Pbf启动子在小麦离体胚乳中的特性分析

为了探明胚乳特异启动子的调控活性，对小麦醇溶蛋白盒结合因子基因（pbf）的启动子序列进行了5´、3´和中间缺失片段-GUS基因嵌合体载体（pbf.a-d）的构建，并在小麦离体胚乳中转化。GUS瞬间表达活性检测和功能缺失试验表明：pbf 的-2510- -1bp全长序列能够在小麦离体胚乳中表现活性，而5´ 端-2510bp- -670bp以及3´端-1288bp- -1bp序列的缺失则使启动子在胚乳中丧失了活性；中间序列-2160bp- -689bp的缺失使GUS的表达强度明显降低。文中对pbf启动子序列中存在的重要基序种类和数量以及基序对启动子表达调控活性的作用也给予了初步分析。本文对小麦胚乳发育状态及GUS瞬间表达体系等对pbf启动子活性检测的影响也作了探讨。

Characterization of Pbf Promoter in Wheat Endosperm In vitro

To insight into the characteristic of its endosperm-specific promoting regulation activity, –2510- -1bp sequence of the wheat pbf (prolamin-box binding factor) promoter was cloned by Polymerase Chain Reaction (PCR) amplification and a series of pbf deletion fragments were fused with the β-glucuronidase (GUS) reporter gene (UidA) and 3΄ non-coding region of the nopaline synthase gene (nos) in pCAMBIA1381z vector. And these fusions of pbf deletion had been transformed into wheat endosperm in vitro and GUS transient expressions regulated by pbf promoter deletions had been assayed subsequently. Results of GUS transient assay in wheat endosperm and Loss of function displayed that –2510- -1bp pbf sequence could show promoting activity in endosperm, however, 5’ and 3’ deletions of –2510- -670bp, –1288- -1bp sequence resulted in undetectable promoter activity; inner deletion of –2160- -689bp evidently decreased the GUS expression activity. Compared with levels of GUS expression of the Cauliflower Mosaic Virus (CaMV) 35S promoter used as a control, the full sequence of pbf promoter had lower activity. In this article, the number and sorts of main motifs in pbf promoter sequence had been described. It was suggested that the presence of a Skn-1-like motif (G/ATCAT) and a –300bp element (TGHAAARK) in the promoter sequence might be important and essential for expressing pbf promoter activity and specificity. Effects of wheat endosperm developing state and GUS-transient assay system on promoter activity assay had been discussed also.