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Application for peanut hairy-root strain in improvement of low-nitrogen resistance and high-salt resistance of peanuts

A technology of hairy roots and peanuts, applied in the field of plant genetic engineering, to achieve the effect of avoiding long breeding cycle, easy operation and short cycle

Active Publication Date: 2020-07-14
SHANDONG PEANUT RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, studies have shown that C-terminally pepetides (CEPs) genes play a very important and unique role in the growth and development of plants and their stress responses, but the research has mainly focused on Arabidopsis and alfalfa. There are very few studies, so the application of developing a peanut hairy root strain to improve the ability of peanuts to tolerate low nitrogen and high salt is an effective solution to solve the shortage of cultivated land in our country, alleviate the conflict between grain and oil, and greatly increase peanut production.

Method used

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  • Application for peanut hairy-root strain in improvement of low-nitrogen resistance and high-salt resistance of peanuts
  • Application for peanut hairy-root strain in improvement of low-nitrogen resistance and high-salt resistance of peanuts
  • Application for peanut hairy-root strain in improvement of low-nitrogen resistance and high-salt resistance of peanuts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Extraction and reverse transcription of peanut RNA

[0040] (1) Experimental materials:

[0041] · Culture medium:

[0042]Escherichia coli LB liquid medium, ingredients: tryptone (Tryptone) 10g, yeast extract (Yeasextract) 5g, sodium chloride (NaCl) 10g, distilled water 1000mL, adjust the pH to 7.0, sterilize by autoclaving at 121°C for 20min.

[0043] Agrobacterium rhizogenes YEP liquid medium, ingredients: tryptone (Tryptone) 10g, yeast extract (Yeasextract) 10g, sodium chloride (NaCl) 5g, distilled water 1000mL, adjust pH to 7.0, sterilize by high pressure steam at 121°C for 20min.

[0044] Hairy root induction and propagation MS medium, component: 25mL 20×MS large amount (3.8g KNO 3 , 3.3 g NH 4 NO 3 , 0.34g KH 2 PO 4 , distilled water 100mL), 5mL 100×MS trace (0.223g MnSO 4 4H 2 O, 0.086g ZnSO 4 ·7H 2 O, 0.062g H 3 BO 3 , 0.0083g KI, 2.5mg Na 2 MoO 4 2H 2 O, 0.25 mg CuSO 4 ·5H 2 O, 0.25 mg CoCl 2 ·6H 2 O, distilled water 100mL), 5mL 1...

Embodiment 2

[0064] Embodiment 2: Cloning and expression vector construction of AhCEP1 gene

[0065] (1) Experimental materials:

[0066] AhCEP1 gene primer sequence:

[0067] Upstream primer 5'- GGATCC ATGGCCAATTCCAAACTTGTGTTG-3';

[0068] Downstream primer 5'- GAGCTC CTAGTTTGGCGTGGGGGAGG-3';

[0069] Wherein the underline is the restriction site, the restriction site of the upstream primer is BamHI, and the restriction site of the downstream primer is SacI.

[0070] (2) Experimental method:

[0071] Cloning of Q1 and AhCEP1 genes

[0072] (Q1-1) Using peanut cDNA as a template, use Novizyme high-fidelity enzyme (2×Phanta Max Master Mix, p515) to amplify AhCEP1. The reaction system is: 2×Phanta Max Master Mix 12.5 μL, upstream primer 1 μL, downstream 1 μL of primers, 1 μL of cDNA template, and make up to 25 μL with water;

[0073] The PCR reaction system is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 25s, annealing at 55°C for 25s, extension at 72°C for 20s...

Embodiment 3

[0082] Example 3: Induction of Transgenic Peanut Hairy Roots

[0083] P1, acquisition of peanut explants

[0084] The peanut seeds of Luhua No. 11 were planted in flower pots (bottom diameter: 36 cm, flower pot height: 26 cm) in an artificial climate chamber. Peanuts were planted as a single seed, with 4 seeds per pot. 8h dark. Peanut seedlings grow to the stage of 3-6 compound leaves, and 2-4 compound leaves are selected from the bottom up as experimental materials. Rinse the peanut leaves with tap water for 10 minutes, wash them gently with sterile water for 2 minutes, soak them in 4% sodium hypochlorite solution for 2 minutes, and take the slightly white incision of the petiole as the standard. Then wash with sterile water for 3-4 times, soak in sterile water for 10 minutes, and dry the water for later use;

[0085] P2, AhCEP1-R1601 strain activation

[0086] Pipette 100 μL of AhCEP1-R1601 Agrobacterium stored at -80°C and inoculate it into 100 mL of YEP liquid medium c...

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Abstract

The invention provides an application of a peanut hairy-root strain in improvement of low-nitrogen resistance and high-salt resistance of peanuts, belonging to the technical field of plant genetic engineering. According to a technical scheme of the invention, the method comprises the following steps: cloning an AhCEP1 gene from the peanuts, constructing a plant gene expression vector, then obtaining peanut hairy roots containing the peanut AhCEP1 genes through an agrobacterium rhizogenes mediation method, identifying whether the peanut AhCEP1 genes in transgenic peanut hairy roots are expressed or not, measuring the expression quantity of the peanut AhCEP1 genes, and screening out a transgenic peanut hairy-root positive strain. By utilization of the method provided by the invention, the low-nitrogen and high-salt resistance of the peanut hairy roots can be effectively improved; the content of the peanut hairy roots in a low-nitrogen and high-salt stress environment can also be improved; and the method has wide application prospect in the field of peanut stress resistance.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of a peanut hairy root strain in improving peanut tolerance to low nitrogen and high salt. Background technique [0002] Peanut is an important oil crop in my country. However, with the development of society and the continuous improvement of people's living standards, the output of edible oil in my country is seriously insufficient. Therefore, the control and utilization of saline-alkali land is an important way to solve the shortage of cultivated land in my country and alleviate the contradiction between grain and oil. Nitrogen and high salinity are the two main hazards of peanut growth in saline-alkali land. Among them, nitrogen is one of the main elements necessary for peanut growth. Insufficient nitrogen supply hinders the synthesis of protein, chlorophyll and nucleic acid in peanuts, resulting in short peanut plants, yellow and t...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/06A01H6/54C12Q1/6895A01H1/04
CPCC12N15/8261C12N15/8273C12N15/8271C07K14/415C12Q1/6895A01H1/04C12Q2600/158C12Q2600/13
Inventor 徐扬于子鹏张智猛戴良香丁红慈敦伟张冠初
Owner SHANDONG PEANUT RES INST
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