Gene RKcrtYB and applications thereof

A technology of gene and gene coding, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of low biological activity, low cost of carotenoids, isomerization, etc.

Inactive Publication Date: 2019-04-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Carotenoids are extremely unstable under the conditions of oxygen, high temperature, light, and strong acid, and are easy to deteriorate, degrade, or isomerize. The usual storage method is to store in isolation from air, low temperature, and dark, because it is relatively stable in an alkaline environment , so the industry usually uses alkaline saponification treatment to extrac

Method used

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  • Gene RKcrtYB and applications thereof
  • Gene RKcrtYB and applications thereof
  • Gene RKcrtYB and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) gene of YM25235 RKcrtYB clone

[0017] The total RNA of Rhodosporidium YM25235 was extracted using the OMEGA kit E.Z.N.A Fungal RNA Kit, followed by the instructions of the TaKaRa kit PrimeScript ®RT reagent Kit With gDNA Eraser (Perfect RealTime), the reverse transcription reaction was used to synthesize cDNA, and 0.5 μL was used as a template. Polymerase chain reaction, based on the RKcrtYB sequence found in the transcriptome sequencing; design specific primers (primer 1 and primer 2) according to the transcriptome sequence of Rhodosporidium YM25235 for PCR amplification; primers, components and amplification used in the reaction Addition conditions are as follows:

[0018] Primer 1: RKcrtYB-F: 5'-CATG CCATGG ATGGGCGGACTGGAC-3' (SEQ ID NO: 3)

[0019] Primer 2: RKcrtYB-R: 5'-CTC GATATC TCACAGCGCCCTCCACA-3' (SEQ ID NO: 4)

[0020] ( CCATGG for Nco Ⅰ Restriction site, GATATC for Eco RV...

Embodiment 2

[0024] Example 2: Construction of RKcrtYB Overexpression Recombinant Expression Plasmid pRHRKcrtYB

[0025] Using cDNA in Example 1 as a template to carry out PCR amplification, the primers used in the reaction, the reaction system and the amplification conditions are as follows:

[0026] Primer 1: RKcrtYB-F: 5'-CATG CCATGG ATGGGCGGACTGGAC-3' (SEQ ID NO: 3)

[0027] Primer 2: RKcrtYB-R: 5'-CTC GATATC TCACAGCGCCCTCCACA-3' (SEQ ID NO: 4)

[0028] ( CCATGG for Nco Ⅰ Restriction site, GATATC for Eco RV restriction site)

[0029] The PCR amplification system is as follows (50 μL):

[0030]

[0031] The plasmid pRH2304 double enzyme digestion system is as follows (50 μL):

[0032]

[0033] QUR The fragment double enzyme digestion system is as follows (50 μL):

[0034]

[0035] PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C ...

Embodiment 3

[0036] Example 3: RKcrtYB Effects of Gene Overexpression on β-Carotene Synthesis in Rhodosporidium YM25235

[0037] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

[0038] The recombinant plasmid pRHRKcrtYB was transformed into the Rhodosporidium YM25235 strain by the Agrobacterium-mediated transformation method, and the transformants were selected with the YPD medium containing the final concentration of Hygromycin B (HygromycinB) at a final concentration of 150 µg / mL; Genomic DNA of yeast transformants was extracted according to the steps in the instructions of DNA Extraction Kit of Engineering Co., Ltd., and then verified by PCR as follows: Figure 4 shown; from Figure 4 It can be seen RKcrtYB The sequence has been integrated into the YM25235 genome. Lane 3 shows that PCR amplification can obtain two bands, one is the genomic fragment containing the intron sequence of the strain itself, and the other is the transformed and integrated cDNA coding se...

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PUM

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Abstract

The invention discloses a gene RKcrtYB. The gene is isolated from rhodosporidium kratochvilovae YM25235. The nucleotide sequence of the gene is as shown in SEQ ID NO:1, and the amino acid sequence encoded by the gene is as shown as EDQ ID NO:2. In the present invention, the gene is linked into a vector and transferred into the YM25235, and the translated protein is involved in the synthesis of beta-carotenoids. The gene can enable the rhodospora erythraea YM25235 to improve the synthesis of the beta-carotenoids, and lays a foundation for the large-scale commercial production of the beta-carotenoids.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering in the field of biotechnology, in particular to Rhodosporidium kratochvilovae ) cloned from YM25235 RKcrtYB Then connect the gene to the carrier and transfer it into YM25235, and use it to regulate the synthesis of key enzymes in the synthesis of carotenoids in YM25235, and then can produce a large amount of β-carotenoids, which is high-yield and high-quality β-carotenoids Provide a reference for the industrial production of element. Background technique [0002] Carotenoids are a class of polyene compounds that widely exist in nature. They are red, orange or yellow lipophilic isoprenoid pigments produced by plants and some photosynthetic microorganisms. The existence of the isoprene palace distructure has strong light absorption, so there is generally an absorption peak between the wavelength of 430-570nm. At present, more than 700 kinds of carotenoids have been found in nature. ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/39C12N15/81C12P23/00
CPCC07K14/39C12N15/815C12P23/00
Inventor 张琦巩江世琪魏云林季秀玲
Owner KUNMING UNIV OF SCI & TECH
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