CN104531894B - A kind of Specific primer pair and application for identifying rice fertility restorer genes - Google Patents
A kind of Specific primer pair and application for identifying rice fertility restorer genes Download PDFInfo
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- CN104531894B CN104531894B CN201510054521.XA CN201510054521A CN104531894B CN 104531894 B CN104531894 B CN 104531894B CN 201510054521 A CN201510054521 A CN 201510054521A CN 104531894 B CN104531894 B CN 104531894B
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Abstract
The invention provides a kind of Specific primer pair for identifying rice fertility restorer genes, it is adaptable to various Three-line rice bag bench-types, the screening and identification of red lotus type restorer.The method comprises the following steps, is first the design of restoring gene specific molecular marker PCR primer；Next to that the extraction of DNA；3rd is the foundation of standard PCR amplification system；4th is amplification of DNA fragments detection and analysis.The inventive method is easy, it is easy to operate, the method is based on rice fertility restorer genes molecular labeling, quickly, laboratory screening rice restorer germplasm materials are accurately and reliably passed through, make Breeding of Ternary more purposive and specific aim, the blindness and the big defect of poorly efficient, workload of traditional breeding way are overcome, improve three is, the Breeding Efficiency of double-line hybrid breeding.
Description
Technical field
Markers for Detection technical field is utilized the present invention relates to one kind, a fast qualitative detection paddy rice is more particularly to
Restoring gene molecular labeling and its application, it is adaptable to various Three-line rice bag bench-types, the screening of red lotus type restorer and mirror
It is fixed.
Background technology
Molecular marker assisted selection breeding is the product that modern biotechnology is combined with traditional breeding technique, and it makes biography
Extensive, empirical breeding technique changes to modern fine, M8003 line in system genetic thremmatology, makes breeding become more to have
Operability.If we know that the DNA sequence dna of a certain label, then just can by simple PCR amplification techniques or
RFLP/AFLP technologies, track specific beneficial gene.Because molecular labeling has uniqueness, and can be by the side such as round pcr
Method, has the advantages that very simple operation, reliable results, efficiency high, is independent of phenotype, extensively should on genetic breeding
With with boundless application prospect.
In series of three-series hybrid rice breeding, according to the difference of Rescued virus, cytoplasmic male sterility type mainly has three classes,
Yebai (WA-CMS), bag bench-type (BT-CMS) and red lotus type (HL-CMS).Most researchers think paddy rice Yebai cytoplasmic
Male sterility is controlled by 2 pairs of recessive genes.Bharaj etc. (1995) thinks WA type CMS by different extensive of the 2 pairs of effect
Multiple genes is controlled, and the strong Restore gene of one pair of which is located on the 7th chromosome, and weak Restore gene is located on the 10th chromosome.
One Restore gene seat Rf3 of IR24 is positioned at the 1st dye by Zhang etc. (1997) using RAPD and RFLP molecular marking techniques
Between colour solid RG532 and RG140, RG458.Yao etc. (1997) is recovered using RFLP molecular marking techniques two of bright extensive 63
Gene is respectively positioned between the 1st chromosome RG532 and RG173 and the 10th chromosome long arm C234 and G4003 between, it has therefore proved that
Restore gene seat of the effect value of the gene locus of the 10th chromosome long arm more than the 1st chromosome.Zhang Guiquan etc. (1994) with
Zhenshan 97a, Zhenshan 97B, IR24 and 9 be the base such as near of recurrent parent seed selection as Restore gene donor, with Zhenshan 97a with IR24
It is material because being, the method expanded with random primer finds 6 RAPD molecular labelings related to fertility restorer；With F2Colony
Be mapping population, by linkage analysis the unnamed gene related to fertility restorer be Rf3, Rf4, and Rf3 be positioned at the 1st dye
Colour solid.Zhang Qunyu etc. (2002) Zhenshan 97a and the hybrid F of Restore gene NIL2117 plants in segregating population are completely
Sterile plant carries out linkage analysis, shows that Rf4 is positioned at the 10th chromosome, the subclone Y38 and Rf4 seats obtained from YAC4892
Linkage distance be 0.9cM, the subclone Y110 obtained from YAC4630 is 3.2cM with the linkage distance at Rf4 seats.To red lotus
The research of type cytoplasmic male sterility fertility restoration there has also been progress, the close positive 23 pairs of red lotus types sterile line Cong Guang 41A of restorer
