牛樟芝，學名為Antrodia cinnamomea，是台灣特有之珍貴藥用真菌，具有抗癌和調節免疫等方面之功能。本試驗希望能藉由農桿菌並配合主動誘變(Activation Tagging Mutagenesis)之方式，進行牛樟芝基因轉殖系統之建立及研究。本試驗利用二元載體（binary vector）之農桿菌品系EHA105以及pTAG-8質體進行牛樟芝基因轉殖。首先針對各種影響轉殖的因子，包括農桿菌濃度、牛樟芝菌絲體重量及成熟期、Acetosyringone ( AS ) 濃度以及共培養時間等因子進行測試。試驗結果顯示0.2 g/ ml 的25天成熟菌絲搭配5X108 cfu/ ml農桿菌液，添加1 mM AS之條件下進行共培養6天，可以獲得較多篩選菌系。
轉殖株的分子鑑定：篩選菌系經聚合酵素連鎖反應確認後，再以南方墨點法做進一步分析，結果顯示有73.3％的轉殖株具有單一拷貝的T-DNA 崁入。在GUS組織染色的分析亦有兩株轉殖菌系呈現藍色反應，而利用plasmid rescue方式也成功分離出4株轉殖菌系的基因體DNA。為確定轉殖菌系是否仍為牛樟芝，本試驗利用18S 核醣體（18S rRNA）基因序列和internal transcribed spacer (ITS) 基因序列，針對食品工業研究所不同品種的牛樟芝菌絲體及本試驗的轉殖細胞系進行牛樟芝分子鑑定；結果顯示不管是18S rRNA 或ITS基因序列，轉殖細胞系和牛樟芝主模式菌株相同度均達到97.5-99％，因此證實轉殖細胞系仍為穩定的牛樟芝菌種。
外觀性狀調查方面：非轉殖菌系與轉殖菌系經測定其生長曲線，發現 15 個轉殖菌系之生長速率明顯下降20 %。而外觀顏色變化之轉殖菌系經HPLC成分分析後，發現外觀顏色變為深紅色之轉殖菌系，其總成分與非轉殖菌系比較有30％成分增加，且與牛樟芝子實體的總成分相當；而白色之轉殖菌系則發現其總含量有40％下降。從HPLC剖面圖發現，有兩個轉殖菌系含有一個成分不同於非轉殖菌系菌絲體，而這個成分則是存在於子實體。上述外觀性狀之改變將有助於後續相關基因功能之研究。

Antrodia cinnamomea is well known in Taiwan, as a source of traditional Chinese medicine for treatment of cancer and inflammation. In the present study, T-DNA Activation Tagging Mutagenesis (ATM) was used to generate transgenic lines of A. cinnamomea. Agrobacterium tumefaciens strain EHA105 harboring a standard binary vector pTAG-8 was used to generate the ATM population. Initially, factors like concentration of Agrobacterium, acetosyringone (AS), age of mycelia, its density and duration of co-culture period were optimized to achieve the maximum transformation efficiency. Results showed that Agrobacterium concentration of 5x108 cfu/ml, 1 mM AS, 25 days old mycelia at 0.2 g/ml and co-cultivation for 6 days were the optimum conditions for the transformation of A. cinnamomea.
Molecular analysis of transgenic lines to confirm transformation was also carried out. After confirmation by PCR, transgenic lines were subjected to Southern blot analysis. Results showed that 73.3% of transformants had single T-DNA insertion. GUS activity was observed in the transformants and the segments of genomic DNA of the transformants flanking the T-DNA from right border could be amplified by plasmid rescue. The identity of transgenic lines of A. cinnamomea was confirmed by first amplification and then sequencing of the 18S ribosome RNA (18S rRNA) and internal transcribed spacer (ITS) regions. Sequence analysis showed that transgenic lines shared 97.5-99% identity to BCRC A. cinnamomea strains.
Growth rate of the transgenic lines was slow. It was observed that 15 transgenic lines showed 20% decrease in growth rates compared to non-transformed mycelia. Color based phenotypic screening of the transgenic lines and analysis of compounds by HPLC was carried out. The white mycelia contained 40% less compounds, while the deep red mycelia had 30% more compounds than the non-transformed original mycelia. Quantities in the later case are almost similar to the quantities detected in fruiting bodies. The compound profile of transgenic mycelia was also similar to the compounds found in fruiting bodies. Interestingly, it was noticed that two transgenic lines had different compound profiles compared to non-transgenic mycelia.
Thus, in the present study, we have optimized a protocol for Agrobacterium tumefaciens-mediated transformation of A. cinnamomea, developed transgenic lines having levels of compounds similar to fruiting bodies, and also isolated two transgenic lines with different compound profiles.

