Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (7): 1351-1361.doi: 10.3864/j.issn.0578-1752.2014.07.012

• Insect chitin metabolism and plant protection • Previous Articles     Next Articles

Molecular Cloning, Expression Patterns and RNAi of UDP-N-acetylglucosamine Pyrophosphorylase in Spodoptera exigua

 CHEN  Jie, CHEN  Hong-Xin, YAO  Qiong, ZHANG  Wen-Qing   

  1. State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275
  • Received:2013-10-24 Online:2014-04-01 Published:2013-11-05

RichHTML

2

PDF

612

Cited

7

Abstract: 【Objective】 In insects, chitin biosynthesis is a key process for their growth and development. Although it has been studied deeply on fungi, little is known for other genes in the chitin biosynthesis pathway of insects, because most of researches were focused in trehalase and chitin synthase. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is one of the enzymes in the chitin biosynthesis pathway of insects. This paper aims to clarify its effect on the expression of chitin synthase genes and survival rates of Spodoptera exigua. 【Method】 With the technique of molecular biology, such as the polymerase chain reaction (PCR) and RACE technique, the SeUAP from S. exigua was cloned and sequenced. Also, in order to clarify the expression pattern of SeUAP, the total RNA of several tissues of the 2nd day of 5th instars of S. exigua and total RNA of everyday of its life cycle were extracted to detect the expression levels of SeUAP by RT-PCR. Finally, dsRNA was injected into the 1st day of 5th instars of S. exigua to survey the survival rate and detect the expression levels of CHSA and CHSB, which are the last enzymes in the pathway. 【Result】 The complete cDNA sequence of SeUAP was 2 098 bp encoding a protein of 491 amino acid residues, which has a close evolutionary relationship with orthologues in Culex quinquefasciatus, Aedes aegypti, Drosophila melanogaster and Anopheles gambiae. RT-PCR results showed that SeUAP was highly expressed in integument and ovaries. The expression was also detected in trachea and midgut but virtually not in Malpighian tubules and fat body. In addition, RT-PCR results showed that SeUAP was highly expressed at egg stage, L22 (2nd day 2nd instars), L31, L42, L51, P0, P5, P7 and A3, which appeared to be related to the high demanding of chitin synthesis. Further research of RNAi showed that the injection of dsRNA of SeUAP into the larvae with 5 μg caused lethal effect and abnormal phenotypes of pupae, which could be classified into two major groups: (1) Abdomen puparium was formed while head puparium was not and the pupa had difficulty in breaking the old cuticle and getting out; (2) The head puparium was formed and the pupa already broke the old cuticle, but the pupation process stopped and never finished. Silencing of target gene could down-regulate the mRNA levels of chitin synthase gene A (CHSA) and CHSB, especially CHSB. 【Conclusion】 UAP influences gene expression in the chitin biosynthesis pathway and play a role in growth and development of S. exigua.

Key words: UDP-N-acetylglucosamine pyrophosphorylase (UAP) , expression patterns , Spodoptera exigua , RNAi , chitin biosynthesis

[1]Mesce K A, Fahrbach S E. Integration of endocrine signals that regulate insect ecdysis. Frontiers in Neuroendocrinology, 2002, 23(2): 179-199.

[2]Glaser L, Brown D H. The synthesis of chitin in cell-free extracts of Neurospora crassa. The Journal of Biological Chemistry, 1957, 228: 729-742.

[3]Candy D J, Kilby B A. Studies on chitin synthesis in the desert locust. Journal of Experimental Biology, 1962, 39: 129-140.

[4]Mio T, Yabe T, Arisawa M, Yamada-Okabe H. The eukaryotic UDP-N-acetylglucosamine pyrophosphorylases. Gene cloning, protein expression, and catalytic mechanism. The Journal of Biological Chemistry, 1998, 273: 14392-14397.

[5]Wang-Gillam A, Pastuszak I, Stewart M, Drake R R, Elbein A D. Identification and modification of the uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase. The Journal of Biological Chemistry, 2000, 275(2): 1433-1438.

[6]Brown K, Pompeo F, Dixon S, Mengin-Lecreulx D, Cambillau C, Bourne Y. Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily. The EMBO Journal, 1999, 18(15): 4096-4107.

[7]Olsen L R, Roderick S L. Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites. Biochemistry, 2001, 40(7): 1913-1921.

[8]Kostrewa D, D'Arcy A, Takacs B, Kamber M. Crystal structures  of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase, GlmU, in apo form at 2.33 Å resolution and in complex with UDP-N-acetylglucosamine and Mg2+ at 1.96 Å resolution. Journal of Molecular Biology, 2001, 305(2): 279-289.

[9]Sulzenbacher G, Gal L, Peneff C, Fassy F, Bourne Y. Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture. The Journal of Biological Chemistry, 2001, 276(15): 11844-11851.

