CN110331229A - A kind of PCR primer and identification method for identifying CC genome wild rice - Google Patents
A kind of PCR primer and identification method for identifying CC genome wild rice Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of identification method of the identification primer and CC genome wild rice of identification CC genome wild rice.The sequence of identification primer of the present invention is as shown in SEQ ID No.5 and SEQ ID No.6.The identification method is: acquiring paddy rice root tip and blade to be identified, extracts blade total DNA；Chromosome number is identified with the tip of a root；The sample blade total DNA for the rice to be identified for being 24 to identify chromosome number treats sample using the primer combination of sequence such as SEQ ID No.5 and SEQ ID No.6 and carries out PCR amplification as template；PCR carries out agarose gel electrophoresis to PCR product after reaction, and the sample for as a result occurring specific amplification band at 317bp is that the positive of CC genome wild rice determines result.Methods and results of the invention are stable, easy to operate, efficient and at low cost.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of identification primer and CC for identifying CC genome wild rice
The identification method of genome wild rice.
Background technique
Polymerase chain reaction (polymerase chain reaction, abbreviation PCR) technology is by Mullis K etc.
(1987) technology (K.Mullis, the F.Faloona.Specific Synthesis of the amplification in vitro target gene initially set up
of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction.Methods in
Enzymology, 1987,155,335-350), this technology can expand specific DNA fragmentation within several hours hundreds of
Wan Bei, this rapid technology for obtaining a large amount of single nucleotide fragments have played important in modern molecular biology research
Effect, because it can largely obtain target DNA fragments in a short time, has convenient and quick feature.
Oryza includes the more wild seed rice in cultivated rice and 20, it has been found that have 10 caryotypes (AA, BB, BBCC,
CC, CCDD, EE, FF, GG, HHJJ, HHKK), wherein with 24 chromosomes diploid wild rice have AA, BB, CC, EE,
6 kinds of chromosome component types of FF, GG.Centromere is specific structural and functional region on eukaryocyte chromosome, it is to thin
The correct distribution of homologue/sister chromosomes plays a very important role during born of the same parents' mitosis and meiosis.
The genomic wild rice of CC includes oryza officinalis (Oryza officinalis), rhizome wild rice (O.rhizomatis)
With Oryza eichingeri (O.eichingeri) three classes.Existing research shows the genomic oryza officinalis of CC and FF genome
Oryza brachyantha (O.brachyantha) there is the special satellite DNA sequence in respective centromere；AA, BB and EE genome
Wild rice and cultivated rice, as Oryza glaberrima Steud (O.Glaberrima, AA genome), Oryza punctata (O.punctata,
BB genome) and Australia wild rice (O.australiensis, EE genome) centromere region all contain and Asia cultivate
Rice (O.sativa, AA genome) centromere repetitive sequence CentO and CRR homologous sequence；The genomic medicinal wild of CC
2 Autosome centromeres of raw rice have the homologous sequence of CentO, while it has also been found that its all centromere region all includes to plant
Train homologous sequence (Bao W, Zhang W, Yang Q, Yu Zhang, Bin Han, MH Gu, the YB Xue, ZK of rice CRR
Cheng, Diversity of centromeric repeats in two closely related wild rice
species,Oryza officinalis,and Oryza rhizomatis.Molecular Genetics&Genomics,
2006,275(5):421-30；HyeRan Lee,Wenli Zhang,Tim Langdon,Weiwei Jin,Huihuang
Yan,Zhukuan Cheng,and Jiming Jiang.Chromatin immunoprecipitation cloning
reveals rapid evolutionary patterns of centromeric DNA in Oryza
species.Proceedings of the National Academy of Sciences of the United States
of America,2005,102(33):11793-11798；Descendants pass lamp, Gong Zhiyun, Liang Guohua, Wang Feihua, Tang Shuzhu, Gu Ming
Flood, the separation and identification of Oryza different chromosomes group centromere BAC clone, heredity, 2007,29 (7): 851-858).Bao W
It includes oryza officinalis centromere BAC clone that et al. (2006) screening, which obtains, includes the weight in oryza officinalis centromere
Complex sequences.These results are between centromere the centromeric sequence of clone's Oryza different chromosomes group, research different chromosomes group
Relationship between evolutionary relationship and Oryza difference centromeric DNA sequence and function all has significance.Meanwhile also be we benefit
Possibility is provided with the chromosome of centromere distinguished sequence identification different chromosomes group.But there is no a kind of identification CC genomes
The report of three classes difference wild rice chromosome.
Summary of the invention
The object of the present invention is to provide a kind of PCR primers and CC genome wild rice for identifying CC genome wild rice
Identification method.
A kind of CC genome wild rice PCR primer of the present invention, can amplify from CC genome wild rice
Special band, the nucleotides sequence of upstream primer are classified as 5 '-TTCGCACCACATAGACTT-3 ' (SEQ ID No.5), under
The nucleotides sequence of trip primer is classified as 5 '-TTGGCTCCATTAGTTGCTA-3 ' (SEQ ID No.6).
