Effects Of Tebufenozide On MRNA Expression Of Dopa Decarboxyiase Gene In Larvae Of Spodoptera Litura Fabricius

In order to investigate the action mechanism of tebufenozide, the full length cDNA of dopa decarboxylase (DDC) gene from Spodoptera litura Fabricius was cloned and the effects of tebufenozide on mRNA expression of dopa decarboxylase gene in the process of cuticular formation were investigated in this paper. The results are summarized as follows.1. RT-PCR and RACE techniques were used to clone the complete cDNA of dopa decarboxylase (DDC) from Spodoptera litura. Bioinformatics tools were applied to analyze both the DNA sequence and protein sequence. Open reading frame (ORF) of DDC was1263bp in length, encoding420amino acids. The5’end and3’end non-coding regions were105bp and79bp in length. The sequence was deposited in GenBank database with accession number of KF620492. Homology and phylogenetic analysis indicated that the DDC of Spodoptera litura had the very high similarity with that of Lepidoptera. We successfully predicted the DDC three-dimensional structure by means of SWISS-MODEL online tools. The structure was similar to the DDC structure of Drosophila. The function structure domain has good conservative property.2. Toxiciry of tebufenozide to different instar larvae of Spodoptera litura was determined by artificial diet dipping method. The results showed that the toxiciry of3th,4th,5th and6th instar larvae’s LC50were33.32,40.28,71.20and71.97mg/L. The results indicated that the toxicity of tebufenozide to Spodoptera litura is reduced with the larvae growth.3. The mRNA expression of the gene in different developmental stages of Spodoptera litura were investigated using real time quantitative PCR. The DDC gene mRNA expression was at a high level before and after molting. Then it kept a low level during12-48h. The activation rates raised with instar increasing. The mRNA of the gene expressed among different developmental stages. The egg stage had the highest expression.4. The mRNA expression of the gene in5th instar larvae of Spodoptera litura which were continuously treated with tebufenozide for different time were investigated using real time quantitative PCR. The DDC gene mRNA expression was significantly higher than the control treatment when the larvae were treated with four tebufenozide sublethal doses for12,24,36 and48h, respectively. The activation rates first raised and then declined with tebufenozide-treated time and tebufenozide sublethal doses increasing. The expression of the larvae treated with LC20～LC30sublethal doses was the highest after24～36h tebufenozide-treated time. The DDC gene mRNA expression had a sharp decrease and was significantly lower than the control treatment after60h treated time. The tebufenozide activated the DDC gene expression and then inhibited it. The interference in the formation of new larval cuticle by tebufenozide is closely related to the effects on DDC gene mRNA expression by tebufenozide.