CN103756997B - The cloning process of Dianthus caryophyllus L. PS1 gene 5 ' flank unknown nucleotide sequence - Google Patents
The cloning process of Dianthus caryophyllus L. PS1 gene 5 ' flank unknown nucleotide sequence Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000002773 nucleotide Substances 0.000 title claims abstract description 19
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 19
- 238000010367 cloning Methods 0.000 title claims abstract description 11
- 230000003321 amplification Effects 0.000 claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 11
- 238000007857 nested PCR Methods 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims description 40
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 208000020584 Polyploidy Diseases 0.000 description 3
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- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
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Abstract
The invention discloses a kind of cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, belong to technical field of bioengineering.The method designs 5 degenerated primers and 2 special primers according to homologous sequence, adopts nested PCR amplification, all can obtain Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence.The inventive method has the features such as easy and simple to handle, performance constraint is few, the segment of clone is long, with strong points and with low cost.
Description
Technical field
The invention belongs to technical field of bioengineering, is that one utilizes degenerated primer and special primer, and by nested PCR amplification technology, the partial dna sequence of having cloned based on Dianthus caryophyllus L. PS1 gene measures the method for its 5' flank unknown nucleotide sequence.
Background technology
Utilizing 2n gamete to carry out Sexual polyploid, have higher adaptability and heterozygosity than asexual polyploidization, is a kind of effective polyploid breeding approach.We study and find that Dianthus caryophyllus L. (Dianthus caryophyllus L) mostly is diploid, and most ofly can produce low frequency 2n gamete, spindle body is the gametogenic major cause of Dianthus caryophyllus L. 2n extremely.The direction of PS1 gene regulating Spindle in Arabidopis thaliana research, PS1 transgenation can produce high frequency 2n gamete.Research Dianthus caryophyllus L. PS1 gene contributes to disclosing the gametogenic molecular genetics mechanism of 2n, and the sequence of Dianthus caryophyllus L. PS1 gene is not also cloned, and this have impact on Dianthus caryophyllus L. polyploid breeding process.
Arabidopis thaliana PS1 gene comprises 7 exons and 6 introns, to encode 1477 amino acid, we have cloned Dianthus caryophyllus L. PS1 Gene Partial DNA segment by the method for homologous clone, but the part DNA segment of having cloned is positioned at 3 ' end of gene, 5 ' end also have lengthy motion picture break sequence be not cloned, and Dianthus caryophyllus L. PS1 gene expression amount is very low, acquired a certain degree of difficulty by 5 ' Race technology clone 5 ' terminal sequence.Therefore, cloning PS1 flanking sequence by DNA sequence dna is become first-selected, Dianthus caryophyllus L. PS1 gene, due to sequential structure, adopts Hi-TAILPCR technology to fail to expand flanking sequence, is therefore extremely necessary the cloning process exploring new simple, fast and efficient PS1 gene flanking sequence.
Summary of the invention
The object of this invention is to provide a kind of easy and simple to handle, with low cost, efficiently and accurately PCR amplification method, can based on its 5 ' side wing unknown nucleotide sequence of known PS1 Gene Partial determined dna sequence.
Main contents of the present invention comprise: homology comparison PS1 gene, according to conserved sequence, and design degenerated primer; According to segment in the middle of the Dianthus caryophyllus L. PS1 gene of having cloned, design special primer; With carnation gene group DNA for template, carry out two with degenerated primer and special primer and take turns PCR(nest-type PRC) amplification, and its amplified fragments is checked order, whether checking is object segment.The cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence provided by the present invention, concrete technical scheme is as follows:
1. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, comprises the following steps:
(1) extract carnation gene group DNA, adopt nested PCR amplification, with the carnation gene group DNA extracted for template, respectively with A1 and B2 be primer, A2 and B2 be primer, A3 and B2 be primer or A4 and B2 is that primer carries out first round pcr amplification;
(2) again with first run A1 and the B2 pcr amplification product that is primer for template, be that primer carries out second and takes turns pcr amplification with A2 and B1; Or with the pcr amplification product that is primer with A2 and B2 for template, be that primer carries out second and takes turns pcr amplification with A3 and B1; Or with the pcr amplification product that is primer with A3 and B2 for template, be that primer carries out second and takes turns pcr amplification with A4 and B1; Or with A4 and the B2 pcr amplification product that is primer for template, be that primer carries out second and takes turns pcr amplification with A5 and B1; Take turns pcr amplification product by second and carry out agarose gel electrophoresis detection, PCR primer checks order after reclaiming purifying, and whether checking is object segment; The base sequence of described A1 primer is as shown in SEQ ID NO:1, the base sequence of described A2 primer is as shown in SEQ ID NO:2, the base sequence of described A3 primer is as shown in SEQ ID NO:3, the base sequence of described A4 primer is as shown in SEQ ID NO:4, the base sequence of described A5 primer is as shown in SEQID NO:5, the base sequence of described B1 primer is as shown in SEQ ID NO:6, and the base sequence of described B2 primer is as shown in SEQ ID NO:7.
