油菜毛状体发育相关基因GLABRA2启动子的克隆与功能验证  

Isolation and Functional Characterization of GLABRA2 Promoter Related to Trichome Development in Brassica napus

作　　者：白泽涛[1] 柴国华[1] 石磊[1] 许李明[1] 汪承刚[1] 郭学兰[1] 董彩华[1] 刘胜毅[1] 

机构地区：[1]中国农业科学院油料作物研究所农业部油料作物遗传改良重点实验室,武汉430062

出　　处：《农业生物技术学报》2010年第2期210-217,共8页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(No.2006CB101607);国家自然科学基金(No.30671344);国家高技术研究与发展计划(863)(No.2006AA10A113)共同资助

摘　　要：GLABRA2(GL2)基因在拟南芥毛状体发育中具有重要作用。实验利用基因组步移巢式PCR的方法,通过两次步移克隆得到一个油菜(Brassica napus)GL2基因启动子序列,序列分析发现它与拟南芥GL2启动子同源性较差,但有一些共同的顺式作用元件如MYB结合位点、Gbox等,也有油菜GL2基因所特有的应答元件如E-box。将其中具有启动子的基本特征的序列GP1与GUS报告基因融合构建重组载体pBI121-GP1,转化拟南芥(Arabidopsis)。用卡那霉素筛选得到10株阳性苗。在T2代转基因株系中有6个株系的绿苗与黄花苗的比例都接近3:1,推定T-DNA是以单拷贝的形式插入拟南芥基因组DNA。GUS组织化学染色表明,GP1调控下报告基因主要在子叶、真叶的表皮毛以及根部的幼嫩组织中表达,与拟南芥GL2基因启动子表达模式基本一致,但也有明显不同:油菜GL2启动子GP1只在表皮毛发育早期强烈表达,而拟南芥GL2启动子调控表皮毛发育的整个过程。GL2 gene plays key roles in trichome development of Arabidopsis.A putative GL2 promoter was isolated from genomic DNA of Brassica napus by nested PCR using genomic walking method.Bioinformatics analysis indicated GL2 promoter had low homology between Arabidopsis and B.napus.Some cis-elements were shared by two species, such as MYB binding site, G-box, etc.However, some elements were specific to B.napus, such as E-box.To study the function of this promoter, a pBI121-GP1 vector containing GP1-GUS fusion gene was constructed and introduced into Arabidopsis by Agrobacterium-mediated transformation method.Ten positive transformants were obtained.Segregation analysis in T2 generation revealed that each of six lines segregated as 3：1 for kanamycin-resistance, suggesting T-DNA insertion a single copy.Histochemical staining analysis showed that the GUS gene mainly expressed in young tissues of Arabidopsis, such as cotyledons, trichomes of true leaves and roots, which are basically consistent with the expression style of Arabidopsis GL2 promoter.However, compared with Arabidopsis GL2 promoter expressed in the whole process of trichome development, B.napus GL2 promoter only expressed in the early stage of trichome development.

关 键 词：甘蓝型油菜 GLABRA2启动子 基因组步移 拟南芥 GUS表达 

分 类 号：S794.1[农业科学—林木遗传育种]