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Modified glyphosate-resistant gene and glyphosate-resistant rice cultivation method

A glyphosate-resistant and glyphosate-resistant technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of low seed purity and low hybrid purity affecting rice yield and quality

Inactive Publication Date: 2017-09-05
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of hybrid rice production, there are problems such as low hybrid purity affecting rice yield and quality. By cultivating glyphosate-resistant rice restorer varieties, the problem of low seed purity in hybrid rice seed production can be effectively solved, and hybrid rice production can be promoted. contribute to the increase in food production

Method used

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  • Modified glyphosate-resistant gene and glyphosate-resistant rice cultivation method

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Embodiment 1

[0039] Example 1: Codon optimization of I. variabilis-EPSPS and CTP sequences

[0040] The original sequence of the target gene I.variabilis-mEPSPS used in the present invention was cloned from the microorganism, the bacteria Isoptericola variabilis, by the research group of Professor Liu Ziduo of Huazhong Agricultural University (the verification of gene function was also verified in microorganisms, and it was not in plants, especially not in said For verification in transformation, see literature: Yi S, et al.Characterization of a newtype of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericolavariabilis.J Mol Catal B:Enzym.2015,111:1-8) , the original gene named I.variabilis-EPSPS, its nucleotide sequence is shown in SEQ ID NO: 17, the sequence of encoded amino acids is shown in SEQ ID NO: 18, and the usage frequency of different codons is shown in Table 1 . The analysis of the original I.variabilis-EPSPS gene and its application to the breedin...

Embodiment 2

[0046] Embodiment 2: Construction of plant expression vector

[0047] The original transformation vector used in the present invention is Agrobacterium Ti binary vector pCAMBIA1300 (provided by Australia pCAMBIA Laboratory, Center for the Application of Molecular Biology to International Agriculture). Digest pCAMBIA1300 ( figure 1 ), and then use T 4 DNA polymerase blunts the ends to form the intermediate vector p130. Then use HindⅢ+BamHI to double-enzyme cut p130 and connect it into the maize Ubiquitin promoter; further use BamHI+SacI to cut p130 into the sequence shown in SEQ ID NO:2 and SEQ ID NO:4 to form the final expression Vector PU130-I.variabilis-mEPSPS (see SEQ ID NO: 5 for the sequence, see figure 2 ). PU130-I.variabilis-mEPSPS was transformed into Agrobacterium EHA105, and the transformed Agrobacterium strain was stored at -70°C until use.

Embodiment 3

[0048] Example 3: Genetic transformation of rice Minghui 86 mediated by Agrobacterium

[0049] Callus induction and subculture mainly refer to the existing literature. The optimum concentration of glyphosate required for the selection of resistant callus of rice variety Minghui 86 (a conventional variety, the source of which is preserved by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University) was determined to be 200 mg / L (agricultural For the steps of the bacillus-mediated transformation of Minghui 86, refer to the descriptions in 3.1-3.8).

[0050] 3.1 Callus induction

[0051] The mature rice Minghui 86 seeds were dehulled, then treated with 75% ethanol for 1 min, and 0.15% mercuric chloride seed surface was sterilized for 15 min; the seeds were washed 5 times with sterilized water; the seeds were placed on the callus induction medium (Add 2.5mg / L 2,4-D, 0.8g / L hydrolyzed casein, 0.6g / L proline, 0.5g / L glutamine, 30g / L maltose and 3g / L p...

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Abstract

The invention belongs to the technical field of plant gene engineering, in particular to a modified glyphosate-resistant gene and a glyphosate-resistant rice cultivation method and comprises modification of glyphosate genes, building of plant expression vectors of the glyphosate-resistant gene and a glyphosate-resistant transgenosis rice cultivation method. Artificially-synthesized modified glyphosate-resistant gene serves as a target gene, the sequence of the gene is as shown in SEQ ID NO: 1, and the target gene is led into rice receptors by an agrobacterium tumefaciens mediated transformation method to obtain high glyphosate-resistant transgenosis rice GT28. the glyphosate-resistant gene of GT28 is recombined by the aid of sexual hybridization and somatic hybridization technique to obtain new rice germplasm, and new glyphosate-resistant varieties (strains) of rice are cultivated.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the modification of a glyphosate-resistant gene derived from microorganisms, the construction of a plant expression vector containing the glyphosate-resistant gene, and a highly glyphosate-resistant gene with commercial potential. Breeding of transgenic rice, and a method for obtaining commercially potential high glyphosate-resistant transgenic rice varieties through hybridization. Background technique [0002] Rice is one of the most important food crops in China, providing the staple food for 60% of the population. Weeds are an important factor affecting rice yield and quality. Traditional agriculture mainly uses management methods such as manual weeding combined with plowing and irrigation to control rice field weeds, which requires a lot of manpower and material resources. With the acceleration of urbanization in China, the shortage of labor in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82C12N15/11C12Q1/68A01H5/00
CPCC12N9/1092C12N15/8205C12N15/8275C12Q1/6895C12Y205/01019
Inventor 林拥军崔莹刘子铎
Owner HUAZHONG AGRI UNIV
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