Ascorbate peroxidase gene in lycium chinense miller cytoplasm and application thereof

A technology of peroxidase and ascorbic acid, applied in plant genetic improvement, application, genetic engineering, etc., can solve problems such as irreversible damage, cell damage, and membrane system damage at the cellular and molecular levels

Inactive Publication Date: 2013-09-18
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excessive ROS has a strong toxic effect on plant cells, causing cells to produce irreversible damage at the cellular and molecular levels, destroying the membrane system, resulting in loss of tissue structure and cell compartmentation, and denaturation of the cell membrane system, eventually leading to cell damage and death

Method used

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  • Ascorbate peroxidase gene in lycium chinense miller cytoplasm and application thereof
  • Ascorbate peroxidase gene in lycium chinense miller cytoplasm and application thereof
  • Ascorbate peroxidase gene in lycium chinense miller cytoplasm and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Lycium barbarum cytoplasmic ascorbate peroxidase gene wxya A clone of:

[0043] RNeasy Plant Mini Kit (QIAGEN, German) was used to extract Total RNA from 100 mg of fresh Lycium barbarum leaves. According to the conservation of tomato (DQ096286), pepper (DQ002888), potato (AB041343), and tobacco (U15933) registered in NCBI The upstream degenerate primer APX-JF: 5'-CAATTRCTATGGGTAAGT-3' and the middle specific upstream primer APX-BF: 5'-AGGATATTGTTGCACTCTCTGGTG-3' were designed from the 5' end and the middle part of the nucleotide sequence respectively. The complete gene sequence was amplified using the 3'-FULL RACE Core Set Ver.2.0 (TaKaRa, Japan) kit. Specific steps:

[0044] ①Using Total RNA as a template and using 3'RACE Adapter primers for reverse transcription reaction to synthesize 1st Strand cDNA, the reaction system is as follows:

[0045] RNA: 2 μl

[0046] 3' RACE Adapter: 1μl

[0047] 5×M-MLV Buffer: 2μl

[0048] 2.5mM dNTP Mixture: 1μl

[0049] RNase I...

Embodiment 2

[0074] Cloning vector pMD18-T- wxya build process

[0075] will be shown in the sequence listing wxya The gene is connected to the pMD18-T vector, and the reaction system is as follows:

[0076] Target PCR fragment: 4μl

[0077] pMD18-T vector: 1 μl

[0078] Solution I: 5μl

[0079] Reaction conditions: 16°C, 30min. Ligation Product Transformation E. Coli TOP10, coated on LB plates containing Amp resistance. Use the target gene as a primer to perform PCR (reaction conditions: 94°C, 4min; 94°C, 30sec; 55°C, 30sec; 72°C, 1min; 72°C, 8min, 30 cycles.) to obtain PCR products with correct electrophoresis bands, Then it was sent to Huada Gene Company for sequencing, and the sequencing results were blasted in NCBI, indicating that it was the gene.

Embodiment 3

[0081] Escherichia coli expression vector pET28a- wxya build process

[0082] First, with pMD18-T- wxya as a template, P3 and P4 are the upstream and downstream primers respectively amplified with high-fidelity Pfu enzyme wxya For fragments, the reaction conditions are 94°C, 4min; 94°C, 30sec; 57°C, 30sec; 72°C, 1min50sec; 72°C, 8min, 32 cycles. Introduced in P3 Bam H I restriction site (CGCGGATCC), introduced in P4 Sal I restriction site (ACGCGTCGAC). Then, the PCR product and the pET28a plasmid were subjected to Bam H I and Sal I double digestion, ligation of the two digestion products: 22°C, 2 hrs, ligation (2 μl carrier DNA, 5 μl foreign DNA, 2 μl 5×T4 buffer, 1 μl T4 DNA ligase). Ligation Product Transformation E. coli BL21( DE3) competent cells were spread on LB plates containing kanamycin. The target gene was used as a primer to perform PCR to obtain a product band of about 750 bp, and the target band was identified by enzyme digestion as follows: image...

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Abstract

The invention relates to an ascorbate peroxidase gene in a lycium chinense miller cytoplasm and an application thereof. The ascorbate peroxidase gene has a nucleotide sequence shown in a sequence of SEQ ID No.1; the ascorbate peroxidase gene LmAPX in the lycium chinense miller cytoplasm is cloned by extracting total RNA (Ribonucleic Acid) of fresh lycium chinense miller leaves through a homology-based cloning strategy and a 3'-RACE (Rapid Amplification of Cdna (complementary deoxyribonucleic acid) Ends) technology to obtain a complete coding gene sequence 753bp. An escherichia coli expression vector Pet28a-LmAPX is constructed, the enzyme activity of the cloned ascorbate peroxidase gene LmAPX code in the lycium chinense miller cytoplasm is identified by using an escherichia coli heterologous expression system, and a recombinant protein has higher activity for substrate ascorbic acid (AsA) and H2O2. Meanwhile, a binary plant expression vector pCAMBIA2300-LmAPX is constructed and is transplanted into an agrobacterium C58 cell by using an electroporation method, and the agrobacterium C58 cell is used for transforming a tobacco to obtain a transgenic tobacco used for subsequent adversity stress research.

Description

technical field [0001] The invention relates to an ascorbate peroxidase gene in Lycium chinense Miller cytoplasm wxya The cloning is specifically a wolfberry cytoplasmic ascorbate peroxidase gene and its application. Background technique [0002] Ascorbate peroxidase (APX, EC1.11.1.11) is a heme protein, also known as vitamin C peroxidase, which belongs to a type of terminal oxidase, mainly found in higher plants, The enzyme activity has been detected in eukaryotic algae and some cyanobacteria, but also in some insects. APX is Clear H 2 o 2 The key enzyme, with reduced ascorbic acid as the reaction substrate, it catalyzes H 2 o 2 Revert to H 2 The reaction of O, with high specificity and affinity to ascorbic acid, consumes H 2 o 2 Monodehydroascorbic acid is produced, which can be reduced to ascorbic acid by different pathways (AsAda, 1992; Shigeoka et al., 1980a; 1980b). In higher plants, APX is a multi-gene family, which can be divided into: stromal APX (sAPX) in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/08C12N15/63C12N1/21C12N5/10C12N15/82A01H5/00
Inventor 王罡季静吴广霞关春峰张旭强
Owner TIANJIN UNIV
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