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Application of gene NgAUREO1 of nannochloropsis gaditana to adjustment and control of metabolism of fatty acid

A technology of Nannochloropsis and genes, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve problems such as genes that have not been proved by research

Inactive Publication Date: 2015-03-18
郑明刚 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no studies have demonstrated that the gene has other functions

Method used

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  • Application of gene NgAUREO1 of nannochloropsis gaditana to adjustment and control of metabolism of fatty acid
  • Application of gene NgAUREO1 of nannochloropsis gaditana to adjustment and control of metabolism of fatty acid
  • Application of gene NgAUREO1 of nannochloropsis gaditana to adjustment and control of metabolism of fatty acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cloning of Example 1 NgAUREO1 Gene

[0026] The aureochromel gene fragment was found from the Nannochloropsis transcriptome database established in our laboratory, and two pairs of gene-specific primers (5GSP1, 5GSP2 and 3GSP1, 3GSP2) were designed based on the fragment. Nannochloropsis RNA was extracted with TransZol Plant total RNA extraction kit, reverse transcribed with TransScriptRT reverse transcription kit to form cDNA, and the full-length NgAUREO1 gene was amplified by PCR. The primer sequences are as follows:

[0027] 5GSP1: 5'-GCTTCCTTGTTCAGTTCCTTCG-3'

[0028] 5GSP2: 5'-CAACCACTACCTCCGCCTCA-3'

[0029] 3GSP1: 5'-TACATCTGGCACTACGGGGC-3'

[0030] 3GSP2: 5'-GCTAACACCGACGCTCAAACT-3'.

[0031] The NgAUREO1 product amplified by PCR was purified and recovered, connected to the pMD19-T vector, and sent to Shanghai Sangong for sequence determination. Sequencing results showed that the ORF of the gene was 1398bp; the 5' non-coding region was 192bp; the 3' non-codi...

Embodiment 2

[0079] Example 2 Construction of yeast expression vector pYES2-NgAUREO1:

[0080] The NgAUREO1 gene was connected to the pMD19-T vector and named pT-NgAUREO1. Digest pT-NgAUREO1 with HindlII and SacI to recover small fragments, and at the same time digest pYES2 with HindlII and SacI to recover large fragments, connect the small fragments and large fragments, transform Escherichia coli competent cells and carry out PCR and enzyme digestion identification Finally, pYES2-NgAUREO1 was obtained.

[0081] During the operation, plasmid extraction, enzyme digestion, recovery, connection, cell transformation, enzyme digestion identification and PCR identification of recombinant plasmids were all carried out according to the steps described in Example 1.

Embodiment 3

[0082] Genetic Transformation of Example 3 Saccharomyces cerevisiae:

[0083] Preparation of yeast competent cells:

[0084] (1) Inoculate Saccharomyces cerevisiae into 100ml of YPD medium overnight at 30°C, 250rpm, until the density reaches 1×108cells / ml;

[0085] (2) Place the cells on an ice bath for 15 minutes to stop growth;

[0086] (3) Divide the cells into two sterilized 250ml centrifuge tubes, and centrifuge at 3000rpm at 4°C for 5 minutes;

[0087] (4) Remove the supernatant, and place the centrifuge tube on ice;

[0088] (5) Add 50ml of sterilized ice-cold water, vortex to resuspend, set the volume to 50ml in each centrifuge tube, and centrifuge at 3000rpm at 4°C for 5 minutes;

[0089] (6) Repeat operation according to (5) with 25ml of sterilized ice-cold water;

[0090] (7) Add 4ml of sterilized ice-cold 1M sorbitol to resuspend the cells and transfer to an iced 30ml centrifuge tube, centrifuge at 3000rpm at 4°C for 5 minutes, discard the supernatant;

[0091...

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Abstract

In the invention, a full-length blue-light induced bZIP type transcription factor (NgAUREO1) of 2080bp of nannochloropsis gaditana is cloned. The gene NgAUREO1 comprises an ORF of 1398bp, a 5'-end noncoding region of 192bp and a 3'-end noncoding region of 490bp and is capable of coding 465 amino acids. The gene NgAUREO1 comprises a bZIP structural domain and an LOV structural domain, wherein the bZIP structural domain can be combined with the promoter region of a specific gene to adjust and control the expression of the specific gene, and the LOV structural domain can sense the blue light of about 470nm, so that the gene NgAUREO1 has the characteristic of being capable of being adjusted and controlled by the blue light. The similarity between the sequence of the amino acid in the gene NgAUREO1 and the sequence of the amino acid in the gene aureochromel of vaucheria is 59%. A yeast expression vector of the gene NgAUREO1 is constructed, and the gene NgAUREO1 is introduced into saccharomyces cerevisiae to increase the content of oil in the yeast. The stimulation of the blue light to the recombinant saccharomyces cerevisiae proves that the blue light can serve as a switch to participate in adjusting and controlling the metabolism pathway of the fatty acid.

Description

1. Technical field: [0001] The invention relates to cloning a new blue-light-regulated transcription factor-related gene (NgAUREO1) with a coding region of 1398 bp from Nannochloropsis, and proves the new function of the gene in regulating fatty acid metabolism. 2. Background technology: [0002] With the increasingly severe energy crisis in the world today, people have gradually turned their attention from traditional petrochemical fuels to biomass energy. However, the level of biological oil content is one of the key issues affecting the development of biomass energy. Many scientists have done a lot of research on this issue. Some use organisms with relatively high oil content as raw materials, but still cannot meet the needs of biomass energy; It will inevitably lead to the reduction of biomass; there are also people who propose to transform the organism through genetic engineering so that it can accumulate a large amount of oil, but the experimental results are mostly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/405C12N15/81C12R1/89C12R1/865
Inventor 郑明刚黄一江王玲郑立李妍仝颜丽赵燕燕王春周晓丽
Owner 郑明刚
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