植物受溫度調控之基因的功能與機制分析

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廖敏杏(Min-Hsing Liao) 查詢紙本館藏

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生命科學系

論文名稱

植物受溫度調控之基因的功能與機制分析
(Mechanism and functional analysis of temperature regulated genes in plants)

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摘要(中)

當環境溫度比正常所生長的溫度高出5-10度時，原核和真核生物都會對高溫產生反應而合成熱休克蛋白質。小分子量熱休克蛋白質的分子量約為15-30 kDa，是高等植物中含量最豐富的蛋白質，因此被視為高等植物體內所特有的一群蛋白質。從高山植物黃菀（senecio nemorensis）中找到葉綠體型熱休克蛋白質，利用葉綠體型熱休克蛋白質基因當成模式基因，瞭解小分子量熱休克蛋白質的調節機制。結果顯示除了熱逆境外，也有許多的逆境，如寒害及H2O2都會誘導這類熱休克蛋白質的表現，同時冬天在當地所採集的樣品中也可以偵測到此基因的表現。
另方面，為改善長時間的低溫處理誘導蝴蝶蘭開花時，對植物產生傷害的現象，利用農桿菌將低溫有關的基因，如大麥的HVA1基因、阿拉伯芥中與春化作用有關的開花基因（At.FLC; At.VRN1）轉殖進入蝴蝶蘭中，以期能獲得低溫穩定調控的蝴蝶蘭品系，但經過長時間的轉殖實驗，並沒有成功的獲得到蝴蝶蘭轉植株。

摘要(英)

Both eukaryotes and prokaryotes respond to high temperature by synthesizing heat shock proteins (HSPs) when environmental temperature were elevated 5℃ to 10℃ above normal growth temperature. Small HSPs (sHSPs) are ranging in size from 15 to 30 kDa. and represent the most abundant family of HSPs in higher plants. We have cloned chloroplast small heat shock proteins (CPsHSPs) from the high mountain plants, Senecio nemorensis. Using CPsHSPs as a model gene, we try to understanding of the regulatory mechanism of sHSPs. The results showed that many other stresses such as chilling, H2O2 can induce CPsHSPs expression. Interestingly, CPsHSPs was detected in the local samples which are growing in winter season. In order to alleviate the damage of Phalaenopsis plants when long term of chilling treatment, which is the condition for inducing flowering in Phalaenopsis, we try to transfer HVA1 (a LEAIII gene from barley), At.FLC, and At.VRN1 (flowering genes from Arabidopsis thaliana) genes into Phalaenopsis plants. The major aim is to control flowering time and alleviate the damage induced by low temperature in Phalaenopsis. However, no transgenic plants are established so far.

關鍵字(中)

★ 溫度
★ 熱休克蛋白質
★ 春化作用
★ 蝴蝶蘭

關鍵字(英)

★ heat shock protein
★ vernalization
★ phalaenopsis
★ temperature

論文目次

中文摘要……………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………Ⅱ
目錄…………………………………………………………………………………Ⅲ
圖表目錄……………………………………………………………………………Ⅵ
縮寫與全名對照表…………………………………………………………………Ⅶ
壹、序論 ……………………………………………………………………………1
※ partⅠ
（一）逆境（stress）。…………………………………………………………… 1
（二）熱休克反應和熱休克蛋白質。……………………………………………1
（三）熱休克蛋白質的種類與功能。……………………………………………2
（四）熱休克基因的表現和調控。………………………………………………2
（五）植物小分子量熱休克蛋白質。……………………………………………3
（六）小分子量熱休克蛋白質的功能。…………………………………………4
（七）其他誘導熱休克蛋白質合成的因子。……………………………………4
（八）葉綠體型小分子量熱休克蛋白質。………………………………………4
（九）高山植物菊科黃菀屬植物。………………………………………………6
（十）研究目的。…………………………………………………………………6
※ partⅡ
（1）植物的生活史。………………………………………………………………8
（2）春化作用。……………………………………………………………………8
（3）春化作用相關基因及其調控。………………………………………………9
（4）與低溫逆境相關的蛋白質。…………………………………………………11
（5）蝴蝶蘭。……………………………………………………………………12
（6）低溫誘導蝴蝶蘭開花所遭遇到的問題和主要研究方向。………………12
貳、材料與方法 ……………………………………………………………………14
※ partⅠ
一.植物材料。……………………………………………………………………14
二.CPsHSPs基因套數（gene copy number）的確定。 ………………………14
2.1 純化黃菀genomic DNA。 ………………………………………………14
2.2 DNA的轉漬分析（transfer）。 …………………………………………15
2.3 探針（isotope probe）的置備。 …………………………………………16
2.4 雜交（Hybridization）。……………………………………………………17
三.非生物性逆境處理（abiotic stress treatment）。 ……………………………18
3.1 不同熱休克（heat shock）溫度處理。 …………………………………18
3.2 冷害（chilling）處理。 …………………………………………………18
3.3 氧化逆境（oxidative stress）。 …………………………………………18
四.RNA的分析方法。……………………………………………………………19
4.1黃菀total RNA的純化。 …………………………………………………19
4.2 RNA電泳分析。……………………………………………………………20
4.3 RNA dot blotting。 …………………………………………………………21
4.3.1 Transfer to membrane。…………………………………………………21
4.3.2探針（DIG probe）的備製。……………………………………………22
4.3.3雜合反應（hybridization）。……………………………………………23
4.4 Optimization RT-PCR。 ……………………………………………………24
4.4.1純化不含genomic DNA的RNA。……………………………………24
4.4.2單股cDNA的合成。……………………………………………………25
4.4.3以PCR合成DNA片段。………………………………………………25
五.使用iPCR的方法確定序列的正確性。………………………………………26
※ partⅡ
一.構築質體（construction）。……………………………………………………-28
1.1以PCR方式合成At.FLC；At.VRN1以及大麥的HVA1基因全長片段。…28
1.1.1引子的設計。……………………………………………………………28
1.1.2 PCR反應液的配置和實驗步驟。………………………………………28
1.2載體DNA（pJD312；pCAMBIA1300）的製備。…………………………29
1.3以pCAMBIA1300當binary載體，建構可以轉殖到植物體內的質體。-30
1.4接合反應（Ligation）。………………………………………………………31
1.5大腸桿菌的轉型作用（Transformation）（E. coli strain：DH5α）。……31
1.6少量純化質體DNA（Mini-preparation）。…………………………………32
1.7核酸序列分析（DNA sequencing）。………………………………………32
1.8菌種的保純。 ………………………………………………………………32
二.蝴蝶蘭的基因轉殖。 …………………………………………………………33
2.1農桿菌勝任細胞的製備（Agrobacterium strain：LBA4404）。……………33
2.2農桿菌的轉型（transformation）。 …………………………………………33
2.3蝴蝶蘭的轉殖與篩選。 ……………………………………………………34
參、結果 ……………………………………………………………………………36
肆、討論 ……………………………………………………………………………40
伍、參考文獻 ………………………………………………………………………44
陸、圖表 ……………………………………………………………………………50
柒、附錄
附錄一：黃菀植物的葉綠體型熱休克蛋白質基因序列。………………………64
附錄二：選殖的基因序列。 ………………………………………………………65
附錄三：載體pJD312（A）和pCAMBIA1300（B）的建構圖。 ………………66
附錄四：農桿菌菌液配置培養液。 ………………………………………………67
附錄五：蝴蝶蘭PLB的培養基配方。 ……………………………………………68

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指導教授

葉靖輝(Ching-Hui Yeh)

審核日期

2006-11-20

推文