麻疯树胚乳类F-盒蛋白基因的克隆和载体构建

通过随机测序从本实验室构建的麻疯树胚乳cDNA文库中获得了一条与多种植物、动物F-盒家族(F-box)序列相似的EST序列,并进一步克隆得到含有完整编码框的cDNA全长和基因组序列(Jc-Fbl1).Jc-Fbl1cDNA全长1672bp,开放阅读框为1224bp,编码的蛋白质含有407个氨基酸残基.Jc-Fbl1推导的氨基酸序列与拟南芥、水稻中的同源F-box蛋白相似性分别为52%和75%;与人、斑马鱼、线虫等动物的F-box蛋白的相似性达到48%~56%.应用Pfam Search寻找功能结构域,发现其除了含有F-盒家族类结构域外,还有富含亮氨酸重复单位(leucine-rich repeats,LRRs).比对Jc-Fbl1的基因组序列和cDNA序列,发现Jc-Fbl1有两个长度分别为331bp和893bp的外显子和一个长362bp的插入序列,符合内含子的GT-AG法则,认为是Jc-Fbl1的内含子.水稻、拟南芥基因组中的同源序列也出现了这种两个外显子、一个内含子的结构,且内含子位置较为保守.为了进一步研究麻疯树F-box蛋白的功能,构建了同时含有Jc-Fbl1完整开放阅读框序列和绿色荧光蛋白标签的真核表达载体pBI121-Jc-Fbl1-GFP,并经酶切鉴定构建成功.

Cloning and Eukaryote Expression Vector Construction of a Novel Protein Gene Containing F-box Motif from Jatropha curcas L. Endosperm

A new gene, Jc-Fbll, was cloned from an endosperm cDNA library of Jatropha curcas L. The cloned cDNA encompasses a 1 224 bp open reading frame, which encodes a 407 amino acid peptide. The alignments with F-box proteins in plants show 52% and 75% identity with Arabidopsis thaliana and Oryza sativa respectively, while the alignments with F-box proteins in animals show 48% -56% identity. Pfam search predicted some conserved domains or motifs in the deduced protein, including F-box and leucine-rich repeat domains. The genomic gene sequence was obtained too, and its analysis indicated that two extrons and an intron were 331 bp, 893 bp and 362 bp long, respectively. Nucleotide sequences of all exon/intron boundaries agreed with the GT/AG rule. The similar genomic character was also found in the A. thaliana and O. sativa homologues. Moreover, a eukaryotic expression vector, pBI121- Jc-Fbll-GFP, was constructed and confirmed, which helps farther studies on this protein.