It is restorative show as 1 pair of dominant major gene resistance control (yellow Qingyang etc., 1999), so that (close positive 23) of Cong Guang 41A//Cong Guang 41B are returned
Friendship colony is assignment of genes gene mapping colony, and using group's point-score, the fertility restorer gene mapping that will be present in close positive 23 is dyeed in paddy rice the 10th
On body, away from SSR marker RM258 about 7.8cM.Using identical method, Liu Xuequn etc. (2004) with YTA//YTB/9311, YTA//
YTB/Milyang23 is fertility restorer gene mapping colony, and the Restore gene Rf6 that will be present in 9311 is positioned at the 10th chromosome
On, isolated with SSR marker RM5373；Milyang23 is positioned at the 10th chromosome to the seat of the Restorer gene Rf5 of YTA
On, isolated with SSR marker RM3510.Shinjyo etc. (1975) is with chromosome trisomy and morphological feature and genetic linkage point
Analysis technology, BT type cytoplasmic male sterility restoring genes Rf1 is positioned on the 10th chromosome, in fg1 (faded
Green leaf) and two genes of pgl (pale green leaf) between.Ichikawa etc. (1997) is further by the gene
The middle part of the 10th chromosome long arm is positioned at, G2155 close linkages are marked with RFLP.Liang Guohua etc. (2001) RFLP and Wei Wei
As a result asterisk note is found between BT types Restoring gene Rf 1 and C16 (72.6) and G291 (53.1) to BT type fertility restorer gene mappings
Cross-over value is respectively 19.3% and 14.0%.In these Restore gene after positioning, by Rf1 gene successful clones,
It was found that one mitochondrial protein (Kazama and containing PPR (Pentatricopeptide repeat) structure of its coding
Toriyama,2003；Komori et al.,2004).Wang etc. (2006) find BT types have two Restoring gene Rf 1 a and
Rf1b, the physical distance difference 90kb of the two Restore genes or so, wherein Rf1a encodes 791 amino acid proteins, with 18
Individual PPR domains；Rf1b encodes 506 amino acid proteins, with 11 PPR domains.
The content of the invention
The invention provides a kind of Specific primer pair for identifying rice fertility restorer genes, in solving background technology
Problem, the restoring gene of Rapid identification paddy rice is capable of using primer combination, to the selection of breeding material from being blindly changed into
Accurately, the waste of human resources is drastically reduce the area, operating efficiency is improve.
Realize technical scheme that above-mentioned purpose of the present invention used for：
A kind of Specific primer pair for identifying rice fertility restorer genes, including forward primer F and reverse primer R,
Wherein the gene order of forward primer F is as shown in SEQ ID NO.1：5 ' CCA TGA CAA GAG GAC CAG 3 ', reversely draw
The gene order of thing R is as shown in SEQ ID NO.2：5’GTT ATA AGT GAC GAC ATC CG 3’.
A kind of method of the Rapid identification rice fertility restorer genes based on the combination of above-mentioned primer, step is as follows：Carry first
Target plant genome is taken, then with genome as template, is entered performing PCR with above-mentioned Specific primer pair and is expanded, be finally directed to
Amplification of DNA fragments is detected.
The method for extracting target plant genome is specific as follows：One section of paddy rice tender leaf and powdered quartz sand are taken first, according to
It is secondary to be put into centrifuge tube, blade is ground to form into slurry, add CTAB extract solutions, 60~70 DEG C of 25~30min of heating water bath；
Secondly it is 24 that volume ratio is added in the centrifuge tube after water-bath：1 chloroform and the mixed liquor of isoamyl alcohol, mix, from
The heart, takes supernatant and is transferred to another centrifuge tube；
Absolute ethyl alcohol is added, in being mixed at -20 DEG C, 20min, 12000rpm centrifugations is placed under the conditions of 4 DEG C
15min, outwells supernatant, is filtered dry alcohol centrifuge tube inversion 10min, is then dissolved in 30 μ l sterilized waters.
The foundation of PCR amplification system is as follows：
The pcr amplification reaction system of standard is 25 μ l, is constituted as follows：
Reactant mixture is covered with mineral oil, and PCR response procedures are as follows：
Amplification of DNA fragments detection method is as follows：
First it is amplified fragments electrophoresis on the agarose gel of 1.0% concentration, extracting recycling box with DNA purifies recovery, gram
Grand sequencing；
Then to the identification of positive restoring gene：
(1) using rice varieties 9311 and Guangdong Thailand A as control, wherein control material 9311 has an amplification of 762bp
Band, Guangdong Thailand A has the band of 424bp；
(2) material with 9311 with identical amplified band just has the restoring gene, consistent just with Guangdong Thailand A
Without the restoring gene.
