简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

中国真菌学杂志　 2011, Vol. 6 　Issue (5): 271-275.

简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

王晓慧¹, 刘伟², 李若瑜²

1. 厦门大学附属中山医院皮肤科, 厦门361004;
2. 北京大学真菌和真菌病研究中心北京大学第一医院皮肤科真菌室, 北京100034

收稿日期:2011-05-24 出版日期:2011-10-28 发布日期:2011-10-28
通讯作者: 李若瑜,E-mail:ryli0660@bjmu.edu.cn E-mail:ryli0660@bjmu.edu.cn
作者简介:王晓慧,女(汉族),博士,副主任医师.E-mail:wxhxmzsh@163.com

Cloning a novel catalase gene of Sporothrix schenckii with degenerate PCR and RACE

WANG Xiao-hui¹, LIU Wei², LI Ruo-yu ²

1. Department of Dermatology, Zhongshan Affiliated Hospital of Xiamen University, Xiamen 361004;
2. Department of Dermatology, Peking University First Hospital and Research Center for Medical Mycology, Peking University, Beijing 100034

Received:2011-05-24 Online:2011-10-28 Published:2011-10-28

摘要/Abstract

摘要： 目的克隆孢子丝菌未知过氧化氢酶基因,命名为Sscat基因。方法根据生物信息库中7种已知真菌过氧化氢酶氨基酸序列的高度保守区域设计简并引物,PCR扩增获得部分Sscat基因cDNA片段,随后应用RACE技术分别扩增其3’端和5’端未知序列。结果 Sscat基因cDNA序列全长1746 bp,其中包括5’端121 bp的非编码区、1500 bp的编码区和109 bp的3’端非编码区。该基因编码499个氨基酸,分子量为56.07 kDa,其氨基酸序列与其他真菌过氧化氢酶氨基酸高度同源,其中与米曲霉、黑曲霉同源性分别为66.3%和56.6%,Sscat基因为申克孢子丝菌过氧化氢酶cDNA。结论简并PCR结合RACE技术成功克隆了孢子丝菌未知过氧化氢酶基因。

关键词: 孢子丝菌, 过氧化氢酶, 简并PCR, 快速cDNA末端扩增

Abstract: Objective To isolate a novel catalase homologous gene from yeast-form Sporothrix schenckii and to make a designation.Methods Oligonucleotide primers were designed according to the conserved areas of the other 7 fungal catalase genes.Partial Sscat cDNA was amplified by PCR,and special primers were designed by RACE method to amplify the 3′cDNA and 5′cDNA.Results The full-length Sscat cDNA sequence was 1746 bp with an open reading frame of 1500 bp encoding 499 amino acids.The predicted molecular mass of Sscat was 56.07 kDa.The deduced amino acid sequence of Sscat showed 66.3% and 56.6% identity with those of Aspergillus oryzae and A.clavatus.An intron was identified within the 933-1063 bp Sscat genomic DNA sequence of S.schenckii.Conclusions Degenerate PCR combined with RACE is effective in searching and isolating novel genes of S.schenckii.

Key words: Sporothrix schenckii, catalase, degenerate PCR, RACE

中图分类号:

R379.9

王晓慧, 刘伟, 李若瑜. 简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因[J]. 中国真菌学杂志, 2011, 6(5): 271-275.

WANG Xiao-hui, LIU Wei, LI Ruo-yu. Cloning a novel catalase gene of Sporothrix schenckii with degenerate PCR and RACE[J]. Chinese Journal of Mycology, 2011, 6(5): 271-275.