Chinese abstract…………………………………………………………………I
Abstract …………………………………………………………………….......III
誌謝......................................................................................................................V
Chapter 1. Introduction……………………………………………………..…1
Chapter 2. Literature Review
1. Introduction - Antrodia cinnamomea…………………………………........3
(1) Taxonomy………………………………………………………………..3
(2) Characteristics and distribution………………………………………….4
(3) Basidomycete life cycle………………………………………………....4
(4) Bioactive compounds and its functions………………………………....5
(5) The constraint- non-availability of sufficient quantities of fruiting bodies…………………………………………………………………..8
2. Transformation of fungi…………………………………………………….8
3. Methods for transformation of fungi………………………………………..9
(1) CaCl2/polyethylene glycol (PEG) method………………………………9
(2) Electroporation………………………………………………………….10
(3) Particle bombardment (Biolistic) method………………………………10
(4) Restriction enzyme mediated integration (REMI)……………………...11
(5) Agrobacterium tumefaciens mediated transformation (ATMT)………..12
4. Factors influencing ATMT efficiency in fungi…………………………….14
(1) Starting materials……………………………………………………….14
(2) Agrobacterium and the host strain……………………………………...15
(3) Acetosyringone concentration………………………………………….16
(4) Co-cultivation conditions…………………………………....................17
5. Research on activation tagging…………………………………………….18
(1) Gene function – Introduction…………………………………………...18
(2) Mutation approach to gene function……………………………………18
(3) Activation tagging - the principle and advantages …………………......21
(4) Activation tagging- its applications…………………………………….22
Chapter 3. Materials and Methods
1. Antrodia cinnamomea strains……………………………………………..25
2. Mycelia culture conditions………………………………………………..25
3. Plasmid construction……………………………………………………....26
4. Preparation of competent cells …………………………………………....26
5. Transformation of E.coli and Agrobacterium...............................................27
6. Solid and liquid cultures of E. coli and Agrobacterium……………………27
(1) E. coli culture……………………………………………………………27
(2) Agrobacterium culture…………………………………………………..28
7. Preservation of the strains………………………………………………….28
8. Plasmid extraction………………………………………………………….28
9. Primer design ………………………………………………………………29
10. Plasmid identification…………………………………………………........29
11. Agrobacterium-mediated transformation ………………………………. ...30
(1) Lethal concentration of antibiotics for selection of A. cinnamomea ......30
(2) The optimum Agrobacterium concentration ……………………….. …30
(3) Co-cultivation and selection………………………………………… ...31
12. Genomic DNA extraction…………………………………………….. ….32
13. PCR analysis of transgenic lines………………………………………….33
14. Southern blot analysis…………………………………………………….33
(1) Preparation of non-radioactive labeled probe………………………….33
(2) Digestion and precipitation of genomic DNA…………………………34
(3) Gel electrophoresis and transfer of genomic DNA…………………….34
(4) Hybridization and detection……………………………………............35
15. GUS assay…………………………………………………………...........36
16. Plasmid rescue……………………………………………………............36
(1) Restriction digestion……………………………………………...........36
(2) Ligation…………………………………………………………….......37
(3) Electroporation…………………………………………………………38
17. Identification of A. cinnamomea strains…………………………….........39
(1) Primers design of 18 rRNA and ITS…………………………………...39
(2) PCR assay………………………………………………………………39
(3) T-A cloning…………………………………………………….............40
(4) Transformation-ligation reactions……………………………………...40
(5) Sequence determination and identity analysis…………………….........41
18. Growth model of A. cinnamomea mycelia…………………………..........41
19. Relative growth rate of transgenic lines …………………………….........41
20. Analysis of secondary metabolites ……………………………………….41
(1)Materials……………………………………………………………........41
(2) Equipment……………………………………………………………….42
(3) Preparation of extracts…………………………………………..............42
(4) HPLC analysis…………………………………………………..............43
21. Data analysis……………………………………………………...............43
Chapter 4. Results
1. PCR amplification of selection and reporter genes in
Agrobacterium strain………………………………………………….........44
2. Optimization of conditions for A. cinnamomea transformation……………44
(1) Lethal concentration of antibiotics for selection of A. cinnamomea.........44
(2) Influence of different factors on transformation efficiency………..........45
3. Molecular analysis of transgenic lines …………………………………….46
(1) Identification of T-DNA integration in transgenic lines ………………...46
A. PCR assays………………………………………………………….…46
B. Southern blot analysis………………………………………………….46
C. GUS assay……………………………………………………………...46
D. Plasmid rescue technique………………………………………...........47
(2) Identification of transgenic lines and A. cinnamomea strains…………...47
4. Phenotypic screening, qualitative and quantitative analysis of bioactive compounds ………………………………………………………………...47
(1) Colony color……………………………………………………..............47
(2) Relative growth rates of A. cinnamomea transgenic lines ………………48
(3) Analysis of bioactive compounds or secondary metabolites…………….48
Chapter 5. Discussion…………………………………………………….......50
References ……………………………………………………………..............57
Figures………………………………………………………………………….72
Tables ………………………………………………………………………….88
Appendix……………………………………………………………………….90

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