Nucleotide sequence, molecular probe and method for identifying taxus media seedling

A technology of Taxus mandia and nucleotide sequences, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as subjective discrimination bias and morphological differences, and achieve the method Simple, high-sensitivity, short-time results

Active Publication Date: 2015-04-01
HANGZHOU NORMAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

Moreover, the shape characteristics are easily affected by habitat, climate, physiological conditions, etc., which often lead to deviations in subjective identification, and it is difficult to accurately identify them only by morphological differences.
Therefore, in the past many years, the seedlings of Taxus chinensis or Taxus chinensis were mistaken for the seedlings of Taxus chinensis and planted from time to time, which brought considerable economic losses to agricultural production.

Method used

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  • Nucleotide sequence, molecular probe and method for identifying taxus media seedling
  • Nucleotide sequence, molecular probe and method for identifying taxus media seedling
  • Nucleotide sequence, molecular probe and method for identifying taxus media seedling

Examples

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Embodiment 1

[0018] Example 1 : Preparation of characteristic nucleotide sequence of Taxus media

[0019] 1. Genomic DNA extraction

[0020] Cut 200 mg of the fresh leaves of Taxus chinensis seedlings and put them in a mortar, immediately add liquid nitrogen to grind to powder, and then use the UNIQ-10 column-type plant genome DNA extraction kit of Shanghai Shenggong Bioengineering Co., Ltd. to extract the yew plant genome For DNA, use 1% agarose gel for electrophoresis detection of the obtained DNA, and use an ultraviolet spectrophotometer to detect the DNA concentration, and dilute to 50ng / μl.

[0021] 2. SCoT-PCR reaction, electrophoresis detection

[0022] SCoT universal primer 3 (5’-CAACAATGGCTACCACCG-3’) was used for PCR amplification. The primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd. The reaction system was: 2μl 10×Buffer, 2μl MgCl 2 (25mM), 0.8μl dNTPs (10mM), 1μl SCoT primer 3 (10μM), 1μl template DNA (50ng / μl), 0.4μl Taq enzyme (2U / μl), 12.8μl ddH 2 O. The t...

Embodiment 2

[0027] Example 2: Preparation of MHSF / MHSR specific nucleotide probes for Taxus media, PCR amplification, and electrophoresis detection

[0028] On the basis of obtaining the specific nucleotide sequence of Taxus media, using Primer Primer 5.0 software to design the nucleotide sequence of MHSF / MHSR (respectively shown in SEQ ID NO.2 and SEQ ID NO.3) ), the primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. Then, the designed and synthesized primers MHSF / MHSR were used to perform amplification detection on different yew samples (see the description of the figure for details).

[0029] The PCR reaction system is 2μl 10×Buffer, 2μl MgCl 2 (25mM), 0.8μl dNTPs (10mM), 1μl primer MHSF (10μM), 1μl primer MHSR (10μM), 1μl template DNA (50ng / μl), 0.4μl Taq enzyme (2U / μl), 11.8μl ddH 2 O. The total volume is 20μl.

[0030] The PCR reaction program was 94°C pre-denaturation for 5 min; 35 cycles (94°C denaturation for 50 s, 58°C annealing for 50 s, 72°C extension...

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Abstract

The invention relates to a nucleotide sequence, a molecular probe and a method for identifying a taxus media seedling. The nucleotide sequence for identifying the taxus media seedling is represented as SEQ ID NO.1; upstream MHSF of the nucleotide molecular probe for identifying the taxus media seedling is represented as SEQ ID NO.2, and downstream MHSR is represented as SEQ ID NO.3; and by means of the nucleotide molecular probe MHSF/MHSR, taxus media is identified with a conventional PCR (polymerase chain reaction) method. The sample consumption is small, and the overall operation can be completed only by a small quantity of samples; accuracy and sensitivity are high; MHSF/MHSR is a specific molecular probe for the taxus media, and negative reaction is caused if T. chinensis or T. cuspidata is detected; and the method is simple, the PCR technology is adopted for detection, time is short, and the detection can be finished by half day.

Description

technical field [0001] The invention belongs to the technical field of identifying yew mandia seedlings by means of molecular biology methods, and relates to a nucleotide sequence, a molecular probe and a method for identifying yew mandia. Background technique [0002] Mandia yew ( Taxus madia ) is a genus of Taxaceae (Taxaceae) ( Taxus Linn ) plants are mainly distributed in Southwest, Northeast, Central China, South China and other regions in my country. There are more in Sichuan, Shaanxi, Jiangsu, Zhejiang and other places. Mandia yew T. madia yew T. cuspidata and European yew T. baccata It is rich in taxane diterpenoids, among which paclitaxel has been proved to be a good new anti-cancer drug through clinical trials, and it has significant effects on ovarian cancer, breast cancer, lung cancer and gastrointestinal cancer. Taxus Mandia is a relict plant from the Quaternary Ice Age and is a rare and endangered species in the world. Mandia yew T. madia , souther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 沈晨佳冯尚国王慧中
Owner HANGZHOU NORMAL UNIVERSITY
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