一个苹果β-微管蛋白基因及其启动子序列特征分析

Sequence Characters Analysis of a β-Tubulin Gene and Its Promoter in Apple

为了探讨来自苹果的β-微管蛋白基因家族成员之一(转录本号:MDP0000749824.1)对茎生长的影响,以普通型苹果品种烟富3号和柱型苹果品种舞姿的茎尖为材料,克隆到了该基因的编码序列,该序列全长1 332 bp,编码443个氨基酸。DNAMAN比对结果表明,烟富3号和舞姿上该基因的编码序列完全一致。以舞姿及烟富3号的DNA为模板,用启动子特异性引物PCR扩增后,均获得一段1 661 bp的核苷酸序列,二者有6处碱基差异。启动子序列分析发现,其中具有赤霉素、脱落酸、茉莉酸甲酯和水杨酸等激素响应元件。构建Cam35S-MDP0000749824-GFP重组载体,在烟草叶片中完成瞬时表达,激光共聚焦显微镜观察结果表明,MDP0000749824.1编码蛋白位于细胞膜和微管上。以普通型苹果品种烟富3号和柱型苹果品种舞姿的茎尖组织为试材,实时荧光定量PCR(q RT-PCR)分析表明,该基因在柱型苹果舞姿茎尖中的转录水平显著低于普通型的烟富3号。因此,推测该基因的表达水平可能受激素调控的作用,进而对茎的伸长生长造成影响。

As the basic component of plant microtubules,tubulins are of great significance to the formation of cytoskeleton. To investigate the effect of one member of the β-tubulin gene family with the transcript number of MDP0000749824.1 on the stem growth,the coding sequence of this gene was cloned from the apple young shoot tips of standard apple variety Yanfu 3 and the columnar variety Telemon,respectively. This sequence was 1 332 bp in length which encoded a peptide with 443 amino acids. The results of DNAMAN alignment showed that the coding sequence of the gene was identical between Yanfu 3 and Telemon.Using DNA of Yanfu 3 and Telemon as templates,1 661 bp nucleotide sequences was obtained by PCR amplification with promoter specific primers. There were six bases differences between them. Promoter sequence analysis revealed that some hormone responsive elements of gibberellin,abscisic acid,methyl jasmonate and salicylic acid existed in it. Cam35S- MDP0000749824 -GFP recombinant vector was constructed. Subcellular localization analysis with tobacco leaves indicated that the protein encoded by MDP0000749824.1 was on the cell membrane and microtubules. qRT-PCR detected that the transcript level of this gene in apical stems of columnar apple Telemon was significantly lower than that of standard apple Yanfu 3. It was speculated that the gene could affect the stem elongation be cause its expression level was regulated by hormones.