靈芝SKN1基因族成員之選殖與分析

Abstract

靈芝(Ganoderma lucidum)在東方國家被視為吉祥寶物和治療百病的靈藥，為中國傳統醫學上重要的藥材。其在分類學上屬於擔子菌綱、無褶菌目、多孔菌科的靈芝屬，可寄生在多種植物鄰近根部的樹幹上，引起木材的白腐朽或根腐。靈芝含有多種化學成分，其中的多醣體、三帖類、腺苷及有機鍺等，被認為具保健療效，多醣體已被證實具有提高免疫能力及抑制腫瘤之活性。靈芝多醣體為細胞壁的主要成分之一，其由β-1,3-D-葡聚醣的主幹加上多個由一、兩個葡萄糖以β-1,6-葡萄糖基鍵結之分支所構成的高分子多醣體。為了探討靈芝多醣生合成之相關機制，利用靈芝基因體網站所提供之序列資料，找尋與酵母菌β-1,6-D-葡聚醣生成基因SKN1之同源性基因序列，本論文針對其中兩個基因分別進行了5’端和3’端的RACE (rapid amplication of cDNA ends)，得到全長的cDNA，此兩基因暫命名為Glp4 (Ganoderma lucidum putative β-glucan associated protein 4)和Glp6。以BLAST進行基因序列搜尋之結果顯示，Glp4及Glp6蛋白質與酵母菌Saccharomyces cerevisiae及白色念珠菌Candida albicans之Skn1p及Kre6p有顯著相似性，最相似區域集中在中間區域及C端。北方雜合分析顯示，兩基因在靈芝子實體的不同生長時期及菌絲培養於不同的碳素源，有不同的表現情形。為了進一步證實所得到基因的功能，利用酵母菌系統進行K1毒質測試，將靈芝基因利用載體轉型到具Kre6基因突變酵母菌中，結果證實靈芝的Glp2基因與酵母菌的KRE6應該有相同的功能。

Lingzhi (Ganoderma lucidum) has been treasured and consumed as a health tonic in easten countries. Ganoderma species belong to the kingdom of Fungi, the division of Basidiomycota, the class of Homobasidiomycetes, the order of Aphyllophorales, the family of Polyporaceae (Ganodermataceae) and the genus of Ganoderma. It is a parasite on many palnts and causes white rot symptoms. Lingzhi has many efficacious components such as polysaccharides, triterpenoids, and adenosine, etc. The main functions of polysaccharides extracted from G. lucidum (PS-G) were confirmed to promote immunity and have antitumor activities. PS-G is a branched-β-glucan, which contains a background chain of (1→3)-linked D-glucose residues, attached mainly with one or two (1→6) D-glucosyl units at O-6 and also with a few short (1→4)-linked D-glucosyl units at O-2 positions. In order to understand the biosynthesis pathway of PS-G, we searched yeast SKN1 gene homolog sequences, which possiblely associated with PG-S synthesis based on the information provided by G. lucidum genome project. In this study, two genes, here here are named Glp4 (Ganoderma lucidum putative β-glucan associated protein 4) and Glp6, were cloned by 5’- and 3’-RACE (rapid amplication of cDNA ends). After sequence analysis and use BLAST to search the amino acid sequences from the database, Glp4 and Glp6 proteins revealed significant similarity with Kre6p and Skn1p from Saccharomyces cerevisiae and Candida albicans. The central region and C terminus are highly conserved among these genes and N terminus has high diversity. In Northern analysis, Glp4 and Glp6 mRNA prepared from fruiting body and hyphae growthing in different carbon source showed different density of band. In order to validate the function of SKN1 gene family of Ganoderma lucidum, we used vectors which contain Glp2 to transform yeast Kre6 mutain strain and taken yeast K1 killer toxin system experiment. The result conform that Glp2 gene and yeast KRE6 gene should has the same function.