甘蔗二氨基庚二酸异构酶基因的克隆与表达分析

以水稻ABA99594序列为探针， 使用电子克隆技术， 获得甘蔗二氨基庚二酸异构酶基因（Diaminopimelate epimerase， DAPE）的1条cDNA全长序列，命名为ScDAPE。经RT-PCR扩增和序列分析验证后， 该基因序列与电子克隆结果一致， 基因全长1 168 bp， 包含1个816 bp的开放阅读框， 编码271个氨基酸残基， 生物信息学分析预测该基因编码蛋白定位于内质网膜， 为可溶性酸性蛋白， 无信号肽， 二级结构元件多为无规卷曲， 含有2个保守功能域， 主要功能为氨基酸合成。电子表达分析结果显示， 该基因在甘蔗侧芽、 花序、 叶片和顶端分生组织中均有表达， 花序中的表达量最高， 且该基因的表达受温度调控。荧光定量PCR分析结果表明，该基因在甘蔗根、 蔗髓、 蔗皮、 侧芽、 叶片、 叶鞘中均有表达， 且在根中的表达量最高。此外， 该基因的表达受水杨酸（salicylic acid, SA）、 脱落酸（abscisic acid, ABA）、 茉莉酸甲酯（methyl jasmonate, MeJA）、 模拟干旱（PEG）、 高盐（NaCl）和氯化镉（cadmium chloride, CdCl2）的胁迫诱导， 其中受水杨酸胁迫后表达量最高， 约为对照组的12.4倍， 其次为聚乙二醇， 约为对照组的2.72倍， 推测该基因的表达与甘蔗抗病性和抗渗透胁迫有关。研究结果为甘蔗中不同DAPE基因的克隆和功能验证以及甘蔗ScDAPE基因在甘蔗基因工程中的应用奠定了一定的基础。

Cloning and Expression Analysis of a Diaminopimelate Epimerase Gene in Sugarcane

Using ABA99594.1 sequence from Oryza sativa. as the probe, the full-length cDNA sequence of a Diaminopimelate epimerase gene was cloned in silico from sugarcane(Saccharum comlex) and named as ScDAPE. The results of RT-PCR amplification and sequence analysis showed that the ScDAPE had a length of 1 168 bp containing the 816 bp open reading frame(ORF) for encoding 271 amino acids residues. The characteristics predicted based on the analysis of bioinformatics showed that the ScDAPE of sugarcane was a soluble acidic protein which had two conserved functional domains with the main function for amino acid synthesis, and was located in endoplasmic reticulum membrane. The mainly secondary structure elements were random roil. The electron expression analysis suggested that ScDAPE was constitutively expressed in different tissues of sugarcane, such as lateral buds, inflorescence, leaf and apical meristem, specifically higher in inflorescence. Real-time quantitative PCR(RT-qPCR) analysis revealed that the expression of ScDAPE was higher in root than in leaf sheath, pith, skin, lateral buds and leaf, and its expression could also be induced by several kinds of exogenous stresses including salicylic acid(SA), abscisic acid(ABA), methyl-jasmonate(MeJA), dehydration(PEG), high salt (NaCl)and cadmium chloride(CdCl2), while the inducible expression level of ScDAPE was most apparent after the stresses of SA and PEG, properly 12.4 times and 2.72 times higher than that of the control respectively, which suggested that ScDAPE involves in the sugarcane resistance to disease and osmotic stress. The results in this study will provide the basis for cloning and functional identification of other members of ScDAPE in sugarcane and promote the use of ScASAPE gene in sugarcane genetic engineering.