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Acyl coenzyme A oxidase 2 gene RKACOX2 and application thereof

A technology of oxidase and coenzyme, applied in the fields of genetic engineering and biology, can solve the problems of low activity of synthetic products, residual solvents and raw materials, and poor physiological functions.

Active Publication Date: 2021-02-26
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low activity of synthetic products, the products generally have solvent and raw material residues, which may have safety problems, so the use is limited; and the physiological functions of chemically synthesized carotenoids are worse than natural carotenoids, and are not easily absorbed by the human body; The extraction of carotenoids by biosynthesis is currently the most promising extraction method

Method used

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  • Acyl coenzyme A oxidase 2 gene RKACOX2 and application thereof
  • Acyl coenzyme A oxidase 2 gene RKACOX2 and application thereof
  • Acyl coenzyme A oxidase 2 gene RKACOX2 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: from Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) Isolation of the acyl-CoA oxidase 2 gene from YM25235 RKACOX2 The nucleotide sequence and the construction of its overexpression vector pRHRKACOX2

[0021] The total RNA of Rhodosporidium YM25235 was extracted using the UNlQ-10 Column Trizol Total RNA Extraction Kit (product number: SK1321) of Sangon Bioengineering (Shanghai) Co., Ltd., and then the PrimeScript ® RT reagent of the TaKaRa company kit was used to extract the total RNA. Kit With gDNA Eraser (Perfect Real Time) for reverse transcription to synthesize cDNA, take 0.5 μL as template for polymerase chain reaction, according to the results found in transcriptome sequencing RKACOX2 Sequence, design specific primers RKACOX2-F and RKACOX2-R (primer 1 and primer 2), use the cDNA template obtained above to carry out PCR amplification on a PCR instrument (BIOER company), primers, components and amplification conditions used in the reaction as...

Embodiment 2

[0027] Example 2: Analysis of the relationship between RKACOX2 gene and carotenoid synthesis in Rhodosporidium yeast

[0028] 1. Transform Rhodosporidium YM25235

[0029] The recombinant vector pRHRKACOX2 was transformed into Rhodosporidium YM25235 by PEG-mediated protoplast method, and the transformants were screened with YPD medium containing Hygromycin B (Hygromycin B) at a final concentration of 150 µg / mL, and then followed the Shanghai Genomic DNA of yeast transformants was extracted according to the steps in the instructions of the DNA extraction kit of Gongbio Engineering Co., Ltd., and then verified by PCR. The results are as follows: image 3 shown.

[0030] 2. Analysis of RKACOX2 gene and total carotenoid content of Rhodosporidium sp.

[0031] The overexpressed strain containing pRHRKACOX2 was cultured at 28°C to extract carotenoids, and compared with the Rhodosporidium strain transformed into the empty plasmid pRH2034, the total carotenoids were measured at 445nm ...

Embodiment 3

[0034] Example 3: Analysis of the relationship between RKACOX2 gene and genes related to carotenoid synthesis in Rhodosporidium yeast

[0035] The transgenic strain YM25235 / pRHRKACOX2 and the control strain YM25235 / pRH2304 were shaken and cultured on a constant temperature shaker at 30°C and 220rpm for 120h, and then the UNlQ-10 column Trizol total RNA extraction kit (product No.: SK1321) Total RNA of Rhodosporidium YM25235 was extracted, and then cDNA was synthesized by reverse transcription according to the TaKaRa kit PrimeScript ®RT reagent Kit With gDNA Eraser (Perfect Real Time), and the transgenic strain YM25235 / pRHRKACOX2 and the control strain YM25235 / pRH2304 Carotenoid Synthesis Related Genes RkCrtI , QUR transcript levels were analyzed. The 5.8SrRNA of Rhodosporidium YM25235 was used as an internal reference gene, and the genes related to carotenoid synthesis were used RkCrtI , QUR As the target gene, the transcription level of the corresponding gene of the ...

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Abstract

The invention discloses an acyl coenzyme A oxidase 2 gene RKACOX2, the nucleotide sequence of the acyl coenzyme A oxidase 2 gene RKACOX2 is shown as SEQ ID NO: 1, and the amino acid sequence coded bythe acyl coenzyme A oxidase 2 gene RKACOX2 is shown as SEQ ID NO: 2. The gene is separated from Rhodosporidium kratochvilovae YM25235, the gene is transferred into the Rhodosporidium kratochvilovae YM25235, experimental results show that overexpression of the RKACOX2 gene can cause improvement of the transcription level of the gene in cells to a certain extent, and overexpression of the RKACOX2 gene can promote synthesis of all components of total carotenoid and carotenoid. Microorganisms are modified through a genetic engineering means, the yield of microorganism carotenoid can be increased,and a foundation is laid for large-scale commercialized production of the carotenoid.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to an acyl-CoA oxidase 2 gene RKACOX2 And application thereof; Specifically relate to from yeast --- rhodosporidium ( Rhodosporidium kratochvilovae ) cloned the gene in YM25235, connected it with the carrier and transferred it into yeast cells to increase the expression level of the gene and ultimately promote the synthesis of carotenoids. Background technique [0002] Carotenoid is a general term for terpenoids and their derivatives composed of isoprenoid as the basic unit. So far, humans have discovered more than 600 natural carotenoids in nature; studies have shown that most carotenoids are compounds containing forty carbons, and their main chain is composed of 4 isoprene units. According to the molecular structure Different elements are divided into two categories: the molecular structure contains only two elements of carbon and hydrogen, such as β-carotene,...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/81C12N1/19C12P23/00C12R1/645
CPCC12N9/001C12Y103/03006C12N15/815C12P23/00
Inventor 张琦刘韬和美霞魏云林季秀玲
Owner KUNMING UNIV OF SCI & TECH
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