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Establish and Application of Agrobacterium - mediated Transformation of Rice and Chloroplast Gene Transformation of Cabbage and Rice
|摘要:||水稻(Oryza sativa L.)為本省栽種面積最廣、產值最高的農作物，本實驗首先以''台農67號''與''台梗9號''水稻成熟種子為材料，嘗試以胚子葉盤誘導癒傷組織再生的方式，建立一個簡單且有效率的組織培養再生系統，並克服農桿菌基因轉移法的瓶頸，建立完善的水稻農桿菌再生系統。子葉盤於全暗的環境下並添加2 mg/L 之 2, 4-D的N6基礎培養基中所誘導的癒傷組織，再移到含有2 mg/L之 kinetin與 1mg/L之 NAA的MS基礎培養基繼代培養3週後，''台農67號''與''台梗9號''的再生率分別為52.7 %與98.9 %，4週後平均每顆癒傷組織能夠分化出8.8與7.8個綠芽體。
同樣利用農桿菌法轉移酵母菌的轉酮醇酵素基因(tkl)或水稻油膜蛋白基因(ole)到再生水稻。以100ppm kanamycin抗生素進行初步篩選，癒傷組織的再生率介於3.5~7.2 %之間，改以100ppm geneticin(G418)抗生素篩選轉殖植株，轉殖再生率降到1.3~2.8 %之間。經抗生素篩選後所得到的再生植株，以PCR反應與南方墨點雜交分析均可在轉殖植株基因組DNA上偵測到目標基因，北方墨點雜交分析的結果顯示目標基因能正確的轉錄出RNA。轉殖ole基因的再生水稻北方墨點雜交分析的結果顯示，ole基因表現會隨著種子逐漸發育而增加，於10天種子中表現最強。西方墨點雜交分析的結果顯示在74 kDa (TKL)與16 kDa (OLE)的位置上，轉殖水稻皆有很明顯的tkl或ole基因雜交訊號。試驗結果顯示以農桿菌法確實能將tkl及ole基因轉移到再生水稻染色體組中，轉殖水稻能正確的合成TKL與OLE蛋白質。
本實驗以目前已發表的水稻、菸草以及阿拉伯芥的葉綠體基因圖譜為藍本，設計數組特殊的引子，以甘藍''初秋''與水稻''台農67號''的葉綠體DNA為模板，以PCR反應的方式增幅葉綠體基因組IR區域中之trnV~rrn23S的核酸序列。所篩選之甘藍rrn16S與rrn23S殖系經核酸定序後合計有4,103個鹼基，包括有完整的trnV、16S ribosomal RNA、trnI、trnA以及部分23S ribosomal RNA的基因序列片段。其核酸序列經NCBI電腦比對之結果顯示所分離片段之基因序列與已發表的阿拉伯芥葉綠體基因組序列有99 %的相似性，與其他高等植物或藻類亦有95 %以上的相似性，顯示其序列具有高度保留。
所篩選之水稻rrn16S與rrn23S殖系經核酸定序後分別為2,193與2,127個鹼基，rrn16S殖系之核酸序列與目前已發表的葉綠體基因組有95 %以上的相似性，而rrn23S的相似性則較低，但亦有88 %以上。其核酸序列與目前已發表的水稻葉綠體基因組序列相比僅有3個鹼基發生差異，皆位於非功能性基因的序列中，其二個殖系的核酸序列亦包括IR區域中完整的trnV、16S ribosomal RNA、trnI、trnA以及部分23S ribosomal RNA的基因序列片段。
分別將所選殖之甘藍或水稻葉綠體基因組trnV~ rrn23S之核酸序列中間之間隔序列(spacer)分開，於trnV－rrn16S(左)及trnI－trnA－rrn23S(右)核酸序列間構築入以prrn為啟動子的aadA基因及gus報導基因，同時作為葉綠體基因轉移之重組位置，成為蕓苔屬或禾本科之葉綠體通用轉殖載體(universal vector)－pASCC201或pASRC202。槍擊後之培殖體經200ppm spectinomycin/streptomycin抗生素持續篩選4~6週後，甘藍轉殖再生率皆不到1 %，水稻轉殖再生率最高為2.7 %。轉殖植株葉片以PCR、南方、北方墨點及GUS活性分析的方式檢測，均可發現有aadA及GUS的存在，並可表現出RNA及GUS蛋白，顯示葉綠體轉殖載體之正確性及可行性。
分別將以prrn為啟動子的bt (pASCCBT, pASRCBT)與sod (pASCCSOD, pASRCSOD)基因構築到水稻及甘藍之葉綠體轉殖載體，利用基因槍將其送入甘藍葉片與水稻癒傷組織中，經抗生素spectinomycin與streptomycin持續篩選4~6週後皆可獲得再生植株，二個品種的甘藍再生率約為0.4 %，三個品種的水稻再生率介於0.9~3.1%之間。轉殖植株葉片DNA以PCR反應、南方轉漬分析均可偵測到 bt 及 sod 的存在，北方墨點雜交分析顯示目標基因可以正確的表現出RNA，顯示轉殖載體之正確性及可行性。SOD電泳膠片酵素活性染色分析之結果顯示，轉殖甘藍與水稻植株之SOD表現量均較對照組高出甚多，SOD酵素活性定量分析的結果指出，大部分的SOD蛋白質活性皆位於葉綠體中。生物檢定的結果顯示轉殖bt基因甘藍葉片，對於所餵食的小菜蛾有100 %的致死率，所產生的Bt蛋白晶體能夠保護植物不受到小菜蛾的危害。以800 ppb的SO2同時處理sod基因轉殖植株與對照組植株，以葉綠體基因轉殖法所得到的再生甘藍或水稻植株，葉片僅呈現輕微的傷害，而對照組葉片組織則呈現嚴重的水浸狀壞疽。以分析型電子顯微鏡(AEM)可以在甘藍與水稻轉殖株葉片葉綠體中觀察到Bt晶體的存在。研究結果顯示本實驗所選殖並構築的甘藍與水稻葉綠體基因轉殖載體之正確性，開發蕓苔屬蔬菜與禾本科水稻之葉綠體基因轉殖技術是可行的。|
The present study describes a simple and highly efficient protocol for plant regeneration from scutellar-derived embryogenic calli of original location rice of cultivation(Oryza sativa L.) transformed with Agrobacterium method. The binary-vector pBI121 (or pBI131) which is a non-virulent plasmid containing β-glucuronidase(gus) and hygromycin phosphotransferase(hpt) chimeric genes was transformation and effective regeneration. Scutellar were cultured on N6 callusing medium supplemented with 2.0 mg/L 2, 4-dichlorophenoxy acetic acid for the enhancement of the cell division, and high frequency regeneration of shoot green buds from scutellar-derived embryogenic calli was achieved with Murashige and Skoog's medium containing 2.0 mg/L kinetin + 1.0 mg/L