Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (7): 259-272.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0116
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SHEN Zhen-hui1,2,3,4(), CAO Yao1,2,3,4, YANG Lin-lei1,2,3,4, LUO Xiang-ying1,2,3,4, ZI Ling-shan1,2, LU Qing-qing1,2, LI Rong-chun1,2,3,4()
Received:
2024-01-31
Online:
2024-07-26
Published:
2024-07-30
Contact:
LI Rong-chun
E-mail:1947843434@qq.com;rongchunli@126.com
SHEN Zhen-hui, CAO Yao, YANG Lin-lei, LUO Xiang-ying, ZI Ling-shan, LU Qing-qing, LI Rong-chun. Cloning and Bioinformatics Analysis of the Ergothioneine Biosynthesis Genes in Naematelia aurantialba and Stereum hirsutum[J]. Biotechnology Bulletin, 2024, 40(7): 259-272.
Fig. 1 Biosynthetic pathways of ergothioneine in microorganism SAM, SAH (or AdoHcy), Cys and PLP refers to s-adenosylmethionine, S-adenosylhomocysteine, cysteine and pyridoxal phosphate respectively
Fig. 2 Morphological characteristics of different samples A: N. aurantialba blastospore (JEYB);B: S. hirsutum liquid culture(ShFJY+ShJST);C: N. aurantialba fruiting body(JEZST)
序号Serial No. | 引物名称 Primer name | 序列 Sequence(5'-3') | 退火温度 Annealing temperature/℃ |
---|---|---|---|
1 | ShEgt1-F | TGCGTCGGTCTCCTTTCTT | 53 |
2 | ShEgt1-R | CGGAGCAGAACAGATTTATCG | |
3 | ShEgt2-F | CACCACCACACATCACAGTC | 56 |
4 | ShEgt2-R | GACAAGTTCACAACGAGCAT | |
5 | NaEgt1-F | TGTCTGTCTCCTCCATCCA | 56 |
6 | NaEgt1-R | TAGTCCCAGGCTCATCACA | |
7 | NaEgt2-F | GTATCGCTGTTTACCACCTGT | 56 |
8 | NaEgt2-F | TCTACTCATCCTTCGTCGG |
Table 1 Primer information for ergothioneine biosynthetic genes of N. aurantialba and S. hirsutum
序号Serial No. | 引物名称 Primer name | 序列 Sequence(5'-3') | 退火温度 Annealing temperature/℃ |
---|---|---|---|
1 | ShEgt1-F | TGCGTCGGTCTCCTTTCTT | 53 |
2 | ShEgt1-R | CGGAGCAGAACAGATTTATCG | |
3 | ShEgt2-F | CACCACCACACATCACAGTC | 56 |
4 | ShEgt2-R | GACAAGTTCACAACGAGCAT | |
5 | NaEgt1-F | TGTCTGTCTCCTCCATCCA | 56 |
6 | NaEgt1-R | TAGTCCCAGGCTCATCACA | |
7 | NaEgt2-F | GTATCGCTGTTTACCACCTGT | 56 |
8 | NaEgt2-F | TCTACTCATCCTTCGTCGG |
序号Serial No. | 分析软件Analysis software | 用途Application | 网址Web site |
---|---|---|---|
1 | ORF | CDS序列预测CDS sequence prediction | |
2 | Gene Structure Display Server | 绘制基因结构图Drawing a genetic structure map | |
3 | NCBI-Blast | 基因同源性分析Gene homology analysis | |
4 | ExPASy- ProtParam | 分析蛋白的理化性质Analyzing the physicochemical properties of proteins | |
5 | SignalP4.1 | 信号肽预测Prediction of signal peptide | |
6 | PSORT II | 亚细胞定位Subcellular localization | |
7 | CCD | 保守结构域预测Conservative structural domain prediction | |
8 | PSIPRED | 二级结构预测Secondary structure of protein prediction | |
9 | SWISS-MODEL | 三级结构预测Tertiary structure prediction | |
10 | Phyre2 | 三级结构配体预测Tertiary structure ligand prediction | |
11 | MEGA7.0 | 系统进化树分析Phylogenetic analysis | |
Table 2 Bioinformatics software used in this study
