甘蔗MYB转录因子基因ScMYB52-1的克隆及表达特性分析

MYB转录因子家族成员广泛参与植物的各种生物学过程,在调控植物适应非生物逆境过程中起关键作用。为分离甘蔗（Saccharum spp.）逆境响应MYB基因并研究其功能,本文应用3°RACE的方法,从甘蔗品种ROC22中克隆到一个R2R3-MYB转录因子基因,命名为ScMYB52-1。生物信息学分析表明,该基因cDNA序列长度为1072 bp,开放阅读框（ORF）长度为696 bp,5°非翻译区长54 bp,3°非翻译区长322 bp。该ORF编码231个氨基酸,推导蛋白的分子量为26.65 kDa,含有2个串联的MYB结构域。ScMYB52-1推导蛋白与模式植物拟南芥中的MYB家族蛋白的进化树聚类分析结果表明,ScMYB52-1蛋白与AtMYB52和AtMYB54的亲缘关系较近;进一步的多序列比对分析结果表明,上述三者蛋白质N端的MYB结构域序列高度一致,且三者的预测蛋白质三级结构类似,表明它们可能具有类似的功能。实时荧光定量PCR结果显示,ScMYB52-1在甘蔗组培苗的根中对PEG处理总体上不敏感,在叶中仅在3 h时短暂上调;ScMYB52-1在叶中受外源ABA和NaCl胁迫的诱导,在处理0.5 h均迅速上调表达,表达量分别为对照的3.35倍和2.75倍,并在处理期维持高于对照的水平;在根中受外源ABA和NaCl胁迫的抑制,在处理0.5 h时就开始下调表达,在处理24 h时表达量分别下调为对照的12%和40%,并在处理周期内总体上维持低于对照的水平。亚细胞定位分析表明,ScMYB52-1-GFP重组蛋白定位在烟草叶肉细胞的细胞核中。酵母双杂交自激活活性鉴定结果表明,ScMYB52-1蛋白无自激活活性。以上结果表明ScMYB52-1参与了甘蔗对高盐胁迫的应答,并可能受ABA信号通路的调控,在叶和根中存在差异的调控机制。本研究结果为进一步理解该基因在甘蔗非生物逆境适应过程中的功能及分子调控机制提供了初步的信息。

Cloning and Expression Analysis of an MYB Transcription Factor Gene ScMYB52-1 from Sugarcane

The MYB transcription factor family members are widely involved in various biological processes in plants and play a vital role in the abiotic stress response. This study aims to isolate the stress response MYB gene from sugarcane and further validate its characteristics and biological function under abiotic stresses. An R2R3-MYB gene, termed ScMYB52-1, was obtained via 3°RACE homologous cloning from the sugarcane variety ROC22 (Saccharum. spp. hybrids. cv. ROC22). The cDNA sequence was 1 072 bp long with a 696 bp open reading frame (ORF), 54 bp 5°-UTR (untranslated region), and 322 bp 3°-UTR. The ORF encoded 231 amino acids with a predicted molecular mass of 26.65 kDa and two repeats of the MYB domain. Phylogenetic analysis of ScMYB52-1 with MYB protein family from Arabidopsis thaliana indicated that ScMYB52-1 was most closely related to AtMYB52 and AtMYB54 from A. thaliana. Multiple sequence alignment of ScMYB52-1, AtMYB52, and AtMYB54 showed that the MYB domain sequences in the N-terminal of the three proteins were highly consistent. Moreover, the protein tertiary structure prediction results showed that they shared very similar structures, suggesting that the functions of ScMYB52-1 might be similar to the other proteins. Real-time fluorescence quantitative PCR (qPCR) analysis showed that the expression of ScMYB52-1 was not sensitive under PEG stress in the root. It was transitorily up-regulated in the leaf at the time point of 3 h only. When treated with ABA or NaCl, ScMYB52-1 was up-regulated rapidly in the leaf. It was 3.35 times and 2.75 times as high as that in the control at 0.5 h, respectively, and kept a higher level than control during the experiment. Conversely, ScMYB52-1 was suppressed under the two treatments in the root at 0.5 h. The expression level was 12% and 40% as much as the control at 24 h, respectively, and generally kept lower than control during the experiment. Subcellular localization analysis showed that the efficient expression of ScMYB52-1-GFP fusion protein was observed, and the fluorescence was mainly located in the nucleus of tobacco cells. The yeast two-hybrid self-activation experiment suggested that the ScMYB52-1 protein did not have transcriptional activation activity. It implied that ScMYB52-1 was involved in sugarcane response to salt stress via ABA signaling pathways and might have different regulatory mechanisms in the leaf and the root. The results would provide preliminary information for understanding the role of ScMYB52-1 in abiotic stress tolerance mechanisms in sugarcane.