CN1570149A - Primer , fragment and method for eucommia shoot and seed sex identification - Google Patents
Primer , fragment and method for eucommia shoot and seed sex identification Download PDFInfo
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- CN1570149A CN1570149A CN 200410037405 CN200410037405A CN1570149A CN 1570149 A CN1570149 A CN 1570149A CN 200410037405 CN200410037405 CN 200410037405 CN 200410037405 A CN200410037405 A CN 200410037405A CN 1570149 A CN1570149 A CN 1570149A
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Abstract
The invention belongs to the molecule labeling technique field. A DNA fragment, a group of primer, and a method for determining the young Eucommia ulmoides Oliv. and its sex are provided. The amplified fragment with the length of 569bp can be obtained from the female Eucommia ulmoides Oliv. by using the primers, but none of the amplified fragment in the male Eucommia ulmoides Oliv. can be obtained, which show that the female is specific and the primers can be used for determining the sex.
Description
[technical field]
The invention belongs to the molecular marking technique field, differentiate bark of eucommia shoot and other primer of seminality, fragment and method for one group specifically.
[background technology]
The bark of eucommia (Eucommia ulmoides Oliv.) belongs to commodity trees for Eucommiaceae (Eucommiaceae) bark of eucommia of China's special product, country's secondary is laid special stress on protecting plant, strict dioecy deciduous tree, its phloem contains the gutta-percha (Cronquist of tool essential industry using value, Columbia University Press, New York, USA.1981; Tippo, Amer J Bot, 1940,27:832-838).As if this tree is strict dioecian plant, and investigation is found in a big way, and the male and female sex ratio was near 1: 1, and this shows its sex do as one likes karyomit(e) decision.Yet, there is not evident difference in bark of eucommia male and female plant karyomit(e) on form, that is to say and do not have tangible sex chromosome (Wang B.W., et al., Acta Bot.Sin.1999,41 (1): 11-15), the laboratory previous work of inventor place has proved that the gel content of female plant blade is apparently higher than staminiferous plant, and contain high-grade nutrient and technical oils in the fruit that female plant is tied, female plant just has higher economic worth than staminiferous plant like this, thereby on producing, plant female tree more and just can increase economic efficiency, this also is feasible actually, as long as because there is 10% staminiferous plant just can satisfy the needs of female plant pollination, but the ripening stage of the bark of eucommia be generally 6～7 years, during have no idea to distinguish male and female plant (Wang B.W., et al., ActaBot.Sin.1999,41 (1): 11-15), therefore find a dna marker to differentiate that the shoot sex of the bark of eucommia has important use and is worth from molecular level.
Molecule marker (as RFLP, RAPD) becomes the very wide Plant Genome heredity mark of application already.RAPD can be by cloning and the stable and reliable mark of order-checking generation, design a pair of deutero-primer by the specific band after the hybridization check of strictness through order-checking, can amplify corresponding band, this method called after sequence signature amplification region (SCAR) mark (Paran I.et al., Theor Appl Genet 1993,85:985-993).
Still have nothing to do both at home and abroad at present in the report of differentiating bark of eucommia shoot and seed sex method with molecule marker, the present invention has filled up the blank in this field.
[summary of the invention]
The object of the invention provides differentiates bark of eucommia shoot and seminality other one group of primer and section of DNA fragment;
Another object of the present invention provides a kind of discriminating bark of eucommia shoot and seminality method for distinguishing.
The present invention uses the RAPD method to amplify a female plant specific band in female plant bark of eucommia genome, having obtained a segment length by clone and order-checking is the dna sequence dna of 569 bp, shown in SEQID NO:1, and designed one group in view of the above and differentiated bark of eucommia shoot and other primer of seminality, preferred SEQ ID NO:1 sequence is the above sequence of 300bp at interval.Primer length is 18～22bp, and non-specific band occurs during wherein less than 18bp, produces the primer self-polymerization during greater than 22bp.The embodiment of the invention provides a pair of primer, and its sequence is shown in SEQ ID NO:2 and SEQ IDNO:3.
The invention provides a kind of discriminating bark of eucommia shoot and seminality method for distinguishing, it is characterized in that method is a PCR reaction product electrophoresis result evaluation sample sex per sample.The PCR primer is no less than the sequences Design synthetic of 300bp at interval in this method by the sequence shown in the SEQ ID NO:1.The sample that occurs one section 300～600bp left and right sides band in this method in the PCR product electrophoresis result is a female plant, and same area the sample of band not occur be staminiferous plant.
