烟草远缘杂种合子中父本特异表达基因EB30的克隆与分析

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.018

摘要/Abstract

摘要： 已知烟草远缘杂种的合子中EB30基因（EST序列号EB426694）的表达具有父本特异性。参考NCBI的EST序列设计引物，克隆得到该基因的CDS序列，并对其编码的蛋白进行生物信息学分析。使用染色体步移和hiTAIR-PCR技术获取EB30基因的5'侧翼序列（启动子和5' UTR）。构建由该基因5'侧翼序列驱动的EB30-EGFP融合蛋白表达载体并转化烟草。结果表明，EB30蛋白由211个氨基酸组成，蛋白分子量为22.616 2 kD，等电点pI 为5.63。该蛋白具有亲水性，可能定位于线粒体中。烟草EB30蛋白与同属茄科的番茄和马铃薯的蛋白序列相似度最高，约为73%。不同物种的同源蛋白的N端和C端都具有一段较为保守的序列区段。观察转基因植株合子和二胞原胚能检测到较强荧光，证实所获EB30基因的5'侧翼序列在早期胚胎发生时期具有启动子活性。

关键词: 烟草, 远缘杂交, 早期胚胎发生, 父本特异表达, 基因克隆

Abstract: As known that EB30（EST accession number EB426694）paternally expressed in zygote of a tobacco interspecies hybrid, its CDS sequence was then cloned by using the EST database of NCBI. Also some characters of EB30 protein were analyzed by the methods of bioinformatics. Genome walking and hiTAIR-PCR were used to obtain the 5' flanking region of EB30 including the promoter and 5'UTR. The EB30-EGFP fusion protein expression vector driven by the 5' flanking region of EB30 was constructed and transformed into the tobacco. The results showed that EB30 gene encoded 211 amino-acid residues with a molecular weight 22.616 2 kD and pI5.63. This protein was hydrophilic, and predicted to locate in the mitochondrion. The phylogenetic tree showed that EB30 protein of tobacco was so closed to the tomato and potato, the amino acid homology was about 73%. There were two conserved regions near the N-terminal and C-terminal in homologous proteins from different species. Finally, the 5' flanking region of EB30 was proved to have a promoter activity in the early embryogenesis by fluorescence observation in the zygote and 2-celled proembryo from transgenic plants.

Key words: tobacco, interspecies hybrid, early embryogenesis, paternal expression, cloning

,罗岸. 烟草远缘杂种合子中父本特异表达基因EB30的克隆与分析[J]. 生物技术通报, 2015, 31(1): 115-121.

Luo An. Cloning and Analysis of Paternally Expressed Gene EB30 in Zygote of a Tobacco Interspecies Hybrid[J]. Biotechnology Bulletin, 2015, 31(1): 115-121.

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