CN106282345B - A kind of molecular labeling identifying rice phytochrome gene PHYB wild type and mutant - Google Patents
A kind of molecular labeling identifying rice phytochrome gene PHYB wild type and mutant Download PDFInfo
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Abstract
The invention discloses a kind of molecular labelings for identifying rice phytochrome gene PHYB wild type and mutant.Label relevant to phyB wild type and mutational site detection, nucleotide sequence is respectively as shown in Seq No.1-2.PCR amplification is carried out using primer PHYBF4/PHYBR1, amplified production carries out I digestion of BamH and carries out electrophoretic analysis: if PCR product cannot be 175bp DNA fragmentation, be then wild type phyB by I digestion of BamH；It is then mutant phyB1 if can be two DNA fragmentations of 25bp and 151bp by I digestion of BamH, digestion products.Molecular labeling of the invention has the advantage that amplification is simple, quick, at low cost.The molecular labeling can effectively, quickly, reliably identify the progress of phyB allelotype, provide phyB mutant to strong tool applied to breeding population screening to be subsequent.
Description
Technical field
The present invention relates to a kind of molecular labelings for identifying rice phytochrome gene PHYB wild type and mutant, belong to plant
Object field of biotechnology.
Background technique
Higher plant utilizes phytochrome (phytochromes), cryptochrome (cryptochromes) and image assesment
(phototropins) etc. light receptors, experience the variation of the conditions such as light quality, light intensity, photoperiod in its growing environment, adjust itself
Growth and development, to adapt to ambient enviroment to greatest extent.Wherein, phytochrome mainly experiences feux rouges (red light, R) and remote
Feux rouges (far-red light, FR).Phytochrome gene family includes 3 members: PHYA, PHYB and PHYC in rice.Wherein
PHYB gene is located at the 3rd the short arm of a chromosome of rice, for single copy.Takano etc. (2005) is sieved by gamma ray induced mutation
Choosing has obtained multiple phyB mutant, is respectively designated as phyB1~phyB5.Wherein phyB1 mutant is in PHYB gene internal
At 1644bp be inserted into a base C, so as to cause translation in advance terminate (Takano etc., 2005, Plant Cell, 2005,17:
3311-3325).By the photomorphogenesis feature of analyzing rice phytochrome mutant, find phyB in rice growth
Play a significant role in Stress responses.Under the conditions of phyB mutant either long-day conditions or short-day, florescence is equal
Obviously in advance (Takano etc., 2005, Plant Cell, 2005,17:3311-3325)；Its drought stress patience is also remarkably reinforced
(Liu et al., 2012, Plant Mol Biol., 78 (3): 289-300)；And there is certain resistance (Xie for rice blast
Deng 2011, Mol.Plant 4 (4): 688-696).And the plant height of phyB mutant, grain number per spike, the economical characters such as setting percentage with
Wild rice OryzasativaLcv.Nipponbare compares no notable difference (Takano etc., 2005, Plant Cell, 2005,17:3311-3325).
Therefore phyB mutant can be used as the important germ plasm resource of one kind and cultivate for precocious, disease-resistant, drought-enduring rice varieties.
In order to phyB mutant is preferably applied in breeding, develop it is a kind of simply and easily distinguish phyB mutant with
The molecular labeling of other conventional rice kinds, to realize the effective, reliable of phyB allelotype, Rapid identification, in subsequent benefit
Carry out that there is important value in breeding population screening with the mutational site phyB.Takano etc. although (2005) " Distinct and
Cooperative Functions of Phytochromes A,B,and C in the Control of
Deetiolation and Flowering in Rice " (The Plant Cell, Vol.17,3311-3325) article in
A kind of molecular labeling for identifying phyB1 mutant is developed, but the label amplified fragments are larger (1Kb), G/C content is higher,
It is expanded with the PCR buffer of high GC, and the PCR amplification time is longer, and is used for the restriction enzyme NlaIV of digestion
Belonging to rare restriction enzyme, purchase is difficult, and expensive (3.49 yuan/U, the Beijing NEB), appraisal cost is improved,
It is not suitable for identifying on a large scale；Often there is phenomena such as no inventory, supply shortage, seriously affects identification work and go on smoothly.
