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Application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway

A technology for analyzing signals and protoplasts, applied in the direction of using vectors to introduce foreign genetic material, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of no genome sequence, time-consuming and labor-intensive research on plant signal transduction pathways, etc.

Active Publication Date: 2020-03-13
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to their complexity, the study of signal transduction pathways in plants is time-consuming and laborious
Moreover, most plants do not have genome sequences, transgenic methods, or mutant libraries, etc., making the study of signaling pathways more challenging

Method used

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  • Application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway
  • Application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway
  • Application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, JAZ1 protein degradation mediated by COI1 in Arabidopsis protoplast cells

[0062] 1. Construction of recombinant plasmids

[0063] 1. Construction of protein fusion reporter gene vector (i.e. recombinant plasmid UBQ10pro: HA-JAZ1-fLuc)

[0064] (1) The pBGWL7.0 vector was digested with restriction enzymes HindIII and XmaI, and a DNA fragment 1 of about 2303 bp was recovered. DNA fragment 1 contains the gene encoding firefly luciferase.

[0065] (2) The UBQ10pro:HA-GW vector was digested with restriction enzymes HindIII and XmaI, and the vector backbone 1 of about 10152 bp was recovered.

[0066] (3) Ligate the DNA fragment 1 and the vector backbone 1 to obtain the recombinant plasmid UBQ10pro: HA-GW-fLuc.

[0067] The nucleotide sequence of the recombinant plasmid UBQ10pro:HA-GW-fLuc is shown in SEQ ID No:1.

[0068] (4) Using the genomic DNA of wild-type Arabidopsis as a template, a primer pair consisting of JAZ1-gw-d1 and JAZ1ohnestop-gw-r1 was used fo...

Embodiment 2

[0112] Example 2, JAZ1 protein degradation mediated by COI1 in protoplasts requires jasmonic acid

[0113] In Arabidopsis, endogenous JA molecules mediate the interaction between COI1 and JAZ. To determine whether JAZ1-fLuc proteolysis by COI1 in protoplasts is dependent on JA, the inventors of the present invention performed JA complementation experiments in protoplasts of aos mutants. The AOS gene, one of the key enzymes encoding the JA biosynthetic pathway, is mutated in the aos mutant, so JA cannot be synthesized.

[0114] 1. JA-Ile triggers the specific degradation of JAZ1 in protoplast cells

[0115] 1. Construction of recombinant plasmids

[0116] (1) The construction of the protein fusion reporter gene vector (ie, the recombinant plasmid UBQ10pro: HA-JAZ1-fLuc) is the same as step 1 in Example 1.

[0117] (2) The construction of the protein fusion reporter gene vector (ie, the recombinant plasmid UBQ10pro: HA-mJAZ1-fLuc) is the same as Step 1 and Step 2 of Example 1...

Embodiment 3

[0156] Example 3, COI1 releases JAZ1-inhibited MYC2 transcriptional activity

[0157] 1. Construction of recombinant plasmids

[0158] 1. Construction of recombinant plasmid JAZ1pro: fLuc

[0159] (1) Using the genomic DNA of wild-type Arabidopsis as a template, the primer pair composed of JAZ1pro-gw-d1 and JAZ1pro-gw-r1 was used for PCR amplification to obtain a promoter fragment of about 1267bp.

[0160] (2) The promoter fragment and the vector pDONR207 were subjected to BP reaction to obtain the intermediate vector pDONR207-JAZ1pro.

[0161] (3) The intermediate vector pDONR207-JAZ1pro and the pBGWL7.0 vector were subjected to LR reaction to obtain the recombinant plasmid JAZ1pro:fLuc.

[0162] In the recombinant plasmid JAZ1pro:fLuc, the gene encoding firefly luciferase is driven by the JAZ1 promoter.

[0163] 2. Construction of recombinant plasmid UBQ10pro: HA-JAZ1 (i.e. effector carrier JAZ1)

[0164] (1) Using the genomic DNA of wild-type Arabidopsis as a template, ...

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Abstract

The invention discloses application of Arabidopsis mutant protoplasts in the analysis of signal transduction pathway. The steps of analyzing the signal transduction pathway are as follows: (1) constructing a protein fusion reporter gene expression vector, an effect vector and an internal control vector, wherein the protein fusion reporter gene expression vector expresses a fusion protein containing a protein tag A, the fusion protein consists of a target protein A and firefly luciferase, the effect vector expresses a target protein B containing a protein tag B, and the internal control vectorexpresses Renilla luciferase; and (2) transiently expressing the protein fusion reporter gene expression vector, the effect vector and the internal control vector in Arabidopsis mutant protoplasts andthen analyzing the signal transduction pathway. The invention provides a new strategy for reconstruction and research of plant hormone signal pathways in Arabidopsis protoplasts and has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of protoplasts of Arabidopsis mutants in analyzing signal transduction pathways. Background technique [0002] Plants are constantly exposed to various environmental stresses during their growth, which can negatively affect plant growth and yield. In response to different environmental stresses, plants have evolved specific signaling networks to coordinate their growth and development and environmental adaptation. Understanding the molecular mechanisms of these signal transduction pathways is important for breeding plants tolerant to environmental stress. Due to their complexity, the study of signal transduction pathways in plants is time-consuming and laborious. Moreover, most plants do not have genome sequences, transgenic methods, or mutant libraries, etc., making the study of signaling pathways more challenging. At present, only in some model plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66
CPCC12N15/8222C12N15/66
Inventor 李宁黄黎君缪文卓江文翰彭明博万文凯
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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