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GhGL3 gene promoter, vector and application thereof

A technology of promoter and gene, applied in the field of plant genetic engineering

Active Publication Date: 2015-05-13
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the transcription factor complex MYB-bHLH-WD40 that controls epidermal hair development, the promoter of the first cloned R2R3 transcription factor GhMYB109 has been cloned and patented, and the promoter of the bHLH gene has also been No report

Method used

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  • GhGL3 gene promoter, vector and application thereof
  • GhGL3 gene promoter, vector and application thereof
  • GhGL3 gene promoter, vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Cloning of cotton fiber specific promoter

[0051] The cotton fiber specific promoter was cloned using the Clontech kit (GenomeWalker TM Universal Kit, article number #638904).

[0052] The genomic DNA of cotton was extracted according to the method of Paterson et al. (1993). Take 2.5 μg of genomic DNA and digest with EcoR V, Dra I, Hpa I, Pvu II, Sac I, Sma I and Stu I (Takara products) overnight (16-18 hours). Take 5 μl of the digested product and perform 0.6% agarose ethidium bromide (EB) gel electrophoresis to check whether the digestion is complete. If the digestion is complete, add an equal volume of Tris saturated phenol and chloroform for extraction, and precipitate with 95% ethanol (containing 3M sodium acetate and 20μg glycogen) on ice, and wash the precipitate with 80% ethanol on ice. The pellet was dissolved in 25 μl TE (10 mM Tris / 0.1 mM EDTA, pH 7.5). Take 1 μl of the digested product to test the purity of the digested product by 0.6% agarose eth...

Embodiment 2

[0075] Example 2 Conversion analysis

[0076] In order to construct a cloning expression vector and transform cotton more effectively, this embodiment takes the nucleotide sequence between 2313bp and 4320bp in Sequence Table 1 (SEQ ID No.1), with a length of 2008bp (labeled as SEQ ID No.2). ) Conduct follow-up research and analysis. Design primers containing Sal I and BamH I restriction sites at the 5'end and 3'end of the GhGL3 promoter (SEQ ID No. 2), using genomic DNA as template 20ng, 5μl 10×LA PCR buffer (Mg 2+ Plus), 4μl dNTPs (2.5mM each), 1μl GhGL3p-SalI-F (10μM), 1μl GhGL3p-BamH IR (10μM), 0.5μl LA Taq (5U / μl, purchased from Takara), plus sterile deionized ultra Pure water H 2 O makes up 50μl reaction system. A total of 35 cycles were carried out for PCR amplification at 94°C for 30s, annealing temperature of 50-62°C for 30s, and extension temperature of 72°C for 2 minutes. Among them, the preferable annealing temperature is 51.5°C.

[0077] Upstream primer GhGL3p-Sal I-...

Embodiment 3

[0082] Example 3 Identification and analysis of transgenic cotton plants

[0083] DNA was extracted from the leaves of transgenic cotton plants, diluted to 20ng and 1μl was used as a template. First use the primers of His3 gene to confirm that the template is intact. Then take 1μl genomic gDNA as a template, and 5μl 10×LA PCR buffer (Mg 2+ Plus), 4μl dNTPs (2.5μM each), 1μl GUS-2553F (10μM), 1μl GUS-4361R (10μM), 0.5μl LA Taq (5U / μl, purchased from Takara), add sterile deionized ultrapure water H 2 O makes up 50μl reaction system. A total of 35 cycles were carried out for PCR amplification at 94°C for 30s, annealing temperature at 57°C for 30s, extension temperature at 72°C for 2min.

[0084] Upstream primer His3F: 5′-GCCAAGCGTGTCACAATTATGC-3′

[0085] Downstream primer His3R: 5′-ACATCACATTGAACCTACCACTACC-3′

[0086] Upstream primer GUS-2553F: 5′-ATGTTACGTCCTGTAGAAACCCCA-3′

[0087] Downstream primer GUS-4361R: 5'-TCATTGTTTGCCTCCCTGCTGCGGT-3'

[0088] After the amplification is comple...

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Abstract

The invention discloses a GhGL3 gene promoter, which comprises a nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2, or is obtained by virtue of substitution, deletion or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 and has the same activity as the activity of the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2. The invention relates to a vector of the promoter, host cells and an application of the promoter or vector in cultivation of new varieties of plants. The promoter disclosed by the invention can drive specific expression of target genes in cotton fibers and epidermal hair and has significance for cultivating novel varieties of fiber plants, particularly cultivating novel varieties of cotton.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a GhGL3 gene promoter, vector and application thereof. Background technique [0002] my country is one of the countries with the highest cotton production and consumption. Cotton is native to the subtropical zone and is the seed fiber of cotton plants in the Malvaceae family. Cotton fiber cells develop from single cells of cotton seed coat, and go through four stages: initiation, primary wall synthesis, secondary wall synthesis and maturity. Mature cotton fiber is white to white with yellowish color, about 2 to 4 cm long, and contains about 87-90% cellulose. Cotton is divided into three types: coarse-staple cotton, long-staple cotton and fine-staple cotton. China's fine-lint cotton accounts for 98% of cotton production, and the most widely cultivated upland cotton belongs to the fine-lint. Therefore, the epidermal hair fiber cells of cotton seeds...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N5/10
Inventor 薛勇彪李群陆春霞普莉
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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