榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

Cloning and expression of the GGPP synthase gene from the paclitaxel-producing endophytic fungus (Penicillium aurantiogriseum)in Corylus avellana

关键词:  内生真菌, GGPP合酶, 原核表达, 基因克隆, 紫杉醇

Key words:  endophytic fungus(Penicillium aurantiogriseum), GGPP synthase, prokaryotic expression, gene cloning, taxol

摘要:

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因PaGGPPS(GenBank登录号为KM881430)的cDNA 开放阅读框(Open reading frame, ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98 kD,等电点为6.168, 表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。PaGGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。

Abstract:

Geranylgeranyl diphosphate synthase is one of the key enzymes in taxol synthesis pathway in paclitaxel-producing endophytic fungus. To study taxol synthesis in paclitaxel-producing endophytic fungus, we need to identify the existence of GGPP synthase in the first place. With RT-PCR method, we cloned the ORF fragment of PaGGPPS (GenBank accession number: KM881430)from the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum in Corylus avellana. The bioinformatics methods were employed to analyze and predict the composition of nucleic acid and amino acid sequence of this gene. The results showed that the length of ORF was 1 113 bp, the molecular weight of the protein was 40.98 kD, and the theoretical isoelectric point was 6.168, which suggested that PaGGPPS protein was acidic. The hydrophilicity analysis found that PaGGPPS was a hydrophilic protien. The α-helix was the dominant secondary structure constructional element of the protein which contained one tpolyprenyl_synt function domain. The identity alignment of GGPPS nucleic acid sequences of taxol producing endophytic fungi in hazelnut and other species showed that the identity were highest between Penicillium roqueforti, Aspergillus clavatus and Neosartorya fischeri, respectively 94%, 76% and 76%. The homologous alignment of Amino acids showed that GGPPS genes were divided into two groups. The GGPPS genes from animal, fungal and yeast were clustered in one group, the GGPPS genes from plant were in the other group. The homologous alignment of nucleic acid sequences showed that the homologous were the highest between GGPPSs in paclitaxel-producing endophytic fungus(Penicillium aurantiogriseum)in Corylus avellana and in Penicillium roqueforti, and GGPPSs in paclitaxel-producing endophytic fungus and plant have the longest distance in the phylogenetic tree. After the bioinformatics analysis, we constructed a pET30a prokaryotic expression vector of PaGGPPS. The protein of PaGGPPS was successfully expressed in the soluble state in Escherichia coli. All the experiments we had done can provide some information for the further research.