Plutella xylostella trypsin-9 gene and application thereof

A technology of trypsin and diamondback moth, which is applied in the field of agricultural biology to achieve the effects of increased mortality, good biological control potential and application prospects

Active Publication Date: 2020-10-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports about trypsin-related genes in diamondback moth

Method used

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  • Plutella xylostella trypsin-9 gene and application thereof
  • Plutella xylostella trypsin-9 gene and application thereof
  • Plutella xylostella trypsin-9 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of the CDS Fragment of Example 1 Plutella xylostella Trypsin-9 Gene

[0037] The total RNA of the midgut of Plutella xylostella was extracted by the Trizol method, and one strand of cDNA was synthesized by reverse transcription, and PCR was cloned using this as a template. The primer sequence of the target gene including the entire CDS region was F: 5'-AGCATAATTCAAATTCAGTTACC-3' , R: 5'-AACATTTCTCCTACGCGTTTGTG-3'; The PCR product was separated by 1% agarose gel electrophoresis, recovered and purified, and its sequence size was 820bp after analysis. The recovered PCR product was ligated into the pMD18-T vector, and the ligated product was transformed into a competent cell DH5α, and the positive cloned strain was sent for sequencing identification. The correct strains identified by sequencing were stored at -80°C as glycerol bacteria.

Embodiment 2

[0038] Example 2 Prokaryotic expression and recombinant protein purification of diamondback moth Trypsin-9 gene

[0039] The cDNA obtained above was used as a template for PCR cloning, and a pair of specific amplification primers were designed according to the ORF sequence of the Plutella xylostella Trypsin-9 gene, and the enzyme cutting sites EcoRI (GAATTC) and HindIII (AAGCTT) were introduced into the Trypsin-9 gene. Primer sequence is F: 5'-CG GAATTC AGGATTGTGGGTGGATCCGC-3' (the underline indicates the designed EcoRI restriction site); R: 5'-CC AAGCTT CGCGTTTGTGCGAATCCAGT-3' (the underline indicates the designed HindIII restriction site). Using the recombinant plasmid carrying the Trypsin-9 CDS fragment as a template, PCR amplification was carried out to obtain the Trypsin-9 gene expression fragment. The PCR product was separated by 1% agarose gel electrophoresis and recovered and purified. The recovered PCR product and the pET-32a empty vector were subjected to double ...

Embodiment 3

[0041] Example 3 Different concentrations of Plutella xylostella midgut enzyme solution degrades Cry1Ac protoxin

[0042] 1. Extraction of Cry1Ac protoxin

[0043] In this experiment, Bt HD-73 strain was used as the material to extract the protoxin protein of Cry1Ac. The specific steps are as follows:

[0044] (1) Pick a single colony from the HD-73 strain colony plate and activate it overnight on LB liquid medium (30°C, 200rpm).

[0045] (2) Transfer to 1 / 2LB liquid medium (yeast powder in LB medium is halved) at a ratio of 1:100 (30°C, 200rpm, 36h) for shaking culture to obtain a crystal vegetative mixture.

[0046] (3) Wash twice with PBS, lyse with lysate, and centrifuge at 3000 rpm for 30 min at 4°C.

[0047] (4) Precipitate the supernatant with NaNc-HAc isoelectric point, then centrifuge (4°C, 3000rpm, 30min)

[0048] (5) The obtained precipitate is crystal protein, which is dissolved in the solution. The concentration of the extracted protoxin was determined by aliq...

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Abstract

The invention discloses a plutella xylostella trypsin-9 gene and application thereof; the nucleotide sequence of the plutella xylostella trypsin-9 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the gene is shown as SEQ ID NO: 2. Researches show that Trypsin-9 plays an important role in activation of Cry1Ac prototoxin in the midgut of plutella xylostella, the activation of Bt Cry1Acprototoxin in the midgut of plutella xylostella can be remarkably improved by feeding Trypsin-9, and the mortality rate of plutella xylostella is remarkably increased; after the expression of the geneTrypsin-9 of the plutella xylostella in the midgut is silenced, the digestion of the midgut of the plutella xylostella to food can be reduced, and pupation malformation is caused, so that the Trypsin-9 can be used as a molecular target for novel biological control, is used for preventing and controlling cruciferous vegetable pests, and has high biological control potential and application prospect.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, more specifically, a Plutella xylostella trypsin-9 gene and application thereof. Background technique [0002] Trypsin belongs to the serine protease family and is produced in the pancreas of animals in the form of zymogen precursor (Pre-trypsinogen). It is a major proteolytic enzyme in the digestive tract of vertebrates. Activated by enterokinase through the intestinal lumen to generate active trypsin, its main function is to digest protein in food; activate other zymogens such as (chymotrypsinogen, shuttlepinase, elastaseogen). [0003] Insect trypsin is produced and secreted into the midgut by the intestinal wall cells of the midgut. It is mainly detected in the midgut of larvae during the feeding period. It is the main serine protease in the alkaline midgut environment of insects. Plays an important role in the regulation of Bt toxin toxicity. Its secretion and activation mechanism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/76C12N15/70C12N1/21A01N63/50A01N63/23A01P7/04C12R1/19
CPCC12N9/6408C12Y304/21004C12N15/70A01N63/50A01N63/23Y02A50/30
Inventor 金丰良许小霞花艳艳李树忠
Owner SOUTH CHINA AGRI UNIV
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