靈芝β-(1,3)-D-葡聚糖合成酶基因之選殖與特性分析

Abstract

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.