薄壳山核桃CiDGAT1基因的克隆及表达模式分析

Cloning and expression analysis of the CiDGAT1 from Carya illinoinensis

【目的】研究薄壳山核桃(Carya illinoinensis)CiDGAT1基因的结构特征和表达模式,为深入分析CiDGAT1基因功能提供理论参考。【方法】以薄壳山核桃‘波尼’果实为材料,利用DGAT1基因保守片段设计特异引物,通过3’RACE和RT-PCR技术,克隆薄壳山核桃的CiDGAT1基因。运用生物信息学方法分析其蛋白性质、亲缘进化关系及基因结构。通过实时荧光定量PCR分析该基因在不同品种以及果实不同发育时期的表达模式。【结果】克隆到1条薄壳山核桃油脂合成相关基因CiDGAT1,该基因开放阅读框(ORF)1617 bp,编码538个氨基酸,5’UTR区包含294个碱基,3’UTR区包含340个碱基。基因结构分析显示,CiDGAT1由15个外显子和14个内含子组成。系统进化分析表明CiDGAT1蛋白聚在双子叶植物分支中,与栎树(Quercus suber)QsDGAT1和榛子(Corylus americana)CaDGAT1的亲缘关系最近。实时荧光定量PCR结果表明,在早熟品种‘波尼’和晚熟品种‘马罕’与‘金华’3个品种果实发育过程中,CiDGAT1在‘波尼’发育时表达量较高,在145 d成熟期时达到最高,而‘马罕’、‘金华’品种在生长过程中,CiDGAT1表达量一直缓慢增长,152 d后迅速升高,至最后成熟时达到最高值。【结论】CiDGAT1属于MBOAT家族,在薄壳山核桃果实发育过程中表达量呈上升趋势,是油脂合成过程中重要的调控因子。

【Objective】The purpose of the study was to elucidate the structure and expression patterns of the CiDGAT1 to provide a theoretical reference for a further analysis of the function of CiDGAT1 gene.【Method】A full-length CiDGAT1 gene was isolated from the fruit of Carya illinoinensis using rapid amplification of cDNA ends with specific primers according to the conserved DGAT1 fragment sequence.The genetic structure,protein character,and evolutionary relationship were analyzed using bioinformatics.In addition,the CiDGAT1 temporal expression pattern was analyzed in different varieties using quantitative real-time PCR.【Result】CiDGAT1 has an open reading frame with 1617 bp and encodes 538 amino acids.The gene structure analysis showed that the CiDGAT1 gene consists of 15 exons and 14 introns.The phylogenetic analysis revealed that the CiDGAT1 protein is clustered in the dicotyledonous branch with a close relationship to oak DGAT1 and filbert DGAT1.The qRT-PCR analysis showed that the expression of CiDGAT1 is higher in the growth process of‘Pawnee’and peaks in the mature stage at 145 d.Whereas,CiDGAT1 expression is lower during the growth process of‘Mahan’and‘Jinhua’varieties;it rapidly increased after 152 d,reaching the highest level at final maturity.【Conclusion】CiDGAT1 belongs to the MBOAT gene family,and its expression tends to increase during the growth process of the fruit of C.illinoinensis,which is a key regulator in the process of oil synthesis in plants.