Method of detecting relative level of tobacco nicotine conversion

A relatively high-level, tobacco technology, applied in the field of molecular biology, can solve problems such as elimination of bad transformants, time-consuming, etc., and achieve the effects of low price, convenient use and small sampling amount.

Inactive Publication Date: 2012-11-28
CHINA TOBACCO ZHEJIANG IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the method has good stability and operability, it can usually only analyze mature tobacco leaves or processed tobacco leaves after harvesting, and requires certain treatment after harvesting, which is time-consuming and cannot be used throughout the growth period. Dynamically understand the level of nicotine transformation in different varieties of tobacco leaves and different populations of the same variety, so that it is impossible to eliminate bad transformed strains in time

Method used

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  • Method of detecting relative level of tobacco nicotine conversion
  • Method of detecting relative level of tobacco nicotine conversion
  • Method of detecting relative level of tobacco nicotine conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Preparation of Standards for Detecting Relative Levels of Tobacco Nicotine Conversion

[0054] The following only takes the recombinant plasmid containing the full-length cDNA of the nicotine demethylase gene CYP82E4 as an example to illustrate its effect on detecting the relative level of tobacco nicotine conversion. All other plasmids containing the full-length cDNA of the nicotine demethylase gene CYP82E4 Or the recombinant vector of partial nucleotide sequence or the transgenic host cell containing the recombinant vector has the same effect on detecting the relative level of tobacco nicotine conversion as the standard product.

[0055] (1) Sampling of tobacco leaves

[0056] Using Burley tobacco B37 as the material, select 2g of the middle leaves at the mature stage, wrap the fresh leaf samples with tinfoil paper and mark them, put them in an ice box filled with liquid nitrogen for storage, and send them to the laboratory for further analysis. work in one...

Embodiment 2

[0100] Example 2 Detecting the Relative Levels of Burley Tobacco Wild Type and Mutant Nicotine Conversion

[0101] Burley tobacco B37 and high nicotine conversion mutant HNC-1 were used as materials, and the relative levels of nicotine conversion were detected by using the primers, and the specific steps were as follows:

[0102] (1) Sampling of tobacco leaves

[0103] Using Burley tobacco B37 and the high-nicotine conversion mutant HNC-1 as materials, select 2 g of the middle leaves of the cluster stage, flourishing stage, and mature stage, respectively, and wrap the fresh leaf samples with tin foil and mark them. Quickly put it in an ice box filled with liquid nitrogen for storage, and quickly send it to the laboratory for the next step.

[0104] (2) Total RNA extraction, content determination and reverse transcription reaction

[0105] Total RNA extraction: Take 200 mg of the tobacco leaf sample in the above-mentioned tinfoil paper, and use the RNeasy Plant Mini kit of Qi...

Embodiment 3

[0122] Example 3 Detection of relative levels of nicotine conversion in flue-cured tobacco at different developmental stages

[0123] Using the flue-cured tobacco variety Honghua Dajinyuan as the material, the method was used to detect the relative levels of nicotine conversion in different developmental stages (group tree stage, flourishing stage, lower mature stage, middle mature stage and upper mature stage). The specific steps are as follows:

[0124] (1) Sampling of tobacco leaves:

[0125] Using Dajinyuan safflower as the material, select 2g each of the leaves of the group tree stage, the flourishing stage, the lower mature stage, the middle mature stage and the upper mature stage. Store in an ice box filled with liquid nitrogen and quickly send to the laboratory for the next step.

[0126] (2) Total RNA extraction, content determination and reverse transcription reaction

[0127] Extraction of total RNA: Take 200 mg of the tobacco leaf samples of Honghua Dajinyuan in ...

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Abstract

The invention discloses a method of detecting relative level of tobacco nicotine conversion. The method comprises the following steps of: (1) collecting leaves at different stages of same type of tobacco, leaves of different parts or leaves of different types of tobacco; (2) extracting total RNA (Ribonucleic Acid) of the leaves respectively and synthesizing cDNA (complementary Deoxyribonucleic Acid) through reverse transcription; (3) designing a specific primer aiming at a nicotine conversion related gene and performing real-time fluorescent quantitative PCR (Polymerase Chain Reaction) by using the specific primer by taking the cDNA as a template to obtain respective Ct value; and (4) calculating by using a standard curve according to the Ct value to obtain template quantity related to the nicotine conversion in the respective cDNA and judging the relative level of the nicotine conversion between the leaves at different stages of same type of tobacco, leaves of different parts or leaves of different types of tobacco. According to the method, real-time monitoring can be performed on the relative level of the tobacco nicotine conversion, the accuracy is high, the flexibility is high, and bad tobacco conversion strains can be timely eliminated.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a method for detecting the relative level of tobacco nicotine conversion. Background technique [0002] Alkaloids are a class of secondary metabolites composed of small molecule nitrogen-containing compounds that widely exist in plants. At present, more than 12,000 alkaloids have been isolated and identified in the world. Alkaloids have a special status in tobacco and its products. It is not only an important quality factor of tobacco, but also defines the characteristics of tobacco as a commodity. Nicotine, nornicotine, anatabine, and anatabine are the four main alkaloids in tobacco. People smoke tobacco mainly to inhale nicotine (nicotine), also known as nicotine. Ordinary tobacco belongs to the nicotine accumulation type, and the nicotine content accounts for 90-95% of the total alkaloid content, and the other three types only account for 5%-10% of the total alkalo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 储国海周国俊孙勃黄芳芳林福呈杨军张芬金立锋
Owner CHINA TOBACCO ZHEJIANG IND
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