本研究主要目的是藉由農桿菌媒介重複轉形法提升菇類轉形系統的異源表現 量，由實驗結果可得以下結論：
1. 以雙質體轉形策略進行重複轉形所得到之二次轉形株可於雙重抗生素的選擇性 培養基上生長，顯示二次轉形株可表現兩種抗藥性，也證實重複轉形之成功。
2. 根據酵素免疫分析的結果，雙質體轉形策略之二次轉形株的綠色螢光蛋白質含
量最高可提升5.5 倍，單質體轉形策略之二次轉形株的綠色螢光蛋白質含量最高
亦可提升4.25 倍。
3. 根據即時定量聚合酶鏈鎖反應的結果，二次轉形株之外源基因拷貝數大多有所 提升，所有的雙質體轉形策略之二次轉形株平均外源基因拷貝數是一次轉形株
的1.66 倍，而單質體轉形策略之二次轉形株平均外源基因拷貝數則是一次轉形
株的1.52 倍，顯示重複轉形確實可以再次把外源基因送入宿主染色體。
以上的結論顯示農桿菌媒介重複轉形法確實可應用於菇類轉形系統，且重複轉形
應可結合其他分生策略，例如刪減後的gpd 啟動子，再更進一步地增強異源基因
表現。
未來，可繼續探討農桿菌媒介重複轉形法，本研究僅進行到第二次轉形，之 後可進一步以單質體轉形策略進行第三次或是更多次的重複轉形並觀察異源蛋白 質是否能更加提升。此外我們還可將農桿菌媒介重複轉形法應用於其他菇類，例 如發展較為完善的金針菇系統，以測試重複轉形在其他菇類可否順利運作。亦可 把重複轉形跟各種分生策略作結合並且表現功能性基因，像是病毒表面抗原或是 醫藥用蛋白質等。
第六章 圖表
表一 異源表達系統之比較
Table 1. Comparison of heterologous expression system [78]
Characteristics E. coli Filamentous fungi
Yeast Insect cells Mammalian cells Plant cells culture
Cell Growth Hours to days Days to 1 week Days to 1 week Days to 1 week weeks Months
Cost of Growth Medium
Low to medium Low to medium Low to medium High High Medium to high
Secretion
capability Secretion to
Periplasm Secretion to
medium Secretion to Medium Secretion to Medium Secretion to Medium Secretion to Medium Post-translational modifications
Protein Folding Refolding Usually
Required Refolding might
be required Refolding might
be required Proper Folding Proper Folding Proper Folding N-linked Glycosylation None Mammalian-type
core, no sialic acid, non-human sugars added
High mannose, no sialic acid, non-human sugars added
Complex, no sialic acid, non-human sugars added
Complex, non-human sugars added, e.g. by murine cells
Complex, no sialic acid, non-human sugars added
O-linked glycosylation, phosphorylation, acetylation, acylation
No Yes Yes Yes Yes Yes
表二 植物生產之醫藥用蛋白質發展現況
Table 2. Plant-derived pharmaceuticals in clinical stages of development or on market [79]
Product Disease Plant Clinical trial
status
Company Source: URL/academic
Vaccines
Hepatitis B antigen (HBsAg)
Hepatitis B Lettuce Potato
Phase I Phase II
Thomas Jefferson University, USA
Arizona State University
Streatfield, 2006
Fusion proteins,
including epitopes from rabies
Rabies Spinach Phase I
completed
Thomas Jefferson University, USA
www.labome.org/expert/usa/.../
hilary-koprowski-233492.html Cancer vaccine Non-Hodgkin's
lymphoma
Tobacco Phase II Large Scale Biology, USA http://www.gmo-safety.eu/article /483.pharma-plants-status-report .html
Vibrio cholerae Cholera Potato Phase I Arizona State University Tacket, 2005 Heat-labile toxin B
subunit
of Escherichia coli
Diarrhea Maize Potato
Phase I Phase I
ProdiGene, USA
Arizona State University
Tacket, 2005
Capsid protein Norwalk virus
Diarrhea Potato, Tomato
Phase I Arizona State University Khalsa et al., 2004
Antigen Feline parvovirus
(Dogs)
Tobacco Advanced Large Scale Biology, USA http://www.lsbc.com
Antigen Papilloma virus
(Rabbit) Tobacco Early Large Scale Biology, USA http://www.lsbc.com HN protein of
Newcastle disease virus
Newcastle disease (Poultry)
Tobacco
suspension cells
USDA Approved
Dow Agro Sciences, USA http://www.dowagro.com/animal health
表二 (續) 植物生產之醫藥用蛋白質發展現況
Table 2. (Continued) Plant-derived pharmaceuticals in clinical stages of development or on market Viral vaccine mixture Diseases of
horses, dogs, and birds
Tobacco
suspension cells
Phase I Dow Agro Sciences, USA http://www.dowagro.com/animal health
Poultry vaccine Coccidiosis infection
Canola Phase II Guardian Biosciences, Canada
Basaran and Rodríguez-Cerezo (2008)
Gastroenteritis virus (TGEV) capsid protein
Piglet
gastroenteritis
Maize Phase I ProdiGene, USA Basaran and Rodríguez-Cerezo (2008)
H5N1 vaccine candidate
H5N1 pandemic influenza
Tobacco Phase I Medicago, USA http://www.medicago.com Antibodies
CaroRX Dental caries Tobacco EU approved as medical advice
Planet Biotechnology, USA http://www.planetbiotechnology.
