CN107236810A - Corn male nuclear sterile gene, its molecular labeling and application - Google Patents
Corn male nuclear sterile gene, its molecular labeling and application Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- A—HUMAN NECESSITIES
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Abstract
The present invention relates to a kind of corn male nuclear sterile gene, its molecular labeling and application.The present invention includes two molecular labelings of IDP797245 and IDP30786, and the nucleotide sequence of the primer pair of its PCR amplifications is as shown in SEQ ID NO.1 to SEQ ID NO.4, and it is chain with corn character, can be accurately positioned and mark corn male nuclear sterile genems‑5, it has important application value in the identification of plant, the genetic background of kind, screening target individual plant in terms of the improvement of kind and yield forming and Clonal differentiation control gene；Male sterility gene ms 5 of the present invention is a new male sterility gene, when carrying out hybrid seeding with it, can remove artificial emasculation from, save manpower, seed costs is reduced, while being to improve hybrid seeding efficiency, seed purity again, it is ensured that a cost-effective approach of seed production quality.
Description
Technical field
The present invention relates to gene engineering technology field, and in particular to a kind of corn male nuclear sterile gene, its molecular labeling
And application.
Background technology
Male sterility is a kind of biological phenomena of generally existing in plant, is also a kind of approach of heterosis utilization.
It is abnormal that plants male sterility refers to plant stamen development, it is impossible to produces normal pollen, flower pesticide and the phenomenon for entailing offspring.
Plants male sterility is probably due to caused by inherent gene mutation, it is also possible to caused by being external condition, because
This, can be sorted out by different affecting factors to male sterile material：Can not genotype and heritable type be male not
Two macrotaxonomies of material are educated, this is that Kaul is proposed；Then, forefathers are again divided into male sterility：Cytoplasmic sterility, nucleus are not
Educate, cytoplasm, core interaction infertility.Cytoplasmic male sterility in plants, the F1 of matter sterile gene is unable to self-fertility, is because cell
The reason for matter maternal inheritance, therefore can not be utilized in the production of hybrid seeds；Cytogene can not influence nuclear male sterility, because its be by
Karyogene control, according to fertility control condition, nuclear male sterility point dominant genic male sterile and recessive cytoblast sterile can be passed through
The research of forefathers it is seen that, most of infertility is all to have recessive cytoblast sterile control；Cytoplasmic-nuclear male sterility, the genoid by
Genetic Sterility and the control of matter sterile gene, but can easily be infected and made by selectivity cause of disease microspecies using the male sterility in production
There is risk in hybrid maize.
Molecular labeling, is the genetic marker based on inhereditary material inner nucleotide sequence variations, is DNA level
The direct reflection of genetic polymorphism.The molecular labeling of broad sense refers to heritable and detectable DNA sequence dna or protein, narrow sense
Molecular labeling refer to the characteristic DNA fragmentation that can reflect the species diversity of certain in genome between bion or population.And corn is weight
The cereal crops wanted, are also that male sterile material is a kind of germ plasm resource being of great rarity, the male flower development mechanism of corn are ground
Study carefully, genetic breeding is applied, hybrid vigour aspect has important value.
With the development of molecular marking technique, the molecular labeling chain with corn character is obtained, to whether identifying corn
There is the gene of Sex Determination；The genetic background of identification, kind to plant, screening target individual plant；Improvement and yield to kind
It is significant in terms of composition and Clonal differentiation control gene.
The content of the invention
Corn male nuclear sterile gene can be accurately positioned and marked the technical problem to be solved in the present invention is to provide one kindms-5Molecular labeling, the molecular labeling and corn character be chain, the gene for whether having Sex Determination for identification corn；To plant
Identification, the genetic background of kind, screening target individual plant；Improvement and yield forming and Clonal differentiation control base to kind
There is important application value because in terms of.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
A kind of molecular labeling of corn male nuclear sterile gene, including two molecular labelings are designed, the primer pair of its PCR amplifications
Nucleotide sequence is respectively（As shown in shown in SEQ ID NO.1 to SEQ ID NO.4）：
IDP30786-U：GATTTCGGTTGGAGGAGT,
IDP30786-L：AGGCAGAGGGTGTTTGTG；
IDP797245-U：GCCCTCAACGGCTTCCTT,
IDP797245-L：CCACGCTGTGCTTACGAC.
