Zinc finger protein transcription factor gene RkMSN4 and application thereof
A technology of transcription factors and zinc finger proteins, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as the lack of polyunsaturated fatty acids
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Embodiment 1
[0015] Embodiment 1: Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) YM25235 zinc finger protein transcription factor RKMSN4 gene cloning
[0016] The total RNA of Rhodosporidium YM25235 was extracted with the OMEGA kit E.Z.N.A Fungal RNA Kit, and the cDNA was synthesized with the reverse transcription kit Takara First Strand cDNA Synthesis Kit. According to the transcriptome sequence of Rhodosporidium YM25235, specific primers (primer 1 and primer 2) were designed for PCR amplification; the primers, components and amplification conditions used in the reaction were as follows:
[0017] Primer 1: RkMSN4-F: 5'-AAT GGATCC ATGGCGCCTGCTCCCCGT-3' (SEQ ID NO: 3)
[0018] Primer 2: RkMSN4-R: 5'-ATC GATATC TCACGGCATCGCCCACACCT-3' (SEQ ID NO: 4)
[0019] ( GGATCC for Bam H I restriction site, GATATC for Eco RV restriction site)
[0020] The PCR amplification system is as follows (50 μL):
[0021]
[0022] PCR amplification conditions were: 95°C pre-denaturat...
Embodiment 2
[0023] Example 2: Construction of recombinant expression plasmid pRH2034RkMSN4
[0024] The cDNA measured in Example 1 is used as a template to carry out PCR amplification, and the reaction primer combination, reaction components and amplification conditions are as follows:
[0025] Primer 1: RkMSN4-F: 5'-AAT GGATCC ATGGCGCCTGCTCCCCGT-3' (SEQ ID NO: 3)
[0026] Primer 2: RkMSN4-R: 5'-ATC GATATC TCACGGCATCGCCCACACCT-3' (SEQ ID NO: 4)
[0027] ( GGATCC for Bam H I restriction site, GATATC for Eco RV restriction site)
[0028] The PCR amplification system is as follows (50 μL):
[0029]
[0030] PCR amplification conditions were: 95°C pre-denaturation for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 3 min, and finally 10 min at 72°C. The gene fragment obtained by the above PCR and the expression vector pRH2034 were used Bam H I and Eco Perform double digestion with RV endonuclease, recover the digeste...
Embodiment 3
[0031] Example 3: RKMSN4 Genes and Polyunsaturated Fatty Acid Synthesis Experiments in Low Temperature Environment
[0032] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235
[0033] The recombinant plasmid pRH2034RkMSN4 was transformed into Rhodosporidium YM25235 strain by Agrobacterium-mediated transformation method, and the transformants were selected with YPD medium containing Hygromycin B (HygromycinB) at a final concentration of 150 μg / mL; Genomic DNA was extracted to verify the transformants by PCR .
[0034] 2, RKMSN4 Analysis of Genes and Changes of Fatty Acid Content in Rhodosporidium Yeast
[0035] After the transgenic strain YM25235 / pRH2034RkMSN4 and the control strain YM25235 / pRH2304 were first cultured at 30°C for 24 h, one was cultured at 30°C for 24 h, and the other was immediately transferred to 15°C for cold treatment for 24 h; The total fatty acids in bacterial cells are methylated. The sample was carried out to gas chromatographic ana...
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