Y系山定子AP1同源基因的克隆及序列分析

Cloning and Analysis of the AP1 Homology Gene from Y Series Malus baccata(L.) Borkh

以Y系山定子为试材研究AP1基因,试图为缩短果树童期提供理论依据。根据已登录的苹果AP1基因保守序列设计引物,以Y系山定子的基因组DNA为模板进行PCR扩增,获得1 010 bp的基因片段;PCR产物纯化后,将其连接到pMD18-T载体上,连接产物转化大肠杆菌DH5α菌株感受态细胞,通过蓝白斑筛选、质粒长度和酶切鉴定,获得重组质粒并测序。测序结果表明,该序列有2个外显子、1个内含子及2个内含子的一部分,编码区共编码81个氨基酸;对氨基酸序列保守性分析发现,第13-81个氨基酸为MADS-box基因家族特有的K区域;其氨基酸序列在GenBank中同源性检索表明,Y系山定子与其他植物AP1同源基因的氨基酸序列同源性为67.9%-100%。研究结果为揭示Y系山定子早花现象的分子机制奠定了基础,对果树育种具有重要的理论价值和实际意义。

The study of AP1 gene has theoretical value on shortening juvenility of woody fruit plants. According to the reported AP1 gene sequence of apple primers were designed to amplify the AP1 gene of M. baccata using PCR method. The fragment obtained was 1 010 bp in length. The purified PCR products were connected to the pMD18-T vector. Then the products were transformed into E. coli strain DH5α competent cells to obtain recombinant plasmid through the blue-white screening plasmid length and restriction enzyme digestion. Sequencing and sequence analysis showed that： we obtained a middle fragment of AP1 gene,which has two exons,one introns and the part of two introns. The code area altogether codes 81 amino acids,and the 13- 81 amino acids is K-region of the MADS-box gene family-specific. After the amino acid sequence carried out on the homology search in GenBank,the results showed that the amino acid sequence homology with other plants is 67. 9%- 100%. These results laid the foundation for the molecular mechanism of early-flowering phenomenon. They might have both theoretical value and practical significance in fruit tree breeding.