Agrobacterium rhizogenes-mediated soybean gene transformation

本論文在進行根毛農桿菌對黃豆之多重基因共轉形之探討。黃豆臺南二號有最高之無菌植株發芽率且可用於農桿菌感染之組織材料數高而被選作後續研究材料。選用兩個廣泛使用之報導蛋白質進行黃豆之多基因共轉形：帶有綠色螢光蛋白質 (GFP) 之質體 pCAMBIA 1302 及帶有beta-葡萄糖酸 (GUS) 之質體 pCAMBIA 1201。首先探討對野生型農桿菌進行單一質體轉形時， pCAMBIA 1201 轉形效率為5.5 x 105，pCAMBIA 1302效率為 8.25 x 105；對已帶有一質體之農桿菌轉形平均轉形效率則降低至9.5 x 102。黃豆多基因共轉形操作分為單一菌體同時攜帶兩質體 (2BV) 或兩質體分別位於兩菌體來同時感染黃豆 (2AR)。黃豆子葉之根狀組織誘導率在野生型農桿菌及2BV、2AR組別分別為 48.0%、39.6%及68.2%。經過液態搖瓶培養後，得到的兩株毛狀根都會表現 GFP 而不表現 GUS。本研究已初步建立黃豆之毛狀根誘導系統、將外源基因送入黃豆染色體並在毛狀根中成功表現出異源蛋白質綠色螢光蛋白質。黃豆之多基因共轉形系統仍未完整建立，後續仍需要許多努力以對其有完整之了解並得以應用。Agrobacterium rhizogenes-mediated soybean multiple genes transformation was investigated in this study. Soybean Tainan #2 was selected as the material for the highest germination rate of sterile plants and high number of tissue segments for root induction. Two reporter genes were used for co-transformation study, gfp from pCAMBIA 1302 and gus from pCAMBIA 1201. Transformation efficiency of pCAMBIA 1201 and pCAMBIA 1302 was 5.5 x 105 and 8.25 x 105 for wild-type A. rhizogenes, and efficiency lowered to an average of 9.5 x 102 for transforming the second vector into pre-transformed A. rhizogenes. Co-transformation was carried out either by two binary vectors in one A. rhizogenes (2BV) or two binary vectors in separated A. rhizogenes and infecting soybean at the same time (2AR). Root induction rate from cotyledon was 48.0% for WT A. rhizogenes, 39.6% for 2BV, and 68.2% for 2AR, independently. We gained two hairy root clones after liquid culture, which both expressing GFP but not GUS. As a result, soybean hairy roots are successfully induced by A. rhizogenes transformants, and foreign gene is successfully transformed and expressed in hairy roots. Soybean multiple genes co-transformation is not yet fully constructed and still needs much effort to get a further understanding and for further applications.口試委員會審定書 IIbstract III文摘要 IVbbreviations Vndex VIontents VIIontents of tables and figures Xhapter 1 Introduction 1.1 Transgenic plant cell cultures 2.1.1 Plant cell cultures 2.1.2 Agrobacterium-mediated gene transformation 4.1.3 Hairy root cultures 7.2 Multiple genes co-expression 8.3 Reporter proteins 9.4 Soybean 10.5 Research aim 12hapter 2 Materials and Methods 13.1 Plasmid constructions 14.1.1 Binary vectors 14.1.2 Two reporter genes in one T-DNA region 15.1.3 Two T-DNA regions in one binary vector 17.2 Bacteria transformation methods 18.2.1 Heat shock method for E. coli transformation 18.2.2 Electroporation for A. rhizogenes transformation 19.2.3 Transformation efficiency of A. rhizogenes 20.2.4 PCR confirmation 21.3 Establishment of hairy root clones 22.3.1 Soybean sterile plants germination 22.3.2 Preparation of A. rhizogenes for induction of soybean hairy roots 23.3.3 Induction of soybean hairy roots 24.3.4 Sterilization of hairy roots 25.3.5 Liquid cultures of hairy roots 25.4 Confirmation of foreign DNA in root clones 26.4.1 Extraction of genomic DNA 26.4.2 Confirmation of foreign genes with PCR 27.5 Functional GFP and GUS confirmation 28.5.1 Direct observation of GFP 28.5.2 GUS activity assay 29.6 Protein quantification 29.6.1 Extraction of cytosol protein 29.6.2 Quantification of total protein 30.6.3 Western blotting for detection of GFP 30hapter 3 Results 33.1 A. rhizogenes transformation 34.2 Selection of soybean materials 34.3 Establishment and selection of transgenic root clones 35.4 Liquid cultures of transgenic root clones 36.5 Transgenic root clones gene confirmation 38hapter 4 Conclusions and Discussions 41.1 A. rhizogenes transformation 42.2 Soybean and transgenic soybean roots 43.3 Perspectives 47ables and Figures 51eferences 71ppendix 79.1 Molecular cloning 80.2 Microbe culture 81.3 Plant culture 81.4 Protein purification and detection 8