CN101285084B - Process for cloning flanking plant DNA sequence of multi- copied T-DNA - Google Patents
Process for cloning flanking plant DNA sequence of multi- copied T-DNA Download PDFInfo
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- CN101285084B CN101285084B CN200810064648XA CN200810064648A CN101285084B CN 101285084 B CN101285084 B CN 101285084B CN 200810064648X A CN200810064648X A CN 200810064648XA CN 200810064648 A CN200810064648 A CN 200810064648A CN 101285084 B CN101285084 B CN 101285084B
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Abstract
The present invention discloses a method for effectively cloning the T-DNA side plant DNA sequence in a heredity modification plant containing multi-copy T-DNA. The method comprises the following main steps: a step of cutting of other T-DNA sequences except the T-DNA sequence connected with the plant DNA through restriction enzyme; then a step of carrying out 2 turns of TAIL-PCR. As the enzyme cutting point is located in the NOS poly A zone and/or 35S poly A zone on the T-DNA and the nesting primer used by TAIL-PCR is also designed according to the sequences in the NOS poly A zone and the 35S poly A zone on the T-DNA, the method is versatile.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method for flanking plant DNA sequence of multi-copied T-DNA in a kind of cloned, transgenic plant.
Background technology
In the plant of genetic modification, T-DNA side DNA of plants sequence can be used for the detection of transgenic plant, the research and the exogenous origin gene integrator Study on Mechanism of gene function.At present, the flanking sequence main method of clone T-DNA has following several: and inverse PCR (inverse PCR), hot asymmetric interlaced PCR (thermal asymmetric interlaced PCR, TAIL-PCR), AL-PCR (adaptor ligation PCR) and T Linker PCR (T-linker-specific ligation PCR) etc.
The ultimate principle of TAIL-PCR is 2-3 nested Auele Specific Primer (the special primer of known array design that utilizes target sequence other, be called for short sp1, sp2, sp3, about 20bp), with they respectively with 1 (arbitrary degenerate prime of degenerated primer at random with weak point of low Tm value, AD, about 14bp) combined, be template with the genomic dna, length and specific difference according to primer design asymmetric temperature cycle, come the amplifying specific primer by fractional order reaction.
When being single copy T-DNA in the transgenic plant, the TAIL-PCR method T-DNA side DNA of plants sequence that can effectively increase.But if in the transgenic plant when containing multiple copied T-DNA, the efficient of TAIL-PCR method amplification T-DNA side DNA of plants sequence then can reduce greatly.The present invention carries out TAIL-PCR then for cut other T-DNA sequence except that the part T-DNA sequence that links to each other with DNA of plants with restriction enzyme, can effectively eliminate the influence of multiple copied T-DNA, improves the efficient of amplification T-DNA side DNA of plants sequence.
Summary of the invention
The technical problem to be solved in the present invention provides the method for flanking plant DNA sequence of multi-copied T-DNA in a kind of effective cloned, transgenic plant.
Method provided by the present invention is to carry out improvedly on the basis of TAIL-PCR, may further comprise the steps:
(1) ordinary method is extracted the transgenic plant genomic dna.
(2) DNA described in the step 1 is carried out endonuclease reaction.
Described endonuclease reaction both can be a single endonuclease digestion, also can be double digestion.Single endonuclease digestion both can carry out in NOS polyA district, also can carry out in 35S polyA district, and double digestion is for carrying out at NOS polyA and 35S polyA district simultaneously.
Carrying out the restriction enzyme that enzyme cuts in NOS polyA district is AflII, TfiI, BslI, HpaII, and their isozyme.Carrying out the restriction enzyme that enzyme cuts in 35S polyA district is MslI, AquI, XhoI, McrI, PvuI, and their isozyme.
Can cut simultaneously in NOS polyA district and 35S polyA district with the SmlI enzyme.
(3) ordinary method is cut product with the enzyme described in the step (2) and is separated into two portions, reclaims the fragment part greater than 3-5kb.
(4) be template with the recovery fragment described in the step (3), carry out TAIL-PCR amplification 2 and take turns.
During amplification T-DNA right margin side DNA of plants, taking turns used nested primers among the TAIL-PCR 2 is the single stranded oligonucleotide sequence with sequence 1 and sequence 2 in the sequence table; During amplification T-DNA left margin side DNA of plants, taking turns used nested primers among the TAIL-PCR 2 is the single stranded oligonucleotide sequence with sequence 4 and sequence 5 in the sequence table.
(4) purifying, clone's TAIL-PCR product according to a conventional method.Ordinary method checks order after using PCR method screening positive clone, obtains the DNA of plants sequence of T-DNA side.
