擔子菌菇類藍光受器基因之選殖、序列分析與表現

Cloning, sequence analysis and expression of the blue-light receptor gene from the basidiomycetous mushrooms

龔巧鈴(Chiau-Ling Gung)

指導教授 ： 許瑞祥

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.01513

摘要

在許多食藥用擔子菌的栽培過程中，要使其形成子實體必須控制多項環境因子，其中光線是影響其子實體發育是否完全的一個重要因素，顯示擔子菌中應有接受光線刺激的受器存在。目前研究顯示灰蓋鬼傘(Corprinus cinereus)、隱球菌(Cryptococcus neoformans)、香菇(Lentinula edodes)與褐腐菌(Postia placenta)等四種擔子菌具有與子囊菌紅麵包黴(Neurospora crassa)藍光受器White Collar-1 (WC-1)同源的蛋白質。本研究從常見食用真菌香菇、金針菇、鮑魚菇、美白菇與雞腿菇中選殖其WC-1同源藍光受器。目前使用專一性引子於香菇的互補DNA (complementary DNA, cDNA)中選殖到兩條與已發表之香菇藍光受器基因LephrA (accession no. AB279630)和LephrB (accession no. AB446463)相似之基因，分別命名為photoreceptor A與photoreceptor B。photoreceptor A全長為2,831 bp，其中包含了長56 bp的intron，與LephrA核酸與胺基酸相同度分別為99.5%和99.2%。photoreceptor B全長為942 bp，與LephrB核酸與胺基酸相同度為99.5%和99.6%。在基因組中，photoreceptor B被四個大小在52~87 bp之間intron分開。使用pET-21a(+)載體於大腸桿菌BL 21 (DE3)異源表現的Photoreceptor B-His x6為水溶性且大小約為37.7 kDa。同時以特定之退化性引子於金針菇cDNA中擴增出一段長420 bp的核酸序列，轉譯的140個胺基酸序列分別與香菇、裂褶菌(Schizophyllum commune)、布拉克鬍鬚黴(Phycomyces blakesleeanus)與灰蓋鬼傘中已發表的WC-1同源藍光受器分別具28%、28%、24%與31%相同度。

關鍵字

擔子菌菇類 ； 感光系統 ； 藍光受器 ； 香菇 ； 金針菇

並列摘要

For the fruiting-body formation, there are different environmental factors that must be controlled on the edible and medical mushroom cultivation. Light is one of the most important elements influencing the fruiting-body development, which indicates that there exists photoreceptor for receiving the light stimulus in the bacidiomycetes. The bacidiomycete Corprinus cinereus, Cryptococcus neoformans, Lentinula edodes and Postia placenta have the blue-light receptors that are homologs of White Collar-1 (WC-1) in ascomycete Neurospora crassa. In this study, we attempted to clone the WC-1 homologs from the edible mushrooms: L. edodes , Flammulina velutipes, Pleurotus ostreatus, Hypsizygus marmoreus variant and Coprinus comatus. We cloned two blue-light receptor genes, photoreceptor A and photoreceptor B, from L. edodes by using specific primers and the complementary DNA template of L. edodes for PCR. The genes photoreceptor A and photoreceptor B are similar to the published blue-light receptor genes LephrA (accession no. AB279630) and LephrB (accession no. AB279630) in L. edodes. The sequence of photoreceptor A is 2,831 bp and contains intron in 56 bp. The identity of nucleotides and amino acids between photoreceptor A and LephrA are 99.5% and 99.6%. The sequence of photoreceptor B is 942 bp and the identity of nucleotides and amino acids compared to LephrB are 99.5% and 99.6%. The photoreceptor B gene is divided by four introns in the genome and the size of introns is between 52 bp and 87 bp. The photoreceptor B gene were subcloned to pET-21a(+) and expressed in Escherichia coli BL 21 (DE3). The protein product Photoreceptor B-His x6 is soluble and the molecular weight is about 37.7 kDa. In the same time, we cloned a 420 bp nucleotide from the complementary DNA of F. velutipes by using the degenerate primers. The deduced 140 amino acids of the cloned nucleotide has 28%, 28%, 24% and 31% identity to the WC-1 homologs of L. edodes, Schizophyllum commune, Phycomyces blakesleeanus and C. cinereus.

並列關鍵字

basidiomycetous mushroom ； light-sensing system ； blue-light receptor ； Lentinula edodes ； Flammuliana velutipes

參考文獻

3. Bender, S., C.N. Dumitrache-Anghel, J. Backhaus, G. Christie, R.F. Cross, G.T. Lonergan, and W.L. Baker. 2003. A Case for Caution in Assessing the Antibiotic Activity of Extracts of Culinary-Medicinal Shiitake Mushroom [ Lentinus edodes (Berk.) Singer] (Agaricomycetideae). Int. J. Med. Mushrooms 5: 31-5.

4. Bieszke, J.A., L. Li, and K.A. Borkovich. 2007. The fungal opsin gene nop-1 is negatively-regulated by a component of the blue light sensing pathway and influences conidiation-specific gene expression in Neurospora crassa. Curr. Genet. 52: 149-57.

5. Bisen, P., R. Baghel, B. Sanodiya, G. Thakur, and G. Prasad. 2010. Lentinus edodes: a macrofungus with pharmacological activities. Curr. Med. Chem. 17: 2419-2430.

6. Bobek, P. and Galbavy. 1999. Hypocholesterolemic and antiatherogenic effect of oyster mushroom (Pleurotus ostreatus) in rabbits. Nahrung 43: 339-342.

7. Bobek, P. and S. Galbavy. 2001. Effect of pleuran (beta-glucan from Pleurotus ostreatus) on the antioxidant status of the organism and on dimethylhydrazine-induced precancerous lesions in rat colon. Br. J. Biomed. Sci. 58: 164-8.