Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii
Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii
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DOI：
10.1023/a:1005746713759
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发表时间：
1997-01-01
影响因子：
5.1
通讯作者：
Thompson, R
中科院分区：
文献类型：
--
作者：
Liu, JQ;Seul, U;Thompson, R
从二倍体马铃薯种 Solanum berthaultii 体外萌发花粉的 cDNA 文库中分离出花粉特异性 cDNA。该 cDNA 克隆被命名为 SB401，与成熟和发芽花粉中长度为 1.2 kb 的信使 RNA 杂交。 SB401 信使 RNA 不存在于植物的其他部分，包括其他花组织。 SB401 cDNA 具有一长段富含 AT 的 5'-非翻译前导序列，编码富含谷氨酸的蛋白 (GARP)，该蛋白自始至终都是亲水性的，并包含序列 V-V-E-K-K-N/E-E 的六个不完美重复基序，其中二碱基氨基酸残基对 (K-K) 作为重复内的核心。这些重复序列以不规则的间隔排列，预计会形成α螺旋结构。 SB401蛋白在大肠杆菌中过表达，纯化后的蛋白用于制备抗血清。花粉和花粉管中大肠杆菌表达的蛋白和内源性 SB401 蛋白在 SDS 聚丙烯酰胺凝胶上看起来比其计算的分子量大得多。免疫印迹显示该蛋白质在发芽花粉中含量最丰富。讨论了 SB401 蛋白的结构特征以及该蛋白在花粉发育、花粉萌发和花粉管生长中的可能作用。
A pollen-specific cDNA was isolated from a cDNA library of in vitro germinated pollen of the diploid potato species Solanum berthaultii. The cDNA clone, designated SB401, hybridizes to a messenger RNA of 1.2 kb length in mature and germinated pollen. SB401 messenger RNA is absent from other parts of the plant, including other flower tissues. SB401 cDNA, which possesses a long stretch of AT-rich 5'-untranslated leader sequence, encodes a glutamic acid-rich protein (GARP) which is hydrophilic throughout and contains six imperfect repeated motifs of the sequence V-V-E-K-K-N/E-E with the di-basic amino acid residue pair (K-K) as the core within the repeats. These repeats are spaced at irregular intervals and predicted to form an alpha-helical structure. The SB401 protein was over-expressed in Escherichia coli and the purified protein was used for raising antiserum. Both E. coli-expressed and the endogenous SB401 proteins in pollen and pollen tubes appear much larger on SDS-polyacrylamide gels than their calculated molecular masses. Immunoblotting revealed the protein to be most abundant in germinated pollen. The structural features of SB401 protein and a possible role for the protein in pollen development, pollen germination, and pollen tube growth are discussed.