棉花ADP-ribosylation factor基因(GhARF1)的克隆与表达分析

Cloning,Expression and Characterization of an ADP-ribosylation Factor Gene from Cotton (Gossypium hirsutum L.)

ADP-ribosylation factor(ARF)是GTP结合蛋白,属于小G蛋白超家族中的ARF亚家族成员,在高尔基体小囊泡形成以及细胞信号传导中起重要作用。棉花纤维的发育需要有高尔基体参与,但ARF基因在棉花纤维发育中的功能尚不清楚。本文利用GHA27作探针,筛选棉花花药的cDNA文库,得到两个序列相同的阳性克隆。核苷酸序列分析表明该基因编码一个有181个氨基酸残基,分子量约20.7kD的蛋白,与动物、酵母及其他植物的ARF基因有很高的相似性,命名为GhARF1。采用PCR技术获得该基因序列,该基因含有5个外显子和4个内含子。进化树分析表明该基因更接近人类的classⅠ类ARF基因,在编码的氨基酸序列中具有保守的GTP结合区域,即P(GLDAAGKT)、G(NKQD)以及G'(DVGGQ)。Northern杂交结果显示GhARF1基因在棉花的纤维、花冠和根中表达量较高,特别是在纤维中的表达量最高,在茎、花蕾中也有表达,而在真叶、子叶和胚珠中的表达较微弱。Southern杂交分析表明,ARF基因在二倍体草棉和四倍体陆地棉基因组中均以多拷贝存在。

Cotton （ Gossypium hirsutum L. ） is the most important fiber crop in the plant kingdom. In addition to their economic importance, cotton fibers are an ideal experimental material to study cell differentiation, cell elongation, cell wall biosynthesis. ADP-ribosylation factors （ARFs）, a subfamily of the small GTP binding proteins superfarnily, are believed to participate in vesicular transport and signal transduction events in the cell. The elongation of cotton fiber requires vesicle trofficking to deposit new wall material and increase plasma membrane area, but the function of ARF within the key developmental processes of fiber is still unknown. The positive clone was screened from cotton anther eDNA library using GHA27 as a probe, and its corresponding genome sequence was obtained by PCR. Southern and Northern blotting were employed to determine the gene copy number and gene expression level of GhARF1 in cotton. The eDNA, designated as GhARF1, encoded a polypeptide of 181 amino acid residues with significant homology to the ADP-ribosylation factor （ARF） of mammalian, plant, yeast. Its deduced amino acid sequence showed that the 20.7 kD protein had consensus with GTPbinding domain P（GLDAAGKT）, G＇ （DVGGQ）, and G（NKQD）. The DNA sequence of GhARF1 is 1.5 kb in size, including five exons and four introns. Northern blotting showed that GhARF1 trancript was detected mainly in root and corolla rather than in shoot, bud, leaf, cotyledon and ovule, and maximally in fiber. Southern hybridization suggested that GhARFI is encoded by multi-copies in the genome of Gossypium herbaceum or Gossypium hirsutum. High transcript level of C, hARFI in fiber cells suggests that GhARF1 may play a role in cotton fiber development, especially in fiber cell elongation.