木毒蛾氨肽酶N1基因克隆与表达分析

林健聪; 孙悦; 刘用垄; 王锦达; 王荣; 张飞萍

doi:10.12171/j.1000-1522.20210186

木毒蛾氨肽酶N1基因克隆与表达分析

doi: 10.12171/j.1000-1522.20210186

1.
福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福建福州 350002
2.
福建农林大学国家甘蔗工程技术研究中心，福建福州 350002

基金项目: 国家自然科学基金项目（31600522、31601363），福建省自然科学基金项目（2017J0106），福建农林大学林学高峰学科建设项目（61201400820）

详细信息

作者简介:
林健聪。主要研究方向：森林昆虫。Email：66470106@qq.com　地址：350002 福建省福州市仓山区上下店路福建农林大学田间914

责任作者:
王荣，博士，讲师。主要研究方向：森林昆虫。Email：wangrongbl0501@126.com　地址：同上

中图分类号: S763.3
计量
- 文章访问数: 107
- HTML全文浏览量: 29
- PDF下载量: 24
- 被引次数: 0
出版历程
- 收稿日期: 2021-05-14
- 修回日期: 2021-07-01
- 网络出版日期: 2021-07-06

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 Agricultural University, Fuzhou 350002, Fujian, China

摘要

摘要: 目的氨肽酶N（APN）是昆虫中肠一类重要的Bt受体蛋白，Bt细菌产生的Cry毒素对昆虫的毒杀作用机理在学术界存在一定争议，但是普遍认为毒素与Bt受体蛋白的结合是产生毒力的必要环节。本研究通过基因克隆、生物信息学分析以及不同龄期组织中的表达对木毒蛾APN1基因进行研究，为后续研究APN基因家族、其他Bt受体蛋白及Cry毒素作用机制提供有益补充。方法以木毒蛾中肠cDNA为模板，对木毒蛾APN1基因进行克隆并进行生物学分析，利用实时定量PCR（qRT-PCR）技术分析该基因在木毒蛾发育阶段及不同组织中的表达模式。结果克隆获得木毒蛾APN1基因的全长DNA，命名为LxAPN1。LxAPN1序列全长为3 159 bp，ORF为3 054 bp，编码1 017个氨基酸，序列比对和进化树分析表明，LxAPN1与舞毒蛾的LdAPN1高度同源，在N-端都具有信号肽，都具有锌结合位点HEXXH（X₁₈）E以及保守区域GAMENWG，在末端具有GPI结合位点；LxAPN1在木毒蛾卵期无表达，在所有幼虫阶段中均有表达，幼虫期过后LxAPN1表达量锐减；LxAPN1在肠道的表达量明显高于头部和表皮。结论 LxAPN1在木毒蛾中肠被成功克隆，LxAPN1与LdAPN1高度同源，并且在进化树的分布上也极其相近，推测两者的APN1功能上近似；LxAPN1在木毒蛾2龄幼虫期表达量最高。并且在6龄幼虫肠道中表达量最高，肠道高表达与LxAPN1作为Bt受体在木毒蛾中肠发挥作用息息相关。
- 木毒蛾 /
- Bt受体 /
- 氨肽酶N /
- 克隆
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 to 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

HTML全文

图 1 LxAPN1基因全长

M. 1 kb marker；1代表LxAPN1基因全长。M, 1 kb marker; 1 represents LxAPN1 full-length gene.

Figure 1. Full length of LxAPN1 gene

下载: 全尺寸图片幻灯片

图 2 木毒蛾APN1编码区核酸序列及氨基酸序列

高度保守的残基用深蓝色背景表示；部分保守的残基用白色和灰色背景显示。信号肽用黑色下划线标出；保守结构GAMENWG用红色空心方框标出；锌结合位点HEXXH（X）₁₈E用绿色空心方框标出；GPI结合位点用黑实心三角标出；末端信号肽用红色下划线标出。Highly conserved residues are indicated in deep blue background and only partially conserved residues are indicated in white and gray background; black underline indicates the signal peptite; the gluzincin aminopeptidase GAMENWG is marked with a red hollow box; the characteristic zinc bingding/gluzincin motif HEXXH（X）₁₈E is indicated with green hollow boxes. The GPI anchor point is showed as an black triangles. The C-terminal pro-peptide sequences are marked with a red underline.

Figure 2. Nucleotide and amino acid sequences of LxAPN1

下载: 全尺寸图片幻灯片

图 3 木毒蛾APN1蛋白质二级结构预测

蓝色代表α-螺旋；紫色代表无规则卷曲；红色代表延伸链；绿色代表β-转角；横坐标表示蛋白大小（kDa）。Blue represents α-helix; purple represents random coli; red represents extend strand; geen represents β-turn; abscissa indicates protein size（kDa）.

Figure 3. Prediction of secondary structure of LxAPN1 protein

下载: 全尺寸图片幻灯片

图 4 木毒蛾APN1蛋白三级结构预测

Figure 4. Prediction of three-dimensional structure of LxAPN1 protein

下载: 全尺寸图片幻灯片

图 5 木毒蛾APN1与其他昆虫APN蛋白系统进化树

Figure 5. Neighbor-joining phylogenetic tree of LxAPN1 and APN in other insects

下载: 全尺寸图片幻灯片

图 6 木毒蛾不同龄期及不同组织中LxAPN1基因的表达

L1~L7为1龄到7龄幼虫。L1—L7 are the 1st to 7th instar larvae.

Figure 6. Expression profiles of LxAPN1 in different ages and tissues of Lymantria xylina

下载: 全尺寸图片幻灯片

表 1 基因克隆中使用引物列表

Table 1. List of primers in gene cloning

引物名称 Primer name	序列（5′—3′） Sequence (5′−3′)	用途 Usage
LxAPN1-F	TTACAATAATCCAGTTGAGGGT	LxAPN1基因扩增 LxAPN1 gene amplification
LxAPN1-R	GAACATCAAACTAGATTTCTTAGTAATTGAT	LxAPN1基因扩增 LxAPN1 gene amplification
qAPN1-F	CGTGATCCGTCCTCAAGATTAC	qRT-PCR
qAPN1-R	TGGATGCGACGTAAAGTAAGG	qRT-PCR
qEF-1a-F	CCGTGTTGAAACTGGTATCCT	qRT-PCR
qEF-1a-R	TAGAGCTTCGTGGTGCATTT	qRT-PCR
qActin-F	GGTAGATAAGGAGGCAAGGATTG	qRT-PCR
qActin-R	ACCACAATGTACCCTGGTATTG	qRT-PCR

下载: 导出CSV

参考文献(32)

[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: 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: The Science Publishing Company, 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