Cloning and expression analysis of <i>APN</i>1 gene from <i>Lymantria xylina</i>

Lin Jiancong; Sun Yue; Liu Yonglong; Wang Jinda; Wang Rong; Zhang Feiping

doi:10.12171/j.1000-1522.20210186

Journal of Beijing Forestry University > 2021 > 43(9): 94-100. > DOI: 10.12171/j.1000-1522.20210186

Lin Jiancong, Sun Yue, Liu Yonglong, Wang Jinda, Wang Rong, Zhang Feiping. Cloning and expression analysis of APN1 gene from Lymantria xylina[J]. Journal of Beijing Forestry University, 2021, 43(9): 94-100. DOI: 10.12171/j.1000-1522.20210186

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Cloning and expression analysis of APN1 gene from Lymantria xylina

1.
Key Laboratory for Prevention and Control of Major Pest in Ecological Public Welfare Forests of Forestry College of Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
2.
National Engineering Research Center of Sugarcane, Fujian Agricuture and Forestry University, Fuzhou 350002, Fujian, China

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Received Date: May 13, 2021
Revised Date: June 30, 2021
Available Online: July 05, 2021
Published Date: October 14, 2021

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Abstract

Abstract

Objective Aminopeptidase N(APN) is a class of important kind of BT receptor protein in insect midgut. The mechanism of Cry toxin produced by Bt bacteria to kill insects has been controversial in the academic research, but it is generally believed that the binding of toxin and Bt receptor protein is the necessary link to its virulence. The APN1 gene of Lymantria xylina was studied by gene cloning, biological information analysis and expression patterns, with the purpose to provid a reference for the subsequent study of APN gene family, other Bt receptor proteins and the mechanism of Cry toxin.
Method APN1 was cloned by cDNA from midgut of the moth as template in a PCR system. Biological analysis and real time quantitative PCR(qRT-PCR) were performed to analyze the expression pattern of LxAPN1 gene in different ages and tissues of Lymantria xylina.
Result The full-length DNA of APN1 gene was cloned from midgut of Lymantria xylina and named LxAPN1. The full-length sequence of LxAPN1 was 3 159 bp, ORF was 3 054 bp, encoding 1 017 amino acids. Sequence alignment and phylogenetic tree analysis showed that LxAPN1 and LdAPN1 were highly homologous, with signal peptide at N-terminal, zinc binding site HEXXH (X₁₈) E and conserved region GAMENWG, and GPI binding site at the end. LxAPN1 was not expressed in egg stage, and expressed in 1−7 instar larvae, the expression of LxAPN1 decreased after larval stage; the expression of LxAPN1 in intestine was significantly higher than that in head and cuticle.
Conclusion LxAPN1 was successfully cloned in the midgut of Lymantria xylina. LxAPN1 and LdAPN1 are highly homologous, and the distribution of phylogenetic tree is also very similar, which not only indicates the similarity between Lymantria xylina and Lymantria dispar, but also the similarity of APN1 function between them; The expression of LxAPN1 was the highest in the second instar larvae of Lymantria xylina. And the expression was the highest in the gut from 6 instar larvae, as an Bt receptor in the intestinal of Lymantria xylina.
- Lymantria xylina,
- Bt receptor,
- APN,
- cloning

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References (32)