com/
DoxoRX Side-effects of
cancer therapy
Tobacco Phase I
completed
Planet Biotechnology, USA http://www.planetbiotechnology.
com/
RhinoRX Common cold Tobacco Phase I
completed Planet Biotechnology, USA http://www.planetbiotechnology.
com/
Fv antibodies Non-Hodgkin's lymphoma
Tobacco Phase I Large Scale Biology, USA http://www.lsbc.com
IgG (ICAM1) Common cold Tobacco Phase I Planet Biotechnology, USA http://www.planetbiotechnology.
com/
Antibody against hepatitis B
Vaccine purification
Tobacco On market CIGB, Cuba Kaiser, 2008 Therapeutic human
proteins Gastric lipase,
Merispase® Cystic fibrosis Maize On market Meristem Therapeutics
France http://www.meristem-therapeutic s.com
表二 (續) 植物生產之醫藥用蛋白質發展現況
Table 2. (Continued) Plant-derived pharmaceuticals in clinical stages of development or on market
α-Galactosidase Fabry disease Tobacco Phase I Planet Biotechnology, USA http://www.planetbiotechnology.
com/
Lactoferon™
(α-interferon)
Hepatitis B and C Duckweed Phase II Biolex, USA http://www.biolex.com/
Fibrinolytic drug (thrombolytic drug)
Blood clot Duckweed Phase I Biolex, USA http://www.biolex.com/
Human
glucocerebrosidase (prGCD)
Gaucher‘s disease Carrot suspension
Awaiting USDA's approval
Protalix Biotherapeutics, Israel
http://www.protalix.com/
glucocerebrosidase.html Insulin Diabetes Safflower Phase III SemBioSys, Canada http://www.sembiosys.com/
Apolipoprotein Cardiovascular Safflower Phase I SemBioSys, Canada http://www.sembiosys.com/
Nutraceuticals ISOkine™ , DERMOkine™
Human growth factor
Barley On market ORF Genetics http://www.orfgenetics.com/
Human intrinsic factor, Coban
Vitamin B12 deficiency
Arabidopsis On market Cobento Biotech AS http://www.cobento.dk/default.a sp?id=76
Human lactoferrin Anti-infection, anti-inflammatory
Rice On market as
fine chemical
Ventria, USA http://www.ventriabio.com/
Human lysozyme Anti-infection,
anti-inflammatory Rice On market as
fine chemical Ventria, USA http://www.ventriabio.com/
Immunosphere™ Food additive for shrimps
Safflower Marketing expected for 2010
SemBioSys, Canada http://www.sembiosys.com/
表三 本研究使用之引子
Table 3. The primers used in this study
Name Sequence (5’ to 3’) Source
egfp-F CTG CAG TAT GGT GAG CAA GGG CG [57]
egfp-R ACT AGT CTT GTA CAG CTC GTC CAT GCC [57]
AseI-HiGPD-F CCA TTA ATC CCG TCG TCA GCG ATA CAT CC This study XmaI-Nos-R TCC CCC CGG GCC TGT CAA ACA CTG ATA GTT TAA TTC CC This study
pCAMBIA-F ACT GAT GGG CTG CCT GTA TC This study
pCAMBIA-R CCG ACG GAT GTT CGA CTA TT This study
HiGPD-mid-site CTA ACG AGG GGC ATT CAC AT This study
HmGPD-F ATT GGC AGC ACC TCG TCG TC This study
HmGPD-R GCA CAC ACG GCG GGA GTA TC This study
egfp362-F egfp468-R ApGPD-F hph-R
