Development Of Biotechnological Approaches To Improve Sterile Insect Technique (SIT) Against The Oriental Fruit Fly, Bactrocera Dorsalis (Diptera: Tephritidae)

Tephritid fruit flies destroy fruits and vegetables, causing tremendous economic losses and bringing a barrier to international trades of fruits, vegetables and their associated products. The Sterile Insect Technique(SIT), a target-specific and environment-friendly pest control method, has been used to combat several insect pests for over five decades and has been proven to be quite effective against tephritids. The oriental fruit fly, Bactrocera dorsalis(Diptera: Tephritidae), is a world-wide quarantine pest. With the characteristics of wide host range, rapid population dispersion and high adaptability, it has brought serious threats to economic important crops such as fruits and vegetables in China. SIT can be a good way for effectively management or even eradication of B. dorsalis, however, sterile insects which classically achieved by irradiation often become weak and unfit to compete with the wild mates.Although SIT has achieved great success, improvements to the various technical aspects are constantly made as that will shape the method into a highly efficient, safer and cheaper program to execute. Biotechnology has a great potential to bring desired improvements to SIT in ways that may be difficult to achieve by other means.In this study, based on the analysis of existed transgenic lethality or sterility system to improve SIT, the strategy for establishment of transgenic approaches to improve SIT against the oriental fruit fly, Bactrocera dorsalis(Diptera: Tephritidae) was developed. The genetic elements required were isolated, and two driver constructs, one effector construct and two sperm-specific marking constructs were cloned, all of which above laid a good foundation for establishing the transgenic lethal strains in B. dorsalis. The main results are displayed as follows:1. Full-length cDNAs of three maternal effect genes, nanos, vasa and hunchback were cloned by RT-PCR and RACE. The results showed their conserved regions and high homology with other insects.2. Based on the newly cloned cDNA sequence of Bdnos, a 1964 bp 5’ upstream region including the 5’UTR region was isolated by two round inverse PCR. The embryonic driver construct pBac{PUbDsRed.T3_Bdnos-t TA}(WQ3) was constructed with the 906 bp predicted promoter sequence.3. Based on the known cDNA sequence of Bdb2 t, a 1591 bp 5’ upstream region including the 5’UTR region was isolated by inverse PCR. The male sterile driver construct pBac{PUb-DsRed.T3_ Bdb2tub-tTA}(WQ4) was cloned.4. The MAPK phosphorylation sites of the putative protein of an apoptosis gene hid(head involution defective) was analyzed and mutated. The leathal effector construct pBac{TREhs43-BdhidAla2_att P_Pub-EGFP}(WQ16) was cloned based on the mutation.5. Two transgenic constructs for fluorescent sperm marking pBac{Bdb2tub-DsRed_PUb-nls-EGFP}(WQ18) and pBac{ Bdb2tub-tGFP_PUb-DsRed.T3}(WQ21) were separately cloned with the newly isolated promoter sequence of sperm-specific expressed gene Bdβ2-tubulin.6. The full-length cDNAs of transformer and transformer-2 were cloned by RT-PCR and RACE. The results show their conserved regions and high homology between B. dorsalis and other insects. Their introns were also isolated by PCR with genomic DNA. The sex-specific spliced versions of transformer were identified.7. A new driver construct pBac{Ccvasa-tTA_Pub-DsRed}(AH437) was transformed in Medfly with the helper plasmid pSL{Dmhsp70-hyPBase}(CO1409) by microinjection into fresh embryos to get a new embryonic driver line. The red fluoresce was observed in some of their progenies.