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Plutella xylostella serine protease inhibitor Serpin7 gene and application thereof

A protease inhibitor and serine protease technology, which is applied in the field of agricultural biology, can solve problems such as the serine protease inhibitor of Plutella xylostella, etc., and achieve the effect of good biological control potential and application prospect.

Active Publication Date: 2020-12-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent CN107523572A discloses serine protease inhibitor CvT-SPI gene and application of Plutella xylostella xylostella teratogenic cells, mainly about serine protease inhibitors secreted extracellularly by parasitic wasp natural enemy teratogenic cells of Plutella xylostella; in addition, Zhu Ni (2013 ) disclosed the research on the cloning, transcription and enzymatic activity of the midgut serine protease gene of Plutella xylostella xylostella, but did not involve serine protease inhibitors in Plutella xylostella; at present, the research on the immune-related serine protease inhibitors contained in Plutella xylostella itself There are few reports

Method used

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  • Plutella xylostella serine protease inhibitor Serpin7 gene and application thereof
  • Plutella xylostella serine protease inhibitor Serpin7 gene and application thereof
  • Plutella xylostella serine protease inhibitor Serpin7 gene and application thereof

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Embodiment 1

[0040] Cloning and sequence analysis of embodiment 1 Plutella xylostella Serpin7 gene

[0041] 1. Extraction of Plutella xylostella total RNA and synthesis of cDNA first strand

[0042] Take 50-100 mg of the 4th instar larvae of Plutella xylostella xylostella, grind it with liquid nitrogen, and use TRIzol reagent (Invitrogen, USA) to extract total RNA. The specific operation is carried out according to the instructions.

[0043] The quality and concentration of the extracted RNA were detected. After the standard concentration was determined, the first-strand cDNA was synthesized according to the instructions of the reverse transcription kit (TaKaRa company) and the 5'-cDNA and 3'-cDNA were synthesized according to the instructions of the RACE kit (Clontech company).

[0044] 2. Cloning of the full-length cDNA sequence of Plutella xylostella Serpin7

[0045] Primers Serpin7-F and Serpin7-R were designed according to the Unigene fragment sequence of Serpin7 obtained from the Pl...

Embodiment 2

[0051] Construction of the prokaryotic expression plasmid of embodiment 2 Plutella xylostella Serpin7

[0052] A pair of specific primers were designed according to the ORF (open reading frame) sequence of Plutella xylostella Serpin7 gene (Table 1). In order to facilitate the cloning of the PCR product into the expression vector pET32a, an EcoRI restriction site was designed in the upstream primer, and an XhoI restriction site was designed in the downstream primer. The PCR product was double-digested with EcoRI and XhoI, recovered and purified by tapping the gel, ligated with the expression plasmid pET32a that had been digested with EcoRI / XhoI to construct the prokaryotic expression plasmid pET32a-Serpin7, and transformed into Escherichia coli DH5α, and the sequence was identified by enzyme digestion and sequencing. Accuracy of enzyme cutting sites. The correct monoclonal was identified by enzyme digestion and sequencing, the plasmid was extracted, transformed into the expres...

Embodiment 3

[0053] Example 3 Prokaryotic Expression of Plutella xylostella Serpin7 and Preparation of Polyclonal Antibody

[0054] The single clone was inoculated in 10 mL LB medium (containing 100 μg / mL ampicillin Amp) for overnight culture, and then diluted 1:100 the next day to continue culture at 37 °C, when A 600= 0.6, adding IPTG (isopropylβ-D-1-thiogalactopyranoside) with a final concentration of 0.5mmol / L to induce the expression of the fusion protein. After culturing at 37°C for 4 hours, the culture was collected by centrifugation at 12,000 rpm at 4°C, and the supernatant was discarded. Use bacterial lysate (1.5% sodium lauryl sulfate, 1mM PMSF, 1% TritonX-100, 1mg / mL lysozyme) for the precipitate, lyse on ice for 30min, and then ultrasonicate intermittently until the bacterial solution is clear; 12000rpm, 4°C The supernatant was collected by centrifugation, and the target protein was purified in one step according to Ni-NTA Sefinose Resin (Sangon Biotech). The purification proc...

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Abstract

The invention discloses a plutella xylostella serine protease inhibitor Serpin7 gene and an application thereof. A nucleotide sequence of the plutella xylostella serine protease inhibitor Serpin7 geneis shown as SEQ ID NO:1, and an amino acid sequence of the plutella xylostella serine protease inhibitor Serpin7 is shown as SEQ ID NO:2. After the recombinant protein Serpin7 is added for eating, the activity of plutella xylostella phenol oxidase PO can be strongly inhibited; after a polyclonal antibody anti-Serpin7 of Serpin7 is added for eating, the activity of phenol oxidase PO can be remarkably improved; and after RNAi silences the expression of the Serpin7 gene, the activity of plutella xylostella phenol oxidase PO is improved, and plutella xylostella larvae generate the strong melanization reaction, so that most plutella xylostella cannot normally pupate, and those which can pupate cannot successfully emerge. The result shows that the plutella xylostella Serpin7 is a negative regulatory factor of innate immunity in insects, can be used as a novel molecular target for biological prevention and treatment, is used for preventing and controlling cruciferous vegetable pests, and hasgood biological prevention and treatment potential and application prospect.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a serine protease inhibitor Serpin7 gene of diamondback moth and application thereof. Background technique [0002] Serine protease inhibitors (Serine protease inhibitors, Serpins) are widely distributed in plants, animals, microorganisms, etc., and are regulators of serine protease activity. According to their sequence homology, cysteine ​​residues and the number of disulfide bonds in the molecule, serine protease inhibitors can be divided into Kunitz, Kazal, Bowman-Birk, Serpin, TIL (Trypsin Inhibitor Like Cysteine ​​Rich Domain), etc. Several families. Serine protease inhibitors participate in the regulation of a series of physiological and pathological processes in organisms, such as blood coagulation, fibrinolysis, complement activation, infection, cell migration, etc., play a key regulatory role, thereby maintaining the homeostasis of life. Serine protease inhibi...

Claims

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Application Information

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IPC IPC(8): C12N15/15C12N15/113C12N15/82C07K14/81A01N57/16A01P7/04A01H5/00A01H6/20
CPCC07K14/8121C12N15/113C12N15/8286A01N57/16C12N2310/14
Inventor 许小霞金丰良张展滔曾路
Owner SOUTH CHINA AGRI UNIV
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