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Method for improving rice straw degradation and transformation efficiency by virtue of exoglucanase

A technology of exoglucanase and conversion efficiency, applied in biochemical equipment and methods, glycosylase, botanical equipment and methods, etc., to achieve the effect of improving conversion efficiency and increasing expression

Active Publication Date: 2017-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the effect of expressing exoglucanase in plants on the degradation and transformation efficiency of plant straw

Method used

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  • Method for improving rice straw degradation and transformation efficiency by virtue of exoglucanase
  • Method for improving rice straw degradation and transformation efficiency by virtue of exoglucanase
  • Method for improving rice straw degradation and transformation efficiency by virtue of exoglucanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Cloning and optimization of CBH Ⅰ gene full-length cDNA

[0044] 1. Clone the full-length cDNA of CBH Ⅰ gene from Trichoderma reesei, and optimize the codon of CBH Ⅰ gene by site-directed mutagenesis.

[0045] Among them, the optimized codons of the full-length cDNA of CBH Ⅰ gene are:

[0046] The codon CTA of leucine at position 77 was optimized as CTC, and the CGA of arginine at position 319 was optimized as CGC;

[0047] 2. The codon-optimized site-directed mutagenesis method of the CBH Ⅰ gene

[0048] ——If there are two sites in the gene that need site-directed mutation, 3 pairs of primers need to be synthesized and cloned into the vector, see figure 2 -A; where primers 1F and 3R are added with restriction enzyme sites required by the carrier, primers 2F and 1R, 3F and 2R are completely complementary to each other, and the base to be mutated is placed in the middle of the primers, and the length of the primers is greater than 20 bp;

[0049] ——Respectiv...

Embodiment 2

[0053] Embodiment 2: Construction of expression vector

[0054] 1. Rice Rbcs promoter overexpresses CBH Ⅰ of Trichoderma reesei and localizes to chloroplast

[0055] Construction of the vector (pC1300T-osrbcs-chl-CBH Ⅰ ful) (see image 3 ):

[0056] ——Taking Nipponbare's genomic DNA as a template, synthesize primers to amplify the rbcs promoter (including signal peptide-47 AAs).

[0057] ——Synthetic primers amplify the full length of CBH Ⅰ (codon optimization, including stop codon, without signal peptide), the sequence is shown in sequence 1

[0058] Forward primer: GGGGTACC(KpnΙ)CAGTCGGCCTGCACTCT

[0059] Reverse primer: ATGATACGGGCTCACCAA

[0060] ——Connect the promoter and CBH Ⅰ full-length cDNA sequence to the T vector respectively, digest with restriction enzymes, and transfer to the expression vector pC1300T.

Embodiment 3

[0061] Embodiment 3: Plant expression vector transforms Agrobacterium

[0062] 1. Agrobacterium activation

[0063] Draw the preserved Agrobacterium (EHA105) on the solid LB medium (add antibiotic: Kan, if no antibiotics are added, the Ti plasmid of these strains may be lost, resulting in the lack of infectivity of Agrobacterium), the antibiotic concentration is: 50μg / mL, culture at 28°C for 1-2 days, then transfer to a new solid LB medium with antibiotics and culture for another 2 days;

[0064] 2. Preparation of Agrobacterium Competent Cells

[0065] Inoculate 100 μL into 1 mL of LB liquid medium, shake at 150 rpm at 28°C overnight;

[0066] Take 1mL of the bacterial liquid and inoculate it into 100mL of liquid medium and cultivate until OD600=0.5;

[0067] Place the bacterial solution on ice for 30 minutes, and cool the culture to 0°C;

[0068] Centrifuge at 5000rpm for 30s at 4°C and discard the supernatant;

[0069] Precipitate with 60mL 0.1M CaCl 2 Suspended, ice-b...

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Abstract

The invention discloses a method for improving rice straw degradation and transformation efficiency by virtue of exoglucanase, and belongs to molecular biological techniques of trans-cellulase rice. The method comprises the following steps: (1) cloning complete-length cDNA of a CBHI gene from trichoderma reesei, and in accordance with a rice codon using frequency table, optimizing codon of the CBHI gene by virtue of a site-specific mutagenesis method; (2) constructing a vector, which achieves expression in chloroplast through photoinduction, of the CBHI gene and transforming rice 'zhonghua 11', so that a transgenic plant is obtained; (3) processing straw powder of an obtained rice trans-CBHI gene material by virtue of exogenous cellulase complex enzyme and 1% Tween-80, and determining yield of hexose; and (4) determining a cell wall cellulose content of obtained transgenic rice CBHI straw. With the application of the method provided by the invention, the codon of the CBHI gene is optimized; an expression amount of the exogenous CBHI gene in the rice is increased, so that the degradation and transformation efficiency of the transgenic rice straw is improved by 84.94%.

Description

technical field [0001] The invention relates to a method for constructing cellulase gene-transformed rice, belongs to the technical field of molecular biology of cellulase-transferred rice, and specifically relates to a method for improving the degradation conversion efficiency of rice straw by using exoglucanase. Background technique [0002] Cellulase is widely found in a variety of microorganisms and consists of the following three enzymes: (1) endo-1,4-β-D-glucanase (endo-1,4-β-D-glucanases, EC 3.2.1.4, EG), it acts on the β-1,4-glucoside bond on the cellulose chain, randomly hydrolyzes amorphous cellulose, releases cellooligosaccharides to form new reducing ends; (2) 1,4- β-D-cellobiohydrolases or exoglucanases (1,4-β-D-cellobiohydrolases or exoglucanases, EC 3.2.1.91, CBH), which travel along cellulose chains, from reducing ends or non- The reducing end hydrolyzes crystalline cellulose to release cellobiose; (3) 1,4-β-D-glucosidase (1,4-β-D-glucosidases, EC 3.2.1.21, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/82A01H5/00C12P7/10
CPCC12N9/2437C12N15/8246C12P7/10C12Y302/01091Y02E50/10
Inventor 彭良才丰胜求谢国生王令强王艳婷李英范春芬孙海燕胡慧贞
Owner HUAZHONG AGRI UNIV
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