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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.

Active Publication Date: 2018-11-16
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 protein PpSPI24 and application
  • Pteromalus puparum venom Kazal-type serine protease inhibitor protein PpSPI24 and application
  • Pteromalus puparum venom Kazal-type serine protease inhibitor protein PpSPI24 and application

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Experimental program
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Effect test

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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Abstract

The invention discloses a pteromalus puparum venom Kazal-type serine protease inhibitor protein PpSPI24 with an amino acid sequence as shown in SEQ ID NO:2. The invention further discloses a gene forcoding the chrysalis nasonia venom Kazal-type serine protease inhibitor protein PpSPI24, and the gene has nucleotide sequences of sites 70-231 in SEQ ID NO:1. The inhibitor can be used for inhibitingactivation of imported cabbageworm or papilio xuthus linnaeus hemolymph PPO. The invention further discloses a method of improving prevention force, on lepidoptera pests, of brassicaceous vegetables,and the method comprises the following steps: converting cells of the brassicaceous vegetables by the gene with the nucleotide sequences as shown in SEQ ID NO:1; and cultivating the converted cells ofthe brassicaceous vegetables 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 PpSPI24 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/15A01H5/00A01H6/20
CPCC07K14/8135C12N15/8286
Inventor 叶恭银杨蕾王嘉乐金虹霞方琦
Owner ZHEJIANG UNIV
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