Gastrodia elata superoxide dismutase gene and application thereof

A superoxide and dismutase technology, which is applied in the fields of molecular biology and genetic engineering to achieve the effects of promoting the growth and development of Gastrodia elata, shortening the cultivation time, and improving the yield and quality

Active Publication Date: 2021-10-22
KUNMING UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report about the effect of Gastrodia elata superoxide dismutase (Superoxide dismutase, SOD) gene on improving the cold resistance of Gastrodia elata or promoting the growth of Arabidopsis

Method used

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  • Gastrodia elata superoxide dismutase gene and application thereof
  • Gastrodia elata superoxide dismutase gene and application thereof
  • Gastrodia elata superoxide dismutase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Acquisition of Gastrodia elata superoxide dismutase (Superoxide dismutase, SOD) gene of the present invention

[0038] The female hemp cultivated at 13°C was selected as the experimental material, and the total RNA of Gastrodia elata was extracted by the Trizol Reagent (Invitrogen) method. Specifically, 0.15 g of the 13°C female hemp sample was ground into powder with a mortar, and 1 mL of TRIzoL extract was added to the experimental sample. Continue to grind in the bowl until the liquid is transparent, let it stand at room temperature for 5 minutes, then transfer it to a centrifuge tube, then add 0.2 mL of chloroform, shake and mix, centrifuge at 4°C, 12000 rpm for 15 minutes, transfer the supernatant to a new tube, add 200 μL of chloroform again to take the supernatant , add 200 μL of isopropanol and 200 μL of sodium citrate high-salt solution (used to remove polysaccharides in Gastrodia elata) equal to the volume of the supernatant, mix well and let stand a...

Embodiment 2

[0047] Embodiment 2: the construction of SOD gene eukaryotic expression vector

[0048] pMD-18T- SOD Plasmid and pENTRTM-2B plasmid were carried out separately Eco R V and Kpn I double enzyme digestion (20μL system), the reaction system and operation process are as follows: take 2μL pMD-18T- SOD or pENTRTM-2B plasmid, add 2μL 10×K buffer, 0.5μL Kpn I, 0.5 μL Eco RV, 15 μL ddH 2 O, mix well and place at 37°C to react for 3h; carry out gel recovery separately.

[0049] will be recycled SOD The target gene fragment was connected with the pENTRTM-2B vector fragment, transformed into DH5α competent cells, spread on LB solid containing 100μg / mL Kan resistance and cultured at 37°C for 12h, and picked a single colony in 20mL of the same resistance LB liquid medium Incubate at 37°C for 12 hours, extract the plasmid for double-enzyme digestion, and send it for testing to see if it is connected to the entry vector.

[0050] will detect the correct entry cloning vector pE...

Embodiment 3

[0053] Embodiment 3: Overexpression SOD eukaryotic vector transforms Armillaria AM02

[0054] Agrobacterium transfection Armillaria AM02 to form genetically engineered Armillaria: ① Expand the positive cloned Agrobacterium in 30mL 100μg / mL Spe-resistant medium to about OD600=1.0, centrifuge at 4°C and 3000rpm for 10min, and remove the precipitate Resuspend in 5 mL of induction medium containing 150 µmol / L acetosyringone (AS), and culture on a shaker at 28°C until OD600=1.2. ②Use an ultrasonic homogenizer to crush and mix Armillaria bacterium balls, and culture them at 4°C in the dark for 3 hours. ③Add the induced Agrobacterium into the light-shielding mycelium at a ratio of 1:1 and mix well, co-cultivate at 25°C for 10 hours, centrifuge to remove the medium, and use sterile purified water containing 400µg / mL cef (cefotaxime sodium) antibiotics After washing several times, spread on PDA solid medium containing 100 μg / mL Spe resistance, and culture at 25°C in the dark until Arm...

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Abstract

The invention discloses a gastrodia elata superoxide dismutase (SOD) gene. The nucleotide sequence of the gastrodia elata superoxide dismutase (SOD) gene is shown as SEQ ID NO: 1; 239 amino acid residues are encoded; a prokaryotic expression vector of the gastrodia elata SOD gene is constructed, prokaryotic expression analysis shows that the gastrodia elata SOD gene is soluble protein, and the molecular weight of the gastrodia elata SOD gene is about 47.4 kDa; an eukaryotic overexpression vector of the gastrodia elata SOD gene is constructed, gastrodia elata symbiotic bacteria armillaria mellea is transfected through an agrobacterium method to obtain gastrodia elata SOD transfected armillaria mellea, the SOD transfected gene engineering armillaria mellea is cultured at 13 DEG C, the growth vigor is better than that of wild armillaria mellea, and the growth speed is high; therefore, the gastrodia elata superoxide dismutase gene is beneficial to shortening the culture time of the armillaria mellea and improving the cold-resistant growth of the armillaria mellea, and an experimental basis for enhancing the cold resistance of the gastrodia elata, expanding the planting distribution range of the gastrodia elata and improving the yield of the gastrodia elata is provided. meanwhile, experiments also prove that the gastrodia elata superoxide dismutase gene can promote the growth of arabidopsis thaliana.

Description

technical field [0001] The invention belongs to the related technical fields of molecular biology and genetic engineering, and relates to a Gastrodia elata superoxide dismutase (Superoxide dismutase, SOD) gene and its application in improving the cold resistance of Gastrodia elata or promoting the growth of Arabidopsis thaliana. Background technique [0002] Tianma ( Gastrodia elata ) is a heterotrophic perennial herb symbiotic with fungi, and it is also a precious traditional Chinese medicinal material. Gastrodin and 4-hydroxybenzyl alcohol are the main medicinal components of Gastrodia elata, which can prevent and treat senile dementia, Alzheimer's disease, depression, stroke, and improve memory. Because Gastrodia elata is rich in nutrition, it is a popular health food at present. In recent years, with the continuous destruction of the ecological environment and manual excavation, wild Gastrodia elata resources have decreased or are on the verge of extinction. At presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/84C12N1/15A01H5/00A01H6/20A01H6/62C12R1/645
CPCC12N9/0089C12N15/8273C12N15/8261C12Y115/01001
Inventor 李昆志周春艳仇全雷周福荣陈必连徐慧妮
Owner KUNMING UNIV OF SCI & TECH
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