CN106497936B - The albumen and its encoding gene of control rice male fertility and application - Google Patents
The albumen and its encoding gene of control rice male fertility and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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Abstract
The invention discloses a kind of albumen for controlling rice male fertility and its encoding gene and applications.The albumen has amino acid sequence shown in SEQ ID NO:3 in sequence table, and the genomic dna sequence and cDNA sequence of encoding gene are respectively as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2.The present invention proves that the gene has the function of controlling rice male fertility by rite-directed mutagenesis and functional verification, and the gene is mutated in wild rice, can get male sterile rice strain, has important application value in rice breeding.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to it is a kind of control rice male fertility gene and should
The purposes of gene.
Background technique
Rice (Oryza sativa L.) is one of most important cereal crops.The population of whole world half or so is with water
Countries in Asia of the rice as staple food, especially population more than total world population's number 60%.The yield for improving rice is full generation
The long term object of boundary rice breeding man.
There are two types of main paths for rice breeding at present.One is by hybridization and backcrossing etc. means, breeding high-yield stable yields
Conventional rice variety.Conventional Rice is not necessarily to hybrid seeding, convenient and strength-saving；Generally, the rice matter of conventional Rice is better than common hybrid paddy rice.But
Being conventional Rice often generates variet complexity decline from reserving seed for planting to plant year after year, influences rice yield.And hybrid paddy rice is another
The successful methods of rice breeding.Hybrid rice refers to the parental rice different with two genetic background, the F obtained by hybridization1
The seed in generation, by F1Peasant planting is supplied directly to for seed.F1In generation, has apparent hybrid vigour, shows after field planting
High yield, high-quality and how anti-etc. merits.
Currently used hybrid rice system has ternary hybrid rice and two kinds of two-line hybrid rice.Ternary hybrid rice includes infertility
System keeps system and three system of restorer.The male organs development of male sterile line is abnormal, is unable to self-fertility, female organ is but
Development can normally receive foreign pollen and fertilization.It is solid normal after the pollen that male sterile line receives restorer is awarded, and
And newly generated first generation of hybrid fertility restorer is normal, can self-fertility, and there is stronger advantage.Sterile line selfing is shaky,
Need solid for its pollination by keeping system, and offspring still keeps male sterile gene.Currently, series of three-series hybrid rice kind is not
Educate be cytoplasmic male sterile line, wherein Yebai, red lotus type and packet bench-type be internationally recognized 3 kinds of cytoplasmatic males not
Educate rice main Types.
In double-linear hybrid rice, sterile line is usually photo-thermo-sensitive genetic male sterile line.Under the external conditions such as certain light temperature, sterile line
Fertility be restored, selfed seed can be harvested, for the production of hybrid seeds of next year, thus no longer need additional holding system.Three systems
The sterile line of hybrid paddy rice is selected generally from cytoplasmic male sterility strain, and photo-thermo-sensitive genetic male sterile line is generally karyogene control fertility.
Hybrid rice is widely applied in production.By the effort of many years, science in rice cytoplasmic male sterile
Regulatory mechanism in terms of have been achieved with more progress, while also having cloned dozens of and having regulated and controled male sterile karyogene.It has reflected
The male sterile karyogene of the adjusting and controlling rice made mainly acts on the middle and later periods of paddy rice stamen development, affects including subtrahend point
The development and degradation of the process, tapetum split, the deposition of exposore, the cracking of pollen bag and the indoor redox state of medicine
Multiple processes Deng including.Although obtained the studies above progress, control rice fertility molecule mechanism still not it is clear that
The especially gene of adjusting and controlling rice early stage stamen development, is even more rarely reported.
Existing research has shown that, the Plant Genome editing technique developed recently is combined using transgenic technology, will be helped
In disclosing the gene function of control Important Agricultural character, breeding reality is potentially possibly used for help more rapidly to excavate
The gene trampled efficiently cultivates the excellent new rice variety with production application value.But existing achievement is also few
It can be directly thrown into production application, there are also to be found for more participation male sterile genes of adjusting and controlling rice.
Summary of the invention
The purpose of the present invention is to provide a kind of albumen and its encoding gene for controlling rice male fertility, for illustrating water
The male sterile regulatory mechanism of rice provides potential breeding resources for the cultivation of hybrid rice.
