Study on the Transformation System of Ganoderma lucidum

靈芝為重要藥用真菌，近年來已有進入分子層次的研究趨勢，而轉形系統有助於分子遺傳研究並具應用於菌種改良的潛力，因此本論文嘗試以泛宿主載體pAN7-1上的抗hygromycin B基因hph為篩選標誌建立其基因標的轉形系統。 在此研究中以PCR選殖靈芝的3個同源片段，並將之併入pAN7-1的HindIII切位以構築成同源性較高的「標的載體」，分別命名為pGL400、pGL600、pGL1k。將10 g載體DNA與由單核菌絲製備的原生質體進行聚乙二醇-氯化鈣法轉形，其中pGL400、pGL600、pGL1k的轉形率分別為16.4、11.5、13.5株/ g，較pAN7-1的3.5株/ g高。而大部份轉形株經無篩選壓力的培養後仍具抗hygromycin B的外表型（HygBr），PCR證據與HygBr外表型證明質體能成功進入靈芝體內。分析不同的轉形株發現其菌落型態、生長速率與抗hygromycin B能力不一，而pGL400轉形株出現高比例的菌落型態變異，推測可能與該400 bp同源序列功能有關。另外分析pGL1k轉形株纖維素分解酵素CBH活性發現其中編號TnpGL1kt3可能是cbhI基因突變株。Ganoderma lucidum is an important medical fungus. It seems be a trend that was studied at the molecular level recently. Transformation system helps studying its molecular genetics and may apply to strain improvement. So we attempt to build up the gene target transformation system based on Hygromycin B resistant gene hph of pAN7-1, a broad range host vector of fungi, as a selection marker. In this study, we cloned 3 homologous DNA fragments of G. lucidum by PCR. Target vectors were constructed by inserting these fragments at HindIII site of pAN7-1, and named pGL400、pGL600、pGL1k individually. PEG/CaCl2 transformation was mediated with 10 g plasmid DNA and protoplasts preparing from monokaryotic mycelia. The transformation frequency was 16.4、11.5、13.5 transforants per g DNA respectively for pGL400、pGL600、pGL1k and higher than pAN7-1,which was 3.5 transforants per g DNA. And most transforants harboring HygBr phenotype still after several subcultures without any selection pressure. The evidence of PCR product and HygBr phenotype proved that the plasmids could be transformed into G. lucidum successfully. Penotypic analysis of these transforants shows the differences in the ability to resist Hygromycin B, colony morphology and growth rate from each. And the results of pGL400 transforants with high proportion of morphological aberration implicated that might involved in the gene function of 400 bp. In addition, we analysis the cellobiohydrolase activity of the pGL1k transforants. And we found that a tranforant TnpGL1kt3 harboring very low CBH activity maybe a cbhI mutant.壹、 中文摘要------------------------------------------------3 貳、 英文摘要------------------------------------------------4 參、 前言----------------------------------------------------5 肆、 前人研究------------------------------------------------6 靈芝菌介紹---------------------------------------------6 靈芝的科學研究與趨勢-----------------------------------6 絲狀真菌轉形系統介紹-----------------------------------8 纖維素分解酵素介紹------------------------------------13 伍、 材料與方法---------------------------------------------13 實驗菌種與質體----------------------------------------15 藥品與儀器--------------------------------------------15 靈芝培養方法------------------------------------------16 利用PCR選殖同源DNA片段--------------------------------16 同源載體構築------------------------------------------21 靈芝原生質體製備--------------------------------------21 靈芝轉形實驗------------------------------------------22 靈芝轉形株分析----------------------------------------23 陸、 結果---------------------------------------------------24 柒、 討論---------------------------------------------------31 捌、 結論---------------------------------------------------39 玖、 參考文獻-----------------------------------------------40 拾、 附圖---------------------------------------------------51 拾壹、 附表-------------------------------------------------76 拾貳、 附錄-------------------------------------------------8

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll- 365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant