劉瑞芬臺灣大學：植物病理與微生物學研究所王誌偉Wang, Chih-WeiChih-WeiWang2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/58062許多研究報告皆指出靈芝多醣體(polysaccharides)具有免疫調節與抑制腫瘤的活性，為了從分子層次了解這些多醣體生合成的途徑，本實驗室利用自靈芝基因體資料庫所獲得的序列資料，設計引子對，以RACE (rapid amplification of cDNA ends )選殖與多醣生合成有關之基因的全長度cDNA序列。本論文為針對其中兩個基因glp5-1與glp5-2進行選殖與分析。glp5全長度cDNA 有1583 bp，可以轉譯359個胺基酸，預期的蛋白質分子量為39.7 kDa。Glp5胺基酸序列和洋菇基因shs4 (AJ271693)有70%之相似度，並可能參與1,6-β-glucan的生合成，序列分析結果顯示，SHS4與Glp5-1之胺基酸序列皆含有一個可能的exo-β-1,3 glucanase domain，其他含有該domain的基因主要有在啤酒酵母菌所發現的scw4、scw10、scw11與bgl2等，這些基因依CAZy分類系統被歸類於Glycoside hydrolase family 17。其中只有Bgl2已確定具有endo-1,3-β-glucanase和glucanosyltransferase酵素活性，可以在1,3-β-glucan長鏈上加入1,6-β鍵結的葡萄糖分子，其他三個基因則只能經由突變分析推測其可能的功能為1,3-β-glucanase或1,3-β-glucanosyltransferase。利用BLAST的方法在許多已完成基因體定序的真菌資料庫與EST資料庫，發現許多擔子菌皆可以找到glp5的同源性基因。glp5-2為glp5的同源性基因，位於glp5-1 DNA下游約0.4 kb的位置，經由RACE的實驗得到全長glp5-2 cDNA 序列共1845 bp，可以轉譯414個胺基酸，預期的蛋白質分子量為44.6 kDa。北方雜合分析的結果顯示，glp5-1基因在靈芝菌絲及子實體都會表現，液態培養時期中隨著培養天數的增加，表現量有下降的趨勢，而在不同子實體分化時期與不同子實體部位之表現量差異不大。glp5-2基因在菌絲時期及子實體時期亦皆有表現，但在子實體時期菌傘生長部位的表現量較菌柄部位高出許多。為了對Glp5蛋白有更深入的研究，本研究在E. coli的系統表現Glp5-1蛋白，將該表現蛋白純化後，施打兔子生產抗Glp5-1抗體。經由西方雜合分析的結果發現，靈芝菌絲其細胞壁與培養液，及子實體細胞壁粗抽蛋白中，均含大小約63 kDa的蛋白可被抗體偵測到，顯示Glp5-1蛋白可能位於細胞壁，而Glp5-1/Glp5-2之功能仍有待進一步的探討。Polysaccharides from Ganoderma lucidum have been reported to exhibit immuno-modulation and antitumor activties. The molecular basis of polysaccharides biosynthetic pathway was not yet clear. Sequences of potentially related genes were retrieved from the G. lucidum genome database and primers were desigened to isolate the corresponding clones. Full-length cDNA sequences of target genes were initially obtained by RACE (rapid amplification of cDNA ends). Among them, two genes named glp5-1 and glp5-2 were further characterized in this sudy. glp5-1 cDNA contained an open reading frame of 1,077 nucleotides, which encodes a protein with 359 amino acid residues and an estimated molecular mass of 39.7 kDa. Glp5 has 70% amino acid sequences similarity with SHS4, reported in Agaricus bisporus, and a putative exo-β-1,3 glucanase domain was also identified. SHS4 has been reported to have 42% amino acid similarity with 1,6-β-glucan synthase gene in Pseudomonas putida KT2400. Expression level of shs4 gene is correlated with the increase of 1,6-β-linked glucan side branches and considered to participate in the biosynthesis of 1,6-β-glucan. Genes with such domain include scw4, scw10, scw11, and bgl2 reported in yeast. These four genes were classified as glycoside hydrolase family 17 by CAZy database. Bgl2 has been reported to have endo-1,3-β-glucanase and glucanosyltransferase activity. Others were reported to have putative 1,3-β-glucanase or 1,3-β-glucanosyltransferase activity by genetic analysis. In searching available genome database of other fungi, we have found that glp5-1 homologous genes were highly conserved in many basidiomycetes. Further study also found that glp5-2, a homologous gene of glp5-1, was located about 400 nucleotides downstream of glp5-1 gene. glp5-2 cDNA had an open reading frame of 1,242 nucleotides encoding a 414-amino acid protein with a predicted size of 44.6 kDa. Northern blot analysis showed that glp5-1 mRNA both expressed in mycelium and fruit body stage of G. lucidum, and was decreased in mycelium stage with increasing culture period. In fruit body stage, glp5-2 mRNA expression was greatly induced in pileus of growth area than in stipe. Glp5-1 recombinat protein expressed by E. coli was purified and used to raise antibody for further experiments. Western blot analysis showed a size of 63 kDa protein was detected in cell wall fraction of both mycelium and fruit body. Indicated Glp5-1 is a cell wall protein. The estimated size of Glp5-1 protein based on western blot signals was larger than that predicted from amino acid sequences. Glycosylation of Glp5-1 protein may occurr in G. lucidum. The functions of Glp5-1 and Glp5-2 proteins need to be further investgated.中文摘要………………………………………………………………..……..…...….1 英文摘要……………………………………………………………..…….………….2 研究動機…………………………………………………………………...………….3 壹、前人研究………………………………………………………………...…...…..4 一、關於靈芝……………….…..………………………………………….........4 二、高等真菌子實體發育之相關研究………………………………………....4 (一)、子實體初期構造的發育(Development of Emergent Structures)........5 (二)、擔子菌子實體蕈柄延伸(stipe elongation)相關之研究……………..6 (三)、擔子菌子實體發育相關基因之研究……………………………...…7 三、靈芝功能性物質..………………………......................................................8 (一)、關於β-glucan........................................................................................8 四、關於SHS4基因:…………………………..……. …....................................9 五、關於Glycoside hydrolase相關基因之研究………………………………..9 (一)、Glycoside hydrolase基因之分類…………………………………….9 (二)、SHS4基因之分類…………………………………………………...10 (三)、含有Exo-1,3-beta-glucanase domain之基因………………………10 (四)、酵母菌中Glucosyl hydrolases(GH) family 17相關基因之研究….10 貳、材料與方法……………………………………………….…….……..….……..12 一、靈芝來源:………………………………………………………….….…....12 二、靈芝核酸製備:……………………………...……......………….......…….12 (一)、靈芝total RNA之抽取……………………………………………..12 (二)、抽取靈芝poly(A)+ RNA………………………………………….…12 三、基因全序列之選殖:……………….…………………………. .….…....….13 (一)、第一股cDNA之合成……………………………………………….13 1.合成5’-RACE-Ready cDNA…………………………………...….13 2.合成3’-RACE-Ready cDNA…………………………………...….13 (二)、5’-RACE及3’-RACE…………………………………………..…...13 (三)、自電泳膠體回收DNA片段……………………………..…………14 (四)、將PCR擴增產物選殖至T-Vector (TA cloning) …………………..14 (五)、質體小量製備(mini-prep) ……………………………………….…15 (六)、核酸定序及序列分析……………………………………………….15 (七)、利用Reverse transcription PCR(RT-PCR)進行Glp5與Glp5-2基因全序列之增幅……………………………………………………….16 四、北方雜合分析 (Northern hybridization analysis)……………......….…...16 (一)、RNA 電泳分析………………………………………………….....16 (二)、RNA 轉漬…………………………………………………………..17 (三)、核酸探針製備………………………………………………………17 (四)、前置雜合反應 (Prehybridization) …………………………..…..…17 (五)、雜合反應 (Hybridization) ……………………………………….…17 (六)、標示核酸探針之冷光偵測(Detection of DIG-labeled DNA probe)..18 五、基因組南方雜合分析(Genomic Southern hybridization)…………………18 (一)、靈芝genomic DNA之抽取………………………………………...18 (二)、Genomic DNA 之酵解與瓊脂膠體電泳………………………..….19 (三)、DNA 毛細管轉漬法(Capillay transfer) …………………………….19 (四)、核酸探針之製備………………………………………………….....19 (五)、雜合前置反應與雜合反應……………………………………….….20 六、Glp5與Glp5-2基因引子延伸實驗(primer extension):………………….20 (一)、磷酸化標定寡核苷酸引子………………………………………….20 (二)、沉澱磷酸化標定之寡核苷酸引子與靈芝total RNA…………...…20 (三)、引子延伸反應 (primer extension reaction) ……………………..…20 (四)、質體DNA定序反應………………………………………………..21 七、以大腸桿菌(Escherichia coli)系統表現Glp5蛋白質………...…....….…21 (一)、表現載體(pQE31-Glp5)之構築……………………………….…….21 (二)、大腸桿菌勝任細胞製備…………………………………………….21 (三)、大腸桿菌細胞轉形………………………………………………....22 (四)、表現Glp5蛋白質…………………………………………………...22 (五)、純化重組Glp5蛋白質……………………………………………...22 八、免疫學試驗……………………………………………………………….23 (一)、抗血清製備……………………………………………………….…23 (二)、Glp5免疫球蛋白(Immunoglobulin G, IgG)之純化…………………23 (三)、西方轉漬反應……………………………………………………....23 九、靈芝細胞質、細胞壁及培養液蛋白質之萃取…………………….……24 (一)、靈芝菌絲培養液蛋白質之抽取…………………………………….24 (二)、靈芝細胞質蛋白質之萃取………………………………….………24 (三)、靈芝細胞壁蛋白質之萃取…………………………………………24 十、篩選fosmid clone………………………………………………….……..24 (一)、自fosmid library初步篩選帶有Glp5-2基因的選殖株…..………25 (二)、以菌落雜合分析(colony hybridization)篩選出帶有Glp5與Glp5-2的 fosmid clone……………………………………………………25 (三)、抽取fosmid clone之質體DNA……………………………………26 十一、Glp5與Glp5-2基因置換載體(gene replacement vector)之構築…….26 參、結果………………………………………………………...………….……......27 一、glp5與glp5-2 cDNA全長度序列選殖結果…………………………….27 二、Glp5與Glp5-2胺基酸序列與其他基因庫中相似基因之序列排併比較28 三、glp5與glp5-2基因在靈芝基因組之存在情形……………………….…28 四、北方雜合反應分析glp5與glp5-2基因訊息RNA的表現情形……….28 五、於大腸桿菌系統表現純化Glp5蛋白………………..…………….....…29 六、西方轉漬分析Glp5蛋白………………….……………………….….…29 七、靈芝glp5與glp5-2 基因置換載體之構築……………………………...29 肆、討論……………………………………………………………….…...….…….30 伍、圖表……………………………………………………………..…..…..…....…36 陸、參考文獻………………………………………………………..…..….…….…50 柒、附錄………………………………………………………………..…..…….….60en-US靈芝葡聚醣Ganoderma lucidumbeta-glucanother