TGA ACC GCA TCG AGC TGA AGG G ACC TTG ATG CCG TTC TTC TGC TTG GGA ATT CTT AAG AGG TCC GCA AGC CTG CAG ACA ACT TAA TAA CAC ATT GCG
This study This study This study This study 引子中之限制酶切點以底線粗體標示，AseI：ATTAAT，PstI：CTGCAG，XmaI：CCCGGG，EcoR I：GAATTC。
表四 PCR 反應條件
Table 4. Primers and PCR conditions used in this study
Primer Condition Cycle
AseI-HiGPD-F /
表五 共培養培養基配置法
Table 5. The composition of IM medium for co-cultivation
g/L
MES (2-(N-morpholino)ethanesulfonic acid) Glucose
Glycerol
*Final pH = 5.3
Note：IM 平板培養基含 Glucose 0.9 g/L，1.5% agar
表六 CTAB buffer 配置法
Table 6. The composition of CTAB buffer
Compound Final concentration
CTAB buffer
CTAB (Hexadecyl trimethyl-ammonium bromide) 2%
20 mM EDTA (Ethylenediaminetetraacetic acid)
NaCl
Tris (tris(hydroxymethyl)aminomethane, pH = 8) PVP – 40 (polyvinylpyrrolidone, Mw 40000) Wash buffer
Ethanol
Ammonium acetate
表七 酵素免疫分析法試劑配置法
Table 7. The composition of ELISA reagent
Compound Final concentration
Coating buffer (pH = 9.5)
Carbonate 0.1 M
0.1 M
0.05%
0.13M 0.01 M
0.25%
50 mM 0.15 M 5 mM 0.05%
Bicarbonate
PBST buffer (pH = 7.2) Tween-20
NaCl NaH2PO4
gelatin-NET (pH = 8) Gelatin
Tris Base NaCl
EDTA‧2Na Tween-20
表八 SDS 聚丙醯胺膠體試劑配置法
Table 8. The composition of SDS-PAGE reagent
Compound Final concentration
SDS 聚丙醯胺分離膠體
Acrylamide/Bis-acrylamide (29:1) Tris-HCl (pH = 8.8)
SDS (Sodium dodecyl sulfate) APS (Ammonium persulfate)
TEMED (Tetramethylethylenediamine) SDS 聚丙醯胺焦集膠體
Acrylamide/Bis-acrylamide (29:1) Tris-HCl (pH = 6.8)
表九 轉形株之綠色螢光蛋白質定量與外源基因拷貝數測定
Table 9. Production of EGFP and relative egfp copy number of transformants
EGFP/TSP (ng/g) Relative egfp copy number (fold) P1 75.47 ± 18.57 1.00 ± 0.17
T1 418.83 ± 37.11 1.38 ± 0.08 T2 377.46 ± 36.05 1.86 ± 0.15 T3 246.32 ± 27.89 1.71 ± 0.22 T4 120.37 ± 10.38 2.28 ± 0.40 O1 319.58 ± 6.96 1.24 ± 0.11 O2 280.78 ± 32.22 2.30 ± 0.24 O3 269.34 ± 41.30 1.46 ± 0.17 O4 224.08 ± 59.94 1.56 ± 0.17 P2 92.16 ± 15.06 1.00 ± 0.22 T5 302.22 ± 22.72 1.43 ±0.21 T6 243.16 ± 30.39 1.74 ±0.19 T7 110.43 ± 6.62 1.20 ±0.13 O5 282.89 ± 5.73 0.94 ±0.11 O6 223.61 ± 6.95 1.70 ±0.52 O7 134.47 ± 37.82 1.47 ±0.19
圖一 擔子菌生活史
Figure 1. Life cycle of basidiomycetes.
(Source: http://kentsimmons.uwinnipeg.ca/16cm05/16labman05/lb2pg24.htm)
圖二 真菌於農桿菌媒介轉形之操作模式
Figure 2. Schematic overview of the experimental set-up of Agrobacterium-mediated transformation of fungi. [80]
圖三 農桿菌轉形的過程
Figure 3. A model for the Agrobacterium-mediated genetic transformation. [71]
圖四 論文架構圖
Figure 4. Schematic framework of this study.
圖五 質體 pCAMBIA0390、pHi-hyg-egfpS、p0390-sdi1-ORFM、p0390-AH-Aiegfp 與 p0390-Cbx-Hiegfp 之圖譜 Figure 5. Map of pCAMBIA0390, pHi-hyg-egfpS, p0390-sdi1-ORFM, p0390-AH-Aiegfp and p0390-Cbx-Hiegfp.
