碱茅(Puccinellia tenuiflora)MYBAS1-like基因的克隆与功能分析

Cloning and Functional Analysis of MYBAS1-like Gene of Puccinellia tenuiflora

为了研究碱茅（Puccinellia tenuiflora）转录因子Put MYBAS1-like的功能及其在非生物胁迫下的表达特性。本研究利用差异显示法,从Na HCO3逆境胁迫下的碱茅（Puccinellia tenuiflora）c DNA文库中克隆得到MYB基因片段。利用实时荧光定量PCR、RACE技术克隆得到Put MYBAS1-like的全长序列。生物信息学表明：该基因序列中存在522 bp的开放阅读框,编码173个氨基酸,预测蛋白分子量为19.68 k D,其N端含有一个约45个氨基酸组成的MYB特征结构域。因与二穗短柄草（Brachypodium distachyon）MYBAS1-like基因同源性较高,命名为Put MYBAS1-like（Alternative splicing MYB transcription factor）。本研究还采用了实时荧光定量PCR的方法分析Put MYBAS1-like基因在几种非生物胁迫下表达的情况,通过酵母单杂交实验验证其转录活性以及分析其转录激活区,同时构建Put MYBAS1-like基因的植物过表达载体,为后续分析Put MYBAS1-like基因功能特性研究提供理论依据。

In order to analyze PutMYBAS1-lilce transcription factor function and gene expression profiling in Puccinellia tenuiflora under abiotic stress, we cloned MYB gene by differential display PCR technologies from cDNA libraries in NaHCO3-treated. The full-length of PutMYBAS1-like gene was obtained using RT-PCR and RACE techniques. Bioinformatics analysis showed that this gene harbored 522 bp opening reading frame and coded 173 amino acids with molecular weight 19.68 kD, which contained a MYB domain structure with 45 amino acids in N-terminal and was named PutMYBA S1-like （Alternative splicing MYB transcription factor） because of high homology with the member ofMYBA S1-like gene in Braehypodium distachyon. Further, gene expression levels of PutMYBAS1-like were analyzed under different abiotic stress conditions by RT-PCR, and its transcriptional activation region was located based on yeast one-hybrid assay, then its overexpression vector was constructed, which laid a foundation for future analysis ofgene function research.