CN107418953A - A kind of method that corn phosphorus signal responsive genes are expanded in teosinte - Google Patents
A kind of method that corn phosphorus signal responsive genes are expanded in teosinte Download PDFInfo
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- CN107418953A CN107418953A CN201710817893.2A CN201710817893A CN107418953A CN 107418953 A CN107418953 A CN 107418953A CN 201710817893 A CN201710817893 A CN 201710817893A CN 107418953 A CN107418953 A CN 107418953A
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a kind of method that corn phosphorus signal responsive genes are expanded in teosinte.This method, which is included in teosinte, extracts corn phosphorus signal responsive genes STb gene to be measured, the primer sets for being utilized respectively 5 primer pair compositions carry out pcr amplification reaction, cloning and sequencing is carried out respectively, finally splices sequence, obtains corn phosphorus signal responsive genes whole genome sequence.The nucleotide sequence of 5 primer pairs is as shown in SEQ ID NO.4 SEQ ID NO.13.The invention also discloses a kind of application of above-mentioned primer sets and method in terms of corn phosphorus signal responsive genes or genome amplification.The present invention amplifies the gene order of corn phosphorus signal responsive genes using PCR amplification techniques in teosinte first.The present invention primer pair amplifies come out purpose band it is bright and single, it was demonstrated that primer pair of the invention it is specific good.
Description
Technical field
The invention belongs to genetic engineering field, specifically, is related to one kind and the response of corn phosphorus signal is expanded in teosinte
The method of gene.
Background technology
Corn is as the multiduty first grande culture thing such as feed, grain, the raw material of industry, to China's industrial expansion and people
The life of the people plays huge impetus.And ancestors of the teosinte as corn, its many gene tame process in corn
In all receive artificial selection, for example, teosinte is the very strong plant of tillering ability, but corn does not have tillering ability, will be with
The relevant tb1 genes of teosinte tiller are transferred in corn, cause the tiller of corn to increase on foot.Illustrate during domestication, teosinte
In gene seriously receive artificial selection.Certainly some genes also by artificial selection, are not protected during long-term domestication
It is left behind.
The content of the invention
In view of this, the invention provides a kind of method that corn phosphorus signal responsive genes are expanded in teosinte.
In order to solve the above-mentioned technical problem, the invention discloses corn phosphorus signal responsive genes are expanded in a kind of teosinte
Primer sets, including following five primer pairs, it is specially：
1-F:5'-TGCCTGAATAAAGATAAGTATG-3'；Its nucleotide sequence is as shown in SEQ ID NO.4；
1-R:5'-AGCAGTACAGTATCCAACAAG-3'；Its nucleotide sequence is as shown in SEQ ID NO.5；
2-1F:5'-ATCGCCGGGAGCATCTCAAAC-3'；Its nucleotide sequence is as shown in SEQ ID NO.6；
2-1R:5'-TGCAGGTCCAGACCAGCAACG-3'；Its nucleotide sequence is as shown in SEQ ID NO.7；
2-2F:5'-TGCTGCTTGACTAGCTTTCTGA-3'；Its nucleotide sequence is as shown in SEQ ID NO.8；
2-2R:5'-TTCCTTCCTTTCCCTTCCCTC-3'；Its nucleotide sequence is as shown in SEQ ID NO.9；
3-1F:5'-TGGAGCGATTTGTGGACCTGG-3'；Its nucleotide sequence is as shown in SEQ ID NO.10；
3-1R:5'-AGCTCACGGACTTGGCGGTTT-3'；Its nucleotide sequence is as shown in SEQ ID NO.11；
3-2F:5'-AGGGAAGGGAAAGGAAGGAAG-3'；Its nucleotide sequence is as shown in SEQ ID NO.12；
3-2R:5'-ATCACATCTCGCACAGGCATC-3'；Its nucleotide sequence is as shown in SEQ ID NO.13.
The invention also discloses a kind of method that corn phosphorus signal responsive genes are expanded in teosinte, including following step
Suddenly：Corn phosphorus signal responsive genes STb gene to be measured is extracted in teosinte, above-mentioned 5 primer pairs is utilized respectively and enters performing PCR amplification
Reaction, carries out cloning and sequencing, finally splices sequence, obtain corn phosphorus signal responsive genes whole genome sequence respectively.
