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Method for identifying anti-disease gene of apple by combining apple callus tissue cell culture and genetic transformation

A callus, disease resistance-related gene technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve difficult disease resistance genes, self-incompatibility, gene function Redundancy and other problems, to improve the screening efficiency, overcome the long conversion cycle, short time-consuming effect

Active Publication Date: 2017-03-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is still unavoidable that apples have long growth cycle and difficult transformation, which are different from the main constraints of model plants.
Moreover, due to the high degree of heterozygosity, redundant gene functions and biological self-incompatibility in apples, it is also difficult to identify disease resistance genes in apples through mutant screening.

Method used

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  • Method for identifying anti-disease gene of apple by combining apple callus tissue cell culture and genetic transformation
  • Method for identifying anti-disease gene of apple by combining apple callus tissue cell culture and genetic transformation
  • Method for identifying anti-disease gene of apple by combining apple callus tissue cell culture and genetic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1, apple callus cell culture and genetic transformation identification of apple disease resistance-related gene MdSYP121

[0056] 1. Obtaining the target gene MdSYP121 to be identified

[0057] 1. Extraction of total RNA from Fuji apple pulp by CTAB method

[0058] CTAB extraction buffer formulation: 0.1M Tris-Cl (pH 8.0), 1.4M NaCl, 20mM EDTA, 2% CTAB, 2% PVP, 2% β-mercaptoethanol

[0059] 1) Adjust the water bath to 65°C and pre-cool the centrifuge at 4°C.

[0060] 2) Prepare CTAB extract, take 4mL and add it to a 10mL centrifuge tube treated with DEPC, and preheat in a 65°C water bath.

[0061] 3) Grinding 0.5-1 g of apple pulp in liquid nitrogen until it becomes powdery.

[0062] 4) Quickly add the ground sample into the preheated centrifuge tube, shake vigorously, vortex, and place in a water bath at 65°C for 5 minutes.

[0063] 5) Cool to room temperature on an ice shelf, add an equal volume of chloroform / isoamyl alcohol (24:1) and vortex to mix, and cen...

Embodiment 2

[0150] Example 2, apple callus cell culture and genetic transformation identification of apple disease resistance related gene MdBAK1

[0151] 1. Acquisition of the target gene MdBAK1 to be identified

[0152] According to the apple genome data released by GDR, the primer design software DNASTAR was used to design the cloning primers of the BAK1 homologous gene in apple:

[0153] MdBAK1-1-NcoI-F:5'-CATGCCATGGACCCAACACTGATGAC-3',

[0154] MdBAK1-1-SmaI-R:5'-TCCCCCGGGTCTGGGACCGGACAACTC-3';

[0155] MdBAK1-2-NcoI-F:5'-CATGCCATGGCGTCCTCCGCCTCTGTT-3',

[0156] MdBAK1-2-SmaI-R:5'-TCCCCCGGGTCTGGGACCGGACAACTC-3'.

[0157] The RNA of the red callus of wild apple in Xinjiang was extracted, the cDNA obtained by reverse transcription was used as a template, and the above two pairs of primers were used for PCR amplification respectively, and the MdBAK1-1 CDS sequence was obtained, and the fragment length was 1851bp ( Figure 4 A, left) and MdBAK1-2CDS sequence, fragment length 1833bp (...

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Abstract

The invention discloses a method for identifying an anti-disease gene of an apple by combining apple callus tissue cell culture and genetic transformation, particularly provides a method for identifying a target gene as a disease resistance-related gene of the apple. An apple callus tissue is taken to serve as a transformation receptor, and the target gene is identified as the disease resistance-related gene of the apple. Proved by tests, apple callus tissue transformation is utilized for functional identification of the anti-disease gene of the apple, technical barriers, including long plant transformation period, difficulties in transformation and the like of the apple, in anti-disease gene function research of the apple are overcome, screening efficiency of the anti-disease gene of the apple is increased greatly, an efficient and simple method for screening and identifying the anti-disease gene of the apple is provided, and exploration of the anti-disease gene of the apple is enabled to be possible. The method has the advantages of efficiency, easiness in implementation and short consumed time, and establishes a base and a possibility for anti-disease gene screening and identification of the apple and in-depth research of a mechanism of action of the anti-disease gene of the apple.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying apple disease-resistant genes by combining apple callus cell culture and genetic transformation. Background technique [0002] Apple is one of the four major fruits in the world. my country is the largest producer and consumer of apples in the world, ranking first in both area and output. Fungal diseases represented by apple ring spot are important diseases that seriously restrict the high-quality and efficient development of my country's apple industry. Taking ring spot as an example, the disease is caused by the pathogenic fungus Botryosphaeria dothidea, and its damage is mainly manifested in two aspects: (1) infecting branches and trunks, resulting in tree weakness, resulting in a decline in apple yield and quality; (2) During the fruit ripening period and post-harvest storage period, the fruit is infected, causing the fruit to rot rapidly and lose its ed...

Claims

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Application Information

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IPC IPC(8): C12Q1/18C12Q1/68C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8282C12Q1/18C12Q1/6895C12Q2600/13
Inventor 何晓文邱化荣刘秀霞李学会陈学森吴树敬
Owner SHANDONG AGRICULTURAL UNIVERSITY
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