Betula platyphylla BplSPL1 gene for promoting precocious flowering and encoded protein thereof

A technology for early flowering and encoding protein, applied in the field of birch BplSPL1 gene and its encoded protein, can solve the problems of unclear SPL gene function, no involvement of birch SPL1 gene sequence and its function, etc., so as to improve plant photoperiod and reduce flowering time. Effect

Inactive Publication Date: 2017-04-26
NORTHEAST FORESTRY UNIVERSITY +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the function of the SPL gene of birch with a relatively long juvenile period is not clea

Method used

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  • Betula platyphylla BplSPL1 gene for promoting precocious flowering and encoded protein thereof
  • Betula platyphylla BplSPL1 gene for promoting precocious flowering and encoded protein thereof
  • Betula platyphylla BplSPL1 gene for promoting precocious flowering and encoded protein thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Cloning of Betula BplSPL1 Gene

[0028] (1) Acquisition of plant material

[0029] The experimental material Birch male flowers were collected from the birch forest of Northeast Forestry University. Immediately after collection, refrigerate to -80°C refrigerator for future use.

[0030] (2) Extraction of birch RNA

[0031] Birch total RNA was performed using the improved CTAB method, and the purity and concentration of total RNA were measured using agarose gel electrophoresis and ultraviolet spectrophotometer. Use QuantScript RT Kit to synthesize the first strand of cDNA, and then use BplSPL1-F and BplSPL1-R for PCR amplification. PCR reaction system: cDNA template 1μl, 10×Buffer 2μl, dNTP (2mM) 2.5μl, forward and reverse primers 1μl each (10μM), KOD-plus-Neo 1μl, MgSO4 (25mM) 1.5μl sterilized distilled water to make up 20μl. PCR amplification conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 62°C for 1 min...

Embodiment 2

[0036] Embodiment 2: The plant expression vector construction of BplSPL1 gene

[0037] (1) Double enzyme digestion and detection of BplSPL1

[0038] The plant expression vector double digestion steps are as follows:

[0039] Both the cloning vector pMD18T-BplSPL and the plant binary vector pROK2 were digested with SacⅠ and BamHI. The digestion system was as follows: DNA 1 μg, 10×FD buffer 2 μl, SacⅠ 0.5 μl, BamHI 0.5 μl, and sterilized distilled water to make up 20 μl. After mixing, incubate at 37°C for 2-3h, and at 65°C for 5min. The double digested product was subjected to 0.8% agarose gel electrophoresis, and the target fragment was recovered according to the steps of the OMEGA recovery kit.

[0040] (2) Ligation of double enzyme digestion products

[0041] Ligate at 16°C overnight, and the ligation system is as follows: 4 μl of target gene, 2 μl of pROK2 vector fragment, 1 μl of T4 ligase, 2 μl of T4 buffer, and 20 μl of sterilized distilled water

[0042] (4) Transforma...

Embodiment 3

[0044] Example 3: Functional Analysis of BplSPL1 Gene Overexpression in Arabidopsis Plants

[0045] (1) Experimental materials: Peat soil and vermiculite were mixed evenly at a ratio of 1:1, and packed in plastic pots with a caliber of 8 cm. Pour water into the tray, and after the mixed soil fully absorbs water, sow several wild-type Colombian ecotype Arabidopsis seeds, cover with a layer of film, and pierce 2 to 3 small holes in the film on the top of each pot. 22 ℃, 16h light, 8h dark culture. After the seeds germinate, remove the film and thin the seedlings, leaving 2 to 3 Arabidopsis plants in each pot.

[0046] (2) Agrobacterium-mediated pollen infecting Arabidopsis

[0047] 1) Preparation of bacterial solution: Dip the bacterial solution with an inoculation loop and streak on the LB medium added with rifampicin and kanamycin (concentrations of 10 μg / mL and 50 μg / mL, respectively) to cultivate a single colony. Pick a single colony and inoculate it in an Erlenmeyer flas...

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Abstract

Belonging to the field of gene technology in molecular biology, the invention relates to a betula platyphylla BplSPL1 gene for promoting precocious flowering and an encoded protein thereof. The invention is characterized in that the betula platyphylla BplSPL1 gene for promoting precocious flowering has a nucleotide sequence shown as SEQ ID NO:1 in a sequence table, and has an amino acid sequence shown as SEQ ID NO:2. The invention provides the betula platyphylla flowering gene BplSPL1 and applies the gene to construction of a BplSPL1 gene's plant expression vector, and the constructed plant expression vector is subjected to agrobacterium impregnation of an arabidopsis thaliana plant so as to obtain a transgenic arabidopsis thaliana plant and achieve precocious flowering. Therefore, the gene has the function of inducing precocious flowering of arabidopsis thaliana and can regulate plant leaf development. The gene provided by the invention utilizes genetic engineering technology to regulate betula platyphylla flowering time and leaf development, provides gene resources and theoretical basis, and has great application value.

Description

[0001] Technical field: the present invention relates to a birch BplSPL1 gene and its encoded protein that promotes early flowering, belonging to the field of gene technology in molecular biology. [0002] Background technique: [0003] SPL (SQUAMOSA promoter-binding protein-like) is a plant-specific SBP transcription factor that exists in all green plants, including unicellular green algae, mosses, gymnosperms and angiosperms. SPL proteins constitute a diverse family of transcription factors, all of which have a highly conserved segment of 76 amino acids, which binds to the promoter region of the downstream target gene to regulate the expression of the target gene, and is named the SBP domain. The SBP domain contains a novel zinc finger structure and a nuclear localization signal, which is involved in the entry of transcription factors into the nucleus and specifically binds to the core motif containing GTAC and its flanking DNA regions. The SPL transcription factor was first ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415
Inventor 刘雪梅赵丽红刘闯
Owner NORTHEAST FORESTRY UNIVERSITY
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