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Method for targeted acquisition of trichoderma reesei cellulase controlling gene

A technology of Trichoderma reesei and cellulase, which is applied in the field of agricultural biology and can solve problems such as unclear regulatory functions

Active Publication Date: 2020-11-10
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the cellulose medium, the enzyme production of Trichoderma reesei is relatively the strongest, so the corresponding regulatory genes must play different important roles on the cellulose medium, which is different from other carbon sources. ;Since the expression of thousands of genes is changed on cellulose media, there is no systematic analysis of these genes, so it is not clear which genes play a regulatory role on cellulose media
In addition, there are many regulatory genes (such as molecular chaperone pdi1, transporter sso, etc.) whose transcription levels did not change significantly in the cellulose medium, so it is impossible to simply analyze whether the transcription level on the cellulose medium is up-regulated or not. Down-regulation of altered genes predicts their involvement in the regulation of cellulase expression

Method used

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  • Method for targeted acquisition of trichoderma reesei cellulase controlling gene
  • Method for targeted acquisition of trichoderma reesei cellulase controlling gene
  • Method for targeted acquisition of trichoderma reesei cellulase controlling gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Suppressive subtractive hybridization technique to obtain the cDNA library of differential expression of Trichoderma reesei under the condition of cellulase-induced secretion

[0026]The differential hybridization experiment was performed with Clontech's PCR-Select cDNA Subtraction Kit. The main process is to reverse transcribe the mRNA of the sample (tester) and the control (driver) and synthesize it into double-stranded cDNA, then digest it with enzymes; the sample (tester) is divided into two groups, and different adapter1 / 2 are added respectively; The two groups of samples (tester) were hybridized with excess control (driver) respectively, the two hybridization results were mixed, and the excess control was added to perform the second round of hybridization; the adapter was complemented and then selectively amplified by PCR. Since the excessive control blocks the genes co-expressed in the sample, the genes specifically expressed in the sample will be selec...

Embodiment 2

[0044] Example 2 Construction of recombinant plasmid pSSH-i for performing RNA interference on differential genes

[0045] Using the genomic DNA of Trichoderma reesei Tu-6 as a template, amplify pdc1 Gene promoters are amplified. Perform electrophoresis on 1% agarose gel, cut out the target band in the PCR product, and purify the DNA.

[0046] Design differential gene secondary amplification primers, which contain the repeat sequence of RNAi plasmid library construction. Differentially expressed cDNA libraries under different culture conditions obtained by differential hybridization were used as templates, and the differential gene fragments were amplified with secondary amplification primers. Perform electrophoresis on 1% agarose gel, cut out the target band in the PCR product, and purify the DNA. Use this primer to amplify the differentially significant gene, and it is found that there is obvious specific amplification, such as figure 1 shown.

[0047] Using the genomi...

Embodiment 3

[0050] Example 3. Transformation of pSSH-i into Trichoderma reesei

[0051] The pSSH-i constructed by the present invention is transformed into Trichoderma reesei, and the bidirectional promoter pdc1 and eno1 The mRNA is transcribed under the drive of the gene, which contains two complementary interfering gene sequences, and can be folded to form a hairpin-shaped double-stranded RNA. Hairpin-shaped double-stranded RNA is recognized and cut by enzymes in the body to form small single-stranded RNA molecules, which are complementary to the mRNA of the subtracted gene in the target strain and mediate the cleavage of the mRNA of the subtracted gene by DICER, thereby inhibiting the subtraction of the target strain. gene expression.

[0052] Trichoderma reesei mp1-DsRed-SUS1 ( -pyr4 ) was inoculated on a potato medium (PDA) plate, cultured statically at 30ºC for 7 days until sporulation, the spores were scraped off and inoculated in MM-glucose medium, and cultured overnight with...

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Abstract

The invention relates to the technical field of agricultural biology, in particular to a method for targeted acquisition of a trichoderma reesei cellulase controlling gene. According to the invention,a differential expression cDNA library of trichoderma reesei with increased or decreased gene expression under the condition of cellulase-induced secretion is obtained through a subtractive hybridization technology, RNA interference is performed on a subtractive gene of Trichoderma reesei, and a transformant with significantly improved cellulase activity is obtained.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a method for targeted acquisition of Trichoderma reesei cellulase regulation gene. Background technique [0002] The cellulase system of Trichoderma reesei includes two exo-cellulase, five endo-cellulase, one b-glucosidase and three lytic polysaccharide monooxygenases (lytic polysaccharides monooxygenases, LPMOs, the original GH61 family members). The regulation of the secretion and expression of these cellulases is very complex, involving multiple stages, such as transcription, translation, transport, protein folding, extracellular degradation, etc., and each regulatory process involves a variety of key regulatory factors, but currently these The understanding of regulatory factors is still very lacking. [0003] The transcriptional regulation and signal transduction mechanism of cellulase expression in Trichoderma reesei are still unclear. Gene regulation involves m...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1072
Inventor 苏小运姚斌高飞罗会颖黄火清王苑柏映国涂涛王亚茹张杰师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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