Identification obtains a kind of corn male nuclear sterile genems-5, it is positioned on No. 4 chromosomes of corn and is in
Male nuclear sterile gene between two molecular labelings of IDP797245 and IDP30786.
The molecular labeling or corn male nuclear sterile gene of the corn male nuclear sterile genems-5In corn breeding
Application.
The method for cultivating corn male nuclear sterile kind using the molecular labeling of the corn male nuclear sterile gene, to have
There is corn male nuclear sterile genems-5Corn variety one of for parent, the method for passing through backcrossing, hybridization or tissue cultures is trained
Corn male nuclear sterile self-mating system is educated, selection has corn male nuclear sterile gene simultaneously in generation behindms-5It is described two
Molecular labeling and economical character meet the plant of cultivation needs, repeatedly until acquisition male nuclear sterile self-mating system.
A kind of primer for being used to expand the molecular labeling of the corn male nuclear sterile gene of design, including following primer
It is right：
IDP30786-U：GATTTCGGTTGGAGGAGT,
IDP30786-L：AGGCAGAGGGTGTTTGTG；
IDP797245-U：GCCCTCAACGGCTTCCTT,
IDP797245-L：CCACGCTGTGCTTACGAC.
A kind of detection kit of the molecular labeling of corn male nuclear sterile gene is designed, contains the primer pair.
The present invention is actively beneficial to be had technical effect that：
1. the Maize mutant sterile gene ms-5 of the present invention shows as holandry infertility, plant flower pesticide does not shed, and ms-5 dashes forward
The meiosis of variant plant is normal, and small robe mother cell is degraded after cell membrane is formed.
2. the present invention, by the genetic analysis and positioning to ms-5 materials, is using ms-5 mutant as main experiment material
The molecular breeding and application for realizing ms-5 mutant are laid a good foundation.
, can when carrying out hybrid seeding with it 3. male sterility gene ms-5 of the present invention is a new male sterility gene
To remove artificial emasculation from, manpower is saved, seed costs is reduced, while being to improve hybrid seeding efficiency, seed purity again, it is ensured that system
Plant a cost-effective approach of quality.
4. molecular labeling of the present invention and corn character are chain, the gene for whether having Sex Determination for identification corn；It is being planted
The identification of strain, the genetic background of kind, screening target individual plant, are controlled in the improvement of kind and yield forming and Clonal differentiation
There is important application value in terms of gene.
Brief description of the drawings
Fig. 1 is ms-5Wild type and the male flower phenotype of mutant compare figure；
Fig. 2 is wild type（It is left）And mutant（It is right）Pollen iodine-potassium iodide staining versus schemes；
Fig. 3 is the primer electrophoretic band figure that part has polymorphism between male parent and female parent；
Fig. 4 is to enter the electrophoresis pattern obtained by performing PCR amplification with primer I DP30786；Wherein M is Marker, and 1 is male parent, and 2 is maternal.
Fig. 5 enters the electrophoresis pattern obtained by performing PCR amplification for primer I DP797245；Wherein M is Marker, and 1 is male parent, 2
It is maternal.
Fig. 6 is to enter electrophoresis patterns of the F2 obtained by performing PCR amplification for sterile individual plant in segregating population with primer I DP30786；
Wherein 1 is male parent, and 2 is maternal.
Fig. 7 is that primer I DP797245 enters electrophoresis patterns of the F2 obtained by performing PCR amplification for sterile individual plant in segregating population；
Wherein 1 is male parent, and 2 is maternal.
