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Aptamer for identifying zeatin through specifity, and screening method and application of aptamer

A nucleic acid aptamer and zeatin technology, applied in biochemical equipment and methods, microbial measurement/testing, organic chemistry, etc., can solve problems such as small molecular weight

Active Publication Date: 2012-10-17
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] 7) Small molecular weight, generally 4-50KD

Method used

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  • Aptamer for identifying zeatin through specifity, and screening method and application of aptamer
  • Aptamer for identifying zeatin through specifity, and screening method and application of aptamer
  • Aptamer for identifying zeatin through specifity, and screening method and application of aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1, the screening of zeatin nucleic acid aptamer

[0096] 1. Fixation of L-histidine and zeatin

[0097] 0.5 g of epoxy-activated Sepharose 6B (GE Healthcare, Sweden) microspheres were repeatedly washed with 100 mL of water to obtain 1.75 mL of wet spheres, which were then washed with 0.2 M Na 2 CO 3 (pH≈12) to replace the water in the swollen microspheres, add 45mM L-histidine or amino-modified trans-zeatin (see below for the synthesis steps) to the 3.5mL reaction system, shake and react at room temperature for 48h . After the reaction, use pH4.5 sodium acetate buffer solution (0.1M sodium acetate, 0.5M NaCl) and pH12 sodium carbonate buffer solution (0.2M NaHCO 3 / Na 2 CO 3 , 0.5M NaCl; that is, use 0.2M NaCl 2 CO 3 Add HCl to the solution to adjust the pH to 12, and at the same time make the concentration of NaCl 0.5M) Repeatedly wash the microspheres alternately, finally wash with water, and dilute to 3.5mL, store in a refrigerator at 4°C.

[0098]...

Embodiment 2

[0141]Embodiment 2, the specific detection of zeatin nucleic acid aptamer

[0142] 1. Nucleic acid aptamer pretreatment

[0143] 0.5 μM 6-carboxyfluorescein (6-FAM)-labeled aptamer 2 (sequence 2) was dissolved in binding buffer, denatured at 95°C for 5 minutes, cooled on ice for 15 minutes, and left at room temperature for 5 minutes.

[0144] 2. Nucleic acid aptamer specific detection

[0145] Mix 5 μL of zeatin microspheres (microspheres immobilized with trans-zeatin obtained in step 1 in Example 1) with 1 mM zeatin (cis-zeatin (CZ), trans-zeatin (TZ), dihydrogen Zeatin (DZ), trans-zeatin nucleoside (ZR)) and its analogues (adenine (A), adenosine (AR), adenosine monophosphate (AMP), adenosine triphosphate (ATP), ethanolamine ( AA), 2-methallyl alcohol (MA)) was added to the aptamer solution pretreated in step 1 (zeatin and its analogues were used as aptamer competitors), and incubated at room temperature 30min, heated at 95°C for 5min with the elution buffer, eluted the DN...

Embodiment 3

[0147] Embodiment 3, the preparation of nucleic acid probe

[0148] The following four sets of nucleic acid probes were synthesized by Shanghai Bioengineering Company:

[0149] Nucleic acid probe A: composed of two DNA fragments (DNA fragment A-1 and DNA fragment A-2); DNA fragment A-1 is nucleic acid aptamer 1 (sequence 1), and its 5' end has 6-FAM ( Fluorescent reporter group), BHQ1 (fluorescent quencher group) at the 3' end, referred to as the aptamer element; DNA fragment A-2 is a partial reverse complementary sequence of the nucleic acid aptamer 1 (sequence 9), referred to as the complementary element;

[0150] Nucleic acid probe B: composed of two DNA fragments (DNA fragment B-1 and DNA fragment B-2); DNA fragment B-1 is nucleic acid aptamer 1 (sequence 1), and its 5' end has 6-FAM ( Fluorescent reporter group), referred to as aptamer element; DNA fragment B-2 is a partial reverse complementary sequence of aptamer 1 (SEQ ID NO: 10), and its 3' end has BHQ1 (fluorescent ...

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Abstract

The invention discloses an aptamer for identifying zeatin through specifity, and a screening method and an application of the aptamer. The aptamer is a single-chain DNA (deoxyribonucleic acid) segment shown by any one from sequence 1 to sequence 8 in a sequence table. The aptamer and the nucleic acid probe based on the aptamer can identify the zeatin through specifity, have better affinity for trans-zeatin and trans-zeatin riboside than cis-zeatin and dihydro zeatin, and are used for detecting whether materials to be detected contain the zeatin and the content of the zeatin. The method has the advantages of being simple, sensitive, rapid and special in operation and low in cost, and the method has high application value on the aspects of separating, enriching and detecting the zeatin.

Description

technical field [0001] The invention relates to a nucleic acid aptamer specifically recognizing zeatin, a screening method and application thereof. Background technique [0002] Zeatin is the first natural cytokinin isolated and identified, and it is the most common cytokinin in plants. In the process of plant life, it can promote cell division, bud formation, axillary bud growth, xylem division, seed germination, root and leaf growth, inhibit stem elongation, prevent aging, maintain nucleic acid, protein and chloroplast content, Enhance the ability to resist high and low temperature, accelerate the movement of nutrients and assimilated substances and other important physiological functions. [0003] Zeatin cytokinins include cis-zeatin, trans-zeatin, dihydro-zeatin and their nucleosides and nucleotide derivatives, among which trans-zeatin and its nucleosides have the highest biological activity. At present, the detection methods of zeatin include GC-MS, LC-MS and ELISA. ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/11C12Q1/68C07D473/34C12N15/10
Inventor 上官棣华乞萃刘祥军邴涛
Owner INST OF CHEM CHINESE ACAD OF SCI
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