Progress 09/01/02 to 08/31/06
Outputs
The overall goal of the project is to study the structure, function and biosynthesis of insect neuropeptides, to improve crop quality and productivity and the sustainability of agriculture by developing novel methods to control insect pests. There are two main objectives in the project: 1) To clone the gene that encodes the proprotein convertase and 2) To study the expression pattern and the functions of the proprotein convertase. Using PCR and a 5-RACE (Rapid Amplification of cDNA Ends) technique, we have cloned two fragments that correspond to the catalytic region and 5-end of a gene that encodes a proprotein convertase-like protein. Further efforts to use a 3-RACE technique to study the 3-end of this gene were unsuccessful. We then isolated the mRNA from 2-days-old virgin female brain and subesophageal ganglion and synthesized cDNA from these mRNA. The cDNAs were subcloned into pDONR222 plasmid library vectors which were then electroporated into DH10B T1 Phage
Resistant cells (CloneMiner cDNA Library Construction Kit, Invitrogen Corporation). The resulting library was screened with digoxygenin-labeled probes that represent the DNA sequence of the catalytic regions of a PCR fragment. Positive clones were identified and we are in the process of characterizing the clones to learn more about the DNA sequence of the gene.
Impacts
Studying processing enzymes would reveal the biosynthesis of neuropeptides in insects that could lead to development of novel methods in insect pest control by interferring with the biosynthesis of neuropeptides at critical periods of insect reproduction.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/30/04
Outputs
The overall goal of the project is to study the structure, function and biosynthesis of insect neuropeptides, to improve crop quality and productivity and the sustainability of agriculture by developing novel methods to control insect pest. The aim of the project is to isolate and characterize a processing enzyme gene that could be involved in the processing of a neuropeptide precusor. Messenger RNA was isolated from the central nervous system of corn earworm female moths. Custom designed oligonucleotide primers were used to perform PCR to isolate the 3' and 5' ends of the gene. Sequence analyses of the isolated ends of the gene indicates that approximately 0.5 kb of the 5' end sequence is missing. To further confirm this observation, we constructed a complementary DNA library of the central nervous system and the library will be screened using labeled 3' and 5' end fragments. Once positive clones are isolated and characterized, we will perform the expression analyzes
as described in the proposal.
Impacts
Information on the proprotein convertase-like enzyme could aid in the design of a specific and enironmentally safe pesticide that would aim to disrupt the pest physiology.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The objective of the project is to clone, express and characterize a proprotein convertase enzyme that could be involved in the biosyntehsis of an insect neruopeptide named PBAN (pheromone biosynthesis activating neruopeptide). Polymerase chain reaction was used to amplify the catalytic region of a proprotein convertase-like enzyme from cDNA that were isolated from the brain-subesophageal ganglion complex of female corn earworm. Five prime and three prime (rapid amplification of cDNA ends) was used to isolate the five prime and three prime ends of the cDNA that overlaps with the DNA sequence of the previously isolated catalytic region of the proprotein convertase-like enzyme. Fragments that correspond to the of the five prime and three prime end of this cDNA sequence were isolated, and sequence analyses of these fragments showed that they overlap with the previously isolated catalytic region DNA sequence. Experiments are currently underway to sequence these DNA
fragments and Northern blotting will be used to determine if the full length cDNA sequence was isolated. Subsequently, the entire cDNA sequence will be subcloned into wild-type baculovirus to express the protein to evaluate the biological activity of the expressed protein.
Impacts
Information on the proprotein convertase-like enzyme could aid in the design of specific and enironmentally safe pesticide that would aim to disrupt the pest physiology
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The objective of this investigation is to isolate and characterize the SPC (subtilisin-like proprotein convertase) encoding gene(s) in the central nervous system of the corn earworm, Helicoverpa zea. Funding was received in 9/02 to hire a postdoctoral associate to take charge of the research. Dr. Jorigtoo Chen, an insect biochemist from Oklahoma State Unviversity is hired to fill the position and he is currently waiting for his employment authorization from the U.S. Immigration Department. At the meantime, 5' RACE (Rapid Amplification of cDNA Ends) was used to isolate the 5' end of a gene fragment that showed high homology to the catalytic region of the SPC encoding gene of other organisms including an insect. Currently, we have a partial sequence of the 5' end of a SPC-encoding gene from H. zea. We are in the process to obtain further sequence information at both the 5' end and 3' end of this SPC-encoding gene.
Impacts
The investigation could lead to development of biorational-insecticide that specifically targets neuroendocrine functions in pest insects.
Publications
- No publications reported this period
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