The present invention is designed according to the ISSR primers extension increasing sequence that can identify series of three-series hybrid rice kind fine horse excellent 522 and its parent
Primer, is expanded by PCR, and series of three-series hybrid rice kind fine horse excellent 522 and its parent can be quickly identified from other rice varieties.
Series of three-series hybrid rice kind fine horse excellent 522 and its authentication method of parent that the present invention is provided, can be used for the pure of above-mentioned rice varieties
Degree identification, and then applied in its breeding is aided in.
Brief description of the drawings
Fig. 1 be the present embodiment in by PCR reaction in the expansion in sterile line Guangdong Thailand A, maintainer Guangdong Thailand B and restorer 9311
Increase schematic diagram；
Fig. 2 be the present embodiment in by PCR reaction hybrid rice restorer line amplification schematic diagram.
Fig. 3 is the extension schematic diagram of filial generation and NIL in the present embodiment.
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment does detailed specific description to the present invention, but protection model of the invention
Enclose and be not limited to following examples.
The experimental technique of unreceipted actual conditions in following examples, generally according to normal condition, such as Sambrook etc.
's《Molecular cloning：Laboratory manual》(New York：Cold Spring Harbor Laboratory Press, 2001) in
Described condition, or according to the condition proposed by instrument or reagent manufacturer.Agents useful for same is commercial goods.
1st, special restoring gene molecular labeling PCR primer design
The distinguished sequence of database and the restoring gene that paddy rice 9311 is primarily based in the present invention is foundation, is carried out
Design of primers, it is designed go out the Specific primer pair for identifying rice fertility restorer genes include forward primer F and reversely
Primer R, the wherein gene order of forward primer F are as shown in SEQ ID NO.1：5 ' CCA TGA CAA GAG GAC CAG 3 ',
The gene order of reverse primer R is as shown in SEQ ID NO.2：5’GTT ATA AGT GAC GAC ATC CG 3’.
2nd, the extraction of extensive minim DNA
Target plant genome is extracted first, takes one section of 1-2cm long, the paddy rice tender leaf of 0.5cm wide and 0.2 gram of powdered stone
Ying Sha, is sequentially placed into a 1.5ml centrifuge tubes, and blade is ground to form into slurry with disposable micro plastics pestle, adds 500 μ l
CTAB extract solutions, 65 DEG C of water-bath 25-30min.
500 μ l chloroform-isoamyl alcohols (24 are added in centrifuge tube after water-bath：1), fully mix, on desk centrifuge
10000rpm is centrifuged 5min, takes the μ l of supernatant 300 and is transferred to another 1.5ml centrifuge tubes.
The domestic absolute ethyl alcohol for adding 600 μ l to freeze, fully mixes, and 4 DEG C or places 20min on ice, 12000rpm centrifugations
15min, to supernatant is fallen, is filtered dry alcohol centrifuge tube inversion 10min, is then dissolved in 30 μ l TE.
3rd, the foundation of standard PCR amplification system
Pcr amplification reaction makees negative control, red lotus restorer with typical red lotus sterile line Guangdong Thailand A and maintainer Guangdong Thailand B
9311 is positive control.
The pcr amplification reaction system of standard：Volume is 25 μ l, is constituted as follows
It is centrifuged after reactant mixing, is covered with mineral oil, PCR amplifications is carried out in MJ-100 type PCR instruments, PCR reaction intervals
Sequence is as follows：
4th, amplification of DNA fragments detection：
First it is amplified fragments electrophoresis on 1.0% agarose gel, recovery is purified with DNA Extraction kit, gram
Grand sequencing；Next to that the identification of positive restoring gene：
(1) Fig. 1 is Specific primer pair in sterile line Guangdong Thailand A (YTA), maintainer Guangdong Thailand B (YTB) and restorer 9311
Amplification schematic diagram, control material 9311 has an amplified band of 762bp, and Guangdong Thailand A has the band of 424bp；
(2) material with 9311 with identical amplified band just has the restoring gene, consistent just with Guangdong Thailand A
Without the restoring gene.
Fig. 2 is the 7 kinds of rice varieties detected in the present embodiment, and the DNA of each rice varieties uses Specific primer pair
Schematic diagram after being expanded, in figure, 1~7 kind is respectively：Bright extensive 63, resist extensive 63, extensive 838, survey 49, polyphyly 1,
Victory is safe No. 1, and 9149.
Fig. 3 is the amplification schematic diagram of filial generation and NIL, wherein 1~5 is respectively：Contain the near of Restore gene
Isogenic line individual plant.