When the PCR reaction system is 25 ul, comprising 1 * damping fluid (200mmol/LTris-HCl (pH8.0), 50mmol/L KCl), each 0.1～0.3mmol/L of dNTP, 40～60pmol/L primer, 0.8～1.2mmol/L MgCl
2, 1UTaq enzyme (Promega company), 20～30ng genomic dna, response procedures are 94 ℃ of pre-sex change of 5min, 35 circulations: 94 ℃ of 30Sec, 60～63 ℃ of 80～100Sec, 72 ℃ of 80～100Sec, then 72 ℃ are extended 8～10min, 4 ℃ of preservations.
The present invention one is shared 560 primers comprise OPFA-Z series 460 (ancient cooking vessel state company) and Genemed 600-700 (matching Parkson company).560 primers all amplify the polymorphic bands about 6, and wherein primer OPF8 (GGGATATCGGG) can amplify a feature band about 600bp in 5 female plant genomic dnas, and do not occur (Fig. 1) in the staminiferous plant genomic dna on the same position.
With above-mentioned feature band clone, order-checking, sequence is shown in SEQ ID NO:1.By relatively finding with gene pool, lower with existing sequence homology, and do not have open read area and promotor.
Make up DIG-11-dUTP (BoehringerMannheim) label probe of above-mentioned feature band with random priming, with male and female genomic dna hybridization through double digestion, on the female plant DNA electrophoretic band hybridization signal is arranged, and do not have hybridization signal (Fig. 2) on the staminiferous plant DNA band.
By 2 oligonucleotide chains of sequence construct:
SEQ?ID?NO：2 5′-GGGATATCGGCACCGTGGAA-3′
SEQ?ID?NO：3 5′-CCCTATAGCCCCCTCGGGTT-3′。
Check this validity to primer with the bark of eucommia of the known sex of 20 strains, the SCAR reaction and display all has the feature band about 600bp in all tested female plant DNA electrophoretic bands, and does not have (Fig. 3) on the corresponding position in the tested staminiferous plant.
In sum, the invention provides a segment length is the special dna sequence dna of bark of eucommia female plant of 569bp, and according to the primer of this sequences Design, can be used for the male and female sex identification of the bark of eucommia.Embodiment 2 results have proved the immature bark of eucommia of discriminating that primer and PCR method can 100%; Embodiment 3 has proved that primer and PCR method can identify the sex of seed, helps like this carrying out early stage plantation planning according to the purpose of producing, and creates best economic worth.
[description of drawings]
Fig. 1, RAPD reaction result wherein have a specific band on the electrophoretic band of 5 female plants (fp1～5), and staminiferous plant on the same position (fs1～5) does not have.Fig. 2, Southern results of hybridization: the male and female genome is cut the rear electrophoresis band through enzyme and is transferred on the nylon membrane probe hybridization with digoxigenin labeled, only on female plant genome (p) band hybridization signal is arranged, and does not have on staminiferous plant genome (s) band.
Fig. 3, SCAR reaction and display all have the feature band about 600bp in all tested female plant DNA electrophoretic bands, and do not have on the corresponding position in the tested staminiferous plant.
About 600bp the feature band is arranged in Fig. 4,1,4,7,10,13,14 the electrophoretic band, remaining does not then have.
Fig. 5, obtained feature band clearly for 6,9, No. 10.
[embodiment]
Embodiment 1:
Extract the genomic dna of 10 strains male (ts1-ts10) and 10 strains female (tp1-tp10) plant respectively with the CTAB method, be diluted to 10ng/ul and use in order to PCR;
The present invention according to the special dna fragmentation design synthetic primer of the bark of eucommia female plant that is obtained is:
5′-GGGATATCGGCACCGTGGAA-3′
5′-CCCTATAGCCCCCTCGGGTT-3′
The PCR reaction system is 25ul, comprises 1 * damping fluid (200mmol/LTris-HCl (pH8.0), 50mmol/L KCl), each 0.2mmol/L of dNTP, 50pmol/L primer, 1mmol/L MgCl
2, 1UTaq enzyme (Promega company), 25ng genomic dna, response procedures are 94 ℃ of pre-sex change of 5min, 35 circulations: 94 ℃ of 30Sec, 63 ℃ of 90Sec, 72 ℃ of 90Sec, then 72 ℃ are extended 10min, 4 ℃ of preservations.Pcr amplification product is 0.5 * tbe buffer liquid (89mmol/L Tris, 89 mmol/L boric acid, 2mmol/L EDTA) on 1.6% agarose gel, 60V electrophoresis 2 hours.The result is shown in figure (3):
From the graph as can be seen, feature band about 600bp is all arranged in all tested female plant DNA electrophoretic bands, and do not have on the corresponding position in the tested staminiferous plant, confirmed according to the special dna fragmentation design synthetic primer female plant of the bark of eucommia female plant that obtains special thus.
