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Optimized α-galactosidase gene aga-f75 suitable for expression in maize and its application

A technology of galactosidase and aga-f75, which is applied in the field of genetic engineering and can solve the problems that the properties of the enzyme cannot meet the requirements and the molecular weight of the protein is large.

Active Publication Date: 2017-12-08
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies on the isolation of various α-galactosidase resources and the recombinant expression of microorganisms have been reported. However, due to the large molecular weight of most enzymes and the unsatisfactory enzyme characteristics, there is no recombinant expression of this enzyme in plants. Reported, therefore, the present invention has successfully carried out the expression in maize through gene modification, vector selection, tissue-specific expression, etc.

Method used

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  • Optimized α-galactosidase gene aga-f75 suitable for expression in maize and its application
  • Optimized α-galactosidase gene aga-f75 suitable for expression in maize and its application
  • Optimized α-galactosidase gene aga-f75 suitable for expression in maize and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of α-galactosidase gene expression vector

[0050] 1) Synthesis of α-galactosidase gene aga-F75m

[0051]The alpha-galactosidase gene aga-F75 (FJ392036) was obtained from Gibberella sp. F75. The codon optimization of aga-F75 makes it more suitable for expression in maize. BamHI and AvrII restriction sites were introduced at both ends of the gene, and cloned into pUC57MCS pellet.

[0052] 2) Gene digestion and ligation

[0053] pUC57-aga-F75m and expression vector pHP20754 were cut with BamHI and AvrII. Using New England Biolabs' T 4 Ligase joins the two, constructing pHP20754-aga-F75m, see figure 1 . Digest pHP20754-aga-F75m with PvuII to obtain the α-galactosidase gene expression cassette fragment, see figure 2 .

[0054] The selection marker gene expression vector pHP20754Bar was digested with HindIII, XhoI and SacI to obtain the selection marker gene expression cassette fragment.

Embodiment 2

[0055] Embodiment 2: Maize transformation mediated by gene gun

[0056] The gene gun transformation method and the preparation of the medium were carried out with reference to the method in "Plant Cell, Tissue and Organ Culture: Basic Methods". After shooting, the recipient material was transformed into positive T0 generation plants through several stages of recovery culture, screening stage, pre-differentiation culture and differentiation into seedlings. Then it was crossed with the commercial line Zheng 58 to obtain T1 generation seeds and offspring.

Embodiment 3

[0057] Example 3: Molecular Detection of Regenerated Genetic Maize Plants

[0058] 1. Molecular detection

[0059] Genomic DNA was extracted from regenerated maize plants. And the regenerated maize plants were detected by PCR.

[0060] Target gene primers: F75m-F (5'-CGTTGAGCTGGACCCATCGGATC-3') and 20754-398R (5'-TTCCTGGCAAATCACTCGGTGTATC-3');

[0061] Internal reference gene primers: AC326F (5'-ATGTTTCCTGGGATTGCCGAT-3') and AC326R (5'-GCATCACAAGCCAGTTTAACC-3'). Positive plants were screened out, see image 3 .

[0062] 2. Southern blot analysis of regenerated corn plants, the results are as follows Figure 4 shown.

[0063] 3. SDS-PAGE and Western blot analysis of regenerated corn plants

[0064] The culture supernatant after transformation of yeast GS115 with pIC9γ empty vector and the extract of Zheng 58 seeds were reacted with goat anti-rabbit IgG serum. Diluted purified primary antibodies were obtained after dialysis against PBS and acetate buffer.

[0065] Take ...

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Abstract

The present invention relates to the field of genetic engineering, in particular to an optimized α-galactosidase gene aga-F75 suitable for expression in maize and its application. The nucleotide sequence of the gene is shown in SEQ ID NO.2. Aiming at the importance of α-galactosidase in production, the present invention utilizes the advantages of high efficiency, simplicity, and environmental protection of transgenic plants compared with microbial fermentation production, and focuses on technical difficulties such as large molecular weight and difficulty in recombinant expression. and vectors, tissue-specific high-efficiency expression, etc., the first study of using transgenic plants to express the enzyme has obtained genetically stable plant materials.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an optimized alpha-galactosidase gene aga-F75 suitable for expression in maize and its application. Background technique [0002] Soybean meal and other soy cakes are widely used as the most important protein raw materials in animal feed, mainly because of their high protein content and wide variety of sources. However, because these bean cakes usually contain many anti-nutritional factors, the ratio of their application in feed is limited. Among them, the oligosaccharides in soybeans of the flatulence factor are mainly oligosaccharides such as α-galactoside. α-galactoside is a class of substances of different lengths formed by a sucrose unit (fructose-glucose) and multiple galactose units connected by α-1,6-glycosidic bonds, such as raffinose and stachyose , Mullein sugar, etc. Monogastric animals lack α-galactosidase and cannot decompose these substances. When these subst...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/40C12N15/82A01H5/10A23K10/30
Inventor 姚斌杨培龙张宇宏杨文霞孟昆袁建华陈茹梅孟庆长张伟周晓今
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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