P-coumaric acid-3-hydroxylase as well as coding gene and application thereof
A technology for coumaric acid and hydroxylase, applied in the fields of biotechnology and plant biology, to achieve good substrate specificity and regioselectivity
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Embodiment 1
[0049] Embodiment 1, cloning of p-coumaric acid-3-hydroxylase LaC3H coding gene
[0050] The two primers synthesized respectively have the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing.
[0051] Using the cDNA obtained by reverse transcription of RNA extracted from Hudixiao as a template, PCR was performed using the above two primers SEQ ID NO: 3 and SEQ ID NO: 4. DNA polymerase was selected from Nanjing Nuoweizan Biotechnology Co., Ltd. Super-Fidelity DNA polymerase. The PCR amplification program was: 95°C for 5 min; 94°C for 45 s, 56°C for 45 s, 72°C for 2 min, a total of 30 cycles; 72°C for 10 min, then lowered to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results were as follows: figure 1 .
[0052] Under the irradiation of ultraviolet light, cut out the target DNA band. Then use the multifunctional DNA purification kit (spin column type) (Beijing Biotech Biotechnology Co., Ltd.) to recover the DNA fro...
Embodiment 2
[0054] Embodiment 2, the construction of the recombinant expression vector of LaC3H gene
[0055] (1) Synthesize two primers respectively having the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 7 in the sequence listing. Two restriction sites, NdeI and XhoI, and their protected base sequences were respectively set at the 5'-ends of the synthesized primers SEQ ID NO: 5 and SEQ ID NO: 7, and PCR amplification was performed using the cDNA of Hudixiao as a template. The PCR amplification procedure is the same as in Example 1. The PCR amplified product was detected by agarose gel electrophoresis, separated, gel-cut and recovered, and then digested with NdeI and XhoI, and then ligated with T4 DNA ligase from Takara Bioengineering (Dalian) Co., Ltd. (TaKaRa) and also passed NdeI and XhoI double enzymes. cut pET28a vector (Novagen). The ligation product was transformed into Escherichia coli (E.coli) DH5α (purchased from Nanjing Novizan Biotechnology Co., Ltd.) competent cells...
Embodiment 3
[0057] Embodiment 3, construction of LaC3H and LaC3H (ΔN29) functional expression vector
[0058] (1) Two primers respectively having the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9 in the sequence table were synthesized, and the CPR coding gene was amplified by PCR using the cDNA of Hudixiao as a template. The PCR amplification procedure is the same as in Example 1. PCR products were separated and recovered by agarose gel electrophoresis. Both ends of the recovered DNA fragments have 25 bp homologous sequences to both sides of the NdeI restriction site of the pET28a-LaC3H vector. The pET28a-LaC3H vector was linearized with NdeI restriction endonuclease (purchased from Bao Bioengineering (Dalian) Co., Ltd. (TaKaRa)). Use the One-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to introduce the CPR-encoding gene into the NdeI restriction site of the pET28a-LaC3H vector according to the product instructions, and use the universal sequencing p...
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