Molecular design and application of a readily degradable plant cell wall

A technology for plant stems and plant expression vectors, which is applied in the directions of botanical equipment and methods, applications, plant products, etc., can solve problems such as the synthesis of 2-phenylethanol, and achieve great research value and application potential, and improve glucose efficiency. Effect

Inactive Publication Date: 2019-03-29
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the synthesis of 2-phenylethanol in Arabidopsis

Method used

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  • Molecular design and application of a readily degradable plant cell wall
  • Molecular design and application of a readily degradable plant cell wall
  • Molecular design and application of a readily degradable plant cell wall

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Experimental program
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Embodiment 1

[0025] The construction of the plant expression vector of embodiment 1 2-phenylethanol synthesis pathway:

[0026] 1. 2-phenylethanol synthesis pathway gene: petunia phenylacetaldehyde synthase gene PAAS and tomato phenylacetaldehyde reductase gene LePAR1 clone

[0027] In order to construct the 2-phenylethanol synthesis pathway in plants, the petunia phenylacetaldehyde synthase gene was selected according to the report on the 2-phenylethanol synthesis pathway in plants PAAS (DQ243784) and tomato phenylacetaldehyde reductase gene LePAR1 (NM_001247894) Construction of 2-phenylethanol synthesis pathway. The petunia (Petunia hybrida) blooming petals and the tomato (Solanum lycopersicum) fully mature fruit were taken, and the total RNA was extracted by CTAB method, and the full-length cDNA sequences of the two were obtained by searching GenBank, and gene-specific primers were designed respectively, and RT-PCR technology amplification PAAS and LePAR1 The full-length cDN...

Embodiment 2

[0034] Example 2 Construction of 2-phenylethanol synthesis pathway to reduce the application of plant lignin synthesis:

[0035] 1. Agrobacterium-mediated transformation of Arabidopsis

[0036]The plant expression vector was transformed into Agrobacterium, and positive clones were picked and cultured overnight until OD=2.0. Centrifuge 300ml of the overnight cultured bacterial solution, collect the bacteria by centrifugation at room temperature, and resuspend in 150ml of transformation buffer (transformation buffer: 0.5% sucrose solution contains 0.02% Silwet). Put the inflorescences of Arabidopsis thaliana upside down and immerse them in the bacterial solution. Try to immerse all the inflorescences in the bacterial solution for 5-6 times. Then put the Arabidopsis thaliana upside down in the water receiving tray, cover it with plastic wrap, and place it in the culture room. (22°C) overnight, and then cultured normally. After one week of transformation, it can be retransformed...

Embodiment 3

[0050] Example 3 Construction of 2-phenylethanol synthesis pathway to improve the efficiency of enzymatic hydrolysis and saccharification of plant cell walls

[0051] Efficiency analysis of cell wall extracts from Arabidopsis thaliana stems hydrolyzed by cellulase. The specific steps are:

[0052] (1) The cell wall extracts were divided into two treatment groups. The first group was directly treated with cellulase (Cellulose) and glucosidase (β-glucosidase), and 20 mg of cell wall extracts from control and transgenic plants were used for enzymatic hydrolysis treatment 24 Hours, take 500ul of the enzymatic hydrolyzate at 1, 3, 6, and 24 hours respectively, and measure the glucose content in the hydrolyzate;

[0053] (2) The second group took the cell wall extract and treated it with hot water (121°C) for 1 hour, then treated it with cellulase and glucosidase at 1, 3, 6, and 24 hours to measure the glucose content in the enzymatic hydrolysis product . According to the instruc...

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Abstract

The invention discloses a molecular design and an application of degradable plant cell walls, relates to the field of genetic engineering, and particularly relates to a method and an application of achieving lignin synthesis reduction of the plant cell walls to more easily degrade the plant cell walls by building a 2-phenylethyl alcohol synthesis pathway in a plant, so as to improve the fiber biomass conversion efficiency. The 2-phenylethyl alcohol synthesis pathway comprises two key genes, which are respectively a phenylacetaldehyde synthetase gene PAAS of petunia and a tomato phenylacetaldehyde reductase gene LePAR1. A pathway for synthesizing 2-phenylethyl alcohol is built in arabidopsis thaliana, a competition starting substrate phenylalanine is synthesized by synthesis of 2-phenylethyl alcohol and lignin, and of carbon metabolisms is caused, so as to achieve lignin synthesis reduction of a transgenic plant. The molecular design mainly aims at improving the degradation efficiency of the plant cell wall and reducing the pretreatment difficulty of the fiber biomass by reducing the lignin synthesis in the plant. At present, the method has the characteristics of reducing the lignin synthesis and simultaneously improving the enzymolysis efficiency of a stem cell wall extract for releasing glucose in the arabidopsis thaliana.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method and application for reducing lignin synthesis by constructing a 2-phenylethanol synthesis pathway in transgenic plants. Background technique [0002] Lignocellulosic biomass (biomass) is the most abundant renewable resource on earth. Its conversion products are abundant and can be converted into various products such as biofuels and bio-based chemicals. With the increasing shortage of mineral resources, as well as the environmental pollution and climate deterioration caused by the use of mineral resources such as petroleum, the development and utilization of fiber biomass resources has attracted widespread attention worldwide. Lignin is one of the main components of lignocellulosic biomass, and its existence has always been the main barrier limiting the efficient conversion and utilization of cellulosic biomass. Previous research results have proved that changing the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/52C12N15/82A01H5/00A01H6/20
Inventor 周功克祁广柴国华孔英珍王殿唐贤丰付春祥贺郭
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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