Further, the primer pair 1,2-1,2-2,3-1,3-2 annealing temperature be respectively 52.00 DEG C, 63.30 DEG C,
60.00 DEG C, 63.90 DEG C and 60.20 DEG C.
Further, the program of the pcr amplification reaction is：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 15s, annealing temperature
Lower annealing 15s, 72 DEG C of extension 45min, is circulated 35 times；72 DEG C extend 5min eventually；It is each that the annealing temperature corresponds to 5 primer pairs
Self-corresponding annealing temperature.
Further, the system of the pcr amplification reaction is：μ L of Phanta Max Buffer 12.5, genomic DNA
2.0 μ L, μ L of dNTP Mix 0.5, μ L of sense primer 1.0, the μ L of anti-sense primer 1.0,1U/ μ L Phanta Max Super-
Fidelity DNA Polymerase0.5μL、ddH2O7.5μL。
The invention also discloses a kind of above-mentioned primer sets in terms of corn phosphorus signal responsive genes or genome amplification
Using.
The invention also discloses a kind of above-mentioned method answering in terms of corn phosphorus signal responsive genes or genome amplification
With.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention amplifies the gene sequence of corn phosphorus signal responsive genes using PCR amplification techniques in teosinte first
Row.
2) the purpose band that primer pair amplifies of the invention come out is bright and single, it was demonstrated that the specificity of primer pair of the invention
It is good.
Certainly, any product for implementing the present invention it is not absolutely required to reach all the above technique effect simultaneously.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, forms the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the first paragraph PCR amplification electrophoretograms of the present invention；Wherein, 1 purpose band is represented, 2 represent different size DNA's
Marker；
Fig. 2 is the second segment PCR amplification electrophoretograms of the present invention；Wherein, the 1 purpose band for representing the segment of second segment first, 2 generations
Table different size DNA Marker, 3 represent the purpose band of the segment of second segment second；
Fig. 3 is the PCR amplification electrophoretograms of the 3rd section of the first segment of the present invention；Wherein, 1 purpose band is represented, 2 represent difference
Size DNA Marker；
Fig. 4 is the PCR amplification electrophoretograms of the 3rd section of the second segment of the present invention；Wherein, 1 represent purpose and arrive, 2 represent difference
Size DNA Marker；
Fig. 5 is the comparison result of the non-full length sequence of PCR amplification genes of the present invention；Wherein, the first row (a) is to represent data
The new gene original reference sequence that storehouse is downloaded, the second row (b) are that the sequence come is amplified in teosinte.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technical effect.
The method that embodiment 1 expands corn phosphorus signal responsive genes in teosinte
1. special primer designs
New gene ZmUBA-like (GRMZM2G160990) gene order is downloaded in MaizeGDB databases.New base
The total length of cause is 2626bp (as shown in SEQ ID NO.1).In order to amplify the non-total length of gene, respectively increase before and after sequence