[10]Maruyama D, Nishitani Y, Nonaka T, Kita A, Fukami T A, Mio T, Yamada-Okabe H, Yamada-Okabe T, Miki K. Crystal structure of uridine-diphospho-N-acetylglucosamine pyrophosphorylase from Candida albicans and catalytic reaction mechanism. The Journal of Biological Chemistry, 2007, 282(23): 17221-17230.

[11]Kato N, Mueller C R, Wessely V, Lan Q, Christensen B M. Aedes aegypti phosphohexomutases and uridine diphosphate-hexose pyrophosphorylases: comparison of primary sequences, substrate specificities and temporal transcription. Insect Molecular Biology, 2005, 14(6): 615-624.

[12]Arakane Y, Baguinon M C, Jasrapuria S, Chaudhari S, Doyungan A, Kramer K J, Muthukrishnan S, Beeman R W. Both UDP N- acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival and fecundity. Insect Biochemistry and Molecular Biology, 2011, 41: 42-50.

[13]Liu X, Li F, Li D, Ma E, Zhang W Q, Zhu K Y, Zhang J Z. Molecular and functional analysis of UDP-N-acetylglucosamine pyrophosphorylases from the migratory locust, Locusta migratoria. PLoS ONE, 2013, 8(8): e71970.

[14]Kramer K J, Koga D. Insect chitin. Physiological state, synthesis, degradation and metabolic regulation. Insect Biochemistry, 1986, 16: 851-877.

[15]Stokes M J, Guther M L S, Turnock D C, Prescott A R, Martin K L, Alphey M S, Ferguson M A J. The synthesis of UDP-N- acetylglucosamine is essential for bloodstream form Trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveal a hierarchy in parasite protein glycosylation. The Journal of Biological Chemistry, 2008, 283: 16147-16161.

[16]Boehmelt G, Wakeham A, Elia A, Sasaki T, Plyte S, Potter J, Yang Y, Tsang E, Ruland J, Iscove N N, Dennis J W, Mak T W. Decreased UDP-GlcNAc levels abrogate proliferation control in EMeg32- deficient cells. The EMBO Journal, 2000, 19(19): 5092-5104.

[17]Arakane Y, Muthukrishnan S, Kramer K J, Specht C A, Tomoyasu Y, Lorenzen M D, Kanost M, Beeman R W. The Tribolium chitin synthease genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix. Insect Molecular Biology, 2005, 14(5): 453-463.

[18]Chen X F, Yang X, Kumar N, Tang B, Sun X J, Qiu X M, Hu J, Zhang W Q. The class A chitin synthase gene of Spodoptera exigua: Molecular cloning and expression patterns. Insect Biochemistry and Molecular Biology, 2007, 37: 409-417.

[19]Kumar N S, Tang B, Chen X F, Tian H G, Zhang W Q. Molecular cloning, expression pattern and comparative analysis of chitin synthase gene B in Spodoptera exigua. Comparative Biochemistry and Physiology -Part B: Biochemistry & Molecular Biology, 2008, 149(3): 447-453.

[20]Moreira M F, dos Santos A S, Marotta H R, Mansur J F, Ramos I B, Machado E A, Souza G H, Eberlin M N, Kaiser C R, Kramer K J, Muthukrishnan S, Vasconcellos A M H. A chitin-like component in Aedes aegypti eggshells, eggs and ovaries. Insect Biochemistry and Molecular Biology, 2007, 37: 1249-1261.

[21]Hogenkamp D G., Arakane Y, Zimoch L, Merzendorfer H, Kramer K J, Beeman R W, Kanost M R, Specht C A, Muthukrishnan S. Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development. Insect Biochemistry and Molecular Biology, 2005, 35: 529-540.

[22]Adams M D, Celniker S E, Holt R A, Evans C A, Gocayne J D, Amanatides P G, Scherer S E, Li P W, Hoskins R A, Galle R F. The genome sequence of Drosophila melanogaster. Science, 2000, 287: 2185-2195.

[23]Graack H R, Cinque U, Kress H. Functional regulation of glutamine: fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner. Biochemical Journal, 2001, 360: 401-412.

[24]Thummel C S. Files on steroids-Drosophila metamorphosis and the mechanisms of steroid hormone action. Trends in Genetics, 1996, 12(8): 306-310.

[25]Kato N, Dasgupta R, Smartt C T, Christensen B M. Glucosamine: fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypit. Insect Molecular Biology, 2002, 11(3): 207-216.

