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

doi:10.7668/hbnxb.20195795

Abstract

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

CLC Number:

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.

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Figures/Tables 11

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

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

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

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

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

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.

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

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

Fig.8 Protein sequence phylogenetic tree

Fig.9 ypvA and yjcD subcellular localization

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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