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White birch CCoAOMT gene for lowering lignin and encoded protein thereof

A technology encoding protein and lignin is applied in the field of birch CCoAOMT gene and its encoded protein, which can solve the problems of unclear CCoAOMT gene function and no involvement of birch CCoAOMT gene sequence.

Inactive Publication Date: 2016-06-01
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 CCoAOMT gene of birch is not clear, and there are no relevant patent reports involving the sequence and function of the CCoAOMT gene of birch

Method used

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  • White birch CCoAOMT gene for lowering lignin and encoded protein thereof
  • White birch CCoAOMT gene for lowering lignin and encoded protein thereof
  • White birch CCoAOMT gene for lowering lignin and encoded protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cloning of Birch CCoAOMT Gene

[0028] (1) Acquisition of plant material

[0029] The experimental material birch secondary xylem was 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 ThermoScript TM RT-PCRSystem reverse transcription kit synthesizes the first strand of cDNA, and then uses CCoAOMT-F and CCoAOMT-R for PCR amplification. PCR reaction system: cDNA template 2 μl, 10×Buffer 2.5 μl, dNTP (2.5mM) 2 μl, positive 1 μl of each reverse primer (20 μM), rTaqDNA polymerase 0.15 μl sterilized distilled water to make up 25 μl. The PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C f...

Embodiment 2

[0036] Embodiment 2: the plant expression vector construction of birch CCoAOMT gene

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

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

[0039] Both the cloning vector and the plant binary vector pBI121 were digested with XbaⅠ and BamHI. The digestion system was as follows: 1 μg of DNA, 2 μl of 10×FD buffer, 0.5 μl of XbaⅠ, 0.5 μl of BamHI, and 20 μl of sterilized distilled water. 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 pBI121 vector fragment, 1 μl of T4 ligase, 2 μl of T4 buffer, 20 μl of sterilized distilled water

[0042] (4) Transfor...

Embodiment 3

[0044] Example 3: Functional Analysis of Antisense Expression of Birch CCoAOMT Gene in Tobacco and Birch Plants

[0045] (1) Experimental materials: Soak tobacco seeds and birch axillary buds in 95% alcohol for 3-5 minutes, then soak them in 5.5% sodium hypochlorite for 15 minutes, wash them with sterile water 4-5 times, and dry the liquid on the surface of the seeds with sterile filter paper. Inoculate on MS medium. When the height of the seedlings is about 1 cm, transfer them to the rooting medium to obtain aseptic seedlings of tobacco and white birch.

[0046] (2) Agrobacterium-mediated leaf disc transformation of tobacco and birch

[0047] a, Agrobacterium culture: pick a single bacterium colony of EHA105 bacterial strain on the flat plate, inoculate in 20m1 the bacterial culture liquid culture medium (pH7.0) that adds corresponding antibiotic, in constant temperature shaker, in 27 ℃, 180r / min cultivates to 0.6-1.0. Transfer the bacterial solution into the newly prepare...

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Abstract

The invention relates to a white birch CCoAOMT gene for lowering lignin synthesis and an encoded protein thereof, belonging to the technical field of genes in molecular biology. The white birch CCoAOMT gene is characterized in lowering lignin synthesis. The sequence of the white birch CCoAOMT gene is disclosed as SEQ ID NO:1 in the sequence table; and the amino acid sequence of the protein encoded by the white birch CCoAOMT gene is disclosed as SEQ ID NO:2 in the sequence table. The invention provides a white birch lignin synthesis gene CCoAOMT and a plant expression vector for establishing the white birch CCoAOMT gene. According to the established plant expression vector, a tobacco plant and a white birch plant are infected by agrobacterium to obtain the transgenic tobacco plant, in the stem of which the lignin content is lowered, which indicates that the gene has the function of lowering the tobacco stem lignin content and can regulate the changes of the lignin monomer components. The analysis on the fiber morphology and chemical components of the heptaennial white birch individual plant with 16 reverse transformed CCoAOMT genes is utilized to screen out four (1, 4, 12 and 16) dominant transgenic individual plants for paper pulp. The white birch CCoAOMT gene provides gene resources and theoretical references for lowering the white birch lignin content by using the gene engineering technique, thereby having high application value.

Description

[0001] Technical field: The present invention relates to a birch CCoAOMT gene and its encoded protein that reduces lignin synthesis, belonging to the field of gene technology in molecular biology. Background technique: [0002] Lignin is an aromatic polymer. It is a kind of high-molecular organic substance in plants, which is second only to cellulose. It mainly exists in the secondary thickened plant cell wall, which can provide mechanical support for plants and protect plants from fungi. In recent decades, studies have clarified the main biosynthetic route of lignin monomers, and the regulation of lignin can improve the pulping rate of woody plants and the digestibility of forage. At the same time, plants can also adapt to large ratio changes among the three lignin monomers (H / G / S). There are more than ten kinds of enzymes involved in the lignin biosynthesis pathway, among which coffee phthalein coenzyme A-3-O-methyltransferase (CCoAOMT) is a key enzyme in the lignin biosynth...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54
CPCC12N9/1007C12Y201/01104
Inventor 刘雪梅尹伟平刘闯
Owner NORTHEAST FORESTRY UNIVERSITY
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