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Clones And Function Studies Of Neuropeptide Y Like Peptide In Ostrinia Furnacalis(Guenée) And Helicoverpa Armigera(Hübner)

Posted on:2017-04-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z YueFull Text:PDF
GTID:1363330482492513Subject:Agricultural Entomology and Pest Control
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Neuropeptides,a group of chemically diverse signal molecules,are important regulators of physiology and behavior in insecta.The Asian corn borer(Ostrinia furnacalis)and cotton bollworm(Helicoverpa armigera)are both important agricultural pest major pest.Thus to explore an alternative way for controlling the pest is necessary.In this study,Illumina sequencing technology and RNAi were used to sequence and explore the function of neuropeptides gene.The results are summarized as follows:1.In this study,Illumina sequencing technology was used and 30,742,176 clean reads were acquired in the Asian com borer,Ostrinia furnacalis(Guene).After a de novo assembly,115,926 transcripts were obtained,and 76,232 transcripts were annotated based on 6 main database.A total of 48,731 unique transcripts were assigned to 66 level-2 GO terms,and 28,138 transcripts were assigned to 298 predicted pathways.Furthermore,14 gene families associated with Insulin Pathways were selected,10 of which could found in O.furnacalis library.A total of 423 transcripts involved in 10 gene families were then assembled into 152 unigenes,and 21 of them were verified in BlastX,including two NPF,four NPFR,one ILP/inR,two PI3K,three PTEN,one S6K,two PKA,one AKHR,tow GPCR,and three PKs,respectively·2.The full O.furnacalis npf/npy open reading frames(ORF)were sequenced.The npf contained two splicing variants,O.furnpf1 and O.furnpf2,which were identified at the cDNA level.O.furnpf1 and O.furnpf2 contain an ORF of 246 bp and 366 bp,respectively.O.furnpy contains an ORF of 273 bp.The spatio-temporal expressions of O.furnpf/npy were explored by qPCR.The results showed that it was significantly higher at the 1th instar larvae,and decreased to a lowest point at the 2th instar larvae.Afterwards,it gradually increased from the 2th and attained to highest at the 5th instar along with increases of feeding amount.Furthermore,the O.furnpf and O.furnpy were mainly expressed in midgut,which was significantly higher than other tissues.Most importantly,when 5th larvae were starved for 6h,the O.furnpf2 expression was significantly increases in midgut which was matched with significant increases of feeding amount in starved larvae.These results together testified that O.furnpf and O.furnpy were involved in modulating feeding behavior.3.When knocked down the expression of O.fur npf/npy gene by feeing or injecting dsRNA,larval food intake and weight gain were inhibited except for npf1.For further understanding the regulatory mechanism of the NPF2 on feeding,we analyzed the relationship between NPF2 and energy metabolism.Our results showed that glycogen and total lipid levels drastically decreased in dsNPF2 treated larvae,On the contrary,trehalose content in dsNPF2 treated larvae signiticantly raised.These results suggest that NPF2 regulates feeding behavior through a pathway of energy metabolism,in which dsNPF2 inhibits biosynthesis and promotes metabolism.4.RNAi expression vector about NPY/NPF2/GFP was respectively constructed for researching on transgenic plant and illuminating how NPY/NPF2 gene function on food consumption.This also provided a new way to cultivating insect-resistant transgenic plant with more environmental friendly.5.Knock down of H.armigera larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight,and dsNPF also caused a decrease of glycogen and an increase of trehalose.Moreover,we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expresseddsNPF.Results showed that H.armigera larvae fed on these transgenic plants or leaves had lower food consumption,body size and body weight compared to controls.These results indicate that NPF is important in the control of feeding of H.armigera and valuable for production of potential transgenic cotton.
Keywords/Search Tags:Ostrinia furnacalis(Guenée), Helicoverpa armigera(Hübner), NPY like Neuropeptides, function, RNAi
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