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Soybean trigonelline synthase as well as coding gene and application thereof

A technology that encodes genes and trigonelline, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of slow research progress and achieve the effect of wide application prospects

Inactive Publication Date: 2014-09-10
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Research on nicotinic acid N-methyltransferase at the molecular level has been slow, although as early as 2004 researchers had purified trigonelline synthase (also known as nicotinic acid-N-methyltransferase, Nicotinate-N-methyltransferase), and preliminary studies on the biochemical activity of the protein have been carried out, but so far there has been no report of its cDNA sequence information in any species

Method used

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  • Soybean trigonelline synthase as well as coding gene and application thereof
  • Soybean trigonelline synthase as well as coding gene and application thereof
  • Soybean trigonelline synthase as well as coding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1, the acquisition of GmTS protein and its coding gene

[0052] 1. The Trizol method was used to extract soybean (Glycine max cv Williams82) roots, cotyledons, stems, meristems, leaf buds, leaves, flowers, pods, pods and grains, and the total RNA of grains was extracted with GoScript TM ReverseTranscription System Reverse Transcription Kit were reverse transcribed into cDNA.

[0053] 2. Design and synthesize the following sequence:

[0054] GmTS F:5'-CCG GAATTC ATGGAGAAAGAGGAGAGCACGG-3' (SEQ ID No.1)

[0055] (The underlined sequence is the recognition site for EcoR I digestion)

[0056] GmTS R:5'-GC TCTAGA TCATTTCTGAAACTCAAGGACAGTGT-3' (SEQ ID No. 2)

[0057] (The underlined sequence is the Xba I restriction recognition site)

[0058] Actin F: 5'-ATCTTGACTGAGCGTGGTTATTCC-3' (SEQ ID No.3)

[0059] Actin R: 5'-GCTGGTCCTGGCTGTCTCC-3' (SEQ ID No.4)

[0060] 3. Use the cDNA of each soybean tissue in step 1 as a template, use Acitin F and Actin R as pri...

Embodiment 2

[0064] Embodiment 2, construction of recombinant plasmid pMal-GmTS

[0065] EcoR I and Xba I double-digest the DNA molecule shown in SEQ ID No.5 to obtain a gene fragment; EcoR I and Xba I double-enzyme digest the pMal-c2x vector to obtain a large vector fragment; connect the gene fragment to the large vector fragment, The recombinant plasmid was obtained, named pMal-GmTS, and pMal-GmTS was sent for sequencing, and the result was correct.

[0066] The structure of the recombinant plasmid pMal-GmTS is described as follows: between the EcoR I and Xba I restriction sites of the pMal-c2x vector, the 10th to 1077th nucleoside from the 5' end of SEQ ID No.5 is inserted The DNA molecule indicated by the acid, the plasmid expresses the GmTS-MBP fusion protein, and the expected molecular weight is about 80KD.

Embodiment 3

[0067] Embodiment 3, preparation of GmTS protein and enzyme activity analysis

[0068] 1. Construction of recombinant bacteria and control bacteria

[0069] The recombinant plasmid pMal-GmTS was introduced into Escherichia coli BL21(DE3)pLysS to obtain recombinant bacteria, and the pMal-c2x empty vector was introduced into Escherichia coli BL21(DE3)pLysS to obtain control bacteria.

[0070] 2. Preparation of GmTS protein

[0071] (1) Inoculate pMal-GmTS recombinant bacteria into LB Rich liquid medium (containing 100 μg / mL ampicillin), and culture at 37°C and 200 rpm until OD 600 =0.8, then add IPTG (to make its final concentration in the culture system 0.2g / L) and change the shaking culture condition to 16°C, and shake at 200rpm for 16 hours to obtain a culture solution.

[0072] (2) Centrifuge the culture solution to collect the bacterial cells.

[0073] (3) The cells were disrupted by ultrasonication, and the protein was purified by affinity chromatography using Amylose r...

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Abstract

The invention discloses a soybean trigonelline synthase (GmTS) as well as a coding gene and application thereof. The protein serving as a substrate of the trigonelline synthase has high specificity; Na<+>, K<+>, NH4<+>, Mg<2+>, Mn<2+> and Ca<2+> do not have significant influence on the enzyme activity; heavy metal ions such as Zn<2+>, Cu<2+>, Fe<2+> and Fe<3+> can significantly inhibit the enzyme activity; when the pH value of the protein is 6.0-7.0, the enzyme activity of the trigonelline synthase is relatively high. The protein is the one of the following (1) or (2): (1) protein shown by SEQ ID No. 6; and (2) proteins which are obtained by substituting and / or deleting and / or adding one or a few amino acid residues to an amino acid sequence shown by SEQ ID No. 6 and have the same function. The trigonelline synthase and the coding gene thereof have wide application prospect in the fields of soybean molecular assisted breeding, human health care and the like.

Description

technical field [0001] The invention relates to a trigonelline synthetase, its encoding gene and application, in particular to a soybean trigonelline synthetase, its encoding gene and application, and belongs to the field of biotechnology. Background technique [0002] Trigonelline is a product of N-methylation of Nicotinate (NA), which belongs to alkaloid compounds. Previous isotopic tracers have shown (mainly using 14 C-labeled nicotinamide, i.e. 14 C-NIM), trigonelline is widely present in higher plants, especially in the seeds of legumes, and in addition, the accumulation of trigonelline is also found in the seeds of crops such as rye, lettuce, sunflower and tomato. Studies have shown that in the cotyledons and hypocotyls of clover and alfalfa (legumes) 14 The main products of C-NIM feeding were trigonelline and part of the metabolites of the compensatory synthesis pathway of Nicotinamide adenine dinucleotide (NAD), and there were no other types of nicotinic acid deri...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/12
CPCC12N9/1007C12Y201/01007
Inventor 王国栋迟光红
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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