甜菜夜蛾UAP的克隆、时空表达及RNAi研究

doi:10.3864/j.issn.0578-1752.2014.07.012

摘要/Abstract

摘要： 【目的】在昆虫中，几丁质的合成对于其生长发育至关重要，虽然几丁质合成通路在真菌中的研究比较深入，但是在昆虫中，目前大多数的研究都集中在海藻糖酶和几丁质合成酶方面，而对于通路上其他基因的研究却非常少。论文旨在阐明昆虫几丁质合成通路中的UDP-N-乙酰氨基葡萄糖焦磷酸化酶（UAP）对甜菜夜蛾（Spodoptera exigua）几丁质合成酶基因和害虫存活的影响。【方法】以甜菜夜蛾5龄第1天幼虫的cDNA为模板，通过简并引物扩增出甜菜夜蛾UAP基因（SeUAP）的中间片段，然后利用特异性引物和RACE技术扩增出3′和5′ race序列，拼接得到cDNA全长。随后提取甜菜夜蛾各组织和各龄期的总RNA，并通过RT-PCR方法分析SeUAP在各个组织和龄期的表达情况。最后通过向甜菜夜蛾5龄第1天的幼虫注射dsUAP观察其生长状况，检测RNAi的效率以及对几丁质合成通路下游基因的影响。【结果】获得全长为2 098 bp的SeUAP cDNA，编码一个491个氨基酸残基的蛋白，与双翅目昆虫致倦库蚊、埃及伊蚊、黑腹果蝇和冈比亚按蚊的UAP亲缘关系较近。该基因在表皮和卵巢中大量表达，在气管和中肠中的表达次之，在马氏管中表达微弱，在脂肪体中则基本检测不到其转录本的存在。该基因在甜菜夜蛾生长发育的各个阶段均有表达，在卵的整个阶段、L22（幼虫2龄第2天）、L31、L42、L51、P0、P5、P7以及A3这些天中的表达量较高，其余天数表达量较低，其表达的高峰主要出现在几丁质需求量较高的时期。RNAi结果表明，通过向甜菜夜蛾5龄第1天的幼虫注射5 µg dsRNA，导致部分甜菜夜蛾出现蛹期畸形及死亡，并出现两种畸形现象：（1）虫体腹部蛹壳已基本形成并已硬化，但头部蛹壳形成有障碍，化蛹时无法突破旧表皮；（2）虫体头部蛹壳形成正常，也能正常突破旧表皮，但蜕皮过程仅到此为止，无法正常进行下去。定量PCR结果显示SeUAP的沉默对几丁质合成酶A基因（SeCHSA）下调不明显，而对几丁质合成酶B（SeCHSB）的表达有明显下调作用。【结论】UAP可以影响几丁质合成通路的下游基因并对甜菜夜蛾幼虫-蛹的生长发育起一定作用。

关键词: UDP-N-乙酰氨基葡萄糖焦磷酸化酶 , 时空表达 , 甜菜夜蛾 , RNA干扰 , 几丁质合成

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

陈洁, 陈宏鑫, 姚琼, 张文庆. 甜菜夜蛾UAP的克隆、时空表达及RNAi研究[J]. 中国农业科学, 2014, 47(7): 1351-1361.

CHEN Jie, CHEN Hong-Xin, YAO Qiong, ZHANG Wen-Qing. Molecular Cloning, Expression Patterns and RNAi of UDP-N-acetylglucosamine Pyrophosphorylase in Spodoptera exigua[J]. Scientia Agricultura Sinica, 2014, 47(7): 1351-1361.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.07.012

https://www.chinaagrisci.com/CN/Y2014/V47/I7/1351

参考文献

[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.