Further, the invention also discloses a kind of methods for identifying CC genome wild rice, comprising the following steps: 1)
The extraction of the extraction acquisition and total DNA of the rice sample tip of a root and leaf harvest and blade total DNA to be identified；2) in step 1)
The tip of a root identifies chromosome number；3) identify that sample total DNA that chromosome number is 24 as template, utilizes sequence using in step 2)
The primer combination of SEQ ID No.5 and SEQ ID No.6 treats sample and carries out PCR amplification；；4) PCR is after reaction, right
PCR product carries out agarose gel electrophoresis, and the sample for as a result occurring specific amplification band at 317bp is CC genome
The positive of wild rice determines result.
Methods and results of the invention are stable, easy to operate, efficient and at low cost.
Detailed description of the invention
Fig. 1 is 5 pairs of primers in the PCR amplification of CC genome difference wild rice as a result, the wherein (upstream primer 39O03-1
The nucleotides sequence of primer is classified as 5 '-TTCGCACCACATAGACTT-3 ' (SEQ ID No.5), downstream the nucleotides sequence of primer
It is classified as 5 '-TTGGCTCCATTAGTTGCTA-3 ' (SEQ ID No.6)) there is special target item to the different wild rices of CC group
Band；(a-e) be respectively primer 39G20-1,39G20-2,39O03-1,39O03-2,39E03-1 PCR testing result.1,2 are
2 Oryza eichingeri EIC-7 (RGC81804) of separate sources and EIC-89 (RGC105415)；3,4 be 2 of separate sources
Rhizome wild rice RHI-24 (86466) and RHI-93 (105445)；It 5, is 6,2 oryza officinalis off-3 of separate sources
(80720) and off-66 (102460)；7,8 be AA genome cultivated rice OryzasativaLcv.Nipponbare (Nipponbare) and 02428.Cultivated rice
OryzasativaLcv.Nipponbare (Nipponbare) and 02428 saves (Hou L, Xu M, Zhang T, et al.Chromosome by this seminar
painting and its applications in cultivated and wild rice.Bmc Plant Biology,
2018,18(1):110.).Fig. 2 primer 39O03-1 is to cultivated rice, CC genome difference wild rice and other genomic
Wild rice PCR amplification result.1. Malaysia wild rice (RID-105, HHJJ, 106028)；2. short anther wild rice (FF,
104979)；3. Australia wild rice (EE, ALT-97,105685)；4. opening up clever wild rice (W-34, AA, 106341)；5. wart grain is wild
Rice (GG, RGC89244)；6. Oryza punctata (PNV-22, BB, 83802)；7. oryza officinalis (off-66, CC,
102460))；8. rhizome wild rice (RHI-24, CC, 86466)；9. Oryza eichingeri (EIC-7, RGC81804).
Fig. 3 be recycle target stripe of the primer 39O03-1 in oryza officinalis be connected to carrier T expand it is numerous after bacterium solution
PCR testing result；It is the testing result of 6 single colonies in figure, it is consistent with target stripe size (317bp) as the result is shown.
Specific embodiment
Embodiment 1: the screening of primer
(1) using the BAC clone BAC39G20 announced on the website NCBI containing oryza officinalis centromere region,
BAC39E03 and BAC39O03 sequence design 5 is to primer (table 1)；5 pairs of primers be respectively (39G20-1,39G20-2,39O03-1,
39O03-2 and 39E03-1), target stripe size is respectively 483bp, 329bp, 317bp, 432bp, 406bp (table 1)；
1. 5 pairs of CC genome wild rice centromere repetitive sequence amplimers of table
(2) 5 pairs of primer pair cultivated rices and CC genome difference wild rice, including oryza officinalis in (1) are utilized
(O.officinalis), rhizome wild rice (O.rhizomatis) and Oryza eichingeri (O.eichingeri) carry out PCR expansion
Increase, amplification is as shown in Figure 1, the amplification of primer 39G20-1,39G20-2,39O03-1,39O03-2 and 39E03-1 divide
Fig. 1 a, b, c, d and the e not being listed in Fig. 1 can be seen that primer 39G20-1 from Fig. 1 a and contaminate in cultivated rice (swimming lane 7 and 8) and CC
Colour solid group wild rice (swimming lane 1 to 6) has similar target stripe；Fig. 1 b is not the result shows that primer 39G20-2 expands in cultivated rice
Increase target stripe out, but the target stripe in CC genome wild rice (swimming lane 1 to 6) is not clear enough；Primer 39O03-2 exists
Target stripe is not amplified in cultivated rice, but the target stripe in CC genome wild rice (swimming lane 1 to 6) is also not clear enough
(Fig. 1 d)；There is band in cultivated rice in primer 39E03-1 near target stripe, and in CC genome wild rice (swimming lane 1
Target stripe in 6) is unintelligible (Fig. 1 e)；Primer 39O03-1 does not amplify target item in cultivated rice (swimming lane 7 and 8)
Band, and target stripe is amplified in genomic different wild rice (swimming lane 1 to the 6) kinds of CC, and band is clear (Fig. 1 c), because
This, we screen a pair does not have target stripe in cultivated rice, but has target item in CC genome difference wild rice
The primer 39O03-1 of band；
(3) then again with primer 39O03-1 to CC genome difference wild rice and other genomic wild rices into
Row PCR amplification further proves that primer 39O03-1 specifically can only amplify specific band in the genomic wild rice of CC
(Fig. 2)；
(4) target stripe of the recycling primer 39O03-1 in oryza officinalis be connected to carrier T expand it is numerous, to its into
Row PCR is detected, as the result is shown consistent (Fig. 3) of target stripe size and expection (317bp).