napthaleneacetic acid. After antibiotic selection, the percent of regeneration of two cultivars were about 3.4~4.2 %. The results of PCR and Southern analyses confirmed that the gus gene was undoubtedly incorporated into the transgenic rice genome by Agrobacterium method. The Northern hybridization assays also confirmed that the gus gene was stably integrated into the genome. The rbcS promoter(pBI131) linked to the GUS reporter gene expressed better than CaMV 35S(pBI121). By the histochemical staining of GUS analyses, the leaves were prove to be a part of the regenerated plants.. TKL gene was isolated from yeast and OLE gene was from rice. Both of them were inserted into the plant transformation vectors droved by CaMV 35S or rbcS promoter, which were then introduced into the scutellar-derived embryogenic calli from original location rice of cultivation (Oryza sativa L.) by Agrobacterium- mediated transformation. After Agrobacterium infection, the co-cultivated calli were subjected to two or to three cycles of selection on ST1C regeneration medium supplemented with 100mg/L kanamycin in the light(12-h photoperiod). After antibiotics selection, the percent of regeneration of two cultivars of rice was about 3.5 and 7.2% respectively. The results of PCR and Southern analyses confirmed tkl or ole genes were undoubtedly inserted into the transgenic rice genome by Agrobacterium method. The Northern hybridization assays also indicated that the tkl and ole genes were integrated into the genome and the western blot analysis showed that the presence of 74kDa TKL monomers or a 16kDa OLE protein on the genome. The tissue-specific Northern hybridization also showed that the expression of ole was at a higher level in 10-day embryos than others plants. The transgenic rice synthesized and the TKL or OLE protein accumulated were then assembled into oligomers and were proved to be antigenically identical with purified proteins. All investigations demonstrated that the expression of a variety of foreign genes in crop plants by Agrobacterium method is very useful. We confirmed stable integration of tkl and ole genes in T1, T2 and T3 progeny by PCR and Southern blot analyses. Overall, the expression levels of the transgenes were stable. After 100µM paraquat treatment, the tkl gene transgenic ''TK9'' T2 seedling progeny showed higher degree of resistance than the controls rice. Furthermore, the ole gene transgenic ''TN67'' T3 plant phenotypes showed that seedling progeny transferred to pots were seen to have no adverse pleiotropic effects. When they were compared with the untransformed plants in green house. Our results clearly suggested that Agrobacterium-mediated system could be use to develop rice with increased stress tolerance and increased, yet to be quantized, nutrient quality. Expression of foreign genes via plastid genomes not only dramatically enhances the level of expression (the present of 5,000-10,000 copies of the prokaryotic chloroplasts per plant cell), but also prevents out cross of the introduced foreign genes via pollen grains (their maternal inheritance in most crops). We attempt to transfer the report and antibiotic genes into the chloroplast