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標題: | 應用文心蘭基因默化轉殖延緩老化及抵抗喜姆比蘭嵌紋病毒與齒舌蘭輪斑病毒 Studies on Transgenic Oncidesa Gower Ramsey for Prolonged Vase Life and Increased Resistance against Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus |
作者: | Shin-Yu Chen 陳欣郁 |
指導教授: | 黃鵬林(Pung-Ling Huang) |
關鍵字: | 文心蘭,ETHYLENE INSENSITIVE 2,ETHYLENE INSENSITIVE 3,乙烯訊息傳導,喜姆比蘭嵌紋病毒,齒舌蘭輪點病毒, ETHYLENE INSENSITIVE 2 (EIN2),ETHYLENE INSENSITIVE 3 (EIN3),Ethylene signal transduction,Oncidesa,Cymbidium mosaic virus,Odontoglossum ringspot virus, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | EIN2 (ETHYLENE INSENSITIVE 2) 於乙烯訊息傳導途徑中,位於中心且扮演重要角色,本論文探討文心蘭 (Oncidesa Gower Ramsey) EIN2基因結構及啟動子活性,期望進一步應用於文心蘭花朵老化之研究。篩選文心蘭基因庫 (Genomic library),獲得Exon 1至 4 序列及Exon 6序列,利用IPCR (Inverse polymerase chain reaction) 方式取得約0.8 kb啟動子序列,亦篩選文心蘭cDNA庫,取得EIN2 5’ 端序列。含OgEIN2pro::GUS 表現構築之兩週及三週大的阿拉伯芥轉殖株,啟動子活性不受逆境與荷爾蒙誘導,但花器的柱頭可偵測到 GUS 活性,分析轉殖菸草花朵及果實不同發育階段,只有花托具微弱活性,而柱頭具有較強啟動子活性,顯示文心蘭 OgEIN2 啟動子具有器官專一性。文心蘭採收後經分級選別處理,易造成花枝糾纏及花藥蓋脫落,使乙烯生成,花朵提早老化。本研究應用RNA干擾 (RNA interference) 技術,將乙烯訊息傳導中之轉錄因子ETHYLENE INSENSITIVE 3 (EIN3) 的默化質體轉入文心蘭中,以阻斷乙烯訊息傳導。文心蘭EIN3 RNAi質體,轉殖到文心蘭中,擬轉殖株具GUS正反應;聚合酶連鎖反應 (polymerase chain reaction, PCR) 及南方氏雜交分析 (Southern analysis) 檢測,皆具預期片段;以Real-time RT-PCR 分析EIN3基因表現量,轉殖株較未轉殖株低;觀察轉殖株開花情形,單朵花可較未轉殖株多開24天,整株花序可多開33天;處理ACC (1-aminocyclopropane-1-carboxylic acid) 後,未轉殖株花朵花青素及基因組DNA降解情形都較轉殖株嚴重;以IPCR (Inverse polymerase chain reaction) 分析轉殖株邊界序列,獲得一段左邊界序列,經序列比對分析發現邊界序列有缺失之現象,但所得到之序列可做為轉殖株之分子標誌;小片段RNA北方雜交結果,顯示轉殖株具有小片段RNA產生。此外,本論文進一步轉殖文心蘭EIN2和EIN3 雙基因RNAi質體到文心蘭中,轉殖株具GUS正反應,利用PCR與南方氏雜交分析顯示,轉殖株具有三種不同拷貝數。感染蘭科植物的病毒,以喜姆比蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 及齒舌蘭輪點病毒 (Odontoglossum ringspot virus, ORSV) 最為普遍,對蘭花產業造成的經濟損失最為嚴重。本研究應用核醣核酸干擾技術,構築喜姆比蘭嵌紋病毒及齒舌蘭輪點病毒外鞘蛋白基因之默化質體,讓植物產生小分子雙股RNA,專一性降解病毒之外鞘蛋白,達到克服病毒病害之目的。以CymMV和ORSV高度保守的鞘蛋白區域21或25 bp,作為迴文序列的髮夾結構,形成不同形式的默化構築,報導質體以CymMV或ORSV鞘蛋白基因全長做為默化目標並連結GFP螢光蛋白,預期RNAi質體將會使鞘蛋白全長融合GFP螢光蛋白之表達量下降。阿拉伯芥原生質體以PEG轉殖法共轉殖默化質體與報導質體並利用流式細胞儀 (flow cytometry) 偵測螢光表達量,此方法默化效率可達60% 至 85%,其中含Spacer (9 nt in length) 及單一髮夾結構的質體默化效率最高,未來可藉此平台作為快速篩檢默化效率的方法。此外,構築以農桿菌注射法 (agroinfiltration) 將雙抗病毒構築暫時性轉殖至蝴蝶蘭花瓣及葉片,並利用定量即時反轉錄聚合酶連鎖反應 (quantitative real-time reverse transcription polymerase chain reaction, Real-time RT-PCR) 及酵素連結免疫反應 (enzyme-linked immunosorbent assay, ELISA) 分析默化效率,結果顯示以具有9個核苷酸的間隔序列及單一髮夾結構的構築型式默化效率最佳,利用流式細胞儀及農桿菌注射,得知具最高效率的默化構築型式,未來可藉此平台作為快速篩檢默化效率的方法。抗病構築轉殖至邊沁菸草 (Nicotiana benthamiana),並進行邊沁菸草轉殖株篩選與再生,以聚合酶連鎖反應及南方氏雜交分析檢測,四個不同默化構築皆取得多株轉殖株,並完成轉殖株病毒接種試驗,證實轉殖株具有抗CymMV及ORSV之能力。為了得到抗病毒CymMV及ORSV的文心蘭轉殖株,使用文心蘭癒傷組織轉殖具最高默化效率的質體pGKU-CyORg,即含Spacer (9 nt in length) 及單一髮夾結構的質體,文心蘭癒傷組織轉殖後進行篩選與再生,以聚合酶連鎖反應及南方氏雜交分析檢測,皆有預期片段產生,完成轉殖株病毒接種試驗,證實轉殖株具有抗CymMV及ORSV之能力。此外,利用奈米金粒子做為增加蘭花抵抗CymMV及ORSV病毒的策略,注射甲殼素修飾的奈米金粒子至蝴蝶蘭葉片中,在照射雷射光促進光熱轉換,檢測CymMV及ORSV的基因表現和蛋白質累積,有注射奈米金粒子的葉片較控制組的病毒量來的低,經雷射光照射更可有效降低病毒量,利用奈米科技在蘭花抗病毒可做為新的策略。 