Cloning,Sequence Analysis and Prokaryotic Expression of Novel Antimicrobial Peptide Gene Mysdiap in Oriental Armyworm,Mythimna separata

doi:10.7668/hbnxb.20193801

Abstract

Abstract:

The full-length sequence of Diapausin,a novel antimicrobial peptide gene,was obtained from the third instar of oriental armyworm(Mythimna separata)by RT-PCR and RACE.And then,the amino acid sequence of Diapausin gene was analyzed by bioinformatics software.Finally,the prokaryotic expression system was used to induce the protein expression.This study would lay a theoretical foundation for further parsing of the function and structure of Diapausin gene in M.separata. Results showed that the novel antimicrobial peptide gene Diapausin was successfully cloned from M.separata and named Mysdiap (GenBank No.AZJ51075).The total length of this gene was 537 bp,with the 5' and 3' non-coding ends of 63,279 bp,respectively.The full length of the open reading frame was 195 bp,encoding 64 amino acid residues.The molecular weight and the isoelectric point of the protein were 7.13 ku and 5.59,respectively.The first 24 N-terminal amino acid residues of Mysdiap were signal peptide sequences.Multiple alignment of amino acid sequences showed that Mysdiap had a highly conserved region ECCRAHG.The recombinant expression plasmid pET-Mysdiap was successfully constructed and induced to express proteins by IPTG in Escherichia coli.The molecular weight of the expressed recombinant protein was about 20 ku,which could exist in both inclusion bodies and soluble proteins.In summary,the full-length sequence of the novel antimicrobial peptide gene Mysdiap was cloned from M.separata and successfully induced to express proteins in E.coli.

Key words: Mythimna separata, Antibacterial peptide, Diapausin gene, Gene cloning, Prokaryotic expression

ZHANG Yuanchen, HU Mengjie, XUE Shuang, GUO Wenjun, SHEN Hong, WANG Jingshun. Cloning,Sequence Analysis and Prokaryotic Expression of Novel Antimicrobial Peptide Gene Mysdiap in Oriental Armyworm,Mythimna separata[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 184-191. doi: 10.7668/hbnxb.20193801.

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Figures/Tables 9

Tab.1 Primers used in this study and their purposes

引物名称 Primer name	引物序列(5'—3') Nucleotide sequence of primer(5'—3')	用途 Purpose
Diap-F1	ATGGCCGCCATCAAGACCGTTT	部分cDNA序列扩增
Diap-R1	GTAGCCGGAGTACCCATGAGC
Diap-5-Outer	CAGCTGCTGTAGCCGGAGTACCCA	5'端RACE
Diap-5-Inner	GCCATTGCAGGCTTCGACGCGGT
Diap-3-Inner	TGATGTTTACCGTGGAGGCAAACC	3'端RACE
Diap-F2	ATGGCCGCCATCAAGACCGTTT	全长序列验证
Diap-R2	TCAGTAACAAATCATTTGGCCG
Diap-F3	GAATTCAACCGCGTCGAAGCCTGCAAT	基因原核表达
Diap-R3	AAGCTTTCAGTAACAAATCATTTGGCC
M13-F	CAGGGTTTTCCCAGTCACG	菌液PCR
M13-R	GAGCGGATAACAATTTCACAC

Fig.1 Electrophoretic result of PCR product from Mysdiap M.DL2000 DNA Marker;1,2.PCR product of partial CDS of Mysdiap;3,4.PCR product of Mysdiap via 3'RACE;5.PCR product of Mysdiap via 5'RACE.

Fig.2 Nucleotide and translated amino acid sequences of Mysdiap The red underline.The sequence of predicted signal peptide;The green underline.Polyadenylation signal domain;Boxed in solid line.The predicted phosphorylation site;*.Stop codon.

Fig.3 Conserved domains prediction of Mysdiap protein

Fig.4 Multiple alignment based on amino acid sequences of Diapausin proteins from M.separata and other insects Area marked in red line is the domain of the amino acid sequence.The black,pink and blue shadows delegate the homology of amino acid at 100%,≥75% and ≥50% levels,respectively.Spldiap.Diapausin protein from Spodoptera litura (GenBank No.ABU96713);SpediapA1.Diapausin protein from S.exigua (GenBank No.AKJ54510);SpediapA4.Diapausin protein from S.exigua (GenBank No.AKJ54513);Osfdiap.Diapausin protein from Ostrinia furnacalis (GenBank No.XP_028158203);SpediapB1.Diapausin protein from S.exigua (GenBank No.AKJ54517); Mysdiap.Diapausin protein from Mythimna separata (GenBank No.AZJ51075);Legldiap.Diapausin protein from Leguminivora glycinivorella (GenBank No.XP_048002774);Masediap.Diapausin protein from Manduca sexta (GenBank No.KAG6465316);Orcdiap.Diapausin protein from Orchesella cincta(GenBank No.ODM87430).

Fig.5 Phylogenetic tree analysis based on amino acid sequences of Diapausin proteins from M.separata and other insects

Fig.6 Identification of pET-Mysdiap plasmids by PCR(A)and double digestion(B) M.DL2000 DNA Marker;1,2.PCR product of pET-Mysdiap;3,4.Double digestion product of pET-Mysdiap.

Fig.7 SDS-PAGE analysis of the recombinant protein of Mysdiap M.Protein Marker;1.Induction of empty vector by 1 μL IPTG;2.Induction of empty vector by 3 μL IPTG;3.Induction of recombinant vector by 0 μL IPTG;4.Induction of recombinant vector by 1 μL IPTG;5.Induction of recombinant vector by 3 μL IPTG.The red arrows indicate the target protein.

Fig.8 Detection of Mysdiap protein in the supernatant and precipitate of lysis solution of expressing bacteria M.Protein Marker;1.The precipitate;2.The supernatant.

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