甘薯Iborf56基因的克隆与表达分析

Cloning and Expression Analysis of Sweet Potato Iborf56 Gene

为了研究甘薯叶绿体基因Iborf56的表达是否受到盐胁迫的诱导以及对生长发育的影响,为甘薯的品种选育提供参考,以栗子香为研究材料,从中克隆Iborf56基因,通过生物信息学方法分析其蛋白序列特征,亚细胞定位预测、系统发育分析、调控元件分析、不同组织及盐胁迫表达模式,以及qPCR验证该基因的表达量。结果表明,Iborf56与已知序列相似度为99.28%,开放阅读框长度为929 bp,编码60个氨基酸,含有8个磷酸化位点,没有信号肽和跨膜区,不具有分泌蛋白的特性,主要由α-螺旋组成;亚细胞定位在叶绿体基质中;进化树分析表明,Iborf56与三浅裂野牵牛(Ipomoea trifida)和三裂叶薯(Ipomoea triloba)的遗传距离最近;启动子元件分析发现,该基因启动子区域含有光响应元件、抗逆和激素应答等功能元件。转录组以及实时荧光qRT-PCR定量分析表明,Iborf56在甘薯叶片中的表达量最高且随生长时间增加呈显著上升趋势,推测该基因参与甘薯的生长发育;此外,Iborf56基因的表达量随盐胁迫时间表现为先升后降的趋势,且该基因在盐胁迫下12 h时达到了临界值,暗示其可能参与甘薯盐胁迫响应。

To investigate whether the expression of the chloroplast gene Iborf56 in sweet potato is induced by salt stress and its effect on growth and development,and provide a reference for sweet potato breeding,in this study,the sweet potato variety,Chestnut Fragrance,was used as the research material to clone the Iborf56 gene.The protein sequence features,subcellular localization prediction,phylogenetic analysis,regulatory element analysis,expression patterns under different tissues and salt stress were analyzed using bioinformatics methods,and qPCR was used to validate the expression level of Iborf56.The results showed that Iborf56 had a 99.28%similarity with known sequences from NCBI and an open reading frame length of 929 bp,encoding 60 amino acids with 8 phosphorylation sites.The Iborf56 protein lacked signal peptides and transmembrane regions and did not possess the characteristics of secreted proteins.It was mainly composed ofα-helices and its subcellular localization was in the chloroplast stroma.Phylogenetic tree analysis showed that Iborf56 had the closest genetic distance to Ipomoea trifida and Ipomoea triloba.Promoter element analysis revealed that the promoter region of this gene contained functional elements such as light response,stress response,and hormone response.Transcriptome and qRT-PCR quantitative analysis showed that the expression level of Iborf56 was the highest in sweet potato leaves and increased significantly with growth time,inferring that this gene may be involved in the growth and development of sweet potatoes.In addition,the expression level of the Iborf56 gene showed an upward trend first and then a downward trend with increasing salt stress time,and the critical value was reached after 12 h under salt stress,indicating that it may be involved in the salt stress response of sweet potatoes.