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Plant selective autophagy receptor small peptide and application thereof

A plant and autophagy technology, applied in the field of plant biology, can solve the problems of unreported virus infection and achieve the effect of improving anti-virus ability and broad-spectrum disease resistance

Active Publication Date: 2021-05-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, people have gradually discovered that there are many small coding frames in the plant genome, which encode small peptides of 30-100 amino acids. These small peptides are divided into secreted peptides and non-secreted peptides. According to predictions, there are more than 8,000 small Peptides, for these known small peptides, it is found that they play an important role in the whole growth and development of plants and in response to abiotic stress. It has been reported in the literature that small peptides play a role in resisting pathogenic infections such as bacteria and fungi, but , the role in virus infection has not been reported, so it is very important to explore small peptides with unknown functions

Method used

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  • Plant selective autophagy receptor small peptide and application thereof
  • Plant selective autophagy receptor small peptide and application thereof
  • Plant selective autophagy receptor small peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Obtaining of AtVISP3 gene and construction of expression vector

[0058] In this example, transcriptome sequencing was performed on Arabidopsis plants inoculated with cucumber mosaic virus (CMV) for one week. The sequencing results showed that, compared with the control (mock), the transcription level of the gene AT1G21525 was up-regulated by more than four times after inoculation with CMV, and the results were verified using realtime (such as figure 1 shown). figure 1 The results show that inoculation of wild-type CMV or attenuated mutant virus CMV-2blm can significantly induce the expression of gene AT1G21525, and this gene is named AtVISP3 gene. The full-length amino acid sequence of VISP3 protein in Arabidopsis is as SEQ ID NO.1 As shown, the nucleotide sequence of the coding frame is shown in SEQ ID NO.12. Amplification primers were designed according to the cDNA nucleotide sequence.

[0059] The amino acid sequence and nucleotide sequence of VISP3 wer...

Embodiment 2

[0060] Example 2 Overexpression of VISP3 gene and construction of knockout mutant plants

[0061] 1. Construction of overexpression plants of VISP3 gene

[0062] (1) Extraction of plant tissue total RNA

[0063] Total RNA was extracted from plant tissues by the Trizol method. The specific method was as follows: collect plant tissues, grind with liquid nitrogen, add 1ml Trizol (Invirtogen) to 0.1g tissue leaves, and then add 200 μL chloroform, let stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm at 4°C for 5 minutes. Take the upper aqueous phase into a centrifuge tube, add 200 μL chloroform to extract again, and repeat the previous step. Take 500 μL of the upper aqueous phase into a centrifuge tube, add an equal volume of isopropanol, mix thoroughly, place at room temperature for 20 min, and centrifuge at 12,000 rpm at 4°C for 20 min. Discard the supernatant, add 500 μL of 70-80% ethanol (prepared with DEPC water), centrifuge at 12,000 rpm for 5 min at 4°...

Embodiment 3

[0075] Example 3 Analysis of autophagy level and SGS3 accumulation level in VISP3 overexpression and knockout plants

[0076] 1. Analysis of autophagy in VISP3 overexpression plants and knockout plants

[0077] Analysis of the autophagy situation analysis of the overexpressed plants and knockout mutant plants of the VISP3 gene constructed in Example 2, the specific methods are as follows:

[0078] (1) The wild-type Col-0, VISP3 overexpression plants and visp3 mutant plants were further transformed into YFP-ATG8e indicator gene, and the positive plants were grown in 1 / 2MS medium for 5 days, and Col-0 / 35S:YFP-ATG8e They were transferred to nitrogen-deficient or non-nitrogen-deficient medium, respectively, and VISP3 / 35S:YFP-ATG8e and visp3 / 35S:YFP-ATG8e were transferred to non-nitrogen-deficient medium for 4 days, and the whole plant was taken for Western blotting experiment. The last Col-0 is a non-transgenic negative control.

[0079] (2) Western blotting detection. Sampling...

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Abstract

The invention relates to a plant selective autophagy receptor small peptide and application thereof. The invention provides a novel selective autophagy receptor VISP3, the VISP3 is a small peptide and is conservatively distributed in cruciferous plants, and the protein sequence of the VISP3 in arabidopsis thaliana is as shown in SEQ ID NO. 1. The VISP3 degrades a key component SGS3 / RDR6 small body of an RNA (Ribonucleic Acid) silencing pathway, interferes with synthesis of exogenous virus small interfering RNAs (vsiRNAs) and endogenous tasiRNAs, promotes virus infection and influences plant development. Infection of cucumber mosaic virus and turnip mosaic virus of plants can be promoted by increasing the expression quantity of the VISP3; by knocking out the VISP3, the antiviral capability of the plant can be enhanced. The knockout mutant can be used for preventing and treating plant diseases independently or in combination with other plant agents, and the yield and quality of plants are improved.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to the principle of selective autophagy receptors regulating the accumulation of antiviral RNA silencing key factor protein and the application of improving plant disease resistance. Background technique [0002] Plants are attacked by a variety of pathogenic microorganisms during their growth, and RNA silencing is a broad-spectrum antiviral process in plants. The double-stranded RNA formed by the virus during replication and transcription can be recognized by the host endonuclease dicer and cut into small double-stranded RNAs (siRNAs), which are assembled into the RISC complex (RNA-induced silencing complex) to target and The paired viral RNA cleaves the target RNA and inhibits translation. The abnormal RNA that is damaged by shearing can be used as a template by the host RNA polymerase complex RDR6 and SGS3 to synthesize double-stranded RNA, which is further cut by dicer to pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8218C12N15/8283
Inventor 王献兵佟昕赵加佳王颖
Owner CHINA AGRI UNIV
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