References

[1]	Fibiger M, Lafonatine J D. A review of the higher classification of the Noctuoidea (Lepidoptera) with special reference to the Holarctic fauna[J]. Experiana, 2005, 11: 7−92.
[2]	Lafontaine J D, Fibiger M. Revised higher classification of the Noctuoidea (Lepidoptera)[J]. Canadian Entomologist, 2006, 138(5): 610−635. doi: 10.4039/n06-012
[3]	Zahiri R, Kitching I J, Lafontaine J D, et al. A new molecular phylogeny offers hope for a stable family level classification of the Noctuoidea (Lepidoptera)[J]. Zoologica Scripta, 2011, 40(2): 158−173. doi: 10.1111/j.1463-6409.2010.00459.x
[4]	Pogue M G, Schaefer P W. A review of selected species of Lymantria Hübner [1819] including three new species (Lepidoptera: Noctuidae: Lymantriinae) from subtropical and temperate regions of Asia, some potentially invasive to North America[M]. Washington D C: United States Department of Agriculture, Forest Health Technology Enterprise Team, 2007.
[5]	Dewaard J R, Mitchell A, Keena M A, et al. Towards a global barcode library for Lymantria (lepidoptera: lymantriinae) tussock moths of biosecurity concern[J/OL]. PLoS One, 2010, 5(12): e14280[2010−12−09]. http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC3000334&blobtype=pdf.
[6]	周志强. 龙眼木毒蛾的发生规律及防治策略探究[J]. 现代农业, 2016(6):52−53. doi: 10.3969/j.issn.1008-0708.2016.06.035 Zhou Z Q. Research on the occurrence and control strategies of Lymantria xylina[J]. Modern Agriculture, 2016(6): 52−53. doi: 10.3969/j.issn.1008-0708.2016.06.035
[7]	De B H, F Lemille. Presence of flagellar antigenic subfactors in serotype 3 of Bacillus thuringiensis[J]. Journal of Invertebrate Pathology, 1970, 15(1): 139. doi: 10.1016/0022-2011(70)90113-8
[8]	Goldberg L J, Margalit J. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univittatus, Aedes aegypti and Culex pipiens[J]. Mosquito News, 1977, 37(3): 355−358.
[9]	Knight P J K, Crickmore N, Ellar D J. The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N[J]. Molecular Microbiology, 1994, 11(3): 429−436. doi: 10.1111/j.1365-2958.1994.tb00324.x
[10]	Pigott C R, Ellar D J. Role of receptor in Bacillus thuringiensis crystal toxin activity[J]. Microbiology and Molecular Biology Reviews, 2007, 71: 255−281. doi: 10.1128/MMBR.00034-06
[11]	Wang G, Kongming W. Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region[J]. Science in China, 2005, 48(4): 346−356. doi: 10.1360/03yc0273
[12]	Bravo A, Gill S S, Soberón M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[J]. Toxicon, 2007, 49(4): 423−435. doi: 10.1016/j.toxicon.2006.11.022
[13]	马文静, 韩兰芝, 尹新明, 等. 鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系[J]. 环境昆虫学报, 2011, 33(3):147−153. Ma W J, Han L Z, Yi X M, et al. Binding of Bt Cry toxins to lepidopteran midgut aminopeptidase N and the relationship between their interactions with Bt resistance[J]. Journal of Environmental Entomology, 2011, 33(3): 147−153.
[14]	Jenkins J L, Dean D H. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors[J]. BMC Biochemistry, 2001, 2(1): 1−8. doi: 10.1186/1471-2091-2-1
[15]	Mi K L, Dean D H. Inconsistencies in determining Bacillus thuringiensis, toxin binding sites relationship by comparing competition assays with Ligand Blotting[J]. Biochemical and Biophysical Research Communications, 1996, 220(3): 575−580. doi: 10.1006/bbrc.1996.0445
[16]	Lorence A, Darszon A, Bravo A. Aminopeptidase dependent pore formation of Bacillus thuringiensis CrylAc toxin on Trichoplusia nimembranes[J]. FEBS Letters, 1997, 414(2): 303−307. doi: 10.1016/S0014-5793(97)01014-4
[17]	Jenkins J L, Lee M K, Valaitis A P, et al. Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor[J]. Journal of Biological Chemistry, 2000, 275(19): 23−31.
