Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (8): 1512-1521.doi: 10.3864/j.issn.0578-1752.2014.08.007

• PLANT PROTECTION • Previous Articles     Next Articles

Effects of Tebufenozide on mRNA Expression of Dopa Decarboxylase Gene in Larvae of Spodoptera litura Fabricius

 ZHANG  Wen-Hui, HOU  Hai-Xia, YANG  Qiong, WANG  Shu-Hui, MAO  Xu-Lian, LIU  Yong-Jie   

  1. College of Plant Protection, Shandong Agricultural University, Taian 271018, Shandong
  • Received:2013-09-29 Online:2014-04-15 Published:2013-11-19

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Abstract: 【Objective】 The objective of this study is to clone the full length cDNA of dopa decarboxylase (DDC) gene from 5th instar larvae of Spodoptera litura Fabricius and to investigate the effects of tebufenozide on mRNA expression of DDC gene in the process of cuticular formation. 【Method】 RT-PCR and RACE techniques were used to clone the complete cDNA of DDC gene from S. litura. Bioinformatics tools were applied to analyze both the DNA sequence and protein sequence. Toxicity of tebufenozide to different instar larvae of S. litura was determined by artificial diet dipping method. The mRNA expression of the gene in 5th instar larvae of S. litura which were continuously treated with tebufenozide for different lengths of time were investigated using real time quantitative PCR. 【Result】 The complete DDC gene from 5th instar larvae of S. litura was cloned. Open reading frame (ORF) of DDC was 1 263 bp in length, encoding 420 amino acids. The 5′ end and 3′ end non-coding regions were 105 and 79 bp in length. The sequence was deposited in GenBank database with accession number of KF620492. Homology and phylogenetic analysis indicated that the DDC of S. litura had very high similarity with that of Lepidoptera. The DDC three-dimensional structure was successfully predicted by means of SWISS-MODEL online tools. The structure was similar to the DDC structure of Drosophila. The function structure domain had good conservative property. The results showed that the toxicity of 3rd, 4th, 5th and 6th instar larvae’s LC50 were 33.32, 40.28, 71.20 and 71.97 mg?L-1, respectively. The DDC gene mRNA expression was significantly higher than CK when the larvae were treated with four tebufenozide sublethal doses (7.50, 16.24, 28.34, 45.63 mg?L-1) for 12, 24, 36 and 48 h, respectively. The activation rates raised and then declined with tebufenozide-treated time increasing. The expression of the larvae treated with 16.24 mg?L-1 sublethal doses was the highest after 24, 36 and 48 h tebufenozide-treated time. Meanwhile, the four tebufenozide sublethal doses had the highest expressions after 36 h. The DDC gene mRNA expression had a sharp decrease and was significantly lower than CK after 60 h treated time. The tebufenozide activated the DDC gene expression and then inhibited it. 【Conclusion】 DDC gene was cloned and characterized from S. litura. The interference in the formation of new larval cuticle by tebufenozide is closely related to the effects on DDC gene mRNA expression by tebufenozide.

Key words: Spodoptera litura , tebufenozide , dopa decarboxylase (DDC) , gene cloning , real time quantitative PCR

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