混合不同葉綠體基因轉殖載體以基因槍法共同轉殖基因到甘藍葉綠體

In this study﹐hsc70﹐bt﹐sod62 and cat78 gene was constructed in the Brassice chloroplast transgenic vector﹐respectively.Mixture of the transgenic vector was co-transferred into cabbage (KY-cross)chloroplast via particle bombardment. The results of PCR﹐Southern and cat78 genes﹐and expressed bt and hsc70 mRNA .One of three co-transfer plant contained cat78 gene and the other plant didn't containing target genes.The bt gene transformed plants exhibited the high degree of resistance to the plutella xylostella.本研究分別將熱休克蛋白(heat shock proteins cognate,HSC);蘇力菌殺蟲晶體蛋白(Bt);超氧化歧化酵素(superoxide dismutase,SOD)與過氧化氫酵素(catalase,CAT)基因構築到蕓苔屬蔬菜葉綠體轉殖載體，並同時混合四種不同葉綠體基因轉殖載體 (bt、hsc70、cat、sod)，利用基因槍轉移到'初秋'甘藍葉綠體中。以PCR、南方墨點與北方墨點分析三棵轉殖株顯示，一株植株同時其有二種基因(bt、cat78)，並可轉錄出bt及cat78之mRNA;一株只有一種基因(cat78)，一株則不帶任何轉殖基因。將含有bt基因的轉殖株餵食小菜蛾幼蟲有明顯的殺蟲效果

構築基因至同一個葉綠基因體轉殖載體以基因槍法共同轉殖基因到甘藍葉綠體

In this study, bt and hsc70 genes as well as sod 62 and cat 78 genes were constructed in the same Brassica-specific-plastid vector which were named pASCCBtHSC and pASCCSODCAT, respectively. The constructed vectors were transferred into cabbage (KY-cross) chloroplast via particle bombardment mediated transformation. The results of PCR, Southern and Northern blot hybridization indicated that seven plants of co-transformed sod 62+ cat 78 plants contained both sod 62 and cat 78 genes, and expressed sod 62 and cat78 mRNA. One of the two co-transformed bt + hsc 70 plants contained both bt and hsc 70 genes, and exhibited the high degree of resistance to the Plutella xylostella.本研究將熱休克蛋白(heat shock proteins cognate, HSC)基因與蘇力菌殺蟲晶體蛋白 (Bt) 基因構築再同一個蕓苔屬蔬菜葉綠體轉殖載體，稱之為pASCCBtHSC；將超氧化歧化酵素(superoxide dismutase, SOD)基因與過氧化氫酵素(catalase, CAT)基因構築到同一個蕓苔屬蔬菜葉綠體轉殖載體，稱之為 pASCCSODCAT。利用基因槍法將兩種載體分別轉殖到'初秋'甘藍葉綠體中。以PCR、南方墨點與北方墨點分析轉殖再生植株，在 pASCCSODCAT (cat+ sod)方面，獲得的七株轉殖甘藍，均同時具有 cat 及 sod 基因並轉錄出其 mRNA；在 pASCCBtHSC (bt+hsc)方面，獲得的兩株轉殖甘藍中，即有一株轉殖甘藍同時具有 bt 及 hsc70 基因，轉錄出 bt 及 hsc70 mRNA，並大量表現HSC70蛋白及具有顯著的抗蟲效果

甘藍葉綠體基因轉殖載體開發之建立

葉綠體的基因屬母系遺傳，不會隨著花粉散播，且植物細胞含多套的葉綠體基因組，有助於外來基因大量表現，因此開發葉綠體基因轉殖技術將可避免基因轉殖作物造成的基因汙染及破壞生物平街。本研究利用PCR的方式，大量增幅並分離出初秋'甘藍葉綠體基因粗中invert repeat (IR)區域中trnV到rrn23S約4.lkb的核酸序列片段，經核酸定存與NCBI核酸資料庫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等葉綠體基因組序列有99%的相似性，包括有IR中完整的trnV、16S ribosomal RNA、trnl、trnA以及部分 23S ribosomal RNA的基因序列片段。將此基因片段均分為二個部分，同時構築到pBluScript II(SK-)載體上成為蕓苔屬葉綠體之通用轉殖載體(universal vector)─pASCC2O1。此載體帶有蕓苔屬葉綠體之lRA區域之trnV-rrn16S(左)及trnI-trnA-rrn23S(右)核酸序列，作為葉綠體基因轉移之重組位置，並含有以prrn為啟動子的aadA基因作為抗生素篩選基因。Expression of foreign genes via plastid genomes not only dramatically enhances the level of expression(5,000-10,000 copies of prokaryotic chloroplasts per plant cell), but also prevents out cross of the introduced foreign genes via pollen grains (their maternal inheritance in most crops). The objectives of the current research are to isolate plastid gene sequences from cabbage(Brassica oleracea L. var. capitata L.), and to construct a universal transplastomic vector for the gene transformation of Brassica vegetables. A 4.1 kb of DNA fragment of cabbage chloroplast between trnV and rrn 23S of invert repeat (IR) was amplified with PCR. This fragment contains trnV, rrn 16S, trnI, trnA, and a part of rrn23S. A universal transformation vector (pASC201) for Brassica chloroplast was constructed with trnV-rrn16S (left) and trnI-trnA-rrn 23S(right) of IRA rergion as recombination site for the insertion of transgene, and a chimeric aadA marker gene (spectinomycin resistance) was also inserted between rrn16S and rrn23S plastid gene sequences

