Method for improving calcium and phosphorus utilization ratio of livestock and poultry

A utilization rate, calcium and phosphorus technology, applied in the biological field, can solve the problems of application and promotion limitation, high temperature resistance, high cost of phytase, and achieve the effects of broad application prospects, low cost and simple use.

Inactive Publication Date: 2015-06-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of producing phytase is relatively high, and it is not resistant to high temperature in feed production, which limits its application and promotion.

Method used

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  • Method for improving calcium and phosphorus utilization ratio of livestock and poultry
  • Method for improving calcium and phosphorus utilization ratio of livestock and poultry
  • Method for improving calcium and phosphorus utilization ratio of livestock and poultry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Carrier construction

[0028] Use conventional PCR technology to amplify the phytase full-length gene of Escherichia coli (its nucleotide sequence is shown in SEQ ID No.1) and add AgeI and SacII restriction site sequences at both ends respectively, and the sequence is correct The PCR product was digested with restriction endonucleases AgeI and SacII, and connected to the AgeI and SacII restriction vector pMDEAAssA (constructed by the inventor's laboratory based on the pBR322 vector, containing the mic2 promoter of Eimeria tenella, Drug screening gene DHFR-TS3m, reporter gene yellow fluorescent protein EYFP, actin promoter and 3' regulatory sequence of Eimeria tenella, signal peptide sequence of Toxoplasma gondii dense granule protein 8, its nucleotide sequence is shown in SEQ ID No.2), and then identify the positive clones obtained. The correctly identified recombinant plasmid was extracted to obtain the constructed coccidian transfection vector pMDEA-phytase (its n...

Embodiment 2

[0039] In steps 2 and 3 of Example 1, we compared the relationship between the number of serial passages and obtaining the transgenic coccidia stably expressing phytase. After the coccidial oocysts of each generation are collected, they are purified and sporulated, and their luminous efficiency is detected under a fluorescent microscope. The calculation formula is: luminous rate = sporulated oocysts expressed by yellow fluorescent protein / all sporulated oocysts ×100%. We found that the luminous efficiencies of the 4th and 5th generations were 54% and 83% respectively, and when the drug pressure screened to the 6th generation, 100% of the transgenic coccidia expressing the yellow fluorescent reporter protein could be obtained, and the subsequent serial passages (7- 9 passages) still expressed 100% fluorescent protein (see image 3 ). Through the identification of step 3 in Example 1, it was confirmed that continuous drug pressure passage for 6 generations is the optimal con...

Embodiment 3

[0041] In a transgenic coccidian comparison test, we compared the calcium and phosphorus utilization effects of the commercialized recombinant phytase and the transgenic coccidia obtained by applying the present invention.

[0042]The 1-day-old AA broiler chickens were randomly divided into blank control group, phytase addition group and transgenic coccidia use group (10 chickens in each group). The feed is full-price pellet feed without phytase (entrusted to Beijing Huadu Feed Co., Ltd. for small-scale production). In the phytase addition group, phytase 5000 (produced by Su Kehan ​​Bioengineering Co., Ltd.) was added to the feed throughout the whole process, and the addition amount was 600g / kg. The chickens of the transgenic coccidia group were orally inoculated with the transgenic Eimeria mellifera expressing phytase at the age of 1 day, and the inoculation dose per chicken was 5×10 3 Sporulated oocysts. Determination of calcium and phosphorus content in feces during the p...

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Abstract

The invention provides a method for improving the calcium and phosphorus utilization ratio of livestock and poultry. The method comprises the following steps: (1) obtaining full-length DNA of a phytase gene; (2) preparing a coccidium transfection vector pMDEA-phytase; (3) performing transfection on the vector and screening transgenic coccidia; and (4) inoculating the transgenic coccidia for expressing phytase into bodies of the livestock and poultry in an oral administration manner to ensure that the coccidia express and release phytase while developing in intestinal tracts, and degrading phytic acid. By adopting the method provided by the invention, phytase genes with different sources are expressed in the coccidia for the first time, so that a recombinant micro biological reactor is obtained and can generate phytase directly applied to the livestock and poultry, and equipment for production and purification and related operations are not needed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for expressing a phytase gene in poultry so as to improve the utilization rate of calcium and phosphorus in poultry. Background technique [0002] Phytic acid serves as the major storage form of phosphorus in various plant tissues (especially seeds), where it exists as calcium, magnesium, and potassium salts. Livestock and poultry (chicken, cattle, sheep, pigs, etc.) feeds with corn, wheat and other grains as the main components contain a large amount of phytic acid. The presence of phytic acid greatly reduces the amount of calcium and phosphorus available in the feed. However, poultry (chicken, duck, etc.) and non-ruminant animals (pig, rabbit, etc.) do not have normal flora expressing phytase in their intestines and cannot digest phytic acid. Therefore, in order to improve the utilization efficiency of calcium and phosphorus in feed, it is necessary to Use phytase to deg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/55C12N9/16A01K67/033A23K1/165
Inventor 刘贤勇索勋罗雪索静霞田秀玲秦梅汤新明
Owner CHINA AGRI UNIV
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