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4-coumarate coenzyme a ligase originated from ornithogalum caudatum, nucleotide sequence, and applications thereof

A technology of coumaric acid and ligase, applied in the field of genetic engineering, can solve problems such as low yield, environmental pollution of chemical synthesis, and shortage of resources

Inactive Publication Date: 2014-12-17
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Facing the problems of resource shortage, serious environmental pollution of chemical synthesis, and low yield, it is not conducive to the development of social medical and health services and the improvement of people's health. Synthetic systems are crucial to the sustainable development of innovative drugs

Method used

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  • 4-coumarate coenzyme a ligase originated from ornithogalum caudatum, nucleotide sequence, and applications thereof
  • 4-coumarate coenzyme a ligase originated from ornithogalum caudatum, nucleotide sequence, and applications thereof
  • 4-coumarate coenzyme a ligase originated from ornithogalum caudatum, nucleotide sequence, and applications thereof

Examples

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Embodiment 1

[0049] Example 1. Transcriptome Sequencing and Sequence Analysis

[0050] After extracting the total RNA from the sterile bulb of Dieffenbachia tiger eye, using the mRNA as a template, the first cDNA strand was synthesized with six base random primers (random hexamers), and then the second strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I After the cDNA chain is purified by QiaQuick PCR kit and eluted with EB buffer, the end is repaired, poly (A) is added, and the sequencing adapter is connected, and then the size of the fragment is selected by agarose gel electrophoresis, and finally PCR amplification is performed. The built sequencing library was used with Illumina HiSeq TM 2000 for sequencing.

[0051] The original image data obtained by sequencing is converted into sequence data through base calling, that is, raw data or raw reads. Perform data filtering on raw reads, remove reads with adapters, duplicates, and low-quality sequencing, and obtai...

Embodiment 2

[0053] Example 2 Osa4CL bioinformatics analysis

[0054] Using NCBI's ORF Finder (http: / / www.ncbi.nlm.nih.gov / gorf / orfig.cgi) software to analyze the ORF of SEQ ID NO.3, it was found that it contained a 1638bp 4- The coumaric acid-CoA ligase ORF is named Osa4CL, and its sequence is shown in SEQ ID NO.2. Using the tool ProtParam in Expasy (http: / / web.expasy.org / protparam / ) to predict the physicochemical properties of the protein encoded by Osa4CL, the results show that the Osa4CL protein encodes 545 amino acids with a molecular mass of 59.5332kDa and a theoretical isoelectric point of 5.37. Analysis by TMpred (http: / / www.ch.embnet.org / software / TMPRED_form.html) shows that the protein has two transmembrane domains, which are composed of 26 amino acids from 82 to 107. The domain from the inside to the outside of the membrane and the domain from the outside to the inside of the membrane composed of positions 232 to 257. Therefore, it is considered that the encoded protein is li...

Embodiment 3

[0057] Embodiment 3 Osa4CL gene cloning

[0058] Take 100mg of aseptic bulbs of Dieffenbachia tiger's eye, freeze them quickly in liquid nitrogen, grind them into a fine powder with a mortar, and extract total RNA from the callus of Dieffenbachia tiger's eye according to the instructions of RNeasy Plant Mini Kit (QIAGEN). RT-PCR Kit (ReverTra-Plus-, TOYOBO) was used to reverse transcribe the total RNA from the callus of Dieffenbachia tiger's eye into cDNA. The reverse transcription system and procedure are as follows: Add 1 μg of total RNA to 20 μL system, RNase Free H 2 O4 μL, Olig(dT) 20 1 μL, after incubating at 65°C for 5 minutes, immediately place it on ice to cool, then add 4 μL of 5×RT buffer, 1 μL of RNase Inhibitor (10U / μL), 1 μL of dNTP Mixture (10mM) and ReverTra Ace to the above tube, and incubate at 30°C for 10 minutes , incubated at 42°C for 60 minutes, denatured at 85°C for 5 minutes, and placed on ice for 5 minutes to complete cDNA synthesis. The cDNA was st...

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Abstract

The invention provides a 4-coumarate coenzyme A ligase originated from ornithogalum caudatum, and the amino acid sequence of the ligase is represented by the SEQ ID No.1. The invention further provides a nucleotide sequence, which encodes the gene of the 4-coumarate coenzyme A ligase and is represented by the SEQ ID No.2, a carrier containing the nucleotide sequence and a host cell. The invention also provides an application of 4-coumarate coenzyme A ligase or cells containing 4-coumarate coenzyme A ligase on catalyzing substrates such as p-coumaric acid, caffeic acid, ferulic acid, cinnamic acid, and the like into corresponding thioester compounds.

Description

technical field [0001] The invention relates to a 4-coumaric acid coenzyme A ligase, its coding gene and its application in catalytic substrate p-coumaric acid, caffeic acid, ferulic acid and cinnamic acid as corresponding thioester compounds, belonging to genetic engineering field. In particular it relates to Dietweed 4-coumarate-CoA ligase, its coding gene and its use in catalyzing the substrates p-coumaric acid, caffeic acid, ferulic acid and cinnamic acid as the corresponding thioester compounds Background technique [0002] Flavonoid natural products are a class of compounds with a wide variety, significant activity and complex structure. They are the direct or indirect sources of many clinical drugs, and are also widely used in industrial and agricultural production. Facing the problems of resource shortage, serious environmental pollution of chemical synthesis, and low yield, it is not conducive to the development of social medical and health services and the improve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N1/21C12N1/19C12N5/10C12P19/32
CPCC12N9/93C12P19/32C12Y602/01012
Inventor 孔建强陈茜王志标李丽娜王伟郭磊程克棣
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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