序号Serial No. | 分析软件Analysis software | 用途Application | 网址Web site |
---|---|---|---|
1 | ORF | CDS序列预测CDS sequence prediction | |
2 | Gene Structure Display Server | 绘制基因结构图Drawing a genetic structure map | |
3 | NCBI-Blast | 基因同源性分析Gene homology analysis | |
4 | ExPASy- ProtParam | 分析蛋白的理化性质Analyzing the physicochemical properties of proteins | |
5 | SignalP4.1 | 信号肽预测Prediction of signal peptide | |
6 | PSORT II | 亚细胞定位Subcellular localization | |
7 | CCD | 保守结构域预测Conservative structural domain prediction | |
8 | PSIPRED | 二级结构预测Secondary structure of protein prediction | |
9 | SWISS-MODEL | 三级结构预测Tertiary structure prediction | |
10 | Phyre2 | 三级结构配体预测Tertiary structure ligand prediction | |
11 | MEGA7.0 | 系统进化树分析Phylogenetic analysis | |
Fig. 3 Verification of PCR amplification of Egt1 and Egt2 genes of N. aurantialba and S. hirsutum(A, B)and gene structure analysis(C) M: DL2000 marker; ShJST: S. hirsutum mycelium; JEYB: N. aurantialba blastospore ; JEZST: N. aurantialba fruiting body
基因 Gene | CDS长度CDS length/bp | 蛋白质长度 Protein length | 蛋白质分子量Molecular weight/kD | 等电点 Isoelectric point | 总平均亲水系数Grand average of hydropathicity (GRAVY) | 信号肽 Signal peptide | 细胞中分布 Location |
---|---|---|---|---|---|---|---|
NaEgt1 | 2952 | 984 | 110.32 | 5.30 | -0.479 | 无No | 细胞质Cytoplasmic |
ShEgt1 | 2565 | 885 | 99.03 | 5.19 | -0.528 | 无No | 细胞质Cytoplasmic |
NaEgt2 | 2178 | 726 | 81.61 | 5.87 | -0.368 | 无No | 细胞质Cytoplasmic |
ShEgt2 | 1383 | 461 | 51.46 | 5.68 | -0.238 | 无No | 细胞质Cytoplasmic |
Table 3 Analysis of physical and chemical properties of Egt1 and Egt2 in two species
基因 Gene | CDS长度CDS length/bp | 蛋白质长度 Protein length | 蛋白质分子量Molecular weight/kD | 等电点 Isoelectric point | 总平均亲水系数Grand average of hydropathicity (GRAVY) | 信号肽 Signal peptide | 细胞中分布 Location |
---|---|---|---|---|---|---|---|
NaEgt1 | 2952 | 984 | 110.32 | 5.30 | -0.479 | 无No | 细胞质Cytoplasmic |
ShEgt1 | 2565 | 885 | 99.03 | 5.19 | -0.528 | 无No | 细胞质Cytoplasmic |
NaEgt2 | 2178 | 726 | 81.61 | 5.87 | -0.368 | 无No | 细胞质Cytoplasmic |
ShEgt2 | 1383 | 461 | 51.46 | 5.68 | -0.238 | 无No | 细胞质Cytoplasmic |
Fig. 4 Tertiary structure and binding site analysis of five fungal Egt1 and Egt2 proteins A: Comparison of the tertiary structures of five fungal Egt1 and Egt2(Egt3); B: Egt1 binds to imidazole(red arrow)and acetate ions(blue arrow); C: binding of Egt1 to s-adenosylmethionine(SAM); D: the combination of EgtD and Fe3+; E: Egt1 binds to cysteine(red arrow); F: Egt2 binds to S-mercaptocysteine; G-H: Egt2 binds to two pyridoxal phosphate(PLP)sites. SpEgt1, NcEgt1, FvEgt1, SpEgt2, NcEgt2, FvEgt2 and FvEgt3 are key enzyme for ergothioneine biosynthesis of S. pombe, N. crassa and F. filiformis, respectively(Accession number: CAA22334.2, XP_956324.3, QBB19872.1, NP_595091.1, A7UX13, QBB19873.1 and QBB19874.1).
