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Chitinasesn gene by Paecilomyces lilacinus and clone method thereof

A technology of chitinase gene and Paecilomyces lilacinus, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of high toxicity of nematicides and environmental pollution, and achieve obvious control effect , the effect of increasing the amount of expression

Inactive Publication Date: 2008-09-17
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, nematicides are mainly used to control plant parasitic nematodes, but nematicides are usually highly toxic and pollute the environment

Method used

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  • Chitinasesn gene by Paecilomyces lilacinus and clone method thereof
  • Chitinasesn gene by Paecilomyces lilacinus and clone method thereof
  • Chitinasesn gene by Paecilomyces lilacinus and clone method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Isolation of Nematode Parasitic Fungi

[0039]Potato dextrose agar medium PDA was first prepared by adopting the method of culturing and separating oocysts and female worms. 200g of potatoes, 20g of glucose, 20g of agar, 1000mL of water, 0.2g of chloramphenicol was added to every 1000mL of PDA medium to inhibit the growth of bacteria; For the oocysts outside the knot and the females in the root knot, 25 oocysts and 15 females were selected for each sample; the oocysts and females were disinfected with 5% NaCl for 3 minutes; the oocysts and females were quickly transferred to sterilization Rinse in water for a total of 3 times; finally transfer the oocysts and females to PDA and culture at 25°C; pick up 3 oocysts or females in each dish; after colonies grow around the oocysts and females, pick the marginal bacteria Silk, purified, cultured on PDA, preserved.

[0040] 2. The identification of the fungus is based on the morphology combined with the means of molecular ...

Embodiment 2

[0041] Cloning of Example 2 Paecilomyces lilacinus chitinase gene

[0042] (1) Use chitin to induce the chitinase activity of Paecilomyces lilacinus, collect fungal hyphae and spores with the highest enzyme activity, use Trizol reagent method to extract total RNA, and use GeneRacer TM Kit and BD SMART RACE cDNA Aplication Kit were reverse transcribed into a strand of cDNA as templates for 3'RACE and 5'RACE respectively;

[0043] 1. Study on Chitinase Activity of Paecilomyces lilacinus

[0044] The chitinase activity of Paecilomyces lilacinus induced by chitin, and the Paecilomyces lilacinus not induced by chitin were used as the control, and the chitin-induced and non-chitin-induced Paecilomyces lilacinus were determined by DNS method The chitinase activity of Paecilomyces after cultured for 3d, 7d, 10d and 15d respectively.

[0045] The results showed that adding chitin induced the highest chitinase activity of Paecilomyces lilacinus on the 7th day. The fungal hyphae and s...

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Abstract

The invention discloses a novel chitinase gene of nematode bio-control fungi lilac paecilomyces with a gene sequence table as seen in SEQ ID NO: 1, and meanwhile discloses a method for cloning the chitinase gene of the lilac paecilomyces. The method comprises the steps of using lilac paecilomyces RNA with highest enzyme activity as a template, designing and expanding a pair of degenerate primers to get the chitinase gene fragments of the lilac paecilomyces, obtaining gene 5' terminal and 3' terminal sequences from a lilac paecilomyces cDNA chain through SMART RACE RT-PCR technology, and connecting the three sequences to obtain a novel cDNA full-length sequence of the chitinase gene (PL Chil). The invention provides important technological base for transforming the chitinase gene of the lilac paecilomyces to other nematode bio-control fungi by using bioengineering technology, realizing the heterologous expression of the gene, and improving the expression quantity of the chitinase gene of the nematode bio-control fungi and the bio-control ability of plant nematodes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Paecilomyces lilacinus chitinase gene and a cloning method thereof. Background technique [0002] Plant parasitic nematodes are one of the most important pathogens that cause crop diseases and cause great harm to agriculture. At present, nematicides are mainly used to control plant parasitic nematodes, but nematicides are usually highly toxic and pollute the environment. At present, the biological control of plant nematodes has attracted more and more attention. At present, there are commercial nematode biocontrol agents including Nebac and others on the market. [0003] Fungi are the most studied natural enemies in the biological control of plant nematodes, and they are also one of the most important biological control factors. Paecilomyces lilacinus is one of the most famous and important nematode biocontrol fungi. Studies have shown that Paecilomyces lilacinus ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/10
Inventor 卓侃廖金铃
Owner SOUTH CHINA AGRI UNIV
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