Molecular Identification and Productivity on Hyaluronic Acid in Submerged Culture of Different Cordyceps cicadae Strains
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摘要: 为准确鉴定收集的4株蝉花（Cordyceps cicadae）并比较其产透明质酸能力，利用内转录间隔区（ITS）序列及随机扩增多态性DNA（RAPD）技术对蝉花菌株CC1502、CC1504、CC1601和CC1602进行分子水平鉴定分析。结果表明：ITS序列不能区分4个蝉花菌株；随机引物AP-A20、AP-D18和AP-H18能够对4个菌株进行差异性扩增，获得RAPD多态性条带。聚类分析结果表明，菌株CC1504与其他3个菌株的遗传相似性最低，而菌株CC1601和CC1602的遗传相似性最高。液体发酵结果显示，菌株CC1502和CC1504能够产生透明质酸，葡萄糖和蛋白胨分别为最佳碳源和氮源。蝉花菌株CC1502产透明质酸的能力最强，产量可达2.45 mg·mL-1，具有良好的开发潜力。Abstract: This study aimed to identify 4 Cordyceps cicadae strains and compare their productivities on hyaluronic acid (HA) in a liquid culture. The molecular identification for CC1502, CC1504, CC1601 and CC1602 was conducted based on their internal transcribed spacer (ITS) sequences and using the randomly amplified polymorphic DNA (RAPD) analysis. The results indicated that there was no difference on ITS sequences among these fungal strains, and that the RAPD patterns were amplified by the primer, AP-A20, AP-D18 or AP-H18. As shown by the dendrogram analysis with UPGMA method, CC1504 had a low genetic similarity with any of the other strains, and CC1601 related closely to CC1602. The liquid fermentation of the strains showed different levels of HA productivity between CC1502 and CC1504, and glucose and peptone were the choice carbon and nitrogen sources. The maximum HA production was up to 2.45 mg·mL-1 generated by CC1502, which, therefore, was considered a candidate for industrial application in the future.
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图 1 不同蝉花菌株在PDA平板上的菌落形态
注：A为CC1502，B为CC1504，C为CC1601，D为CC1602。
Figure 1. Colony morphologies of C. cicadae strains on PDA plates
图 2 蝉花菌株的ITS序列系统进化树
Figure 2. Phylogenetic trees of C. cicadae strains based on ITS sequences (neighbor-joining method)
图 3 蝉花菌株的RAPD分析
注：A~E分别为引物AP-A20、AP-D18、AP-G05、AP-H18和AP-I07的扩增图谱；1~4分别为蝉花菌株CC1502、CC1504、CC1601和CC1602；M为1 kb DNA分子量; NC为无模板对照。
Figure 3. RAPD analysis on 4 C. cicadae strains
图 4 4株蝉花基于RAPD分析的UPGMA聚类
Figure 4. UPGMA dendrograms of 4 C. cicadae strains based on RAPD polymorphisms
图 5 不同碳源对蝉花发酵产透明质酸的影响
Figure 5. Effect of carbon sources on hyaluronic acid production by C. cicadae strains
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