500bp fragments (as shown in SEQ ID NO.2) expand to enter performing PCR.Sequence is divided into 3 sections, because sequence variations are larger, again will
Second segment, the 3rd section is respectively divided into two segments, and the segment of second segment first is from 993-1968bp, and the second segment is from 1724-2812bp；The
Three section of first segment is from 2187-3148bp, and the second segment is from 2793-3449bp；The present invention, which needs to be segmented to expand just expand, to be come, no
Segmentation can not expand fragment；It is and as follows to each section of design special primer, primer specifying information using Premier5.0：
1-F:5'-TGCCTGAATAAAGATAAGTATG-3'；1-R:5'-AGCAGTACAGTATCCAACAAG-3'；
2-1F:5'-ATCGCCGGGAGCATCTCAAAC-3'；2-1R:5'-TGCAGGTCCAGACCAGCAACG-3'；
2-2F:5'-TGCTGCTTGACTAGCTTTCTGA-3'；2-2R:5'-TTCCTTCCTTTCCCTTCCCTC-3'；
3-1F:5'-TGGAGCGATTTGTGGACCTGG-3'；3-1R:5'-AGCTCACGGACTTGGCGGTTT-3'；
3-2F:5'-AGGGAAGGGAAAGGAAGGAAG-3'；3-2R:5'-ATCACATCTCGCACAGGCATC-3'；
The primer annealing temperature of table 1
| Primer numbers | Annealing temperature (DEG C) |
| 1 | 52.00 |
| 2-1 | 63.30 |
| 2-2 | 60.00 |
| 3-1 | 63.90 |
| 3-2 | 60.20 |
2.PCR is expanded and detection
2.1PCR reaction system
PCR reactions are using 25 μ L system, specific composition：
The PCR reaction systems of table 2
2.2PCR amplification program
The PCR of table 3 amplification program
2.3 electrophoresis detections and sequencing analysis
Product is splined on 2% agarose gel electrophoresis, and 170V electrophoresis 30min, observation electrophoresis result is (such as Fig. 1-Fig. 4 institutes
Show), its result shows that primer 1, primer 3-1 specificity are good, and it is bright and single to amplify the purpose band come；Primer 2-1,2-2 and 3-2
The purpose band amplified is bright but has the miscellaneous band that can be separated to have little to no effect result.The PCR primer of purposeful band is sent
Company is sequenced.Sequencing result is spliced (splicing result SEQ ID NO.3) with software DNASTAR-SeqMan, uses software
DANMAN and the new gene sequence of download carry out contrast and see the sequence correctness for amplifying and (shown in result figure 5)；Compare knot
Fruit shows that similarity reaches 83.62%, illustrates that amplification is correct, it was demonstrated that the gene in teosinte be present, also in certain journey
Illustrate that teosinte exists with corn in evolution on degree to contact.
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification
And environment, and can be carried out in the scope of the invention is set forth herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in power appended by invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit requires.
Sequence table
<110>Sichuan Agricultural University
<120>A kind of method that corn phosphorus signal responsive genes are expanded in teosinte
<130> 2017
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2626
<212> DNA
<213>Corn phosphorus signal responsive genes (ZmUBA-like)
<400> 1
ggtaacaggt gtccgtacct ggtgggaaat tttctattga catctctgtt gcagaggatg 60
