Molecular biological method for identifying seedling stage of nicotine transformation tobacco plant

A technology of molecular biology and identification method, which is applied in the field of molecular biological identification of tobacco nicotine transformed plant seedlings, can solve the problems that the identification results are affected by environmental factors, it is difficult to ensure the size of the target group, it is not efficient and timely, and achieves Reduce the average nicotine conversion rate, easy to promote, and easy to operate

Inactive Publication Date: 2011-11-02
TOBACCO RES INST OF HUBEI PROVINCE
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Problems solved by technology

Although this method is effective, there are following deficiencies: 1, the sampling period is still late in the group tree stage after transplanting, and the field was wasted after removing the transformed strains, and it is difficult to ensure the size of the target group; 2, it needs to be induced by chemicals. ;3. The identification results are affected by environmental factors: such as drug concentration, modulation temperature and time, chemical detection factors, etc.
Therefore, although there is a good reference, it is still not efficient and timely enough to meet the actual needs

Method used

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Examples

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Embodiment 1

[0019] Embodiment 1, the seedling stage molecular biological identification of burley tobacco nicotine transformed strain, its specific steps are as follows in order:

[0020] A. Tobacco Seedling Identification and Fresh Leaf Sample Acquisition: List the Burley tobacco seedlings to be identified one by one for identification, and then test each marked tobacco seedling at the seedling stage (as long as one leaf can be obtained during the growth period). Sampling of fresh leaves, the sampling volume of each tobacco seedling is 1 leaf, the fresh leaf samples taken are packed in sealed bags, and the labels consistent with the original tobacco seedlings are done, and they are quickly stored in an ice box;

[0021] B. Tobacco seedling genomic DNA extraction: take out the fresh leaf samples in each sealed bag, and use the CTAB method to extract the tobacco seedling genomic DNA of each sample. The specific extraction methods are as follows:

[0022] a. Preheat the CTAB extraction buff...

example 2

[0043] Example 2, the seedling stage molecular biology identification of Maryland nicotine nicotine transformed strain, its specific steps are as follows:

[0044] A. Tobacco Seedling Identification and Fresh Leaf Sample Acquisition: Mark the Maryland tobacco seedlings to be identified one by one, and then perform a fresh test on each marked tobacco seedling at the seedling stage (as long as two leaves can be obtained). Leaf sampling, the sampling amount of each tobacco seedling is 2 leaves, the fresh leaf sample taken is packed in a sealed bag, and the label is consistent with the original tobacco seedling, and it is quickly stored in an ice box, and the fresh leaf sample is returned to the experiment. Leaf samples were stored in an ultra-low temperature refrigerator for 20 days and then taken out to extract genomic DNA;

[0045] B. Tobacco seedling genomic DNA extraction: take out the fresh leaf samples in each sealed bag, and use the CTAB method to extract tobacco seedling ...

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Abstract

The invention relates to a molecular biological method for identifying the seedling stage of a nicotine transformation tobacco plant. The method comprises the following main steps of: extracting the genomic DNA from a fresh leave sample of the plant in the seedling stage by a CTAB (hexadecyltrimethylammonium bromide) method, performing PCR (polymerase chain reaction) using the genomic DNA as an amplification template in combination with specific primers, then enabling the PCR products to be subjected to agarose gel electrophoresis, observing and reading a molecular marker spectrum in the gel imaging system, and subsequently comparing the molecular marker spectrum with the target spectrum to determine the transformation characteristics of the sample. The method in the invention is free from environmental factor influences, is in no need of artificial chemistry to induce nicotine to transform, and is capable of sampling operation during the seedling stage to bring forward the identification and screening of a transformation plant ahead of tobacco plant implantation, thus greatly saving time, being accurate and efficient, simple and convenient to operate, easy to popularize, and applicable to purification of tobacco parent seeds and identification of nicotine transformation traits as well as identification and removal of nicotine transformation plants of other tobacco types.

Description

technical field [0001] The invention relates to an identification method for tobacco nicotine transformed strains, in particular to a molecular biological identification method for tobacco nicotine transformed strains at seedling stage. Background technique [0002] The current research results show that there are four main types of alkaloids in tobacco: nicotine, nornicotine, anatabine, and anatabine. Common tobacco belongs to the nicotine accumulation type, mainly nicotine, its content accounts for more than 90% of the total alkaloids, nornicotine is the second largest alkaloid, the content generally only accounts for 3-5% of the total alkaloids, the other two Alkaloids account for only a small part. The level of nicotine content is an important factor to determine the sensory quality of tobacco leaves. Generally, it is required that the content of nicotine should be appropriate to achieve the purpose of moderate physiological strength, mellow taste and low irritation. N...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 蔡长春李宗平李进平杨树杨春雷林国平朱天罗晓敏
Owner TOBACCO RES INST OF HUBEI PROVINCE
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