Summary of the invention
The primary purpose of the present invention is that overcoming the deficiencies of the prior art and provide a kind of simple, effective, lower-cost mirror
The molecular labeling and primer of other rice phytochrome gene PHYB wild type and mutant.Another object of the present invention is to provide
The application of above-mentioned molecular labeling.
The technical scheme is that a kind of molecule mark for identifying rice phytochrome gene PHYB wild type and mutant
Note, characterized in that
Nucleotide sequence relevant to the detection of phyB wild type site is (Seq No.1) as follows:
TGTCACACAAAGCCCCAGCATCATGGACCTTGTGAAGTGTGATGGTGCTGCTCTGTATTACCATGGGA
AGTACTACCCTCTTGGTGTCACTCCCACAGAAGTTCAGATTAAGGACATCATCGAGTGGTTGACTATGTGCCATGG
AGACTCCACAGGGCTCAGCACAGATAGCCTT。
Nucleotide sequence relevant to the detection of phyB mutant site is (Seq No.2) as follows:
TGTCACACAAAGCCCCAGCATCATGGACCCTTGTGAAGTGTGATGGTGCTGCTCTGTATTACCATGGG
AAGTACTACCCTCTTGGTGTCACTCCCACAGAAGTTCAGATTAAGGACATCATCGAGTGGTTGACTATGTGCCATG
GAGACTCCACAGGGCTCAGCACAGATAGCCTT。
The phytochrome mutant phyB mutant is phyB1 mutant.
The invention also discloses a kind of for identifying point of above-mentioned rice phytochrome gene PHYB wild type and mutant
The primer of son label, specifically:
Upstream primer PHYBF4:5 '-TGTCACACAAAGCCCCAGCATCATGGATC-3 ' (Seq No.3)；
Downstream primer PHYBR1:5 '-AAGGCTATCTGTGCTGAGCC-3 ' (Seq No.4).
The molecular labeling for identifying rice phytochrome gene PHYB wild type and mutant is in rice germplasm
Application in resource identification or breeding assist-breeding.
Using the method for above-mentioned primer identification rice phytochrome gene PHYB wild type and mutant, characterized in that packet
Include following steps:
(1) PCR amplification is carried out to oryza sativa genomic dna using upstream primer PHYBF4 and downstream primer PHYBR1；
(2) electrophoretic analysis is carried out to the pcr amplification product that step (1) obtains:
If pcr amplification product size is 175bp DNA fragmentation, for wild type phyB；
If pcr amplification product size is 176bp DNA fragmentation, for mutant phyB1；
(3) I digestion of BamH is carried out to the PCR product that step (1) obtains and carries out electrophoretic analysis:
It is then wild type phyB if PCR product cannot be still 175bp DNA fragmentation by I digestion of BamH；
It is then prominent if PCR product can be two DNA fragmentations of 25bp and 151bp by I digestion of BamH, digestion products
Variant phyB1.
PCR amplification system described in step (1) can use regular-PCR buffer and be expanded, and can also use PCR
Mix is expanded, and preferred system is 20 μ L systems, is expanded with PCR mix.PCR mix used in this experiment is that full formula gold is public
It takes charge of 2 × EasyTaq PCR SuperMix (+dye), PCR reaction system includes following component:
Pcr amplification reaction program described in step (1) is preferred are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 60 DEG C of 30s, 72
DEG C 30s, 35 circulations；Extend 5min after 72 DEG C.
Above-mentioned rice strain can for the OryzasativaLcv.Nipponbare kind of report (open), new rice 18 (state examines rice 2008028) and
Face the rice strains such as rice 11 (Lu Nong examines word [2004] 014).
The present invention compared with the prior art have following advantages and effects
(1) it develops a kind of for distinguishing point of rice phytochrome gene phyB mutant and other conventional rice kinds
Sub- labeled primer PHYBF4/PHYBR1, thus to phyB allelotype carry out effectively, quickly, reliable identification, for it is subsequent will
PhyB mutant is applied to breeding population screening and provides powerful.
