Molecular cloning and characterization of the actin-depolymerizing factor gene in Gossypium barbadense
Molecular cloning and characterization of the actin-depolymerizing factor gene in Gossypium barbadense
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海岛棉肌动蛋白解聚因子基因的分子克隆与表征
DOI：
10.1266/ggs.83.383
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发表时间：
2008-10-01
影响因子：
1.1
通讯作者：
Ma, Zhiying
中科院分区：
文献类型：
--
作者：
Chi, Jina;Wang, Xingfen;Ma, Zhiying
海岛棉(Gossypium barbadense L.)在抗黄萎病和许多纤维品质方面具有很高的价值，包括纤维长度、强度和细度。本研究克隆了一个肌动蛋白解聚因子(ADF)基因，并对其与陆地棉纤维发育相关基因进行了鉴定。该基因编码139个氨基酸，其中非极性氨基酸占39.57%，酸性氨基酸占17.27%，碱性氨基酸占15.83%，疏水性氨基酸占31.92%。其分子量约为15 kDa，等电点为5.04。GbADF1含有两个保守结构域，即6-Ser和PIP2/肌动蛋白结合位点。其氨基酸序列与其他植物的ADF/cofilin家族相似。与cDNA3‘端比较，GbADF1基因在基因组序列的3’端附近含有1个内含子。半定量RT-PCR结果表明，GbADF1在棉花中是一个结构性表达基因，在纤维中的表达水平高于在营养组织中的表达水平。成功地在大肠杆菌BL21(DE3)中表达了GbADF1融合蛋白。首先用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定其相对分子质量。Western blotting分析证实存在与GbADF1相对应的蛋白质。虽然所有克隆的ADF的编码序列都高度保守，但GbADF1的结构与高等植物中其他ADF基因的结构不同。
The Sea Island cotton (Gossypium barbadense L.) has been highly valued in verticillium wilt resistance and many fiber qualities including fiber length, strength and fineness. To identify whether it had some special genes in fiber development in comparison with the Upland cotton (G. hirsutum L.), an actin-depolymerizing factor (ADF) gene was cloned and characterized in this research. A 420 bp open reading frame of the cloned gene, named GbADF1, encoded a protein of 139 amino acids, including 39.57% nonpolar amino acids, 17.27% acidic amino acids, 15.83% basic amino acids and 31.92% hydrophobic amino acids. Its molecular weight was about 15 kDa, and pI 5.04. GbADF1 contained two conserved domains, 6-Ser and the PIP2/actin binding site. Its amino acid sequence was similar to the ADF/cofilin family of other plants. Compared with cDNA sequence, the GbADF1 gene contained one intron near the 3' end in genomic sequence. Semi-quantitative RTPCR result showed that GbADF1 was a constitutive expression gene in cotton, and higher expression level was detected in fibers than in trophic tissues. The GbADF1 was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3). The molecular weight was firstly calculated by SDS-PAGE. Western blotting analysis confirmed the existence of a protein corresponding to GbADF1. The structure of GbADF1 was different from that of other ADF genes in higher plant, although the coding sequences of all cloned ADFs were highly conserved.