苹果果实β-半乳糖苷酶基因启动子的克隆与功能分析

摘要

根据已发表的苹果基因组序列设计特异引物,克隆得到苹果品种'嘎拉'中β-半乳糖苷酶基因(Md-gal)的启动子.序列分析表明,该启动子除含有大多数高等植物启动子具有的保守元件外,还含有大量光响应元件和与激素相关的顺式作用元件,主要有赤霉素响应元件(GARE-motif和P-box)、茉莉酸甲酯(MeJA)应答元件(CGT-CA-motif和TGACG-motif)和生长素响应元件(TGA-element);构建5个不同长度Md-gal启动子和GUS基因融合的瞬时表达载体,并进行苹果果实和烟草叶片转化试验,结果显示,5个启动子均具有启动子活性,在Md-gal启动子序列中激活与抑制调节元件并存,正调控元件位于-1206~-754 bp区域,负调控元件位于-754~-597 bp区域;对Md-gal启动子进行激素响应分析结果显示,激素处理可对其产生诱导作用,且MeJA处理后GUS活性最高.研究表明,Md-gal启动子具有真核基因启动子的基本结构特征和正负调控特性,可响应外源激素处理,可能在苹果果实生长发育和成熟软化方面具有一定的作用.%Primers were designed according to the published sequence of the apple genome and a Md-gal gene promoter of Malus× domestica Borkh 'Gala'were cloned.Online function prediction by PLACE and Plant CARE showed that Md-gal promoter contained a large number of phytohormone responsive ele-ments,such as gibberellin-responsive elements (GARE-motif,P-box),auxin-responsive element (TGA-element),MeJA responsive elements (CGTCA-motif,TGACG-motif)and some stress responsive ele-ments.Meanwhile,to identify the upstream region of the Md-gal gene required for promoter activity,we constructed five Md-gal promoter deletion derivatives.Each deletion construct was analyzed by Agrobac-terium-mediated transient transformation in apple fruits and tobacco leaves.Then the transgenic apple and tobacco with GUS histochemical staining and quantitative detection of GUS enzyme activity were analyzed. The results indicated that all the five promoters had the promoter activity,and both activation and repres-sion regulatory elements co-existed in the Md-gal promoter sequence.Positive regulatory elements may be located in a region from -1206 to -754 bp and negative regulatory elements are present at position from-754 to -597 bp.Finally,hormone treatment assay suggested that the cis-regulatory regions in the Md-gal promoter was responsive to different exogenous hormones in tobacco,and the highest level of GUS ac-tivity was observed in MeJA treatment.Taking together,our results demonstrate that Md-gal promoter with the basic structural characteristics of eukaryote's gene promoters has the up-and down-regulation function and hormonal treatment responsiveness,which might be involved in apple fruit development and ripening.