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Recombinant Paecilomyces lilacinus strain pnvt8 and its application

A technology of Paecilomyces lilacinus and Paecilomyces lilacinus is applied in the field of agricultural microbial genetic engineering, which can solve different problems and achieve the effects of increasing growth rate, increasing sporulation rate, and increasing biomass and cell yield.

Inactive Publication Date: 2015-10-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, studies have found that different segments and positions of vgb on the chromosome have different effects on the host bacteria, and there is no absolute relationship between the number of copies of vgb and the expression of VHb, the promotion of cell growth and the expression of target proteins

Method used

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  • Recombinant Paecilomyces lilacinus strain pnvt8 and its application
  • Recombinant Paecilomyces lilacinus strain pnvt8 and its application
  • Recombinant Paecilomyces lilacinus strain pnvt8 and its application

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: recombinant Paecilomyces lilacinus strain

[0047] 1. Artificial design of vgb gene

[0048] Find the amino acid sequence of Vitreum hyaline hemoglobin (VHb) from GenBank (GenBank accession number: M27061), and then according to the codon degeneracy and the codon preference of Paecilomyces lilacinus (see URL

[0049] http: / / www.kazusa.or.jp / codon / codon usage database) principle, select the appropriate codon corresponding to each amino acid, and convert the amino acid sequence of VHb into the original nucleotide sequence. Then, use DNASIS software to analyze its repeat sequence and stem-loop structure, etc., through the replacement of local codons to make the length of the repeat sequence ≤ 7 bases, and the stem length in the stem-loop structure ≤ 7 bases, the final The nucleotide sequence of the vgb gene (such as figure 1 shown).

[0050] 2. Construction of fungal expression vector pUPNVT

[0051]The list of primers required for construction of the exp...

Embodiment 2

[0186] Embodiment 2: the fermentation performance detection of PNVT8

[0187] 1. The relationship between the expression of VHb protein and the amount of dissolved oxygen

[0188] In order to further determine the influence of dissolved oxygen conditions on the expression of VHb protein, the present invention determines the number of spores of ACSS and PNVT8 by a hemocytometer, and inserts 2.4×10 5 1 spore was cultured with gradient medium (20mL, 40mL, 60mL, 80mL, 100mL), sealed with a sealer, 200r / min, 28°C for 7 days, the experimental results are as follows Figure 14 shown.

[0189] Shake the above-cultured mycelia in turn, draw 5mL into a graduated plate, add 5mL of distilled water containing 1% Tween 80, mix well and count the mycelium balls that can be clearly seen by the naked eye. is the abscissa, and the number of hyphae balls is plotted as the ordinate. The experimental results are as follows Figure 15 shown

[0190] Depend on Figure 14 and Figure 15 It can ...

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Abstract

The invention relates to a paecilomyces lilacinus strain PNVT8 for expressing the hemoglobin VHb of vitreoscilla and the application of the paecilomyces lilacinus strain PNVT8. The recombinant paecilomyces lilacinus strain PNVT8 is collected in the China center for type culture collection with the collection number of CCTCC No:M 2013296. The paecilomyces lilacinus strain PNVT8 is obtained by optimizing a codon, a promoter and a gene structure, successfully integrating the hemoglobin VHb from the vitreoscilla into a chromosome of a wild paecilomyces lilacinus strain ACSS which has a plant parasitic nematode killing effect and is provided by the agricultural culture collection of China, and performing screening. Compared with the wild paecilomyces lilacinus strain ACSS, the paecilomyces lilacinus strain PNVT8 has the advantages that more extracellular proteases, chitinase, alpha-amylases and the like can be generated, and meanwhile, the biomass and the cell yield are improved; after the paecilomyces lilacinus strain PNVT8 is applied as a product, the growth rate during field planting under a soil micro-aerobic condition also is increased to further improve the nematode larva killing activity.

Description

technical field [0001] The invention belongs to the technical field of agricultural microbial genetic engineering, and relates to the breeding and application of microbial pesticide genetic engineering bacteria, in particular to a Paecilomyces lilacinus strain PNVT8 expressing VHb protein and its application. technical background [0002] The research of American nematologists shows that the annual crop loss rate caused by plant pathogenic nematodes is 12.3% in the world, that is, plant pathogenic nematodes can cause direct economic losses of about 100 billion U.S. dollars per year, while my country’s annual loss is about 13.3 billion U.S. dollars. Soybeans alone lost $100 million due to nematode damage, which had a great impact on crop yields and farmers' income. After the nematode invades the plant, it absorbs the nutrients in the plant and affects its normal growth and development. At the same time, the secretion of the nematode can also stimulate the cells and tissues of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15A01N63/04A01P5/00C12R1/79
Inventor 何进危雅乐王阶平张艳郝勃
Owner HUAZHONG AGRI UNIV
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