[18]	Yaoi K, Nakanishi K, Kadotani T, et al. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori[J]. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1999, 1444(1): 131−137. doi: 10.1016/S0167-4781(98)00250-4
[19]	Ferre J, VanRie J. Biochemistry and genetics of insect resistance to Bacillus thuringiensis[J]. Annual Review Entomology, 2002, 47: 501−533. doi: 10.1146/annurev.ento.47.091201.145234
[20]	Kumar S, Tamura K, Nei M. MEGA: molecular evolutionary genetics analysis software for microcomputers[J]. Bioinformatics, 1994, 10(2): 189−191. doi: 10.1093/bioinformatics/10.2.189
[21]	喻子牛. 苏云金杆菌[M]. 北京: 科学出版社, 1990: 20−21. Yu Z N. Bacillus thuringiensis[M]. Beijing: Science Press, 1990: 20−21.
[22]	Schnepf E, Crickmore N, Van R J, et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiology and Molecular Biology Reviews, 1998, 62(3): 775−806. doi: 10.1128/MMBR.62.3.775-806.1998
[23]	Cristofoletti P T, Terra W R. The role of amino acid residues in the active site of a midgut microvillar aminopeptidase from the beetle Tenebrio molitor[J]. Biochimica et Biophysica Acta, 2000, 1479(1): 185−195.
[24]	Zhu Y C, Kramer K J, Oppert B, et al. cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins[J]. Insect Biochemistry and Molecular Biology, 2000, 30(3): 215−224. doi: 10.1016/S0965-1748(99)00118-6
[25]	Crave C M, Bel Y, Lee S, et al. Study of aminopeptidase N gene family in the Lepidopterans Ostrinia nubilalis and Bombyx mori: Sequences, mapping and expression[J]. Insect Biochemistry and Molecular Biology, 2010, 40: 506−515. doi: 10.1016/j.ibmb.2010.04.010
[26]	Chauhan V K, Dhania N K, Lokya V, et al. Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure[J]. Journal of Biosciences, 2021, 46(1): 209−213.
[27]	Knight P J K, Carroll J, Ellar D J. Analysis of glycan structures on the 120 kDa aminopeptidase N of Manduca sexta and their interactions with Bacillus thuringiensis Cry1Ac toxin[J]. Insect Biochemistry and Molecular Biology, 2004, 34(1): 101−112. doi: 10.1016/j.ibmb.2003.09.007
[28]	展恩玲. 梨小食心虫中肠氨肽酶N3(GmolAPN3)基因的克隆、表达及功能分析[D]. 杨凌: 西北农林科技大学, 2018. Zhan E L. Gene cloning, expression and functional analysis of aminopeptidase N3(GmolAPN3) from midgut of the oriental fruit moth, Grapholitha molesta[D]. Yangling: Northwest A&F University, 2018.
[29]	Zhang Y, Dan Z, Yan X, et al. Identification and characterization of Hyphantria cunea aminopeptidase N as a binding protein of Bacillus thuringiensis Cry1Ab toxin[J]. International Journal of Molecular Sciences, 2017, 18(12): 2575. doi: 10.3390/ijms18122575
[30]	牛琳琳, ZAW Lin Naing, 张彩虹, 等. 棉铃虫APN基因家族系统进化与功能分析[J]. 中国生物防治学报, 2021, 37(1):91−101. Niu L L, Zaw L N, Zhang C H, et al. Phylogenetic evolution and function of APN gene family in Helicoverpa armigera[J]. Chinese Journal of Biological Control, 2021, 37(1): 91−101.
[31]	王兴云, 马文静, 韩兰芝, 等. 大螟中肠氨肽酶N基因的克隆及表达谱分析[J]. 昆虫学报, 2012, 55(9):1022−1030. Wang X Y, Ma W J, Han Z L, et al. Cloning and expression profiling of aminopeptidase N encoding gene in larval midgut of Sesamia inferens[J]. Acta Entomologica Sinica, 2012, 55(9): 1022−1030.
[32]	Wang J D, Zhang J S, Guo Y F, et al. Molecular cloning, characterization, and expression profiling analysis of Cry toxin receptor genes from sugarcane shoot borer Chilo infuscatellus (Snellen)[J]. Pesticide Biochemistry and Physiology, 2019, 157: 186−195. doi: 10.1016/j.pestbp.2019.03.023

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Cloning and expression analysis of APN1 gene from Lymantria xylina

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