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 direc

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
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Example Embodiment

[0040] Example 1

[0041] Mulberry AKR2A antigen ELISA test:

[0042] (1) Antibody production: Search the cDNA sequence of AKR2A in the Arabidopsis Tair Database, remove the N-terminal 1-198 bases, and add the start base ATG to connect the 199th base of AKR2A to the stop code sequence; use PET21 as Plasmid, BL21 is a strain for producing Arabidopsis AKR2A target fragment protein; producing Arabidopsis AKR2A target fragment antibody;

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

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

[0045] (4) Preparation of mulberry AKR2A antigen: Take 0.2g of Xinjiang white mulberry and the subsequent 30-day cutting mulberry seedlings of 17 different mulberry variet...

Example Embodiment

[0054] Example 2

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

[0056] Take 0.2g of Husang 32 30-day cutting mulberry seedling top 1 to 2 leaf tissue samples, after grinding in a mortar under liquid nitrogen freezing, 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 well, and allowed to stand on ice for 0.5h; after taking it out, centrifuge at 12000r / min for 8min at 4°C, transfer the supernatant to another test tube, and use 0.2mL of the precipitate to contain 50mmol / L Phosphate buffer, 2% (v / v) Triton X-100 and 2% (m / v) SDS, the second extract of pH 6.8 is suspended to obtain a suspension; the above suspension is boiled for 10 min, and placed Cool on ice, centrifuge at 12000r / min for 8min at 4℃, take the supernatant; combine the above two supernatants to obtain the mulberry AKR2A antigen;

[0057] The remaining steps are exactly the same as in Example 1.

Example Embodiment

[0058] Example 3

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

[0060] Take 0.2g of Yunnan Changsui Mulberry 30-day cutting mulberry seedling top 1 to 2 leaf tissue samples to be tested, ground in a mortar under liquid nitrogen freezing, and 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 well, and allowed to stand on ice for 0.5h; after taking it out, centrifuge at 15000r / min for 9min at 4℃, transfer the supernatant to another test tube, and use 0.2mL of the precipitate to contain The second extract with 50mmol / L phosphate buffer, 2% (v / v) Triton X-100 and 2% (m / v) SDS, pH 7.2 was suspended to obtain a suspension; the above suspension was boiled for 10 minutes, and then set aside Cool on ice, centrifuge at 15000r / min for 9min at 4°C, take the supernatant; combine the above two supernatants to obtain the mulberry AKR2A antigen;

[0061] The remaining steps are exactly the sam...

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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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