内生枯草芽孢杆菌ZA1解旋酶<i>ypvA</i>和<i>yjcD</i>基因的克隆及生信分析

doi:10.7668/hbnxb.20195795

摘要/Abstract

摘要：

枯草芽孢杆菌ZA1具有良好的抑菌和促生作用。解旋酶已被证实为核酸代谢关键蛋白复合体,且解旋酶超家族成员通过特异性切割DNA/RNA分子中的磷酸二酯键,在遗传信息传递过程中执行关键催化功能。为明确解旋酶基因ypvA和yjcD在枯草芽孢杆菌ZA1抑菌过程中的潜在作用,以枯草芽孢杆菌ZA1为材料,克隆ypvA和yjcD基因,利用生物信息学分析它们的序列特征,通过亚细胞定位检测2个基因的位置,对2个基因及其编码蛋白的结构与功能特性进行初步解析。结果表明,克隆获得的ypvA和yjcD基因编码区全长分别为1 791,1 767 bp,分别编码596,588个氨基酸,其编码蛋白的预测相对分子质量分别为68.804 01,68.275 08 ku,理论等电点分别为4.81,8.25,且亲水性较弱。ypvA和yjcD的蛋白空间结构主要由α-螺旋、β-折叠和大量无规则延伸链组成的紧密结构。亚细胞定位于细胞质和细胞核中,不存在跨膜区及信号肽,为非分泌蛋白。分析保守结构域得到ypvA属于Rad3相关DNA解旋酶DinG家族,基因yjcD属于UvrD超家族I DNA或RNA解旋酶,通过进一步的蛋白质系统进化树分析明确将这2个蛋白鉴定为ATP依赖性解旋酶。因此,通过克隆枯草芽孢杆菌ZA1中的ypvA和yjcD基因并分析序列为非分泌性ATP依赖性解旋酶。

关键词: 枯草芽孢杆菌, 解旋酶基因, 克隆, 生物信息分析, 亚细胞定位

Abstract:

Bacillus subtilis ZA1 exhibits notable antibacterial and growth-promoting activities. Helicases have been established as crucial protein complexes in nucleic acid metabolism, with members of the helicase superfamily performing key catalytic functions during genetic information transfer by specifically cleaving phosphodiester bonds in DNA/RNA molecules. To elucidate the potential roles of the helicase genes ypvA and yjcD in the antibacterial process of B.subtilis ZA1, we used B.subtilis ZA1 as the experimental material, cloned the ypvA and yjcD genes, analyzed their sequence characteristics through bioinformatics, and determined their subcellular localization. A preliminary investigation was conducted on the structural and functional properties of these two genes and their encoded proteins. The results showed that the cloned coding sequences (CDS) of ypvA and yjcD were 1 791, 1 767 bp in length, encoding 596, 588 amino acids, respectively. The predicted relative molecular masses of the encoded proteins were 68.804 01, 68.275 08 ku, with theoretical isoelectric points (pI) of 4.81, 8.25, respectively, and both exhibited weak hydrophilicity. The spatial structures of the ypvA and yjcD proteins consisted of a compact architecture primarily composed of α-helices, β-sheets, and extensive random coils. Subcellular localization analysis indicated that both proteins were localized in the cytoplasm and nucleus, lacking transmembrane domains and signal peptides, thus classifying them as non-secretory proteins. Analysis of conserved domains revealed that ypvA belonged to the DinG family of Rad3-related DNA helicases, while yjcD belongs to the UvrD superfamily I DNA/RNA helicases. Further phylogenetic analysis confirmed that both proteins are ATP-dependent helicases. In summary, cloning and sequence analysis of the ypvA and yjcD genes from B.subtilis ZA1 identified them as non-secretory ATP-dependent helicases.

