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

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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

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