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Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus

A technology of unknown sequence and cloning method, applied in the field of bioengineering, can solve the problem of low expression of carnation PS1 gene, and achieve the effect of saving manpower, low cost and strong pertinence

Inactive Publication Date: 2014-04-30
YUNNAN YUNKE FLOWER +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Arabidopsis PS1 gene contains 7 exons and 6 introns, encoding 1477 amino acids. We cloned a partial DNA fragment of Carnation PS1 gene by homologous cloning method, but the cloned partial DNA fragment is located in the gene There are still long fragments of sequences at the 3' end and 5' end that have not been cloned, and the expression level of carnation PS1 gene is very low. It is difficult to clone the 5' end sequence by 5'Race technology
Therefore, cloning the flanking sequence of PS1 through DNA sequence is the first choice. Due to the characteristics of the sequence structure of the carnation PS1 gene, the flanking sequence cannot be expanded by Hi-TAILPCR technology. Therefore, it is very necessary to explore a new simple, fast and efficient PS1 Cloning method of gene flanking sequence

Method used

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  • Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus
  • Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus
  • Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus

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Experimental program
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Effect test

Embodiment 1

[0067] The design of embodiment 1 degenerate primer and specific primer

[0068] (1) Design of degenerate primers for amplifying the unknown sequence on the 5' flank of PS1 gene: through homologous comparison of PS1 gene, design 5 degenerate primers according to the conserved sequence, named A1, A2, A3, A4 and A5 , the base sequence of the A1 primer is shown in SEQ ID NO:1. The base sequence of the A2 primer is shown in SEQ ID NO:2. The base sequence of the A3 primer is shown in SEQ ID NO:3. The base sequence of the A4 primer is shown in SEQ ID NO:4. The base sequence of the A5 primer is shown in SEQ ID NO:5.

[0069] (2) Based on the middle fragment of the cloned PS1 gene of Carnation, two specific primers were designed, named B1 and B2 respectively. The base sequence of the B1 primer is shown in SEQ ID NO:6. The base sequence of the B2 primer is shown in SEQ ID NO:7.

[0070] The above primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.

Embodiment 2

[0071] Example 2 Cloning of Carnation PS1 Gene 5' flanking Unknown Sequence

[0072] (1) Carnation genomic DNA was extracted by CTAB method, amplified by nested PCR, using the extracted Carnation genomic DNA as a template, and A1 and B2 as primers for the first round of PCR amplification: the total reaction volume was 25 μL, of which 10×Ex TaqBuffer2.5μL, 25mM MgCl 2 1.5 μL, 2.5 mM dNTP 2 μL, 10 μM A1 primer 1 μL, 10 μM B2 primer 1 μL, 5 U / μL Ex Taq 0.15 μL, template DNA 1 μL, the rest is sterile water;

[0073] The amplification procedure is:

[0074] (1), 1min at 95°C,

[0075] (2), 94°C for 30s,

[0076] (3), 60°C for 1min,

[0077] (4), 72°C 2min30s,

[0078] (5), from step (2) to step (4), carry out a total of 10 rings,

[0079] (6), 94°C for 30s,

[0080] (7) 2min at 25°C, rising to 72°C at 0.5°C / s,

[0081] (8), 72°C 2min30s,

[0082] (9), 94°C for 30s,

[0083] (10), 58°C for 1min,

[0084] (11), 72°C for 2min30s,

[0085] (12), from steps (9) to (11) a tot...

Embodiment 3— Embodiment 5

[0108] Example 3-Example 5 are all clones of the unknown sequence of the 5' flank of Carnation PS1 gene, except for the operations listed in Table 1, the rest of the operations are the same as in Example 2, and will not be repeated. See Table 1 for details.

[0109] Embodiment 1-Example 5 adopts the nested PCR amplified agarose gel electrophoresis to detect PCR product fragment results see Figure 2-Figure 3 ,Table 2.

[0110] Table 2 embodiment 1-embodiment 5 adopts nested PCR amplified agarose gel electrophoresis to detect PCR product fragment result

[0111]

[0112] The time used in each of the above embodiments is 6-8 hours, and the sequencing results show that the obtained fragment is the target fragment including the known sequence, and the method of the present invention can be conveniently, quickly, efficiently and accurately cloned into Carnation PS1 gene 5 'flanking unknown sequences.

[0113] Table 1 Example 3-Example 5 Carnation PS1 gene 5' flanking unknown ...

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Abstract

The invention discloses a method for cloning unknown flanking sequences of a PS1 gene 5' of dianthus caryophyllus, belonging to the technical field of bioengineering. According to the method, five degenerate primers and two specific primers are designed according to homologous sequences, and the unknown flanking sequences of the PS1 gene 5' of dianthus caryophyllus can be obtained by adopting nested PCR (Polymerase Chain Reaction) amplification. The method disclosed by the invention has the characteristics of simplicity and convenience in operation, few operating restrictions, long cloned fragments, strong pertinence, low cost and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for determining the unknown 5' flank sequence based on the cloned partial DNA sequence of carnation PS1 gene by using degenerate primers and specific primers through nested PCR amplification technology. Background technique [0002] Using 2n gametes for sexual polyploidy has higher adaptability and heterozygosity than asexual polyploidy, and is an effective polyploid breeding approach. We found that carnation (Dianthus caryophyllus L) is mostly diploid, and most of them can produce low-frequency 2n gametes. The abnormal spindle is the main reason for the formation of caryophyllus 2n gametes. In Arabidopsis, the PS1 gene regulates the orientation of the spindle, and mutations in the PS1 gene can produce high-frequency 2n gametes. The study of carnation PS1 gene will help to reveal the molecular genetic mechanism of 2n gamete formation, but the sequence of carnation ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 周旭红莫锡君桂敏吴学尉卢珍红田敏余蓉培龙江
Owner YUNNAN YUNKE FLOWER
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