生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 60-70.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0141

• 研究报告 • 上一篇    下一篇

扩展青霉erg4的生物信息学、亚细胞定位及表达分析

韩占红1(), 宗元元1, 张学梅1, 王斌1, PRUSKY Dov2, 毕阳1()   

  1. 1.甘肃农业大学食品科学与工程学院,兰州 730070
    2.Department of Postharvest Science of Fresh Produce,Agricultural Research Organization,Volcani Center,Bet Dagan 50250,Israel
  • 收稿日期:2021-02-04 出版日期:2021-12-26 发布日期:2022-01-19
  • 作者简介:韩占红,硕士研究生,研究方向:采后生物学与技术;E-mail: hanzhanhong009@163.com
  • 基金资助:
    国家自然科学基金国际(地区)合作与交流项目(31861143046)

Bioinformatics,Subcellular Localization and Expression Analysis of erg4 in Penicillium expansum

HAN Zhan-hong1(), ZONG Yuan-yuan1, ZHANG Xue-mei1, WANG Bin1, PRUSKY Dov2, BI Yang1()   

  1. 1. College of Food Science and Engineering,Gansu Agricultural University,Lanzhou 730070
    2. Department of Postharvest Science of Fresh Produce,Agricultural Research Organization,Volcani Center,Bet Dagan Israel 50250,Israel
  • Received:2021-02-04 Published:2021-12-26 Online:2022-01-19

摘要:

麦角甾醇是真菌细胞质膜的特有组分,在真菌生长发育中具有重要作用。erg4是参与麦角甾醇生物合成最后一步反应的基因,但扩展青霉中该基因的功能未知。本文通过RT-PCR方法克隆了扩展青霉3个erg4erg4Aerg4Berg4C)基因的 CDS全长,对基因结构、编码蛋白的跨膜螺旋和亲疏水性进行了生物信息学分析,通过融合绿色荧光蛋白定位的方法进行了亚细胞定位,测定了3个基因在不同生长发育阶段、不同培养基状态以及黑暗和蓝光条件下的表达差异。扩展青霉erg4Aeerg4Berg4C的 CDS全长分别为1 476 bp、1 491 bp和 1 596 bp,分别编码491、496和 531个氨基酸;编码蛋白均属于跨膜蛋白,且表现出疏水性。绿色荧光蛋白与内质网红色荧光探针染色共定位结果显示,Erg4A、Erg4B和Erg4C均定位于内质网。erg4Aerg4Berg4C在孢子阶段、孢子萌发阶段及成熟菌丝阶段的表达水平存在显著差异,其中,erg4A在3个阶段的表达量无明显变化,而erg4Berg4C的表达量均显著上调,以erg4B的上调幅度最为明显。erg4A在 CY液体和固体培养条件下的表达量无显著变化,erg4Berg4C在CY液体培养条件下的表达量显著高于固体培养,以erg4B的上调幅度最为明显。erg4Aerg4B在蓝光条件下的表达量显著高于黑暗条件,以erg4B的上调幅度最为明显。erg4C对蓝光条件不敏感。扩展青霉Erg4A、Erg4B和 Erg4C均定位于内质网,erg4Aerg4Berg4C在不同生长发育阶段、固体和液体以及黑暗与蓝光培养条件下的表达存在较大差异,其中,以erg4B的响应最为活跃。

关键词: 扩展青霉erg4, 生物信息学分析, 亚细胞定位, 表达差异

Abstract:

Ergosterol,a fungus-specific component in cell plasma membranes,plays an essential role in fungal growth and development. The erg4 gene encodes the catalytic enzyme for the last step of ergosterol synthesis,whereas,the function of this gene in Penicillium expansum is unknown. In this study,the full-length CDSs of erg4A,erg4B and erg4C in P. expansum were cloned by RT-PCR,and their genes’ structures,transmembrane domain and hydrophobicity of the erg4 encoded proteins were analyzed via bioinformatics. The subcellular localizations of 3 proteins were also analyzed by fusion green fiuorescent protein method. In addition,the expression differences of 3 genes in different growth stages,different medium conditions,and dark and blue light conditions were measured. The full-length CDSs of erg4A,erg4B and erg4C were 1 476 bp,1 491 bp and 1 596 bp,respectively,and the number of encoded amino acids were 491,496 and 531,respectively. The encoded proteins were all transmembrane proteins and presented hydrophobicity. The co-localization results of green fluorescent protein and endoplasmic reticulum fluorescent probe showed that Erg4A,Erg4B and Erg4C were all located in endoplasmic reticulum. There were significant differences in the expressions of erg4A,erg4B and erg4C in spore,spore germination and mature mycelium stages. The expression of erg4A had no significant variation in the three stages,while the expressions of erg4B and erg4C were significantly up-regulated,and that of erg4B was the most obvious. There was no significant variation in the expression of erg4A under CY liquid and solid culture conditions. The expressions of erg4B and erg4C under CY liquid culture conditions were significantly higher than those under solid culture conditions,and the up-regulation of erg4B was the most obvious. The expressions of erg4A and erg4B under blue light were significantly higher than those under dark conditions,and the up-regulation of erg4B was the most obvious. erg4C was not sensitive to blue light condition. In conclusion,Erg4A,Erg4B and Erg4C were all located in the endoplasmic reticulum,and the expressions of erg4A,erg4B and erg4C vaied significantly in different growth stages,solid and liquid cultural conditions,and dark and blue light culture conditions,among which erg4B was the most active.

Key words: Penicillium expansum erg4, bioinformatics analysis, subcellular localization, expression differences