Molecular cloning, functional expression and characterization of two serine/threonine-specific protein kinases from Nicotiana tabacum pollen
Molecular cloning, functional expression and characterization of two serine/threonine-specific protein kinases from Nicotiana tabacum pollen
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DOI：
10.1007/s00497-004-0228-6
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发表时间：
2004-12-01
影响因子：
--
通讯作者：
Norioka, S
中科院分区：
文献类型：
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作者：
Dissanayake, K;Castillo, C;Norioka, S
花粉和花粉管中存在不同种类的激酶，这表明在高等植物的生活史中，激酶介导的信号通路可能参与调节花粉萌发和花粉管生长。本研究利用RT-PCR和RACE技术从烟草花粉中分离到了两个激酶基因NtPK 1和NtPK 2的全长cDNA。NtPK 1和NtPK 2编码365和369个氨基酸的蛋白质，计算分子量分别为39.2 kDa和39.5 kDa，并且两种蛋白质都具有在蛋白激酶中保守的12个亚结构域。NtPK 1和NtPK 2的核苷酸和推导的氨基酸序列分别有88%和91%的同源性，其中C-末端区域是最保守的。RT-PCR分析表明，NtPK 1在花粉和花粉管中特异表达，NtPK 2在雌蕊和花瓣中也有表达。使用抗NtPK 1和抗NtPK 2抗体的免疫印迹分析证实，NtPK 1和NtPK 2都产生于花粉和花粉管中，并且NtPK 2也产生于发育中的雄配子体和其他花组织中。生化分馏实验表明，在所有的组织检查，NtPK 1和NtPK 2存在于胞质组分，而不是在微粒体组分。发现NtPK 1和NtPK 2在苏氨酸上自磷酸化，对于NtPK 2，在丝氨酸上也自磷酸化。这些结果表明，NtPK 1和NtPK 2是一种新型的受体样胞质丝氨酸/苏氨酸激酶，可能介导花粉萌发和/或花粉管生长所需的信号通路。
The existence of different kinds of kinases in pollen and pollen tubes suggests that kinase-mediated signaling pathways are likely involved in regulating pollen germination and pollen tube growth during the life cycle of higher plants. We have used RT-PCR and RACE to isolate full-length cDNAs for two pollen-expressed kinases, named NtPK1 and NtPK2, of Nicotiana tabacum. NtPK1 and NtPK2 encode proteins of 365 and 369 amino acids with calculated molecular masses of 39.2 kDa and 39.5 kDa, respectively, and both proteins possess the 12 sub-domains that are conserved among protein kinases. The nucleotide and deduced amino acid sequences of NtPK1 and NtPK2 share 88% and 91% identity, respectively, with the C-terminal region being the most conserved. RT-PCR analysis revealed that NtPK1 was specifically expressed in pollen and pollen tubes, and that NtPK2 was also expressed in pistil and petal. Immunoblot analysis using anti-NtPK1 and anti-NtPK2 antibodies confirmed that both NtPK1 and NtPK2 were produced in pollen and pollen tubes, and that NtPK2 was also produced in developing male gametophytes and other floral tissues. Biochemical fractionation experiments showed that, in all the tissues examined, NtPK1 and NtPK2 were present in the cytosolic fraction and not in the microsomal fraction. NtPK1 and NtPK2 were found to autophosphorylate on threonine and, for NtPK2, on serine as well. All the results taken together suggest that NtPK1 and NtPK2 are novel receptor-like cytosolic serine/threonine kinases, and could mediate signaling pathways required for pollen germination and/or pollen tube growth.