(2) functional label primer PHYBF4/PHYBR1 of the invention utilizes regular-PCR technology, is capable of the detection of specificity
PhyB mutant.Compared with having label, the amplification of this label is simpler, quick；The PCR amplification program time (1h) well below
Former markd time (2h) (Takano etc., 2005, Plant Cell, 2005,17:3311-3325)；PCR product digestion institute
Restriction enzyme BamH I is that molecular biology often uses enzyme, and all routinely stock is not in because supply shortage causes for each company
Test disruption；(3.49 yuan/U, NEB is northern well below NlaIV for the price (0.0478 yuan/U, the Beijing NEB) of BamH I simultaneously
Capital), substantially reduce cost.The label can carry out Genotyping to different rice material phyB1 mutant alleles, and
It can be used as a kind of molecular labeling and be applied to breeding cross, backcrossing segregating population identification, improve molecular marker assisted selection breeding effect
Rate.
Detailed description of the invention
Fig. 1 rice phytochrome PHYB wild-type sequence and phyB1 mutant sequence difference site comparison result；
Fig. 2 is to utilize 2 × GC buffer (TAKARA), 2 × PCR Mix using this labeled primer PHYBF4/PHYBR1
(TRANSGEN) and 10 × PCR buffer (TAKARA) carry out PCR amplification result；Wherein M:500bp DNA ladder；1:
OryzasativaLcv.Nipponbare；2:phyB1 mutant；3: new rice 18；4: new rice 18/phyB1 mutant F1；5: facing rice 11；6: facing rice 11/phyB1
Mutant F1；
Fig. 3 is using this labeled primer PHYBF4/PHYBR1, with 2 × GC buffer (TAKARA), 2 × PCR Mix
(TRANSGEN) and 10 × PCR buffer (TAKARA) carries out PCR amplification, and amplified production carries out the result of digestion with BamH I；
Middle M:500bp DNA ladder；1: OryzasativaLcv.Nipponbare；2:phyB1 mutant；3: new rice 18；4: new rice 18/phyB1 mutant F1；
5: facing rice 11；6: facing rice 11/phyB1 mutant F1；
Fig. 4 is to utilize original labeled primer PHYBF1/PHYBR2, utilizes 2 × GC buffer (TAKARA), 2 × PCR
Mix (TRANSGEN) and 10 × PCR buffer (TAKARA) carries out the result of PCR amplification；Wherein M:2Kb DNA ladder；
1: OryzasativaLcv.Nipponbare；2:phyB1 mutant；3: new rice 18；4: new rice 18/phyB1 mutant F1；5: facing rice 11；6: facing rice 11/
PhyB1 mutant F1；
Fig. 5 is to utilize original label PHYBF1/PHYBR2, with 2 × GC buffer (TAKARA) and 2 × PCR Mix
(TRANSGEN) PCR amplification is carried out, amplified production carries out the result of digestion with NlaIV；Wherein M:2Kb DNA ladder；1: day
This is fine；2:phyB1 mutant；3: new rice 18；4: new rice 18/phyB1 mutant F1；5: facing rice 11；6: it is prominent to face rice 11/phyB1
Variant F1。
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings.According to description below and these realities
Example is applied, those skilled in the art can determine essential characteristic of the invention, and embodiments of the present invention are not limited thereto.
A kind of embodiment 1: foundation for the molecular labeling identifying rice phytochrome gene PHYB wild type and mutant
(1) design of primers
Known phyB1 mutant is inserted into a base C at PHYB gene internal 1644bp, shifts to an earlier date eventually so as to cause translation
Only (Takano etc., 2005, Plant Cell, 2005,17:3311-3325).Before and after mutational site choose 175bp sequence into
Row compares (Fig. 1), according to sequence difference, practical 2.0 Photographing On-line software of dCAPS Finder (http:// helix.wustl.edu/dcaps/dcaps.html) design primer, primer sequence is as follows:
Upstream primer PHYBF4:5 '-TGTCACACAAAGCCCCAGCATCATGGATC-3 ' (Seq No.3)；
Downstream primer PHYBR1:5 '-AAGGCTATCTGTGCTGAGCC-3 ' (Seq No.4).
