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Brassica napobrassica growth regulatory factor gene GRF2 and application thereof

A technology of growth regulation and genetics, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of unknown mechanism of action, increase the complexity of regulating oil content, etc., and achieve the goal of increasing thousand-grain weight, oil content, and oil production Effect

Inactive Publication Date: 2012-12-12
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the functional redundancy of most transcription factors increases the complexity of studying their regulation of oil content, the mechanism of action of some transcription factors is still unknown (To 2008, Gao 2009)
[0006] Although the above studies have theoretically made beneficial explorations on improving oil content through the genetic manipulation of some key enzymes in the fatty acid metabolism pathway, there is still a certain distance in production practice.

Method used

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  • Brassica napobrassica growth regulatory factor gene GRF2 and application thereof
  • Brassica napobrassica growth regulatory factor gene GRF2 and application thereof
  • Brassica napobrassica growth regulatory factor gene GRF2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: BnGRF2, AraGRF2cDNA coding region sequence

[0036] Search the published Arabidopsis gene sequence in NCBI database, BLAST to rapeseed EST sequence and compare the applicant's sequenced rapeseed genome sequence, and design primers on both sides of the gene coding sequence (BnGRF2F: forward primer: [5'- atggatcttgggtcggtaactgg-3'], reverse primer: [5'-TCAGGTTGTGTAATGAAAGTA-3'] is used to amplify the corresponding sequence from the rape leaf cDNA. The amplification primers in Arabidopsis are: AraGRF2 forward primer: [5' -atggatattggtgttcatgttc-3'], reverse primer: [5'-TCAGGTTGTGTAATGAAAGTAAT-3'], amplified directly in Arabidopsis leaf cDNA.

[0037] 1. Extract rapeseed and Arabidopsis mRNA.

[0038] For RNA extraction (TRIZOL™ Kit for RNA extraction), 100 mg of material was ground with liquid nitrogen.

[0039] A. Add 1ml TRIZOL and place it at room temperature (20-25°C, the same below) for 5min.

[0040] B. Add 200ul chloroform, shake vigorously for 30s,...

Embodiment 2

[0047] Embodiment 2: Construction of transgene expression vector and transformation of Arabidopsis:

[0048] After the gene sequence amplified by PCR was connected to the TOPO entry vector (invitrogen company), it was transformed into a competent cell DH5α (invitrogen company), and spectinase screening was carried out, and the carrier primer (T7 primer) and gene primer (gene upstream primer) Amplify and identify forward inserting clones. The plasmid was recombined with Pearleygate100 (Invitrogen Company) after a small amount of preparation, and transformed into competent cells DH5α, screened with kanamycin, and the inserted fragment was subjected to vector primers (35S promoter sequence primers) ) and gene primers (gene downstream primers) for PCR identification, the schematic diagram is shown in figure 1 .

[0049] Arabidopsis transformation process:

[0050] Reagent preparation:

[0051] Infiltration medium (1L): 1 / 2xMurashige-Skoog; 5% (mass ratio) sucrose; 0.5 g of MES...

Embodiment 3

[0058] Example 3: Screening and verification of transgenic Arabidopsis:

[0059] Screening of transformants:

[0060] The vernalized Arabidopsis seeds were planted in artificial soil watered with supersaturated PNS nutrient solution, and covered with plastic wrap. After two days, the light was exposed, and the film was removed after three days.

[0061] Conditions of artificial culture room: relative humidity 80%, constant temperature 20-24°C, light intensity 80-200umol / M2 / S, light cycle 8h dark, 16h light culture. About a week, spray herbicide (glufosinate) to screen positive plants.

[0062] PCR identification:

[0063] (1) Extraction of transformed plant total DNA for PCR:

[0064] A. 70% (volume ratio) ethanol scrub blade, weigh about 100mg

[0065] B. Add 600ul of extraction buffer (0.2M Tris-Cl, 0.25NaCl, 25mM EDTA, 0.5% (mass ratio) SDS, pH 7.5), and grind rapidly at room temperature.

[0066] C. Vortex in a 1.5ml Ependorff tube for 5-10s.

[0067] D.12000rpm, 25mi...

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Abstract

The invention discloses a brassica napobrassica growth regulatory factor gene GRF2 and an application thereof. A base sequence of the gene is a nucleotide sequence represented by SEQ ID NO:1; or a gene sequence of another crop, wherein the gene sequence has no less than 70% homology with that represented by SEQ ID NO:1; or a mutant allele or a derivative obtained after one or more nucleotides areprocessed through addition, substitution, insertion or deletion. As a result of researches, the expression level of the gene in brassica napobrassica high-oil parents and high-oil mixture crops is higher than that in low-oil parents and low-oil mixture crops. A transgene result obtained by using arabidopsis thaliana as an acceptor verified that, with the over-expression of the gene, arabidopsis thaliana oil content is improved, and a 1000-seed weight of arabidopsis thaliana is increased. The invention also provides an application of brassica napobrassica growth regulatory factor gene BnGRF2 in improving the 1000-seed weight and oil content of plants such as arabidopsis thaliana. Therefore, the gene has good application prospect in oil-yield seed breeding of brassica napobrassica and otheroil crops.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, more specifically relates to a rapeseed growth regulator gene GRF2, and also relates to the application of a rapeseed growth regulator gene GRF2. Background technique [0002] The production of oil crops not only plays an important role in national economic and social development, but also is one of the main factors to promote the sustainable development of China's agriculture. At present, China's vegetable oil production can only meet 1 / 2-2 / 3 of the domestic consumption demand, relying heavily on imports. For example, in 2005, China imported more than 6 million tons of vegetable oil and 24 million tons of soybeans, making it the world's largest oil importer. With the growth of population and the improvement of people's living standards, it is estimated that the consumption of edible vegetable oil in China will increase to 25 million tons by 2010. According to the current production scal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415A01H5/10
Inventor 王汉中华玮刘静刘贵华王新发杨庆
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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