Cloning and Prokaryotic Expression of a clip-type Serine Protease Gene from Mythimna separate

doi:10.7668/hbnxb.20192510

Abstract

Abstract:

To clone the full gene sequence of Mythimna separate serine protease,and express it in vitro,Mythimna separata was used as the research object,the partial gene of serine protease was amplified by RT-PCR,and further the whole gene was obtained by RACE technology.The obtained gene sequence and deduced protein sequence of serine protease were analyzed using Blast software and other softwares.The gene of serine protease was expressed using the Escherichia coli expression system.The results showed that the full serine protease gene from Mythimna separate was successfully cloned and named as MsPG,with the sequence length of 2 010 bp,and the open reading frame of 1 593 bp,encoding 530 amino acids.Its protein had the molecular weight of 67.6 ku,and the isoelectrical point of 7.63,and owned a clip-type domain containing 6 cysteine,which was speculated as a clip-type serine protease.The amino acid sequence of MsPG had identities of 70.00%—91.92% to those of other Lepidoptera insects,and showed the highest identity(91.92%)to that of Heliothis virescens(GenBank No.PCG78401.1).The recombinant plasmid pET30a(+)-MsPG was successfully constructed and expressed by 0.1 mmol/L IPTG induction in E.coli prokaryotic expression system,with the highest expression at 25 ℃.In conclusion,MsPG has conserved functional sites in the serine protease family and can be prokaryotically expressed in vitro.

Key words: Mythimna separate, Serine protease, Gene cloning, Sequence analysis, Prokaryotic expression

LIAN Kaiqi, JI Shuang, ZHOU Lingling, SHI Zhiqi, WANG Fei, ZHANG Yuanchen. Cloning and Prokaryotic Expression of a clip-type Serine Protease Gene from Mythimna separate[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 195-201. doi: 10.7668/hbnxb.20192510.

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http://www.hbnxb.net/EN/Y2022/V37/I1/195

Figures/Tables 7

Tab.1 Primer sequences used in the study

引物名称 Primer name	引物序列(5'—3') Primer sequence	引物用途 Primer function
Ser-PRF	AGGACTTTCAGGCGGTGTTCTAT	克隆部分基因
Ser-PRR	AGCGACCATTCTCGTTCACCATTA
5GSP	ATTGCCGTCCAGCGTTGAACAGAG	5'端扩增
5NGSP	TACTCGGTGGCTTAGTTACGCCCG
3GSP	TACCCTACGAAGCCGAGTGTGCCTG	3'端扩增
3NGSP	AGAGAGTCGTGAGACACCGAGGCT
pr1F	GAATTCAGTGACTCCGACGTCAAG	引入酶切位点
pr1R	AAGCTTTTACTGGGCGTTTTTCTGGATCC

Fig.1 PCR products of MsPG gene
M.DL2000 DNA Marker;1.PCR product of partial MsPG gene;2.PCR product of MsPG by 5' RACE;4.PCR product of MsPG by 3' RACE;3,5.The negative control of 5' RACE and 3' RACE.

Fig.2 Nucleotide and deduced amino acid sequences of MsPG gene
The signal peptide sequence is indicated by light blue shading;the active center(GDSGGP)is indicated by red shading;the underlined part is the clip domain;six cysteines are boxed.

Fig.3 Multiple alignment of amino acid sequences for serine protease between Mythimna separate and other insects
The underlined amino acid sequence GDSGGP is the active center.The dark background indicates that the consistency is 100%,the red background indicates that the consistency is greater than or equal to 75%,and the light blue background indicates that the consistency is greater than or equal to 50%.HvB5.Heliothis virescens(GenBank No.PCG78401.1);HaPE.Helicoverpa armigera(GenBank No.XP_021187378.1);S1TS.Spodoptera litura(GenBank No.XP_022821357.1);AtX1.Amyelois transitella(GenBank No.XP_013191307.1);BaPS.Bicyclus anynana(GenBank No.XP_023945684.1);PxPZ.Papilio xuthus(GenBank No.XP_013176912.1); PpPE.Papilio polytes(GenBank No.XP_013135138.1);DpPE.Danaus plexippus(GenBank No.XP_032527749.1);GmPE.Galleria mellonella(GenBank No.XP_026758758.1);AtX2.Amyelois transitella(GenBank No.XP_013191309.1);ApUN.Arctia plantaginis(GenBank No.CAB3239924.1);MsPE.Manduca sexta(GenBank No.XP_030031627.1).

