CN103756997A - Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus - Google Patents
Method for cloning unknown flanking sequences of PS1 gene 5' of dianthus caryophyllus Download PDFInfo
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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
The invention belongs to technical field of bioengineering, is a kind of degenerated primer and special primer of utilizing, and by nest-type PRC amplification technique, the partial dna sequence of having cloned based on Dianthus caryophyllus L. PS1 gene is measured the method for its 5' flank unknown nucleotide sequence.
Background technology
Utilizing 2n gamete to carry out Sexual polyploid, than asexual polyploidization, have higher adaptability and heterozygosity, is a kind of effective polyploid breeding approach.We study and find that Dianthus caryophyllus L. (Dianthus caryophyllus L) mostly is diploid, most ofly can produce low frequency 2n gamete, and 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 disclose the gametogenic molecular genetics mechanism of 2n, and the sequence of Dianthus caryophyllus L. PS1 gene is not also cloned, and this has affected Dianthus caryophyllus L. polyploid breeding process.
Arabidopis thaliana PS1 gene comprises 7 exons and 6 introns, 1477 amino acid of encoding, we have cloned Dianthus caryophyllus L. PS1 Gene Partial DNA segment by the method for homologous clone, yet the part DNA segment of having cloned is positioned at 3 ' end of gene, 5 ' end also have lengthy motion picture disconnected sequence be not cloned, and Dianthus caryophyllus L. PS1 gene expression amount is very low, by 5 ' Race technology clone 5 ' terminal sequence, acquire a certain degree of difficulty.Therefore, by DNA sequence dna, cloning PS1 flanking sequence is to become first-selection, Dianthus caryophyllus L. PS1 gene, due to sequential structure, adopts Hi-TAILPCR technology to fail to expand flanking sequence, therefore is extremely necessary to explore the cloning process of 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 be based on its 5 ' distolateral wing unknown nucleotide sequence of known PS1 Gene Partial determined dna sequence.
Main contents of the present invention comprise: homology comparison PS1 gene, and according to conserved sequence, design degenerated primer; According to segment in the middle of the Dianthus caryophyllus L. PS1 gene of having cloned, design special primer; Take Dianthus caryophyllus L. genomic dna as template, with degenerated primer and special primer, carry out two 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 Dianthus caryophyllus L. genomic dna, adopt nest-type PRC amplification, the Dianthus caryophyllus L. genomic dna extracting of take is template, is that primer, A2 and B2 are that primer, A3 and B2 are that primer or A4 and B2 are that primer carries out first round pcr amplification respectively with A1 and B2;
(2) take the pcr amplification product that first run A1 and B2 be primer is template again, with A2 and B1, is that primer carries out second and takes turns pcr amplification; Or to take with the pcr amplification product that A2 and B2 are primer be template, with A3 and B1, be that primer carries out second and takes turns pcr amplification; Or to take with the pcr amplification product that A3 and B2 are primer be template, with A4 and B1, be that primer carries out second and takes turns pcr amplification; Or to take the pcr amplification product that A4 and B2 be primer be template, with A5 and B1, be that primer carries out second and takes turns pcr amplification; By second, take turns pcr amplification product and carry out agarose gel electrophoresis detection, PCR product 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.
The described first round pcr amplification of step (): reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A1, A2, A3 or A4 primer are respectively 0.5-2 μ L, 10 μ M B2 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, genomic dna 1 μ L, 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) to step (4), carry out altogether 10 Xun ring,
（6）、94℃30s，
(7), 25 ℃ of 2～3min, with 0.5 ℃/s, rise to 72 ℃,
（8）、72℃2min30s～3min，
（9）、94℃30s，
（10）、58℃45s～1min，
（11）、72℃2min30s～3min，
(12), from step (9) to (11), carry out altogether 25 Xun ring,
（13）、72℃5min，
(14), 4 ℃ of terminations;
Step (two) described second is taken turns pcr amplification: reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A2, A3, A4 or A5 primer are respectively 0.5-2 μ L, 10 μ M B1 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, template DNA 1 μ L, 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) to step (13), carry out altogether 13 Xun ring,
（15）、72℃5min，
(16), 4 ℃ of terminations.
In step (two), the second template of taking turns pcr amplification is first run pcr amplification product, and this template need be diluted 10-100 doubly.
The present invention compares with art methods, has advantages of as follows:
1, with strong points, easy and simple to handle, efficiently and accurately, overcome because Dianthus caryophyllus L. PS1 gene expression amount is very low and segment length is difficult to the difficulty with existing 5 ' Race technology clone 5 ' terminal sequence, and adopted the not strong defect of Hi-TAILPCR technology specific amplification with 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 of segment design in the middle of known Dianthus caryophyllus L. PS1 gene, with strong points, can effectively 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 obtaining is longer, and the length that can obtain 2000-5000bp is the fragment of grade not.Improved the accuracy of sequence, adopted the inventive method only to need one day just can obtain flanking sequence, thereby saved a large amount of manpowers, time and financial resources.
2. with low cost.The present invention adopts simple pcr amplification, without expensive instrument and test kit.
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
Fig. 1: clone's principle schematic of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence.
Fig. 2: first round PCR product gel electrophorogram.In figure:
M：Marker；
Fig. 3: second takes turns PCR product gel electrophorogram.In figure:
M：Marker；
Embodiment
Below in conjunction with embodiment, the inventive method is described in detail.The experimental technique using 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: compare PS1 gene by homology, according to conserved sequence, design 5 degenerated primers, difference 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 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.
(2) in the middle of the PS1 gene of having cloned according to Dianthus caryophyllus L., segment has designed 2 special primers, difference called after B1 and B2, and 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 synthetic by Shanghai Sheng Gong biotechnology company limited.