(2) amplified fragments theory analysis
Rice wild type gene group DNA can amplify the segment (sequence 1) of a 175bp；PhyB mutant gene group DNA
Year can amplify the segment (sequence 2) of 1 176bp；Wild type gene group DNA cloning segment cannot be by I digestion of BamH, and phyB is prominent
Variant gene group amplified fragments can be by I digestion of BamH at two DNA fragmentations of 25bp and 151bp.
Seq No.1:
TGTCACACAAAGCCCCAGCATCATGGACCTTGTGAAGTGTGATGGTGCTGCTCTGTATTACCATGGGA
AGTACTACCCTCTTGGTGTCACTCCCACAGAAGTTCAGATTAAGGACATCATCGAGTGGTTGACTATGTGCCATGG
AGACTCCACAGGGCTCAGCACAGATAGCCTT。
Seq No.2:
TGTCACACAAAGCCCCAGCATCATGGACCCTTGTGAAGTGTGATGGTGCTGCTCTGTATTACCATGGG
AAGTACTACCCTCTTGGTGTCACTCCCACAGAAGTTCAGATTAAGGACATCATCGAGTGGTTGACTATGTGCCATG
GAGACTCCACAGGGCTCAGCACAGATAGCCTT。
Embodiment 2: 6 rice materials of molecular marker analysis of rice phytochrome gene PHYB wild type and mutant are utilized
Expect PHYB genotype
(1) extraction of rice leaf genomic DNA
Test material include rice wild type OryzasativaLcv.Nipponbare (number 1), phyB1 mutant (number 2), new rice 18 (number 3),
New rice 18/phyB1 hybridizes F1(number 4) faces rice 11 (number 5), faces rice 11/phyB1 hybridization F1(number 6).
The young leaflet tablet for choosing rice single plant extracts the genomic DNA of rice using SDS method, the specific steps are as follows:
1. appropriate blade is taken to be placed in 2mL centrifuge tube, 300 μ L of DNA extracting solution is added, is added one piece of steel ball, it is broken with tissue
Breakdown mill instrument shakes 30s or so；
2. centrifuge tube is placed in 65 DEG C of water-bath 30min, during which turns upside down and mix 2-3 times；
3. standing to room temperature, isometric chloroform is added, is aggressively shaken up and down, mixes well；
4. 12000rpm is centrifuged 10min, honest and upright and thrifty 200 μ L is sucted into the 1.5mL centrifuge tube of new sterilizing；
The mixing 5. isopropanol that isometric pre-cooling is added turns upside down, -20 DEG C of placement 30min or so keep DNA sufficiently heavy
It forms sediment；
6. 12000rpm is centrifuged 10min, supernatant is outwelled, it is primary that the rinsing of 500 μ L, 75% ethyl alcohol is added；
7. 12000rpm moment is centrifuged, centrifuge tube is upside down on paper handkerchief after outwelling supernatant, stands 2min；
8. appropriate 1 × TE buffer dissolving DNA is added in ventilating kitchen after dry DNA；
9. -20 DEG C of preservations are placed in, it is spare.
(2) the 6 rice materials of molecular marker analysis for identifying rice phytochrome gene PHYB wild type and mutant are utilized
Expect PHYB genotype
PCR amplification, reaction system difference are carried out using identification primer PHYBF4 (Seq No.3)/PHYBR1 (Seq No.4)
For 2 × GC buffer (TAKARA), 2 × PCR Mix (TRANSGEN) and 10 × PCR buffer (TAKARA), wherein 20 μ L
2 × PCR Mix (TRANSGEN) system are as follows: DNA profiling 2 μ L, 10 μM of primer (PHYBF4 and PHYBR1) each 0.3 μ L, 2 ×
10 μ L of EasyTaq PCR SuperMix, uses ddH20 supplies 20 μ L；PCR response procedures are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of changes
Property 30s, 60 DEG C of renaturation 30s, 72 DEG C of extension 30s, totally 35 circulations, then extend 5min after 72 DEG C.
