Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof
An acetyltransferase, acetyl coenzyme technology, applied in the directions of acyltransferase, transferase, application, etc., can solve the problems of complex process, low biological activity and high extraction cost
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Embodiment 1
[0017] Embodiment 1: from Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) Acetyl-CoA acetyltransferase gene isolated from YM25235 RKAcaT2 Nucleotide sequence
[0018] 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) was used for reverse transcription to synthesize cDNA, and 0.5 μL was used as a template for polymerase chain reaction. According to the RKAcaT2 sequence found in the transcriptome sequencing, specific primers RKAcaT2-F and RKAcaT2-R ( Primer 1 and primer 2), carry out PCR amplification on the PCR instrument (BIOER company) with the cDNA template obtained above, the primers, components and amplification conditions used in the reaction are as follows:
[0019] Primer 1: RKAcaT2-F: 5'-TCA...
Embodiment 2
[0025] Example 2: Construction of overexpression vector pRHRKAcaT2
[0026] Using the reverse-transcribed YM25235 cDNA as a template, RKAcaT2-F and RKAcaT2-R were used as primers to amplify the coding sequence of RKAcaT2, and the obtained RKAcaT2 fragment was about 1200bp in size. Noc I. Eco After RV two restriction endonucleases were digested, it was connected to the expression vector pRH2034 to obtain the recombinant plasmid pRHRKAcaT2 ( figure 2 ). The obtained recombinant plasmid was transformed into Escherichia coli DH5α for amplification, and then verified by colony PCR to extract the recombinant plasmid and use Noc I. Eco RⅤ carried out double enzyme digestion verification on pRHRKAcaT2. The results showed that the recombinant plasmid pRHRKAcaT2 produced two bands of about 1.2 kb and 10.7 kb after double enzyme digestion (the third lane in Figure 3), which were respectively related to RKAcaT2 The size of the fragment was the same as that of the pRH2034 vector ...
Embodiment 3
[0027] Example 3: RKAcaT2 Effect of Gene Overexpression on Carotenoid Synthesis in Rhodosporidium YM25235
[0028] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235
[0029] The recombinant plasmid pRHRKAcaT2 was transformed into Rhodosporidium YM25235 by the Agrobacterium-mediated method, and the transformants were selected with the YPD medium containing Hygromycin B (Hygromycin B) at a final concentration of 150 µg / mL, and then the Shanghai Sangon Bioengineering The steps in the instructions of the DNA Extraction Kit of Co., Ltd. extract the genomic DNA of the yeast transformant, and then perform PCR verification. The results are as follows: Figure 4 shown.
[0030] 2, RKAcaT2 Analysis of Carotenoid Content in Gene Overexpressed Rhodosporidium YM25235
[0031] The overexpressed strain YM25235 / pRHRKAcaT2 containing pRHRKAcaT2 was cultured at 30°C for 144h to extract carotenoids, and the Rhodosporidium strain YM25235 / pRH2304 transformed into the empty pla...
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