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Method for accurately detecting degree of homogenization of chloroplast transformation

A chloroplast and homogenization technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of protecting integrity, high failure rate, and high purity

Active Publication Date: 2017-05-31
三杰牧草(杨凌)研究院有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In recent years, fluorescent quantitative PCR technology has been continuously improved, and has been widely used in basic scientific research, drug development, clinical diagnosis, genetic testing and other fields due to its advantages of simple operation, fast and convenient, high sensitivity, good repeatability and low contamination rate. During the research, but it has not been found that the fluorescent quantitative PCR method is applied to the detection of the degree of homogeneity of chloroplast transformation

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  • Method for accurately detecting degree of homogenization of chloroplast transformation
  • Method for accurately detecting degree of homogenization of chloroplast transformation
  • Method for accurately detecting degree of homogenization of chloroplast transformation

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1 Transfer the yellow fluorescent protein (LanYFP-mod, hereinafter referred to as LY) gene into the tobacco chloroplast genome, and use this method to detect the degree of homogeneity of the tobacco chloroplast yellow fluorescent protein gene

[0073] Step (1) Obtain the positive callus that the laboratory has successfully introduced the yellow fluorescent protein gene into the tobacco chloroplast genome. The backbone vector of the target gene is p-DK2, and the backbone vector map is as follows: figure 1 As shown, the whole genome DNA of positive callus was extracted: the obtained whole genome DNA needs to contain as much chloroplast genomic DNA as possible to ensure that the error between the detected degree of homogeneity of chloroplast transformation and the actual degree of homogeneity is small. During the operation, due to the small amount of the sample, it could not be ground in the mortar (the loss was huge), and because the water content of the callus was...

Embodiment 2

[0131] Embodiment 2 The green luciferase (SLG) gene is transferred to the tobacco chloroplast genome, and this method is used to detect the degree of homogeneity of the tobacco chloroplast green luciferase gene

[0132] Step (1) Import the plastid vector p-DK4 containing the green luciferase gene SLG into the tobacco chloroplast genome, and successfully obtain positive callus. The plastid vector p-DK4 map is as follows: Figure 17 Shown, the whole genome DNA method of extracting positive callus is the same as embodiment 1, Figure 18 For the gel running results of the whole genome DNA of the extracted samples, Figure 18 Middle M is 1kb maker, the main bands of samples 4, 6, 8, 9, and 10 are relatively clear and less dragged, and are suitable as template DNA; the main bands of samples 1, 2, 3, 5, 7, and 11 are not clear enough and dragged less Much, not recommended as template DNA.

[0133] In step (2), the target gene is selected to be located on the backbone carrier screen...

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Abstract

The invention discloses a method for accurately detecting the degree of homogenization of chloroplast transformation. A leaf blade into which an exogenous gene-plasmid vector is introduced is cultured in a culture medium to obtain a positive callus; a gene of the plasmid vector is selected as a target gene; a gene of a tobacco chloroplast genome is selected as a reference gene; a primer of the target gene and a primer of the reference gene are designed; a whole genomic DNA of the positive callus is extracted as a template DNA, then a realtime fluorescence quantitative PCR experiment is carried out to verify the amplification efficiency and a dissolution curve so as to determine effective data, and the percentage of homogeneity of the chloroplast transformation is calculated. The detection method provided by the invention is applicable to the known species of a chloroplast genome sequence, and primers are convenient to design and verify. The experimental operation is simple and not cumbersome and the calculation method is simple and accurate. Under normal circumstances, accurate data of the degree of homogenization can be obtained within 3 days, the method is the most accurate method for detecting the degree of the homogenization of the chloroplast transformation, and thus the method has great potential for promotion and application.

Description

technical field [0001] The invention belongs to the technical field of detecting the homogeneity degree of chloroplast transformation, in particular to a method for accurately detecting the homogeneity degree of chloroplast transformation. Background technique [0002] In the past 20 to 30 years, chloroplast transgenic technology has developed rapidly. Compared with nuclear transgenic technology, chloroplast transgenic technology has the advantages of high expression, fixed-point transformation, no gene silencing phenomenon, and high biological safety. It has been widely used in research and application at home and abroad. However, for a long time, chloroplast transgenic technology has not surpassed nuclear transgenic technology to become the most widely used transgenic technology. Not stable enough; the third is that homogenization is difficult. In the current literature and patents, no method for quantitatively detecting the degree of homogeneity of chloroplast transforma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2545/101C12Q2563/107Y02A50/30
Inventor 段康张晨赵娟张业胜董扬
Owner 三杰牧草(杨凌)研究院有限公司
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