Pteromalus puparum venom Kazal-type serine protease inhibitor protein PpSPI24 and application
A technology of serine protease and golden bee, applied in the direction of protease inhibitors, applications, angiosperms/flowering plants, etc.
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Embodiment 1
[0046] 1. Gene synthesis of the Kazal-type serine protease inhibitor PpSPI24 from the chrysalis golden wasp venom:
[0047] According to the gene sequence obtained from the genome data of Chrysalis chrysalis ----- the sequence described in SEQ ID NO: 1, the signal peptide was predicted online by using SignalP 4.1. Gene sequence design and synthesis of single-stranded oligo, using PCR to splice the synthesized oligo into a complete gene sequence. Connected to the carrier PGEM-Teasy (Promega), screened by blue and white spots, picked positive clones, and sent them to Shanghai Boshang Company for sequencing inspection.
[0048] 2. Prokaryotic expression and purification of PpSPI24
[0049] After obtaining the full length of PpSPI24, use the full-length ORF (remove the signal peptide) to design primers for constructing prokaryotic expression vectors:
[0050] Forward primer pGEX-4T-2-PpSPI24-SP:
[0051] 5'-GATCTGGTTCCGCGTGGATCCTTTTACCGACCAGGACGGCA-3',
[0052] Reverse primer ...
Embodiment 2
[0075] 1. Construction of PpSPI24 gene plant binary expression vector
[0076] The cauliflower mosaic virus CaMV 35S promoter and NOS terminator on both sides of the GUS gene in the plant binary expression vector pBI121 were used to insert the PpSPI24 gene to form a complete expression framework. In this experiment, BamHI and SacⅠ were selected as restriction sites to replace the GUS gene with PpSPI24, so that the expression cassettes on both sides of the GUS gene were used to control the expression of the PpSPI24 gene in Arabidopsis plants.
[0077] Primers with BamHI and SacⅠ restriction sites were designed according to the ORF of the serine protease inhibitor of the venom of A. chrysalis, and the plant expression vector was constructed by PCR amplification using the cDNA of the venom gland of A. chrysalis as a template. The primer sequences are:
[0078] PpSPI24-SP:5'-ACGCGTCGACTTTACCGACCAGGACGGCA-3',
[0079] PpSPI24-AP:5'-CGCGGATCCTCAGCATTTGCCTTCGAACC-3',
[0080] Amon...
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