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Heat-resistant phytase, clone and expression of gene

A phytase and gene technology, applied in the field of phytase

Inactive Publication Date: 2006-07-19
TIANJIN NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Full sequence analysis of the target gene carried by positive clones

Method used

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  • Heat-resistant phytase, clone and expression of gene
  • Heat-resistant phytase, clone and expression of gene
  • Heat-resistant phytase, clone and expression of gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] 1. Source of materials

[0127] Biological enzymes: restriction endonucleases and ligases were purchased from Promega; Tag enzymes and DNA recovery kits were purchased from Shanghai Bioengineering Company;

[0128] Chemical reagents: calcium phytate and sodium phytate were purchased from Sigma Company, and IPTG was purchased from Lianxing Biological Company;

[0129] Medium: Gene Donor Bacteria Screening Medium: D-glucose 20%, Sodium phytate 0.01%, CaCl 2 0.2%, NH 4 NO 3 0.5%, KCl 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.001%, MnSO 4 ·H 2 O0.001%, agar 20%;

[0130] Others: primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd. and Shanghai Sangon Biotechnology Co., Ltd.; DNA sequence was determined by Dalian Baobio Co., Ltd. and United Gene Technology Co., Ltd.; E.coli JM109 and pMD18-T were purchased from Dalian Baobio Co., Ltd.

[0131] 2. Screening of natural strains producing heat-resistant phytase:

[0132] (1) Take 1 gram of soil sa...

Embodiment 2

[0141] Establishment of heat-resistant phytase expression system: The high-efficiency expression system E.Coli M15 was selected as the bioreactor for expressing heat-resistant phytase. The thermostable phytase gene that embodiment 1 obtains is excised from pMD18-TP, is connected on the pQE30 of expression vector again and forms recombinant DNA and is pQE30-P, see image 3 .

[0142] Pick a single colony of E.Coli M15 and inoculate it in LB (Km50μg / mlAp100μg / ml) medium, LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7; 250rpm, 37°C, shaker Incubate for 3 hours. Take it out and bathe in ice for 10 minutes, transfer it to a pre-cooled centrifuge tube, refrigerate and centrifuge, collect the precipitate, and wash it with cold CaCl 2 Solution washed pellet and resuspended in CaCl 2 In the solution, ice-bath for 30 minutes, add pQE30-P, ice-bath for 30 minutes, 42°C water bath for 1 minute and 30 seconds, ice-bath for 2 minutes, add fresh LB culture medium, culture at 37°...

Embodiment 3

[0145] Heat-resistant phytase characteristics: SDLiuTP01 was cultured in LB (Km25ug / ml, Ap50ug / ml) medium, 37°C, 250rpm, overnight. IPTG induces product formation. To measure the biological activity of heat-resistant phytase, the temperature range of the reaction is 25-95°C, all heat-resistant phytases have biological activity within this reaction temperature range, and the optimum reaction temperature is 65°C, see Figure 5 .

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Abstract

Heat resistant phytase and its gene clone and expression are disclosed to solve low heat resistance of the prior phytase, loss of its nutrition and waste on adding surplus nutrition. The technological scheme includes screening natural heat resistant strain of phytase, cloning its gene that has 1152 nucleotides, 383 encoding amino acids with 26 amino acids at the end N as signal peptides in total length, cut point of the signal peptides locating at rear of +26 lactamine, establishing engineering strain of heat resistant phytase gene (SDLiuTP01) to express gene products. The said phytase has biological activity at wide temperature range to promote plant growth, to increase soil fertility, not to damage crop quality, to degrate insoluble phosphorus in food and feed to increase use of phosphorus and to reduce organic phosphorous pollution.

Description

technical field [0001] The invention relates to a phytase, in particular to the cloning and expression of a phytase and its gene. Background technique [0002] For a long time, chemical fertilizers have been widely used in rural areas. Chemical fertilizers can increase soil fertility. However, with continuous use of chemical fertilizers, the efficiency of fertilizers decreases day by day, and the amount of fertilizer application has to be increased. Excessive application of chemical fertilizers will cause soil compaction, weaken the soil fertility, and damage the quality of crops. At the same time, it causes chemical residual pollution, pollutes the environment (such as eutrophication of water bodies), leads to a vicious circle, and endangers future generations. In the 1970s, the "Organic Agriculture Alliance" was established internationally, which called for the use of chemical fertilizers not to be used or to minimize the use of chemical fertilizers, and strongly advocate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21C12N9/16A01N37/18C05B21/00A23K1/00A23L1/305A01P21/00A23K20/189A23K50/00A23L33/17
Inventor 刘丽丽陈立新杨文博
Owner TIANJIN NORMAL UNIVERSITY
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