纤枝短月藓BeLEA2基因的克隆及表达分析

为探究纤枝短月藓LEA2基因的结构和表达特征,该研究以纤枝短月藓为材料,首次利用PCR克隆技术得到纤枝短月藓BeLEA2基因序列,并对该基因进行分析。结果表明:(1)该基因序列中含有2个外显子和1个内含子,其开放阅读框(ORF)为456 bp,编码151个氨基酸,预测其相对分子质量为16515.96 Da。(2)将纤枝短月藓与其他植物LEA2基因氨基酸序列进行比对,构建系统进化树,结果显示纤枝短月藓与小立碗藓的亲缘关系最近。(3)利用HiTail-PCR技术克隆获得1072 bp的BeLEA2启动子序列,用PlantCARE在线工具对该启动子的顺式作用元件进行预测,结果表明该启动子除了含有核心启动子元件TATA-box和CAAT-box外,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他顺式元件。(4)实时荧光定量PCR分析表明,BeLEA2基因在纤枝短月藓不同发育时期和不同组织中都有表达,且对脱水胁迫有响应。以上结果为进一步探究LEA2基因在苔藓植物中的功能及作用机制奠定了基础。

Cloning and expression analysis of BeLEA2 gene from Brachymenium exile

The purpose of this study was to explore the structural and expression characteristics of LEA2 genes from Brachymenium exile. BeLEA2 gene was firstly isolated and analyzed by polymerase chain reaction(PCR). The results were as follows:(1)Gene structure analysis showed that BeLEA2 gene contained 2 exons and 1 intron and contained an open reading frame(ORF)of 456 bp encoding a protein of 151 amino acids, and its molecular mass was predicted to be 16 515.96 Da.(2)The phylogenetic analysis of LEA2 with other LEA2 in different plants revealed that BeLEA2 from B. exile and LEA2 from Physcomitrella patens belonged to the same branch of evolutionary distance.(3)The promoter sequence of the BeLEA2 gene of 1 072 bp was isolated from Brachymenium exile by high-efficiency hermal asymmetric interlaced polymerase chain reaction(HiTail-PCR)and analyzed by PlantCARE, the results showed that it had TATA-box, CAAT-box, ABRE, MYB, MYC, MYB binding site and other cis-acting elements.(4)Quantitative real-time PCR analysis indicated that BeLEA2 expressed in different stages and tissues of B. exile, and BeLEA2 responded to dehydration stress. These results lay a foundation for further study on the function of LEA2 gene in bryophytes.