Fig. 8 is corn male nuclear sterile genems-5With the relative position schematic diagram of molecular labeling on chromosome.
Embodiment
Illustrate the embodiment of the present invention with reference to the accompanying drawings and examples, but following examples are used only in detail
Describe the bright present invention in detail, the scope of the present invention is not limited in any way.
The explanation of involved materials and methods in following examples（It without special instruction is then conventional material and side
Method）：
1. corn male sterility mutant material：The present invention material to be tested be with C7-2, Zheng 58 be for self-mating system sterile plant, its
Show as：The atrophy of flower pesticide volume is 1/3rd of normal plant, and without loose powder phenomenon, plant shows as holandry
Sterile line, is named as ms-5 by infertility, is by the unnamed gene for causing male-sterile characterms-5。
2. maize leaf DNA is extracted
Extraction for the southern DNA hybridized：
（a）2 × CTAB of 10ml are added in 50ml centrifuge tube with cover（Use β-thioglycol of preceding addition 2%）Buffer solution,
Preheated in 65 DEG C of water-baths.
（b）The fresh blades of 5g or so are taken, are placed in the mortar of precooling, liquid nitrogen is added, powdery is ground to form rapidly（It must protect
Card sample is in liquid nitrogen）, it is transferred to rapidly in above-mentioned buffer solution afterwards, it is reverse it is mixed completely（Do again next sample it
Before to ensure being sufficiently mixed for a upper sample because sample and buffer solution are not thoroughly mixed and can cause DNA degradation）.
（c）65 DEG C of water-bath 60min, are overturned once per 10min.
（d）Add isometric chloroform：Isoamyl alcohol（24:1）, 20min is constantly rocked clockwise, to reach abundant mixing,
It is put into centrifuge（4℃ 12000rpm）Centrifuge 15min.
（e）Supernatant is taken, isometric isopropanol is added（Put -20 DEG C of precoolings）, slight wobble, it is seen that white flock sinks
Form sediment, more than 30min is placed in -20 DEG C.
（f）Choose white flock precipitate, outwell supernatant, plus the alcohol flushings of 5ml 70% are twice.
（g）Plus absolute ethyl alcohol 1ml, precipitation is transferred in 2ml centrifuge tubes, alcohol is outwelled, TE 1ml are added after drying.
（h）Often pipe adds 15 μ l RNases, 37 DEG C of water-bath 30min.
（i）Add isometric phenol：Chloroform：Isoamyl alcohol（25:24:1）, slight reverse mixing, 4 DEG C of 12000rpm centrifugations
10min。
（j）Supernatant is drawn onto in another new 2ml centrifuge tube, repeat step（i）Twice.
（k）Supernatant is drawn onto in another new 2ml centrifuge tube, plus isometric chloroform：Isoamyl alcohol（24:1）, slight top
Mix, 4 DEG C of 12000rpm centrifuge 10min.
（l）Supernatant is taken, 2 times of volume absolute ethyl alcohols and the sodium acetate of 10% volume are added（pH=5.2）In -20 DEG C of placements
More than 30min, takes out choose white precipitate afterwards.
（m）With 70% alcohol flushing twice, then with absolute ethyl alcohol rinse once, dry（Color is transparence）, add 50 μ l
The ultra-pure water of sterilizing, is saved backup in -20 DEG C.
3. PCR reaction systems
PCR amplification system is formulated as follows.
With mixed system
Component volume
The μ l of water 12
The μ l of sense primer 2
The μ l of anti-sense primer 2
Mix 20µl
The μ l of masterplate DNA 2
Note：Mix is 2 × Tap Plus Master Mix
PCR amplification programs
Program temperature, reaction time
95 DEG C of 5min of pre-degeneration
It is denatured 95 DEG C of 30s
56 DEG C of 30s of annealing are denatured to 33 circulations of extension
Extend 72 DEG C of 50s
Extend 72 DEG C of 10min afterwards
Preserve 4 DEG C.
