CN1425770A - Plant cytoplasmic male sterile recovering gene and its use - Google Patents
Plant cytoplasmic male sterile recovering gene and its use Download PDFInfo
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Abstract
The present invention provides the nucleotide sequence and encoded amino acid polypeptide sequence of one cytoplamis male sterile restoring gene OsRf1 originating from rice. The gene is member of PPR gene family, and is one constitutive expression gene. The present invention also relates to the application of utilizing the gene in transforming rice and other plant breeding restorer and the application of the molecular label produced based on the gene sequence in breeding.
Description
Technical field
The present invention relates to the clone and the application thereof of plant gene, the rice cytoplasmic male sterile that particularly relates to clone's acquisition recovers the dna sequence dna and the encoded protein matter sequence thereof of gene.The invention further relates to and recover the application of gene in plant breeding.
Background technology
Plant hybridizes by the allogamy between not of the same race or different varieties, and hybrid has powerful advantage at aspects such as growth potential, adaptability, output, is called hybrid vigour.Utilize hybrid vigour can improve output, quality and the resistance of farm crop.The microgamete that the male plant reproductive organ can not produce normal function is that the phenomenon of pollen is called male sterile.Plant or crop varieties with male sterile characteristic are called sterile line.On the other hand, the hybrid that some plant or crop varieties and sterile line hybridization produce has normal fertility, and these plants or crop varieties are called recovery system.Utilizing sterile line to make female parent and recover is to make paternal hybrid can economize the operation of removing hero, the highly purified cenospecies of convenient preparation, so have important production application to be worth.
Multiple kinds of crops such as paddy rice, wheat, corn, rape etc. are used for cross-fertilize seed on a large scale and produce about the existing many reports of the research of male infertility and fertility restorer thereof on producing.In China, hybrid rice establishing in large scale since the 1970's, wherein major part is to utilize " three series " preparing hybrid seed." three are " in " three series " is meant sterile line, recovers system and maintenance line.Wherein sterile line has cytoplasmic male sterility (cytoplasmic male sterility the abbreviates CMS as) characteristic by the cytoplasmic sterility Gene Handling.Recover cording normal tenuigenin or sterile cytoplasm are arranged, pollen normally can be educated, but self-fertility is characterized in carrying in the nuclear gene group cytoplasmic male sterility and recovers gene (fertility restorer gene for CMS), abbreviates as and recovers gene or Rf gene.Maintenance line has normal tenuigenin, and pollen normally can be educated, but self-fertility, but its nuclear gene group does not carry the recovery gene of function.In supporting " three are " of using in the production, maintenance line has identical nuclear gene group with sterile line, and its difference is the tenuigenin difference.
Rice cytoplasmic male sterile is divided into broad variety, wherein mainly contains: (1) bag platform (BT) type, and its male sterile cytoplasm derives from India long-grained nonglutinous rice Chisurah Boro II (Shinjyo, 1975), and it is sterile to belong to gametophyte; (2) Yebai (wild abortive is called for short the WA type), its male sterile cytoplasm derives from the wild-rice in Chinese Hainan Province, and it is sterile to belong to sporophyte.(3) red Lotus (HL) type, its male sterile cytoplasm derives from the red awns wild-rice in Chinese Hainan Province, and it is sterile to belong to gametophyte.To dissimilar rice cytoplasmic male steriles, found corresponding recovery gene.For the male sterile of bag bench-type, it recovers gene Rf1 and derives from India long-grained nonglutinous rice Chisurah Boro II and other long-grained nonglutinous rice.For Yebai male sterile, finding has 2 couples of main efficient recovery gene Rf3 and Rf4, is mainly derived from the late long-grained nonglutinous rice of low latitudes.For the male sterile of red Lotus type, it recovers gene Rf5 and is mainly derived from red awns wild-rice and tropical long-grained nonglutinous rice.
Existing rice restorer breeding method mainly contains 2 kinds: (1) is use as recovering directly with having kind and sterile line test cross now, select the kind that recovery fertility ability is arranged; (2) be genetic donor parent and good parent's hybridization of not carrying the recovery gene and how form to carry the kind or the product that recover gene for backcross breeding.Because the recovery gene of paddy rice mainly is present in the minority kind, most rice varieties does not carry the recovery gene of function, especially japonica rice variety does not carry the recovery gene of function basically, therefore (2) kind method is mainly used in the breeding of rice restorer, needs years just can finish.
