Nucleotide sequence, molecular probe and method for discriminating paphiopedilum micranthum

A technology of nucleotide sequence and P. sclera, applied in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc. Simple, low sample volume, short time effect

Active Publication Date: 2014-11-19
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Traditional morphological research methods cannot essentially accurately reflect the genetic variation of species and the genetic relationship between

Method used

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  • Nucleotide sequence, molecular probe and method for discriminating paphiopedilum micranthum
  • Nucleotide sequence, molecular probe and method for discriminating paphiopedilum micranthum
  • Nucleotide sequence, molecular probe and method for discriminating paphiopedilum micranthum

Examples

Experimental program
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Example Embodiment

[0026] Example 1: Preparation of specific nucleotide sequence of Paphiopedilum sclerophylla

[0027] 1. Genomic DNA extraction

[0028] Cut 0.2 g of Paphiopedilum leaves stored at -80°C into a mortar, immediately add liquid nitrogen to grind to powder, and then use UNIQ-10 column plant genomic DNA extraction kit from Shanghai Shenggong Bioengineering Co., Ltd. The genomic DNA of Paphiopedilum is electrophoresed with 1% agarose gel, and the DNA concentration is detected with an ultraviolet spectrophotometer, diluted to 50ng / μl.

[0029] 2. SCoT-PCR reaction and electrophoresis detection

[0030] Use SCoT universal primer 2 (5’-CAACAATGGCTACCACCC-3’) for PCR amplification, the amplification system is: 2μl 10×Buffer, 2μl MgCl 2 (25mM), 0.8μl dNTPs (10mM), 1μl SCoT primer 2 (10μM), 1μl template DNA (50ng / μl), 0.5μl Taq enzyme (2U / μl), 12.7μl ddH 2 O. The total volume is 20μl.

[0031] The PCR program was: 94°C pre-denaturation 4 min; 35 cycles (94°C denaturation 45 s, 50°C annealing 45 ...

Example Embodiment

[0035] Example 2: Preparation of YYF / YYR specific nucleotide probe for Paphiopedilum sclerophylla, PCR reaction and electrophoresis detection

[0036] On the basis of obtaining the specific nucleotide sequence of Paphiopedilum sclerophylla, the YYF / YYR nucleotide sequence (shown in SEQ ID NO.2 and SEQ ID NO.3 respectively) was designed using Primer Primer 5.0 software. , The primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd. Then use the designed and synthesized primers YYF / YYR to perform amplification detection on different Paphiopedilum samples (see the description of the figure for details).

[0037] PCR amplification system is 2μl 10×Buffer, 2μl MgCl 2 (25mM), 0.8μl dNTPs (10mM), 1μl primer YYF (10μM), 1μl primer YYR (10μM), 1μl template DNA (50ng / μl), 0.5μl Taq enzyme (2U / μl), 11.7μl ddH 2 O. The total volume is 20μl.

[0038] The reaction program is 94°C pre-denaturation for 4 min; 35 cycles (94°C denaturation for 45 s, 57°C annealing for 45 s, 7...

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Abstract

The invention relates to a nucleotide sequence, a molecular probe and a method for discriminating paphiopedilum micranthum. The nucleotide sequence for discriminating paphiopedilum micranthum is shown as a SEQ ID NO.1. A nucleotide molecule probe upstream YYF: for discriminating paphiopedilum micranthum is shown as a SEQ ID NO.2, and a nucleotide molecule probe downstream YYR: is shown as a SEQ ID NO.3. The nucleotide molecular probe YYF/YYR can be used for discriminating paphiopedilum micranthum through a conventional PCR method. According to the invention, sample amount is less, only a few of sample is required for completing the whole process; accuracy and sensitivity are high, the YYF/YYR are specificity molecular probes of paphiopedilum micranthum, the molecular probe presents negative response if the paphiopedilum is other types; the method is simple, by using the PCR method for detection, the detection time is short, and the method can be competed in half day.

Description

technical field [0001] The invention belongs to the technical field of using molecular biological methods to identify Pauplophyllum sclerophyllum, and relates to a nucleotide sequence, a molecular probe and a method for identifying Pauplophyllum sclerophyllum. Background technique [0002] Paphiopediphyllum ( Paphiopedilum micranthum ) is a member of the Orchidaceae family (Orchidaceae) Paphiopedilum ) plants, mainly distributed in my country's Yunnan, Guangxi, Guizhou and other provinces and northern Vietnam. Pedrophyllum sclerophyllum has a high ornamental value because of its unique flowers and beautiful posture, so it is called "Magnolia" and is known as one of the most beautiful flowers in the world. Due to its very high ornamental value and economic value, artificial overharvesting and serious habitat destruction, the wild population of P. CITES is listed as a first-class protected plant. Therefore, in order to effectively protect and search for new P. sclerophyll...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王慧中冯尚国何仁锋
Owner HANGZHOU NORMAL UNIVERSITY
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