ETHYLENE INSENSITIVE 2 (EIN2) is an important protein ethylene ein signal transduction. The goal of this study is to prolong the postharvest life of cut flowers. We got exon 1 to exon 4 and exon 6 of EIN2 gene in screen a genomic library of Oncidesa. We use IPCR method to identify a promoter region of about 0.8 kb. We also screen the Oncidesa cDNA library and get partial EIN2 sequence. No expression of OgEIN2 promoter was detected in 5 to 20 day-old transgenic plants and no any stress or hormone induction for OgEIN2pro::GUS transgenic Arabidopsis. Promoter activity was only fould in the flower stigma of transgenic Arabidopsis and tobacco. In OgEIN2pro::GUS transgenic tobacco no promoter activity in different stage of flowers and fruits, only slightly GUS activity was found in receptacle region. Overall, OgEIN2 promoter is a tissue-specific promoter. The operation of Oncidesa flowers after harvest results in the dislodgment of pollinia cap and petal senescence induced by ethylene production. OgEIN3 RNA-mediated silencing construct was transformed into Oncidesa by Agrobacterium tumefaciens. Confirmation of integration of T-DNA into leave was performed by positive GUS staining. Then, the transgenic lines were confirmed by polymerase chain reaction (PCR) and Southern analysis. Transgenic plant EIN3 gene expression detected by Real-time RT-PCR and EIN3 gene expression was lower than untrasformant. In the transgenic plants the longevity was approximately 24 days longer for individual cut flowers and 33 days longer for flowers within inflorescences compared with those of the non-transgenic plants. After treatment of cutting flower with ACC (1-aminocyclopropane-1-carboxylic acid), we detected the degradation phenomenon of DNA and anthocyanin content of plant, but transgenic plant did not severely degraded than control. To exploit the flanking sequences as molecular markers, IPCR (Inverse polymerase chain reaction) analysis were carried out to determine the border sequences of transgenic lines. One left border sequence was obtained. The sequence alignment analysis showed that some conserved sequence were missing in the border sequence. But the flanking sequences of transgenic plant still can be used as molecular marker. To elucidate the mechanism of OgEIN3 silencing transgenic lines, we used northern blot to determine the small RNA accumulation. Further, OgEIN2 and OgEIN3 RNA-mediated silencing construct was transformed into Oncidesa by Agrobacterium tumefaciens. Detection of T-DNA was performed by positive GUS staining. The transgenic lines were confirmed by polymerase chain reaction (PCR) and Southern analysis. Based on the results of Southern blot, the copy numbers of transgenic plants were classified into 3 groups. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most common and serious diseases affecting the development of the orchid industry. To enhance the resistance of orchids against CymMV and ORSV, RNA interference (RNAi)-mediated resistance was employed. Highly conserved regions of 21 or 25 nt derived from the coat protein (CP) gene of CymMV and ORSV were chosen to construct the inverted repeat region that may form a hairpin structure. Various hairpin-type small interfering RNA expression RNAi vectors were constructed to facilitate CP mRNA degradation and subsequent