Embodiment 1：ms-5The genetic analysis of corn male sterility mutant material
Cornms-5Mutant material is the sterile individual plant found in Sanya, Hainan reinforcement sweet tea seed farm, is natural mutation,
The same year made of fertile plant in plant male parent withms-5It is sisters and hands over conservation, after excessively handing over conservation purification for sisters, with corn certainly
Friendship be C7-2 for male parent withms-5Colony is assembled, F1 offspring's fertility is analyzed and identified, all plant are normal, group is separated in F2
Fertile plant and sterile plant are identified in body, it meets Mendel's law of segregation 3:1 segregation ratio (is shown in Table 1), therefore deducibility controls this not
Fertility shape is controlled by a pair of Recessive Male sterilities.
The table normal strain of 1 F2 colonies and mutant strain segregation ratio
| Combination | Total strain number | Normal strain | Sterile plant | Normally/infertility theoretical value（3：1） | χ2 |
| ms-5/C7-2 | 359 | 276 | 83 | 3.33：1 | 0.676 |
χ2(0.05,1)=3.84
Embodiment 2：ms-5The phenotypic analysis of corn male sterility mutant material
ms-5Mutant plant is in plant early stage nutrient growth process and normal Fertile material completelyms-5 In plant type substantially without
Difference；Arrived reproductive growth period, fertile plant can normally take out hero, flower pesticide can normal crack, loose powder, pollen connects with filigree
Touching can be normally solid；Compared with fertile plant,ms-5Sterile plant can normally take out hero, but can not normally bloom, and flower pesticide glume is not opened
Split, flower pesticide is not shrivelled exposed, no pollen grain is formed（Fig. 1）.The observation developed by female fringe is sent out, mutant strain and the female fringe of normal strain
Can be normally solid, illustrate that mutator does not influence female fringe fertility.
Embodiment 3：ms-5The iodine-potassium iodide dyeing of corn male sterility mutant material pollen
Iodine-potassium iodide dyeing (Fig. 2) is carried out to normal plant flower pesticide and mutant plants flower pesticide to find, normal strain pollen grain warp
In dark brown (Fig. 2 is right) after dyeing, show that normal strain pollen fertility is normal, and under the microscope, mutant flower pesticide is without pollen grain
In the presence of, it is impossible to iodine-potassium iodide dyeing (Fig. 2 left) is carried out, illustrate to occur during mutant anther development to cause extremely can not be just
Pollen grain is commonly formed.
Embodiment 4ms-5The assignment of genes gene mapping of corn male sterility mutant material
（1）The structure of crop groups
Utilizems-5Sterile line is maternal and self-mating system C7-2 and hybridized, and F1 generation plant selfing obtains F2 seeds, and plantation F2 generations obtain
Trait segregation colony, chooses and sterile plant is identified in F2 segregating populations, and clip young leaflet tablet extracts DNA, the positioning for gene.
Specific building process is as follows：
In August, 2014, Yuanyang, plantationms-5Parent material, F1 generation is assembled using C7-2 self-mating systems and sterile plant hybridization.
In December, 2014, Hainan, plantationms-5The F1 generation seed assembled with C7-2, and selfing is carried out, F2 is produced for group
Body.
In June, 2015, Yuanyang, plantationms-5F2 colonies, use sterile individual plant, extract DNA and are positioned.
（2）The screening of polymorphism primer
Using existing full-length genome 372 to SSR primer pairsms-5Mutant and C7-2 carry out polymorphism primer screening, altogether sieve
65 SSR primers parent with polymorphism are selected, have covering on 10 chromosomes of corn.
In the 65 pairs of primers filtered out, electrophoresis result band is selected clearly, band difference is obvious between Parent and F1
Primer is as the primer used in the assignment of genes gene mapping, and part polymorphism primer electrophoretic band is as shown in Figure 3.
