Neutral phytase transgenic recombinant lactobacillus and product and application thereof

A lactobacillus and gene recombination technology, applied in the application, recombinant DNA technology, bacteria and other directions, can solve the problems of waste of phosphorus resources, low enzyme activity per unit, difficult to use in monogastric animals, etc., to increase economic benefits, reduce residues, Metabolism-boosting effect

Inactive Publication Date: 2012-02-01
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the seeds of grains, beans, and oil crops, the basic storage form of phosphorus is phytate phosphorus, which accounts for 60%-80% of the total phosphorus in plants[3], but it is difficult to be utilized by monogastric animals, resulting in great Problem: First is the waste of phosphorus resources (Ehrlich KC, Montalbano B G, Mullaney E J, et al. Identification and cloning of a second phytase gene (phyB) from Aspergillus niger (ficuum) . Biochemical and Biophysical Research Communications, 1993, 195:53 ~57.)
[0011] At present, aci

Method used

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  • Neutral phytase transgenic recombinant lactobacillus and product and application thereof
  • Neutral phytase transgenic recombinant lactobacillus and product and application thereof
  • Neutral phytase transgenic recombinant lactobacillus and product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Example 1: Construction of recombinant lactobacillus that secretes and expresses neutral phytase

[0187] 1. Cloning of neutral phytase gene

[0188] Using the Bacillus subtilis genome as a template, using P1 and P2 as primers to amplify the phy gene (see attached image 3 ), there is a specific band at about 1150bp. The purified product was ligated with pMD18-T to obtain the plasmid pMD-phy, and its enzyme digestion and PCR identification results are shown in the appendix Figure 4 (XbaI / SmaI double digestion can produce a band of about 2700bp and a band of about 1150bp, and SmaI single digestion can produce a band of about 3850bp).

[0189] 2. Cloning of signal peptide sp gene of Lactobacillus pumilus

[0190] Lactobacillus pumilus L.brevis1.2028 genome was used as template, and sp gene was amplified with P3 and P4 as primers (see attached Figure 5 ), there is a specific band at about 90bp. The purified product was ligated with pMD18-T to obtain the pl...

Embodiment 2

[0195] Embodiment 2: Experiment of the influence of recombinant lactobacillus of the present invention on pig production performance

[0196]1. Selection, grouping and feeding management of experimental animals

[0197] The experiment was carried out in the same double-row closed piggery, and all the methods of free feeding and drinking were adopted.

[0198] Select 90 Du×Chang×Da Sanyuan cross-growing pigs about 20kg in weight, half male and half female, castration and epidemic prevention according to routine. The initial weight was recorded, and they were randomly divided into 3 groups, with 3 repetitions in each group and 10 pigs in each repetition. The treatments of each group were as follows: Group I (control group), basal diet + 0.10% secondary meal; Non-recombinant lactobacillus preparation (wherein 30% calcium hydrogen phosphate is replaced with 15% calcium carbonate+15% bran, the same below); Group III (recombinant lactobacillus group): basal diet (remove 30% hydrog...

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Abstract

The invention relates to a neutral phytase transgenic recombinant lactobacillus and a product and application thereof. Neutral phytase genes in AY220075 Bacillus subtilis with lactobacillus signal peptides are integrated in a genome, and an amplified sequence is a 1-1152bp basic group; signal peptide genes are 260-349th site basic groups of Z14250; and the neutral phytase genes and the signal peptide genes are inserted into multiple cloning sites after being connected and are integrated into ribosome binding sites of Lactobacillus casei L.CECT5276. The neutral phytase transgenic recombinant lactobacillus provided by the invention can be used as a feed additive for pig husbandry, can make up the defect that the intestinal tract of a pig cannot secrete phytase, can promote the metabolism of phytate phosphorus in a feedstuff raw material and reduce discharge of inorganic phosphorus, which are favorable for the protection of the environment. A host bacteria lactobacillus also has beneficial function to animal bodies, and has important application value in production of pig, poultry and other monogastric animals.

Description

technical field [0001] The invention relates to a gene recombination technology, in particular to a neutral phytase gene-transferred lactobacillus and its product and application. Background technique [0002] Phytase (Phytase) is myo-inositol hexaphosphate phosphor-hydrotase, which catalyzes the hydrolysis of phytic acid (ie, phytic acid) and phytate into inositol and phosphoric acid (or phosphate). A general term for a class of enzymes. The chemical name for phytic acid is cyclohexyl hexaphosphate. The molecular formula is C6H18O24P6, and the structural formula is C6H6 [OPO(OH) 2 ] 6 , it is a light yellow or tan viscous liquid. Soluble in water, ethanol, acetone, almost insoluble in benzene, chloroform, ether and hexane. Although phytic acid itself has little toxicity, it has strong chelating ability, so it has anti-nutritional effect. It will interact with a variety of metal ions such as Zn in the gastrointestinal tract of animals 2+ , Ca 2+ 、Cu 2+ , Fe 2+ Pla...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A23K1/16A23K1/18C12R1/125C12R1/24C12R1/245A23K10/18
Inventor 丁轲赵振升钟社普王国永余祖华白东英周变华孔涛
Owner HENAN UNIV OF SCI & TECH
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