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A kind of 3-phosphate glyceraldehyde dehydrogenase and its application

A technology of glyceraldehyde phosphate dehydrogenase and triglyceride, applied in the field of glyceraldehyde 3-phosphate dehydrogenase, can solve the problems of high cost of animal and plant cultivation, long growth cycle and the like, and achieve the effect of improving the ability of microorganisms to resist acid stress

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the growth cycle of animals and plants is too long and the cultivation cost of animals and plants is relatively high, only by extracting polyunsaturated fatty acids (PUFAs) from these animals and plants can no longer meet the growing market demand. Methods to increase production of polyunsaturated fatty acids (PUFAs)

Method used

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  • A kind of 3-phosphate glyceraldehyde dehydrogenase and its application
  • A kind of 3-phosphate glyceraldehyde dehydrogenase and its application
  • A kind of 3-phosphate glyceraldehyde dehydrogenase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Screening of genes encoding MaGAPDHA and MaGAPDHB

[0068] Specific steps are as follows:

[0069] In NCBI, the gapdh gene sequences with identified functions in different species were selected as templates (Table 1), and BLAST comparison was performed in the gene bank of the sequenced M.alpinaATCC 32222 strain to obtain alternative target genes; The selected genes were compared and screened for the second time in the NCBI library, and the finally obtained target genes were named Magapdha (nucleotide sequence as shown in SEQ ID No.3) and Magapdhb (nucleotide sequence as shown in SEQ ID No.4) ), and the corresponding proteins are named MaGAPDHA (amino acid sequence shown in SEQ ID No.1) and MaGAPDHB (amino acid sequence shown in SEQ ID No.2).

[0070] In order to further determine whether the screened MaGAPDHA and MaGAPDHB belong to GAPDH enzymes, they were compared with the amino acid sequences of five typical GAPDHs in Table 1, which were derived from anima...

Embodiment 2

[0075] Example 2: Cloning of Magapdha and Magapdhb

[0076] Specific steps are as follows:

[0077] The total RNA of Mortierella alpina (Mortierella alpina) ATCC 32222 was extracted using the Trizol method, and cDNA was obtained by reverse transcription according to the instructions of the Takara reverse transcription kit. The reaction amplifies Magapdha and Magapdhb, and the primers used for amplifying Magapdha and Magapdhb are shown in Table 2.

[0078] The PCR instrument used is BIO-RAD T100 Thermal Cycler, using KOD plus high-fidelity DNA polymerase, the reaction system is 50 μL, and the content of the system is carried out according to the instructions of the DNA polymerase; the reaction process is as follows: 95°C for 3min, 95°C for 30s, 55°C for 30s , 68°C 1min 5s, 30 cycles, 68°C 5min.

[0079] After the reaction is completed, the amplified product is obtained, and after the amplified product is purified, the band size of the amplified product is verified by 1% agaro...

Embodiment 3

[0082] Example 3: Expression of Magapdha and Magapdhb in Mortierella alpina

[0083] Specific steps are as follows:

[0084] (1) Construction of Mortierella alpina expression vector

[0085] Such as figure 1 , the Magapdha obtained in Example 2 and the expression vector pBIG2-ura5s-ITs were digested with the restriction endonuclease Hind III, and the digested product was digested with the restriction endonuclease Sac I. 4The ligase connects the digested products that have been digested by HindIII and SacI successively to obtain the ligated product;

[0086] The Magapdhb obtained in Example 2 and the expression vector pBIG2-ura5s-ITs were digested using restriction endonuclease Hind III, and the digested product was digested using restriction endonuclease Sac I. 4 The ligase connects the digested products that have been digested by HindIII and SacI successively to obtain the ligated product;

[0087] Among them, HindIII digestion system (20 μL): 2 μL HindIII-FD, 2 μg plasmi...

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Abstract

The invention discloses a glyceraldehyde-3-phosphate dehydrogenase and applications thereof, and belongs to the technical field of genetic engineering and microbial engineering. The glyceraldehydes-3-phosphate dehydrogenase with an amino acid sequence as shown in SEQ ID No.1 has the function of helping microorganisms produce triacylglycerol (TAG). By culturing the recombinant Mortierella alpina containing the glyceraldehydes-3-phosphate dehydrogenase in a shaking table for 7 days, the total fatty acid content and the polyunsaturated fatty acid content of the Mortierella alpina containing the glyceraldehydes-3-phosphate dehydrogenase can be increased by 12.56% and 12.79% respectively, compared with the Mortierella alpina containing no glyceraldehydes-3-phosphate dehydrogenase. The result provides abundant theoretical support for further improving the capability of oil-producing microorganisms such as Mortierella alpina to produce triacylglycerol (TAG) and further improving the capability of microorganisms to produce polyunsaturated fatty acid (PUFAs) through a genetic engineering means.

Description

technical field [0001] The invention relates to a 3-phosphoglyceraldehyde dehydrogenase and application thereof, belonging to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing two or more double bonds and a carbon chain length of 18-22 carbon atoms. Because it has the function of reducing cardiovascular and cerebrovascular diseases and plays a very important role in maintaining health and preventing diseases, polyunsaturated fatty acids (PUFAs), as a health product, have a huge domestic and international reputation. market. [0003] At present, most of the polyunsaturated fatty acids (PUFAs) on the market are derived from aquatic phytoplankton, and the viscera of deep-sea fish such as wild cod, herring, and salmon that eat such plants are also rich in polyunsaturated fatty acids (PUFAs) . However, because the growth cycle of animals and plan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/80C12N1/21C12N1/15C12P7/64C12R1/19C12R1/01C12R1/645
CPCC12N9/0008C12N15/70C12N15/80C12P7/6427C12Y102/01012
Inventor 陈海琴陈卫王顺贤唐鑫赵建新张灏陈永泉
Owner JIANGNAN UNIV
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