A method for positioning and expressing foreign protein in mitochondria

A technology of exogenous protein and mitochondria, applied in the field of molecular genetics, can solve difficult problems such as the location of signal expression

Active Publication Date: 2020-09-15
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the experiment, it is difficult to determine the expression position of the signal for the point-like distribution of the signal in the cell

Method used

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  • A method for positioning and expressing foreign protein in mitochondria
  • A method for positioning and expressing foreign protein in mitochondria
  • A method for positioning and expressing foreign protein in mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] This embodiment provides a signal peptide and its coding sequence, the sequence of which is as follows:

[0069] 1. PSBR1 DNA sequence:

[0070] ATGGAGGAAGATGGTAGTGGTACGCTTCTACGGCGTATGCTGCATAAGGCA GTATGGGCAGCGAGAGCCATAGCTCACCTCCGTCCATCTCCGACGACGAC GACGACACCGGCAACGACGCCCAGCCGGCTCCCACCGAGCCTGCTGGAC TGCACCGACGACGACGACGCCGCGTCGACTGCCGGGTGTCCGAGCTTCCA CACGGCGAGCAGCACCCCGGACTGGGCCGTCCACTCGTGGCTGCCCAGCC CCGGCGCCGTCGAGGTCGACGGCGACAGGCGCGCCGAGGAGTTCATCGA GAGGTTCTGGCGCAACGTGTCCCTGGAGCTGCGGTACTGCTCGCCGGTTA CGCCCGCCAGGCCGCCCGTGTCGCCGGACACGTACTTCAACCTCTCGAGG CTTAGTCATAGGATCTGCCTTGATTGA

[0071] 2. PSBR1 amino acid sequence

[0072] MEEDGSGTLLRRMLHKAVWAARAIAHLRPSPTTTTTPATTPSRLPPSLLDCTDDDDAASTAGCPSFHTASSTPDWAVHSWLPSPGAVEVDGDRRAEEFIERFWRN VSLELRYCSPVTPARPPVSPDTYFNLSRLSHRICLD

Embodiment 2

[0074] This embodiment provides a method for localized expression of foreign proteins in mitochondria: comprising the following steps:

[0075] 1. Construction of the vector PSBR1-YFP with YFP yellow fluorescence linked to the C-terminus of the signal peptide

[0076] Clone PSBR1: Search the PH01000831G0490 sequence in the BambooGDB database (http: / / www.bamboogdb.org / ), and name it PSBR1. The primers PSBR1-F: 5'-ATGGAGGAAGATGGTAGTGG-3' and PSBR1-R: 5'-ATCAAGGCAGATCCTATGAC-3' were designed according to the open reading frame (ORF) of PSBR1 sequence in Moso bamboo. Total RNA from Moso bamboo leaves was prepared using a total RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

[0077] First-strand cDNA was synthesized from 1 μg of total RNA using the GoScript™ Reverse Transcription System (Promega Biotech, Co., Ltd., Beijing, China). Using cDNA as a template for PCR, amplification was performed with primers PSBR1-F and PSBR1...

Embodiment 3

[0116] Application of DNA sequence and amino acid sequence of signal peptide PSBR1 to localize and express foreign protein in mitochondria.

[0117] Beneficial effects: The subcellular localization information of proteins predicted by amino acid sequences is not yet very accurate, and large-scale proteomics methods are difficult to separate low-abundance proteins, and contamination cannot be avoided in the preparation of protein samples. Certain limitations. It is difficult to judge the specific organelle for the point-like structure in the cell, and because the mitochondria are very high in the cells of some tissues and are distributed anywhere in the cell, this is often misjudged as expressed in any area of ​​the cell. Therefore, it is very important to have a mitochondrial localized marker gene as a control. In this study, a sequence of a Phyllostachys pubescens gene was cloned, constructed into an expression vector, and transformed into Agrobacterium to express transientl...

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Abstract

The invention discloses a method for locating and expressing foreign protein in mitochondria, belongs to the field of molecular genetics, provides a DNA sequence for encoding polypeptides, applicationof the DNA sequence in location and expressing of the foreign protein in the mitochondria and a related approach, and particularly relates to a sequence from a phyllostachys edulis genome. A polypeptide encoded by the sequence can express the foreign protein in the mitochondria, serve as a marker gene for co-location of the mitochondria and provide a reliable comparison approach for subcell location. The method comprises the steps of firstly, cloning a sequence of a phyllostachys edulis gene, and introducing yellow fluorescent protein connected to a C end of the sequence into the mitochondria. Transfer peptides and the fluorescent protein are fused, observation can be carried out in living cells, time and labor are saved, and the method is a basic method for researching the functions of the protein.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and provides a DNA sequence encoding a polypeptide and its application and related methods for positioning and expressing foreign proteins in mitochondria. Background technique [0002] Mitochondria are essential organelles found in almost all eukaryotic cells, involved in a variety of key cellular processes such as energy conversion, cell signaling, cell cycle regulation and cell differentiation [1] . The vast majority of the mitochondrial proteome is encoded in the nucleus, translated in the cytoplasm, and subsequently entered into the mitochondria. Targeting newly synthesized proteins and importing them into the correct organelles requires the presence of organelle-specific targeting signals and involves a complex intracellular protein trafficking machinery involving different cytosolic factors, chaperones and organelle-specific translocases [1 ]. Most proteins for import into the mitochon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/65C07K14/415
Inventor 王文斐郭泽军杨雪莲张哲
Owner FUJIAN AGRI & FORESTRY UNIV
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