(3) agarose gel electrophoresis detection, digestion and the genotype of amplified production determine
Pcr amplification product passes through 2% agarose gel electrophoresis (1 × TAE, 120V, time 20min), is anticipated using monarch solidifying
The observation of glue imaging system, as a result, it has been found that this primer can be amplified in wild type and mutant it is single, clearly, very bright
Bright band.Clip size and expected consistent (Fig. 2).Wherein the expanding effect of 2 × PCR Mix (TRANSGEN) system is best,
Band is clearest.
Pcr amplification product carries out digestion, digestion system with BamH I (TAKARA) are as follows: 10 × buffer, 2 μ L, PCR product
10 I 1U of μ L, BamH are taken, ddH is used2O polishing is to 20 μ L, 30 DEG C of digestion 1h.Then electrophoresis detection is carried out with 3% Ago-Gel
(1 × TAE, 120V, time 25min) is observed using monarch's meaning gel imaging system, as a result, it has been found that wild type remains as a list
One, clearly band, and phyB mutant band is significantly less than wild type, this is because 25bp is fallen in digestion, but due to 25bp
Band is too small can not to be shown on gel；Hybridization F1 single plant has two apparent bands, and size is big with wild type and mutant respectively
Small consistent (Fig. 3).Wherein the digestion effect of the amplified production of 2 × PCR Mix (TRANSGEN) system is best, and band is clearest.
The above-mentioned cementing fruit of genotype race is consistent with corresponding material banding pattern, consistent with expected results.Prove the mark of exploitation
Note can effectively distinguish wild type and mutant.
(4) with the comparative analysis of original identification marking
According to Takano etc., 2005, Plant Cell, the primer PHYBF1/ in 2005,17:3311-3325 articles
The above-mentioned primer of PHYBR2 sequent synthesis, primer sequence are as follows:
Upstream primer PHYBF1:5 '-GGGTTCCATTGCATCTCTTG-3 ' (Seq No.5)；
Downstream primer PHYBR2:5 '-TTGCCCATTgCTTCCTCAAC-3 ' (Seq No.6).
Using above-mentioned primer PHYBF1 (Seq No.5)/PHYBR2 (Seq No.6), PCR is carried out to wild type and mutant
Amplification, reaction system are 20 μ L systems: DNA profiling 2 μ L, 10 μM of primers (PHYBF1 and PHYBR2) each 0.3 μ L, 2 × EasyTaq
10 μ L of PCR SuperMix, uses ddH2O supplies 20 μ L；PCR response procedures are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 1min,
60 DEG C of renaturation 1min, 72 DEG C of extension 1min, totally 35 circulations, then extend 5min after 72 DEG C.As a result see Fig. 4, can be seen by Fig. 4
Out, primer PHYBF1/PHYBR2 amplification condition is more demanding, and the GC buffer of only high GC and efficient PCR mix can
Smoothly amplification, and common 10 × PCR buffer not can be carried out effective amplification.The PCR program reaction time is longer simultaneously, PCR instrument
Actual run time is more than 2 hours.The time is greatly saved in new label in contrast, can utilize commonly with stylish label
10 × PCR buffer is expanded, and cost is saved.
Original identification marking PHYBF1/PHYBR2 amplification wild type and the PCR product of phyB mutant can be by NlaIV
(NEB) digestion (Fig. 5)；Digestion system are as follows: 10 × buffer, 2 μ L, PCR product take 10 μ L, NlaIV 1U, use ddH2O polishing is extremely
20 μ L, 37 DEG C of digestion 1h.Then it is carried out electrophoresis detection (1 × TAE, 120V, time 25min), is utilized with 1% Ago-Gel
Monarch's meaning gel imaging system observation.It is respectively 564bp and 437bp that wherein wild type, which is two clip sizes, and phyB is mutated physical efficiency
It is enough three bar segments by NlaIV (NEB) digestion, size is respectively 437bp, 323bp and 242bp；And heterozygote banding pattern then has four
Bar segment, size are 564bp, 437bp, 323bp and 242bp (Fig. 5) respectively.New label is in contrast, new to mark enzyme used
BamH I is that molecular biology often uses enzyme, and there are stock or product in each Reagent Company；And former label restriction enzyme used
NlaIV is rare restriction enzyme, it is difficult to buy (such as: TAKARA company does not have relevant product).Used in new label
I price of enzyme BamH (0.0478 yuan/U, the Beijing NEB) well below NlaIV (3.49 yuan/U, the Beijing NEB), more save at
This；In addition new marker bands: wild type 175bp, saltant type 151bp, heterozygosis are two 175bp and 151bp, are marked relative to original
Banding pattern is simpler, it is easier to interpretation (Fig. 2).
