Gene mapping expression vector for detecting plant autophagy phenomenon and application

A technology of gene localization and expression vector, applied in the biological field, can solve the problem of lack of identification and detection materials, and achieve the effect of accurate localization

Inactive Publication Date: 2016-03-30
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology

It is certain that the Atg8 gene plays an important role in the autophagy process, but to clarify a series of molecular mechanisms by which Atg8 participates in the formation of autophagy, there is still a lack of relevant identification and detection materials

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  • Gene mapping expression vector for detecting plant autophagy phenomenon and application
  • Gene mapping expression vector for detecting plant autophagy phenomenon and application
  • Gene mapping expression vector for detecting plant autophagy phenomenon and application

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Embodiment Construction

[0020] A method for constructing gene-localized expression vectors for detecting plant autophagy. Using Honghua Dajinyuan tobacco varieties as materials, RNA is extracted, reverse-transcribed to obtain cDNA, and NtAtg8-F / NtAtg8-R is used as a primer pair for PCR Amplify the NtAtg8 gene, process and recover the DNA fragment with the double enzyme digestion system, process and recover the pFF19-GFP plasmid with the same double enzyme digestion system; connect the NtAtg8 gene and the pFF19-GFP vector containing the same sticky end, and transform the ligation product into In DH5α competent cells, select positive colonies for sequencing verification.

[0021] The system for linking the NtAtg8 gene and the pFF19-GFP vector is as follows: 1 μl of each plasmid after enzyme digestion, 1 μl of Buffer, 1 μl of T4 ligase, replenished with water to 10 μl, and ligated overnight at 16°C.

[0022] The sequence of the NtAtg8-F / NtAtg8-R primer pair is shown in SEQ ID NO: 1 / SEQ ID NO: 2, wherein...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to construction of a gene vector for detecting a cell autophagy phenomenon. A protein of an Atg8 gene mapping expression vector is expressed in a suspension cell after being fused with a green fluorescence protein (GFP), and subcellular localization is performed by detecting the position of a fused protein through a laser scanning confocal microscope. Through a simple, convenient and accurate method for locating in living cells, a reference basis is laid for the research of the molecular mechanism of the tobacco cell autophagy phenomenon.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction of a gene carrier for detecting the phenomenon of cell autophagy, and using the gene positioning expression carrier to study the phenomenon of cell autophagy. Background technique [0002] Autophagy is an important degradation mechanism in cells, through which aging proteins in cells can be transported to lysosomes for degradation. The phenomenon of autophagy widely exists in the cells of most eukaryotes, including yeast, fruit fly, plant and human body. It is an important way for cells to recycle their damaged proteins, amino acids and other nutrients contained in organelles. An adaptation to the environment formed during the evolution of eukaryotes. [0003] The process of cell autophagy can be roughly divided into four stages: First, after cells receive autophagy-inducing signals such as starvation, lack of nutrient factors, or invasion by diseases an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66G01N21/64
CPCC12N15/66G01N21/6402
Inventor 周会娜陈千思刘萍萍陈霞金立锋翟妞郑庆霞张慧林福呈徐国云
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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