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Method for preparing inhibitor for reducing tobacco nicotine synthesis by using double-stranded RNA interference technology, and applications of inhibitor

A technology of RNA interference and inhibitors, applied in botany equipment and methods, chemicals for biological control, applications, etc., can solve problems such as difficult promotion and application, and achieve low cost, broad industrialization prospects, no residue and Effects of Environmental Pollution

Inactive Publication Date: 2014-01-22
HUBEI CHINA TOBACCO IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the greater risks of genetically modified tobacco varieties in planting and consumption, it is difficult to popularize and apply genetically modified tobacco varieties with controllable nicotine content

Method used

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  • Method for preparing inhibitor for reducing tobacco nicotine synthesis by using double-stranded RNA interference technology, and applications of inhibitor
  • Method for preparing inhibitor for reducing tobacco nicotine synthesis by using double-stranded RNA interference technology, and applications of inhibitor
  • Method for preparing inhibitor for reducing tobacco nicotine synthesis by using double-stranded RNA interference technology, and applications of inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Extraction of total RNA from tobacco roots

[0027] The main tobacco variety Yunyan 87 in my country was used as the material. After washing the young roots with sterile water, the total RNA of tobacco was extracted with TRNzol Total RNA Reagent.

[0028] 1) Take 0.1 g of young tobacco root and grind it sufficiently in liquid nitrogen for 55 seconds, and add 1 mL of TRNzol;

[0029] 2) Place the homogenate sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex; 3) Centrifuge at 12,000rpm for 10m at 4°C, and take the supernatant;

[0030] 4) Add 0.2mL chloroform, cover the tube cap, shake vigorously for 15s, and place at room temperature for 3min;

[0031] 5) Centrifuge at 4°C, 12000rpm for 10~15m, the sample will be divided into three layers: the yellow organic phase, the middle layer and the upper colorless water phase, RNA is mainly in the water phase, transfer the water phase to a new centrifuge tube;

[0032] 6) Add an equal vol...

Embodiment 2

[0189] application of the inhibitor nornicotine,

[0190] Two days before tobacco topping, the inhibitor containing 30ug / mL dsRNA was sprayed on the root surface of the tobacco in the experimental group before 9 am or after 5 pm, and the application rate was 10mL / plant. The experimental group and the control group each have 4 groups, and each group has 100 tobacco plants, which are harvested and baked after normal maturity, and the nicotine content in the tobacco leaves is sampled to determine the content of nicotine. The experimental results showed that compared with the control, the nicotine content in the middle leaves decreased by 43.36%, and the nicotine content in the upper leaves decreased by 55.42%, see Table 1.

[0191] Table 1 Nicotine content in tobacco leaves of root-applied PMT gene partial sequence hairpin dsRNA preparation (%)

[0192]

[0193] Note: * indicates that the difference between the data in the same column and the control reaches a significant lev...

Embodiment 3

[0195] In this example, 10 mL / plant of the inhibitor containing 30 ug / mL dsRNA was applied to the leaves of tobacco. Others are identical with embodiment 2. The experimental results showed that compared with the control, the nicotine content in the middle leaves decreased by 18.92%, and the nicotine content in the upper leaves decreased by 23.31%.

[0196] Table 2 Nicotine content (%) in tobacco leaves of hairpin dsRNA preparation of leaf-applied PMT gene partial sequence

[0197]

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Abstract

The present invention discloses a method for preparing an inhibitor for reducing tobacco nicotine synthesis by using a double-stranded RNA interference technology, and applications of the inhibitor. The method comprises: extracting tobacco RNA; carrying out reverse transcription to obtain cDNA of tobacco putrescine N-methyltransferase (PMT) gene; adopting a PCR technology to amplify a fragment in the cDNA of the gene; linking the fragment to a plasmid vector PET-21a capable of expressing RNA; transforming the resulting plasmid into escherichia coli cells with RNAE3 enzyme deletion to express hairpin dsRNA; and breaking the cells to obtain a dsRNA crude preparation, and sequentially adding glycerol, a protection agent and a dispersing agent to the dsRNA crude preparation to obtain the inhibitor for reducing tobacco nicotine synthesis, wherein per strain of the tobacco is applied with 5-15 mL of the prepared inhibitor to reduce tobacco nicotine synthesis. The inhibitor of the present invention has characteristics of convenient preparation, low cost, and no harm on humans and animals, is suitable for a large amount application on tobacco, and has broad industrialization prospects.

Description

technical field [0001] The invention belongs to plant cultivation technology, and in particular relates to a method for preparing an inhibitor for reducing tobacco nicotine synthesis by double-stranded RNA interference technology and its application. Background technique [0002] Nicotine, also known as nicotine, belongs to the pyridine family of alkaloids and is the main component of alkaloids in tobacco. Its content is closely related to the quality and availability of tobacco leaves, and it is also one of the main quality indicators in cigarettes. The nicotine content in high-quality tobacco leaves is generally 1.5-3.5%. The nicotine content of tobacco leaves is low, the smoke has a low strength and little aroma; the nicotine content is high, the smoke strength is too strong, the irritation is enhanced, and a spicy taste is produced. In the main production areas of flue-cured tobacco in my country, the problem of high nicotine content, especially in the upper leaves, is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N63/02A01P21/00
Inventor 李琳吴风光张明月王豹祥何结望邱立友
Owner HUBEI CHINA TOBACCO IND
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