5th, specific fragment clone reclaims and nucleotide sequence analysis
(1) on ultraviolet transillumination instrument, the object tape on Ago-Gel is dug out with sterile razor blade and is put into aseptic Eppendorf
Pipe, weigh with scale Ago-Gel weight, and estimates volume (1g ≈ 1ml), adds 3 times of Binding of volume
Solution。
(2) 55 DEG C of water-bath 5-10min, until Ago-Gel is completely melt.
(3) 5 μ l silica gel solutions are added, if DNA >=2.5 μ g, is often increased 1 μ g DNA and is added 1 μ l silicone fluids.
(4) 55 DEG C of water-bath 5min make DNA be combined with silica gel, and flicking Eppendorf pipes frequently therebetween makes silica gel suspend.
(5) room temperature (20-25 DEG C) 10000rpm centrifugations 20sec, removes supernatant.
(6) 0.5ml Washing buffer liquid is added in Eppendorf pipes, flicking makes silica gel suspend.
(7) room temperature (20-25 DEG C) 10000rpm centrifugations 20sec, removes supernatant, with Washing buffer repeated washings two
It is secondary, air-dry precipitation.
(8) with 20 μ l TE or aseptic ddH2O suspends precipitation completely, and 55 DEG C of water-bath 5min make DNA be released into TE or ddH2O
In.
(9) room temperature (20-25 DEG C) 14000rpm centrifugations 1min, Aspirate supernatant obtains final product the DNA fragmentation for having purified, and takes few
Amount electrophoretic examinations recovery purifying effect.
(10) DNA clone that the standardization program that fragment provides according to Promega companies will reclaim to pGEM-T loads will be reclaimed
Body (Promega A3600, USA), selects the positive colony of white, and by Shanghai, connection polygene Co., Ltd carries out DNA sequencing.
(11) sequence alignment：Using the rice fertility restorer genes that release has been cloned in distinguished sequence and Rice database
Sequence is compared, and embodies the specificity and practicality of the molecular labeling.
Claims (3)
1. it is a kind of based on primer combination Rapid identification rice fertility restorer genes method, it is characterised in that step is as follows：Institute
Stating primer combination includes forward primer F and reverse primer R, and wherein the gene order of forward primer F is as shown in SEQ ID NO.1：
5 ' CCA TGA CAA GAG GAC CAG 3 ', the gene order of reverse primer R is as shown in SEQ ID NO.2：5’GTT ATA
AGT GAC GAC ATC CG 3’；
Target plant genome is extracted first, and then with genome as template, being combined with above-mentioned primer carries out PCR amplifications, most
Detected for amplification of DNA fragments afterwards, detection method is as follows：
Next to that amplified fragments electrophoresis on the agarose gel of 1.0% concentration, extract recycling box with DNA and purify recoverys, clone survey
Sequence；
Then to the identification of positive restoring gene：
(1) using rice varieties 9311 and Guangdong Thailand A as control, wherein control material 9311 has an amplified band of 762bp,
Guangdong Thailand A has the band of 424bp；
(2) material with 9311 with identical amplified band just has the restoring gene, and the just nothing consistent with Guangdong Thailand A should
Restoring gene.
2. the method for Rapid identification rice fertility restorer genes according to claim 1, it is characterised in that：Target is extracted to plant
The method of thing genome is specific as follows：One section of paddy rice tender leaf and powdered quartz sand are taken first, is sequentially placed into centrifuge tube, by leaf
Slice lapping adds CTAB extract solutions, 60~70 DEG C of 25~30min of heating water bath into slurry；
Secondly it is 24 that volume ratio is added in the centrifuge tube after water-bath：1 chloroform and the mixed liquor of isoamyl alcohol, mix, and centrifugation takes
Supernatant is transferred to another centrifuge tube；
Absolute ethyl alcohol is added, in being mixed at -20 DEG C, 20min, 12000rpm centrifugation 15min is placed under the conditions of 4 DEG C,
Fall supernatant, be filtered dry alcohol centrifuge tube inversion 10min, be then dissolved in 30 μ l sterilized waters.
3. the method for Rapid identification rice fertility restorer genes according to claim 1, it is characterised in that：PCR expands body
The foundation of system is as follows：
The pcr amplification reaction system of standard is 25 μ l, is constituted as follows：
Reactant mixture is covered with mineral oil, and PCR response procedures are as follows：
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| CN101899522A (en) * | 2010-08-03 | 2010-12-01 | 武汉大学 | Method for screening novel restoring gene for HL cytoplasm male sterile rice |
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