生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 60-70.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0141
韩占红1(), 宗元元1, 张学梅1, 王斌1, PRUSKY Dov2, 毕阳1()
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: 基金资助:
HAN Zhan-hong1(), ZONG Yuan-yuan1, ZHANG Xue-mei1, WANG Bin1, PRUSKY Dov2, BI Yang1()
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erg4(erg4A、erg4B 和 erg4C)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erg4A、eerg4B 和 erg4C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。erg4A、erg4B 和erg4C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erg4B和 erg4C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,erg4B和erg4C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。erg4A和 erg4B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,erg4A、erg4B和 erg4C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。
韩占红, 宗元元, 张学梅, 王斌, PRUSKY Dov, 毕阳. 扩展青霉erg4的生物信息学、亚细胞定位及表达分析[J]. 生物技术通报, 2021, 37(12): 60-70.
HAN Zhan-hong, ZONG Yuan-yuan, ZHANG Xue-mei, WANG Bin, PRUSKY Dov, BI Yang. Bioinformatics,Subcellular Localization and Expression Analysis of erg4 in Penicillium expansum[J]. Biotechnology Bulletin, 2021, 37(12): 60-70.
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | F:ATGGAGGACCAAGTCAAATC |
R:AAAAAGAGAAATTGCAGTAG | |
erg4B | F:ATGCCCTCCAAAAAGGACTCTC |
R:GTAAATTCCAGGAATGATGC | |
erg4C | F:ATGGATCGTCCCGGCTTCATT |
R:TTAGAAGACATAAGGAATGA |
表1 erg4A、erg4B和erg4C基因CDS的克隆引物序列
Table 1 Primers sequence for the clone of CDS of gene erg4A,erg4B and erg4C
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | F:ATGGAGGACCAAGTCAAATC |
R:AAAAAGAGAAATTGCAGTAG | |
erg4B | F:ATGCCCTCCAAAAAGGACTCTC |
R:GTAAATTCCAGGAATGATGC | |
erg4C | F:ATGGATCGTCCCGGCTTCATT |
R:TTAGAAGACATAAGGAATGA |
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | CF:TCAACTCCATCACATCACAAGAGCTC ATGGAGG- ACC AAGTCAAATC |
CR:AGCTCCTCGCCCTTGCTCACTCTAGAAAAAAGAG- AA ATTGCAGTAG | |
erg4B | CF:TCAACTCCATCACATCACAA GAGCTC ATGCCCTC- CA AAAAGGACTC |
CR:AGCTCCTCGCCCTTGCTCAC TCTAGA GTAAATTC- CA GGAATGATGC | |
erg4C | NF:GCATGGACGAGCTGTACAAG GAGCTC ATGGATC- GTCCCGGCTTCAT |
NR:ATGGAGCTATTAAATCACTA TCTAGA TTAGAAG- ACA TACTATAGGA |
表2 用于构建GFP融合表达载体的引物序列
Table 2 Primer sequences for GFP fusion vector construction
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | CF:TCAACTCCATCACATCACAAGAGCTC ATGGAGG- ACC AAGTCAAATC |
CR:AGCTCCTCGCCCTTGCTCACTCTAGAAAAAAGAG- AA ATTGCAGTAG | |
erg4B | CF:TCAACTCCATCACATCACAA GAGCTC ATGCCCTC- CA AAAAGGACTC |
CR:AGCTCCTCGCCCTTGCTCAC TCTAGA GTAAATTC- CA GGAATGATGC | |
erg4C | NF:GCATGGACGAGCTGTACAAG GAGCTC ATGGATC- GTCCCGGCTTCAT |
NR:ATGGAGCTATTAAATCACTA TCTAGA TTAGAAG- ACA TACTATAGGA |
Gene | Primer sequence(5' - 3') |