The described first round pcr amplification of step (): reaction is totally 25 μ L, wherein 10 × Ex TaqBuffer2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μMs of A1, A2, A3 or A4 primers are respectively 0.5-2 μ L, 10 μMs of B2 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, genomic dna 1 μ L, and surplus is sterilized water; Amplification program is:
（1）、95℃1min，
（2）、94℃30s，
（3）、60℃45s～1min，
（4）、72℃2min30s～3min，
(5), from step (2) 10 Xun rings are carried out altogether to step (4),
（6）、94℃30s，
(7), 25 DEG C of 2 ~ 3min, rise to 72 DEG C with 0.5 DEG C/s,
（8）、72℃2min30s～3min，
（9）、94℃30s，
（10）、58℃45s～1min，
（11）、72℃2min30s～3min，
(12), from step (9) to (11) 25 Xun rings are carried out altogether,
（13）、72℃5min，
(14), 4 DEG C of terminations;
Step (two) described second takes turns pcr amplification: reaction is totally 25 μ L, wherein 10 × Ex TaqBuffer2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μMs of A2, A3, A4 or A5 primers are respectively 0.5-2 μ L, 10 μMs of B1 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, template DNA 1 μ L, and surplus is sterilized water; Amplification program is:
（1）、94℃3min，
（2）、94℃20s，
（3）、65℃45s～1min，
（4）、72℃2min30s～3min，
（5）、94℃20s，
（6）、68℃45s～1min，
（7）、72℃2min30s～3min，
（8）、94℃20s，
（9）、68℃45s～1min，
（10）、72℃2min30s～3min，
（11）、94℃20s，
（12）、50℃45s～1min，
（13）、72℃2min30s～3min，
(14), from step (2) 13 Xun rings are carried out altogether to step (13),
（15）、72℃5min，
(16), 4 DEG C of terminations.
In step (two), the second template of taking turns pcr amplification is first run pcr amplification product, and this template need dilute 10-100 doubly.
The present invention, compared with art methods, has following advantage:
1, with strong points, easy and simple to handle, efficiently and accurately, overcome the difficulty be difficult to because the very low and segment of Dianthus caryophyllus L. PS1 gene expression amount is long with existing 5 ' Race technology clone 5 ' terminal sequence, and adopt with the not strong defect of the Hi-TAILPCR technology specific amplification of random primer amplification.
The present invention is based on homology comparison PS1 gene, according to conserved sequence, the degenerated primer of design, and according to the special primer that segment in the middle of known Dianthus caryophyllus L. PS1 gene designs, with strong points, effectively can amplify Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, it is easy and simple to handle, use two-wheeled PCR just can amplify the 5' flank unknown nucleotide sequence of Dianthus caryophyllus L. PS1 gene, the flanking sequence obtained is longer, the fragment that the length that can obtain 2000-5000bp does not wait.Improve the accuracy of sequence, adopt the inventive method only to need one day just can obtain flanking sequence, thus save a large amount of manpowers, time and financial resources.
2. with low cost.The present invention adopts simple pcr amplification, without the need to instrument and the test kit of costliness.
In sequence table shown in SEQ ID NO:1 is the base sequence of A1 primer.
In sequence table shown in SEQ ID NO:2 is the base sequence of A2 primer.
In sequence table shown in SEQ ID NO:3 is the base sequence of A3 primer.
In sequence table shown in SEQ ID NO:4 is the base sequence of A4 primer.
In sequence table shown in SEQ ID NO:5 is the base sequence of A5 primer.
In sequence table shown in SEQ ID NO:6 is the base sequence of B1 primer.
In sequence table shown in SEQ ID NO:7 is the base sequence of B2 primer.
Accompanying drawing explanation
The cloning mechanisms schematic diagram of Fig. 1: Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence.
Fig. 2: first round PCR primer gel electrophoresis figure.In figure:
M：Marker；
Swimming lane 1,2: with A1 and B2 for primer PCR amplified production;
Swimming lane 3,4: with A2 and B2 for primer PCR amplified production;
Swimming lane 5,6: with A3 and B2 for primer PCR amplified production;
Swimming lane 7,8: with A4 and B2 for primer PCR amplified production.