The clone of embodiment 2 Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequences
(1) adopt CTAB method to extract Dianthus caryophyllus L. genomic dna, employing nest-type PRC amplification, the Dianthus caryophyllus L. genomic dna extracting of take is template, take A1 and B2 to carry out first round pcr amplification as primer: reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A1 primer 1 μ L, 10 μ M 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) to step (4), carry out altogether 10 Xun ring,
（6）、94℃30s，
(7), 25 ℃ of 2min, with 0.5 ℃/s, rise to 72 ℃,
（8）、72℃2min30s，
（9）、94℃30s，
（10）、58℃1min，
（11）、72℃2min30s，
(12), from step (9) to (11), carry out altogether 25 Xun ring,
（13）、72℃5min，
(14), 4 ℃ 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) take the pcr amplification product that first run A1 and B2 be primer is template again, and 10 times of template dilutions are that primer carries out second and takes turns pcr amplification with A2 and B1; By second, take turns pcr amplification product and carry out agarose gel electrophoresis detection, PCR product checks order after reclaiming purifying, and the segment obtaining is the object segment that comprises known array.The object fragment obtaining is as swimming lane in Fig. 31,2.
Described second takes turns pcr amplification: reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A2 primer 1 μ L, 10 μ M 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) to step (13), carry out altogether 13 Xun ring,
（15）、72℃5min，
(16), 4 ℃ 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 listed operation difference 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 nest-type PRC amplification to detect PCR product 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 nest-type PRC amplification to detect PCR product fragment result
Each embodiment time used is 6-8 hour above, and sequencing result shows, the segment obtaining is the object segment that comprises known array, adopts the inventive method can be cloned into easily and fast, efficiently and accurately Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence.
The operation steps of distinguishing 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> /
<160> 7
<170> PatentIn version 3.3
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Claims (3)
1. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence, is characterized in that:
(1) extract Dianthus caryophyllus L. genome DNA, adopt nest-type PRC amplification, the Dianthus caryophyllus L. genome DNA of extracting of take is template, is that primer, A2 and B2 are that primer, A3 and B2 are that primer or A4 and B2 are that primer carries out first round pcr amplification respectively with A1 and B2;
(2) take the pcr amplification product that first run A1 and B2 be primer is template again, take A2 and B1 to take turns pcr amplification as primer carries out second; Or to take with the pcr amplification product that A2 and B2 are primer be template, with A3 and B1, be that primer carries out second and takes turns pcr amplification; Or to take with the pcr amplification product that A3 and B2 are primer be template, with A4 and B1, be that primer carries out second and takes turns pcr amplification; Or to take with the pcr amplification product that A4 and B2 are primer be template, with A5 and B1, be that primer carries out second and takes turns pcr amplification; By second, take turns pcr amplification product and carry out agarose gel electrophoresis detection, PCR product 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.
2. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence according to claim 1, is characterized in that: the described first round pcr amplification of step (): reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A1, A2, A3 or A4 primer are respectively 0.5-2 μ L, 10 μ M B2 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, genomic dna 1 μ L, 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) to step (4), carry out altogether 10 Xun ring,
（6）、94℃30s，
(7), 25 ℃ of 2～3min, with 0.5 ℃/s, rise to 72 ℃,
（8）、72℃2min30s～3min，
（9）、94℃30s，
（10）、58℃45s～1min，
（11）、72℃2min30s～3min，
(12), from step (9) to (11), carry out altogether 25 Xun ring,
（13）、72℃5min，
(14), 4 ℃ of terminations;
Step (two) described second is taken turns pcr amplification: reaction is totally 25 μ L, 10 * Ex TaqBuffer2.5 μ L wherein, 25mM MgCl
21.5 μ L, 2.5mM dNTP2 μ L, 10 μ M A2, A3, A4 or A5 primer are respectively 0.5-2 μ L, 10 μ M B1 primer 0.5-2 μ L, 5U/ μ L Ex Taq0.1-0.2 μ L, template DNA 1 μ L, 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) to step (13), carry out altogether 13 Xun ring,
（15）、72℃5min，
(16), 4 ℃ of terminations.
3. the cloning process of Dianthus caryophyllus L. PS1 gene 5' flank unknown nucleotide sequence according to claim 1 and 2,
It is characterized in that: in step (two), the second template of taking turns pcr amplification is first run pcr amplification product,
This template need be diluted 10-100 doubly.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195142A (en) * | 2014-08-29 | 2014-12-10 | 云南省农业科学院花卉研究所 | Method for building ihpRNA carrier of Dianthus caryophyllus PS1 gene |
| CN111304214A (en) * | 2020-02-18 | 2020-06-19 | 云南省农业科学院花卉研究所 | Gene for increasing quantity of carnation petals and application |
| CN114540341A (en) * | 2022-01-12 | 2022-05-27 | 西北农林科技大学 | Rapid and efficient DNA 5' flanking sequence cloning method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195142A (en) * | 2014-08-29 | 2014-12-10 | 云南省农业科学院花卉研究所 | Method for building ihpRNA carrier of Dianthus caryophyllus PS1 gene |
| CN111304214A (en) * | 2020-02-18 | 2020-06-19 | 云南省农业科学院花卉研究所 | Gene for increasing quantity of carnation petals and application |
| CN111304214B (en) * | 2020-02-18 | 2023-05-30 | 云南省农业科学院花卉研究所 | Gene for increasing number of carnation petals and application |
| CN114540341A (en) * | 2022-01-12 | 2022-05-27 | 西北农林科技大学 | Rapid and efficient DNA 5' flanking sequence cloning method |
| CN114540341B (en) * | 2022-01-12 | 2023-10-27 | 西北农林科技大学 | Quick and efficient DNA5' flanking sequence cloning method |
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