A previous team has produced two moth sex pheremones (Z)-11-hexadecen-1-ol and (Z)-11-hexadencyl acetate using N. benthamiana. They had shown successful implementation of the gene sequences necessary to produce the core enzymes in the biosynthetic pathway that produces their group’s target pheromone, but utilizing agroinfiltration in N. benthamiana meant that pheromone expression was temporary. This could create logistical problems with the implementation of their modified plant, especially in environments where consistent implementation over a large area would be desired, like fruit tree farms. Consequently, the Missouri Miners sought to develop two composite parts that would consist of the two vectors necessary for disparlure production, which would be able to be stably expressed in yeast. The vectors contain a constitutive promoter (TDH3), terminator (ADH1), and their respective gene sequences (coding for reductase and desaturase transcription, the two main enzymes in the biosynthetic pathway). The gene sequence for desaturase transcription (see gene sequence one) was obtained by blasting the turnip moth annotated gene sequence for desaturase production1 against the whole spongy moth genome2. The gene sequence for reductase transcription (see gene sequence two) was obtained by blasting sequence from the cotton bollworm annotated for reductase production3 against the same spongy moth genome2. Each completed vector was then placed inside of a backbone consisting of both E.coli and yeast transcriptional replication sites. Upon completion, the Missouri Miners would have developed a composite part that allowed for rapid replication of the desired gene sequences in E.coli utilizing their high replication rates, then transformation to yeast cultures that naturally produce the glycoproteins critical to the biosynthetic pathway for disparlure production for field implementation.
Gene Sequence One - Desaturase:
TTGGCAGAAATAGGAATAACGGCTGGAGCACATCGCCTTTGGACACATAAGGCATACAAAGCAAAGTTGCCCTTACAAATTCTTCTAATGCTTATG AACTCTGTGGCTTTTCAAAATACTGTAACAGATTGGGTCAGAGATCATCGTCTTCATCATAAATGTAGCGATACAGATGCTGATCCTCATAATGCTAGT CGGGGATTCTTCTTCTCTCATATTGGATGGTTATTAACGAGAAAGCATCCTGATGTTAAGAAGAATGGAAAAAATATTGATATGTCTGATATTTTAAATA ACCCAGTGTTAAAATTTCAAAAAAAGTGAG-TATGAGCCTTCAT
Gene Sequence Two - Reductase:
ATGCTCATTATTTGTTTCAGTACTGACGCAACTAAAAATATGCATTCTAATGACAAGCCTGTTAAAATCAAAAGCGAATCTGTTGCTGACTTTTATGCGGG ACAATCAGTATTTATCACAGGCGGCACCGGTTTCCTTGGAAAGGTAA