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Method for detecting cold resistance of mulberry tree variety

A mulberry tree and ability technology, applied in the field of testing the cold resistance ability of mulberry varieties, can solve the problems of lack of molecular chaperones and protein orientation, and achieve the effect of great academic and economic value.

Active Publication Date: 2018-07-06
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Without chaperones, many proteins cannot be directed to target regions to carry out their functions

Method used

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  • Method for detecting cold resistance of mulberry tree variety
  • Method for detecting cold resistance of mulberry tree variety
  • Method for detecting cold resistance of mulberry tree variety

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Mulberry AKR2A Antigen ELISA Detection:

[0042] (1) Antibody production: Retrieve the cDNA sequence of AKR2A in the Arabidopsis Tair Database, remove the N-terminal 1-198 bases, and add the starting base ATG to connect the 199th base of AKR2A to the stop codon sequence; PET21 is used as the Plasmid, BL21 is used to produce Arabidopsis thaliana AKR2A target fragment protein; produce Arabidopsis thaliana AKR2A target fragment antibody;

[0043] (2) Antibody coating: Dilute the above-mentioned Arabidopsis thaliana AKR2A target antibody, and coat a 96-well polystyrene ELISA reaction plate at a concentration of 100 μL / well to obtain an antibody matrix for ELISA reaction, and store it at 4°C for later use;

[0044] (3) Block the antibody-coated plate with 5% skimmed milk powder, 200 μL per well, and incubate at room temperature for 1 h;

[0045] (4) Preparation of mulberry AKR2A antigen: take 0.2g of Xinjiang white mulberry and subsequent 17 different mulberry varieties of ...

Embodiment 2

[0055] The difference between embodiment 2 and embodiment 1 is in step (4):

[0056] Take 0.2g of Husang 32 30d cuttage mulberry seedling top 1~2 leaf tissue samples, after grinding in a mortar under liquid nitrogen freezing state, use 0.2mL containing 50mmol / L sodium phosphate and 1mmol / L EDTA, The first extract with a pH of 6.8 was shaken for 30s, mixed evenly, and kept on ice for 0.5h; after taking it out, it was centrifuged at 12000r / min for 8min at 4°C, and the supernatant was transferred to another test tube, and the precipitate was mixed with 0.2mL containing 50mmol / L phosphate buffer saline, 2% (v / v) Triton X-100 and 2% (m / v) SDS, pH is the suspension of the second extraction solution of 6.8 to obtain a suspension; the above-mentioned suspension is boiled for 10min, placed in Cool on ice, centrifuge at 12000r / min for 8min at 4°C, and take the supernatant; combine the above two supernatants to obtain the Morus alba AKR2A antigen;

[0057] All the other steps are ident...

Embodiment 3

[0059] The difference between embodiment 3 and embodiment 1 is in step (4):

[0060] Take 0.2g of the 30-day cuttage mulberry seedlings to be tested of 0.2g leaf tissue samples from the top of mulberry seedlings, grind them in a mortar in a liquid nitrogen frozen state, and use 0.2mL containing 50mmol / L sodium phosphate and 1mmol / L EDTA , the first extract with a pH of 7.2 was shaken for 30s, mixed evenly, and left to stand on ice for 0.5h; after taking it out, it was centrifuged at 15000r / min for 9min at 4°C, and the supernatant was transferred to another test tube, and the precipitate was mixed with 0.2mL containing 50mmol / L phosphate buffer, 2% (v / v) Triton X-100 and 2% (m / v) SDS, the pH is the suspension of the second extraction solution of 7.2 to obtain a suspension; the above-mentioned suspension is boiled for 10min, placed Cool on ice, centrifuge at 15,000 r / min for 9 minutes at 4°C, and take the supernatant; combine the above two supernatants to obtain the Morus alba A...

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Abstract

The invention relates to the technical field of biologics, in particular to a method for detecting cold resistance of mulberry tree varieties. The method comprises the following steps: preparing an antibody through a specific AKR2A protein sequence, and coating a 96-pore polystyrene reaction plate with the arabidopsis thaliana AKR2A antibody. After specific mulberry leaf tissue treatment, an AKR2Aprotein antigen is prepared, the AKR2A protein antigen is put into an anti-body coated reaction plate according to serial numbers, is subjected to immunoreactions with the AKR2A antibody, and is further subjected to a reaction with an second antibody marked by horseradish peroxidase, and comparison analysis shows that the low-temperature stress resistance of a mulberry tree is in a positive correlative relationship with the content of an AKR2A gene or protein, and the cold resistance of a mulberry tree can be indicated by detecting the content of the AKR2A protein in 1-2 leaves at the top endof the mulberry tree. A direct, rapid and reliable mulberry tree cold resistance detection method can be established, and the method can be applied to screening of cold-resistant mulberry tree variety, molecular seed breeding and long-distance and super long-distance seed introduction of mulberry germplasm (varieties), and has great academic and economic values.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting cold resistance of mulberry varieties. Background technique [0002] The natural distribution area of ​​mulberry is very wide. There are mulberry trees found in Asia, North America, Europe and Africa. The mulberry resources are found in tropical, subtropical, temperate to cold temperate zones. Due to long-term growth in different natural environments, extremely rich genetic diversity has been formed. Some mulberry trees can grow in arid and semi-arid desert areas with less than 150mm of precipitation, some can resist a low temperature of minus 30°C, and can also endure a high temperature of 40°C, while some mulberry trees are more adaptable to soil pH. It can grow under the condition of 4.5~8.5, and the mulberry tree can also grow normally when the soil salinity is 0.2%. There are more than 7000 copies of mulberry germplasm preserved in the world's major mulb...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/535
CPCG01N33/535G01N33/543G01N33/6857G01N33/686G01N2333/415
Inventor 陈琳张彩萍于少芳胡文君裘晓云卢红伶沈国新
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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