The present invention has found the albumen of an adjusting and controlling rice stamen early development by genetic research, when the encoding gene strikes
After removing, the stamen development of rice can be made abnormal, and pollen cannot be generated completely, eventually lead to male sterility.
The albumen of control rice male fertility provided by the present invention, is named as OsMPL, derives from rice (Oryza
Sativa) kind OryzasativaLcv.Nipponbare has one of following amino acid sequences:
1) the SEQ ID NO:3 in sequence table；
2) the SEQ ID NO:3 amino acid sequence in sequence table passes through the substitution of one or more amino acid residues, missing
Or it adds and the derivative amino acid sequence with same function.
SEQ ID NO:3 in sequence table is made of 349 amino acid residues.Amino acid residue is replaced, is lacked
Or addition method be it is well-known to those skilled in the art, usually using genetic engineering means to its encoding gene into
Row mutation, then gives expression to corresponding albumen again.By expressing the albumen in plant, and test these plants anther and
The developmental state of pollen, it can be determined that whether the albumen after these variations occur also has the function of controlling rice male fertility.
It is provided by the invention control rice male fertility albumen OsMPL expression quantity and/or activity will affect anther and
The development of pollen, so as to the male fertile of adjusting and controlling rice.
The encoding gene OsMPL for encoding above-mentioned control rice fertility albumen can be the cDNA sequence of the gene, can also
To be the genomic dna sequence of the gene, or there is 90% or more homology with these sequences and encode identical function
The DNA sequence dna of albumen.Such as in sequence table shown in genomic dna sequence shown in SEQ ID NO:1 and SEQ ID NO:2
CDNA sequence.
The nucleotide sequence for knocking out or changing the encoding gene is allowed to that the albumen of the control rice fertility cannot be expressed
Or expression is reduced, or make the amino acid sequence of expression that missing or variation occur, so that the amino acid sequence is corresponding
Polypeptide loss of activity, it is abnormal to can lead to pollen and anther development.
It is provided by the present invention control rice male fertility albumen or its encoding gene purposes, including but not limited under
State several aspects:
1) fertility of rice male organ is controlled；
2) molecular labeling as rice male fertility；
3) rice male-sterile plants system is formulated；
4) new heterosis utilization approach is created, new varieties are cultivated.
The method of present invention initiative rice male-sterile plants system, comprising: knock out the OsMPL gene of purpose rice, or make mesh
Rice the gene mutation cause expression protein inactivation, or inhibit purpose rice in OsMPL gene expression, obtain water
Rice male sterility strain；Wherein the purpose rice is the rice for carrying OsMPL gene.
It can be realized the knockout or mutation of OsMPL gene using conventional gene engineering method, and inhibit or reduce
The expression of OsMPL gene.It can be realized by any known site-directed point mutation or interference system, such as CRISPR/
The skills such as Cas9 system, TALEN system, zinc finger enzyme system, RNA interference and artificial tiny RNA technology (artificialmicroRNA)
Art means.
In some embodiments of the invention, by the CRISPR/Cas9 vector introduction plant cell of target OsMPL gene,
Tissue or organ, then the plant cell being converted, tissue or organ are cultivated into plant, it obtains male sterile transgenosis and plants
Object.
Expression vector, the expression cassette, transgenosis of rite-directed mutagenesis can be carried out to the OsMPL gene on rice genome
Cell line and host strain also belong to protection scope of the present invention.
The element of rite-directed mutagenesis paddy gene OsMPL of the present invention or its homologous sequence can be imported by plant expression vector plants
Object tissue, cell or organ.Carrier for constructing the plant expression vector can be used for agrobacterium tumefaciens for any one
Or agrobacterium rhizogenes converts the binary vector of plant or can be used for the carrier etc. of plant micropellet bombardment, can also be can be in prokaryotes
The carrier of middle duplication, such as pUC serial carrier or pBluescript serial carrier.
For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out
Processing, as be added the coding that can be expressed in plant can produce color change enzyme or luminophor gene (gus gene,
GFP gene, luciferase genes etc.), resistant antibiotic marker (hygromycin phosphotransferase gene, carboxylic benzyl mould
Plain marker or kanamycins marker etc.) or anti-chemical reagent marker gene (such as anti-herbicide gene).