of cabbage or rice. The objectives of the current research are to isolate plastid gene sequences from Brassica oleracea L. var. capitata L. or Oryza sativa L., and to explore the possibility for the establish more of the gene transformation system of Brassica or monocots chloroplast. A 4.1kb(cabbage) and 4.3kb(rice) of chloroplast DNA fragments from leaves, which were between trnV and rrn 23S of invert repeat were amplified by PCR. These fragments contain trnV, rrn 16S, trnI, trnA, and a part of rrn 23S. A universal transformation vector-pASCC201 or pASRC202 for Brassica or Oryza chloroplast were constructed with trnV—rrn16S (left) and trnI—trnA—rrn23S (right) of IRA region as recombination site for transformed gene. The chimeric aadA or gus genes are cloned between rrn16S and rrn23S plastid gene spacer sequences. After biolistic delivery of cabbage- or rice- chloroplast transformation vectors into callus, regenerated plantlets were selected by their resistance to 200 ppm of spectinomycin and 200 ppm of streptomycin. The PCR and Southern analyses of DNA confirmed that the gus and aadA genes were undoubtedly inserted into the chloroplast genome by two homologous recombination events via the flanking plastid gene sequences. The Northern hybridization and GUS histochemical assays indicated that the aadA and gus genes were integrated into the chloroplast genome suggesting that the expression of a variety of foreign genes in rice or cabbage plants by chloroplast leads is very useful. In chapter 7, we attempted to transfer the Bt and SOD genes into the chloroplast of cabbage and rice. The objectives of this study are to establish the gene transformation system of Brassica and Oryza chloroplast, and to study the possibility for improvement of dicot vegetables and monocot crops with insect and stresses resistance by chloroplast gene transformation. The genes of aadA, gus and bt were inserted into the pASCC201 (cabbage) or pASRC202(rice) vector and droved by prrn promoter. The cabbage and rice specific plastid vectors transferred into the chloroplast of cabbage or rice via particle gun mediated transformation. After antibiotics selection, the percent of regeneration of two cultivars of cabbage and three cultivars of rice was about 0.4 % and 0.9~3.1 % respectively. The results of PCR, Southern and Northern hybridization analyses depicted that the aadA, bt and sod genes were integrated into the chloroplast genome and expressed their RNAs properly. The results of isozyme profile assay revealed that the thicker bands of transformed sod was present in the transformed plants rather than in that of the non-transformed plants. Furthermore, the protein native PAGE analysis showed that the protein level was higher in regenerated plants than that in control plants. The SOD activity in leaves and isolated chloroplast protein of transformed plants were significantly higher than that of the control plants. The establishment of a plastid transformation system in cabbage and rice, which has several advantages over the conventional nuclear transformation, offers new hopes for the genetic improvement of the crops.
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