（3）Target group determines
Using 600 plants of F2 target groups, wherein 150 sterile plants pairms-5Gene just position.First extract sterile plant and
The DNA of corresponding parent, is expanded followed by PCR.
（4）Linksystem is analyzed
The inclined segregation phenomenon of banding pattern, most of banding pattern and infertility are occurred on certain chromosome according to sterile character and polymorphism mark
Parent's banding pattern is identical, and it is heterozygosis banding pattern to have sub-fraction in addition, it is determined that mark has linkage relationship with sterile gene, by the gene
Navigate on this chromosome.
（5）ms-5The first positioning of gene
PCR primer is passed through after polyacrylamide gels, reads the linkage analysis that band carries out gene, all by corn
Colony's polymorphism primer screening of 10 chromosomes is found, ms-5Gene is located at the SSR marker Bnlg1621 on No. 4 chromosomes
（49：40：10）Near, it is maternal many compared with normal gene segregation ratio, and male parent is few, illustrates gene loci close to parental gene
Site, developing deeply mark, is screened by polymorphism primer and found, gene is located at the SSR marker p-umc1775 on No. 4 chromosomes
Near, occur the inclined segregation phenomenon of banding pattern, maternal, heterozygosis in the colony（123: 27）, most of banding pattern and sterile parentms-5
The banding pattern of gene is identical, and it is heterozygosis banding pattern to have part in addition, and this shows that the mark position may be withms-5Mutator is deposited
In linkage relationship, and on other chromosomes the amplified band result of all 150 sterile individual plants there is sterile banding pattern, it is fertile
The random separation of banding pattern and heterozygosis banding pattern, thus can primarily determine that influencems-5The sterile gene of mutant material fertility is located at 4
On number chromosome.
（6）ms-5The finely positioning of gene
Target group is expanded and more polymorphism marks are developed on the chromosome just positioned, target gene place is determined
Chromosome segment.DAN amplifications are carried out to 150 sterile individual plants, at SSR marker p-umc1775, there are 27 exchanges
, at SSR marker p-umc1808, there are 22 exchange individual plants, at SSR marker Bnlg2162, there is 1 exchange in individual plant
Individual plant, illustrates that sterile gene, closer to Bnlg2162, carries out DNA cloning, in SSR marker p- again to 300 sterile individual plants
At umc2188, discovery has 25 differences to exchange individual plant with above SSR marker Bnlg2162, at mark mmc0321, finds 13
Individual heterozygosis individual plant, further proves mark Bnlg2162 and mmc0321 that the sterile gene is located on corn rice chromosome
Between.
For the further finely positioning gene, according to the genome sequence being sequenced, development and location marker interval
Bnlg2162 and mmc032 mark density, develops 6 pairs of marks altogether, as a result finds 2 distance objective genes closer in parent
Between have polymorphism mark, i.e., the present invention mark（As shown in shown in SEQ ID NO.1 to SEQ ID NO.4）, it is respectively
IDP30786（Fig. 4）And IDP797245（Fig. 5）；Target group is further expanded to 4500 plants simultaneously（In F2 segregating populations not
Educate individual plant）.4500 sterile individual plant DNA are extracted, DNA cloning and gel electrophoresis are carried out again to 4500 sterile individual plants, as a result sent out
It is existing：In 4500 sterile individual plants in F2 segregating populations, there are 4485 sterile individual plants and sterile parent band at Tag ID P30786
Type is consistent（Fig. 6）, only 15 exchange individual plants；And at Tag ID P797245, the 4500 sterile individual plants in F2 segregating populations
In, there are 4486 sterile individual plants consistent with sterile parent banding pattern（Fig. 7）, and there is difference to be exchanged with 14 at Tag ID P30786
Individual plant.The result illustrates sterile genems-5Between Tag ID P30786 and Tag ID P797245；According to maize database
（http://www.maizegdb.org）, Tag ID P30786 is near rice chromosome 185,760Kb, and is marked
IDP797245 is near rice chromosome 186,526Kb；The distance between two marks about 76.6Kb（Fig. 8）.
