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Pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein and application

A serine protease, golden bee technology, applied in the direction of protease inhibitors, applications, biocides, etc.

Active Publication Date: 2018-11-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, with the continuous planting of insect-resistant Bt crops with only a single Bt gene, the problem of target pest resistance to it has attracted increasing attention, and this concern has been reported on the American cotton bollworm Helicopera zea in Bt cotton fields. signs

Method used

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  • Pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein and application
  • Pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein and application
  • Pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein and application

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Experimental program
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Embodiment 1

[0046] 1. Gene synthesis of the Kazal-type serine protease inhibitor PpSPI20 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 PpSPI20

[0049] After obtaining the full length of PpSPI20, use the full-length ORF (remove the signal peptide) to design primers for constructing prokaryotic expression vectors:

[0050] Forward primer pGEX-4T-2-PpSPI20-SP:

[0051] 5'-GATCTGGTTCCGCGTGGATCCGAGGGTGAGCCCAAACACT-3',

[0052] Reverse primer p...

Embodiment 2

[0075] 1. Construction of PpSPI20 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 PpSPI20 gene to form a complete expression framework. In this experiment, BamHI and SacⅠ were selected as restriction sites to replace the GUS gene with PpSPI20, so that the expression cassettes on both sides of the GUS gene were used to control the expression of the PpSPI20 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] PpSPI20-SP:5'-ACGCGTCGACGAGGGTGAGCCCAAACACT-3',

[0079] PpSPI20-AP:5'-CGCGGATCCTCAGTCGTCGCTCGTGTCT-3',

[0080] Among...

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Abstract

The invention discloses a pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein. The protein comprises an amino acid sequence shown as SEQ ID NO: 2. The invention further discloses genes coding the pteromalus puparum venom Kazal-type serine protease Inhibitor PpSPI20 protein. The genes comprise a nucleotide sequence at sites 70-246 in SEQ ID NO:1. The inhibitor disclosed bythe invention can be used for inhibiting activation of hemolymph PPO (Poly Phenylene Oxide) of imported cabbageworms or papilio xuthus. The invention further discloses a method for improving prevention ability of brassicaceous vegetables on lepidoptera pests. The method comprises the following steps: transforming brassicaceous vegetable cells by using the genes comprising the nucleotide sequenceshown as SEQ ID NO:1, and cultivating the transformed brassicaceous vegetable cells into plants.

Description

technical field [0001] The invention relates to the fields of molecular biology, genetic engineering and protein engineering. In particular, a Kazal-type serine protease inhibitor protein PpSPI20 expressed in the chrysalis chrysalis venom and its encoded nucleic acid sequence and application. Background technique [0002] Pests have always been a serious threat to the safe production of global crops, causing about a quarter of global food production to be reduced every year, resulting in huge economic losses. In my country, agricultural pests are also an important factor restricting the increase in agricultural production and the improvement of the quality of agricultural products. According to statistics, in 2014, the annual area of ​​pests and diseases in the country was about 5.5 billion mu per time. The annual grain loss restored through the prevention and control of diseases and insect pests accounts for about 15%-20% of the total output. Even so, the annual grain loss...

Claims

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Application Information

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IPC IPC(8): C07K14/81C12N15/15A01N47/44A01P7/04C12N15/82A01H5/00A01H6/20
CPCA01N47/44C07K14/8135C12N15/8286
Inventor 叶恭银杨蕾王嘉乐金虹霞方琦
Owner ZHEJIANG UNIV
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