To recovering discovering of gene, the bag bench-type recovers system has 1 pair to recover gene Rf1, is positioned at the 10th karyomit(e) (Shinjyo, 1975).Ichikawa etc. (1997) have further located Rf1 in the 10th chromosomal approximate location with molecule marker.And Yebai is recovered system, most researchs think that 2 pairs of main efficient recovery genes are arranged, and are positioned at the 1st and the 10th karyomit(e) (Zhang et al., 1997 respectively; Zhang Qunyu etc. 2002).Red lotus type recovers system has 1 pair to recover gene, is positioned in the 10th karyomit(e).
About the clone that plants male sterility recovers gene, the T type cytoplasmic male sterility of having reported at present of having only corn (Maize) is recovered the cytoplasmic male sterility of gene Rf2 and petunia (Petunia) and is recovered gene Rf-PPR592 (Cui et al., 1996; Bentolila et al., 2002).Before proposing, the present patent application also do not have the male sterile of paddy rice to recover the report of gene clone.
The present invention seeks to clone a kind of new cytoplasmic male sterility from paddy rice and recover gene, particularly paddy rice seed selection new restorer line and cross-breeding provide the gene and the molecule marker of usefulness for plant.
Summary of the invention
Recovery gene OsRf1 provided by the invention recovers system with map based cloning (map-based cloning) method separation acquisition from containing the allelic paddy rice bag of Rf1 bench-type cytoplasmic male sterility, it is characterized by:
(1) has the nucleotide sequence of sequence 1 in the sequence table, wherein the 1-1459 bit base is for containing the sequence of promoter regulation element and 5 ' end non-translational region, the 1460-2980 bit base is 1521 base opening code-reading frames (ORF) that do not have intron, and the 2981-3453 bit base is the sequence of 3 ' end non-translational region.
(2) the amino acid polypeptide sequence shown in the sequence 2 in the code sequence tabulation, this sequence is made up of 506 amino-acid residues, have the functional domain (Fig. 4) that 7 PPR repeating units (pentatricopeptide repeat) form, its biological function is for recovering the fertility of cytoplasmic male sterility in plants.
PPR is the repeating unit that is made of 35 amino-acid residues, forms functional domain in different containing in the PPR gene with the repeating unit in the same way more than 2, and there is variation in various degree in the aminoacid sequence of different repeating units.Therefore, the minimum length that constitutes the pairing dna sequence dna of PPR multiple functional domain is 210 bases, promptly corresponding to the sequence of 2 PPR repeating units.
According to OsRf1 gene order information provided by the invention (sequence 1, sequence 2), those skilled in the art can easily obtain the gene that is equal to OsRf1 by the following method: (1) obtains by database retrieval; (2) with the OsRf1 gene fragment be the genomic library acquisition of probe Screening of Rice or other plant; (3), obtain with the genome of pcr amplification method from paddy rice or other plant according to OsRf1 gene order information design oligonucleotides primer (containing degenerated primers); (4) on the basis of OsRf1 gene order, obtain with the gene engineering method transformation; (5) method with chemosynthesis obtains.
Term OsRf1 of the present invention etc. are homogenic, be to be defined as with the nucleotide sequence or the amino acid residue sequence of OsRf1 gene to compare the difference that one or several Nucleotide or amino-acid residue are arranged, comprise change, disappearance or the insertion of base or amino-acid residue, or with the OsRf1 gene order in continuous 210 bases more than section homology more than 85% and the function of its expression product gene identical with the function of OsRf1 expression product are arranged.
Therefore, recovery gene provided by the invention comprises that also OsRf1 etc. is homogenic, and they are one of following nucleotide sequences, or the nucleotide sequence of one of the following amino acid polypeptide sequence of encoding:
(1) with replacement, disappearance or the interpolation of the nucleotide sequence of sequence 1 through one or several Nucleotide, and coding and the proteinic derivatized nucleotide sequence of sequence 2 same functions;
(2) with the 1460-2980 bit base sequence of sequence 1 in the above section of continuous 210 bases have homology more than 85% and coding and the proteinic nucleotide sequence of sequence 2 same functions.