The plant expression vector for carrying the paddy gene OsMPL of mutation present invention control male fertile can be by using original
Raw plastid-chemistry mediation method (Ca2+, PEG), Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube import,
The combination of any one of conventional biology methods such as microinjection, electric shock, particle gun, mediated by agriculture bacillus or several method converts
Plant cell, tissue or organ, and the plant cell, tissue or organ of conversion are cultivated into plant；The tissue and organ can
Fruit pod, callus, stem apex, blade and seed including host plant etc..
In addition, being carried out by the transgenic plant of the paddy gene OsMPL of mutation present invention control male fertile after being commissioned to train
After supporting, can therefrom further screening gene pure mutant plant.
The rice male-sterile plants system that the present invention obtains can be used as female parent and be cross-breeding.Using conventional genetic means
The OsMPL gene is transferred in the cell or tissue of the rice male-sterile plants system, is regenerated, recovery male can be cultivated into and educated
The plant of property.
Present invention finds the albumen for regulating and controlling its male fertile in rice and its encoding gene OsMPL, are mutated the gene and obtain
Obtain male sterile phenotype.Therefore, OsMPL gene of the present invention has in plant hybridization breeding potentially applies valence
Value.
Detailed description of the invention
Fig. 1 shows the sequence variation situation for passing through CRISPR/Cas9 system rite-directed mutagenesis OsMPL gene in embodiment 3,
Compared with wild type, in the representative strain of two target sites, the difference that there is 1 base at target site is inserted
Enter, causes translation frameshift.
Fig. 2 shows the phenotype of wild type (left side) and Osmpl-1 afunction mutant (right side) appearance in embodiment 3,
Osmpl-1 shows as to bear seeds, but other organs and wild type control ratio do not have notable difference.Scale bar=10 centimetre.
Fig. 3 shows the anther phenotype of Osmpl-1 and Osmpl-2 afunction mutant in embodiment 3.Wherein, A is to sweep
Retouch electromicroscopic photograph: left side is wild type, and centre is Osmpl-1 mutant, and right side is Osmpl-2 mutant；B is I2- KI dyeing
Microphoto afterwards: left side is wild type, and centre is Osmpl-1 mutant；Right side is Osmpl-2 mutant.Scale bar=100 are micro-
Rice.
Specific embodiment
Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in:
" Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rdEdition, 2001, NY, Cold Spring Harbor).
The rite-directed mutagenesis of embodiment 1, OsMPL gene
(1) entry vector of OsMPL rite-directed mutagenesis is constructed:
Two pairs of specific spacer sequences for capableing of target OsMPL gene, synthesis following two are selected according to sequence-specific
To primer:
F1:5 '-GGCAAGCAAGAGGGCGCCGCAGCG-3 ' (SEQ ID NO:4)；
R1:5 '-AAACCGCTGCGGCGCCCTCTTGCT-3 ' (SEQ ID NO:5)；
F2:5 '-GGCACGGCGTTGAGGTCGCGGAAC-3 ' (SEQ ID NO:6)；
R2:5 '-AAACGTTCCGCGACCTCAACGCCG-3 ' (SEQ ID NO:7).
After two pairs of primer annealings, will be connected into respectively the pOs-sgRNA plasmid after BsaI digestion (Miao et al.,
2013, Cell Research 23 (10): 1233-1236.) in.Screen the correct carrier of spacer direction of insertion (using R1 and
M13R sequencing primer can amplify target stripe) and be sequenced, be sequenced correct plasmid be named as pOs-sgRNA-MPL-1 and
POs-sgRNA-MPL-2 is for constructing OsMPL rite-directed mutagenesis end carrier.
(2) building of OsMPL rite-directed mutagenesis end carrier:
In order to construct OsMPL rite-directed mutagenesis end carrier, pOs-sgRNA-MPL-1 and pOs-sgRNA-MPL-2 is carried first
Body and pH-Ubi-cas9-7 carrier (Miao et al., 2013, Cell Research 23 (10): 1233-1236.) carry out LR
It reacts (Invitrogen company), connection product is converted into bacillus coli DH 5 alpha competent cell, carry out positive gram of PCR screening
It is grand, the plasmid containing the element for capableing of target OsMPL gene is extracted, sequence verification is correctly converted for the callus of rice afterwards.
Embodiment 2, rite-directed mutagenesis OsMPL gene transgenic paddy rice acquisition:
(1) rice callus induces:
Full OryzasativaLcv.Nipponbare rice paddy seed is chosen, kind of a skin is peelled off and uniformly clicks and enters after sterilizing washing with 2 mg/litres
The sterilizing MS solid medium of 2,4- dichlorphenoxyacetic acids (2,4-D), 32 DEG C of continuous lights, 5 days callus inductions are formed.
