Croceic acid glycosyltransferase and coding gene thereof and application

A technology of glycosyltransferase and crocetin, applied in the direction of glycosyltransferase, transferase, application, etc., can solve the problem of inability to obtain high-purity crocetin glycosyltransferase

Inactive Publication Date: 2016-07-20
ECONOMIC CROPS RES INST XINJIANG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But up to now, it is still impossible to obtain high-purity crocetin glycosyltransferase

Method used

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  • Croceic acid glycosyltransferase and coding gene thereof and application
  • Croceic acid glycosyltransferase and coding gene thereof and application
  • Croceic acid glycosyltransferase and coding gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, the preparation of recombinant crocetin acid glycosyltransferase

[0074] 1. Construction of recombinant plasmids

[0075] 1. The total RNA of the saffron suspension cells was extracted by the Trizol method, and the first-strand cDNA was reverse-transcribed from the total RNA by reverse transcriptase.

[0076] 2. Synthetic primer F1: 5'-C CATATG GAGCAGAAAGATGAGAACGGAA-3' (the underline is the recognition sequence of restriction endonuclease NdeI) and R1: 5'-C GTC GAC TTTACAACAATGATCAATGAACTCCT-3' (the underline is the recognition sequence of restriction endonuclease SalI).

[0077] 3. Using the cDNA obtained in step 1 as a template and F1 and R1 synthesized in step 2 as primers, perform PCR amplification to obtain a PCR amplification product of about 1388 bp. Detected by 1% agarose gel electrophoresis, the results are shown in figure 1 (M is DNAMarker, lane 1 is the PCR amplification product, and the arrow points to the target PCR amplification produ...

Embodiment 2

[0140] Example 2. Using HPLC to detect the glycosyltransferase activity of recombinant crocetin glycosyltransferase under different reaction conditions

[0141] 1. Dialysis

[0142]At 4°C, the recombinant crocetin glycosyltransferase solution obtained in Example 1 was dialyzed to desalt, then replaced into pH 7.5, 5mM Tris-HCl buffer, and finally ultrafiltration was performed using an ultrafiltration tube with a molecular weight of 30MW. The protein was concentrated by filtration to obtain a protein sample of the recombinant crocetin glycosyltransferase with a purity of 98%. The protein sample was sequenced for the 25 amino acid residues at the N-terminus, and the results showed that the sequence of the 25 amino acid residues at the N-terminus of the protein was as shown in positions 1 to 25 from the N-terminus of Sequence 4 in the sequence listing.

[0143] 2. Using HPLC to detect the glycosyltransferase activity of recombinant crocetin glycosyltransferase under different re...

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Abstract

The invention discloses croceic acid glycosyltransferase and a coding gene thereof and application.The croceic acid glycosyltransferase is shown as a1 or a2 or a3 or a4; a1, the amino acid sequence refers to protein shown as the sequence 2 in a sequence table; a2, the amino acid sequence refers to fused protein obtained after the end N or / and C of the protein shown as the sequence 2 in the sequence table is / are connected with a label; a3, the amino acid sequence refers to protein shown as the sequence 4 in the sequence table; a4, the amino acid sequence refers to protein with the activity of the croceic acid glycosyltransferase, and the protein is obtained after the protein shown as a1 or a2 or a3 is subjected to substitution and / or deletion and / or addition of one or more amino acid residues.Experiments prove that the croceic acid glycosyltransferase has the activity of glycosyltransferase with croceic acid as a substrate, the croceic acid can be subjected to sequential glycosylation to form crocin glycoside 5 and crocin glycoside 3, and high application value is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a crocetin glycosyltransferase, its coding gene and application. Background technique [0002] Saffron is also called crocus and saffron, which is the dry stigma of the perennial Iridaceae plant saffron (Crocussativus L.), and is a kind of famous and precious Chinese medicine in my country. Crocetin is a pigment compound extracted from saffron. Its main components are crocus, crocetin and its derivatives. Crocetin is a series of ester glycosides formed by combining crocetin with different sugars. , is one of the main active ingredients of saffron, has anti-platelet aggregation, anti-thrombosis, anti-myocardial ischemia, anti-cancer and other pharmacological effects, and almost no toxic side effects. [0003] Existing studies have shown that crocetin glycosyltransferase has the function of glycosylation crocetin to form crocin. For example, in 2004, Morgan et al. isolated...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/44
CPCC12N9/1051C12P19/44C12Y204/01271
Inventor赖成霞
OwnerECONOMIC CROPS RES INST XINJIANG ACAD OF AGRI SCI