- 學位論文
水稻花藥發育早期專一性啟動子之選殖與分析
Characterization and Analysis of Rice Promoter Specific for Early Stage of Anther Development
摘要
本試驗根據前人研究資料，篩選在水稻花藥前期具高轉錄量基因，並從水稻基因體中選殖出啟動子片段，並接上報導基因gus及gfp，進一步了解啟動子專一性表現；最後將啟動子接上致死基因barnase建立專一性致死系統，應用於植物基因轉殖，期望建立雄不稔水稻轉植株。本試驗主要可以分為三部分：第一部分，根據7個水稻花藥發育時期SBS轉錄體資料，篩選在花藥發育前期有高TPM（transcripts per million）值的基因，並根據NCBI水稻基因體資料庫中啟動子DNA序列設計專一性引子，利用PCR選殖出ATG起始碼前1.5 Kb啟動子DNA片段。第二部分，建構兩個系統共五個構築，利用農桿菌轉殖法，轉基因於水稻、菸草與阿拉伯芥，其系統為：（一）C1301與C1303報導基因系統：將啟動子接入pCAMBIA1301與pCAMBIA1303載體中，預期在水稻花藥專一性表現GUS、GFP。（二）barnase致死系統：將啟動子接入barnase基因前，預期觀察到雄不稔。第三部分，對pCAMBIA水稻及阿拉伯芥轉殖株進行TAIL PCR，研究T-DNA插入後水稻及阿拉伯芥側翼序列，並對阿拉伯芥轉殖株進行南方墨點分析。本次選殖的1.5 Kb啟動子命名為P13、P41，分別在減數分裂（meiosis）與液胞前期（early vacuolated）大量轉錄。報導基因系統C1301-P13與C1303-P13之水稻、菸草與阿拉伯芥轉殖株皆有GUS表現，在轉殖水稻花藥及小孢子有部分GUS表現；在轉殖菸草的花藥、花萼與柱頭有部分GUS表現；在轉殖阿拉伯芥只有在幼苗有部分GUS表現。而C1301-P41與C1303-P41轉殖水稻之小孢子及花藥內壁有GUS表現。barnase致死系統的P13bar阿拉伯芥轉殖株花粉進行TTC染色確認花粉仍有活性。阿拉伯芥TAIL PCR結果發現T-DNA插入基因體之中，與阿拉伯芥的南方墨點分析結果相呼應。未來將進一步分析P13及P41啟動子確切的表現時期與組織，建構P41bar觀察barnase是否造成花藥敗育導致雄不稔，並將P13與P41啟動子用於雙重調控系統，應用於雜交水稻研究。
並列摘要
In this study, anther-specific promoters were cloned from rice which highly express in rice anther. In order to characterize the functions of promoters, the reporter gene (gus and gfp) was constructed. Then, a lethal system was established with lethal gene barnase to obtain male sterile transgenic plants.This study comprises three parts. Previously, high TPM (transcripts per million) genes of seven rice anther development stages were selected by Huang. Based on NCBI rice genomic DNA database, specific primers were designed to clone 1.5Kb putative DNA fragment of anther-specific promoters by PCR, which were named P13 and P41. Second, the 35S promoters in pCAMBIA1301 and pCAMBIA1303 plasmids were replaced by P13 and P41 promoter to construct reporter gene system, C1301-P13, C1303-P13, C1301-P41 and C1303-41. Third, the lethal system, P13bar, was achieved by inserting P13 promoter flanking the barnase gene.The resulted five constructs were introduced into rice, tobacco and Arabidopsis plants by Agrobacterium-mediated transformation system. Reporter gene containing transgenic rice, tobacco and Arabidopsis plants were subjected to GUS expression assays. GUS staining results revealed that P13 promoter expressed GUS in microspores of rice and anther, sepals and stigma of tobacco. P41 promoter expressed GUS in tapetum and microspores of rice. Furthermore, GUS activity was not dected in flowers but in seeding of Arabidopsis Pollen activity of lethal system transgenic Arabidopsis was confirmed by TTC staining. Transgenic Arabidopsis pollens containing P13bar showed no lethal effect. In the future, expression stage and tissue of P13 and P41 promoters will be further characterized and P41bar lethal system will be constructed. This two anther-specific promoters many be applied in dual inducible system for hybrid rice experiments.