Kit for quickly extracting plant genome and applications thereof

Abstract

The invention discloses a kit which is suitable for PCR amplification and used for quickly extracting plant genomes, comprising a lysate, a neutralizer and a precipitation liquid. The kit improves and optimizes the traditional alkaline lysis method used for preparing genome and enlarges the applicable range of the alkaline lysis method used for preparing genome. The kit is widely applied to the extraction on the genome of various plants and can quickly extract the genome used for PCR template directly from plant seeds. The kit has obvious detection effect on the target gene of 600bp, has excellent amplification effects on endogenous gene and exogenous gene and can discover the samples with the components to be measured more than 1%. The extraction time for the whole genome is controlled within 21min, thus greatly shortening the extraction time of the genome.

Description

technical field

[0001] The invention belongs to the technical field of molecular biology, and develops a method for quickly extracting various plant genomes suitable for PCR amplification and a kit suitable for the method by optimizing the traditional method for preparing genomes by alkaline lysis. Background technique

[0002] Since the first batch of genetically modified crops such as extended ripening tomatoes and herbicide-resistant cotton were approved for commercial production in 1994, there have been 16 types of commercialized genetically modified crops reported at home and abroad, with more than 70 kinds [James: Brief No. 21-2000: Global status of commercialized transgenic crops. 2000]. Genetically modified crops and genetically modified foods have quickly occupied the market because of their incomparable advantages in stress resistance, nutritional value and improvement of food flavor, etc., and have greatly met the needs of people's lives. However, at the same tim...

Examples

Embodiment 1

[0054] Embodiment 1 uses the method of the present invention to extract various plant genomes

[0055] As a rapid preparation method of plant genome for molecular detection, we hope that this method has wide applicability and can be widely used in the molecular detection of genetically modified products in the food field. Edible tissues or organs in plants can be roughly divided into the following categories: roots, stems, leaves, flowers, fruits, and seeds. In order to test the universality of the method, we must not only cover edible tissues of plants when selecting samples Partly, try to select plants of different families and genera as research objects. Therefore, when selecting samples, this example selects (1) cereals: corn, soybeans, peanuts; (2) stems; Chrysanthemum chrysanthemum, celery; (3) Leaves: rapeseed; (4) roots: carrots; (5) flowers: broccoli; (6) fruits: tomatoes.

[0056] The specific operation is as follows:

[0057] a. Cereals: BT176 transgenic corn and ...

Embodiment 2

[0080] Example 2 Using the method of the present invention to amplify target genes of different fragment sizes

[0081] The plant genome obtained by the method of the present invention presents a diffuse state due to degradation, and the genome electrophoresis bands mainly focus on 1000bp to 200bp, while the genes amplified in the above examples are all about 200bp plant internal standard genes, so there is better amplification. increase the effect. In order to compare the effect of this method on the amplification of genes with different fragment sizes, in this example, three genes in BT176 corn were selected as the amplification objects. The specific operations are as follows:

[0082] The BT176 transgenic corn was crushed with a grinder and passed through a 60-mesh sieve.

[0083] According to the method of the present invention of embodiment 1 and CTAB method (CTAB method is referring to: Xu Fang, Li Xin, Luo Xin, Liu Zhiguo. Qualitative PCR detection [J] of genetically mod...

Embodiment 3

[0088] The detection result of the sensitivity of embodiment 3 the present invention

[0089] After mixing BT176 transgenic corn flour and soybean flour at a mass ratio of 50%, 10%, 1%, and 0.1%, the genome was extracted according to the method of the present invention in Example 1, and the extracted DNA was used as a template for PCR electrophoresis detection. The maize internal standard gene IVR226 was selected as the primer for PCR detection, and the target fragment amplified by the reaction was about 226bp: the base sequence of the primer IVR226 was:

[0090] F5′-CCG CTG TAT CAC AAG GGC TGG TCA C-3′

[0091] R5′-GGA GCC CGT GTA GAG CAT GAC GAT C-3′

[0092] The PCR reaction system, 10×PCR buffer, PCR reaction conditions and detection conditions are all consistent with Example 1.