Fig. 3: the second takes turns PCR primer gel electrophoresis figure.In figure:
M：Marker；
Swimming lane 1,2: with the pcr amplification product that is primer with A1 and B2 for template, with A2 and B1 for primer PCR amplified production;
Swimming lane 3,4: with the pcr amplification product that is primer with A2 and B2 for template, with A3 and B1 for primer PCR amplified production;
Swimming lane 5,6: with the pcr amplification product that is primer with A3 and B2 for template, with A4 and B1 for primer PCR amplified production;
Swimming lane 7,8: with the pcr amplification product that is primer with A4 and B2 for template, with A5 and B1 for primer PCR amplified production.
Embodiment
Below in conjunction with embodiment, the inventive method is described in detail.The experimental technique used in following embodiment, without specified otherwise, is ordinary method.Material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The design of embodiment 1 degenerated primer and special primer
(1) design of the degenerated primer of amplification PS1 gene 5' flank unknown nucleotide sequence: by homology comparison PS1 gene, according to conserved sequence, design 5 degenerated primers, respectively called after A1, A2, A3, A4 and A5, the base sequence of described A1 primer is as shown in SEQ ID NO:1.The base sequence of described A2 primer is as shown in SEQID NO:2.The base sequence of described A3 primer is as shown in SEQ ID NO:3.The base sequence of described A4 primer is as shown in SEQ ID NO:4.The base sequence of described A5 primer is as shown in SEQ ID NO:5.
(2) the middle segment of PS1 gene of having cloned according to Dianthus caryophyllus L. devises 2 special primers, and called after B1 and B2 respectively, the base sequence of described B1 primer is as shown in SEQ ID NO:6.The base sequence of described B2 primer is as shown in SEQ ID NO:7.
Above-mentioned primer is synthesized by Shanghai Sheng Gong biotechnology company limited.
The clone of embodiment 2 Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence
(1) CTAB method is adopted to extract carnation gene group DNA, adopt nested PCR amplification, with the carnation gene group DNA extracted for template, with A1 and B2 for primer carries out first round pcr amplification: reaction is totally 25 μ L, wherein 10 × Ex TaqBuffer2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mMdNTP2 μ L, 10 μMs of A1 primer 1 μ L, 10 μMs of B2 primer 1 μ L, 5U/ μ L Ex Taq0.15 μ L, template DNA 1 μ L, surplus is sterilized water;
Amplification program is:
（1）、95℃1min，
（2）、94℃30s，
（3）、60℃1min，
（4）、72℃2min30s，
(5), from step (2) 10 Xun rings are carried out altogether to step (4),
（6）、94℃30s，
(7), 25 DEG C of 2min, rise to 72 DEG C with 0.5 DEG C/s,
（8）、72℃2min30s，
（9）、94℃30s，
（10）、58℃1min，
（11）、72℃2min30s，
(12), from step (9) to (11) 25 Xun rings are carried out altogether,
（13）、72℃5min，
(14), 4 DEG C of terminations;
First round pcr amplification carries out agarose gel electrophoresis detection, and the fragment of acquisition is as swimming lane in Fig. 21,2.
(2) again with first run A1 and the B2 pcr amplification product that is primer for template, template dilutes 10 times, is that primer carries out second and takes turns pcr amplification with A2 and B1; Take turns pcr amplification product by second and carry out agarose gel electrophoresis detection, PCR primer checks order after reclaiming purifying, and the segment obtained is the object segment comprising known array.The object fragment obtained is as swimming lane in Fig. 31,2.
Described second takes turns pcr amplification: reaction is totally 25 μ L, wherein 10 × Ex TaqBuffer2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μMs of A2 primer 1 μ L, 10 μMs of B1 primer 1 μ L, 5U/ μ L Ex Taq0.15 μ L, template DNA 1 μ L, surplus is sterilized water; Amplification program is:
（1）、94℃3min，
（2）、94℃20s，
（3）、65℃1min，
（4）、72℃2min30s，
（5）、94℃20s，
（6）、68℃1min，
（7）、72℃2min30s，
（8）、94℃20s，
（9）、68℃1min，
（10）、72℃2min30s，
（11）、94℃20s，
（12）、50℃1min，
（13）、72℃2min30s，
(14), from step (2) 13 Xun rings are carried out altogether to step (13),
（15）、72℃5min，
(16), 4 DEG C of terminations.
Embodiment 3-embodiment 5
Embodiment 3-embodiment 5 is the clone of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, and except operation difference listed in table 1, all the other operations are identical with embodiment 2, repeat no more.Refer to table 1.
Embodiment 1-embodiment 5 adopts the agarose gel electrophoresis of nested PCR amplification to detect PCR primer fragment and the results are shown in Figure 2-Fig. 3, table 2.