共同轉殖蘇力菌殺蟲結晶蛋白、熱休克蛋白、超氧化歧化酵素及過氧化氫酵素基因至甘藍之研究

Attempts had been made to co-transfer the Bt-toxin gene (bt), superoxide dismutase gene (sod62) of Chinese cabbage, catalase gene (cat78) of Chinese cabbage, and heat shock protein gene (hsc70) of tomato into the cabbage (Brassica oleracea L. var. K-Y cross). The objectivesof this study are to establish the technology of gene co-transferand targeting the foreign proteins into the chloroplast by chloroplast trainsit peptide, and to study the possibility for improvement of cabbage with insect, disease, and stresses resistance by genes co-trans-formation. The results of PCR, Southern nad Northern hybridization indicated that the bt, hsc70, sod62 and cat78 genes had been inserted into the genome of cabbage, and expressed. The result of isozyme profiles assay revealed that the transformed SOD62 and CAT78 were present in the transformed plants. The SOD and CAT activities in leaves and chloroplast of transformed plants were higher than those of the controls. Increases in amounts of HSC70 protein were found in the transformed plants s analyzing by western blot hybridization. Preliminary results of bioassay indicated that the increases in tolerance to high temperature (42℃, 4 days), SO2 (800 ppb, 3 days), and insect resistance (Plutella xylostella) were found in the transgenic plants. This study demonstrated that two, three and four foreign genes could be co-transgenic plants. This study demonstrated that two, three and four foreign genes could be co-transferred into a cabbage plant simultaneously via Agrobacterium mediated transformation.本研究是利用農桿菌共同轉移法，研究同時轉移帶有 rbcS 為啟動子且攜帶有大豆葉綠體訊息 transit peptide 之蘇力菌殺蟲晶體蛋白 (bt)、結球白菜之超氧化歧化酵素 (sod62)、結球白菜之過氧化氫酵素 (cat78) 及番茄之熱休克蛋白 (hsc70) 等四種基因至同一棵之'初秋'甘藍，並探討轉運蛋白質到葉綠體之可行性。 以PCR、南方墨點及北方墨點雜交分析再生之bt、hsc70、sod62及cat78基因共同轉移的甘藍植株，試驗結果顯示轉移之基因已插入到轉殖植株染色體上，並表現出bt、hsc70、sod62及cat78基因之mRNA。電泳膠片酵素活性染色分析之結果顯示，轉殖植林之SOD及CAT條帶，均較未轉殖植株之條帶濃，其SOD及CAT酵素活性之對照組顯著增加。西方墨點分析結果顯示，轉殖植株之HSC70顯著較未轉殖植株之對照組多，且能轉連到葉綠體內。 具有轉殖bt基因之甘藍再生植株葉片經餵食小菜蛾幼蟲，證實具有抗蟲之效果。以800ppb之SO2燻蒸轉殖植株三天，具有sod及cat基因之甘藍再生植株，對於SO2 之危害較未轉殖或只轉殖單一sod或cat基因之轉殖株具有抗性。本研究證實用農桿菌混合感染培殖體可達到多基因同時轉移到同一棵甘藍，且能正常表現有功能之蛋白質，使轉殖植株具有抗蟲、耐高溫及耐逆境之特性

水稻子葉盤癒傷組織再生系統的建立

本研究之目的為建立台灣栽培水稻品種之成熟胚誘導子葉盤癒傷組織的再生盤系。水稻成熟種子以無菌播種於含有2mg/L之2 ,4-D的N6基礎培養基10天後，置於全暗的環境下，子葉盤所誘導的癒傷組織分裂速度最快。添加2mg/L之kinetin與m/L 之NAA 的MS基礎培養基培養3週後，約52.7%的'台農67號'與98.9%的'台梗9號'水稻癒傷組織能夠分化出芽體。'台梗l號'與'台梗8號'於完全黑暗的環境下誘導的癒傷組織，移到STl培養基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，在16小時明期與8小時暗期的環境中，平均綠芽體數則增加到12與15.2個。'台梗8號'於MSD培養基中16小時明期與8小時暗期的環境下3週後，於再生培養基27天後平均有11.2個綠芽體，'台農67號'則要全暗處理3週後，平均每顆癒傷組織能夠得到8.7個芽體。The purpose of this study is to develop a simple and highly efficient protocol for plant regeneration from scutella-derived embryogenic calli of rice (Oryza sativa L.). Vigorous growth of scutella-derived embryogenic calli was obtained from the mature rice seeds germinated in the N6 medium containing 2.0 mg/L of kinetin and 1.0 mg/L of napthaleneacetic acid for 10 days, and followed by cultivation in the dark for 10 days. Fifty-three and ninety-nine percentages of regeneration rate of green buds from embryogenic calli was achieved in the 'TN67' and 'TK9' rice, respectively, after three weeks of cultivationin the MS medium containing 2.0 mg/L of kinetin and 1.0 mg/L of napthaleneacetic acid. The average number of green buds emerged form each callus (GBP/callus ) was 8.8 and 7.8 in 'TK1' and 'TK8' rice, respectively, after four weeks of cultivation in the ST1 medium, while 12 and 15.2 GBP/callus, respectively, after supply with a 16-hr photoperiod. The GBP/callus was 11.2 in 'TK8' rice after four weeks of cultivation in the MSD medium supply with 16-hr photoperiod, but only 8.7 GBP/callus in 'TN67' rice after three weeks of cultivation in the dark

