Engineering neurospora crassa, preparation method thereof and application of neurospora crassa

A technology of engineering bacteria and bacteria, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem that the cell wall of plants cannot be rapidly degraded, and achieve the effect of reducing the fermentation time

Inactive Publication Date: 2019-10-25
威海惟农生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims to solve the technical problem that the existing bacterial strains cannot rapidly degrade the cell

Method used

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  • Engineering neurospora crassa, preparation method thereof and application of neurospora crassa
  • Engineering neurospora crassa, preparation method thereof and application of neurospora crassa

Examples

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Embodiment 1

[0013] Example 1 Construction of Neurospora crassa strains with positive feedback regulation of cellulase genes (1) extract the total RNA of Neurospora crassa (Neurospora crassa) FGSC 4200 (purchased from Fungal Genetics Stock Center), reverse transcription cDNA was generated and used as a template, using the upstream primer 5'-GAGGCTGAAGCTGAATTCATGGCACCATCAATGTCG-3' and the downstream primer 5'-ATGAGTTTTTGTTTCTAGAGTAGGTGTACCCATCCATA-3' to amplify the cellulase regulatory gene sequence clr -2; PCR amplification conditions are as follows: pre-denaturation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 2.5 minutes; finally, extension at 72°C for 10 minutes.

[0014] (2) The PCR product obtained in the above step (1) was purified using a small DNA purification kit (purchased from Tiangen Company). At the same time, the vector pPICZa-A was digested with EcoRI and XbaI, and the corresponding vector fragment was recovered using...

Embodiment 2

[0019] The comparison of cellulase and xylanase activity of embodiment 2 Neurospora crassa bacterial strains

[0020] 1. Analysis of clr-2 protein expression

[0021] Neurospora crassa (Neurospora crassa) Pcbh-2-clr2 was inoculated into vogel's medium, with 0.5% CMC as the sole carbon source, and cultured at 30°C for 5 days. The cells were disrupted with glass beads, and the expression of clr-2 protein (antibody: c-myc) was detected by western blot. The results showed that clr-2 was highly expressed under this condition. Its protein size is about 91 kDa.

[0022] 2. Analysis of cellulase activity

[0023]Configure 50mL Vogel's medium, use the treated straw as the single carbon source, inoculate N.crassa FGSC4200 and Neurospora crassa (Neurospora crassa) Pcbh-2-clr2 into the medium (the final concentration of spores is 10 6 individual / mL). Take 500 uL of the day 3 culture supernatant, centrifuge, and transfer the supernatant to a new storage tube. Take 100uL of supernatan...

Embodiment 3

[0026] Example 3 The effect of accelerating the composting of straw after adding the mixed bacterial solution

[0027] 1. Analysis of temperature shock period

[0028] In the fermentation process without the addition of the bacterial liquid, there is a period of temperature fluctuations in the early fermentation stage. Usually when the temperature reaches 70 degrees Celsius, the compost pile needs to be turned over to increase sufficient oxygen and maintain the normal operation of microorganisms. temperature. It is generally about 7 days from the beginning of fermentation to when the temperature reaches 70 degrees Celsius. The period from the first turning to the second turning is also about 7 days, the temperature from the second turning to the third turning is about 10 days, and the period from the third to the fourth is about 15 days. The overall temperature shock period is around 40.

[0029] However, after the bacterial solution was added, the time from the beginning t...

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Abstract

The invention relates to an engineering neurospora crassa, a preparation method thereof and an application of the neurospora crassa, and solves the technical problem that an existing strain cannot rapidly degrade cell walls of plants. The engineering neurospora crassa is classified and named as neurospora crassa Pcbh-2-clr2, and the collection number of the engineering neurospora crassa is CGMCC No. 18102. The engineering neurospora crassa can be widely applied to the field of branch fermentation.

Description

technical field [0001] The invention relates to an engineering bacterium and its preparation method and application, in particular to a Neurospora crassa engineering bacterium and its preparation method and application. Background technique [0002] With the rapid development of my country's agricultural level, agricultural and forestry waste such as pruning straw has become an urgent problem to be solved in my country's agriculture. The traditional method of burning straw not only does not improve the soil, but also releases a large amount of toxic and harmful gases, which seriously increases the content of PM2.5 and other pollutants in the air. In addition, in the context of advocating green economic development, straw burning will dramatically increase greenhouse gas emissions and aggravate the effect of global warming. In developed countries, pruning straws are mainly discarded by crushing, and the crushed straws can be used to make compost or improve soil through surfa...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/56C12N15/80C05F17/00C12R1/645
CPCC12N9/2437C12N9/248C12N15/80C05F17/20Y02W30/40
Inventor 刘恩玺王瑞杰
Owner 威海惟农生物科技有限公司
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