Fig.4 Phylogenetic tree of serine protease among Mythimna separate and other species based on amino acid sequences

Fig.5 PCR product of MsPG ORF gene without signal peptide sequence and double digestion identification of the recombinant plasmid
M.DL2000 DNA Marker;1,2.PCR product of MsPG ORF gene without signal peptide sequence;3.Double digestion product of the recombinant plasmid pET30a(+)-MsPG.

Fig.6 SDS-PAGE analysis of fusion protein MsPG expressed by induction
M.Protein Marker;1—5.Induced expression of pET30a(+)-MsPG at 40,35,30,25,20 ℃,respectively;6.Induced expression of pET30a(+).

References 20

[1]	唐琦,周倩,邱立鹏,李东,李国辉.丝氨酸蛋白酶在昆虫先天免疫中的功能和作用机制研究进展[J].蚕业科学,2017,43(3):502-508.doi:10.13441/j.cnki.cykx.2017.03.022.
	Tang Q,Zhou Q,Qiu L P,Li D,Li G H.Advances in functions and action mechanism of serine protease in insect innate immunity[J].Science of Sericulture, 2017,43(3):502-508.
[2]	季星来,孙之荣.基于结构的丝氨酸蛋白酶超家族进化分析[J].电子学报,2001,29(S1):1756-1758.doi:10.3321/j.issn:0372-2112.2001.21.007.
	Ji X L,Sun Z R.Structure based evolution analysis of the serine protease superfamily[J].Acta Electronica Sinica,2001,29(S1):1756-1758.
[3]	Heinen R,Biere A,Bezemer T M.Plant traits shape soil legacy effects on individual plant insect interactions[J].Oikos,2020,129(2):261-273.doi:10.1111/oik.06812.
[4]	Dhania N K,Chauhan V K,Chaitanya R K,Dutta-Gupta A.Midgut de novo transcriptome analysis and gene expression profiling of Achaea janata larvae exposed with Bacillus thuringiensis(Bt)-based biopesticide formulation[J].Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,2019,30:81-90.doi:10.1016/j.cbd.2019.02.005.
[5]	Little N S,Elkins B H,Mullen R M,Perera O P,Parys K A,Allen K C,Boykin D L.Differences between two populations of bollworm,Helicoverpa Zea(Lepidoptera:Noctuidae),with variable measurements of laboratory susceptibilities to Bt toxins exposed to non-Bt and Bt cottons in large field cages[J].PLoS One,2019,14(3):e0212567.doi:10.1371/journal.pone.0212567.
[6]	Bhongade B A,Amnerkar N D,Gadad A K.3D-QSAR studies on 4,5-dihydro-1H-pyrazolo[4,3-h]quinazolines as Plk-1,CDK2/A and Aur-A serine/threonine kinase inhibitors[J].Letters in Drug Design & Discovery,2020,17(4):388-395.doi:10.2174/1570180816666190611161332.
[7]	Lange J,Demir F,Huesgen P F,Baumann U,von Elert E,Pichlo C.Heterologous expression and characterization of a novel serine protease from Daphnia magna:A possible role in susceptibility to toxic cyanobacteria[J].Aquatic Toxicology,2018,205:140-147.doi:10.1016/j.aquatox.2018.09.013.
[8]	周晓群,高艳玲,赵奎军,樊东.苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达[J].昆虫学报,2014,57(9):1008-1017.doi:10.16380/j.kcxb.2014.09.001.
	Zhou X Q,Gao Y L,Zhao K J,Fan D.cDNA cloning,sequence analysis and prokaryotic expression of a serine protease from the midgut of Heliothis viriplaca(Lepidoptera:Noctuidae)[J].Acta Entomologica Sinica,2014,57(9):1008-1017.