SEQUENCE LISTING
<110>Henan Academy of Agricultural Sciences
<120>Corn male nuclear sterile gene, its molecular labeling and application
<130> /
<160> 4
<170> PatentIn version 3.2
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
gatttcggtt ggaggagt 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
aggcagaggg tgtttgtg 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
gccctcaacg gcttcctt 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
ccacgctgtg cttacgac 18
Claims (6)
1. a kind of molecular labeling of corn male nuclear sterile gene, including two molecular labelings of IDP797245 and IDP30786, its
The nucleotide sequence of primer pair of PCR amplifications is respectively：
IDP30786-U：GATTTCGGTTGGAGGAGT,
IDP30786-L：AGGCAGAGGGTGTTTGTG；
IDP797245-U：GCCCTCAACGGCTTCCTT,
IDP797245-L：CCACGCTGTGCTTACGAC.
2. a kind of corn male nuclear sterile genems-5, it is characterised in that it is positioned at cornms-5On rice chromosome and
Male nuclear sterile gene between two molecular labelings of IDP797245 and IDP30786.
3. corn male nuclear sterile described in the molecular labeling or claim 2 of corn male nuclear sterile gene described in claim 1
Genems-5Application in corn breeding.
4. cultivate the side of corn male nuclear sterile kind using the molecular labeling of corn male nuclear sterile gene described in claim 1
Method, it is characterised in that with corn male nuclear sterile genems-5Corn variety one of for parent, pass through backcrossing, hybridization
Or the method for tissue cultures cultivates corn male nuclear sterile self-mating system, selection has corn male nuclear sterile simultaneously in generation behind
Genems-5Described two molecular labelings and economical character meet cultivation needs plant, repeatedly until obtain hero
Property Genetic Sterility self-mating system.
5. a kind of be used to expand the primer of the molecular labeling of corn male nuclear sterile gene described in claim 1, including following draw
Thing pair：
IDP30786-U：GATTTCGGTTGGAGGAGT,
IDP30786-L：AGGCAGAGGGTGTTTGTG；
IDP797245-U：GCCCTCAACGGCTTCCTT,
IDP797245-L：CCACGCTGTGCTTACGAC.
6. a kind of detection kit of corn male nuclear sterile gene molecule marker, contains the primer pair described in claim 5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988420A (en) * | 2018-01-24 | 2018-05-04 | 四川农业大学 | The molecular labeling of corn male nuclear sterile gene ms39 and its application |
| CN108588273A (en) * | 2018-08-02 | 2018-09-28 | 北京科技大学 | The functional label of corn recessive nucleus male sterility mutator ms30 and its application |
| CN108950046A (en) * | 2018-08-02 | 2018-12-07 | 北京科技大学 | The functional label of corn recessive nucleus male sterility mutated gene ms1 and its application |
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| CN107988420A (en) * | 2018-01-24 | 2018-05-04 | 四川农业大学 | The molecular labeling of corn male nuclear sterile gene ms39 and its application |
| CN107988420B (en) * | 2018-01-24 | 2021-02-02 | 四川农业大学 | Molecular marker of maize male nuclear sterility gene ms39 and application thereof |
| CN108588273A (en) * | 2018-08-02 | 2018-09-28 | 北京科技大学 | The functional label of corn recessive nucleus male sterility mutator ms30 and its application |
| CN108950046A (en) * | 2018-08-02 | 2018-12-07 | 北京科技大学 | The functional label of corn recessive nucleus male sterility mutated gene ms1 and its application |
| CN108588273B (en) * | 2018-08-02 | 2020-10-13 | 北京科技大学 | Maize recessive nuclear male sterility mutant genems30Functional mark and application thereof |
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