Functional Plant Biology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India.
Protoplasma. 2011 Jul;248(3):613-21. doi: 10.1007/s00709-010-0207-8. Epub 2010 Sep 18.
Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e(-108)), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling.
鉴定那些使植物能够适应各种胁迫的基因对于培育具有胁迫耐受性的作物至关重要。在本研究中，我们使用基于 PCR 的 cDNA 抑制性消减杂交技术构建了甘蔗盐（NaCl）胁迫特异的正向和反向消减 cDNA 文库。为此，我们将受到 NaCl（200mM）胁迫的茎和根组织的 mRNA 混合，在不同时间间隔（0.5 到 18 小时）进行处理。对正向消减 cDNA 文库中的克隆进行测序，我们鉴定了一个类卷曲蛋白激酶（以下称为甘蔗类卷曲蛋白激酶，SuSK；NCBI GenBank EST 数据库 Acc: FG804674）。SuSK 的序列分析显示与拟南芥卷曲相关蛋白激酶 delta（E 值，1e(-108)）、dzeta 和 iota 具有同源性。使用 ClustalW 工具对 GSK-3/类卷曲激酶的催化结构域序列与 SuSK 的部分序列进行比对，显示激酶活性位点特征序列。基于实时 PCR 的 SuSK 基因时空转录表达谱分析表明，该基因在甘蔗叶片和茎中对短期盐（NaCl 200mM）或聚乙二醇-8000（PEG；20%w/v）诱导的渗透胁迫表现出显著的转录诱导。在盐胁迫下，转录表达逐渐增加，在 8 小时处理时达到对照的 1.5 倍。在 PEG 胁迫下，叶片中对照的转录表达增加了 1.5 倍，而在茎中，各种处理下的表达保持不变。与对照、未胁迫的植物相比，短期盐或 PEG 胁迫下植物的生长参数、相对含水量和膜损伤率没有统计学差异。表达分析表明，该基因在盐和 PEG 胁迫下表现出差异和时间调节，其早期诱导可能表明其参与了胁迫信号转导。