(2), and have derivative amino peptide sequence with sequence 2 same functions with replacement, disappearance or the interpolation of the amino acid polypeptide sequence of sequence 2 through one or several amino-acid residue, or its conservative property variation polypeptide, or its active fragments.
OsRf1 gene of the present invention or its etc. are homogenic, also comprise the carrier that contains whole prefaces of these genes or partial sequence.
Recovery gene provided by the invention has important use and is worth.One of application is that described recovery gene order is connected to any plant conversion carrier, will recover gene with any method for transformation and import paddy rice or other vegetable cell, can obtain the transgenic restorer line of energy expressing said gene.With transgenic restorer line and cytoplasmic male sterile line hybridization, can produce cenospecies and be used for producing.With of the present invention gene constructed in plant conversion carrier the time, can do suitably to modify to described gene or its regulating and controlling sequence, also can before its transcription initiation codon, replace the original promotor of described gene with other promotor.
The Another application of recovery gene provided by the invention is to produce specific molecule marker according to described gene order information, includes but not limited to SNP (mononucleotide polymorphic), SSR (simple sequence repeats polymorphic), RFLP (restriction enzyme length is polymorphic), CAP (the cutting amplified fragments is polymorphic).Can identify the male sterile restoring line of paddy rice or other plant or recover plant and the genotype of non-recovery system or non-recovery plant with this mark, be used for the molecular marker assisted selection breeding.Described non-recovery is kind and the strain that does not carry the recovery gene gene of function, can be sterile line or maintenance line.
The present invention has obvious advantage and the effect of getting.Utilize cloned genes of the present invention can carry out the breeding of molecular level.Compare with the back cross breeding method with the hybridization of routine, cultivate recovery system with transgenic method and can shorten breeding time, can also import other useful gene simultaneously,, cultivate the recovery system of disease-resistant worm as disease-resistant anti insect gene.In addition, compare with the breeding system of selection of routine, the molecular marker assisted selection breeding can improve the breeding efficiency of selection.
Below in conjunction with drawings and Examples clone's process, gene function analysis and the purposes that male sterile provided by the invention recovers gene specified.
Description of drawings
Fig. 1 recovers the map based cloning synoptic diagram of gene Rf1 for paddy rice;
Fig. 2 is the rice sterile line PCR test of resistant plants Seven detected result figure of Rf1 candidate gene;
Fig. 3 detects figure for the pollen fertility that changes Rf1 candidate gene sterile line transformant;
Fig. 4 recovers gene OsRf1 amino acids coding peptide sequence structure iron for paddy rice;
Fig. 5 is that the RT-PCR of OsRf1 gene and allelotrope Osrf1 expression characterization thereof detects figure;
Rice restorer and the hybridization F thereof of Fig. 6 for changeing the OsRf1 gene
1Seville orange flower powder fertility detects figure;
Fig. 7 is for identifying parent and hybrid F with molecule marker Rf1m
2For the Rf1 loci gene type of plant figure as a result.
Y3-8 among Fig. 1,01-2.5,01-2, Rf1m, C1361, S10019 are dna molecular marker, Rf1 is for recovering gene locus, A24 is for can transform artificial chromosome (Transformation-competent artificial chromosome, TAC) clone, ORF is the gene opening code-reading frame of prediction.
Swimming lane 1 is for containing the vector plasmid PCR product of Rf1 candidate gene among Fig. 2, swimming lane 2 is the PCR product of DNA for recovering, swimming lane 3-7 is the PCR product of the sterile line transformant DNA of importing Rf1 candidate gene, swimming lane 8 is no template DNA negative control, swimming lane 9 be the non-transformant DNA of sterile line as negative control, swimming lane M is a molecular weight marker.
Pollen fertility detection method shown in Figure 3 is pollen to be placed on the substratum sprout, the pollen tube of arrow indication for sprouting.Wherein A figure is the pollen that recovers system, and B figure is the pollen of sterile line, and C figure and D figure are the pollen of transformant.
Partly for this albumen of location arrives mitochondrial signal peptide, it is 7 PPR repeating units that mark rolls off the production line partly to tilted letter among Fig. 4.
