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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[1] 刘爱英.中国蝉花资源研究应用[M].贵阳:贵州科学技术出版社, 2012. [2] 贺亮, 马素云, 程俊文, 等.药用真菌蝉拟青霉生物活性物质的研究进展[J].食品与生物技术学报, 2012, 31(1):8-16. http://www.cqvip.com/QK/92286X/200404/9615672.html [3] 朱碧纯, 柴一秋, 章思思.蝉花虫草活性成分的抗惊厥作用[J].菌物学报, 2016, 35(5):619-627. http://manu40.magtech.com.cn/Jwxb/CN/abstract/abstract3315.shtml [4] 武立琨. 虫生真菌蝉拟青霉发酵法生产透明质酸[D]. 贵阳: 贵州大学, 2007. http://cdmd.cnki.com.cn/Article/CDMD-10657-2007133058.htm [5] 张洪梅, 史晓飒, 刘腾飞, 等.不同产地蝉花中腺苷、虫草素和麦角甾醇的含量比较[J].环球中医药, 2017, 10(3):297-301. http://www.cqvip.com/QK/88879X/201703 [6] 邓旺秋, 杨小兵, 李泰辉, 等.广东省草菇主要栽培菌株RAPD多态性分析[J].食用菌学报, 2004, 11(3):1-6. http://www.docin.com/p-512078914.html [7] 应正河, 林衍铨, 马璐, 等.绣球菌栽培菌株RAPD和SRAP传多样性分析[J].西南农业学报, 2014, 27(2):777-780. http://industry.wanfangdata.com.cn/dl/Detail/Periodical?id=Periodical_xnnyxb201402059 [8] KERMANI F, SHAMS-GHAHFAROKHI M, GHOLAMI-SHABANI M, et al. Diversity, molecular phylogeny and fingerprint profiles of airborne Aspergillus species using random amplified polymorphic DNA[J]. World J Microbiol Biotechnol, 2016, 32:96. doi: 10.1007/s11274-016-2052-1 [9] YANG C l, WU X P, CHEN B, et al. Comparative analysis of genetic polymorphisms among Monascus strains by ISSR and RAPD markers[J]. J Sci Food Agric, 2017, 97:636-640. doi: 10.1002/jsfa.2017.97.issue-2 [10] WHITE T J, BRUNS T, LEE S. et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols:a guide to methods and applications[M]. Academic Press, Inc., San Diego, Calif, 1990:315-322. [11] KUMAR S, STECHER G, TAMURA K. MEGA7:Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[J]. Mol Biol Evol, 2016, 33:1870-1874. doi: 10.1093/molbev/msw054 [12] FENG K, WANG S, HU D J, et al. Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis[J]. J Pharm Biomed Anal, 2009, 50:522-526. doi: 10.1016/j.jpba.2009.04.029 [13] 张传博, 王艳丽, 易萌, 等.江苏省天王镇磨盘山金蝉花分离菌株鉴定及系统发育分析[J].广东农业科学, 2013, 15:152-154. http://mall.cnki.net/magazine/magadetail/GDNY201315.htm [14] 陈成, 许佳伟, 孙细涓, 等.茅山地区蝉花菌株的分离及其多糖生物活性的研究[J].江苏农业科学, 2015, 43(4):347-352. http://mall.cnki.net/magazine/magadetail/JSNY201504.htm [15] SHARMA S K, GAUTAM N, ATRI N S. Optimized extraction, composition, antioxidant and antimicrobial activities of exo and intracellular polysaccharides from submerged culture of Cordyceps cicadae[J]. BMC Comple-ment Altern Med, 2015, 15:446. doi: 10.1186/s12906-015-0967-y [16] WEI C Y, LI W Q, SHAO S S, et al. Structure and chain conformation of a neutral intracellular heteropolysaccharide from mycelium of Paecilomyces cicadae[J]. Carbohyd Polym, 2016, 136:728-737. doi: 10.1016/j.carbpol.2015.09.088 [17] 黄锴, 谭艾娟, 潘名志.蝉拟青霉产透明质酸的粗提方法及工艺条件研究[J].凯里学院学报, 2009, 27(3):33-36. http://www.doc88.com/p-909534284841.html [18] 彭凡, 李春如, 耿德贵, 等.传统药用菌蝉花退化菌株虫体活体复壮的研究[J].安徽农业大学学报, 2014, 41(2):294-298. http://www.cqvip.com/QK/95557A/201402/49057778.html -
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