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通过聚合酶链式反应(PCR)鉴定拟南芥花中的类受体蛋白激酶

Identification by PCR of receptor-like protein kinases from Arabidopsis flowers.

作者信息

Takahashi T, Mu J H, Gasch A, Chua N H

机构信息

Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10021, USA.

出版信息

Plant Mol Biol. 1998 Jul;37(4):587-96. doi: 10.1023/a:1005924817190.

Abstract

We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.

摘要

我们利用针对蛋白激酶保守结构域的简并寡核苷酸引物,通过聚合酶链反应(PCR)从拟南芥花的cDNA文库中分离出了三个类受体激酶cDNA。对全长cDNA(命名为RKF1至3,即花中的类受体激酶)进行克隆和测序后发现,RKF1蛋白假定的胞外结构域含有13个富含亮氨酸序列的串联重复,而RKF2和RKF3与其他植物序列没有明显的同源性。RNA印迹分析表明,RKF1 mRNA在雄蕊中高度表达,而RKF2和RKF3 mRNA在所检测的所有器官中表达水平较低。原位定位实验表明,在早期花原基和雄蕊发育过程中可检测到RKF1 mRNA。此外,当与GUS报告基因融合时,RKF1启动子在花粉粒中指导高水平的GUS表达。在大肠杆菌中产生的重组RKF1在体外被发现具有丝氨酸/苏氨酸特异性的激酶活性。

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