参考文献

[1] 李珊山,刘鹤松,郑华,等.皮肤型孢子丝菌病585例临床分析[J].中华皮肤科杂志,2011,44(3):161-164.
[2] 吴昌昊,吴旭,张立溪,等.肺部孢子丝菌病1例[J].南方医科大学学报,2009,10:2147.
[3] Callens SF,Kitetele F,Lukun P,et al.Pulmonary Sporothrix schenckii infection in a HIV positive child[J].J Trop Pediatr,2006,52:144-146.
[4] Vilela R,Souza GF,Fernandes Cota G,et al.Cutaneous and meningeal sporotrichosis in a HIV patient[J].Rev lberoam Micol,2007,24:161-163.
[5] 朱衡,瞿峰.利用氯化苄提取适于分子生物学分析的真菌DNA[J].真菌学报,1994,13:34-40.
[6] Frohman MA,Dush MK,Martin GR,et al.Rapid production of full-length cDNA,from rare transcripts:amplification using a single gene-specific oligonucleotide primer[J].Proc Nat Acad Sci USA,1998,85:8998-9002.
[7] Feng P,Xie Z,Sun J,et al.Molecular cloning,characterization and expression of PmRsrl,a Ras-related gene from yeast form of Penicillium marneffei[J].Mol Biol Rep,2010,37 (7):3533-3540.
[8] Cao C,Liu W,Li R,et al.Penicillium marneffeiSKN7,a novel gene,could complement the hypersensitivity of S.cerevisiae skn7 disruptant strain to oxidative stress[J].Mycopathologia,2009,168(1):23-30.
[9] Carmo JP,Dias-Melicio LA,Calvi SA,et al.TNF-alpha activates human monocytes for paracoccidioides brasiliensis killing by an H202-dependent mechanism[J].Med Mycol,2006,44 (4):363-368.
[10] Howard,DH.Comparative sensitivity of histoplasma capsulatum conidiospores and blastospores to oxidative antifungal systems[J].Infect Immun,1981,32(1):381-387.
[11] Brummer E,Sugar AM,Stevens DA.Immunological activation of polymorphonuclear neutrophils for fungal killingl studies with murine cells and Blastomyces dermatitidis in vitro[J].J Leukoc Biol,1984,36(4):505-520.
[12] 张军民,章慧,孙九峰,等.过氧化氢酶家族部分成员在马尔尼非青霉的相关研究[J].中国人兽共患病学报,2008,24(9):824-826.
[13] Wang XH,Li RY,Cao CW,et al.Differential mRNA expression of Sporothrix schenckii catalase gene in two growth phases and effect factors[J].Chin Med J (Engl),2008,121 (20):2100-2102.

[1]	吕莎, 齐琳, 刘士瑞, 武寒飞, 张明瑞, 李福秋. 沿肋间神经走向分布的孢子丝菌病1例[J]. 中国真菌学杂志, 2020, 15(4): 238-239.
[2]	曲芯瑶, 李珊山, 曲悦, Abdulhakim Salad Nageye, 宋洋, 崔岩. 10种中药单体对球形孢子丝菌的体外抗真菌作用研究[J]. 中国真菌学杂志, 2020, 15(3): 145-149.
[3]	王晓东, 迪丽努尔·塔西买买提, 迪丽达尔·地里夏提, 哈地利亚·哈斯木, 帕丽达·阿布利孜. 球形孢子丝菌刺激小鼠树突状细胞表达前炎症细胞因子的研究[J]. 中国真菌学杂志, 2019, 14(6): 321-325.
[4]	张明瑞, 周盈, 李福秋, 姚春丽, 杨鑫, 龚杰, 赵飞. 实时荧光PCR检测巴西孢子丝菌的实验方法的建立[J]. 中国真菌学杂志, 2019, 14(5): 297-302.
[5]	赵林, 王林会, 郑方亮, 侯彬彬. 申克孢子丝菌STE20基因RNA干扰菌株的构建方法[J]. 中国真菌学杂志, 2019, 14(3): 129-133.
[6]	张一男, 刘晓娜, 杨晶, 刘玥姣, 石铭, 薛文成. 子宫颈感染孢子丝菌1例[J]. 中国真菌学杂志, 2019, 14(1): 47-49.
[7]	张振颖, 胡媛媛, 陈凯丽, 侯彬彬. 初探Ste-20激酶在调控孢子丝菌双相生长过程中的作用[J]. 中国真菌学杂志, 2018, 13(5): 268-271,261.
[8]	崔岩, 李珊山, 侯媛媛, 宋洋, 陈瑞丽, 李洪霞. 球形孢子丝菌黑素抵御抗真菌药物杀伤作用的研究[J]. 中国真菌学杂志, 2018, 13(4): 193-196,212.
[9]	刘雯, 邵丽芳, 赵庆利, 赵广. 皮肤播散型孢子丝菌病1例并文献复习[J]. 中国真菌学杂志, 2018, 13(1): 30-33.
[10]	黄梦雅, 周汛. 孢子丝菌相关复合种需加强生物安全检控[J]. 中国真菌学杂志, 2017, 12(6): 371-374.
[11]	姜媛, 孔庆涛, 张征, 曾秋琼, 桑红. 皮肤淋巴管型孢子丝菌病1例[J]. 中国真菌学杂志, 2016, 11(6): 376-377.
[12]	赵莉佩, 崔岩, 李珊山. 申克孢子丝菌基因分型的研究现状与展望[J]. 中国真菌学杂志, 2016, 11(5): 316-320.
[13]	贺羽, 黄宜进, 赵秘密, 周汛. 中国临床球形孢子丝菌菌丝相药物敏感性分析[J]. 中国真菌学杂志, 2016, 11(4): 209-212.
[14]	史希武, 许静. 面部皮肤型孢子丝菌病1例误诊分析[J]. 中国真菌学杂志, 2016, 11(4): 223-225.
[15]	贺羽, 黄梦雅, 胡青碧, 周汛. 中国南北方地区临床孢子丝菌菌种鉴定[J]. 中国真菌学杂志, 2015, 10(5): 279-282.