loss of target CP expression. To test the efficiency of these constructs, reporter plasmids containing the CP gene of CymMV or ORSV were used. A fast and reliable quantitative analysis for gene silencing could be achieved by attaching the target CP gene to the gene encoding green fluorescent protein (GFP) and assaying the inactivation of GFP expression. Transient expression of RNAi vectors was performed in Arabidopsis protoplasts by polyethylene glycol (PEG) transformation and analyzed by flow cytometry. The silencing efficiency for these vectors ranged from 60% to 85% as determined by flow cytometric analysis. The strongest RNAi effect was obtained in RNAi constructs with a spacer (9 nt in length). In Phalaenopsis experiments using agroinfiltration, we further demonstrated that RNAi constructs specifically targeted the CP in Phalaenopsis petals and leaves by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). The use of a spacer (9 nt in length) is more effective as compared to the use of an intron sequence as a spacer for the establishment of efficient silencing. In conclusion, the constructed vectors could effectively trigger a silencing mechanism in plant cells, which will provide a solid basis for transgenic orchids conferring resistance against CymMV and ORSV. The construct with different structure of RNAi effect will be obtained by transforming into Nicotiana benthamiana through Agrobacterium tumefaciens, and the transgenic lines will be selected by antibiotics. Transgenic lines were confirmed by PCR and Southern analysis. Transgenic tobaccos have been proven resistant against CymMV and ORSV after inoculation with viruses. To obtain transgenic Oncidesa plants with dual resistance against CymMV and ORSV. Oncidesa callus transformed with strongest RNAi effect plasmid pGKU-CyORg, an RNAi construct with a spacer (9 nt in length) by Agrobacterium tumefaciens. The transgenic lines will be selected by antibiotics. Transgenic plants were confirmed by PCR and Southern analysis. After inoculation with viruses, transgenic plants have been proven resistant against CymMV and ORSV. We treated nanoparticles as a strategy for enhancing the resistance of orchids against CymMV and ORSV. After chitosan-modified gold nanoparticles (Au NPs) were injected into Phalaenopsis leaves, the injected leaves were exposed to 980 nm laser for light–heat conversion. To evaluate virus elimination in the treated Phalaenopsis leaves, the expression of coat protein genes and amount of coat proteins for both CymMV and ORSV was significantly lower in the chitosan-modified Au NP-treated Phalaenopsis leaves than in the control. We propose that the temperature increase in the chitosan-modified Au NP-treated Phalaenopsis tissues after laser exposure reduces the viral population, consequently conferring resistance against CymMV and ORSV. Our findings suggest that the application of chitosan-modified Au NPs is a promising new strategy for orchid virus therapy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17980 |
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