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Transformation method utilizing red fluorescent protein as selection marker of rice transformation

A genetic transformation method and a technology for screening marker genes, which are applied in the biological field to achieve high reliability results

Active Publication Date: 2011-09-28
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, red fluorescent protein as a selection marker for transgenic plants has not been reported

Method used

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  • Transformation method utilizing red fluorescent protein as selection marker of rice transformation
  • Transformation method utilizing red fluorescent protein as selection marker of rice transformation
  • Transformation method utilizing red fluorescent protein as selection marker of rice transformation

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 pSPT18 ( figure 1 ). The vector is artificially constructed on the basis of pPZP. There are two gene expression cassettes on the expression vector: one is the OsCYP704B2 gene expression cassette, which is composed of the OsCYP704B2 gene and its own endogenous promoter and terminator. In order to distinguish the endogenous OsCYP704B2 gene of rice itself from the OsCYP704B2 gene transformed into rice through an expression vector, three single nucleotide mutation sites (SNP) were introduced into the wild-type allele OsCYP704B2 and transformed into rice. The two SNPs were located at the 1468th, 1470th and 1473rd base respectively, all of which were mutated from G to C. None of these changes affected the encoded amino acid sequence. The nucleotide sequence of the modified OsCYP704B2 gene is shown in SEQ ID NO.3, wherein the SNP is m...

Embodiment 2

[0027] Embodiment 2: rice transformation

[0028]The following steps have been taken:

[0029] 1. Seed disinfection

[0030] 1. Soak rice seeds with skin removed in 95% ethanol for 2-3 minutes;

[0031] 2. Rinse in 40% sodium hypochlorite solution containing Tween (40ul, 20%Tween / 100ml) for 15 minutes, replace the rinse solution once, and continue rinsing for 15 minutes;

[0032] 3. Rinse in 40% sodium hypochlorite solution without Tween for 15 minutes, replace the rinse solution, and continue rinsing for 15 minutes;

[0033] 4. Rinse the seeds 4 times with sterile water.

[0034] 2. Callus induction

[0035] Calli were induced for 11 days in a light incubator at 32°C.

[0036] 3. Agrobacterium transformation

[0037] 1. Pick a single colony, connect it to 25ml of freshly prepared Agrobacterium culture medium (with antibiotics added), shake culture overnight at 250-300 RPM at 28°C, and expand the Agrobacterium to 0.3<OD550<1.0;

[0038] 2. Centrifuge the bacterial solut...

Embodiment 3

[0052] Embodiment 3: PCR detection of transgenic plants

[0053] The leaves of the transgenic rice plants in Example 4 were taken, and the total DNA was extracted. Using the FP gene sequence as a template, primers were designed to amplify the genomic DNA of T0 transgenic rice 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 7 It is the PCR amplification result of some transgenic plants, indicating that the exogenous gene has been integrated into the rice recipient genome.

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Abstract

The invention provides a transformation method utilizing a red fluorescent protein as a selection marker. The method comprises the following steps of: synthesizing a fluorescent protein (FP) gene and modifying according to the bias of a rice codon, wherein the modified FP gene is used as a selective marker gene of rice transformation and plant regeneration; driving the FP gene by a callus / seed coat-specific promoter, closely connecting with a target gene, and transferring to rice embryonic calli under the mediation of Agrobacterium tumefaciens; screening for three times by a fluorescence microscope in 30 days after coculture; and further verifying an obtained transgenic seedling by utilizing a polymerase chain reaction (PCR) and Southern hybridization. The result shows that the positive rate of the transgenic seedling is up to 80 percent, which proves that FP serving as the selection marker of plant transformation is completely feasible. By the method, the potential hazard of the traditional selection markers such as antibiotic and herbicide resistance genes and the like to environment and food safety is effectively reduced. The invention provides a novel safe plant genetic transformation screening system.

Description

technical field [0001] The invention provides a new safe screening marker for plant transformation and a screening method using a fluorescent microscope, belonging to the field of biotechnology. Background technique [0002] The use of transgenic technology can introduce the target gene with excellent traits into the plant genome, so that the genetic traits of the plant can be improved. However, only a few plant cells are able to absorb foreign DNA and integrate into the plant genome during the transformation process, and most cells are untransformed. Therefore, the introduction of the target gene often requires the aid of a selection marker gene to endow the transformed cells with a specific selectable marker in order to identify and identify transgenic plants. [0003] Most of the traditional methods for identifying and isolating transformed cells are "negative selection". Commonly used screening genes such as antibiotic marker NptⅡ gene (produce neomycin phosphotransfera...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65
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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