蛋白 Protein | 物种 Species | 模板 Template | 覆盖度 Coverage/% | 可信度 Confidence/% | 功能域一致性 Functional domain identity/% | 结合位点 Binding site |
---|---|---|---|---|---|---|
Egt1 | 耻垢分枝杆菌 M smegmatis | c4uy5A | 51/46/55/36/73 | 100/100/100/100/100 | 34/32/26/24/35 | 咪唑和醋酸根离子 Imidazole and acetate ion |
大利什曼原虫 Leishmania major | d1xtpa | 23/35/38/-/59 | 96/98/99/-/98 | 31/30/18/-/19 | s-腺苷甲硫氨酸 S-adenosyl-methionine(SAM) | |
耐热分枝杆菌 Mycobacterium thermoresistibile | c4x8bA | 44/49/55/57/- | 100/100/100/100/- | 27/26/23/24/- | Fe3+离子 Fe3+ ion | |
嗜热氢单胞菌 Hydrogenimonas thermophila | c8khqD | 44/48/55/57/- | 100/100/100/100/- | 26/26/24/24/- | 半胱氨酸 Cysteine(Cys) | |
Egt2 | 粗糙脉孢霉 N. crassa | c5utsC | 59/91/97/94/93/92 | 100/100/100/100/100/100 | 39/35/31/97/21/36 | - |
集胞藻属 Synechocystis sp. | d1elua | 56/88/95/90/90/89 | 99.9/100/100/100/100/100 | 20/19/16/16/18/16 | 磷酸吡哆醛和S-巯基半胱氨酸 Pyridoxal phosphate and S-mercaptocysteine |
Table 4 Predicted information on the tertiary structure of Egt1 and Egt2 proteins from five fungal species
蛋白 Protein | 物种 Species | 模板 Template | 覆盖度 Coverage/% | 可信度 Confidence/% | 功能域一致性 Functional domain identity/% | 结合位点 Binding site |
---|---|---|---|---|---|---|
Egt1 | 耻垢分枝杆菌 M smegmatis | c4uy5A | 51/46/55/36/73 | 100/100/100/100/100 | 34/32/26/24/35 | 咪唑和醋酸根离子 Imidazole and acetate ion |
大利什曼原虫 Leishmania major | d1xtpa | 23/35/38/-/59 | 96/98/99/-/98 | 31/30/18/-/19 | s-腺苷甲硫氨酸 S-adenosyl-methionine(SAM) | |
耐热分枝杆菌 Mycobacterium thermoresistibile | c4x8bA | 44/49/55/57/- | 100/100/100/100/- | 27/26/23/24/- | Fe3+离子 Fe3+ ion | |
嗜热氢单胞菌 Hydrogenimonas thermophila | c8khqD | 44/48/55/57/- | 100/100/100/100/- | 26/26/24/24/- | 半胱氨酸 Cysteine(Cys) | |
Egt2 | 粗糙脉孢霉 N. crassa | c5utsC | 59/91/97/94/93/92 | 100/100/100/100/100/100 | 39/35/31/97/21/36 | - |
集胞藻属 Synechocystis sp. | d1elua | 56/88/95/90/90/89 | 99.9/100/100/100/100/100 | 20/19/16/16/18/16 | 磷酸吡哆醛和S-巯基半胱氨酸 Pyridoxal phosphate and S-mercaptocysteine |
Fig. 5 Phylogenetic tree analysis of Egt1(A)and Egt2(B)in different species Purple colours indicate the results of Egt2 sequence comparison between S. hirsutum and N. aurantialba; red colours indicate the results of Egt2 sequence comparison between S. hirsutum and other fungi; blue colours indicate the results of Egt2 sequence comparison between N. aurantialba and other fungi
Fig. 7 Determination of hercynine and ergothioneine content in different samples A: Liquid sample; B: solid sample. YTPDA-CK: PDA liquid medium(control); ShFJY: S. hirsutum fermentation broth; ShJST: S. hirsutum mycelium; JSJ-JEYB: N. aurantialba blastospa; JEZST: N. aurantialba fruiting body. *, ** and *** indicate a significant difference at P<0.05, P<0.01 and P<0.0001, respectively
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