Method for simply and rapidly identifying transgenic seeds and estimating copy numbers

A technology for transgenic and screening marker genes, applied in the field of plant breeding and biology, it can solve the problems of mixing non-transgenic seedlings, killing of transgenic seedlings, and difficulty in screening transgenic plants, and achieves the effect of high accuracy.

Inactive Publication Date: 2011-11-02
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many problems with this method. First, different stages of seedling development have different degrees of resistance to herbicides and antibiotics. It is difficult to control the dosage of screening agents. If it is too high, the transgenic seedlings may be killed, and if it is too low, it may kill non- Transgenic seedlings mixed in
Second, the potential risks of herbicides and antibiotics to the environment and food safety have received more and more attention. Therefore, people have tried to use safer screening markers during the transformation process, such as key enzymes in the carbohydrate metabolism process such as PMI, xylA, etc., but it is difficult to screen transgenic plants with such screening markers in the field
Currently, there is no fast, stable, and secure way to do this

Method used

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  • Method for simply and rapidly identifying transgenic seeds and estimating copy numbers
  • Method for simply and rapidly identifying transgenic seeds and estimating copy numbers
  • Method for simply and rapidly identifying transgenic seeds and estimating copy numbers

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Construction of expression vectors

[0025] The name of the expression vector used for transformation in the present invention is pSPT51 ( figure 1 ). The vector is artificially constructed on the basis of pPZP. There are 2 gene expression cassettes on the expression vector: one is OsCYP704B2 gene expression cassette, the expression cassette consists of OsCYP704B2 Genes and their own endogenous promoters and terminators. In order to distinguish the endogenous OsCYP704B2 Genes and expression vectors transformed into rice OsCYP704B2 gene, in the wild-type allele OsCYP704B2 The three SNPs were mutated from G to C at bases 1468, 1470 and 1473, respectively. None of these changes affected the encoded amino acid sequence. Modified OsCYP704B2 The nucleotide sequence of the gene is shown in SEQ ID NO.3, wherein the SNP is marked by a box; the second is the reporter / screening marker gene FP Gene (SEQ ID NO.1) expression cassette, the gene is driven ...

Embodiment 2

[0027] Example 2: Transformation of rice ms26 male sterile mutant material to obtain transgenic rice plants

[0028] The expression vector in Example 1 was introduced into the Agrobacterium strain AGLO by electric shock method, and used to transform the callus of rice ms26 male sterile material. Transformed callus was screened by red fluorescent protein, attached figure 2 In order to express the callus of exogenous gene, it was further differentiated to regenerate transformed plants expressing exogenous gene (attached image 3 ).

[0029]

Embodiment 3

[0030] Embodiment 3: PCR detection of transgenic plants

[0031] The leaves of the transgenic rice plants in Example 2 were taken, and the total DNA was extracted. by FP The gene sequence is used as a template to design primers, for T 0 Genomic DNA of transgenic rice was amplified by PCR. The fragment of the amplified product is 789bp. The amplification program was: 94°C 10min; 94°C 1min, 60°C 1min, 72°C 1min; 37 cycles; 72°C 10min. The forward primer sequence is 5'-GGACTTGAACTCCACCAGG-3'; the reverse primer sequence is: 5'-ATAATGCCAATACGACACC-3'. attached Figure 4 It is the PCR amplification result of some transgenic plants, indicating that the exogenous gene has been integrated into the rice recipient genome.

[0032]

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Abstract

The invention provides a method for simply and rapidly identifying transgenic seeds and estimating copy numbers. According to the invention, the red fluorescent protein (FP) gene (driven by callus/seed coat-specific promoters) is closely connected in series with the target gene and then introduced into the rice male sterile mutant ms26 by means of Agrobacterium tumefaciens-mediated transformation. The transgenic plants are self-crossed to generate the following two types of seeds: non-fluorescent seeds free of transgenic components and fluorescent seeds containing transgenic components. The two types of seeds can be directly separated with the naked eye or with the aid of a fluorescence microscopy, and the separation method is simple and has high accuracy up to 100%. Further, according to the proportions of the fluorescent seeds and the non-fluorescent seeds, the copy numbers of the exogenous gene in the transgenic plant line can be estimated. Since the method provided by the invention does not need the molecular detection of the germinated seeds and young seedlings, the identified seeds can be conveniently applied to the late-stage scientific researches or production.

Description

technical field [0001] The invention belongs to the field of biological technology and the technical field of plant breeding. The invention provides a simple and effective method for quickly identifying transgenic seeds and non-transgenic seeds. The method does not need to germinate the seeds, and can conveniently distinguish the transgenic and non-transgenic seeds for scientific research or production. Background technique [0002] Transgenic technology has shown great application potential in crop quality improvement and resistance enhancement. However, the transgenic T0 generation is often transgenic heterozygous, and its selfing produces seeds of three genotypes of 1:2:1, a quarter of which are homozygous non-transgenic seeds, a quarter of which are homozygous transgenic seeds, and two One-third of the seeds were heterozygous transgenic seeds. In traditional transformation methods, transgenic seeds and non-transgenic seeds cannot be identified at the seed stage, so eac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02G01N21/64C12N15/11A01H5/00A01H5/10
Inventor 邓兴旺王海洋万向元周君莉
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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