彩色辣椒果實轉色過程中色素及基因表現之變化

The colored chili pepper (Capsicum annum L.) is native to Central and South America, and belongs to Solanaceae family and Capsicum genus. Colored chili pepper is unique in the colors transition from pale green, purple, orange, to red within 15 to 20 days. Our studies indicated that the colors transition of the chili pepper fruits was probably not related to the pH of fruit sap. The results of the measurements of total anthocyanins and carotenoids contents, and the expression of psy and lcy-b genes indicated that a dramatic shit from anthocyanins metabolism to carotenoids biosynthesis occurred during the purple color transformed into orange color of chili pepper fruit. Changes in the profiles of protein synthesis and the populations of translatable mRNA of chili pepper fruits were also found during colors transition. Our results reveal that the regulation of genes expression was involved during colors transition of chili pepper fruits.本研究觀察到'PBCl328'品系之彩色辣椒其果實轉色之順序為淡白綠色、紫色、橙色、紅色，約在15~20天內完成，並發現該轉色與細胞液之pH值因素無關。分析總花青素及總類胡蘿蔔素含量，及psy與lcy-b(類胡蘿蔔合成相關酵素)等基因的表現結果顯示在彩色辣椒果實轉色過程中有明顯的花青素合成先增加再減少而按著類胡籮蔔素被誘導大量合成的現象。此種轉變在in vivo放射性蛋白質標記及in vitro translation 的分析都可顯示彩色辣椒果實轉色過程中與合成新蛋白質或/且降解有關，且轉色的過程也牽涉到mRNA層次的調控

不同灌溉水溫對番茄苗生育之影響

本研究以'花蓮亞蔬五號'番茄為材料，調查不同水溫、植株部位、冷水灌溉時間對番茄苗矮化效果及其品質之影響，其目的為建立冷水灌溉矮化番茄苗之系統，以作為種苗產業之應用的參考依據，並作為深入研究矮化苗之基礎資訊。試驗結果顯示當灌溉水溫較室溫水愈低(5°C)或愈高(65°C)均可抑制番茄苗莖伸長，以冷水處理(5°C)能獲得較高莖硬度(g/mm)及壯苗指數(seedhng index;地上部乾物重/株高比值)之番茄苗。冷水處理主要抑制番茄苗之部位為第一節間莖長。冷水灌溉時間於每日清晨(8:00)進行較中午(13:00)及下午(16:00)能夠獲得更矮之植株;每日植株最大生長速率為夜晚至清晨期間。冷水處理四週後，矮化植株的效果最佳，且冷水處理之溫度愈低或處理持續時間愈長，矮化植株效果愈明顯。冷水澆施至栽培介質內，介質土溫能夠在60-90秒內降至最低點，土溫回復至正常溫度約需60分鐘;葉溫鞍上溫變化程度大且葉片回溫時間較介質為短。綜合上述結果顯示5°C冷水灌溉能夠有效的矮化番茄苗及提升種苗品質，可作為種苗產業之參考及應用。In this study,‘Hwalien Yasu No.5'tomato was used to study the effects of different temperatures of irrigation water, parts of plant, timing of irrigation on the shortening and quality of plg-seedling. The purposes of this study were to establish the cold-water irrigation system for plug-seedlings production, and to provide the basic knowledge of shortening seedling for advanced study. The results indicated that shortest seedling was found in the seeding irrigated with the lowest (5∘C) or highest (65∘C) water temperature.However, highest stem strength and seedling index was only found in the seedling irrigated with the 5∘C water. Decrease in the length of first internode was primary responsible for the shortening seedling by cold-water irrigation.Irrigation of cold water at the morning (8:00) or evening (16:00) showed the best results in shortening seedling. After the cold water irrigation, the soil temperature drop to minimum within 60 to 90 sec, and recovered to the room temperature after 60 min. Longer period of cold-water irrigation and lower the cold-water temperature resulted shorter the seedlings. In conclusion, cold-water irrigation system for plug-seedlings production could apply in the commercial usages