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Yeast recombinant bacterium and application thereof in reducing production of ammonia gas in caecum of laying hens

A technology of recombinant bacteria and yeast, applied in the biological field, can solve the problems of less probiotics and low ammonia emission reduction effect, and achieve the effects of easy cultivation, low cost, and reduced ammonia emission

Active Publication Date: 2021-04-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been a lot of basic research on the screening and cultivation of ammonia nitrogen degrading bacteria, there are few studies on probiotics applied to reducing ammonia emissions in animals, and the ammonia emission reduction effect is low

Method used

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  • Yeast recombinant bacterium and application thereof in reducing production of ammonia gas in caecum of laying hens
  • Yeast recombinant bacterium and application thereof in reducing production of ammonia gas in caecum of laying hens
  • Yeast recombinant bacterium and application thereof in reducing production of ammonia gas in caecum of laying hens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Construction of double-gene recombinant bacteria

[0021] (1) Vector selection and primer design: The yeast expression vector used is pPICZA. According to the multi-cloning site of the vector, the enzyme cutting sites that are not contained in the gdhA gene and glnA gene coding regions are used to determine the enzymes that can be added at both ends of the gene. Restriction sites. According to the gene sequences of gdhA gene and glnA gene, primers GDH(F), GDH(R) and GS(F), GS(R) were designed and synthesized by using Primer5 software. Add EcoR I and Kpn I restriction site sequences at the upstream and downstream of the gdhA gene, respectively, and add Kpn I and Not I restriction site sequences at the upstream and downstream of the glnA gene.

[0022] Table 1: Synthetic Primer Information

[0023]

[0024] (2) Using Enterococcus faecium and Bacillus coagulans as templates, using GDH(F) and GDH(R) as primers to synthesize the gdhA gene sequence, and usi...

Embodiment 2

[0035] Example 2: Functional detection of the double-gene recombinant bacteria constructed in Example 1

[0036] The double-gene recombinant bacteria obtained in Example 1 were inoculated in the YPDA liquid medium containing Zeocin, and cultivated for 1 day at 30° C. on a shaker at 200 rpm; Cultivate in a bottle at 30°C with a 200rpm shaker until the OD600 is 2-6; transfer the bacterial solution to a 50mL sterilized centrifuge tube, and collect the bacterial cells by centrifugation; transfer the collected bacterial cells to a 500mL Add methanol to the Erlenmeyer flask so that the concentration of methanol is 1%, and cultivate at 30° C. on a shaker at 200 rpm for 144 hours. Samples were taken every 24 hours and methanol was added to ensure a final concentration of 1%. After induced expression, the double-gene recombinant bacteria can effectively express glutamate dehydrogenase and glutamine synthetase, and the molecular weights of the expressed products are about 50kDa and 55k...

Embodiment 3

[0039] Example 3: The effect of the double-gene recombinant bacteria constructed in Example 1 on reducing ammonia emissions from the cecum of laying hens

[0040] Using 33-week-old Roman powder laying hens as the test material, and laying hen feed (Table 2) as the fermentation substrate (purchased from Huizhou Lefu Agricultural Technology Co., Ltd.), in vitro fermentation verification double-gene recombinant bacteria simulated the effect of laying hens. Effects of ammonia gas production in the cecal environment.

[0041] Table 2: Component composition and nutritional level of laying hen feed

[0042]

[0043] The test set up a blank group (CK), original bacteria group, empty bacteria group, gdhA gene recombinant bacteria, glnA gene recombinant bacteria and double gene recombinant bacteria groups, and each test group had 6 replicates (Table 3). The added bacterial strains are yeast original bacteria after induced expression, empty bacteria, gdhA gene recombinant bacteria, gln...

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Abstract

The invention discloses a yeast recombinant bacterium and application thereof in reducing production of ammonia gas in the cecum of laying hens. The yeast recombinant bacterium is obtained by transforming a recombinant expression vector containing a gdhA gene and a glnA gene into a pichia pastoris GS115 strain. The glnA gene in bacillus coagulans and the gdhA gene in enterococcus faecium are introduced into pichia pastoris through a genetic engineering means to construct a double-gene recombinant bacterium; and the double-gene recombinant bacterium can efficiently express glutamate dehydrogenase and glutamine synthetase, and can reduce 75% of ammonia gas emission in the cecum of laying hens. The yeast recombinant bacterium is low in cost and easy to culture, has the advantages of no residue, no side effect, no resistance and the like, and has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a yeast recombinant bacterium and its application in reducing the caecal ammonia gas production of laying hens. Background technique [0002] About 70% of the odor produced by livestock and poultry houses comes from the cecum fermentation and excrement fermentation of livestock and poultry. Therefore, regulating the intestinal microorganisms of livestock and poultry is the main means to reduce the emission of livestock and poultry body odor. Probiotics have the advantages of low cost, simple operation, non-toxic, harmless and pollution-free, high safety, and no resistance, and are widely used in livestock and poultry production. Although there have been a lot of basic research on the screening and cultivation of ammonia nitrogen degrading bacteria, there are few studies on probiotics applied to reduce ammonia emission in animals and the ammonia emission reduction effect ...

Claims

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Application Information

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IPC IPC(8): C12N1/19A23K50/75A23K10/18A01K67/02C12R1/84
CPCC12N9/0016C12Y104/01002C12N9/93C12Y603/01002A23K50/75A23K10/18A01K67/02
Inventor 王燕冯坤娴王玮吴银宝米见对
Owner SOUTH CHINA AGRI UNIV
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