Effects Of Perilocosides On Trypsin Promoter From The Midgut Of Mythimna Separata

Previous study showed that the novel insecticidal compounds Periplocoside P(PsP) and Periplocoside T(PsT) can up-regulate the expression level of trypsin gene significantly in the midgut and increase the activity of trypsin in Mythimna separata(Walker)(Lepidoptera: Noctuidae)in vivo. These changes will lead insects to death by destroying the midgut cells of M. separata. In order to clarify the regulatory mechanism of PsP and PsT on trypsin gene expression, the 5′flanking promoter sequence of trypsin gene was cloned from the midgut of M. separata and their functions were studied. These results will provide the basic foundation for further exploration of the regulatory mechanism of activity of insect trypsin. The main results were as follows:1. The promoter of trypsin gene was cloned successfully from the midgut of M. separata.According to the study, the nested specific primers were designed basing on the 5′ end of trypsin gene coding region, and the 1 863 bp 5′flanking promoter sequences of trypsin gene were cloned by Genome Walking from the midgut of M. separata after constructing Genome Walking Library. The sequence analysis from NCBI and online software NNPP v.2.2 indicated that this sequence had a strong homology of 73% with the transposon gene TRAS4 of silkworm, and contained TATA box, CAAT box, and other core promoter sequence. Furthermore, GATA, STAT, C/EBP and other transcriptional regulatory elements can also be searched in this sequence.2. The function of trypsin promoter from the midgut of M. separata was analized.The trypsin gene promoter fragment was cloned into the firefly luciferase reporter gene vector, and transfected into Spodoptera frugiperda(JE Smith)(Lepidoptera: Noctuidae) Sf21 cell lines companied with the renilla luciferase reporter gene vector with dual Luciferase Reporter Assay system. The transient expression analysis showed that the recombinant p(-1 673/+25)had a significant promoter activity, compaired with the pGL3-Basic vector, which also indicated the inserted fragment was indeed trypsin promoter from the midgut of M. separata.3. The effects of periplocosides on trypsin promoter from the midgut of M. separata were detected.The recombinant p(-1 673/+ 25)was transiently transfected into insect cells Sf21, then which were treated with different concentrations of periplocosides. Levels of reporter gene expression were detected after 5 hours, with the untreated group as a control. The results showed that the pharmaceutical treatments had no significant effects on the level of reporter gene expression, which indicated that these compounds had no significant effects on the activity of trypsin promoter, either.The sequence of nucleotide of trypsin promoter and its function were identified and clarified, and some structure features of the promoter fragment and possible transcription factor binding sites were also clarified. This research indicated trypsin promoter might not be the sites of action of periplocoside compounds directly, thereby laying the foundation for further research on the mechanism of action periplocoside compounds to trypsin in the level of transcription factors.