花藥特異基因起動子片段之分離及其對GUS基因在轉殖菸草表現之調控

Genomic Amplification of an Anther-specific Promoter Fragment and Its Regulation for GUS Gene Expression in Transgenic Tobacco

本試驗利用菸草花藥特異基因TA29的核酸序列，設計兩段引子AS29A及AS28B，以聚合酶連鎖反應的技術，從菸草染色體中分離出366 bp的TA29起動子區域。將此起動子接於含GUS基因的植物表現載體上，構築成pBIA129，經由農桿菌將pBIA129送入菸草中，其中的5株再生植株以聚合酶連鎖反應及南方氏雜交分析，確定GUS基因已嵌入轉殖菸草的染色體中。以組織化學法分析GUS酵素的活性，證實GUS在TA29起動子的調控下，僅在轉殖菸草花藥的營養層細胞表現，而在葉子等其他組織並不表現。這結果顯示，利用聚合酶連鎖反應分離出來的TA29起動子366 bp小片段，具有控制組織特異表現的能力。

Based on the sequence information for the promoter region of TA29, a pair of primers AS29A and AS28B that could specifically amplify this promoter region was synthesized. DNA fragment of 366 bp containing the promoter region of T A29 was amplified from tobacco genomic DNA by polymerase chain reaction. To test the tissue specific function of this PCR amplified promoter fragment, a chimaeric gene construct pBIA129 that contains this 366 bp promoter region fused to GUS reporter gene was obtained. Five kanamycin resistant tobacco plants were selected. The existence of the chimaeric gene, TA29-366/ GUS/ NOS-T, in each kanamycin resistant tobacco plant was demonstrated by PCR and Southern analysis. The histochemical analysis of GUS activity showed that the GUS was appeared only in the tapetal cells of tobacco anther. These results indicated that the PCR amplified promoter fragment could program the specific expression of GUS gene in transgenic tobaccos.