过表达腺苷生物合成正相关基因GlPNP提高灵芝腺苷的含量

Overexpression of GlPNP, a gene positively correlated with adenosine biosynthesis, increases the adenosine content of Ganoderma lucidum

摘要:

【背景】灵芝被纳入我国“药食同源”试点名单，腺苷作为其主要活性物质之一，在免疫调节、抗炎、抗癌等方面发挥着重要作用。【目的】调控腺苷生物合成关键酶基因的表达来提高灵芝腺苷产量。【方法】将不同培养时间阶段腺苷合成酶基因(包括5-氨基咪唑-4-甲酰胺核糖核苷酸甲酰转移酶GlATIC、嘌呤核苷磷酸化酶GlPNP、腺苷激酶GlADK)的表达量与腺苷含量相关联，筛选出与灵芝腺苷含量呈正相关的关键酶基因。克隆关键酶基因并在灵芝中过表达，探究关键酶基因过表达对灵芝腺苷积累的影响。【结果】GlPNP的表达与灵芝腺苷含量呈正相关。GlPNP的cDNA全长为969 bp，预测GlPNP蛋白的相对分子量为34.6 kDa，呈三聚体的四元结构。研究结果表明，过表达菌株中GlPNP的表达量在第4天比野生型菌株(WT)上调了2.9−3.9倍，与含空载体的菌株(CK)相比，腺苷含量分别提高了78%和63%。【结论】过表达嘌呤核苷磷酸化酶是提高灵芝腺苷产量的一种有效手段。

关键词:灵芝;腺苷;嘌呤核苷磷酸化酶;过表达

Abstract:

[Background] Ganoderma lucidum is a precious Chinese medicinal herb with both medicinal and edible values. Adenosine, one of the active components, endows the herb with immunomodulatory, anti-inflammatory, and anti-tumor activities. [Objective] To regulate the expression of key genes involved in adenosine biosynthesis to increase the adenosine production of G. lucidum. [Methods] We studied the correlations of the expression of adenosine synthase genes (GlATIC,GlPNP, and GlADK) with the adenosine content. The key enzyme genes that were positively correlated with the adenosine content of G. lucidum were screened out. The key enzyme genes in G. lucidum were cloned and overexpressed to investigate the effect of key enzyme gene overexpression on adenosine accumulation. [Results] The expression of GlPNP was positively correlated with adenosine content. The key gene GlPNP was cloned and overexpressed in G. lucidum. The cDNA of GlPNP was 969 bp in length. GlPNP displayed a trimeric quaternary structure, with a predicted molecular weight of 34.6 kDa. The expression levels of GlPNP in the transformants were 2.9–3.9 folds higher than that of the wild type on day 4. The adenosine content in the transformants increased by 78% and 63%, respectively, compared with that in the strain transformed with the blank vector. [Conclusion] Overexpression of GlPNP is an effective strategy to increase the production of adenosine.

Key words:Ganoderma lucidum;adenosine;purine nucleoside phosphorylase;overexpression