薏苡油脂合成关键基因克隆及其生物信息学分析

Cloning and bioinformatic analysis of key lipid synthesis genes from Coix lacryma-jobi

【目的】掌握薏苡二酰甘油酰基转移酶基因(ClDGAT)、内质网油酸脱饱和酶基因(ClFAD2)和硬脂酸脱饱和酶基因(ClSAD)的生物信息学特性,明确薏苡油脂代谢调控机制,为今后开展以提高薏苡含油量和改良脂肪酸组成为目标的育种研究提供理论依据。【方法】利用RT-PCR和RACE克隆薏苡油脂合成关键基因(ClDGAT、ClFAD2和ClSAD),通过染色体步移技术获得ClFAD2和ClSAD基因启动子序列,并以ExPASy ProtParam tool、SMART、TMHMM Server v.2.0和SOPMA等在线软件进行生物信息学分析。【结果】从薏苡叶片中成功克隆获得ClDGAT、ClFAD2和ClSAD基因,NCBI登录号分别为MK589802、MK589803和MK589804。其中,ClDGAT基因cDNA序列全长1842 bp,其开放阅读框(ORF)为1539 bp,编码512个氨基酸;ClFAD2基因cDNA序列全长1768 bp,其ORF为1164 bp,编码387个氨基酸,无内含子;ClSAD基因cDNA序列全长1727 bp,其ORF为1182 bp,编码393个氨基酸,有2个内含子。ClDGAT和ClFAD2为不稳定蛋白,ClSAD为稳定蛋白;ClDGAT为疏水性蛋白,ClFAD2和ClSAD为亲水性蛋白。ClDGAT蛋白含有MBOAT功能域,有9个跨膜区;ClFAD2蛋白含有2个功能域(DUF3473和FA_desaturase),有3个跨膜区;ClSAD蛋白含有FA_desaturase_2功能域,无跨膜区。基于蛋白氨基酸序列相似性构建的系统发育进化树显示,ClDGAT、ClFAD2和ClSAD均与高粱对应的蛋白聚类在同一分支上,即薏苡在进化关系上与高粱的亲缘关系最近。ClFAD2和ClSAD基因启动子序列上均含有光响应元件(Light responsive element)、生长素响应元件(Auxin-responsive element)、茉莉酸甲酯响应元件(MeJA-responsive element)和ABA响应元件(ABA responsive element)。【结论】ClFAD2和ClSAD基因启动子序列上含有抗逆性相关顺式作用元件和植物激素响应元件,说明ClFAD2和ClSAD基因不仅参与薏苡的籽粒发育及油脂合成,还可能与植株的抗逆响应相关。

【Objective】This paper aimed to gain bioinformatic characteristics of diacylglycerol acyltransferase gene(ClDGAT),fatty acid desaturation 2 gene(ClFAD2)and stearoyl-ACP desaturase gene(ClSAD)from Coix lacryma-jobi,learn the mechanism of lipid metabolism regulation in C.lacryma-jobi and provide theoretical basis for the future breeding targeting increasing oil content and improving the fatty acid composition.【Method】Three key lipid synthesis genes in C.lacryma-jobi,ClDGAT,ClFAD2 and ClSAD,were cloned by RT-PCR and RACE technique.The promoter sequences of ClFAD2 and ClSAD were obtained by genome walking.All sequences were comprehensively analyzed by online bioinformatic tools such as ExPASy ProtParam tool,SMART,TMHMM Server v.2.0 and SOPMA.【Result】ClDGAT,ClFAD2 and ClSAD were successfully cloned from C.lacryma-jobi leaf and assigned to GenBank accession numbers MK589802,MK589803 and MK589804,respectively.The full-length cDNA of ClDGAT was 1842 bp and its open reading frame(ORF)was 1539 bp encoding 512 amino acids.The full-length cDNA of ClFAD2 was 1768 bp and its ORF was 1164 bp encoding 387 amino acids with no intron.The full-length cDNA of ClSAD was 1727 bp and its ORF was 1182 bp encoding 393 amino acids with 2 introns.ClDGAT and ClFAD2 were unstable proteins while ClSAD was a stable protein.ClDGAT was a hydrophobic protein while ClFAD2 and ClSAD were hydrophilic proteins.ClDGAT contained MBOAT domain with nine transmembrane domains,ClFAD2 protein contained two functional domains(DUF3473 and FA_desaturase)with three transmembrane domains and ClSAD protein contained functional domain FA_desaturase_2 without transmembrane domains.Phylogenetic tree constructed based on the amino acid sequences showed ClDGAT,ClFAD2 and ClSAD were clustered on the same branch with the corresponding proteins of sorghum,that was,C.lacryma-jobi was the closest to sorghum evolutionally.The promoter sequences of genes ClFAD2 and ClSAD contained auxin-responsive element,MeJA-responsive element and ABA responsive element.【Conclusion】The promoter sequences of both ClFAD2 and ClSAD contain stress-resistant cis-acting elements and plant hormone response elements,indicating ClFAD2 and ClSAD are not only involved in grain development and oil synthesis in C.lacryma-jobi,but may also be related to the plant stress response.