甘薯IbGL3的克隆和表达分析

Cloning and expression analysis of IbGL3 in Ipomoea batatas

紫色甘薯富含花青素,具有较高的食用和药用价值。花青素的生物合成受到结构基因和调节基因的控制。bHLH(basic helix-loop-helix protein)转录因子能够调节多个花青素结构基因的表达,在花青素生物合成途径中具有重要的调控作用,但目前在甘薯中还没有关于bHLH调控花青素生物合成的相关报道。为进一步了解IbGL3基因在甘薯花青素生物合成的功能和作用机理,该研究根据甘薯转录组数据,利用RTPCR技术在甘薯中克隆了一个2 120 bp的bHLH基因IbGL3,该基因包含一个1 878 bp的开放阅读框,编码625个氨基酸,蛋白质分子量69.08 kD,理论等电点(pI) 5.20。IbGL3蛋白和其他植物中类黄酮合成相关的bHLH蛋白具有较高的同源性,都包含保守的MIR区、bHLH结构域和ACT类似结构域。系统发育进化树分析结果显示,IbGL3与其他植物类黄酮相关bHLH蛋白聚为一类,属于Ⅲf亚类成员。表达结果显示,IbGL3基因在紫色甘薯中的表达量最高,在浅紫色甘薯中的表达量次之,在白色甘薯中表达量最低,与花青素积累正相关,因此推测其在甘薯花青素生物合成途径中具有重要的调控作用。

Anthocyanin-rich purple sweet potatos have high edible and medicinal values.Anthocyanin biosynthesis is controlled by structural genes and regulatory genes.The basic helix-loop-helix protein(bHLH)transcription factor plays an important role in anthocyanin biosynthesis by regulating multiple structural genes.However,there are no reports about bHLH regulating anthocyanin biosynthesis in sweet potato.In order to further understand the function and molecular mechanism of IbGL3 in the biosynthesis of anthocyanins in sweet potato,in this study,a bHLH gene named IbGL3 was cloned in Ipomoea batatas based on transcriptome data and RT-PCR technology.The full-length cDNA of IbGL3 was 2 120 bp,containing 1 878 bp opening reading frame(ORF),and encoding 625 amino acids.The encoded protein of IbGL3 has a molecular weight of 69.08 kD and the theoretical isoelectric point(pI)of 5.20.The IbGL3 protein had highly conserved MIR motif,bHLH domain and ACT domain,shared high identities and similar domains with bHLH proteins involved in anthocyanin biosynthesis from other plants.Phylogenetic analysis showed that IbGL3 was clustered in theⅢf bHLH subgroup together with other anthocyanin-related bHLH proteins.The expression of IbGL3 in the storage root of different sweet potato varieties was detected by quantitative polymerase chain reaction(qPCR).The results indicated that IbGL3 was mainly expressed in purple-fleshed sweet potato,followed by light purple-fleshed sweet potato and weakly expressed in white-fleshed sweet potato,and its expression was positively related to the accumulation of anthocyanin Ipomoea batatas.The results showed that IbGL3 may be involved in regulating anthocyanin biosynthesis in Ipomoea batatas.