agtaatgcaa ttcacagtta caaaaaaaca tctgacaagc tgatgtaaac ccgtcatgta 120
ctcatataag aaaggcatat aatataagct cgcacctggt tacaagcaca tatgcccatt 180
aaaagtgagg gcatatgcca acagcgacca gtgatcactc agccaaccta cttatcttat 240
tccataccga ccttcagctg aaaaaggtag aaggatccgt tctaatctgc tggctgatgg 300
gggaaaaaag agccagataa aatagacccg acgagctccc agctgcaata cgtatagtag 360
aatgctgtgt ggaagcagtg aaatgcaaga ccgcgcaccg tccaccgcta tgctgaccat 420
tccctagggg acgatgttac attgctgcca gcattgagct tatcaaccgg tagtacaggt 480
tgggcggcag taatcgccgg gagcatctca aactgatccc gtgggtaccc taccacgctg 540
gacctggacg tttcaaccat agtatttcca aatggatggt tgtgcacgac ctgtggtccc 600
aagggatggt acgccataca ggcgatggca ggaggctgag gcagaagagc tacaacggct 660
gcagttgcag ggcagttaga agggttcttg tatgcggagc tgtaggtctg gccgtttcct 720
tgaacagcat agggtgcggc gctgcagtga ctgggcttgc ctggctgagg tggacaggag 780
gggccctggc tgtgtagctg cactggttgc ggttgctgat gctgagcgtt atgatctgct 840
acaggaagca atactgtgtt gccttggcca tagaatgaat gggcaacttc atgctgctgc 900
tgctggtatt gctgctgtga tgaagagtat ggaggctgcc cagccattct tcttgtaatt 960
gtctccgtgt tacaactcga aggtttcacg gaactgaatg gaggaaacaa gggtgtggct 1020
tgtggagcgc tcagctgaga ctgaggagac gaggacggct gctgagccac caccatatgt 1080
cgtgaaactt gttggatact gtactgctgc tgttgcactt gcaccatggg ctgctgggga 1140
tgccatgtat gcagatcaac ctgctgggta ggcgtctgca gcctgtgact ctgagtctga 1200
ggctgagggt acagttcagc tgctgctgct tgactagctt tctgaggcag ctcgacttgc 1260
accacggcat cagggctgtg cgcaagcggc tgcacaacct gaaccagagg ctccaacgcc 1320
gccttgccct cggtgcattt agcacggtcc cggctttgca cgtagaacac gggagcctgg 1380
ttcagctgct gctgctcatg ctgctgcgtg ctcaaagcga cgggcaccag gtgctgcaga 1440
acgaagccgt tgctggtctg gacctgcacg ggcacggcag gggagtgcat cgtcccattg 1500
acctggtgaa gcggcaaaag ggcgacctcg tgttttgggg ctcgcgtgct gtggccgttt 1560
tcccttgccg cgagcatggg gaaaccggcg gcctcaccct tcgtctgaga aggcgcctcg 1620
tgtgctatct gaagcttgct gagttctttt gcagtttcgg ctaagtcttg tctatcctga 1680
agaagctgga gcgatttgtg gacctggcaa cccaaaatgt agatctagta gttatcaaaa 1740
gatgatgtta gtctcacatc caaagtgatg cagaatctca tctccgggaa ccataagttg 1800
actccactga atgtactagg tttcagggaa acagactatc attttactac accaaaccaa 1860
acataagtga gaagtccaaa tcgcactgcc attgcactcg tcggcgcgag aagccggtgc 1920
accgggatcc agtctttatc tacctgtctg aggttcctgt cgatgcattt gaagttggcg 1980
ttgctgtcgc tgtggtagtc catgacgtcg ctgcgcagct ccccgatgga cctctcgagc 2040
ttgtagcagt agagctcgag ctgggagagc ctgcccccga tggcgccgag cgagtgcagc 2100
agtgcgtcgg cctgcttccg cgtgcactcc tccacggccg ccaccacggc gtccttgggg 2160
tacaagaaac cgtcctgctg ctgctgctgc tgctgcggcg gcggcggctg gccgtaggcg 2220
gccacgaacg tccgcaccag cctcccgtcc tgcaagccct gtgcccggcg cgagtcggcc 2280
tgtcaggcgc cgagggaagg gaaaggaagg aagaaaattc ggaatcgaga gagcgcgacg 2340
agagcagagc ggagcggagc ggagtgcggg cacctcgcgg cggcggggca ggggctgatc 2400
gtaggagcag cgcgcgtccg ggggcaggcc gaaggcgccg gagacggagg cggcagcggg 2460
gcgcgcgggg gacgccatgc gcgggctaga gttttgggtt ggtgccgtga ggtgaggagc 2520
cggagctggg ggtgggaatc cgaggcggga ggaggaagcg gcggggaaag agagggagga 2580
gtggagggcg gctatgcact tgcagttgca ggcagccccc caatgt 2626
<210> 2
<211> 3626
<212> DNA
<213>Corn phosphorus signal responsive genes (ZmUBA-like)
<400> 2
cttacggtta gagtatgaaa agattttaat cctcgtaggt gcaaatttgg gagaaaatct 60
ttttccattg ggtatggtgg gtatgggtat gagaaaccag aatccgaatc cgattagaaa 120