Embodiment 2: the not clear plant of sex is not checked this validity to primer by blooming
Gathered 15 bark of eucommia seedling samples (bark of eucommia life in 8 years is bloomed) that life in 7 years is not bloomed from Luoyang, extracted their genomic dna respectively, be diluted to 10ng/ul and use in order to PCR with the CTAB method.
The present invention according to the special dna fragmentation design synthetic primer of the bark of eucommia female plant that is obtained is:
5′-GGGATATCGGCACCGTGGAA-3′
5′-CCCTATAGCCCCCTCGGGTT-3′
The PCR reaction system is 25ul, comprises 1 * damping fluid (200mmol/LTris-HCl (pH8.0), 50mmol/L KCl), each 0.2mmol/L of dNTP, 50pmol/L primer, 1mmol/L MgCl
2, 1UTaq enzyme (Promega company), 25ng genomic dna, response procedures are 94 ℃ of pre-sex change of 5min, 35 circulations: 94 ℃ of 30Sec, 63 ℃ of 90Sec, 72 ℃ of 90Sec, then 72 ℃ are extended 10min, 4 ℃ of preservations.Pcr amplification product is 0.5 * tbe buffer liquid (89mmol/L Tris, 89mmol/L boric acid, 2mmol/L EDTA) on 1.6% agarose gel, 60V electrophoresis 2 hours.The result is shown in Figure 4:
As can be seen from Figure 4, the feature band is arranged about 600bp in 1,4,7,10,13,14 the electrophoretic band, remaining does not then have.Went the Luoyang verification result in 1 year, except that 10 and No. 15 trees do not bloom can't confirm, other plant are the same with experimental result, what numbering 1,4,7,13,14 plant opened is female flower, what numbering 2,3,5,6,8,9,11,12 was opened is male flower, fits like a glove with experimental result.The primer bark of eucommia female plant that further confirms design thus is special, and can be used for the male and female sex identification of bark of eucommia shoot.
Embodiment 3: detect this primer is used for the potentiality that the seed sex detects
Extract the genomic dna of 10 eucommia bark seed, be diluted to 10ng/ul and use in order to PCR.
The present invention according to the special dna fragmentation design synthetic primer of the bark of eucommia female plant that is obtained is:
5′-GGGATATCGGCACCGTGGAA-3′
5′-CCCTATA?GCCCCCTCGGGTT-3′
The PCR reaction system is 25ul, comprises 1 * damping fluid (200mmol/LTris-HCl (pH8.0), 50mmol/L KCl), each 0.2mmol/L of dNTP, 50pmol/L primer, 1mmol/L MgCl
2, 1UTaq enzyme (Promega company), 25ng genomic dna, response procedures are 94 ℃ of pre-sex change of 5min, 35 circulations: 94 ℃ of 30Sec, 63 ℃ of 90Sec, 72 ℃ of 90Sec, then 72 ℃ are extended 10min, 4 ℃ of preservations.Pcr amplification product is 0.5 ' tbe buffer liquid (89mmol/L Tris, 89mmol/L boric acid, 2mmol/L EDTA) on 1.6% agarose gel, 60V electrophoresis 2 hours.The result is shown in figure below (5):
See from the graph to have obtained feature band clearly for 6,9, No. 10, this primer that shows that the present invention designs can be applied to the evaluation of eucommia bark seed sex.
