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Apple MdARF5 gene beneficial for apple callusing, and applications thereof

An apple and gene technology, applied in the field of molecular biology, can solve problems such as unclear mechanism, improve salt resistance and drought resistance, mature technical process, and improve the effect of plant salt resistance

Inactive Publication Date: 2017-09-15
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Recent studies have shown that H + - Increased ATPase activity contributes to the secretion of organic acids, thereby inhibiting the absorption of aluminum ions, and AHAs belong to a class of H + -ATPase, they play an important role in the secretion of organic acids and aluminum ion resistance. Previous studies have also found that auxin signaling also plays an important role in regulating the secretion of organic acids, but the mechanism is unclear

Method used

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  • Apple MdARF5 gene beneficial for apple callusing, and applications thereof
  • Apple MdARF5 gene beneficial for apple callusing, and applications thereof
  • Apple MdARF5 gene beneficial for apple callusing, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the cloning of apple MdARF5 gene

[0028] 1. Gala tissue culture leaf RNA extraction and reverse transcription

[0029] 1. Extract total RNA by CTAB method:

[0030] (1) Take 1.5 g of Gala apple tissue culture seedlings treated with 200 mM NaCl salt for 24 hours, put them into a pre-cooled mortar, add liquid nitrogen to grind, and transfer to a pre-cooled 50 mL centrifuge tube;

[0031] (2) Quickly add 10 mL of extraction buffer preheated to 65°C (in which PVP and mercaptoethanol are added immediately), mix gently, and place in a water bath at 65°C for 0.5 hour. The composition of the extraction buffer is shown in Table 1;

[0032] Table 1 Extraction buffer

[0033] CTAB

20% (w / v)

Tris-HCl

0.1mmol / L

EDTA

25mmol / L

NaCl

2mmol / L

Mercaptoethanol

2% (w / v)

pvp

2% (w / v)

RNase free ddH 2 o

Dilute to 100mL

[0034] (3) Add a water-saturated phenol / chloroform / isoamyl alcohol (25...

Embodiment 2

[0074] Embodiment 2: the acquisition of transgenic apple callus

[0075] 1) Pick a monoclonal colony of Agrobacterium and inoculate it in 10 mL of YEP liquid medium (containing 50 mg / L hygromycin), at 28° C., 200 rpm, and shake it until the OD600 is 06-0.8 (about 48 hours). Take 1 mL of the bacterial liquid and add it to 20 mL of YEP liquid medium, and culture it with shaking at 28°C and 200 rpm until the OD600 is 06-0.8 (about 5 hours). Bacteria were collected by centrifugation, ddH 2 O diluted to prepare the infection solution.

[0076] 2) Take wild-type apple callus in good growth state, place the callus in the infection solution and incubate for 30 minutes. After blotting dry with absorbent paper, pre-incubate for 1 day.

[0077] 3) Spread the apple callus pre-cultivated for 1 day on the selective medium (100 mg·L -1 Hyg) for 1-2 months.

[0078] 4) Screen the transgenic callus by gene detection.

Embodiment 3

[0079] Embodiment 3: Al ion stress resistance observation of transgenic apple callus

[0080] Use apple tissue culture seedlings that have grown for about 3 weeks and are growing well, and use different concentrations of Al 3+ Treatment, expression analysis experiments verified the expression response of MdARF5 to external aluminum ion stress, and the expression analysis results found that Al 3+ Significantly promote the expression of MdARF5 gene. At the same time, MdARF5 was genetically transformed into apple callus, and in Al 3+ The apple callus was processed under stress, and the growth curve was observed. The results showed that compared with the wild-type apple callus, the transgenic apple callus had a higher growth rate in Al 3+ faster growth rate under stress, such as figure 1 shown.

[0081] 3. Improvement of MdARF5 transgenic apple callus

[0082] The apple MdARF5 and wild-type calluses that were subcultured for about two weeks and grown well were grown on the su...

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Abstract

The invention discloses apple MdARF5 gene beneficial for apple callusing, and applications thereof, and belongs to the technical field of molecular biology. The nucleotide sequence of the apple MdARF5 gene is represented by SEQ.ID.NO.1. The invention also discloses an operation method of the apple MdARF5 gene, and applications of the apple MdARF5 gene in callusing of genetically modified apples. According to the applications, beneficial properties, such as plant Al<3+> stress resistance, are improved via plant genetic engineering technology, DNA fragments capable of responding Al<3+> stress-related gene complete coding fragments are obtained via separating and cloning from apples, the functions of the apple MdARF5 gene are verified, and it is found that the sensitivity on Al<3+> stress of genetically modified plants obtained via overexpression of the apple MdARF5 gene in apple callusing and arabidopis thaliana is improved.

Description

technical field [0001] The invention relates to an apple MdARF5 gene beneficial to apple wound healing and application thereof, belonging to the technical field of molecular biology. Background technique [0002] Aluminum ions are toxic to plants and are the main limiting factor for plant growth. Under aluminum stress, the main way for plants to resist aluminum ion stress is to chelate rhizosphere active aluminum through the secretion of organic acids from roots. In recent decades, excessive nitrogen application in my country's apple industry has led to soil acidification. In acidic soil, aluminum ions are activated and easily absorbed by plants, thereby inhibiting the growth and development of fruit trees and reducing the safety of edible horticultural products. . Therefore, it is urgent to study the mechanism of plants to improve the resistance to aluminum ion stress, which plays a key role in the resistance and quality of fruit trees. [0003] Recent studies have shown ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C07K14/415C12N15/82A01H5/00
CPCC07K14/415C12N15/8273
Inventor 王小非郝玉金安建平
Owner SHANDONG AGRICULTURAL UNIVERSITY
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