A kind of nitrilase gene nit2 and its overexpression and application

A nitrilase and overexpression technology, applied in the field of genetic engineering, can solve the problems of reduced IAA biosynthesis and redundant biosynthesis, and achieve the effect of improving the growth-promoting effect

Active Publication Date: 2022-05-10
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the key enzymes encoding rate-limiting steps in bacteria and plants include indole-3-pyruvate decarboxylase (IPDC) and indole-3-acetaldehyde dehydrogenase (IAAld), which are missing in bacteria and Azotobacter brasiliensis. All genes can lead to a decrease in IAA biosynthesis, but no IAA complete deletion mutants have been obtained so far, indicating that IAA biosynthesis has redundant functions
However, there are still relatively few studies on the metabolic pathway of Trichoderma to synthesize IAA and its key genes, and most studies only involve a specific gene or specific enzyme in a biosynthetic pathway

Method used

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  • A kind of nitrilase gene nit2 and its overexpression and application
  • A kind of nitrilase gene nit2 and its overexpression and application
  • A kind of nitrilase gene nit2 and its overexpression and application

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 strain activation and preparation

[0033] 1. Observation of strains: NJAU4742 strain is Trichoderma guizhouense, which has the following characteristics: filamentous fungus, hyphae with septal arbuscular shape, conidiophores are smooth, green, the top is enlarged and spherical, and there are small growths on it. Stems produce spores, conidia are smooth spherical, green.

[0034] 2. Culture of strains

[0035] 1) Inoculate NJAU4742 into a solid medium PDA glass petri dish, culture conditions: 28°C, 7 days, wash and scrape the green spores on the petri dish with 5mL sterile water, filter with sterile gauze to sterilize A spore suspension was prepared in a glass bottle for use.

[0036] 2) Inoculate the Trichoderma harzianum NJAU4742 spore liquid into the configured PDA medium according to 1% and shake the flask for culture, the liquid volume is 20%-50% of the volume of the Erlenmeyer glass bottle, and the culture conditions are: temperature is 28°C, 180rmp...

Embodiment 2

[0037] Example 2 The growth-promoting effect of Trichoderma harzianum NJAU4742 on cucumber under hydroponic aseptic conditions

[0038] 1. Growth-promoting effect

[0039]Cucumber seeds used in this study were Lufeng cucumbers from Jiangsu Academy of Agricultural Sciences. Seeds were sterilized in 70% (v / v) ethanol for about 5 minutes, then soaked in 20% sodium hypochlorite (NaClO) for 20 minutes, and then rinsed with sterile water at least 4 times. The sterilized aseptic seeds were germinated in the dark at 30°C for 24 hours, and then the germinated seeds were placed on the beaker column containing the nutrient solution for aseptic culture for 7 days, and the aseptic seedlings with the same growth were transferred into the 50mL container containing 50mL Hoagland's nutrient solution was cultured in a triangular flask, and Trichoderma cells were inserted for interaction experiments. On the 10th day of the interaction, the results of significant growth differences among the tr...

Embodiment 3

[0044] Embodiment 3 Cucumber Root System IAA Content Determination

[0045] Cucumber roots were cut and ground in liquid nitrogen, frozen and ground, dissolved in methanol for extraction, and determined by ELISA method:

[0046] 1) Adding samples: set blank wells, standard wells, and sample wells to be tested respectively. Add 50 μl of sample diluent to the blank well, and add 50 μl of standard substance or sample to be tested in the remaining wells. Be careful not to have air bubbles. Add 50 μl of Detection Reagent A (prepared just before use) to the well, cover the microtiter plate with a membrane, and incubate at 37°C for 1 hour.

[0047] 2) Discard the liquid, shake dry, add 350 μl of 1x Wash Solution, soak for 1-2 minutes, and shake dry. Wash the plate 3 times, put the microplate upside down on absorbent paper for the last time, and blot the liquid.

[0048] 3) Add 100 μl of Detection Reagent B working solution (prepared just before use) to each well, add a membrane, a...

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Abstract

The invention discloses a nitrilase gene nit2 and its overexpression method and application. A key gene for IAA biosynthesis in Trichoderma harzianum NJAU4742 is a nitrilase gene nit2, the nucleotide sequence of which is shown in SEQ ID NO.1. A method for overexpression containing the nitrilase gene nit2. Application of nitrilase gene nit2 and its overexpression fragment in constructing Trichoderma harzianum NJAU4742 genetically engineered bacteria with significantly improved IAA yield. The newly discovered nit2 gene in the Trichoderma harzianum NJAU4742 of the present invention plays an important role in the IAA synthesis process, and the overexpression of the gene can significantly increase the IAA content at the pericycle in the root hair region of the main root system of the plant. The invention provides a theoretical basis and technical support for the development of novel trichoderma bio-organic fertilizer and the development of ecological agriculture.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a nitrilase gene nit2 and its overexpression and application. Background technique [0002] Indole-3-acetic acid, also known as auxin (IAA), is a type of plant endogenous hormone containing an indole ring structure, which can regulate the growth of plant cells, the differentiation of roots, stems and leaves, and the development of fruits. Each stage plays an important role. Studies have shown that beneficial microorganisms in the rhizosphere can also synthesize IAA, although the amount of synthesis is relatively small, and the beneficial microorganisms in the rhizosphere of the genus Trichoderma (Trichoderma spp.) have become a research hotspot. Trichoderma is an important class of plant growth-promoting bacteria, which not only has strong rhizosphere colonization ability, but also can significantly promote plant growth and development. It has been reported that Trichoderma has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N1/15C12R1/885
CPCC12N1/14C12N9/78C12Y305/05001
Inventor 沈其荣刘东阳刘秋梅孟晓慧黄启为
Owner NANJING AGRICULTURAL UNIVERSITY
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