(2) Agrobacterium-mediated Transformation:
The plasmid of OsMPL rite-directed mutagenesis end carrier electrization is converted into Agrobacterium EHA105 competent cell, coating zone
There is a solid LB media of 50 micrograms per litre spectinomycins and 50 micrograms per litre rifampins, 28 DEG C after dark culturing 2 days, with gene spy
Different primer U1 and C1 screening positive clone.Obtained positive colony is containing 50 micrograms per litre spectinomycins and 50 micrograms per litre rifampins
Fluid nutrient medium in 28 DEG C of cultures to OD600=0.8, it is used for rice conversion.
U1:5 '-GCGGTCGTTCATTCGTTCTAGATCG-3 ' (SEQ ID NO:8)；
C1:5 '-AGTTGGGCGATCAGATTCTC-3 ' (SEQ ID NO:9).
(3) rice callus converts:
The good callus of upgrowth situation is chosen, impregnates 2 minutes in bacterium solution, is dried on aseptic filter paper later, then
Callus is moved into the NB co-culture medium containing 100 micromoles per liter acetosyringones, is co-cultured 3 days under 25 DEG C of dark.
(4) rice callus is screened:
Co-culture 3 days after, with sterile water washing callus 5 times, then with 200 milliliters contain 500 mg/litre carbenicillins
The sterile washing of sodium one time, carefully removes liquid, clamps to aseptic filter paper callus with aseptic nipper, shifts after drying
(the carboxylic benzyl mould of the 2,4-D containing 2 mg/litres, the hygromycin of 50 mg/litres and 400 mg/litres on to NB screening and culturing medium
Plain sodium), 32 DEG C continuous light 2 weeks.
(5) differentiation of positive callus:
The positive callus that well-grown is in bright yellow is chosen, the pre- differential medium of NB is moved to aseptic nipper and (contains 1
Mg/litre methyl α-naphthyl acetate (NAA), 5 mg/litre abscisic acids (ABA), 2 mg/litre kinetins (kinetin), 25 mg/litres tide
The carbapen of enzyme element and 200 mg/litres), 32 DEG C of continuous light cultures.Eugonic callus is selected after 2 weeks
Be transferred to MS differential medium (containing 0.02 mg/litre NAA, 2 mg/litre kinetin, 50 mg/litres damp enzyme element and 200 milli
The carbapen of grams per liter), 32 DEG C of continuous light cultures.It is long to 2 to 5 millimeters wait differentiate the seedling come, it is transferred to without sharp
It in 2 to 3 week of MS culture medium culture of element and antibiotic, moves into soil be placed in a greenhouse (28-30 DEG C of temperature, 16 hours of growth later
Illumination/8 hour dark).
The Molecular Identification and phenotypic evaluation of embodiment 3, OsMPL afunction mutant
(1) in transgenic paddy rice OsMPL gene rite-directed mutagenesis:
It is total to extract plant with plant genomic DNA extracts kit (QIAGEN company) for the blade of clip Transgenic Rice Seedlings
DNA.Gene-specific primer MPL-F and MPL-R are designed according to the locus DNA sequence of OsMPL gene and carries out PCR reaction, are expanded
Increase the segment containing OsMPL targeted mutagenesis site, and the catastrophe for checking OsMPL gene be sequenced:
MPL-F:5'-GGAGACAGCCTCCCTTCGAGTACACTA-3'(SEQ ID NO:10)；
MPL-R:5'-GGCAGGAGGAGGAGGAAGAAGAAGA-3'(SEQ ID NO:11).
The catastrophe of sequencing detection OsMPL gene, discovery detect the different insertions an of base at target site
The two class genes mutant, be respectively designated as Osmpl-1 and Osmpl-2, as shown in Figure 1.Osmpl-1 and Osmpl-2 are removed
It does not bear seeds, the rice of appearance and wild type does not have notable difference, as shown in Figure 2.