---|---|
C-CX | CX1:GGAGA CGTAT TTAGGTGCTA |
CX2:TGAACTTCA GGGTCAGCTT | |
N-CX | NX1:CGACAACCAC TACCTGAGCA |
NX2:TGAAGGGCGT ACTAGGGTTG | |
erg4A-GFP | F:ATGGAGGACCAAGTCAAATC |
R:AAAAAGAGAA ATTGCAGTAG | |
erg4B-GFP | F:ATGCCCTCCAAAAAGGACTC |
R:GTAAATTCCA GGAATGATGC | |
erg4C-GFP | F:ATGGATCGTC CCGGCTTCAT |
R:TTAGAAGACA TAAGGAATGA |
表3 转化子验证所用引物序列
Table 3 Primer sequences for transformant validation
Gene | Primer sequence(5' - 3') |
---|---|
C-CX | CX1:GGAGA CGTAT TTAGGTGCTA |
CX2:TGAACTTCA GGGTCAGCTT | |
N-CX | NX1:CGACAACCAC TACCTGAGCA |
NX2:TGAAGGGCGT ACTAGGGTTG | |
erg4A-GFP | F:ATGGAGGACCAAGTCAAATC |
R:AAAAAGAGAA ATTGCAGTAG | |
erg4B-GFP | F:ATGCCCTCCAAAAAGGACTC |
R:GTAAATTCCA GGAATGATGC | |
erg4C-GFP | F:ATGGATCGTC CCGGCTTCAT |
R:TTAGAAGACA TAAGGAATGA |
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | F:TCCACGAATGTTCGGAATCC |
R:AGCTCAAACCCAAGAGCATGA | |
erg4B | F:CGGCGCTTTATGATACCAATG |
R:GGGAAGGAAGACCTGCAAAAA | |
erg4C | F:GCATTCCCATCAATCAAAGCA |
R:CCGGGAAGAAGCACGTAACA | |
β-tubulin | F:CTCCAGCTCGAGCGTATGAAC |
R:GGCTCCAAATCGACGAGAAC |
表4 实时荧光定量PCR引物序列
Table 4 Primers for real-time fluorescent quantitative PCR
Gene | Primer sequence(5' - 3') |
---|---|
erg4A | F:TCCACGAATGTTCGGAATCC |
R:AGCTCAAACCCAAGAGCATGA | |
erg4B | F:CGGCGCTTTATGATACCAATG |
R:GGGAAGGAAGACCTGCAAAAA | |
erg4C | F:GCATTCCCATCAATCAAAGCA |
R:CCGGGAAGAAGCACGTAACA | |
β-tubulin | F:CTCCAGCTCGAGCGTATGAAC |
R:GGCTCCAAATCGACGAGAAC |
图3 Erg4A、Erg4B和Erg4C的跨膜螺旋 图中的红色矩形为跨膜螺旋结构,蓝色线段是位于膜内的结构,而红色线段是位于膜外的结构
Fig. 3 Transmembrane helices of Erg4A,Erg4B and Erg4C Red rectangle indicates the transmembrane,blue line segment indicates the structure inside the membrane,and the red line segment indicates the structure outside the membrane
图4 Erg4A、Erg4B和Erg4C蛋白亲疏水性 图中的正值区域代表疏水氨基酸,负值区域代表亲水氨基酸
Fig. 4 Hydrophilicity/Hydrophobicity of Erg4A,Erg4B and Erg4C protein Positive areas represent the hydrophobic,the negative areas represent the hydrophilic
图6 野生型与荧光标记菌株erg4A-GFP、erg4B-GFP及erg4C-GFP的菌落形态比较
Fig. 6 Comparison of colony morphologies of erg4A-GFP,erg4B-GFP and erg4C-GFP in wild type and fluor-esence labelled strains
图10 扩展青霉中erg4A、erg4B和erg4C在孢子阶段、孢子萌发阶段以及成熟菌丝阶段的相对表达量 竖线表示标准误(±SE). 不同字母代表显著性差异(P<0.05),下同
Fig. 10 Relative expressions of erg4A,erg4B and erg4C in the spore,germinated spore and mycelium stages in P. expansum Vertical line indicates standard error(±SE). Different letters refer to significant differences(P<0.05). The same below
图11 扩展青霉中erg4A、erg4B和erg4C在CY固体和液体培养条件下的相对表达量
Fig. 11 Relative expressions of erg4A,erg4B and erg4C in P. expansum in Czapek yeast extract solid medium and Czapek yeast extract liquid medium
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