Eleusine indica PFK protein polyclonal antibody as well as preparation method and application thereof

A polyclonal antibody and goosegrass technology, applied in the biological field, can solve problems such as cumbersome processes, high environmental and operational requirements, and large quantities

Active Publication Date: 2020-06-30
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of EPSPS enzyme activity requires a large amount of reagents, the process is cumber

Method used

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  • Eleusine indica PFK protein polyclonal antibody as well as preparation method and application thereof
  • Eleusine indica PFK protein polyclonal antibody as well as preparation method and application thereof
  • Eleusine indica PFK protein polyclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 finds that the content of PFK protein and EPSPS protein has a positive correlation linear relationship

[0040] Select mature, plump and uniform goosegrass seeds of resistant populations, sow them into flowerpots filled with soil topsoil and organic matter (3:1) mixed soil, and place them in the greenhouse to cultivate them until the 5-6 leaf stage (reserve one piece of goosegrass in each pot). strain). 20 resistant plants were selected, and 0.2 g of the leaves of each plant were cut and frozen in liquid nitrogen and stored at -80 °C for detection of EPSPS and PFK enzyme activities.

[0041] The detection of phosphofructokinase (PFK) activity was determined by the cuvette method using a kit (MAK093) (Sigma-Aldrich). According to the steps in the instructions, 0.1 g of goosegrass leaf tissue was homogenized in an ice bath, centrifuged at 8000 g for 10 min at 4 ° C, Take 30 μL of the supernatant, catalyze it with the reaction reagent in a 1 mL quartz cuvette...

Embodiment 2

[0042] The cloning of embodiment 2 goosegrass PFK gene and the construction of prokaryotic expression vector

[0043] (1) Preparation of lyophilized plasmid of goosegrass PFK gene. First, codon optimization analysis was performed on the gene sequence of goosegrass PFK (see SEQ ID NO.5 for the specific nucleotide sequence), and no codons affecting subsequent reactions were found. Beijing Qingke Xinye Biotechnology Co., Ltd. was entrusted to artificially synthesize the goosegrass PFK gene (SEQ ID NO.5) using the standard vector pUC57, and obtain the PFK gene freeze-dried plasmid.

[0044] (2) The amino acid sequence of goosegrass PFK was analyzed using the CFSSP protein secondary structure online analysis tool, TopPred 1.10 protein hydrophobicity online analysis tool and Parker Antigenicity Plot protein immunogenicity online analysis tool. Select a relatively loose structure, no large α-helix or β-sheet region, and a region with strong hydrophilicity and immunogenicity, and des...

Embodiment 3

[0051] Embodiment 3 Induced expression and purification of goosegrass PFK recombinant protein of the present invention

[0052] Inoculate the Escherichia coli liquid containing pGEX-4T-1-PFK into LB culture liquid, and cultivate it on a constant temperature shaker at 37°C and 180 rpm. When the OD value of the cells reaches 0.5, add IPTG with a final concentration of 0.8 mM, and Adjust the temperature to 37°C and continue culturing at 180rpm for 4h. Take 50ml of E. coli bacterial liquid, place it in a centrifuge, 10000rpm, 1min, discard the supernatant to collect the bacterial cells, add 10ml of PBS, and resuspend the bacterial cells. Place the resuspended cells in an ice bath, and use an ultrasonic breaker to disrupt the cells. Place the crushed bacterial solution in a centrifuge at 12000rpm for 10min, collect the supernatant and precipitate respectively, and resuspend the precipitate with 10ml PBS, take 10μl of the two solutions respectively, add 10μl 2×SDS buffer, boil in b...

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Abstract

The invention discloses a eleusine indica PFK protein polyclonal antibody as well as a preparation method and application thereof. The invention provides an application of the polyclonal antibody in identification of drug resistance to herbicide glyphosate of eleusine indica. The application has the advantages of short detection period, high sensitivity and strong specificity, and has important application prospects in detection and research of glyphosate target resistance of eleusine indica. The relation between the eleusine indica PFK protein and the herbicide glyphosate resistance is disclosed for the first time, and whether the eleusine indica generates target amplification resistance to the herbicide glyphosate or not can be identified by detecting the content of the PFK protein. Themethod can be used for rapidly identifying the resistance and has guiding significance for scientifically selecting medicines to prevent and eliminate eleusine indica and relieving the generation andspreading of the resistance to glyphosate of eleusine indica.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a goosegrass PFK protein polyclonal antibody, a preparation method and application of the polyclonal antibody. Background technique [0002] Goosegrass is one of the top ten vicious weeds in the world. It belongs to the annual Gramineae plant. It is distributed in temperate and tropical regions all over the world. It is distributed in all provinces in the north and south of my country. crop field. Goosegrass has a well-developed root system and strong reproductive ability. A single mature goosegrass plant can produce nearly 140,000 seeds. The above characteristics make goosegrass cause serious economic losses to crops. [0003] Glyphosate is a broad-spectrum killing, systemic herbicide, which can effectively control annual and perennial weeds such as goosegrass. Glyphosate has a history of more than 40 years of application in my country, and is mainly used in non-cultivated land an...

Claims

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Application Information

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IPC IPC(8): G01N33/573C12N9/12C12N15/54C07K16/40C07K16/06
CPCC07K16/06C07K16/40C12N9/1205C12Y207/01011C12Y207/01056G01N33/573G01N2333/91215
Inventor 陈景超李香菊张朝贤崔海兰魏守辉黄红娟
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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