Jiménez Jesús A, Rodríguez Dolores, Lorenzo Oscar, Nicolás Gregorio, Nicolás Carlos
Departamento de Fisiología Vegetal, Centro Hispano-Luso de Investigaciones Agrarias, Facultad de Biología, Universidad de Salamanca, Plaza de los Doctores de la Reina s/n, 37007 Salamanca, Spain.
J Plant Physiol. 2006 May;163(7):761-9. doi: 10.1016/j.jplph.2005.07.010. Epub 2005 Oct 26.
An abscisic acid (ABA)-induced cDNA fragment encoding a putative serine/threonine protein kinase (PK) was obtained by means of differential reverse transcriptase-polymerase chain reaction (RT-PCR). The full-length clone (FsPK4) was isolated from a cDNA library constructed using mRNA from ABA-treated Fagus sylvatica L. seeds. This clone contained the 11 catalytic domains present in all PKs and a highly acidic domain in the C-terminus. By expressing FsPK4 in Escherichia coli as a His tag fusion protein, we obtained direct biochemical evidence supporting Ca2+-dependent kinase activity of this protein. The expression of FsPK4 increased after ABA treatment or warm pre-treatment, when seeds are maintained dormant, but decreased and tended to disappear when dormancy was released by stratification or under gibberellic acid (GA3) treatment, and when seeds were artificially dried. Further, FsPK4 transcript expression is tissue specific, and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the expression of the corresponding protein could be more closely related with the maintenance of seed dormancy than with responses to drought stress mediated by ABA.
通过差异逆转录聚合酶链反应（RT-PCR）获得了一个编码假定丝氨酸/苏氨酸蛋白激酶（PK）的脱落酸（ABA）诱导的cDNA片段。从使用ABA处理的欧洲山毛榉种子的mRNA构建的cDNA文库中分离出全长克隆（FsPK4）。该克隆包含所有PK中存在的11个催化结构域以及C末端的一个高酸性结构域。通过在大肠杆菌中表达FsPK4作为His标签融合蛋白，我们获得了支持该蛋白Ca2+依赖性激酶活性的直接生化证据。在ABA处理或温暖预处理后，当种子保持休眠时，FsPK4的表达增加，但在通过层积或赤霉素（GA3）处理解除休眠以及种子人工干燥时，其表达下降并趋于消失。此外，FsPK4转录本表达具有组织特异性，发现在ABA处理的种子中积累，而不是在所检测的其他ABA处理的营养组织中积累。这些结果表明，相应蛋白的表达可能与种子休眠的维持比与ABA介导的干旱胁迫反应更密切相关。
