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Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers

A technology without selectable markers and disease-resistant genes, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of heavy analysis work and difficult screening of transgenic individuals without selectable markers, and achieve improved screening Efficiency and workload reduction effects

Inactive Publication Date: 2012-10-03
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

In addition, in the genome of about 50% of the co-transformed plants, the T-DNA of the marker gene and the target gene are linked, and it is difficult to obtain isolated transgenic individuals without selection markers from their progeny populations
Therefore, the analysis of the progeny population of double-strain co-transformation is very heavy, and it is urgent to improve the screening efficiency of transgenic individuals without selection markers, so as to cultivate new germplasm of crops that meet the breeding goals on the basis of efficient and safe transgenic technology

Method used

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  • Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers
  • Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers
  • Method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers

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Embodiment

[0021] Example: Taking the transformation of rice broad-spectrum rice blast resistance gene Pi9 as an example, through an improved double-strain (double-vector) co-transformation system, a rice germplasm material without a selection marker for the disease-resistant gene was created.

[0022] Vector construction: co-transformation vector system includes marker gene vector (pRB01) and target gene vector (pLJ42), such as figure 1 . The green fluorescent protein (GFP) gene controlled by the maize ubiquitin promoter Ubi was cloned into the HindIII and EcoRI restriction sites of the Agrobacterium transformation vector pCAMBIA1300 (Genbank AF234296) to generate the marker gene vector pRB01, which also carries the cauliflower mosaic virus Hygromycin phosphotransferase gene (HPT) controlled by the CaMV35S promoter. HPT is used as a hygromycin resistance selection marker to screen transformed rice cells, and GFP gene is used as a visible genetic marker to track transformed rice cells, ...

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Abstract

The invention relates to a method for converting disease-resistance genes of rice and obtaining transgenic descendants without selective markers. The method comprises the following steps: first, cloning green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT) on a marker gene carrier, placing a target gene in other T-DNA carrier, mixing strains of agrobacterium tumefaciens carrying the marker gene carrier and strains of agrobacterium tumefaciens carrying the target gene carrier and converting callus of the rice, performing PCR (polymerase chain reaction) detection on the disease-resistance genes of the target gene carrier by a specific primer, and screening to obtain co-transformation plants (T0); then, screening the marker gene plants with positive GFP rapidly and massively by means of a desk lamp fluorescence detector in segregative generations (T1 or T2) of the co-transformation plant for removing, and performing PCR detection for disease-resistance genes to the plants with negative GFP so as to obtain the individuals without selective markers of the transgenic disease-resistance genes. The method can be applied to transgenic breeding without selective markers of rice blast-resistant genes or other functional genes of the rice, and enhances the disease resistance of the rice or improves other agronomic traits.

Description

technical field [0001] The invention relates to a rice transgenic technology, in particular to a method for transforming rice disease resistance genes and obtaining transgenic offspring without selection markers. Background technique [0002] Breeding disease-resistant varieties is one of the main goals of crop breeding. As an important supplement to traditional breeding methods, transgenic technology can avoid cumbersome processes such as hybridization and multi-generation selection, transform the disease-resistant gene cloned from the plant genome into susceptible varieties, and improve the disease resistance of recipient varieties in a short period of time . In terms of the breeding application of rice disease resistance genes, with the improvement of rice genomics and map-based cloning technology, a series of rice disease resistance genes have been isolated and studied one after another, providing rich genetic resources for transgenic rice research. [0003] Breeding d...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68A01H4/00A01H5/00
Inventor 瞿绍洪李军王歆赵建华何海燕刘中来
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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