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Polypeptide sequence for specifically regulating and controlling plant protein stability under adversity condition and application of polypeptide sequence

A plant protein and polypeptide sequence technology, which is applied in the field of plant genetic engineering, can solve problems such as hindering normal growth of plants, waste of nutrition and energy, and breaking metabolic balance, so as to improve crop yield and quality traits, increase yield and/or Quality, the effect of improving adversity resistance

Inactive Publication Date: 2015-04-08
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that in the existing plant genetic engineering technology, the overexpression of foreign genes in plants leads to a large amount of continuous accumulation of heterologous proteins in the process of plant growth and development, which breaks the original metabolic balance of plants; The metabolic process regulated by protein is not always required for plant growth, but the continuous activation causes waste of nutrients and energy, hinders the normal growth of plants, and even leads to death. It provides a specific way to regulate the stability of plant proteins under stress Polypeptide sequence and its application

Method used

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  • Polypeptide sequence for specifically regulating and controlling plant protein stability under adversity condition and application of polypeptide sequence
  • Polypeptide sequence for specifically regulating and controlling plant protein stability under adversity condition and application of polypeptide sequence
  • Polypeptide sequence for specifically regulating and controlling plant protein stability under adversity condition and application of polypeptide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: The coding sequence of WX01 polypeptide and GUS Reporter gene fusion, using pCAMBIA 1301 as the basic vector to construct the " WX01 - GUS Plant binary expression vector of fusion gene

[0032] First, use the pCAMBIA 1301 plasmid as a template to amplify the 2108 bp " WX01 - GUS "Fuse the gene, recover the amplified product and perform TA cloning.

[0033] (1) PCR amplification of the target fragment

[0034] According to the known pCAMBIA 1301 vector GUS Design specific primers for the sequence of the reporter gene and introduce them in sequence in the upstream primer 1 Nco I restriction site and the coding sequence of WX01 (as shown in lowercase letters), the upstream primer 2 contains part of the WX01 coding sequence, and the downstream primer introduces Bst E II restriction site.

[0035] Upstream primer 1: 5’- CCATGG atgggtcttcctctaatgatggagagatcatcaaacaacaacATGG-3’ (introduced Nco I cut point)

[0036] Upstream primer 2: 5’-ggagagatcatcaaacaacaacATGG...

Embodiment 2

[0067] Example 2: Preparation of Transgenic Arabidopsis

[0068] (1) Constructed with Example 1 containing " WX01 - GUS The binary expression vector of the fusion gene is used to transform wild-type Arabidopsis thaliana. The specific transformation method adopts Agrobacterium-mediated bud soaking method. The obtained seeds are screened for 30 mg / L hygromycin resistance, and normal-growing plants are transferred to soil for culture .

[0069] (2) PCR detection of transgenic plants: respectively cut the leaves of transgenic plants and wild-type plants, refer to the "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002) method to extract leaf genomic DNA, and perform PCR with the following primers The reaction system is the same as in Example 1:

[0070] Upstream primer: 5’-TGGGTCTTCCTCTAATGATGGA-3’

[0071] Downstream primer: 5’- GGTCACC TCACACGTGGTGGTGGTGG-3’

[0072] The PCR products were subjected to agarose gel electrophoresis, and the transgenic plant...

Embodiment 3

[0074] Example 3: Detection of Specific Accumulation of "WX01-GUS" Fusion Protein in Transgenic Arabidopsis

[0075] (1) Using GUS histochemical staining method to detect “WX01” in transgenic Arabidopsis at different developmental stages - GUS" fusion protein accumulation level. Will " WX01-GUS "The 11-day, 26-day and 28-day seedlings of transgenic Arabidopsis thaliana were used for staining analysis and driven by the same promoter." GUS "Gene expression of transgenic Arabidopsis thaliana is a control. The blue in each organ or tissue of the transgenic plant is the histochemical staining of the reporter gene GUS, the stronger the staining represents the accumulation level of "GUS" or "WX01-GUS" fusion protein The higher the dyeing time is about 6 hours, the dyeing result of the material is scanned into a picture and saved.

[0076] The staining result is like figure 2 : Under the condition of 16 h light / 8 h dark photoperiod, in vigorously growing seedlings (including 11-day seed...

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Abstract

The invention discloses a polypeptide sequence for specifically regulating and controlling plant protein stability under adversity condition and application of the polypeptide sequence. The polypeptide sequence provided by the invention contains 14 amino acids and is named as WX01 as shown in SEQ ID No.1. Researches show that the WX01 contains a unique protein degradation signal which is capable of responding development and environment signals and horizontally regulating and controlling the stability of a target protein (such as GUS) fused with the WX01 after transcription. By applying the polypeptide sequence provided by the invention, a fusion gene of a WX01-target gene to be expressed can be constructed; through carrying out plant conversion, transgenic plants which specifically accumulate the target proteins with adversity resistance only under the adversity stress conditions such as ageing, high salinity, high temperature and water loss and limit the accumulation level when the plants grow normally and vigorously can be obtained, so that the normal growth and the excellent character such as relatively strong adversity resistance of the transgenic plants are ensured and important value is brought to the research and application of plant genetic engineering technology.

Description

technical field [0001] The present invention belongs to the field of plant genetic engineering, and specifically uses molecular biology techniques to fuse a polypeptide with a target protein to regulate the expression level of the target protein in transgenic plants, so that it can only be specifically accumulated under various stress conditions, but It is degraded when the plant grows normally and vigorously, so as to ensure that the transgenic plant can not only grow normally but also have strong adversity resistance, and the application of the peptide segment in the field of scientific research and genetic engineering technology. Background technique [0002] my country is a country based on agriculture, but with the increasing population, decreasing arable land and increasingly tight agricultural resources, food supply is facing a huge challenge. Improving and stabilizing grain production has become the demand of my country's current major national strategic development ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/84A01H5/00
CPCC12N9/88C12N15/8243C12Y404/01014
Inventor 王宁宁熊莉孙立方王丹
Owner NANKAI UNIV