Method for improving content of peanut secretory peptide of peanut hairy roots

A technology for secreting peptides and hairy roots, applied in the field of genetic engineering, can solve the problems of no biological activity of the product, high cost of chemical synthesis, uneven final yield, etc., and achieves less space utilization, good induction efficiency and low cost. Effect

Pending Publication Date: 2019-11-05
SHANDONG PEANUT RES INST
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AI Technical Summary

Problems solved by technology

[0003] At present, chemical synthesis and DNA recombination synthesis are generally used to synthesize peanut secretory peptides. However, chemical synthesis has the disadvantages of high cost, many by-products, and no biological activity of the product. DNA recombination synthesis is due to the transformation of different plant receptors. Large differences in efficiency, resulting in uneven final output
It is reported in the prior art that the hairy root culture system is used to obtain transgenic hairy roots to achieve high yields of some functional substances, but there is no report on the hairy root culture system in the field of peanut secretion peptides, so how to cultivate transgenic peanut hairs root to increase the content of active peanut secretory peptides, which is of great significance to improve the ability of peanuts to resist environmental stress and increase peanut production

Method used

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  • Method for improving content of peanut secretory peptide of peanut hairy roots
  • Method for improving content of peanut secretory peptide of peanut hairy roots
  • Method for improving content of peanut secretory peptide of peanut hairy roots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Acquisition of Peanut Secreted Peptide Gene

[0049] (S1) Peanut RNA Extraction

[0050] Use Tiangen Plant Total RNA Extraction Kit (DP432) to extract the total RNA of peanuts, the specific steps are as follows:

[0051]S1-1: Take peanut seedlings that have grown for about 15 days and grind them fully in liquid nitrogen, add 400 μL of RL reagent in the kit for every 50-100 mg of tissue, vortex and oscillate to mix, and incubate at 56°C for 1-3 minutes to completely lyse the plant tissue;

[0052] S1-2: Transfer the solution in step S1-1 to the filter column CS (the filter column CS is placed in a collection tube), and centrifuge at 12,000 rpm for 5 min. Carefully pipette the supernatant from the collection tube into a new RNase-Free 1.5mL centrifuge tube with a tip with a part of the end cut off, and try to avoid contacting the cell debris in the collection tube with the tip;

[0053] S1-3: Slowly add 0.5 times the supernatant volume of absolute ethanol, mi...

Embodiment 2

[0063] Example 2: Peanut Secretory Peptide Gene Cloning and Vector Construction

[0064] The peanut cDNA obtained in Example 1 was used as a template for gene cloning and vector construction.

[0065] (P1) Target Peanut Peptide Gene Cloning

[0066] P1-1: Using peanut cDNA as a template, use Novizyme high-fidelity enzyme (2×Phanta Max Master Mix, p515) to amplify the target small peptide. The reaction system is: 2×Phanta Max Master Mix 12.5 μL, upstream primer 1 μL, 1 μL of downstream primers, 1 μL of cDNA template, and make up to 25 μL with water;

[0067] The PCR reaction system is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 25s, annealing at 55-60°C for 25s (according to the primer annealing temperature), extension at 72°C for 15-30s (according to the amplification length), 35 cycles, and post-extension at 72°C 5min;

[0068] P1-2: After the PCR reaction, perform agarose gel electrophoresis, cut the target band and perform gel recovery. The gel reco...

Embodiment 3

[0076] Embodiment 3: Induction of peanut hairy roots

[0077] The peptide-R1601 obtained in Example 2 was transformed into Agrobacterium rhizogenes to infect peanut leaves.

[0078] (M1) Peanut explant acquisition

[0079] The seeds of the peanut varieties to be tested (including 2 large peanut varieties HY25 Huayu No. 25, Luhua No. 11 and 2 small peanut varieties HY20 Huayu No. 20 and Luhua No. 12) were planted in pots in the artificial climate chamber (The diameter of the bottom is 36cm, and the height of the flowerpot is 26cm). The cultivation temperature is 28°C during the day, 25°C at night, 16h light / 8h darkness, and single peanuts are planted, 4 seeds per pot. 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 t...

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Abstract

The invention provides a method for improving the content of peanut secretory peptide of peanut hairy roots, and belongs to the field of genetic engineering. A secretory peptide gene is cloned from peanut, a plant gene expression vector is constructed, the peanut hairy roots containing the peanut secretory peptide gene are obtained by an agrobacterium rhizogenes mediated method, the expression ofthe peanut secretory peptide gene in the transgenic peanut hairy roots is identified, the biomass of the peanut hairy roots is determined, the expression level of the peanut secretory peptide is determined by a Tricine SDS-PAGE method, and the content of the peanut secretory peptide is determined by a BCA protein quantitative detection method. The method adopts the peanut hairy root system to produce the peanut secretory peptide, has the advantages of simple operation, short cycle, high output, low cost, biological activity and the like, and is a relatively economic and efficient preparation method of the peanut secretory peptide.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a method for increasing the content of peanut secreted peptide in peanut hairy roots. Background technique [0002] Peanut is an important oil crop, but its yield is easily affected by environmental stresses such as external bacteria, fungi, drought or salinity. Studies have shown that peanut secreted peptides play an important role in plant growth and development, antibacterial, and salt tolerance. This provides a new research direction for improving the stress and disease resistance of peanuts. [0003] At present, chemical synthesis and DNA recombination synthesis are generally used to synthesize peanut secretory peptides. However, chemical synthesis has the disadvantages of high cost, many by-products, and no biological activity of the product. DNA recombination synthesis is due to the transformation of different plant receptors. Efficiency varies greatly, resulting in une...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06A01H6/54
CPCC12N15/8251C12N15/8205
Inventor 徐扬于子鹏张智猛戴良香慈敦伟丁红秦斐斐
Owner SHANDONG PEANUT RES INST
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