靈芝β-(1,3)-D-葡聚糖合成酶基因之選殖與特性分析

Abstract

靈芝(Ganoderma lucidum)雖是樹木的病原真菌，也一直是中國醫藥中一種極為珍貴的藥用真菌。靈芝具有抗腫瘤活性、降血糖作用、清除自由基與增加免疫調節等保健功效，而其中最受矚目的便是抗腫瘤活性，故成為現今研究抗癌的重要材料之ㄧ。近年來對靈芝有效成分之分析研究，主要包括多醣(polysaccharides)、三萜類(triterpenes)、腺嘌呤核苷與有機鍺，並且發現其中具抗腫瘤活性的主要成份為多醣體。靈芝的多醣體為細胞壁的主要成份，除α-(1,4)-link的基丁質(chitin)外，還有β-(1,3)-D-葡聚醣(glucan)及β-(1,6)-D-葡聚醣及其它少量的醣類，而許多的研究已指出β-(1,3)-D-葡聚醣具有抗腫瘤活性與增強免疫力的效應，所以找出β-(1,3)-D-葡聚醣相關合成酶變成一很重要的課題。β-(1,3)-D-葡聚醣合成酶為具有相當保守性之酵素，真菌與植物中均普遍的發現其酵素之存在，如在Saccharomyces cerevisiae、Neurospora crassa、Aspergllius niger及Hordeum vulgare cv. Clipper等物種上都發現了相關的基因與蛋白質，其主要功能是將UDP-Glucose聚合成長鏈的β-(1,3)-D-葡聚醣，為構成細胞壁之主要成份。為找出靈芝的β-(1,3)-D-葡聚醣合成酶之基因，利用靈芝基因體網站所提共之資料，搜尋與S. cerevisiae相關之β-(1,3)-D-葡聚醣合成酶基因fks相似之序列，並將其選殖出來，分別得到glfks1與glfks2，利用hydropathy分析，顯示GlFks1與GlFks2分別具有16個與14個transmembrane helix，且推測GlFks1與GlFks2應也是屬於嵌入的membrane protein。另外利用南方雜合分析發現glfks1與glfks2於靈芝的基因體中各只有一個copy number。利用Real-Time RT-PCR來分析gfks1與glfks2的表現，gfks1與glfks2在添加Ca+之情況下，都無被誘導表現。在缺乏碳素原葡萄糖與有galactose時，可誘導glfks2表現。若將培養溫度升至39℃，已可誘導glfks2表現，但若升至42℃，可使誘導的表現量更高。若添加重金屬元素，發現glfks1在有重金屬鎘(Cd)與(Pb)鉛存在之情況下被誘導，而鋅(Zn)可以短暫的誘導增加glfks2表現，接著便會使glfks2的表現量減少，此外，在子實體之glfks1與glfks2表現量均比菌絲之表現量高。為進一步求證glfks1與glfks2基因之功能，利用S. cerevisiae的fks1突變株進行互補性分析實驗，來確認GlFks1與GlFks2具有合成β-(1,3)-D-葡聚醣的能力，實驗結果glfks1與glfks2基因分別都能部份地使S. cerevisiae fks1突變株缺失的Fks1的功能恢復，證實靈芝的β-(1,3)-D-葡聚醣合成酶可在S. cerevisiae中表現以合成 β-(1,3)-D-葡聚醣。

Ganoderma lucidum is a Chinese tranditional herbal fungi, tough it is also a pathogenic fungi of trees. The immunomodulation function of G. lucidum was reported to be effective in the treatment of hypertension, hyperglycemia, neoplasia, eliminating harmful free redicals and stimulatimg immunity. The anti-tumor efficacy of G. lucidum is much interested in the modern times. For the past few years, the enchancing immune matter of G. lucidum was separated and characterized, including polysaccharides, triterpenes,nucleic acid and organic germanium. Polysaccharides had anti-tumor activity was reported. Polysaccharides was the major component of the cell wall in G. lucidum. Excluding the α-(14)-link chitin, polysaccharides contained β-(1,3)-D-glucan, β-(1,6)-glucan and other small content saccharides.Since β-(1,3)- D-glucan had immunostimulating and antitumoral properties was reported, it is interest to find and study β-(1,3)-D-glucan synthase. β-(1,3)-D-glucan synthase was very conserved. The gene and protein of β-(1,3)-D-glucan synthase was identified and found in fungi and plant, for example, Saccharomyces cerevisiae、Neurospora crassa、Aspergllius niger and Hordeum vulgare cv. Clipper etc.. The major function of the enzyme was to transfer glucose from UDP-Glucose in plasma to elongate the poly-β-(1,3)-D-glucosides chain, the major component of the cell wall. I used the fks gene sequence of S. cerevisiae to research the homologous gene of G. lucidum in the network station of G. lucidum. Then we clone the gene, glfks1 and glfks2. Hydropathy analysis predicts that GlFks1 and GlFks2 are integral membrane protein with about 16 and 14 transmembrane helices (TMHs).In genomic southern blot analysis, glfks1 and glfks2 both are single copy gene. I detect the different expression of glfks1 and glfks2 by Real-Time RT-PCR. Both glfks1 and glfks2 cannot be induced by the addition of Ca+ to the growth medium. The glfks2 can be induced in the absence of glucose, and by galactose. The expression of glfks2 is induced at 39℃. At 42℃,the inducble amount is more high. In the addition of the heavy metal, the cadmium (Cd) and lead (Pb) can be to induce the expression of glfks1; the zinc (Zn) can be to induce transiently the expression of glfks2, then decrease the expression. The express quantity of the glfks1 and glfks2 in fruit body were much more than in mycelium. To further establish a functional role of GlFks1 and GlFksS2 in β-(1,3)- glucan synthesis, I find that each glfks1 and glfks2 can complement partially the S. cerevisiae fks1 mutant. The result proves that the β-(1,3)-D-glucan synthase of G. lucidum can express in S. cerevisiae to synthesize β-(1,3)-D-glucan.