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Liriodendron cellulose synthase gene lccesa1 and its expression protein and application

A technology of cellulose synthase and Liriodendron, applied in the field of plant gene technology, can solve the problem of insufficient research on catalytic synthesis products, and achieve the effect of promoting the accumulation and wide application of cellulose

Active Publication Date: 2019-12-31
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the research on cellulose synthase is relatively mature, the research on all catalytic synthesis products of cellulose and cellulose-like enzymes between the two is not enough.

Method used

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  • Liriodendron cellulose synthase gene lccesa1 and its expression protein and application
  • Liriodendron cellulose synthase gene lccesa1 and its expression protein and application
  • Liriodendron cellulose synthase gene lccesa1 and its expression protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 LcCESA1 clone of and LcCESA1 Overexpression vector construction

[0039] 1. Primer design: According to the transcriptome data of Liriodendron thaliana, compared with that of Arabidopsis CESA1 CDS sequence prediction of genes LcCESA1 Gene sequence, and design specific CDS full-length primers suitable for Gibson Assembly.

[0040] LcCESA1 CDS full-length primers: Primer-F: 5'-GAGAACACGGGGGACTCTAGAATGGAGGCGAATGCTGGAATGGT-3'; Primer-R: 5'-ATAAGGGACTGACCACCCGGGGCAGTTGATGCCACATTGGTTTGC-3'.

[0041] 2. Cloning of the target gene: using Q5 ultra-fidelity enzyme to perform PCR amplification on the cDNA sample of Liriodendron tulipifera to obtain the band of the target gene.

[0042] (1) System: 3 μL ddH 2 O, 5 μL Q5 ® Ultra-fidelity 2×Master Mix, 0.5 μL Primer-F (10 μM), 0.5 μL Primer-R (10 μM), 1 μL cDNA.

[0043] (2) PCR reaction program: 98°C for 30s; 98°C for 10s, 72°C for 30s, 72°C for 2min, 35 cycles;

[0044] 2min at 72°C; store at 4°C.

[0045] 3. C...

Embodiment 2L

[0058] Example 2 LcCESA1 Identification and Screening of Transgenic Arabidopsis

[0059] 1. Arabidopsis culture

[0060] 1) Disinfection of Arabidopsis seeds: put an appropriate amount of wild-type Arabidopsis seeds into a sterilized EP tube, add an appropriate amount of 75% ethanol, oscillate and sterilize for 30 seconds, and then suck it out; add the same amount of 0.1% mercury liter, and sterilize for 2.5 minutes Aspirate (discard in special waste liquid tank); add the same amount of deionized water, shake and wash, aspirate and put the seeds in a new EP tube; repeat washing with water four times.

[0061] 2) Cultivation of Arabidopsis thaliana: Put the sterilized seeds in the suspension, and use a pipette gun to evenly sow them into 1 / 2MS medium; seal the medium, and put it in a refrigerator at 4°C for vernalization for 3 days; The vernalized medium was placed in a constant temperature culture room at 23°C for 7 days; when the Arabidopsis seedlings grew 2 true leaves, th...

Embodiment 3L

[0092] Example 3 LcCESA1 Observation on Microstructure of Transgenic Arabidopsis Regenerated Plants

[0093] Prepared with Example 2 LcCESA1 Transgenic Arabidopsis T3 seeds were used as plant material, and wild-type Arabidopsis was used as control. LcCESA1 Microstructural observation and comparison of regenerated plants of transgenic Arabidopsis thaliana.

[0094] 1. Arabidopsis culture

[0095] 1) Disinfection of Arabidopsis seeds: Take an appropriate amount of wild-type Arabidopsis seeds and put them into the sterilized EP tube, add an appropriate amount of 75% ethanol, oscillate for 30 seconds and then suck out; add the same amount of 0.1% mercury liter, and oscillate for 2.5 minutes Then suck it out (discard it in a special waste liquid tank); add an equal amount of deionized water, shake and wash it, suck it out and put the seeds in a new EP tube, and repeat the water washing four times.

[0096] 2) Cultivation of Arabidopsis thaliana: Put the sterilized seeds in the ...

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Abstract

The invention discloses a Liriodendron cellulose synthase gene LcCESA1 and its expression protein and application, LcCESA1 The nucleotide sequence of the protein is shown in SEQ ID NO.1; the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. The present invention takes the model plant Arabidopsis thaliana as the research object, and clones the cellulose synthase gene of Liriodendron chinensis LcCESA1 Using Agrobacterium-mediated transformation into Arabidopsis thaliana, through screening and cultivation, the homozygous transgenic plants of the T3 generation were finally obtained; by observing the microstructure of wild-type Arabidopsis and transgenic Arabidopsis, it was found that the stem of transgenic Arabidopsis The thickness of the cell wall of segmental vascular bundle tissue cells and leaf tissue cells is different from that of wild type Arabidopsis. It can be seen that LcCESA1 The gene affects both the cell wall of Arabidopsis stem vascular bundle tissue cells and leaf tissue cells, can promote the accumulation of cellulose, and will have a wide range of applications.

Description

technical field [0001] The invention belongs to the technical field of plant genes. Specifically relate to a kind of Liriodendron cellulose synthase gene LcCESA1 And its expression protein and application. Background technique [0002] Cellulose in plants is synthesized from polyprotein complexes consisting of hexamers, rosette-like structures in the plasma membrane. A single unit of each hexamer component is called cellulose synthase-A (cellulose synthase-A, CESA ), where the letter A represents the catalytic subunit. The basic structural unit of cellulose molecule is D-glucopyranose, which is a high molecular polymer connected by β-1,4 glycosidic bonds, and forms a helical structure through intramolecular hydrogen bonds. Its glucose residues are about 2000-25000. Cellulose is synthesized at the plasma membrane by the production of hemicellulose and pectin precursor oligomers in the Golgi apparatus in the cell, which are then secreted to the plasma membrane by exocyto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10A01H5/00A01H5/12A01H6/20
CPCC12N9/1059C12N15/8261
Inventor 陈金慧金磊磊施季森钟山成铁龙杨立明
Owner NANJING FORESTRY UNIV
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