水稻花粉專一啟動子之分析

Abstract

為了瞭解水稻花粉專一性基因表現之功能，本試驗根據前人研究針對水稻花粉時期表現量高但功能未知的基因，從中得到啟動子的片段，透過接上報導基因GUS、GFP以及致死基因barnase，來探討啟動子的專一性表現。本試驗分為兩大部份：第一部份為從水稻花粉發育時期中SBS轉錄體資料，選出花粉時期高TPM（transcripts per million）值的基因，在其上游約1.5kb片段處設計專一性引子，藉由PCR分別得到P124與P128啟動子。另外為了利於啟動子分析，將P124啟動子從5’端逐漸刪除（Deletion）為1.2 kb、1 kb、0.9 kb、0.6 kb、0.3 kb之片段。並置於pCAMBIA1301與pCAMBIA1303載體內，建立C1301/C1303報導基因表現系統構築，隨後透過農桿菌轉殖於水稻與阿拉伯芥中。最後將所得水稻轉植株進行TAIL-PCR，藉此合成T-DNA插入側翼序列後，與NCBI資料庫進行比對。第二部分則將P128啟動子接上barnase，預期專一性表現於雄不稔。經由上述所設計的專一性啟動子，在C1301-P124F/C1303-P124F的水稻轉植株中發現GUS明顯表現於花粉時期之花藥維管束及穎花花瓣，其餘根莖葉表現不明顯；阿拉伯芥中GUS出現於開花後的花粉及花藥維管束；C1301-P128F的轉植株水稻的GUS則明顯表現於花粉時期之花藥及花粉。試驗至此，推測P124、P128之啟動子可啟動水稻花粉的相關基因之表現，且具有相當程度的專一性，而P128的刪除分析更有待未來研究深入探討，期望產生無活性之花粉以利雄不稔發展。

In order to illustrate the functions of pollen-specific genes, putative promoter sequences were isolated from genes in rice pollen with high expression. The putative promoters were constructed with reporter genes, GUS, GFP or a lethal gene (barnase), to explore the specificity of the promoter. First, we screened for the genes with high TPM during the development stages of the pollen based on SBS transcription database. Specific primers were designed to amplify the DNA fragments of 1.5 kb upstream the target genes by PCR, which were named P124 and P128. Also, to characterize the P124 sequences, they were sequentially deleted from the 5’ end to acquire fragments of 1.2, 1.0, 0.9, 0.6, and 0.3 kb. The cloned DNA fragments were then constructed into pCAMBIA1301 and pCAMBIA1303 vectors, and introduced into the rice and Arabidopsis plant via Agrobacteria. Genomic DNA of the transgenic rice was subjected to amplify the flanking sequences by TAIL-PCR. In order to assess the ability of triggering male sterility, P128 was constructed with the barnase gene from Bacillus amyloliquefaciens. The GUS expression was observed mainly in vascular bundle of the anther and petal of the C1301/C1303-P124F transgenic rice as well as in vascular bundle of the anther and pollen of the transgenic Arabidopsis. Additionally, GUS was significantly expressed in the anther and pollen of the transgenic rice containing C1301-P128. The sequential deletion analysis of P128 contributes to further research in male sterility from inactive pollens.