚基组。CeAGP家族蛋白分子质量范围为38753.22~59743.05kDa,等电点范围为5.64~8.82,亚细胞定位为叶绿体、淀粉体和细胞质等。蛋白的保守motif分析发现,CeAGP家族蛋白共预测到11个保守motif,6个motif功能注释为NTP_transferase,除CeAGPS2外,另外5个CeAGP蛋白均包含11个motif。在不同组织中,6个CeAGP基因均能在所有组织中表达,其中CeAGPL3和CeAGPS1基因在叶片和叶柄中高表达,CeAGPL1和CeAGPS1基因在球茎中高表达,6个CeAGP基因在根的表达量均较低。在球茎不同发育阶段中,CeAGPL1和CeAGPS1高表达且均表现先升高后降低的趋势,2个基因分别在4月龄和5月龄阶段的表达量最高。该研究结果为后续阐明芋淀粉合成的分子机制提供依据。

Taro(Colocasia esculenta) is a member of the genus Colocasia in the family Tenaxaceae and is widely grown worldwide,and the main storage material of its corm is starch.ADP-glucose pyrophosphorylase(AGPase) is the first key and rate-limiting enzyme for starch synthesis and plays a critical role in plant starch synthesis,but the molecular structural features and expression patterns of the taro AGPase gene family are still unclear.In this study,six AGPase genes were selected from the full-length transcriptome annotations using the new taro variety ‘Lipuyu No.1’ as the test material.The coding regions of the six genes were cloned by RT-PCR,and the physicochemical properties,evolutionary relationships and conserved motifs were analyzed by bioinformatics.The results showed that the cDNA sequences of the coding regions of six taro AGPase genes were ranged from 1398 bp to 2028 bp and the proteins encoded were ranged from 350 aa to 543 aa in size,of which four genes(CeAGPL1 to CeAGPL4) encoded large subunits of AGPase and two genes(CeAGPS1,CeAGPS2) encoded small subunits of AGPase.Phylogenetic analysis showed that all AGPases were divided into two groups,large and small subunits,with four large subunits in the large subunit group and two small subunits in the small subunit group.The molecular mass of CeAGP family proteins ranged from 38 753.22 kDa to 59 743.05 kDa,with an isoelectric point range of 5.64 to 8.82 and subcellular localization to chloroplasts,amyloplasts and cytoplasm,etc.The conserved motif analysis of the proteins revealed that a total of 11 conserved motifs were predicted for CeAGP family proteins,six motifs were functionally annotated as NTP_transferase,and the other five CeAGP proteins contained 11 motifs except for CeAGPS2.In different tissues,all six CeAGP genes were expressed in all tissues,with high expression of CeAGPL3 and CeAGPS1 in leaves and petioles,high expression of CeAGPL1 and CeAGPS1 in corms,and low expression of all six CeAGP genes in roots.CeAGPL1 and CeAGPS1 were highly expressed in different developmental stages of the corm and both showed a tendency to increase and then decrease,with the highest expression of both genes at 4 and 5 months of age,respectively.These results provide a basis for the subsequent elucidation of the mechanism of taro starch synthesis.