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A kind of serine protease inhibitor serpin7 gene of diamondback moth and its application

A protease inhibitor, moth serine technology, which is applied in the field of agricultural biology, can solve the problem of not involving the diamondback moth serine protease inhibitor, etc., and achieves the effect of good biological control potential and application prospect.

Active Publication Date: 2022-04-29
SOUTH CHINA AGRI UNIV
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  • 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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  • A kind of serine protease inhibitor serpin7 gene of diamondback moth and its application
  • A kind of serine protease inhibitor serpin7 gene of diamondback moth and its application
  • A kind of serine protease inhibitor serpin7 gene of diamondback moth and its application

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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 with liquid nitrogen, and then use TRIzol reagent (Invitrogen, USA) to extract total RNA, and 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 ...

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 monoclonals were identified by enzyme digestion and sequencing, the plasmids were extracted, transformed into 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 Serpin7 gene of a diamondback moth serine protease inhibitor and an application thereof. The nucleotide sequence of the Serpin7 gene of the diamondback moth serine protease inhibitor is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2 shown. Feeding the recombinant protein Serpin7 can strongly inhibit the PO activity of the diamondback moth phenol oxidase; feeding the Serpin7 polyclonal antibody anti-Serpin7 can significantly increase the phenol oxidase PO activity; after RNAi silences the Serpin7 gene expression, the diamondback moth phenol oxidase PO activity increases High, the larvae of the diamondback moth produced a strong blackening reaction, which caused most of the diamondback moths to fail to pupate normally, and those who could pupate could not successfully emerge. It is indicated that Serpin7 of diamondback moth is a negative regulator of innate immunity in insects, and can be used as a molecular target for new biological control for the control of cruciferous vegetable pests, and has good biological control potential and application prospects.

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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Patent Type & Authority Patents(China)
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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