Orotidine-5'-phosphate decarboxylase gene, and protein and use thereof

A technology of phosphate decarboxylase and orotic acid, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that Rhodosporidium toruloides has not been publicly reported, and achieves improved competitiveness, increased production intensity, and reduced production costs Effect

Active Publication Date: 2009-05-27
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Description
  • Claims
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Information about the orotidine-5′-phosphate decarboxylase ge

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  • Orotidine-5'-phosphate decarboxylase gene, and protein and use thereof
  • Orotidine-5'-phosphate decarboxylase gene, and protein and use thereof
  • Orotidine-5'-phosphate decarboxylase gene, and protein and use thereof

Examples

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Embodiment 1

[0041] Embodiment 1: Extraction of Rhodosporidium toruloides AS2.1389 total RNA

[0042] Inoculate fresh Rhodosporidium toruloides AS 2.1389 into 50ml YEPD liquid medium from a slant, culture on a shaker at 30°C for 12 hours, and then transfer the bacterial liquid to 100ml YEPD liquid culture at a volume ratio of 1:25 culture medium at 30°C for 12 hours to reach the logarithmic growth phase. Centrifuge at 4000 rpm for 5 min at 4°C to collect the cells, freeze the cells quickly with liquid nitrogen, and grind to break the wall. Total RNA was extracted using the RNAiso kit from TaKaRa Company and following its standard procedures.

[0043] The RNA was electrophoresed on a 1.5% agarose gel, observed and identified using a fluorescence-ultraviolet analyzer, and two clear bands ( figure 1 ). Analyze the total RNA sample with a UV / Vis spectrometer and measure the OD 260 / OD 280 =2.03, indicating good quality of total RNA. Total RNA samples were frozen at -80°C for later use. ...

Embodiment 2

[0044] Example 2: Reverse transcription synthesis of Rhodosporidium toruloides AS 2.1389 cDNA first strand and degenerate PCR

[0045] Using the total RNA of Rhodosporidium toruloides AS 2.1389 as a template, the first strand of cDNA was synthesized by reverse transcription. First, 1.0 μl RNA (about 2 μg), 1.0 μl primer SMART IV: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ and 1.0 μl primer CDSIII / 3′: 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30N-1 N-3′, 2.0 μl of DEPC-treated water (diethyl pyrocarbonate-treated water, purchased from Dalian TaKaRa Company), was added to the PCR tube and mixed evenly, kept at 72°C for 2 minutes, immediately placed on ice for 2 minutes, and 2.0 μl 5×first strand buffer, 1.0 μl DTT (20 mM), 1.0 μl dNTP (10 mM), 1.0 μl powerscript reverse transcriptase (Clontech Company) were added to the system, and mixed. Extend the reaction at 42°C for 60 minutes, and end the reaction at 4°C, and store it at -20°C for later use.

[0046] Design and synthesize tw...

Embodiment 3

[0047] Example 3: Obtaining the full-length cDNA of Rhodosporidium toruloides AS2.1389 ura3

[0048]1. According to the ura3 cDNA intermediate sequence cloned in Example 2, design primers 5'-NUP: 5'-AAGCAGTGGTATCAACGCAGAGT-3' and ura3-GSP-anti: 5'-ATGCCCCGACCGACGATGATAACG-3', synthesized in Example 2 The first strand of cDNA was used as a template, and 5'-RACE was performed with reference to the BD SMART RACE cDNA Amplification kit operation manual (www.bdbiosciences.com) provided by Clontech, to obtain a PCR product of about 0.9 kb ( figure 2 B); Design primer ura3-GSP-sense: 5'-GGCACACATCACCAACGCCCACCT-3' and CDSIII / 3'PCR primer (Example 2), with the first strand of cDNA synthesized in Example 2 as a template, with reference to the one provided by Clontech BD SMART RACE cDNA Amplification kit operation manual (www.bdbiosciences.com) was used for 3'-RACE to obtain a PCR product of about 0.6 kb ( figure 2 C). The PCR amplification product was recovered according to the ope...

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Abstract

The invention relates to an orotidine-5'-phosphoric acid decarboxylase gene separated from rhodosporidum toruloides and coding albumen and a recombined vector thereof. The rhodosporidum toruloides AS2.0389 total RNA is taken as a template, the degeneracy PCR and RACE methods are used, Rt-ura3 and cDNA sequences which are as shown in SEQ ID No:1 are obtained by separation, a genome DNA sequence is as shown in SEQ ID No:3, and an amino acid sequence of the coding albumen is as shown in SEQ ID No:2. The obtained vector pYES2/CT-Rt-ura3 can be used for compensating biosynthesis way of pyrimidine of auxotroph distillers yeast BY4741, and makes constructed engineering strain normally grow in a culture medium without containing uracil. When the Rt-ura3 gene is used as auxotroph marker gene or counter selection marker gene, a new yeast genetic operation system and a new recombined engineering strain can be constructed.

Description

technical field [0001] The present invention relates to the orotidine-5'-phosphate decarboxylase encoding gene ura3, specifically the cloning of the orotidine-5'-phosphate decarboxylase gene of Rhodosporidium toruloides, the construction of a recombinant vector, and Applications in genetic engineering research. Background technique [0002] Living organisms are organisms composed of biological macromolecules such as nucleic acids and proteins. Among them, nucleotides are the constituent units of nucleic acids, which are widely distributed in the body and have various biological functions. The normal biosynthesis of nucleotides is the prerequisite for the maintenance of life characteristics. In the absence of remedial pathways, the abnormal or suspension of nucleotide biosynthesis will lead to the loss of life characteristics of living organisms. [0003] [0004] As shown in the figure above, the de novo biosynthesis of yeast pyrimidine nucleotides uses glutamine as a r...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/65
Inventor 张素芳赵宗保杨帆
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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