Family shuffling technology system for improvement of multiple genes related to phytase

A phytase and gene technology, applied in plant genetic improvement, genetic engineering, hydrolytic enzymes, etc., can solve the problems of low mutation rate, difficult target shape, and small mutation potential

Inactive Publication Date: 2009-07-01
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the deficiencies such as low mutation rate, small mutation potential, and difficulty in obtaining the target shape caused by shuffling of a single gene in the existing phytase transformation technology, and provides a technology for directional molecular evolution through gene in vitro system

Method used

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  • Family shuffling technology system for improvement of multiple genes related to phytase
  • Family shuffling technology system for improvement of multiple genes related to phytase
  • Family shuffling technology system for improvement of multiple genes related to phytase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: PAS methanol yeast preferred codon design and synthesis of AY136631

[0037] (1) Test method:

[0038] 1. Design and synthesis of genes and oligonucleotide primers

[0039] 1) Design a new nucleotide sequence AY136631 according to the phytase gene nucleotide sequence published by GenBank U59804 (refer to website: http: / / www.ncbi.nlm.nih.gov) and the preferred codons of Methylotrophic yeast.

[0040] 2) Design primers according to the new phytase nucleotide sequence AY136631. Primer synthesis was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0041] 3) Purify by PAGE referring to the molecular cloning literature method.

[0042] 2. Synthesis of phytase gene AY136631

[0043] The method for synthesizing genes is the PAS method (specific references Xiong AS, Yao QH, PengRH, Duan H, Li X, Fan HQ, Cheng ZM, Li Y.PCR-based accurate synthesis of long DNA sequences.Nat Protoc.2006; 1( 2): 791-797.).

[0044] The reaction program was ...

Embodiment 2

[0051] Example 2: Design and Synthesis of PAS Methanol Yeast Preference Codons for AF547224

[0052] (1) Test method:

[0053] 1. Design and synthesis of genes and oligonucleotide primers

[0054] 1) Design a new nucleotide sequence AF547224 according to the phytase gene nucleotide sequence published in the gene bank AY150806 (refer to website: http: / / www.ncbi.nlm.nih.gov) and the preferred codons of Methylotrophic yeast.

[0055] 2) Design primers according to the new phytase nucleotide sequence AF547224. Primer synthesis was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0056] 3) Carry out polyacrylamide gel electrophoresis (PAGE) according to the molecular cloning literature method, and recover the electrophoresis band of the primer with the target size.

[0057] 2. Synthesis of phytase gene AF547224

[0058] The method for synthesizing genes is the PAS method (specific references Xiong AS, Yao QH, PengRH, Duan H, Li X, Fan HQ, Cheng ZM, Li ...

Embodiment 3

[0066] Example 3: Design and synthesis of AF542235 by PAS methanolic yeast preference codon

[0067] (1) Test method:

[0068] 1. Design and synthesis of genes and oligonucleotide primers

[0069] 1) Design a new nucleotide sequence AF542235 according to the phytase gene nucleotide sequence published in the gene bank AB022700 (refer to website: http: / / www.ncbi.nlm.nih.gov) and the preferred codons of M. methanolica.

[0070] 2) Design primers according to the new phytase nucleotide sequence AF542235. Primer synthesis was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0071] 3) Purify by PAGE referring to the molecular cloning literature method.

[0072] 2. Synthesis of phytase gene AF542235

[0073] The method for synthesizing genes is the PAS method (specific references Xiong AS, Yao QH, PengRH, Duan H, Li X, Fan HQ, Cheng ZM, Li Y.PCR-based accurate synthesis of long DNA sequences.Nat Protoc.2006; 1( 2): 791-797.).

[0074] The reaction prog...

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Abstract

The invention relates to a system for modifying phytase through synthesis of a plurality of related phytase genes and combination of in vitro oriented molecular evolution. In the system, four different types of phytase genes are designed and synthesized according to methanol yeast favored codon, and a plurality of genes participate in family shuffling, so the system can obtain mutation potential which is larger than single-gene shuffling and mutation, and develops oriented molecular evolution research of the phytase and promotes the marketization process of the phytase in China through gene family shuffling and a high-flux screening system. The phytase can promote utilization of animal nutritive materials and reduce phosphorus emission, and has large potential in reducing environmental pollution, and large ecological benefit.

Description

technical field [0001] The invention relates to an enzyme preparation for green, environment-friendly and pollution-free feed industry, in particular to a gene in vitro directional molecular evolution technology system improved by a plurality of phytase-related genes, especially the technology system in the in vitro directional transformation of phytase Applications. Background technique [0002] Phytic acid (1.2.3.4.5.6-inositol hexaphosphate) widely exists in the plant kingdom, and phytic acid combines with phosphorus to form phytate phosphorus, which is a storage of phosphorus. Phytate phosphorus is a potentially useful phosphorus source, but it is difficult to be utilized by monogastric animals. Only 1 / 3 of the phosphorus in most plant feeds can be effectively utilized by poultry. In addition, phytate can bind to different cations and proteins, it reduces the bioavailability of proteins and important mineral nutrients such as calcium, zinc, magnesium, iron, etc. Phytic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16C40B40/08
Inventor 姚泉洪熊爱生彭日荷高峰付晓燕薛永李贤田永生赵伟
Owner SHANGHAI ACAD OF AGRI SCI
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