agttgggtat gtagctcaat agaaaataga aaataagcct aacaactagc tctatatctc 180
ctaacccttt taagcaccca gctacccaac ctagacgatt gactaccagt cgtcctgccc 240
accggtccac atgctaattt gtgttttgct atctgtggtt aaacaatatt tttttgttac 300
atgtcagatt tattgatgat gtattatcgt tgggtgtcgc atgattatta atattatata 360
atgtgatcat gtttttcttg ttagggtctt agggatggag gcccattaaa gagccaatgc 420
ctgaataaag ataagtatgg gatgaacctt atacccatga cggatacgaa gatgtagatg 480
agtgtaactt cataggaatg ggtaacaggt gtccgtacct ggtgggaaat tttctattga 540
catctctgtt gcagaggatg agtaatgcaa ttcacagtta caaaaaaaca tctgacaagc 600
tgatgtaaac ccgtcatgta ctcatataag aaaggcatat aatataagct cgcacctggt 660
tacaagcaca tatgcccatt aaaagtgagg gcatatgcca acagcgacca gtgatcactc 720
agccaaccta cttatcttat tccataccga ccttcagctg aaaaaggtag aaggatccgt 780
tctaatctgc tggctgatgg gggaaaaaag agccagataa aatagacccg acgagctccc 840
agctgcaata cgtatagtag aatgctgtgt ggaagcagtg aaatgcaaga ccgcgcaccg 900
tccaccgcta tgctgaccat tccctagggg acgatgttac attgctgcca gcattgagct 960
tatcaaccgg tagtacaggt tgggcggcag taatcgccgg gagcatctca aactgatccc 1020
gtgggtaccc taccacgctg gacctggacg tttcaaccat agtatttcca aatggatggt 1080
tgtgcacgac ctgtggtccc aagggatggt acgccataca ggcgatggca ggaggctgag 1140
gcagaagagc tacaacggct gcagttgcag ggcagttaga agggttcttg tatgcggagc 1200
tgtaggtctg gccgtttcct tgaacagcat agggtgcggc gctgcagtga ctgggcttgc 1260
ctggctgagg tggacaggag gggccctggc tgtgtagctg cactggttgc ggttgctgat 1320
gctgagcgtt atgatctgct acaggaagca atactgtgtt gccttggcca tagaatgaat 1380
gggcaacttc atgctgctgc tgctggtatt gctgctgtga tgaagagtat ggaggctgcc 1440
cagccattct tcttgtaatt gtctccgtgt tacaactcga aggtttcacg gaactgaatg 1500
gaggaaacaa gggtgtggct tgtggagcgc tcagctgaga ctgaggagac gaggacggct 1560
gctgagccac caccatatgt cgtgaaactt gttggatact gtactgctgc tgttgcactt 1620
gcaccatggg ctgctgggga tgccatgtat gcagatcaac ctgctgggta ggcgtctgca 1680
gcctgtgact ctgagtctga ggctgagggt acagttcagc tgctgctgct tgactagctt 1740
tctgaggcag ctcgacttgc accacggcat cagggctgtg cgcaagcggc tgcacaacct 1800
gaaccagagg ctccaacgcc gccttgccct cggtgcattt agcacggtcc cggctttgca 1860
cgtagaacac gggagcctgg ttcagctgct gctgctcatg ctgctgcgtg ctcaaagcga 1920
cgggcaccag gtgctgcaga acgaagccgt tgctggtctg gacctgcacg ggcacggcag 1980
gggagtgcat cgtcccattg acctggtgaa gcggcaaaag ggcgacctcg tgttttgggg 2040
ctcgcgtgct gtggccgttt tcccttgccg cgagcatggg gaaaccggcg gcctcaccct 2100
tcgtctgaga aggcgcctcg tgtgctatct gaagcttgct gagttctttt gcagtttcgg 2160
ctaagtcttg tctatcctga agaagctgga gcgatttgtg gacctggcaa cccaaaatgt 2220
agatctagta gttatcaaaa gatgatgtta gtctcacatc caaagtgatg cagaatctca 2280
tctccgggaa ccataagttg actccactga atgtactagg tttcagggaa acagactatc 2340
attttactac accaaaccaa acataagtga gaagtccaaa tcgcactgcc attgcactcg 2400
tcggcgcgag aagccggtgc accgggatcc agtctttatc tacctgtctg aggttcctgt 2460
cgatgcattt gaagttggcg ttgctgtcgc tgtggtagtc catgacgtcg ctgcgcagct 2520
ccccgatgga cctctcgagc ttgtagcagt agagctcgag ctgggagagc ctgcccccga 2580
tggcgccgag cgagtgcagc agtgcgtcgg cctgcttccg cgtgcactcc tccacggccg 2640
ccaccacggc gtccttgggg tacaagaaac cgtcctgctg ctgctgctgc tgctgcggcg 2700
gcggcggctg gccgtaggcg gccacgaacg tccgcaccag cctcccgtcc tgcaagccct 2760
gtgcccggcg cgagtcggcc tgtcaggcgc cgagggaagg gaaaggaagg aagaaaattc 2820