圖五 (續) 質體 pCAMBIA0390、pHi-hyg-egfpS、p0390-AH-Aiegfp、p0390-sdi1-ORFM 與 p0390-Cbx-Hiegfp 之圖譜 Figure 5. (continued) Map of pCAMBIA0390, pHi-hyg-egfpS, p0390-AH-Aiegfp, p0390-sdi1-ORFM and p0390-Cbx-Hiegfp.
圖六 質體 p0390-Cbx-Hiegfp 建構流程圖
Figure 6. Construction of the plasmid p0390-Cbx-Hiegfp.
Ase I, Xma I digestion
圖七 以限制酶截切確認質體 p0390-Cbx-Hiegfp 之大小
(A)質體 p0390-Cbx-Hiegfp 之限制酶圖譜，以 BglII 與 AseI 截切後預期得到片段大 小為1.2 kb、2.2 kb 以及 7.6 kb；(B) 質體 p0390-Cbx-Hiegfp 以 BglII 與 AseI 進行 截切。
Figure 7. Confirm the size of p0390-Cbx-Hiegfp by restriction enzyme digestion. (A) Map sites for restriction enzymes, expecting fragments including 1.2 kb, 2.2 kb and 7.6 kb after digestion by BglII, AseI. (B) Digestion of p0390-Cbx-Hiegfp by BglII, AseI.
圖八 質體 p0390-Cbx-Hiegfp 定序結果
(A) GPD 啟動子定序結果；(B) egfp 與 NOS 終止子定序結果。上排代表正確之序列，
下排代表質體p0390-Cbx-Hiegfp 定序結果，若比對結果與上排相同便以圓點表示。
箭頭代表起始密碼子，星號代表終止密碼子。
Figure 8. Sequencing results of p0390-Cbx-Hiegfp.
(A) Sequencing for GPD promoter of p0390-Cbx-Hiegfp; (B) Sequencing for egfp and NOS terminator of p0390-Cbx-Hiegfp. The upper row represents correct sequence and the lower row represents sequencing result of p0390-Cbx-Hiegfp. The dot in lower row means alignment was matched. Arrow represents start codon of egfp. Asterisk represents stop codon of egfp.
圖八 (續) 質體 p0390-Cbx-Hiegfp 定序結果
Figure 8. (Continued) Sequencing results of p0390-Cbx-Hiegfp.
圖九 農桿菌 GV3101 菌落聚合酶連鎖反應
使用引子對egfp-F 與 egfp-R 於農桿菌轉形株進行 PCR。(A)以電穿孔法轉入質體 p0390-Cbx-Hiegfp 之農桿菌菌株 GV3101；(B)以電穿孔法轉入質體
p0390-AH-Aiegfp 之農桿菌菌株 GV3101
Figure 9. Identification of putative A. tumefaciens GV3101 transformants by PCR using primers egfp-F and egfp-R.
Lanes: 1 to 5, putative A. tumefaciens GV3101 transformants harboring
p0390-Cbx-Hiegfp (A) or p0390-AH-Aiegfp (B); N, negative control without template;
P, positive control; M, 100 bp marker.
圖十 農桿菌 LBA4404 菌落聚合酶連鎖反應
使用引子對egfp-F 與 egfp-R 於農桿菌轉形株進行 PCR。(A)以電穿孔法轉入質體 p0390-Cbx-Hiegfp 之農桿菌菌株 LBA4404；(B)以電穿孔法轉入質體
p0390-AH-Aiegfp 之農桿菌菌株 LBA4404
Figure 10. Identification of putative A. tumefaciens LBA4404 transformants by PCR using primers egfp-F and egfp-R.
Lanes: 1 to 5, putative A. tumefaciens LBA4404 transformants harboring
p0390-Cbx-Hiegfp (A) or p0390-AH-Aiegfp (B); N, negative control without template;
P, positive control; M, 100 bp marker.
圖十一 農桿菌轉形株與美白菇共培養過程
Figure 11. Co-cultivation of A. tumefaciens transformants and H. marumoreus MMP.
圖十二 p0390-Cbx-Hiegfp 轉形株篩選過程
(A)負控制組野生型美白菇；(B) p0390-Cbx-Hiegfp 轉形株
Figure 12. Screening of p0390-Cbx-Hiegfp transformants in selective medium. (A) wild type H. marumoreus; (B) p0390-Cbx-Hiegfp transformants
圖十三 p0390-AH-Aiegfp 轉形株篩選過程
(A)負控制組野生型美白菇；(B) p0390-AH-Aiegfp 轉形株
Figure 13. Screening of p0390-AH-Aiegfp transformants in selective medium. (A) wild type H. marumoreus; (B) p0390-AH-Aiegfp transformants
圖十四 轉形株繼代後生長情形
(A) p0390-Cbx-Hiegfp 轉形株；(B) p0390-AH-Aiegfp 轉形株 Figure 14. Subculture of transformants into selective medium.
