陆地棉GhRACK1启动子的克隆与缺失分析

Cloning and Deletion Analysis of GhRACK1 Promoter from Gossypium hirsutum

在基因工程发展中,有应用前景的纤维特异表达启动子的缺乏是制约棉花纤维品质改良的主要因素之一。本研究利用反向PCR方法克隆到陆地棉纤维优势表达基因GhRACK1的上游启动子GhRACK1-P。GhRACK1-P全长为1987 bp,含有TATA-box、CAAT-box、MYB2和I-box等顺式作用元件和调控元件。根据调控元件的分布对GhRACK1-P进行不同程度的缺失,获得了p1、p2、p3和p4缺失体;构建不同缺失体和全长GhRACK1-P的植物表达载体,并通过农杆菌介导法导入烟草,获得不同类型转基因烟草。GUS组织化学染色结果表明,全长启动子GhRACK1-P只能驱动gus基因在转基因烟草的幼根及根毛中表达,其他缺失体驱动gus基因在转基因烟草的花粉、叶片和根中表达,为组成型表达启动子。由于根毛与棉花纤维具有相似的发育机制,推测全长GhRACK1-P可能为纤维优势表达启动子。本研究结果为棉花纤维品质改良基因工程提供了新的调控元件。

The absence of fiber specific promoter with future prospect is one of the main factors to restrict the development of genetic engineering in cotton fiber improvement. A 1987 bp length promoter sequence of Gossypium hirsutum GhRACK1 gene, which encodes receptor for activated C kinase 1 and precedantly expresses in fiber, was cloned by combination of inverse PCR and touchdown PCR method. Sequence analysis showed there were lots of promoter regulation elements such as cis acting factor and the tissue specific regulation elements. The full-length GhRACK1-P and truncations from -600 to -1 bp, 1036 to -1 bp, -1260 to -1 bp and -1620 to -1 bp were obtained by PCR method. Each of the truncations was fused with gus gene and inserted into plant expression vectors pCamBIA2300. All constructs were transformed into Nicotiana tabacum var. NC89 through Agrobacterium-mediated transformation method. GUS histochemical assay showed that the full-length GhRACK1-P promoter was expressed in root and exhibited a tissue-specific expression manner. All of the truncations were expressed in root, leaf and pollen and exhibited a constitutive expression manner. Because there is the similar developmental mechanism between cotton fiber and tobacco young root or trichome, the results indicate that GhRACK1- P may be a fiber specific expression promoter.