In conclusion the exploitation newly marked can not only effectively distinguish wild type and mutant, and with original label
Compared to more saving time and cost.It is the molecule mark of an effective, quick, cheap identification phytochrome PHYB mutant
Note.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes made without departing from the spirit and principles of the present invention, modification, substitution, simplification etc. should all
For equivalent substitute mode, it is included within the scope of the present invention.
Claims (6)
1. a kind of for identifying the primer of the molecular labeling of rice phytochrome gene PHYB wild type and mutant, feature
It is the primer are as follows:
Upstream primer PHYBF4:5 '-TGTCACACAAAGCCCCAGCATCATGGATC-3 '；
Downstream primer PHYBR1:5 '-AAGGCTATCTGTGCTGAGCC-3 '.
2. primer described in claim 1 is identifying the application in rice phytochrome gene PHYB wild type and mutant.
3. a kind of method for identifying rice phytochrome gene PHYB wild type and mutant, characterized in that
(1) oryza sativa genomic dna is carried out using upstream primer PHYBF4 and downstream primer PHYBR1 described in claim 1
PCR amplification；
(2) electrophoretic analysis is carried out to the pcr amplification product that step (1) obtains:
If pcr amplification product size is 175bp DNA fragmentation, for wild typephyB；
If pcr amplification product size is 176bp DNA fragmentation, for mutantphyB1。
4. a kind of method for identifying rice phytochrome gene PHYB wild type and mutant, characterized in that
(1) oryza sativa genomic dna is carried out using upstream primer PHYBF4 and downstream primer PHYBR1 described in claim 1
PCR amplification；
(2) I digestion of BamH is carried out to the PCR product that step (1) obtains and carries out electrophoretic analysis:
If PCR product cannot be 175bp DNA fragmentation, be then wild type by I digestion of BamHphyB；
It is then mutant if PCR product can be two DNA fragmentations of 25bp and 151bp by I digestion of BamH, digestion productsphyB1。
5. the method for a kind of identification rice phytochrome gene PHYB wild type and mutant as described in claim 3 or 4,
It is characterized in, the PCR amplification system that PCR amplification described in step (1) uses is 20 μ L system, including following component: 50ng/ μ L
2 μ L of genomic DNA；10 μM of 0.3 μ L of upstream primer PHYBF4；10 μM of 0.3 μ L of downstream primer PHYBR1；2×EasyTaq
PCR SuperMix 10μL；ddH2O supplies 20 μ L.
6. the method for a kind of identification rice phytochrome gene PHYB wild type and mutant as described in claim 3 or 4,
It is characterized in, the response procedures of PCR amplification described in step (1) are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C
30s, 35 circulations；Extend 5min after 72 DEG C.
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| CN107475254A (en) * | 2017-09-29 | 2017-12-15 | 江苏丘陵地区镇江农业科学研究所 | A kind of eary maturity of rice allele, its molecular labeling and application |
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| CN105154531A (en) * | 2015-07-28 | 2015-12-16 | 华南农业大学 | Molecular marker for identifying wild type and mutant of 1000-grain weight gene TGW6 of rice |
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| CN103497948A (en) * | 2013-09-12 | 2014-01-08 | 山东省农业科学院蔬菜研究所 | Molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1/B2 and application of molecular marker |
| CN105154531A (en) * | 2015-07-28 | 2015-12-16 | 华南农业大学 | Molecular marker for identifying wild type and mutant of 1000-grain weight gene TGW6 of rice |
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| Distinct and Cooperative Functions of Phytochromes A, B, and C in the Control of Deetiolation and Flowering in Rice;Makoto等;《The Plant Cell》;20051231;第17卷;第3319页左栏最后1段，第3323页左栏第1段、右栏第4段，补充图1 |
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