Osmotic stress ubiquitination modified protein screening method

A technique for ubiquitination modification and osmotic stress, which is applied in the field of screening ubiquitination modified proteins under potato osmotic stress, and can solve problems such as screening methods for ubiquitination modification proteins that have not been seen

Active Publication Date: 2021-05-18
GANSU AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Problems in the prior art: There is no screening method for u

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  • Osmotic stress ubiquitination modified protein screening method
  • Osmotic stress ubiquitination modified protein screening method
  • Osmotic stress ubiquitination modified protein screening method

Examples

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Embodiment 1

[0013] A method for screening ubiquitination-modified proteins under potato osmotic stress, specifically comprising the following steps:

[0014] 1. Breeding and grouping of shoot tip virus-free tissue culture seedlings and stress treatment

[0015] The tubers of potato cultivar "Atlantic" were taken out from the cold storage, and kept in the dark at room temperature for 30 days to germinate. When the potato sprouts grow to 2 cm, wash the sprouts with sterile water for 10 min, transfer them to a sterile Erlenmeyer flask, and use 0.1% HgCl 2 Sterilize for 1 min, transfer to a new sterile Erlenmeyer flask, sterilize with 75% ethanol for 5 min, then transfer to a new sterile Erlenmeyer flask, sterilize with 0.05% NaClO for 15 min, rinse with sterile water 3 times , and finally cut off the bottom of the buds with sterilized scissors, transferred to solid MS medium, and cultured at 20°C under light (2000 lx, 16 h / d) for 20 days. When the buds differentiated into seedlings for the...

Embodiment 2

[0048] This example provides a method for extracting plant total RNA, which is operated according to the instructions of the TIANGEN plasmid mini-extraction kit (with deletions and modifications). The specific steps are as follows:

[0049] 1. Soak round-bottom centrifuge tubes, tweezers, scissors, pipette tips, grinding rods and other utensils in 0.1% DEPC solution for 24 hours, take them out, put them in a plastic box, wrap them in newspaper, and place them in an autoclave at 120°C for 30 minutes Sterilize twice, ventilate and dry in an ultra-clean bench, and set aside.

[0050] 2. Take about 200 mg of plant tissue and place it in a round-bottomed centrifuge tube. The outer wall of the round-bottomed centrifuge tube is immersed in liquid nitrogen to freeze (to avoid contamination caused by liquid nitrogen entering the centrifuge tube). Use a grinding rod to grind until the sample is powdery. The bottom centrifuge tube was continuously extended into liquid nitrogen for freezi...

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Abstract

The invention provides a method for screening ubiquitinated modified proteins under potato osmotic stress, comprising: (1) stem tip virus-free tissue culture seedling breeding and grouping and stress treatment; (2) ubiquitin (Ub) and 26S proteasome (26S ) expression level determination and sampling time; (3) protein sample preparation; (4) protein sample trypsinization; (5) ubiquitinated peptide enrichment; (6) LC-MS/MS analysis; (7) Mass spectrometry data analysis. This method can accurately and maximize the screening of ubiquitination modified proteins under osmotic stress in potato, and a total of 25 differential ubiquitination modification sites under osmotic stress were obtained. These significantly changed ubiquitination modified proteins are related to osmotic stress response. Therefore, the method for screening ubiquitination-modified proteins described in this application is of great significance and significant progress in discovering genes responding to osmotic stress and clarifying the mechanism of ubiquitination modification involved in osmotic stress.

Description

technical field [0001] The invention belongs to the field of molecular genetic breeding and provides a method for screening ubiquitinated modified proteins under potato osmotic stress. Background technique [0002] Potato (Solanum tuberosum L.) belongs to the Solanaceae tuber crop, which originated in the Andes and has high economic value. Humans have domesticated and grown potatoes for more than 8,000 years. According to statistics from the World Food and Agriculture Organization (FAO) in 2016, the global planting area of ​​potatoes reached 19,246,462 hectares, with an annual output of 376,826,967 tons (http: / / www.fao.org / home / en / ), second only to corn, wheat and rice , is the fourth largest food crop. As of May 2018, a total of 163 countries around the world grow potatoes (http: / / www.fao.org / faostat / en / #data / QC), and the planting area has continued to increase in recent years. The food and nutrition of residents in many poor areas mainly Derived from potatoes. Potatoes...

Claims

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Application Information

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IPC IPC(8): G01N30/08G01N30/88
CPCG01N30/08G01N30/88
Inventor 唐勋司怀军张宁
Owner GANSU AGRI UNIV
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