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研究生:楊育瑋
研究生(外文):Yu-Wei Yang
論文名稱:不同光質處理對油點百合(Ledebouria socialis)組織培養生長分化反應之基因表現探討
論文名稱(外文):Transcriptome and epigenetic analysis of In vitro cellular differentiation of silver squill (Ledebouria socialis) under different light quality
指導教授:張岳隆
指導教授(外文):Yueh-Long Chang
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物農業科技學系碩士班
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
畢業學年度:100
語文別:英文
中文關鍵詞:油點百合光質組織培養cDNA-AFLP甲基化敏感擴增多型性
外文關鍵詞:Silver squillLight qualityTissue cultureTranscriptomecDNA-AFLPEpigeneticMSAP
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摘要:

植物是自營性生物,需光進行光合作用以維持正常生理機能。光線除供應植物細胞行光合作用外,不同光質成份對植物細胞分化及形態發生亦扮演重要角色。植物以不同的光感受器(photoreceptors)以傳遞光線對植物細胞生長分化的影響。本試驗擬藉由不同光源包含白光、紅光或藍光等處理小葉油點百合(silver squill, Ledebouria socialis)組織培養,進而探討不同光質處理下之油點百合生長發育反應與基因表現的差異及探討DNA甲基化情形。
為瞭解光質對於培植體分子層面上生長的影響,利用Western blot檢測光合作用及氧化逆境相關蛋白的表現情形;另外利用cDNA (complementary DNA)-AFLP(amplification fragment length polymorphism;擴增片段長度多型性)方法,獲得基因表現的DNA片段多型性差異資料,經由定序方法獲取序列資料,接著比對資料庫得知個別序列之基因可能性的功能,再以RT-PCR方法來確認所挑選的基因在不同光質處理下之基因表現的差異。而且利用MSAP(methylation-sensitive amplification polymorphism)方法來檢測genome的甲基化程度探討不同光質處理下DNA甲基化情形與基因表達的差異性。藉由這些研究結果可作為未來油點百合的基因組結構分析的基礎及其他作物在組織培養或作物栽培上的實際應用。

在試驗結果中發現,油點百合組織培養在不同光質處理下,紅光及白光照射可以加速癒傷組織分化形成,而紅光亦有助於不定根的分化,藍光有助於芽的分化形成。在蛋白質表現分析上發現光誘導的光合作用相關蛋白RuBisCO和PEPC(phosphoenolpyruvate carboxylase)對於藍光刺激誘導有較高的敏感性,而逆境相關蛋白Cu/ZnSOD(Cu/Zn superoxide dismutase)與APX(ascorbate peroxidase)的表達發現在紅光與藍光的照射下有較高的表現情形。而在cDNA-AFLP基因表現檢測發現油點百合的基因體含有大量的retrotransposons的序列並會大量表現此類基因;另外,也發現與胚發育相關的(UBP14 ubiquitin-specific protease 14)基因表現會受到藍光的刺激誘導。而在MSAP檢測DNA甲基化程度發現油點百合在紅光照射下會產生高程度的甲基化與去甲基化變化,由此推測經紅光處理會造成大規模基因表現的改變。另外,經由生物資訊分析發現目前油點百合的分子資料相當少,以至於在基因功能性比對上造成許多未知的序列片段不易於進一步的分析。

Abstract:

Plants are autotrophic organisms; they need light for photosynthesis to maintain their normal developmental and physiological processes. In addition to its effect on photosynthesis, light quality (different wavelengths) also plays an important role in plant differentiation and morphogenesis. Therefore, plants have evolved different photoreceptors that absorb specific wavelengths of light to affect plant growth and differentiation.
This study focuses on the effects of different light quality on in vitro differentiation and gene expression profiling of silver squill (Ledebouria socialis). For carrying out this research, western blot analysis was used to detect photosynthetic and stress proteins expression levels, cDNA-AFLP (complementary DNA-amplification fragment length polymorphism) method to detect gene expression difference, and then the RT-PCR method to confirm the expression diversity of target genes under different light conditions. The MSAP (methylation-sensitive amplification polymorphism) method was used to detect DNA methylated modification to know the gene expression patterns under different light treatments.
Silver squill tissue culture results showed that the callus formation was found under red and white treatments, whereas red light induced high root formation and blue light promoted bud formation. Based on the protein expression analysis results indicated that photosynthesis-related proteins of RuBisCO and PEPC (phosphoenolpyruvate carboxylase) was more sensitive to blue light induction, whereas the stress related proteins of Cu/ZnSOD (Cu/Zn superoxide dismutase) and APX (ascorbate peroxidase) had a high expression under the red or blue treatment. The genome-wide analysis of gene expression by cDNA-AFLP revealed that the genome of silver squill could host a high content of retrotransposon elements and there high expression was confirmed in all of light treatments. RT-PCR evidence showed that the early embryo development essential protein of UBP14 (UBP14 ubiquitin-specific protease 14) was induced by blue LED light. The DNA methylation assay by MSAP showed that the explants irradiated by red LED light had high percentage methylation and demethylation changes to up-regulate or repress gene expression. However molecular annotation, derived from the genome-wide analysis of gene expression and epigenesis analysis of DNA methylation in our study, was limited by non-available and non-sufficient molecular information of silver squill in present gene databases such as NCBI.

List of Contents

Abstract…………………………………………………………………I
Acknowledgements……………………………………………………V
List of content…………………………………………………………VI
List of figures…………………………………………………………VII
List of tables…………………………………………………………VIII

Introduction……………………………………………………………1

Material and Method…………………………………………………10
Plant material……………………………………………………………10
Protein expression analysis by western blot method……………………10
Transcriptional analysis by cDNA-Amplification Fragment Length Polymorphism method…………………………………………………13
Methylation-Sensitive Amplification Polymorphism analysis…………22

Result and Discussion…………………………………………………30
Silver squill tissue culture and protein western blot analysis…………30
cDNA-AFLP analysis…………………………………………………33
MASP analysis…………………………………………………………36

Conclusion……………………………………………………………39
Reference………………………………………………………………58
Appendix………………………………………………………………66

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