Trichoderma reesei derived reductase and coding gene and application thereof

A reductase and coding technology, applied in the directions of oxidoreductase, genetic engineering, plant genetic improvement, etc., can solve the problems of little research on sporulation, affecting the competitive growth of Trichoderma reesei, etc.

Inactive Publication Date: 2020-09-11
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few studies on how these metabolic pathways affect th...

Method used

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  • Trichoderma reesei derived reductase and coding gene and application thereof
  • Trichoderma reesei derived reductase and coding gene and application thereof
  • Trichoderma reesei derived reductase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Homologous comparison and target gene acquisition

[0074] 1. Acquisition and homology comparison of Saccharopine reductase coding gene in Trichoderma reesei

[0075] Lys9p catalyzes the seventh step reaction in the lysine synthesis pathway, which is to catalyze the formation of ε-N-(1 glutaryl 2)-L-lysine from L-alpha-aminoadipate6-semialdehyde. Align Lys9p in Saccharomyces cerevisiae in the Trichoderma reesei genome database (https: / / mycocosm.jgi.doe.gov / pages / blast-query.jsf?db=Trire2), protein ID 123631 and Lys9p in Saccharomyces cerevisiae There is a high similarity, the similarity is 64%. Therefore, the protein ID123631 was named as TrLys9, and its amino acid sequence is shown in SEQ ID NO:3. At the same time, the Lys9p protein sequence in Trichoderma reesei and other filamentous fungi were analyzed by phylogenetic tree, such as figure 1 As shown, Lys9p in Trichoderma reesei has strong conservation in filamentous fungi, such as Fusarium tritici (E-val...

Embodiment 2

[0097] Embodiment 2 mutant obtains

[0098] 1. Construction of knockout vector and complementary vector

[0099] Trichoderma reesei genome extraction: the culture conditions are the same as the above-mentioned RNA extraction culture conditions. Methods for extraction of genomic DNA are described in (J Sambrook et al. 2001).

[0100] Construction of the knockout vector: PCR amplification of the upstream and downstream fragments of the TrLys9 gene, each fragment is about 1200bp in length, the upstream uses Kpn I and Sal I restriction endonuclease double enzyme cutting sites, and the downstream uses Hind III and Xba I restriction Endonuclease double restriction site. The resulting PCR fragment was directly digested, and the upstream fragment was first connected to pBS-HPH with the same restriction site (Liu et al., 2007), and then the above vector was digested with Hind III and Xba I, and the T vector was digested After the downstream fragment is connected to the restriction s...

Embodiment 3

[0123] Effect of embodiment 3 TrLys9 gene on the growth and development of Trichoderma reesei

[0124] PDA solid medium: potato 200g / L, glucose 20g / L, agar powder 15-20g / L.

[0125] MM solid medium: glucose 20g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 15g / L, magnesium sulfate 0.6g / L, calcium chloride 0.6g / L, ferrous sulfate 0.005g / L, manganese sulfate 0.016 g / L, zinc sulfate 0.0014g / L, cobalt chloride 0.002g / L, agar powder 15-20g / L, pH5.5

[0126] 1. Colony morphology observation

[0127] The strains were respectively inoculated on CM, PDA and MM plates, and cultured at 30°C for 3 days to observe the colony morphology. Among them, QM9414 is a wild-type strain, ΔTrLys9-11 is a gene deletion mutant strain, and 11HB is a gene complementation mutant strain. Such as Figure 4 As shown, there is no difference in the colony morphology of each strain on the CM medium. On the PDA medium, the mycelial growth of the mutant is slower than that of the wild-type strain, an...

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Abstract

The invention discloses an enzyme, which has the activity of epsilon-N-(l glutarate 2)-L-lysine reductase, and provides a function of catalyzing 6-oxo-DL-norleucine (L-alpha-aminoadipate 6-semialdehyde) to form epsilon-N-(l glutarate 2)-L-lysine in a diaminopimelic acid pathway. The invention discloses a DNA molecule for encoding the enzyme. The invention discloses a recombinant vector. The invention discloses a host cell. The invention discloses a construction method of a lysine auxotrophic mutant. The invention discloses an application of the DNA molecule as an auxotrophic marker gene of lysine. The invention discloses an application of protein or the DNA molecule in growth and development and actual production optimization of trichoderma reesei. A TrLys9 gene provided by the invention can be used as the auxotrophic marker gene of the lysine, and is used as a marker gene for subsequent transgenic operation of the Trichoderma reesei.

Description

technical field [0001] The invention belongs to the application field of functional genes related to filamentous fungi, and relates to Saccharopine reductase Trlys9 and its cDNA sequence cloned from the filamentous fungus Trichoderma reesei. The invention also provides a recombinant expression vector related to the Saccharopine reesei cDNA sequence and Recombinant host cells, and their use in influencing Trichoderma reesei hyphae growth, conidia production, etc. Background technique [0002] Lysine is an essential amino acid for humans and animals that can only be obtained from dietary protein. In bacteria, fungi and some plants, lysine can be synthesized by itself. Of the 20 common proteinogenic amino acids, only lysine has two distinct biosynthetic pathways. In bacteria, lower fungi (such as Phycomycetes) and some plants, lysine is synthesized through the diaminopimelate pathway (DAP pathway); while in higher fungi (the cell wall contains chitin components) and eyes In ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/80C12N1/15C12R1/885
CPCC12N9/0028C12N15/80C12Y105/0101
Inventor 赫荣琳贾文娣张东远
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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