2013 Annual Report
1a.Objectives (from AD-416):
To isolate new antioxidant gene sequences from tephritid species and to evaluate them by functional genomic analysis for eventual use in transgenic strains for improved biological control.
1b.Approach (from AD-416):
New antioxidant gene sequences, including those for superoxide dismutase I, superoxide dismutase II, and catalase, will be isolated by degenerate polymerase chain reaction (PCR) protocols in Anastrepha suspensa and Ceratitis capitata, based on homologous sequences in dipteran species. Complete genomic sequences placed under conditional regulation will inserted into recombinase-based vectors and individually transformed into the tephritid species by recombination into genomic target sites. With assistance from the Cooperator, lines homozygous for the transgenes will be created and adult males will be assayed for antioxidant gene expression and enzymatic activity. Untreated males and males sterilized by gamma-irradiation will be tested for life fitness and reproductive competitiveness parameters.
Specifically, antioxidant genes will be isolated from the Caribbean and Mediterranean fruit fly genomes and their expression and enzymatic activity tested after genetic transformation in host species. Fitness and reproductive competitiveness parameters will be evaluated in adult transgenic males to assess whether ectopic expression of these genes have a beneficial effect on viability and mating behavior in males sterilized for the sterile insect technique.
This research relates directly to Objective 1. Genetics: Identify developmentally significant genes from whole genome and transcriptome sequencing projects that may be targeted or manipulated in transgenic and nontransgenic insect strains for biological control. Test conditional lethal systems using cell death genes and microRNAs targeted to embryos and vital processes in tephritids and lepidopterans and develop germ-line transformation for the cactus moth and Asian citrus psyllid.
To study antioxidant gene activity and its effect on irradiated males used in the sterile insect technique, cognates to the Drosophila melanogaster superoxide dismutase I and II genes were isolated by degenerate PCR from the Caribbean fruit fly, Anastrepha suspensa. The AsSOD I gene was then placed under heat shock promoter control and transformed into wild type A. suspensa flies. Analysis of the transgene structure and expression have been initiated.