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Atg 5 transient silencing vector

A silencing and carrier technology, applied in the field of plant genetic engineering, can solve the problems of low expression of target protein, restricting the industrialization of plant expression systems, etc., and achieve the effect of alleviating degradation and promoting expression

Inactive Publication Date: 2019-11-22
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
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  • Application Information

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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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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 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 Verification that GFP-Atg8f can be used as an indicator protein of the 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 ben...

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Abstract

The present invention utilizes a principle of silencing target genes by RNA silencing pathways. Two specific silencing fragments of SL and SS of Atg 5 gene are connected to a binary expression vectorpCV 1300, and agrobacterium transient expression constructs an Atg 5 transient silencing vector, namely a siatg 5 silencing vector. An analysis by quantification and use of GFP-Atg8f demonstrates thatsiatg 5 can lead to silence of the Atg 5 gene, effectively inhibits autophagy pathway, alleviates degradation of proteins of cell organelle localization, and thus promotes accumulation of foreign proteins of cell organelles. The siatg 5 silencing autophagy pathway relates to most of cell sites such as endoplasmic reticulum, plastids, mitochondrion, peroxisome, etc. The siatg 5 silencing vector can have regulating effects on the various sites and autophagy pathway of the cell organelles in plant cells.

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 Atg5 gene silencing, inhibit the autophagy pathway, and relieve the degradation of proteins located in different organelles, thereby promoting the accumulation of foreign 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 system, 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, the plant expression system has the following advantages: (1) As a eukaryote, plants h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66
CPCC12N15/66C12N15/8218
Inventor 彭杰军鲁宇文燕飞钱靖郑红英林林程晔陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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