[9]	Zhou X Q,Fan D,Zhao K J.Characterization of trypsin-like and chymotrypsin-like serine proteases from midgut of Mythimna separata walker[J].Archives of Insect Biochemistry and Physiology,2016,92(3):173-191.doi:10.1002/arch.21324.
[10]	张元臣,蔡新,张国强,董丽丽,王景顺.黏虫溶菌酶基因Mswly的克隆与真核表达[J].河南农业科学,2019,48(5):70-77.doi:10.15933/j.cnki.1004-3268.2019.05.011.
	Zhang Y C,Cai X,Zhang G Q,Dong L L,Wang J S.Cloning and eukaryotic expression of Mswly in oriental armyworm,Mythimna separata[J].Journal of Henan Agricultural Sciences,2019,48(5):70-77.
[11]	于洁,王登杰,张林雅,雷仲仁,王海鸿.参与烟粉虱免疫反应的clip丝氨酸蛋白酶基因分析[J].植物保护学报,2016,43(1):55-61.doi:10.13802/j.cnki.zwbhxb.2016.01.008.
	Yu J,Wang D J,Zhang L Y,Lei Z R,Wang H O.Analysis on clip serine protease genes involved in innate immunity of Bemisia tabaci(Hemiptera:Aleyrodidae)[J].Journal of Plant Protection,2016,43(1):55-61.
[12]	周倩.家蚕丝氨酸蛋白酶BmSP142在抗病毒侵染中的作用[D].镇江:江苏大学,2016.
	Zhou Q.Characterization of Bomyx mori serine protease142(BmSP142)against viral infection[D].Zhenjiang:Jiangsu University,2016.
[13]	刘碧朗,李玉欣,亓希武,向仲怀,何宁佳.家蚕丝氨酸蛋白酶基因BmHP21的克隆及表达分析[J].蚕业科学,2011,37(3):419-424.doi:10.3969/j.issn.0257-4799.2011.03.007.
	Liu B L,Li Y X,Qi X W,Xiang Z H,He N J.Molecular cloning and expression analysis of serine protease gene BmHP21 in silkworm,Bombyx mori[J].Science of Sericulture,2011,37(3):419-424.
[14]	Li H R,Oppert B,Higgins R A,Huang F N,Zhu K Y,Buschman L L.Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and-susceptible Ostrinia nubilalis(Lepidoptera:Crambidae)[J].Insect Biochemistry and Molecular Biology,2004,34(8):753-762.doi:10.1016/j.ibmb.2004.03.010.
[15]	Yao J X,Buschman L L,Oppert B,Khajuria C,Zhu K Y.Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae[J].PLoS One,2012,7(8):e44090.doi:10.1371/journal.pone.0044090.
[16]	Brackney D E,Isoe J,Black W C IV,Zamora J,Foy B D,Miesfeld R L,Olson K E.Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito,Aedes aegypti[J].Journal of Insect Physiology,2010,56(7):736-744.doi:10.1016/j.jinsphys.2010.01.003.
[17]	Patankar A G, Giri A P, Harsulkar A M, Sainani M N, Deshpande V V, Ranjekar P K, Gupta V S.Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest[J].Insect Biochemistry and Molecular Biology,2001,31(4/5):453-464.doi:10.1016/s0965-1748(00)00150-8.
[18]	Budatha M, Meur G, Dutta-Gupta A.Identification and characterization of midgut proteases in Achaea janata and their implications[J].Biotechnology Letter, 2008, 30(2): 305-310. doi: 10.1007/s10529-007-9539-7.
[19]	Cao X L,He Y,Hu Y X,Zhang X F,Wang Y,Zou Z,Chen Y R,Blissard G W,Kanost M R,Jiang H.Sequence conservation,phylogenetic relationships,and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta[J].Insect Biochem Mol Biol,2015,62:51-63.doi:10.1016/j.ibmb.2014.10.006.
[20]	Liu H W,Wang L L,Meng Z,Tang X,Li Y S,Xia Q Y,Zhao P.A clip domain serine protease involved in moulting in the silkworm,Bombyx mori:Cloning,characterization,expression patterns and functional analysis[J].Insect Molecular Biology,2017,26(5):507-521.doi:10.1111/imb.12312.

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