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ghgl3 gene promoter, vector and application thereof

A promoter and gene technology, applied in the direction of introducing foreign genetic material using vectors, cells modified by introducing foreign genetic material, DNA/RNA fragments, etc.

Active Publication Date: 2018-11-16
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] Cloning of Example 1 Cotton Fiber-Specific Promoter

[0051] Cotton fiber-specific promoters were cloned using the Clontech kit (GenomeWalker TM UniversalKit, Cat. No. #638904).

[0052] Cotton genomic DNA was extracted according to the method of Paterson et al. (1993). 2.5 μg of genomic DNA was digested with EcoR V, Dra I, Hpa I, Pvu II, Sac I, Sma I and Stu I (Takara products) overnight (16-18 hours). 5 μl of the digested product was subjected to 0.6% agarose ethidium bromide (EB) gel electrophoresis to detect whether the digested product was 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 20 μg glycogen) pre-cooled on ice, wash the precipitate with 80% ethanol pre-cooled on ice, and finally The pellet was dissolved in 25 μl TE (10 mM Tris / 0.1 mM EDTA, pH 7.5). 1 μl of the digested product was subjected to 0.6% agarose ethidium br...

Embodiment 2

[0075] Embodiment 2 conversion analysis

[0076] In order to more effectively construct the cloning expression vector and transform cotton, this embodiment takes the nucleotide sequence between the 2313bp and 4320bp of Sequence Table 1 (SEQ ID No.1), and the length is 2008bp (marked as SEQ ID No.2) Conduct follow-up research and analysis. Primers containing Sal I and BamHI restriction sites were designed at the 5' end and 3' end of the promoter (SEQ ID No.2) of GhGL3 respectively, using genomic DNA as template 20ng, 5 μl 10×LA PCR buffer (Mg 2+ Plus), 4μldNTPs (2.5mM each), 1μl GhGL3p-SalI-F (10μM), 1μl GhGL3p-BamH I-R (10μM), 0.5μl LATaq (5U / μl, purchased from Takara), plus sterile deionized ultrapure water h 2 O to make up 50 μl reaction system. 94°C for 30s, annealing temperature at 50-62°C for 30s, extension temperature at 72°C for 2min, a total of 35 cycles for PCR amplification. Among them, the preferred annealing temperature is 51.5°C.

[0077] Upstream primer GhGL...

Embodiment 3

[0082] Identification analysis of embodiment 3 transgenic cotton plants

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

[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 amplificati...

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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, carrier and application thereof. Background technique [0002] my country is one of the countries with the highest cotton production and consumption. Cotton originates in the subtropical zone and is the seed fiber of the Malvaceae Cotton plant. Cotton fiber cells are developed from single cells of the cotton seed coat, and go through four stages: initiation, primary wall synthesis, secondary wall synthesis, and maturation. The 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-staple cotton accounts for 98% of cotton production, and the most widely cultivated upland cotton belongs to fine-staple cotton. Therefore, the epidermal hair fiber c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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