Key words: Bacillus subtilis, Helicase gene, Cloning, Bioinformation analysis, Subcellular localization

中图分类号:

任姝锦, 蔡锋锋, 马婷, 王婷, 施玉安, 王一丹, 杨成德. 内生枯草芽孢杆菌ZA1解旋酶ypvA和yjcD基因的克隆及生信分析[J]. 华北农学报, 2025, 40(S1): 285-291. doi: 10.7668/hbnxb.20195795.

REN Shujin, CAI Fengfeng, MA Ting, WANG Ting, SHI Yu'an, WANG Yidan, YANG Chengde. Cloning and Bioinformatics Analysis of Endophytic Bacillus subtilis ZA1 Helicase ypvA and yjcD Genes[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(S1): 285-291. doi: 10.7668/hbnxb.20195795.

http://www.hbnxb.net/CN/Y2025/V40/IS1/285

图/表 11

表1 引物序列

Tab.1 Primer sequence

引物名称 Primer name	序列(5'-3') Sequence (5'-3')	作用 Function
ypvA	CGAGCTCATGGCTTTTCAGCTGGA	克隆
	CGGATCCCATCATGTGCGGTTCAA
yjcD	CGAGCTCATGATCGAACACTTAGGC	克隆
	CGGATCCATGATGCAAGGGCTGTCTT
ypvA-Y	ATTTGGTCATTGCCGTTCCC	定量引物
	ATCATGTGCGGTTCAACAGG
yjcD-Y	TTCTTGCCCACGTAGACCAT	定量引物
	CTCGTCTCCTTTTCTTGCCG
16S	CGGTCTCAGTTCGGATTGTAGG	内参引物
	GCTGGCTCCTTGCGGTTAC

图1 ypvA和yjcD基因PCR扩增 M.Maker;1-2.ypvA克隆;3-4.yjcD克隆。

Fig.1 PCR of ypvA and yjcD gene M.Maker;1-2.ypvA clone; 3-4.yjcD clone.

图2 ypvA和yjcD基因开放阅读框 A.ypvA开放阅读框;B.yjcD开放阅读框。

Fig.2 Open reading frame of ypvA and yjcD gene A. ypvA open reading frame; B.yjcD open reading frame.

图3 ypvA(A)和yjcD(B)信号肽预测

Fig.3 Signal peptide prediction of ypvA(A)and yjcD(B)

图4 ypvA(A)和yjcD(B)跨膜结构预测

Fig.4 Transmembrane structure prediction of ypvA(A)and yjcD(B)

图5 ypvA(A)和yjcD(B)蛋白二级结构预测蓝色.α-螺旋;橘色.无规则卷曲;紫色.延伸链;绿色.β-折叠。

Fig.5 Prediction of secondary structure of ypvA(A)and yjcD(B)proteins Blue.α-helix;Orange.Random coil;Purple.Extended stand;Green.Beta turn.

图6 ypvA(A)和yjcD(B)蛋白三级结构预测

Fig.6 Tertiary structure prediction of ypvA(A)and yjcD(B)protein

图7 ypvA(A)和yjcD(B)保守结构域分析

Fig.7 A putative conserved domain of ypvA(A)and yjcD(B)amino acid sequence

图8 蛋白序列系统进化树

Fig.8 Protein sequence phylogenetic tree

图9 ypvA和yjcD亚细胞定位

Fig.9 ypvA and yjcD subcellular localization

图10 ypvA和yjcD在烟草中7 d的表达量不同字母表示0.05水平差异显著性(P<0.05)。

Fig.10 Expression of ypvA and yjcD in tobacco on 7 days Different letters represent a significant difference at the 0.05 level(P<0.05).

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内生枯草芽孢杆菌ZA1解旋酶ypvA和yjcD基因的克隆及生信分析

Cloning and Bioinformatics Analysis of Endophytic Bacillus subtilis ZA1 Helicase ypvA and yjcD Genes

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内生枯草芽孢杆菌ZA1解旋酶ypvA和yjcD基因的克隆及生信分析

Cloning and Bioinformatics Analysis of Endophytic Bacillus subtilis ZA1 Helicase ypvA and yjcD Genes

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