(A) p0390-Cbx-Hiegfp transformants; (B) p0390-AH-Aiegfp transformants.
圖十五 農桿菌媒介重複轉形法的二次轉形株篩選
(A)負控制組 p0390-Cbx-Hiegfp 轉形株；(B)負控制組 p0390-AH-Aiegfp 轉形株；(C) 雙質體轉形策略之二次轉形株
Figure 15. Screening of double transformants from multiple ATMT in selective medium. (A) negative control, p0390-Cbx-Hiegfp transformants; (B) negative control, p0390-AH-Aiegfp transformants; (C) two-vector double transformants.
圖十六 染色體 DNA 之聚合酶連鎖反應檢測
(A) 以引子對 egfp-F/egfp-R 檢測 p0390-Cbx-Hiegfp 轉形株；(B) 以引子對 ApGPD-F/hph-R 檢測 p0390-AH-Aiegfp 轉形株。
Figure 16. PCR analysis for genomic DNA isolated from transformants. (A) PCR for p0390-Cbx-Hiegfp transformants using primers egfp-F and egfp-R; (B) PCR for p0390-AH-Aiegfp transformants using primers ApGPD-F/hph-R. Lane 1: wild type H.
marumoreus DNA; lane 2 to 4: transformants; N, negative control without template; P, positive control; M, 100 bp marker.
圖十七 二次轉形株染色體 DNA 之聚合酶連鎖反應檢測
Figure 17. PCR analysis for genomic DNA isolated from double transformants. Lane 1: wild type H. marumoreus DNA; lane 2 to 5: two-vector double transformants; N, negative control without template; P, positive control; M, 100 bp marker.
圖十八 螢光顯微鏡觀察
右側為菌絲於藍光激發下的照片，左側為菌絲於明視野下的照片。圖中刻度為50
μm。
Figure 18. The mycelia of transformants under fluorescent microscope.
圖十九 轉形株綠色螢光蛋白質定量
測定美白菇轉形株每克總可溶性蛋白質中綠色螢光蛋白質之含量。(A)一次轉形株 母體(P1 與 P2)、雙質體轉形策略之二次轉形株(T1~T7)；(B)一次轉形株母體(P1 與 P2)與單質體轉形策略之二次轉形株(O1~O7)。P1、T1~T4 與 O1~O4 轉形株係利用 農桿菌LBA4404 進行轉形，P2、T5~T7 與 O5~O7 轉形株係利用農桿菌 GV3101 進行轉形。
Figure 19. Production of EGFP. (A) Parental single transformant (P1 and P2) and two-vector double transformants (T1 to T7); (B) parental single transformant (P1 and P2) and one-vector double transformants (O1 to O7). P1, T1~T4 and O1~O4 were
transformed by A. tumefaciens LBA4404; P2, T5~T7 and O5~O7 were transformed by A. tumefaciens GV3101.
圖二十 美白菇轉形株之西方墨點法分析
Figure 20. Western blotting analysis of H. marumoreus transformants. 1~6: randomly selected transformants; M, protein ladder; P, positive control; W, wild type H.
marumoreus.
圖二十一 即時定量聚合酶鏈鎖反應
利用即時定量聚合酶鏈鎖反應進行相對定量以分析轉形株中外源基因拷貝數。(A) 一次轉形株母體(P1 與 P2)、雙質體轉形策略之二次轉形株(T1~T7)；(B)一次轉形 株母體(P1 與 P2)與單質體轉形策略之二次轉形株(O1~O7)。P1、T1~T4 與 O1~O4 轉形株係利用農桿菌LBA4404 進行轉形，P2、T5~T7 與 O5~O7 轉形株係利用農 桿菌GV3101 進行轉形。
Figure 21. The egfp copy numbers of transformants. (A) Relative egfp copy number in parental single transformant (P1 and P2) and two-vector double transformants (T1 to T7). (B) Relative egfp copy number in parental single transformant (P1 and P2) and one-vector double transformants (O1 to O7). P1, T1~T4 and O1~O4 were transformed by A. tumefaciens LBA4404; P2, T5~T7 and O5~O7 were transformed by A.
tumefaciens GV3101.