ggaatcgaga gagcgcgacg agagcagagc ggagcggagc ggagtgcggg cacctcgcgg 2880
cggcggggca ggggctgatc gtaggagcag cgcgcgtccg ggggcaggcc gaaggcgccg 2940
gagacggagg cggcagcggg gcgcgcgggg gacgccatgc gcgggctaga gttttgggtt 3000
ggtgccgtga ggtgaggagc cggagctggg ggtgggaatc cgaggcggga ggaggaagcg 3060
gcggggaaag agagggagga gtggagggcg gctatgcact tgcagttgca ggcagccccc 3120
caatgtaaaa ccgccaagtc cgtgagctgc ctatagcgcc gagagcgagc tcgaggtgcg 3180
gagcggggag gaataaaaac gggtgtgggg ctggggaagg cgggaagcgg agagcgagcg 3240
tggtggacct cggagtgcga gacggatatg ccaaggtatt tgcttgagct ctcggcggag 3300
tccgtccgca tgtgggctga gatccggcga aatctggtgg gggcgtggcg gtggataggc 3360
ctctaggggt gacctgccgt gctcgtgctc tgctgctcct gcgggctgcg gctgtggcgc 3420
ggattcggga tgcctgtgcg agatgtgatc ggtttccgtt ccgatgacgt tttgttgggt 3480
gctccatcgc tgtccgcctc gtgcgccatg tgcgtgatta ttaagaaatg taaacgcatc 3540
aggcctccaa gaaggtactc tggttttctt tcccaggacc ggttagctgg tactcccttt 3600
ccctccatca ctcatctttt tcaagt 3626
<210> 3
<211> 2997
<212> DNA
<213>Corn phosphorus signal responsive genes (ZmUBA-like)
<400> 3
atacacatct cgcacaggca tcccgaatcc acgccacagc tgcaggagcg tgcagcagag 60
cacgagcacg gcaggtcatc cctagaggcc ctatccaccg ccacgccccc accagatttc 120
gccggatctc agcccacatg cggacggact ccgccgagag ctcaagcaaa taccttggca 180
tatccgtctc gctctccgag gtccaccacg ctcgctctcc gcttccccag ctccataccc 240
gtttttattc ctccccgccc cacacctcga gctcgctctt cggcgctata ggcagctcac 300
ggacttgcgg ttttacatcg gggctgcctg caastgcatc gcatagccgc cctccactcc 360
tccctctctt tccccgccgc ttcctcctcc cgcctcggat tcccacccaa ccccccagct 420
csggctcctc acctcacrgc accaacccaa aaccctagcc cgcgcatggc rtcccccgcg 480
cgccccgctg ccgcctccgt ctccggcgcc ttcggcctgc ccccggacgc gcgctgctcc 540
tacgaccatc ccctgccccg ccgccgcgag gtgcccgcrs tccgctccgc tctgcyctcg 600
tcgcgctctc tcgattccga attttcttcc ttccyttccc ttccctcrrc gcctgacagg 660
ccgactcgcg ccgggcacag ggcttgcagg acgggaggct grtgcggacg ttcgtggccg 720
sctacggcca gccgcagcag ccgccgccgc cgccgcagca gcagcagcag caacagcagg 780
acggtttctt ttacccccag gacgccgtgg tggcggcctt ggaggartgc acgcggaagc 840
aggccgacgc gctgctgcac tcgatcggcg ccatcggggg caggctctcc cagctcgagc 900
tctactgcta caagctcgag aggtccatcg gggagctgcg cagcgacgtc atggactacc 960
acagcgacag cgccgccaac ttcagatgca tcgacaggaa cctcagacag gtagataaag 1020
attggatccc ggtgcaccgg cttctcgcgc cgacgacgat tgcaatgrca gtgcgatttg 1080
gacttctcac tyatgtttgg tttggtgtag taaaatgatt gtctgtttcc ctgaaaccta 1140
gtacattcag tggagtcaac ttatggttcc cggagatgag attctgcatc actttgcatg 1200
tgagactaac agcatctttt gataactact agatctacat tttggttgcc aggtccacaa 1260
atcgctccag cttcttcagg atagacaaga cttagccgaa actgcaaaag aactcagcaa 1320
gcttcagata gcacacgagg cgccttctca gaagggtgag ggtggggccg ccggtttccc 1380
catgcccgcg gcaagggaaa acggccacag cacgcgagct ccaaaacacg aggtcgccct 1440
tttgccgctt caccaggtca atgggacgat gcactcccct gccgtgcccg tgcaggtcca 1500
gaccagcaac ggcttcgttc tgcagcacct ggtgcccgtc tctttgagca cgcagcagca 1560
tgagcagcag cagctgaacc aggctcccgt gttctacgtg caaagccggg accgtgctaa 1620
atgcaccgag ggcaaggcgg cgttggagcc tctggttcag gttgtgcagc cgcttgcgca 1680
cagccctgat gccatggtgc aagtcgagct gcctcagaaa gctagtcaag cagcagcagc 1740
agcagcagct gaactgtacc ctcagcctca gactcasagk cacaggctgc atacgcctac 1800
ccagcaagtt gatctgcrta catggcatca tcagcagccc atggtgcaag tgcaacagcr 1860
