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Method for driving AhRESS to produce resveratrol in peanut capillary root systems through specific promoters NtR2

A specific promoter, resveratrol technology, applied in the field of molecular biology, can solve problems such as no report on resveratrol research, and achieve the effects of rapid growth, stable genetic traits, and simple culture

Inactive Publication Date: 2016-04-20
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is no report on the production of resveratrol through the root-specific promoter to drive the expression of resveratrol synthase gene in peanut hairy roots

Method used

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  • Method for driving AhRESS to produce resveratrol in peanut capillary root systems through specific promoters NtR2
  • Method for driving AhRESS to produce resveratrol in peanut capillary root systems through specific promoters NtR2
  • Method for driving AhRESS to produce resveratrol in peanut capillary root systems through specific promoters NtR2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] [Example 1] Construction of pBI121-NtR2-AhRESS vector

[0036] 1. Cloning of tobacco root-specific promoter NtR2

[0037] Weigh about 0.1g of tobacco leaves, put them in a mortar and freeze-thaw them with liquid nitrogen, and quickly grind them into powder; use the CTAB method to extract DNA from tobacco, dissolve them in 40 μL, 10 ng / μL RNase sterile water, and place in Dissolve at 37°C for about 1 hour. Take 1 μL of extracted tobacco DNA and spot it for electrophoresis detection. Electrophoresis conditions: electrophoresis buffer 1×TAE, 1.0% agarose gel, voltage 120V, electrophoresis time about 20 minutes; concentration.

[0038] According to the splicing sequence of the NtR2 promoter obtained by the previous cloning in our laboratory, design primers NtR2-HindⅢ-promter-F (5'accaaagcttGAATATCTTCAGTATATTCGTTGTG3'), NtR2-XbaⅠ-promter-R (5'accatctagaGTCTCCTTCTTTAATTTTCTATCAAAG3'), according to the following PCR system Amplify. The system included 32.5 μL ddH 2 O, 10 ...

Embodiment 2

[0053] [Example 2] Transformation of Agrobacterium rhizogenes with pBI121-NTR2-AhRESS vector

[0054] Take about 2 μg (volume less than 20 μl) of pBI121-NTR2-AhRESS plasmid DNA and add it to 200 μL Agrobacterium rhizogenes competent cells, gently pipette and mix with a pipette gun; ice bath for 30 minutes, then place in liquid nitrogen for 5 minutes, Then bathe in a constant temperature water bath at 42°C for 1min, repeat 3 times; then place on ice for 5min, add 800μL YEB liquid medium, shake and culture in a constant temperature oscillator at 28°C, 175rpm, after 8h, centrifuge at 3000rpm for 3min, discard 800μl Clear, mix the remaining bacterial liquid, spread it on the YEB plate containing 50mg / lKan, and cultivate it in a constant temperature incubator at 28°C until a single colony is formed, about 72h; pick a single clone in the medium of 400μlYEB+50mg / l medium, 220rpm, 28°C shaking culture for 16h; for bacterial liquid PCR verification, if it is verified as a positive clon...

Embodiment 3

[0059] [Example 3] Induction of Peanut Hairy Roots

[0060] The aseptic seedlings and engineering bacteria of Xinhui small-grained peanut variety were prepared respectively, and peanuts were genetically transformed by Agrobacterium rhizogenes.

[0061] (1) Preparation of sterile peanut seedlings: prepare 0.1% mercuric chloride, dilute the corresponding amount of 1% mercuric chloride according to the ratio, store in a brown bottle, keep away from light, and seal; take 60 peanut seeds in a tissue culture bottle medium, soak in 75% ethanol for 1 min, shake vigorously, soak in 0.1% mercuric chloride for 10 min, shake vigorously, wash 5 times with sterile water, 5 min each time, soak in sterile water for 2 hours, remove the seed coat, insert the radicle down into MS culture Base, 3 grains per bottle; 28°C, dark culture for 48h, then move to 16h photoperiod, 8h dark condition until 2 true leaves grow (about 8-10d).

[0062] (2) Preparation of engineering bacteria: Take out the prev...

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Abstract

The invention relates to a method for driving AhRESS to produce resveratrol in peanut capillary root systems through specific promoters NtR2 and belongs to the field of plant genetic engineering. The tobacco root specific promoters NTR2 drive a peanut resveratrol synthase gene AhRESS carrier pBI121-NTR2-AhRESS to carry out genetic transformation on peanuts through mediation of agrobacterium rhizogenes, and the transgene peanut capillary roots for specifically expressing the AhRESS are obtained. Resveratrol in the transgene peanut capillary roots is extracted through an organic solvent extraction method, and the content of resveratrol in the transgene peanut capillary roots is 66.16 micrograms per gram (FW) through high performance liquid chromatography (HPLC) measurement to the maximum degree and is three times of the content of resveratrol in non-transgene capillary roots. The tobacco root specific promoter NTR2 drives the peanut resveratrol synthase gene AhRESS to be specifically expressed in the peanut capillary roots, and then a good foundation is laid for producing resveratrol.

Description

technical field [0001] The invention relates to a method for driving AhRESS to produce resveratrol in peanut hairy roots by a specific promoter NtR2, and belongs to the field of molecular biology. technical background [0002] Resveratrol (Res for short) is an important phytoalexin that exists in plants such as knotweed, grapes, and peanuts, and is particularly abundant in grape peels and peanut roots. It has a variety of biological activities, is a natural antioxidant, can reduce blood lipids, inhibit platelet aggregation, anti-cancer, anti-inflammatory, anti-radiation, anti-aging, prevention and treatment of cardiovascular diseases and so on. Both it and paclitaxel are known as green anticancer drugs. However, the content of resveratrol in nature is small, and the efficient production of resveratrol by plant genetic engineering technology is an important way to obtain a large amount of resveratrol. [0003] Resveratrol widely exists in seed plants, and as a stilbene seco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/06
CPCC12N15/8205C12N15/8243
Inventor 陈华庄伟建马世伟张冲蔡铁城邓烨
Owner FUJIAN AGRI & FORESTRY UNIV