[26]Chen J, Tang B, Chen H X, Yao Q, Huang X F, Chen J, Zhang D W, Zhang W Q. Different functions of the insect soluble and membrane- bound trehalase genes in chitin biosynthesis revealed by RNA interference. PLoS ONE, 2010, 5(4): e10133.
[1] ZHANG Qi, CHEN ErHu, SUN DeHong, TANG PeiAn. Relationship Between Glutathione S-Transferase Genes CfGSTe1 and CfGSTd1 and Ethyl Formate Tolerance in Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2026, 59(5): 1008-1019.
[2] YAN WenYing, ZHANG YuanZhen, WU HongXin, PANG Rui, CHEN ZePeng, JIN FengLiang, XU XiaoXia. Construction of an RNAi-Enhanced Metarhizium anisopliae Targeting PxGNBP3 and Its Immunoregulatory Mechanism in Plutella xylostella [J]. Scientia Agricultura Sinica, 2026, 59(3): 556-574.
[3] CHEN ErHu, TANG JingJie, HU ShunJie, TANG PeiAn. The Roles of Heat Shock Protein Genes CfHsp70-1 and CfHsp70-2 in Enhancing the High-Temperature Tolerance after Heat Acclimation in Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2025, 58(5): 918-928.
[4] XIAO ZhuoDan, QIAO JiaZheng, GAO YuLan, SHANG ZhangYin, LIU Huai, WANG Jia. Silencing of Cytochrome P450 Genes CYP6CY53 and CYP302A1 in Aphis craccivora Enhances the Sensitivity to Flonicamid [J]. Scientia Agricultura Sinica, 2025, 58(18): 3664-3675.
[5] YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley [J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.
[6] CHEH ErHu, YUAN GuoQing, CHEN Yan, CHEN MengQiu, SUN ShengYuan, TANG PeiAn. Mitochondrial Protein-Coding Genes Nad5, Nad6 and Atp6 are Involved in Phosphine Resistance of Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2024, 57(9): 1722-1733.
[7] LUO LiDan, CHEN JiaMing, AN Qi, LIU Lei, SUN QinZhe, LIU Huan, WANG SenShan, SONG LiWen. Effects of Extreme High Temperature on Trehalose Content and Trehalose Transporter Gene in Tetranychus truncatus [J]. Scientia Agricultura Sinica, 2024, 57(6): 1091-1101.
[8] ZHAO YiYan, GUO HongFang, LIU WeiMin, ZHAO XiaoMing, ZHANG JianZhen. Effects of Apolipophorin on Ovarian Development and Lipid Deposition in Locusta migratoria [J]. Scientia Agricultura Sinica, 2024, 57(4): 711-720.
[9] YUAN GuoQing, CHEN ErHu, TANG PeiAn. The Mechanisms of Mitochondrial Protein-Coding Genes ND6 and ATP6 in Regulating Cold Tolerance of Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2024, 57(22): 4483-4494.
[10] LI ChuXin, SONG ChenHu, ZHOU JinHuan, LI JiaXin, WANG XinLiang, TIAN XuBin, SONG Zhen. Research on Prevention and Control Technology of Citrus Yellow Vein Clearing Virus Based on VIGS [J]. Scientia Agricultura Sinica, 2024, 57(22): 4473-4482.
[11] WANG Ni, SHI ZheYi, YOU YuanZheng, ZHANG Chao, ZHOU WenWu, ZHOU Ying, ZHU ZengRong. Effects of miRNA on Gene Expression of Sphingolipids Metabolism and Small RNA Analysis of Silencing NlSPT1 and NlSMase4 in Nilaparvata lugens [J]. Scientia Agricultura Sinica, 2024, 57(20): 4022-4034.
[12] CHEN FeiEr, ZHANG ZhiPeng, JIANG QingXue, MA Lin, WANG XueMin. Cloning and Biological Function Verification of Alfalfa MsSPL17 [J]. Scientia Agricultura Sinica, 2024, 57(17): 3335-3349.
[13] ZHU BingLin, YU JiaLi, CHEN JiaYue, TIAN Yuan, WAN Yuan, LIU ChenYang, WANG XiaoYu, WANG MiaoLi, CHENG Gong. Cloning, Expression Characterization, and Functional Analysis of the Snail1 in Qinchuan Cattle and Its Impact on Proliferation of Bovine Adipocytes [J]. Scientia Agricultura Sinica, 2024, 57(13): 2674-2686.
[14] CHEH ErHu, SHEN DanRong, DU WenWei, MENG HongJie, TANG PeiAn. Cuticle Protein Genes are Involved in Phosphine Resistance of Cryptolestes ferrugineus [J]. Scientia Agricultura Sinica, 2023, 56(9): 1696-1707.
[15] SHAO HongYang, MENG Xiang, ZHANG Tao, CHEN Min. Analysis of Cytochrome P450 Genes in Response to Quercetin and Function of CYP6ZB2 in Hyphantria cunea [J]. Scientia Agricultura Sinica, 2023, 56(7): 1322-1332.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd