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Trichoderma viride engineering bacterium and uses thereof

A technology of Trichoderma viride and engineering bacteria is applied in the application field of vegetable biological control to achieve the effects of high control efficiency, increased yield and strong antagonism.

Inactive Publication Date: 2009-07-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is no work in China to construct high-efficiency Trichoderma viride engineering bacteria by overexpressing the endochitinase gene

Method used

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  • Trichoderma viride engineering bacterium and uses thereof
  • Trichoderma viride engineering bacterium and uses thereof
  • Trichoderma viride engineering bacterium and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1. Construction of engineering strain Trichoderma viride (Trichoderma viride) R65CCTCC NO: M 208265

[0026] The endochitinase gene overexpression vector pG-ech42 was constructed by Wang Xiaodong, State Key Laboratory of Microbial Technology (Jinan) (Wang Xiaodong, antifungal protein and chitinase gene cloning and expression research, Shandong University master thesis, 2006) The upstream of the vector endochitinase ech42 gene contains a strong promoter of the gpd gene from Aspergillus nidulans. The physical map of the endochitinase gene overexpression vector pG-ech42 is shown in Figure 4 , its construction process is as follows:

[0027] Use pAN7-1 as a template and use primer p1 / p2 to PCR amplify the gpd promoter of about 1.1k. The primer p1 / p2 and PCR process are as follows:

[0028] p1: 5′-GCGC GAATTC AGACCTAATACAGCCCCTAC-3′

[0029] EcoRI

[0030] p2: 5′-GCGC GGTACC TGTCTGCTCAAGCGGGGTAG-3′

[0031] KpnI

[0032] PCR con...

Embodiment 2

[0058] ] Embodiment 2. Trichoderma viride (Trichoderma viride) R65CCTCC NO: M 208265 morphology observation

[0059] Trichoderma viride (Trichoderma viride) R65CCTCC NO: M 208265 was inoculated into PDA medium, cultured at 30°C for 3 to 4 days, and the characteristics of the colonies were observed as follows: figure 1 ; Mycelium cotton blue staining observation, and use NIKONALPHAPHOT-2 system photography, mycelium and spore morphology as figure 2 .

[0060] On the PDA medium, the colony is colorless or light-colored fluffy, and the surface of the colony begins to appear green in the later stage, and concentric ring patterns can be seen in the spore-forming bundle area; the hyphae are separated and the branches are complicated. Opposite or alternate branches on short mycelial branches, branches can continue to branch, the branches are acute or nearly right-angled, fascicles, opposite or solitary terminal branches; conidia are produced successively from the final terminal bra...

Embodiment 3

[0061] Example 3. Trichoderma viride (Trichoderma viride) R65CCTCC NO: M 208265 chitinase activity assay

[0062] With reference to Tan Jianxin (Tan Jianxin, Bt cry gene cloning and Bacillus circulans chitinase research. Graduate School of Chinese Academy of Agricultural Sciences doctoral dissertation, 2001) chitinase activity assay method for Trichoderma viride R65CCTCC M208265 several Tinase activity assay. The specific method is as follows:

[0063] Preparation of snail enzyme liquid: weigh 1g of snail enzyme dry powder, 0.2mL of EDTA (0.5mol / L), 1mL of KCl (1mol / L), put them in a volumetric flask and mix well, add double distilled water until 100ml is completely dissolved, then you can get 1% helicase, stored at low temperature.

[0064] Preparation of N-acetylglucosamine standard curve: configure 100ug / ml N-acetylglucosamine (GlcNAc) mother solution, and then dilute the mother solution to different concentrations (0, 20, 40, 60, 80, 100ug / ml); then Take several clean tes...

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Abstract

The invention discloses Trichoderma viride engineering bacteria, which comprises endochitinase genes, was named as green Trichoderma viride R65 and was preserved in 'CCTCC' on December 29, 2008. The preserving number is CCTCC NO: M 208265. The invention also discloses the applications of the strains in controlling vegetables. The highest vigor of the chitinase of the strains can achieve 77.4U / ml, which is 3.67 times of the strains of an original host. The strains have stronger antagonism on a plurality of plant pathogenic fungi like cucumber gray mold, Aspergillus niger, rhizoctonia solani kuhn, wheat red mould, cotton Phytophthora, and the like, and is an engineering bacteria which have more application values in controlling vegetable.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering and biological control; specifically, the invention relates to an engineering strain Trichoderma viride engineering strain overexpressing an endochitinase gene, and also relates to the application of the bacterial strain in vegetable biological control. Background technique [0002] Trichoderma (Trichoderma spp.) belongs to the subphylum Deuteromycotina, Hyphospora, Synchosporales, and Synchosporaceae. It is a widely distributed soil-dwelling fungus, which is often found on plant residues and animal feces. Except for a few weak pathogenic bacteria, most of them have antagonistic effect on plant pathogenic bacteria. Due to Trichoderma's wide adaptability, diversity of antagonists and broad-spectrum parasitism, it has been extensively studied as an antagonistic agent for biological control of plant diseases. [0003] The biocontrol mechanisms of Trichoderma against plant pathogens inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15A01N63/04A01P3/00C12R1/885
Inventor 汪天虹皇传华肖鹏李秀深
Owner SHANDONG UNIV
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