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Phytoene dehydrogenase gene RKcrtI and application thereof

A technology of phytoene and dehydrogenase, which is applied to the phytoene dehydrogenase gene RKcrtI and its application field, and can solve the problems of unseen gene sequence disclosure and the like

Inactive Publication Date: 2019-03-29
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no disclosure of gene sequences related to the present invention

Method used

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  • Phytoene dehydrogenase gene RKcrtI and application thereof
  • Phytoene dehydrogenase gene RKcrtI and application thereof
  • Phytoene dehydrogenase gene RKcrtI and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: From Rhodospora yeast ( Rhodosporidiumkratochvilovae ) The nucleotide sequence of phytoene dehydrogenase RKcrtI, an enzyme related to carotenoid synthesis isolated from YM25235

[0017] The total RNA of Rhodosporidium YM25235 was extracted with the UNlQ-10 column Trizol total RNA extraction kit (product code: SK1321) of Sanggong Bioengineering (Shanghai) Co., Ltd., and then the PrimeScript ®RT reagent Kit With gDNA Eraser (Perfect Real Time) was used for reverse transcription to synthesize cDNA. 0.5μL was used as a template for polymerase chain reaction. According to the RKcrtI sequence found in transcriptome sequencing, specific primers RKcrtI-F and RKcrtI-R ( Primer 1 and Primer 2), using the cDNA template obtained above, perform PCR amplification on a PCR machine (BIOER). The primers, amplification system and amplification conditions used in the reaction are as follows:

[0018] Primer 1: RKcrtI-F: 5`-CGGGATCCATGAACGGCCACGCCAAG-3` (SEQ ID NO: 3)

[0019] Primer...

Embodiment 2

[0024] Example 2: RKcrtI Construction of gene overexpression vector pRHRKcrtI

[0025] Using reverse-transcribed YM25235 cDNA as a template, using RKcrtI-F and RKcrtI-R as primers to amplify the coding sequence of RKcrtI, RKcrtI The size of the fragment is about 1600bp, and the amplified RKcrtI Fragment Bam HⅠ, Eco After the two restriction enzymes of RⅤ were digested, they were ligated to the expression vector pRH2304 to obtain the recombinant plasmid pRHRKcrtI ( figure 2 ); Transform the obtained recombinant plasmid into E. coli DH5α for amplification, and then extract the recombinant plasmid after colony PCR verification, and use Bam HⅠ, Eco RⅤ double-enzyme digestion of pRHRKcrtI was verified; the results showed that the recombinant plasmid pRHRKcrtI produced two bands of about 1.6 kb and 10.7 kb after double-enzyme digestion (Figure 3, lane 3). These two bands were respectively the same as the RKcrtI fragment. The fragment size is the same as the fragment size of pRH2304 ...

Embodiment 3

[0026] Example 3: RKcrtI Effect of gene overexpression on β-carotene synthesis in Rhodosporidium YM25235

[0027] 1. Agrobacterium-mediated transformation of Rhodosporidium yeast YM25235

[0028] The recombinant plasmid pRHRKcrtI was transformed into Rhodosporidium yeast YM25235 using Agrobacterium-mediated method, and the transformants were selected using YPD medium containing hygromycin B (HygromycinB) at a final concentration of 150 µg / mL, and then according to Shanghai Shenggong Bioengineering The steps in the instructions of the DNA extraction kit of Co., Ltd. extract the genomic DNA of yeast transformants, and then perform PCR verification as follows: Figure 4 Shown. From Figure 4 Knowable RKcrtI The sequence has been integrated into the YM25235 genome. Lane 3 shows that two bands can be obtained from PCR amplification, one is the genome fragment containing the intron sequence of the strain itself, and the other is the transformed integrated cDNA coding sequence fragment. R...

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Abstract

The invention discloses a phytoene dehydrogenase gene RKcrtI having the nucleotide sequence shown in SEQ ID NO:1; the amino acid sequence encoded by the gene is shown in SEQ ID NO:2. The gene is derived from rhodosporidium kratochvilovae YM25235; the gene is connected with a vector and then is transferred into yeast cells. The rhodosporidium kratochvilovae YM25235 can be promoted to produce beta-carotenoid, and a foundation is laid for large-scale commercial production of beta-carotenoid.

Description

Technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to a phytoene dehydrogenase gene RKcrtI , Which is derived from Rhodosporidium ( Rhodosporidium kratochvilovae ) The gene cloned in YM25235 is directly connected to the vector and transferred into yeast cells to increase the expression level of this gene and ultimately promote the synthesis of carotenoids. Background technique [0002] As a class of fat-soluble pigments with important functions, carotenoids are widely distributed in animals, plants, fungi, algae and bacteria in nature. They have high nutritional value and have anti-cancer, anti-oxidation, and anti-cardio-cerebrovascular diseases effects. Therefore, it has been widely used in food, feed, health care products and cosmetics industries. Although many researchers have done a lot of research on how to increase the yield of carotenoids, the yield and quality of carotenoids are still far from meeting people's ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/81C12P23/00C12R1/645
CPCC12N9/001C12P23/00C12Y103/99029
Inventor 张琦胡丽魏云林季秀玲
Owner KUNMING UNIV OF SCI & TECH
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