Use of an atg5 transient silencing carrier to alleviate the degradation of organelle-localized proteins

A protein degradation and organelle technology, applied in the field of transient silencing vectors, can solve the problems of low target protein expression and restrict the industrialization of plant expression systems, and achieve the effect of promoting expression

Active Publication Date: 2021-11-09
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the plant expression system still has the problem of low expression of the target protein, which is one of the main factors restricting the industrialization of the plant expression system

Method used

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  • Use of an atg5 transient silencing carrier to alleviate the degradation of organelle-localized proteins
  • Use of an atg5 transient silencing carrier to alleviate the degradation of organelle-localized proteins
  • Use of an atg5 transient silencing carrier to alleviate the degradation of organelle-localized proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Transformation of pCV1300 binary expression vector

[0045] The present invention selects pCV1300 as the binary expression vector constructed by the silencing vector, and its structure diagram is as follows figure 2 shown.

[0046] Plasmids pCAMBIA1300 and pBI121 were digested with HindIII and EcoRI, and the 35S-GUS-NOS segment of pBI121 was ligated into pCAMBIA1300 vector to form pCV1300 vector.

Embodiment 2

[0047] Example 2 Construction of siatg5 silencing vector

[0048] 1. Screening of target fragments

[0049] The method for screening RNA interference gene silencing fragments with a hairpin structure is:

[0050] According to the Nb-Atg5 gene sequence (SEQ ID No.3) of NCBI (KX369397.1, full length 1116bp), it was segmented into short continuous nucleotide fragments with a length of about 300-500bp, through http: / / vigs. solgenomics.net / Analysis of the possible specific silence segment is a 1-300bp sequence, but the sequence analysis shows that Niben101Scf02433g01001.1 (referred to as Nb1001, this sequence has a high homology with Atg5, and also has the Autophagy protein Apg5 domain, but the gene function is unknown ) gene sequence (SEQ ID No.4) is highly conserved with NbAtg5 from about 450-1000 segments ( image 3 ). And according to the analysis of NCBI website (https: / / blast.ncbi.nlm.nih.gov), it was further confirmed that the conserved region of NbAtg5 and Niben101Scf0...

Embodiment 3

[0070] Example 3 Validation of GFP-Atg8f as an indicator protein of autophagy pathway

[0071] Atg8 has different homologues in different species, and Atg8f in Arabidopsis and N. benthamiana is an effective protein for monitoring the autophagy pathway. Because GFP is more sensitive to pH, after the transient expression of GFP-Atg8f, the modified GFP-Atg8f cooperates with other autophagy proteins to form autophagic vesicles, and then the GFP protein fused with Atg8f is cleaved and released into the cytoplasm in an acidic environment. The activation level of the autophagy pathway can be detected by detecting the protein expression of free GFP (Cleave GFP).

[0072] MV (methyl viologen) is an inducer of the autophagy pathway, and MV treatment can activate the autophagy pathway. Therefore, MV treatment was used to verify whether GFP-Atg8f can be used as an indicator protein of the autophagy pathway. According to the XM_016638904.1 sequence, the Nicotiana benthamiana Atg8f seque...

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Abstract

The invention relates to the field of plant genetic engineering, in particular to the use of an Atg5 transient silencing vector. The present invention discloses the use of the Atg5 transient silencing vector in alleviating protein degradation in different organelles by co-infiltrating the Atg5 transient silencing vector with endoplasmic reticulum, mitochondria, peroxisome or plastid GFP expression vector. The Atg5 transient silencing carrier of the present invention can act on the organelle region and promote the accumulation of exogenous protein in the organelle. The present invention shows that the autophagy pathway of siatg5 silencing involves most cellular sites such as endoplasmic reticulum, plastids, mitochondria, peroxisomes, etc., and the siatg5 silencing vector may have certain effects on autophagy pathways of various sites and organelles in plant cells Regulatory effect. The invention is of great significance for elucidating the molecular mechanism of the autophagy pathway regulated by the Atg5 gene in affecting the expression of foreign proteins, and has guiding significance for improving the expression of foreign proteins using plants as bioreactors.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a transient silencing vector of Atg5, a key gene of autophagy pathway. The silencing vector can lead to silencing of the Atg5 gene, inhibit the autophagy pathway, alleviate the degradation of proteins located in different organelles, and thus promote the accumulation of exogenous proteins. Background technique [0002] Using gene recombination technology to express and produce foreign proteins by using organisms as bioreactors is a hot issue in current biotechnology research. Commonly used exogenous protein expression systems mainly include prokaryotic expression, yeast expression system, insect expression system, mammalian expression system and plant expression system, etc. Different expression systems have different expression characteristics. Compared with other expression systems, plant expression systems have the following advantages: (1) Plants, as eukaryotes, have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/67C12N15/29
CPCC07K14/415C12N15/67C12N15/8218C12N15/8222
Inventor 彭杰军鲁宇文燕飞陈思涵郑红英林林程晔陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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