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Primers for amplifying paphiopedilum type B MADS-box genes

A gene and B-type technology, which is applied in the application of separation of B-type MADS-box genes, genetic relationship analysis, the above-mentioned primers and the kits including the primers, can solve the problem of no cloning and the like

Active Publication Date: 2017-05-31
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

We have not cloned the PI-1, AP3B2 gene of Paphiopediphyllum using the existing primers published in the literature, nor have we cloned any B class MADS-box genes of Paphiopedilum henryi

Method used

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  • Primers for amplifying paphiopedilum type B MADS-box genes
  • Primers for amplifying paphiopedilum type B MADS-box genes
  • Primers for amplifying paphiopedilum type B MADS-box genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Design of primers for isolating Paphiopedilum class B MADS-box genes

[0069] Download the B gene sequences of other orchids from Genbank, compare the sequences with MEGA 6.0, select the gene conservation region but relatively specific sequence region in Paphiopedilum relatives to design primers. The length of the primer is 18-24bp, and the Tm value is generally controlled at 55-60°C. Avoid using base A at the 3' end of the primer; try not to have a hairpin structure for a single primer, and try not to have a dimer between primers; The GC content of the primer is generally 40-60%, and the GC content of the upstream and downstream primers cannot be too different; the distribution of the four bases in the primer is preferably random, and there is no polypurine and polypyrimidine, especially in the 3' of the primer There should be no more than 3 consecutive G's or C's. Design as many primers as possible.

[0070] Fifteen pairs were selected from the designed...

Embodiment 2

[0071] Example 2. Screening primers for isolating Paphiopedilum class B MADS-box genes

[0072] With reference to the experimental method and materials shown in Example 3 below, the following experiments were performed to screen suitable primer sets:

[0073]Utilize existing Oligo(dT) 17 The primers are combined with different F1 primers (AP3A1_F1, AP3A2_F1, AP3B1_F1, AP3B2_F1 or PI_F1) in Table 1-1, and the first round of 3'RACE PCR reaction is carried out. The PCR products are detected by 1% agarose gel electrophoresis, selected and recovered for electrophoresis. Gel DNA with clear and single bands and lengths within the expected range to screen for suitable F1 primers;

[0074] Using Oligo(dT) 17 Combined with different F2 primers (AP3A1_F2, AP3A2_F2, AP3B1_F2, AP3B2_F2 or PI_F2) in Table 1-1, carry out the second round of 3'RACE PCR reaction, detect the PCR product by 1% agarose gel electrophoresis, select and recover the electrophoresis strip Sequence the gel DNA wit...

Embodiment 3

[0080] Example 3. Isolation of Paphiopedilum class B MADS-box gene

[0081] 1. Test material

[0082] (1) Plant material: Henry's Paphiopedilum.

[0083] (2) Cloning vectors: pEASY-T1 and pEASY-T3 (Beijing Quanshijin Biotechnology Co., Ltd.).

[0084] (3) Reagents: RNA extraction was performed using RNA prep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (spin column type, DP441, Beijing Tiangen Biochemical Technology Co., Ltd.); reverse transcription was used to synthesize first-strand cDNA using Invitrogen’s Superscript III reverse transcription kit. Transcriptase and related system (18080-044), purchased from Beijing Huazhong Haiwei Technology Co., Ltd.; 5'RACE uses Invitrogen's Kit (L150202), purchased from Beijing Juhua Tektronix Technology Co., Ltd.; gel recovery using ordinary agarose gel DNA recovery kit (DP209-02), purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Rnase Inhibitor (2313A), Takara Taq enzyme and dNTP were purchased fr...

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Abstract

The invention provides a group of primers for separating paphiopedilum type B MADS-box genes comprising PahePI, PaheAP3Al, PaheAP3A2, PaheAP3B1 and PaheAP3B2 and a kit comprising the primers. The invention also provides a method for developing the primers. In addition, the invention provides a method of separating the type B MADS-box genes from a paphiopedilum plant through a nested PCR method by means of the primers.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers for amplifying Paheopedilum class B MADS-box genes, including PahePI, PaheAP3A1, PaheAP3A2, PaheAP3B1 and PaheAP3B2. Specifically, the present invention relates to the above-mentioned primers and kits comprising said primers. The present invention also relates to a method for developing the above primers and a method for isolating class B MADS-box genes using the above primers. The present invention also relates to the application of genetic relationship analysis using the gene sequence amplified by the above primers. Background technique [0002] The name of the MADS-box gene is derived from the first letters of the four family members first discovered: MCM1 (MiniChromosom Maintenancel, yeast microchromosome maintenance gene), AGAMOUS (Arabidopsis whorl organ deterministic gene), DEFICIENS (Snapdragon whorling Organ deterministic gene) and SRF (Serum Response Factor, human...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2549/119C12Q2531/113
Inventor 贾瑞冬周妍慧葛红杨树华赵鑫徐玉凤
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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