Embodiment 2: primer and its application
1, synthesis CC genome wild rice identifies that primer 39O03-1, the nucleotides sequence of upstream primer are classified as such as SEQ
ID No.5, the downstream nucleotide sequence of primer such as SEQ ID No.6；
2, paddy rice root tip and leaf sample to be detected are collected, determines that its chromosome number is 24 by the tip of a root, extraction has
24 chromosome wild rice blade total DNAs；
3, using the total DNA of primer and the middle extraction of step 2) in step 1) as template, survey is treated using the combination of above-mentioned primer
Sample DNA carry out PCR amplification, reaction condition are as follows: 95 DEG C 4 minutes, 95 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 32 circulation；
72 DEG C 6 minutes；
4, PCR after reaction, carries out agarose gel electrophoresis to PCR product, as a result occurs specificity at 317bp and expands
The sample for increasing band is that the positive of CC genome wild rice determines result (stripe size is as shown in Fig. 27,8 and 9).This
Methods and results are stable, easy to operate, efficient and at low cost.
SEQUENCE LISTING
<110>Yangzhou University
<120>a kind of PCR primer and identification method for identifying CC genome wild rice
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (manual sequence)
<400> 1
aattccttca cgagttggt 19
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 2
tgccgaacac attctcat 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 3
atgtgttcgg caccattg 18
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (manual sequence)
<400> 4
ccaccaactt gtgtaggaat 20
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 5
ttcgcaccac atagactt 18
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (manual sequence)
<400> 6
ttggctccat tagttgcta 19
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 7
atgaccaccg actcaagt 18
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 8
ggcatccagg aatgtgtt 18
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (manual sequence)
<400> 9
ttacggcagg ctcactaa 18
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (manual sequence)
<400> 10
gttcttacac gagttgatgg 20
Claims (2)
1. it is a kind of identify CC genome wild rice PCR primer, which is characterized in that the primer sequence such as SEQ ID No.5 and
Shown in SEQ ID No.6.
2. a kind of method for identifying CC genome wild rice, which comprises the following steps: the following steps are included: 1)
Paddy rice root tip and blade to be identified are acquired, blade total DNA is extracted；2) chromosome number is identified with the tip of a root in step 1)；3) with step
It is rapid 2) in identify that the sample blade total DNA of rice to be identified that chromosome number is 24 is template, utilize sequence such as SEQ ID
The primer combination of No.5 and SEQ ID No.6 treats sample and carries out PCR amplification；4) PCR after reaction, to PCR product into
Row agarose gel electrophoresis, the sample for as a result occurring specific amplification band at 317bp is CC genome wild rice
The positive determines result.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254160A (en) * | 2020-03-30 | 2020-06-09 | 扬州大学 | Protoplast verification method for efficiently identifying rice enhancer |
| CN113308563A (en) * | 2021-05-28 | 2021-08-27 | 扬州大学 | Method for rapidly developing Australian wild rice specific molecular marker |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701257A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
| CN107475426A (en) * | 2017-09-25 | 2017-12-15 | 山东省水稻研究所 | A kind of molecular labeling for differentiating cultivation rice varieties indica rice type and application |
| CN108949925A (en) * | 2018-07-06 | 2018-12-07 | 中国水稻研究所 | A kind of molecular detecting method for quick and precisely identifying Weedy Rice and cultivated rice |
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2019
- 2019-08-07 CN CN201910725242.XA patent/CN110331229A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701257A (en) * | 2009-11-13 | 2010-05-05 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
| CN107475426A (en) * | 2017-09-25 | 2017-12-15 | 山东省水稻研究所 | A kind of molecular labeling for differentiating cultivation rice varieties indica rice type and application |
| CN108949925A (en) * | 2018-07-06 | 2018-12-07 | 中国水稻研究所 | A kind of molecular detecting method for quick and precisely identifying Weedy Rice and cultivated rice |
Non-Patent Citations (1)
| Title |
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| 王为云: "不同野生稻的鉴定及紧穗野生稻分子标记图谱的构建", 《中国优秀博硕士学位论文全文数据库(硕士)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254160A (en) * | 2020-03-30 | 2020-06-09 | 扬州大学 | Protoplast verification method for efficiently identifying rice enhancer |
| CN113308563A (en) * | 2021-05-28 | 2021-08-27 | 扬州大学 | Method for rapidly developing Australian wild rice specific molecular marker |
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