小麦蛋白磷酸酶2A基因TaPP2AbB″-α启动子的克隆及表达分析

Cloning and Expression Analysis of Protein Phosphatase 2A Gene TaPP2AbB″-α Promoter in Wheat

植物蛋白磷酸酶2A(protein phosphatase 2A,PP2A)由结构亚基A、调节亚基B和催化亚基C组成,在应答逆境胁迫途径中发挥着重要作用。小麦基因Ta PP2Ab B″-α是调节亚基亚家族B″的成员,过量表达该基因可以促进拟南芥的根系生长及侧根发育,在盐胁迫和渗透胁迫条件下的作用更显著。本研究从小麦(Triticum aestivum L.)抗旱品种"旱选10号"基因组中克隆了Ta PP2Ab B″-α基因的启动子PB″α,序列长度为1899 bp,含有TATA-box和CAAT-box,以及响应干旱和渗透胁迫的顺式作用元件EECCRCAH1(?1058 bp至?1052 bp)、GCCCORE(?1073 bp至?1068 bp)和MYCCONSE(?1179 bp至?1174 bp)。将启动子PB″α和5种5′端缺失启动子片段与报告基因GUS(β-glucuronidase)连接后转化拟南芥,组织化学染色结果显示PB″α在植株的叶片和根中均有表达。5′端缺失分析表明缺失片段PB″α-1545和PB″α-1389具有启动子活性,活性区域位于?1389 bp和?946 bp之间。GUS定量分析结果显示,在盐和渗透胁迫条件下,PB″α、PB″α-1545和PB″α-1389的活性显著上升。本研究表明PB″α具有较强的启动子基本活性,并且在盐胁迫及渗透胁迫条件下活性显著上升,该结果为合理选用启动子改良作物提供了依据。

Protein phosphatase 2A（PP2A） is a heterotrimeric protein, consisting of a scaffolding subunit（A）, a catalytic subunit（C）, and a member of four families of regulatory subunits（B）. PP2 A plays significant roles in the pathway responding to abiotic stresses in plants. Ta PP2 Ab B″-α, a member of regulatory subunit B″ in wheat（Triticum aestivum L.）, enhanced root development and could develop more lateral roots in the gene overexpressed Arabidopsis, especially under the osmotic stresses of mannitol and Na Cl. In order to elucidate transcriptional regulatory mechanism of the promoter PB″α of Ta PP2 Ab B″-α, we isolated an 1899 bp full-length sequence of promoter PB″α from a drought-tolerant wheat cultivar Hanxuan 10. The PB″α sequence contained TATA-box, CAAT-box and a series of cis-acting elements responding to drought and osmotic stresses, such as elements of EECCRCAH1（from ？1058 to ？1052 bp）, GCCCORE（from ？1073 to ？1068 bp） and MYCCONSE（from ？1179 to ？1174 bp）. The full-length promoter PB″α and five 5′-end truncated PB″α promoters in different lengths fused with the reporter gene β-glucuronidas（GUS） were transformed into Arabidopsis, respectively. The histochemical staining results showed that the full-length promoter PB″α, and the deletion fragments of PB″α-1545 and PB″α-1389 could drive GUS gene expression in shoots and roots of transgenic Arabidopsis seedlings. As the result of quantitative fluorometric GUS assay, only the expression of PB″α, PB″α-1545 and PB″α-1389 could be up-regulated by salt and osmotic stresses in transgenic Arabidopsis lines, and the active region of PB″α promoter was located in the interval between ？1389 bp and ？946 bp. In conclusion, PB″α has strong basic promoter activity which is up-regulated significantly by salt and osmotic stresses. These findings contribute to the selection of a suitable promoter for crop improvement.