gcagtacagt atccaacaag tttcacgaca tatggtggtg ggtcagcagg cgtcctcgtc 1920
tcctcagtct cagctgagcg ctccacaagc cacacccttg tttcctccat tcagttccgy 1980
gaaaccttcg agttgtaaca ccgagacaat tacaagaaga atggctgggc agcctccata 2040
cccttcatca cagcagcart accascagca gcagcatgaa gttgcccatt cattctttgg 2100
ccaaggcaac acagtaytgc ttcctgtasc agatcataac ggtcagcatc agcaaccrca 2160
rccagtgcag ctacacagcc agcaggcccc yyttgtccgc ctcagccagg caggcccagg 2220
cactgttgcg ccgcatcatc ctatgctgtc caaggaaatg gccagaacta cwkctccaga 2280
tacaagaacc cttctaactg ccctgcaact gcagccgttg tagctcttct gcctcagcct 2340
cctgccaccg cctgtatggc gtaccrtccc ttgggaccac aggtcgtgca cacccatcca 2400
tttggaaata ctaaggctga aacgtccagc gtggtagggt acccacggga tcagtttgag 2460
atgctcccgg cgattactgc cgcccaacct gtactaccgg ttgataagct caatgctggc 2520
agcaatgtag catcgtcccc tcgggaatgg ttagcatagt ggtggacggt gcgaggtctt 2580
gcatttcact gcttccacac agcattctac tatacgtatt gcagctggga gctcgtcggg 2640
tctattttat ctggctcttt ttccccccat cagccagcag attagaacgg atccttctac 2700
cttttttcag ctgaaggtcg gtatggaata agataagtag gttggctgag tgatcactgg 2760
tcgctgttgg catatgccct cacttttaat gggcatatgt gcttgtaacc aggtgcgagc 2820
ttatattata tgcctttctt atatgagtac atgacgggtt tacatcagct tgtcagatgt 2880
ttttttgtaa ctgtgaattg cattactcat cctctgcaac agagatgtca atagaaaatt 2940
tcccaccagg tacggacacc tgttacccat tcctatgaag ttacactcat ctacatc 2997
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tgcctgaata aagataagta tg 22
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
agcagtacag tatccaacaa g 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
atcgccggga gcatctcaaa c 21
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
tgcaggtcca gaccagcaac g 21
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
tgctgcttga ctagctttct ga 22
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ttccttcctt tcccttccct c 21
<210> 10
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
tggagcgatt tgtggacctg g 21
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
agctcacgga cttggcggtt t 21
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
agggaaggga aaggaaggaa g 21
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
atcacatctc gcacaggcat c 21
Claims (7)
1. the primer sets of corn phosphorus signal responsive genes are expanded in a kind of teosinte, it is characterised in that including following five primers
It is right, be specially：
1-F:5'-TGCCTGAATAAAGATAAGTATG-3'；Its nucleotide sequence is as shown in SEQ ID NO.4；
1-R:5'-AGCAGTACAGTATCCAACAAG-3'；Its nucleotide sequence is as shown in SEQ ID NO.5；
2-1F:5'-ATCGCCGGGAGCATCTCAAAC-3'；Its nucleotide sequence is as shown in SEQ ID NO.6；
2-1R:5'-TGCAGGTCCAGACCAGCAACG-3'；Its nucleotide sequence is as shown in SEQ ID NO.7；
2-2F:5'-TGCTGCTTGACTAGCTTTCTGA-3'；Its nucleotide sequence is as shown in SEQ ID NO.8；
2-2R:5'-TTCCTTCCTTTCCCTTCCCTC-3'；Its nucleotide sequence is as shown in SEQ ID NO.9；
3-1F:5'-TGGAGCGATTTGTGGACCTGG-3'；Its nucleotide sequence is as shown in SEQ ID NO.10；
3-1R:5'-AGCTCACGGACTTGGCGGTTT-3'；Its nucleotide sequence is as shown in SEQ ID NO.11；
3-2F:5'-AGGGAAGGGAAAGGAAGGAAG-3'；Its nucleotide sequence is as shown in SEQ ID NO.12；
3-2R:5'-ATCACATCTCGCACAGGCATC-3'；Its nucleotide sequence is as shown in SEQ ID NO.13.
