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Prawn QM protein for regulating phenol oxidizing enzymes (PO) activity by interactively acting with hemocyanin

A technology of hemocyanin and prawns, applied in medical raw materials and antiviral agents derived from Ginkgo biloba, can solve problems such as decreased yeast protein synthesis, abnormal cytoskeleton, growth and division arrest, etc.

Inactive Publication Date: 2008-09-03
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In yeast, the QM homologous gene GRC5-Q.R1 is related to mitochondrial respiration, and QM gene mutations can lead to decreased protein synthesis, growth and division arrest, and cytoskeleton abnormalities in yeast, and the deletion of QM genes can lead to yeast death; In chicken, the QM homologous gene Jif-1 can bind to the protein product of the oncogene c-Jun to inhibit the transactivation transcriptional regulation activity of c-Jun, affecting cell growth and proliferation
Although the functions of many QM proteins have been reported, their specific functions in the immune response are not very clear

Method used

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  • Prawn QM protein for regulating phenol oxidizing enzymes (PO) activity by interactively acting with hemocyanin
  • Prawn QM protein for regulating phenol oxidizing enzymes (PO) activity by interactively acting with hemocyanin
  • Prawn QM protein for regulating phenol oxidizing enzymes (PO) activity by interactively acting with hemocyanin

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Embodiment 1

[0035] Structure of the QM gene

[0036] According to the results of suppression subtractive hybridization (SSH) in our laboratory, a gene fragment with high homology to QM was up-regulated in the disease-resistant shrimp. In order to study the function of the QM gene, the RACE technique ( figure 1 ), obtained the full-length cDNA sequence of QM gene, GenBank accession No.EU004069 ( figure 2 ). Since this gene comes from Peneaus japonicus, we named it PjQM according to the source. After sequencing the obtained DNA fragment of about 1000bp, homology comparison analysis found that the homology of shrimp QM protein with human, mouse, Drosophila and brewer's yeast was 76.23%, 76.82%, 78.64% and 62.44%, respectively.

[0037] See attached figure 1 RACE analysis to obtain QM cDNA A: 5'RACE product, B: Colony PCR

[0038] After analyzing the cDNA, it was found that the PjQM gene contained an open reading frame (open reading frame, ORF) with a length of 663bp, which was predicte...

Embodiment 2

[0041] Transcription and expression analysis of 2PjQM gene in shrimp

[0042] After extracting total RNA from the muscle, heart, hepatopancreas, digestive tract, blood, and gills of Penaeus japonicus, reverse transcription was performed using OligdT(18) as a reverse transcription primer, and the cDNA of each tissue was used as a template to amplify the PjQM gene , the results showed that the PjQM gene could be amplified in muscle, heart, hepatopancreas, digestive tract, blood and gills, indicating that PjQM was transcribed in every detected tissue ( image 3 ), without tissue specificity at the transcriptional level.

[0043] attached image 3 RT-PCR analysis of PjQM gene transcription tissue specificity, in which

[0044] 1: DNA marker; 2: Negative control; 3-8: Muscle, heart, hepatopancreas, digestive tract, blood, gill, respectively.

[0045] Muscle, heart, hepatopancreas, digestive tract, blood, gills and other tissues of Penaeus japonicus were homogenized, then SDS-PAG...

Embodiment 3

[0048] 1. The role of QM protein in shrimp immunity

[0049] 3.1 The effect of virus stimulation on the expression of QM in shrimp

[0050]Penaeus japonicus was injected with WSSV, and the total RNA of the hepatopancreas was extracted at 0h, 4h, 8h, 12h, 24h, 48h and 72h after infection, and the cDNA was reverse-transcribed with Olig(dT) as a primer. Using 18S RNA as a control, the real-time PCR method was used to study the changes in the transcriptional level of PjQM gene after the shrimp was infected with WSSV virus. It was found that the transcription of PjQM was up-regulated 4 hours after being infected by the virus, and its expression level was about 150% of that of normal prawns after 72 hours of infection ( Figure 5 ). At the same time, the results of Real-time PCR showed that in the disease-resistant shrimp, the expression of PjQM gene was 180% of the normal level ( Figure 5 ), showing a significantly up-regulated expression. At the same time, using the Weste...

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Abstract

Since it was proposed to be a tumor suppressor, QM protein has attracted intensive studies in plants, animals and fungi. So far, however, the function of the QM protein remains unknown. In this investigation, it is found that the shrimp QM gene (designated as PjQM) is significantly up-regulated in virus-resistant shrimp, thereby suggesting that the PjQM is involved in shrimp immunity. The GST pull-down assays show that the PjQM protein interactes with shrimp hemocyanin and myosin, thus indicating that the PjQM protein might participate in prophenoloxidation (proPO) activation system of shrimp immunity. As revealed by the RNAi assays, it is demonstrated for the first time that the QM protein could regulate the activity of phenol oxidase (PO) through interaction with hemocyanin, a key enzyme in the proPO activation system of invertebrate antiviral immunity.

Description

[0001] The invention relates to the function of a QM protein, especially the QM protein in prawns. Background technique [0002] The QM gene is a new gene that Weissman et al. first isolated from a cell line related to human Wilms' tumor, and then found that the gene exists widely in many species in nature. In addition to humans, it has been found in mice QM homologous genes have been cloned in organisms such as chicken, fruit fly, silkworm, moth, yeast, corn and rice, with a molecular weight of about 24-26kDa. The nucleotide and amino acid sequences encoded by these genes are highly conserved. Current research shows that QM is a multifunctional protein involved in basic life activities such as cell growth, differentiation, development and apoptosis. In yeast, the QM homologous gene GRC5-Q.R1 is related to mitochondrial respiration, and QM gene mutations can lead to decreased protein synthesis, growth and division arrest, and cytoskeleton abnormalities in yeast, and the deleti...

Claims

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Application Information

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IPC IPC(8): A61K36/16A61P31/12
Inventor 章晓波吴穗洁许建阳
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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