Table 2 embodiment 1-embodiment 5 adopts the agarose gel electrophoresis of nested PCR amplification to detect PCR primer fragment result
Each embodiment time used is 6-8 hour above, and sequencing result shows, the segment obtained is the object segment comprising known array, adopts the inventive method can be cloned into Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence easily and fast, efficiently and accurately.
The operation steps distinguished in the clone of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence described in table 1 embodiment 3-embodiment 5
Sequence table
<110> Yunnan Yunke Flower Co., Ltd
Yunnan Ji Chuan gardening Science and Technology Ltd.
The cloning process of <120> Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence
<130> /
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<170> PatentIn version 3.3
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Claims (2)
1. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, is characterized in that:
(1) carnation gene group STb gene is extracted, adopt nested PCR amplification, with the carnation gene group STb gene extracted for template, respectively with A1 and B2 be primer, A2 and B2 be primer, A3 and B2 be primer or A4 and B2 is that primer carries out first round pcr amplification, described first round pcr amplification: reaction is totally 25 μ L, wherein 10 × Ex Taq Buffer 2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mM dNTP 2 μ L, 10 μMs of A1, A2, A3 or A4 primers are respectively 0.5-2 μ L, 10 μMs of B2 primer 0.5-2 μ L, 5U/ μ L Ex Taq enzyme 0.1-0.2 μ L, genomic dna 1 μ L, and surplus is sterilized water; Amplification program is:
(1)、95℃1min，
(2)、94℃30s，
(3)、60℃45s～1min，
(4)、72℃2min 30s～3min，
(5), from step (2) 10 Xun rings are carried out altogether to step (4),
(6)、94℃30s，
(7), 25 DEG C of 2 ~ 3min, rise to 72 DEG C with 0.5 DEG C/s,
(8)、72℃2min 30s～3min，
(9)、94℃30s，
(10)、58℃45s～1min，
(11)、72℃2min 30s～3min，
(12), from step (9) to (11) 25 Xun rings are carried out altogether,
(13)、72℃5min，
(14), 4 DEG C of terminations;
(2) again with first run A1 and the B2 pcr amplification product that is primer for template, take turns pcr amplification with A2 and B1 for primer carries out second; Or with the pcr amplification product that is primer with A2 and B2 for template, be that primer carries out second and takes turns pcr amplification with A3 and B1; Or with the pcr amplification product that is primer with A3 and B2 for template, be that primer carries out second and takes turns pcr amplification with A4 and B1; Or with the pcr amplification product that is primer with A4 and B2 for template, be that primer carries out second and takes turns pcr amplification with A5 and B1; Take turns pcr amplification product by second and carry out agarose gel electrophoresis detection, PCR primer checks order after reclaiming purifying, and whether checking is object segment; The base sequence of described A1 primer is as shown in SEQ ID NO:1, the base sequence of described A2 primer is as shown in SEQ ID NO:2, the base sequence of described A3 primer is as shown in SEQ ID NO:3, the base sequence of described A4 primer is as shown in SEQ ID NO:4, the base sequence of described A5 primer is as shown in SEQ ID NO:5, the base sequence of described B1 primer is as shown in SEQ ID NO:6, and the base sequence of described B2 primer is as shown in SEQ ID NO:7; Described second takes turns pcr amplification: reaction is totally 25 μ L, wherein 10 × Ex Taq Buffer 2.5 μ L, 25mM MgCl
21.5 μ L, 2.5mM dNTP 2 μ L, 10 μMs of A2, A3, A4 or A5 primers are respectively 0.5-2 μ L, 10 μMs of B1 primer 0.5-2 μ L, 5U/ μ L Ex Taq enzyme 0.1-0.2 μ L, template DNA 1 μ L, and surplus is sterilized water; Amplification program is:
(1)、94℃3min，
(2)、94℃20s，
(3)、65℃45s～1min，
(4)、72℃2min 30s～3min，
(5)、94℃20s，
(6)、68℃45s～1min，
(7)、72℃2min 30s～3min，
(8)、94℃20s，
(9)、68℃45s～1min，
(10)、72℃2min 30s～3min，
(11)、94℃20s，
(12)、50℃45s～1min，
(13)、72℃2min 30s～3min，
(14), from step (2) 13 Xun rings are carried out altogether to step (13),
(15)、72℃5min，
(16), 4 DEG C of terminations.
2. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence according to claim 1, is characterized in that: in step (two), the second template of taking turns pcr amplification is first run pcr amplification product, and this template need dilute 10-100 doubly.
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| WO2001032932A2 (en) * | 1999-11-02 | 2001-05-10 | Curagen Corporation | Method of identifying a nucleic acid sequence |
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| CN102140500A (en) * | 2010-11-19 | 2011-08-03 | 江南大学 | T-vector-mediated method for determining unknown sequence of 5'-end flanking region |
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