谷子肌动蛋白基因的克隆及序列分析

Cloning and Sequence Analysis of Actin Gene from Millet (Setaria italica)

以谷子 (Setariaitalica)为材料 ,提取总RNA。根据植物肌动蛋白基因编码区的两端的保守序列设计了简并引物 ,用 5’RACE方法扩增出了谷子肌动蛋白基因编码区序列。以豌豆肌动蛋白cDNA作探针进行的Southern杂交分析表明扩增出了目的基因。将所获得的片段克隆到T载体后进行测序 ,序列分析结果表明 :谷子肌动蛋白基因的编码区长 1 1 3 1个核苷酸 ,编码了 3 77个氨基酸 ;所得序列 (命名为MIAc)与GenBank中注册的肌动蛋白基因序列的相似性均在 6 0 %以上 ,与其它肌动蛋白氨基酸序列的相似性达 89%以上。根据高等植物肌动蛋白序列相似性重建了进化树 ,表明谷子肌动蛋白与水稻肌动蛋白异型体RAc2和RAc3之间的亲缘关系最为密切 。

Total RNA was isolated from millet (Setaria italica Beauv.) seedlings. Degenerate primers of actins' coding region were designed and 5'RACE technique was employed to obtain the actin cDNA of Setaria italica. Southern blotting analysis with probe from pea tendril actin gene coding region shows that there is a high similarity between the actin of Setaria italica and pea. The putative actin gene was sequenced after cloning into pGEM_T vector and sequencing result revealed that it contains an open reading frame of 1131 bp, encoding a protein of 377 amino acids. Similarity comparison with other actin gene sequences in the GenBank shows that it shares over 60% nucleotide sequence similarity and over 89% amino acid sequence similarity with other actins. The phylogenetic tree reconstructed on the base of amino acid sequences suggests that the relationship between millet actin and rice actins(RAc2, RAc3) is most intimate and they might have diverged at the same time.