Diamondback moth peptidoglycan recognition protein, preparation method and application thereof

A technology for moth peptidoglycan and recognition protein, which is applied in the field of diamondback moth peptidoglycan recognition protein and its preparation, and can solve the problems of no related reports and the like

Active Publication Date: 2015-04-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the peptidoglycan recognition protein and its recognition mechanism of Plutella xylostella

Method used

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  • Diamondback moth peptidoglycan recognition protein, preparation method and application thereof
  • Diamondback moth peptidoglycan recognition protein, preparation method and application thereof
  • Diamondback moth peptidoglycan recognition protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Cloning of Plutella xylostella peptidoglycan recognition protein (PGRP-S1) gene

[0029] S1. laboratory rearing diamondback moth ( Plutella xylostella ) larvae, Paecilomyces fumigatus ( Paecilomyces Fumosoroseus ) as the tested strain.

[0030] S2. Determination of the transcriptome sequence of Plutella xylostella: Collect the 1st to 4th instar larvae, prepupa, pupae, and adults of Plutella xylostella, extract the total RNA, and use RNA-seq technology to determine the transcriptome sequence. The extraction method of total RNA refers to Trizol (Invitrogen, USA) kit manual operation. A total of 34,522,812 clean reads were detected, the average length of the reads was 90bp, and 3,107,053,080 bases were obtained. Finally, 107710 Unigenes were assembled, with a length ranging from 200 to 3000 bp. After COG, GO, KEGG annotation and analysis, a total of 68,984 Unigenes with high annotation reliability were obtained, and 725 Unigenes directly related to inse...

Embodiment 2

[0050] Example 2 Temporal and spatial expression pattern of Plutella xylostella peptidoglycan recognition protein PGRP-S1

[0051] S1. Collect Plutella xylostella at different development stages, including eggs, 1st instar larvae; 2nd instar larvae; 3rd instar larvae; 4th instar larvae;

[0052] S2. The 4th instar larva was dissected, and the immune organs of fat body, blood cells, epidermis, midgut and Malpighian duct were collected respectively.

[0053] S3. Collect Plutella xylostella at different ages and tissues, add liquid nitrogen, use TAKARA’s RNAiso Plus extraction reagent, and follow the instructions to obtain total RNA at different ages and tissues.

[0054] S4. Synthesize cDNA of different ages according to the RACE kit instructions of CLONTECH company, add the following reagents at a time: 10 micrograms of total RNA, add 20 microliters of reaction solution and react at 42°C for 90 minutes. The reaction was terminated at 72°C for 10 minutes. Reaction composition:...

Embodiment 3

[0062] Example 3 Plutella xylostella PGRP-S1 is against Paecilomyces fumigatus ( P. fumosoroseus ) of the recognition effect

[0063] S1. Get the 4th instar Plutella xylostella larvae, collect the spores of I. fumigatus and dilute to 10 with sterile water. 5 Spores / mL, inject diamondback moth with a micro-syringe, about 8uL per head, and take 6 diamondback moths at 0h, 3h, 6h, 12h, 18h, 24h and 36h, respectively. Total RNA was extracted, cDNA was reverse transcribed, and it was used as a template to detect the expression of PGRP-S1 at different time points after infection by I. fumigatus. The experiment was repeated 3 times.

[0064] S2. Using the β-Actin gene (AB282645) of Plutella xylostella as an internal reference gene, fluorescent quantitative PCR primers were designed according to the sequences of PGRP-S1 and β-Actin of Plutella xylostella. The quantitative primer sequence for PGRP-S1 is:

[0065] qF: 5'-TCATCAACCACTCCGTGTCCCCC-3' (shown in SEQ ID NO: 17).

[00...

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Abstract

The present invention discloses diamondback moth peptidoglycan recognition protein, a preparation method and an application thereof, wherein the nucleotide sequence of the diamondback moth peptidoglycan recognition protein gene is represented by SEQIDNO:1-2, and semi-quantitative RT-PCR is adopted to detect temporal and spatial expression of diamondback moth PGRP-S1 in the body. According to the present invention, isaria fumosorosea is adopted to treat diamondback moth larva, quantitative RT-PCR is adopted to detect an immune recognition effect of the diamondback moth PGRP-SA gene on fungi paecilomyces fumosoroseus isaria, and the PGRP-S1 is the innate immune recognition receptor of diamondback moth, activates innate immunity, regulates antibacterial peptide expression, and provides a new target for prevention and control of diamondback moth from immunology.

Description

technical field [0001] The invention relates to the field of molecular microbiology, in particular to a diamondback moth peptidoglycan recognition protein and its preparation method and application. Background technique [0002] Plutella xylostella is a worldwide migratory pest, which mainly harms Brassicaceae plants such as cabbage, purple cabbage, broccoli, mustard greens, cauliflower, cabbage, rape, and radish. It has typical insect evolutionary advantages, such as small body, strong reproductive ability, strong ecological adaptability, etc. The most important thing is that it has developed strong resistance to pesticides. At present, it has developed resistance to many pesticides, such as imidacloprid and chlorpyrifos. , BT, Abamectin, etc. In the 1990s, diamondback moths were rampant in many places, and there was no cure. Due to the large occurrence area, long damage time and difficulty in control, diamondback moth gradually replaces cabbage caterpillar and becomes th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C07K14/435
Inventor 金丰良郑志华许小霞
Owner SOUTH CHINA AGRI UNIV
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