Use PCR method screening positive clone before the order-checking.For the clone of T-DNA right margin side DNA of plants, the primer is the single stranded oligonucleotide sequence with sequence 2 and sequence 3 in the sequence table; For the clone of T-DNA left margin side DNA of plants, the primer is the single stranded oligonucleotide sequence with sequence 5 and sequence 6 in the sequence table.
Embodiment
The clone of embodiment 1, multiple copied T-DNA transgenic poplar T-DNA right side DNA of plants sequence
(1) ordinary method is extracted transgenosis poplar genomic dna.Willow transform used carrier be pYHY (reference: woods is equal. transform spider insecticidal peptide toxin gene to little black poplar. insect journal, 2006,49 (4): 593-598).
(2) endonuclease reaction.
Cut the DNA of gained in the step (1) at NOS polyA district enzyme.The restriction enzyme of selecting for use is Hpa II (a MBI company), operates by the specification sheets that the manufacturer provides.
(3) utilize agarose electrophoresis to carry out the separation that enzyme is cut product in the step (2), utilize Shanghai to give birth to worker UNIQ-10 pillar DNA glue and reclaim the fragment of test kit (SK1131) recovery greater than 5kbp.Operate by the specification sheets that the manufacturer provides.
(4)TAIL-PCR。Recovery product with step (3) is that template is carried out TAIL-PCR.TAIL-PCR carries out 2 and takes turns half nested PCR.The 1st takes turns that used nested primers is the single stranded oligonucleotide sequence with sequence 1 in the sequence table in the amplification, and the sequence of random primer is the single stranded oligonucleotide sequence with sequence 7,8,9 in the sequence table and 10, and program is:
The 2nd takes turns that used nested primers is the single stranded oligonucleotide sequence with sequence 2 in the sequence table in the amplification, and the sequence of random primer is the single stranded oligonucleotide sequence with sequence 7,8,9 in the sequence table and 10, and program is:
(5) according to a conventional method the TAIL-PCR product in the step (4) is carried out T-A clone, order-checking.Use PCR method screening positive clone, the primer is the single stranded oligonucleotide sequence with sequence 2 and sequence 3 in the sequence table.
The clone of embodiment 2, multiple copied T-DNA transgenic poplar T-DNA left side DNA of plants sequence
(1) ordinary method is extracted transgenosis poplar genomic dna.Willow transform used carrier be pYHY (reference: woods is equal. transform spider insecticidal peptide toxin gene to little black poplar. insect journal, 2006,49 (4): 593-598).
(2) endonuclease reaction.
Cut the DNA of gained in the step (1) at 35S poly A district enzyme.The restriction enzyme of selecting for use is Xho I (a MBI company), operates by the specification sheets that the manufacturer provides.
(3) utilize agarose electrophoresis to carry out the separation that enzyme is cut product in the step (2), utilize Shanghai to give birth to worker UNIQ-10 pillar DNA glue and reclaim the fragment of test kit (SK1131) recovery greater than 5kbp.Operate by the specification sheets that the manufacturer provides.
(4)TAIL-PCR。Recovery product with step (3) is that template is carried out TAIL-PCR.TAIL-PCR carries out 2 and takes turns half nested PCR.The 1st takes turns that used nested primers is the single stranded oligonucleotide sequence with sequence 4 in the sequence table in the amplification, and the sequence of random primer is the single stranded oligonucleotide sequence with sequence 7,8,9 in the sequence table and 10, and program is:
The 2nd takes turns that used nested primers is the single stranded oligonucleotide sequence with sequence 5 in the sequence table in the amplification, and the sequence of random primer is the single stranded oligonucleotide sequence with sequence 7,8,9 in the sequence table and 10, and program is:
(5) according to a conventional method the TAIL-PCR product in the step (4) is carried out T-A clone, order-checking.Use PCR method screening positive clone, the primer is the single stranded oligonucleotide sequence with sequence 5 and sequence 6 in the sequence table.
Sequence table
＜110〉Northeast Forestry University
＜120〉a kind of method of cloning flanking plant DNA sequence of multi-copied T-DNA
<160>10
<170>PatentIn?version?3.3
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<220>
Forward primer when＜223〉the TAIL-PCR first round is increased T-DNA right margin side DNA of plants sequence
<400>1
ttgccggtct?tgcgatgatt?at 22
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
Forward primer when＜223〉TAIL-PCR second takes turns amplification T-DNA right margin side DNA of plants sequence; PCR method sieve
Forward primer when selecting positive colony.
<400>2
gtttttatga?ttagagtccc?gcaa 24
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse primer during PCR method screening positive clone.
<400>3
ctagtaacat?agatgacacc?gc 22
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
Forward primer when＜223〉the TAIL-PCR first round is increased T-DNA left margin side DNA of plants sequence.