(2) observation of OsMPL rite-directed mutagenesis transgenic paddy rice anther phenotype:
This research uses scanning electron microscopic observation anther appearance, and photographs to record；Use I2Inside-KI dyeing observation pollen bag
Dividing condition；And the pollen with wild rice is awarded to OsMPL afunction mutant, whether observation mutant ovary develops just
Often.As shown in figure 3, scanning electron microscope analysis shows that Osmpl-1 and Osmpl-2 afunction mutant anther appearance all distorts, with
Quite different (see the A in Fig. 3) of wild type.I2Micro- sem observation shows Osmpl-1 and Osmpl-2 afunction after-KI dyeing
The inside of mutant anther is free of pollen grain, and being full of in the anther of wild type can be by I2The mature flower powder of-KI dyeing is (see figure
B in 3).When awarding with wildtype pollens, Osmpl-1 and Osmpl-2 mutant can be normal solid, illustrates the function of OsMPL
Missing leads to male sterility of rice, and Stamen development is normal, illustrates that knocking out OsMPL can lead to male sterility of rice.
Claims (9)
1. the application of rice Os MPL albumen or its encoding gene in control rice male fertility, which is characterized in that described
The sequence of OsMPL albumen is the SEQ ID NO:3 amino acid sequence in sequence table.
2. application as described in claim 1, which is characterized in that the sequence of the encoding gene of rice Os MPL albumen is its gene
Group DNA sequence dna or cDNA sequence.
3. application as claimed in claim 2, which is characterized in that the sequence such as sequence of the encoding gene of the rice Os MPL albumen
In list shown in SEQ ID NO:1 or SEQ ID NO:2.
4. application as described in claim 1, which is characterized in that the nucleotide sequence for knocking out or changing the encoding gene makes
Cannot express the OsMPL albumen or expression reduce, or make the amino acid sequence of expression that missing or variation occur, into
And make the corresponding polypeptide loss of activity of the amino acid sequence, lead to male sterility of rice.
5. a kind of method for formulating rice male-sterile plants system, comprising: knock out the OsMPL gene of purpose rice, or make purpose water
The OsMPL gene mutation of rice leads to the protein inactivation of expression, or inhibits the expression of OsMPL gene in purpose rice, obtains water
Rice male sterility strain；Wherein the purpose rice is the rice for carrying OsMPL gene, the OsMPL gene coded sequence table
In SEQ ID NO:3 amino acid sequence shown in protein.
6. method as claimed in claim 5, which is characterized in that SEQ ID in the sequence such as sequence table of the OsMPL gene
Shown in NO:1 or SEQ ID NO:2.
7. method as claimed in claim 5, which is characterized in that pass through CRISPR/Cas9 system, TALEN system, zinc finger enzyme system
System, RNA interference or artificial tiny RNA technology realize the knockout or mutation of OsMPL gene.
8. the method for claim 7, which is characterized in that plant the CRISPR/Cas9 vector introduction of target OsMPL gene
Object cell, tissue or organ make OsMPL gene mutate, then the plant cell being converted, tissue or organ are cultivated into plant
Strain, obtains male sterile transgenic line.
9. a kind of rice breeding method, the rice male-sterile plants system formulated using claim 5 is cross-breeding as female parent.
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| WO1992013956A1 (en) * | 1991-02-08 | 1992-08-20 | Plant Genetic Systems, N.V. | Stamen-specific promoters from rice |
| WO2002014506A1 (en) * | 2000-08-17 | 2002-02-21 | Japan Tobacco Inc. | Method of estimating the genotype of fertility recovery locus to rice bt type male fertility cytoplasm |
| CN101704881B (en) * | 2009-10-29 | 2012-03-14 | 中国农业科学院作物科学研究所 | Plant male fertility-associated protein, coding gene and application thereof |
| CN102796184A (en) * | 2012-07-10 | 2012-11-28 | 四川农业大学 | Rice male fertility-related protein, encoding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992013956A1 (en) * | 1991-02-08 | 1992-08-20 | Plant Genetic Systems, N.V. | Stamen-specific promoters from rice |
| WO2002014506A1 (en) * | 2000-08-17 | 2002-02-21 | Japan Tobacco Inc. | Method of estimating the genotype of fertility recovery locus to rice bt type male fertility cytoplasm |
| CN101704881B (en) * | 2009-10-29 | 2012-03-14 | 中国农业科学院作物科学研究所 | Plant male fertility-associated protein, coding gene and application thereof |
| CN102796184A (en) * | 2012-07-10 | 2012-11-28 | 四川农业大学 | Rice male fertility-related protein, encoding gene and application thereof |
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