Sequence table
＜110〉Peking University
＜120〉differentiate bark of eucommia shoot and other primer of seminality, fragment and method
<160>3
<210>1
<211>569
<212>DNA
＜213〉artificial sequence
<400>1
GGGATATCGG?GGGAGCCCAA?CATTAATGTG?GGGTTCAACA?AGTTCAACAT?CAGACAAAAA 60
CCCCAACTCT?GATACCACGT?AGGAACCCAA?TAGGCCTCAC?TCACTCTCAA?AAGACGCCTT 120
GTAAGAGAAG?GATTGCCTTA?GGCTTTATAT?ATAGCTCAGG?ATTAGGTAAC?ACAAGCGATG 180
TGGAATTCAA?CATGATTCAG?GAACACCTGT?AAAAATCTAA?TTCCGTGCAA?CCAAACATGA 240
CCTTAATGAA?AAATGTACCT?CGAATAGATC?ATCCAGAATC?TTATTCCTGT?ACTCTATCTC 300
CAAAAACCGA?CTCCTTTCAT?GGGTTATAGC?TTCGGTTTCA?CTAGAAGTGC?TTTGAACAAC 360
TGCACTAGGT?GCAGGGCCGG?TATCATCATT?CCCATCTAAT?TGAGGATTAT?CAACATTAAT 420
ATCCCAAGAA?ACATCCTCTG?TATTACTCAA?CAAAATCTTG?TCTGATTCTT?CACCGTTATT 480
TTGAGAATCA?ACCTCGCTGG?CATTAACGAT?TTCGTAAGGA?CCTAATCCAT?TTGCAGCATC 540
TTCTGTTTCT?TCCACGGTGC?CGATATCCC 569
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<400>2
GGGATATCGG?CACCGTGGAA 20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
CCCTATAGCC?CCCTCGGGTT 20
Claims (8)
1. differentiate bark of eucommia shoot and other primer of seminality for one group, the sequence that it is characterized in that this group primer is by sequences Design synthetic shown in the SEQ ID NO:1.
2. want 1 described primer according to right, its sequence length is 18～22bp.
3. want 1 or 2 described primers according to right, its sequence is the sequences Design synthetic that is no less than 300bp by the interval of sequence shown in the SEQ ID NO:1.
4. want 1 or 2 described primers according to right, wherein a pair of primer sequence is the sequence shown in SEQ IDNO:2 and the SEQ ID NO:3.
5. differentiate bark of eucommia shoot and other dna fragmentation of seminality for one section, it is characterized in that this fragments sequence is shown in SEQ ID NO:1.
6. differentiate bark of eucommia shoot and seminality method for distinguishing for one kind, it is characterized in that this method is the electrophoresis result evaluation sample sex of PCR reaction product per sample.
7. want 6 described methods according to right, it is characterized in that PCR primer in this method is no less than the sequences Design synthetic of 300bp by the interval of sequence shown in the SEQ ID NO:1.
8. want 6 or 7 described methods according to right, the sample that it is characterized in that in this method occurring in the PCR product electrophoresis result one section 300～600bp size strip is a female plant, and same area the sample of band not occur be staminiferous plant.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100374059A CN1295346C (en) | 2004-04-30 | 2004-04-30 | Primer , fragment and method for eucommia shoot and seed sex identification |
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| CNB2004100374059A CN1295346C (en) | 2004-04-30 | 2004-04-30 | Primer , fragment and method for eucommia shoot and seed sex identification |
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| CN1570149A true CN1570149A (en) | 2005-01-26 |
| CN1295346C CN1295346C (en) | 2007-01-17 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433389A (en) * | 2012-01-06 | 2012-05-02 | 中国林业科学研究院热带林业研究所 | DNA (deoxyribonucleic acid) fragment, primers and method for identifying gender of salak |
| CN111549162A (en) * | 2020-04-16 | 2020-08-18 | 仲恺农业工程学院 | Primer, fragment and method for identifying sex of eucommia ulmoides |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2225600A1 (en) * | 1995-06-23 | 1997-01-09 | Commonwealth Scientific And Industrial Research Organisation | Gibberellin-regulated myb polypepetides |
| CA2336487A1 (en) * | 1998-07-22 | 2000-02-03 | The Trustees Of The University Of Pennsylvania | Copper transporter in ethylene signaling pathway |
| AU2001234916A1 (en) * | 2000-02-07 | 2001-08-14 | The Trustees Of The University Of Pennsylvania | Eto1 and related proteins, and methods of regulating ethylene biosynthesis |
| CN100371457C (en) * | 2001-01-16 | 2008-02-27 | 中国科学院海洋研究所 | Reagent and method for identifying laver idioplasm |
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- 2004-04-30 CN CNB2004100374059A patent/CN1295346C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433389A (en) * | 2012-01-06 | 2012-05-02 | 中国林业科学研究院热带林业研究所 | DNA (deoxyribonucleic acid) fragment, primers and method for identifying gender of salak |
| CN102433389B (en) * | 2012-01-06 | 2013-06-19 | 中国林业科学研究院热带林业研究所 | DNA (deoxyribonucleic acid) fragment, primers and method for identifying gender of salak |
| CN111549162A (en) * | 2020-04-16 | 2020-08-18 | 仲恺农业工程学院 | Primer, fragment and method for identifying sex of eucommia ulmoides |
| CN111549162B (en) * | 2020-04-16 | 2022-05-06 | 仲恺农业工程学院 | Primer, fragment and method for identifying sex of eucommia ulmoides |
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