<400>4
atgtgtgagt?agttcccaga?taagg 25
<210>5
<211>24
<212>DNA
＜213〉artificial sequence
<220>
Forward primer when＜223〉TAIL-PCR second takes turns amplification T-DNA left margin side DNA of plants sequence; PCR method sieve
Reverse primer when selecting positive colony.
<400>5
tcgctcatgt?gttgagcata?taag 24
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward primer during PCR method screening positive clone.
<400>6
attcggggga?tctggatttt?agtac 25
<210>7
<211>16
<212>DNA
＜213〉artificial sequence
<220>
Random primer when＜223〉TAIL-PCR increases.
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉s=g or c
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉w=a or t
<220>
<221>misc_feature
<222>(11)..(11)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉w=a or t
<400>7
ngtcgaswga?nawgaa 16
<210>8
<211>16
<212>DNA
＜213〉artificial sequence
<220>
Random primer when＜223〉TAIL-PCR increases.
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉w=a or t
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉s=g or c
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉s=g or c
<400>8
tgwgnagsan?casaga 16
<210>9
<211>16
<212>DNA
＜213〉artificial sequence
<220>
Random primer when＜223〉TAIL-PCR increases.
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉w=a or t
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉w=a or t
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉w=a or t
<400>9
agwgnagwan?cawagg 16
<210>10
<211>16
<212>DNA
＜213〉artificial sequence
<220>
Random primer when＜223〉TAIL-PCR increases.
<220>
<221>misc_feature
<222>(1)..(1)
＜223〉s=g or c
<220>
<221>misc_feature
<222>(5)..(5)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(8)..(8)
＜223〉s=g or c
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉n=a or g or c or t
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉n=a or g or c or t
<400>10
sttgntastn?ctntgc 16
Claims (1)
1. flanking plant DNA sequence of multi-copied T-DNA method in the cloned, transgenic plant may further comprise the steps:
(1) ordinary method is extracted the transgenic plant genomic dna;
(2) DNA described in the step (1) is carried out endonuclease reaction, described endonuclease reaction is single endonuclease digestion or double digestion, single endonuclease digestion carries out in NOS poly A district or in 35S poly A district, double digestion is for carrying out at NOS poly A and 35S poly A district simultaneously, carrying out the restriction enzyme that enzyme cuts in NOS poly A district is AflII or TfiI or BslI or HpaII or their isozyme, carrying out the restriction enzyme that enzyme cuts in 35S poly A district is MslI or AquI or XhoI or McrI or PvuI or their isozyme, cuts simultaneously in NOS polyA district and 35S polyA district with the SmlI enzyme;
(3) ordinary method is cut product with the enzyme described in the step (2) and is separated into two portions, reclaims the fragment part greater than 5kb;
(4) be template with the recovery fragment described in the step (3), carry out TAIL-PCR amplification 2 and take turns;
(5) the TAIL-PCR product described in purifying, clone step (4) according to a conventional method, use PCR method screening positive clone after ordinary method check order, obtain the DNA of plants sequence of T-DNA side.
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| CN101285084B true CN101285084B (en) | 2011-08-17 |
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| CN103757120A (en) * | 2014-01-23 | 2014-04-30 | 宁夏林业研究所股份有限公司 | DNA (deoxyribonucleic acid) detection method for quickly distinguishing transgenic plant and product thereof |
| CN104059932B (en) * | 2014-06-05 | 2016-05-18 | 中国农业大学 | The construction method of single endonuclease digestion carrier |
| CN108103169A (en) * | 2017-12-11 | 2018-06-01 | 西北大学 | A kind of PCR method of the connector mediation based on hot asymmetric reaction |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995348A (en) * | 2006-12-22 | 2007-07-11 | 浙江大学 | Cabbage pollen outer wall protein gene BcMF5 promotor and its separation method |
| CN101037705A (en) * | 2006-03-15 | 2007-09-19 | 华中农业大学 | Method for appraising albino seeding gene type wound curing |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037705A (en) * | 2006-03-15 | 2007-09-19 | 华中农业大学 | Method for appraising albino seeding gene type wound curing |
| CN1995348A (en) * | 2006-12-22 | 2007-07-11 | 浙江大学 | Cabbage pollen outer wall protein gene BcMF5 promotor and its separation method |
Non-Patent Citations (4)
| Title |
|---|
| Jeong Hoe Kim et al.Analysis of junctions between T-DNA and plant genome in transgenic Arabidopsis thaliana.《Journal of Biology》.2007,第50卷(第4期),455-460. * |
| JP特开2006-115813A 2006.05.11 |
| 罗丽娟等.一种DNA侧翼序列分离技术—TAIL-PCR.《南京林业大学学报（自然科学版）》.2003,第27卷(第4期),87-90. * |
| 蒋红等.蜘蛛杀虫肽基因的合成及其在植物中表达质粒的构建.《植物学报》.1995,第37卷(第4期),321-325. * |
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