[0093] The results show that: when the content of corn flour is less than or equal to 1%, the target gene bands that can be distinguished by naked eyes can be obtained after PCR amplification, ...

technical field

[0001] The invention belongs to the technical field of molecular biology, and develops a method for quickly extracting various plant genomes suitable for PCR amplification and a kit suitable for the method by optimizing the traditional method for preparing genomes by alkaline lysis. Background technique

[0002] Since the first batch of genetically modified crops such as extended ripening tomatoes and herbicide-resistant cotton were approved for commercial production in 1994, there have been 16 types of commercialized genetically modified crops reported at home and abroad, with more than 70 kinds [James: Brief No. 21-2000: Global status of commercialized transgenic crops. 2000]. Genetically modified crops and genetically modified foods have quickly occupied the market because of their incomparable advantages in stress resistance, nutritional value and improvement of food flavor, etc., and have greatly met the needs of people's lives. However, at the same tim...

Examples

Embodiment 1

[0054] Embodiment 1 uses the method of the present invention to extract various plant genomes

[0055] As a rapid preparation method of plant genome for molecular detection, we hope that this method has wide applicability and can be widely used in the molecular detection of genetically modified products in the food field. Edible tissues or organs in plants can be roughly divided into the following categories: roots, stems, leaves, flowers, fruits, and seeds. In order to test the universality of the method, we must not only cover edible tissues of plants when selecting samples Partly, try to select plants of different families and genera as research objects. Therefore, when selecting samples, this example selects (1) cereals: corn, soybeans, peanuts; (2) stems; Chrysanthemum chrysanthemum, celery; (3) Leaves: rapeseed; (4) roots: carrots; (5) flowers: broccoli; (6) fruits: tomatoes.

[0056] The specific operation is as follows:

[0057] a. Cereals: BT176 transgenic corn and ...

Embodiment 2

[0080] Example 2 Using the method of the present invention to amplify target genes of different fragment sizes

[0081] The plant genome obtained by the method of the present invention presents a diffuse state due to degradation, and the genome electrophoresis bands mainly focus on 1000bp to 200bp, while the genes amplified in the above examples are all about 200bp plant internal standard genes, so there is better amplification. increase the effect. In order to compare the effect of this method on the amplification of genes with different fragment sizes, in this example, three genes in BT176 corn were selected as the amplification objects. The specific operations are as follows:

[0082] The BT176 transgenic corn was crushed with a grinder and passed through a 60-mesh sieve.

[0083] According to the method of the present invention of embodiment 1 and CTAB method (CTAB method is referring to: Xu Fang, Li Xin, Luo Xin, Liu Zhiguo. Qualitative PCR detection [J] of genetically mod...

Embodiment 3

[0088] The detection result of the sensitivity of embodiment 3 the present invention

[0089] After mixing BT176 transgenic corn flour and soybean flour at a mass ratio of 50%, 10%, 1%, and 0.1%, the genome was extracted according to the method of the present invention in Example 1, and the extracted DNA was used as a template for PCR electrophoresis detection. The maize internal standard gene IVR226 was selected as the primer for PCR detection, and the target fragment amplified by the reaction was about 226bp: the base sequence of the primer IVR226 was:

[0090] F5′-CCG CTG TAT CAC AAG GGC TGG TCA C-3′

[0091] R5′-GGA GCC CGT GTA GAG CAT GAC GAT C-3′

[0092] The PCR reaction system, 10×PCR buffer, PCR reaction conditions and detection conditions are all consistent with Example 1.

[0093] The results show that: when the content of corn flour is less than or equal to 1%, the target gene bands that can be distinguished by naked eyes can be obtained after PCR amplification, ...

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