A kind of 2. method that corn phosphorus signal responsive genes are expanded in teosinte, it is characterised in that comprise the following steps：Big
Corn phosphorus signal responsive genes STb gene to be measured is extracted in hay grass, 5 primer pairs described in claim 1 is utilized respectively and enters performing PCR expansion
Increase reaction, carry out cloning and sequencing respectively, finally splice sequence, obtain corn phosphorus signal responsive genes whole genome sequence.
3. the method according to claim 2 that corn phosphorus signal responsive genes are expanded in teosinte, it is characterised in that institute
State primer pair 1,2-1,2-2,3-1,3-2 annealing temperature be respectively 52.00 DEG C, 63.30 DEG C, 60.00 DEG C, 63.90 DEG C and
60.20℃。
4. the method according to claim 3 that corn phosphorus signal responsive genes are expanded in teosinte, it is characterised in that institute
The program for stating pcr amplification reaction is：95 DEG C of pre-degeneration 3min；95 DEG C are denatured 15s, and anneal 15s under annealing temperature, 72 DEG C of extensions
45min, circulate 35 times；72 DEG C extend 5min eventually；The annealing temperature corresponds to each self-corresponding annealing temperature of 5 primer pairs.
5. the method according to claim 3 that corn phosphorus signal responsive genes are expanded in teosinte, it is characterised in that institute
The system for stating pcr amplification reaction is：μ L of Phanta Max B uffer 12.5, the μ L of genomic DNA 2.0, the μ of dNTP Mix 0.5
L, μ L of sense primer 1.0, the μ L of anti-sense primer 1.0,1U/ μ L Phanta Max Super-Fidelity DNA Pol
ymerase0.5μL、ddH2O7.5μL。
6. application of the primer sets described in claim 1 in terms of corn phosphorus signal responsive genes or genome amplification.
7. any claim methods described is in terms of corn phosphorus signal responsive genes or genome amplification in claim 2-5
Using.
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| CN102577926A (en) * | 2012-02-20 | 2012-07-18 | 唐祈林 | Method for breeding novel waterlogging-tolerant maize variety by using special waterlogging tolerance of teosinte |
| CN106434708A (en) * | 2016-08-24 | 2017-02-22 | 海南波莲水稻基因科技有限公司 | Rice MSP1 gene mutant, and molecular identification method and application thereof |
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|---|---|---|---|---|
| CN102577926A (en) * | 2012-02-20 | 2012-07-18 | 唐祈林 | Method for breeding novel waterlogging-tolerant maize variety by using special waterlogging tolerance of teosinte |
| CN106434708A (en) * | 2016-08-24 | 2017-02-22 | 海南波莲水稻基因科技有限公司 | Rice MSP1 gene mutant, and molecular identification method and application thereof |
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| ENSEMBL: "Gene： Zm00001d011562,Zea mays(B73_RefGen_v4)", 《ENSEMBL》 * |
| NCBI: "PREDICTED: Zea mays uncharacterized LOC103636164 (LOC103636164), mRNA，XM_008658513.2", 《NCBI GENBANK》 * |
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