Development Of A Transgenic Sexing System Based On Female-specific Embryonic Lethality For The Oriental Fruit Fly,Bactrocera Dorsalis(Diptera: Tephritidae)

Insect pest control programs incorporating the sterile insect technique(SIT) has proven to be one of the most effective methods to eradicate Tephritid flies or prevent the colonization of the invasive species. To eliminate all females at rearing stage for male-only release, we would target on Bactrocera dorsalis, an economic important pest to the world agriculture, the genetic sexing systems based on female-specific embryonic lethality would be developed following the isolation and identification of specific regulatory elments, the construction of embryonic driver and female-specific effector using those specific regulatory elements and the development of genetic transformation technology of B. dorsalis. The main results are as follows:(1) The expression patterns of Bdvasa transcript measured by RT-q PCR indicated that Bdvasa transcript was a maternal effect gene, which was highly expressed in early embryos. A total of 1996 bp fragment from the translation start codons of Bdvasa was isolated by i PCR. Two drivers based on two-component tetracycline-repressible letal system were constructed using two different fragments of candidate promoter sequences of Bdvasa.(2) In order to isolate candidate dominant lethal gene, homologous cloning combined with RACE PCR strategy was performed to isolate cell death protease caspase-1 gene. Caspase-1, which was named as Bdcp-1, was 1679 bp in length, encoded 328 amino acids. The expression patterns of Bdcp-1 measured by RT-q PCR indicated that Bdcp-1 was transcribed during the different developmental stages and in different tissue of adults. The relative expression of Bdcp-1 transcript was highly related to the developmental stages. Besides, the expression level of Bdcp-1 increased by moderate UV stimuli, as the expression level of 8 days old pupae after irradiation by UV for 1 hours peaked to 5.6 folds, then followed by a decline after 1.5 hours irradiation with 5.2 folds. These results suggested that cell could maintain its normal activity in a way by sweeping away the damaged cells, but this mechanism was destroyed this mechanism by 2 hours irradiation. The female abdominal RNAi indicated that Bdcp-1 play an important role in female fertility, as knock-down of Bdcp-1 delayed ovarian development, inhibited the expression of Bdyp1, delayed female spawning and significantly reduced the accumulative amount of eggs.(3) The genomic and c DNA structure of Bdtra and Bdtra-2 amplified by PCR and RACE PCRs showed that Bdtra demonstrated sex-specific transcripts: one transcript in females and two transcripts in males. In contrast, Bdtra-2 only had one transcript that was common to males and females. The expression patterns of Bdtra and Bdtra-2 analyzed by semi-quantiative RT-PCR showed that Bdtra-2 and the female form of Bdtra were maternally inherited in eggs, whereas the male form of Bdtra was not detectable until embryos of 1 and 2 h after egg laying. Function analyses of Bdtra and Bdtra-2 indicated that both were indispensable for female development, as nearly 100% males were obtained with embryonic RNAi against either Bdtra or Bdtra-2. The female-specific splicing intron was 1068 bp in length, which was successfully used to the construction of female-specific lethal effector for transgenic sexing system.(4) In order to optimize the transformation technology and develop fluorescent sperm marking strain for B. dorsalis, the plasmid 1260 for fluorescent sperm marking of Ceratitis capitata was transformed to B. dorsalis by embryonic injection. Two kinds of fluorescent expression patterns were screened by fluorescent microscope. The transcripts of t GEP and Ds Red were detected in transformed individuals.