Swimming lane 1-7 is the amplified production that uses OsRf1 special primer Rf1m-3R and Rf1m-2 in the 2nd the PCR reaction among Fig. 5, wherein swimming lane 1 is that the RNA of fringe portion of sterile line is a template, swimming lane 2 is that the RNA of fringe portion that recovers to be is a template, swimming lane 3-4 is that the RNA of fringe portion of the sterile line transformant of importing OsRf1 gene is a template, swimming lane 5 is that the RNA of leaf portion that recovers to be is a template, and swimming lane 6-7 is that the RNA of leaf portion of the sterile line of importing OsRf1 gene is a template.Swimming lane 8-14 is the amplified production that uses Osrf1 special primer Rf1m-3B and Rf1m-2 in the 2nd the PCR reaction, wherein swimming lane 8 is that the RNA of fringe portion of sterile line is a template, swimming lane 9 is that the RNA of fringe portion that recovers to be is a template, swimming lane 10 is that the RNA of fringe portion of the sterile line transformant of importing OsRf1 gene is a template, swimming lane 11 is that the RNA of leaf portion that recovers to be is a template, swimming lane 12 is that the RNA of leaf portion of sterile line is a template, and swimming lane 13-14 is that the RNA of leaf portion of the sterile line transformant of importing OsRf1 gene is a template.
The pollen fertility detection method of Fig. 6 is with using microscopic examination after the potassiumiodide dyeing.Wherein A figure changes the pollen that the OsRf1 gene recovers system, and B figure is the pollen of sterile line, and C figure is the pollen of maintenance line, and D figure is hybrid F
1Pollen for plant.
Swimming lane 1 is the sterile line parent among Fig. 7, and genotype is rf1rf1; Swimming lane 2 is the parent for recovering, and genotype is Rf1Rf1; Swimming lane 3-9 is F
2Pollen can be educated strain entirely, and genotype is Rf1Rf1; Swimming lane 10-16 is F
2Pollen semi-sterility strain, genotype are Rf1rf1.
Embodiment 1. paddy rice recover Fine Mapping and the clone of gene Rf1
The present invention clones the Rf1 gene with the map based cloning method.Be C9083 hybridization with paddy rice bag bench-type cytoplasmic male sterile line 731A and recovery at first, having set up one has 600 strain F
2Individual segregating population.According on paddy rice the 10th karyomit(e) of having reported the approximate location (Ichikawa, 1997) in localized Rf1 site, selected should the zone molecule marker carried out polymorphism and detected and linkage analysis.Http:// rgp.dna.affrc.go.jp) and polymorphism probe 01-2.5, the 01-2 and the Rf1m that obtain with pcr amplification at the design of BAC cloned sequence OSJNBa0078O01 position and synthetic primer according to the data in Public Rice Genome Sequence Data storehouse these marks comprise Y3-8 (Zhang Qunyu etc., 2002), C1361, S10019 (Japanese RGP institute:.Carry out linkage analysis with these marks, made up the linkage map (Fig. 1) of a Rf1 site areas.It is to have only 1 recombination event respectively between 01-2.5 and C1361 and the Rf1 site that 2 marks are arranged among the figure, is equivalent to the genetic distance of 0.08cM (cM), and in addition is zero to recombinate i.e. complete linkage between 2 mark 01-2 and Rf1m and the Rf1 site.01-2 is a RFLP mark, and Rf1m is a SNP mark, and both are at a distance of about 10kb.Therefore the Rf1 site by Fine Mapping at mark 01-2 and Rf1m near zone (Fig. 1).Made up a genomic library that recovery is C9083 can transform artificial chromosome carrier TAC (Liu et al., 2002).With 01-2 and Rf1m is that probe screens this genomic library, has obtained 1 TAC clone, is numbered A24, and it inserts the son size is 36kb (Fig. 1).
To clone A24 has carried out dna sequencing and has carried out the predictive genes analysis with predictive genes software Autopredgeneset.With mark Rf1m position overlapped on predict a gene opening code-reading frame (ORF).Show that with computer analysis software PSORT prediction result this ORF coding expressed protein is positioned to plastosome.Because the cytoplasmic male sterility in plants gene is present in plastosome, recover gene expression product and need enter the function that plastosome could be exercised the recovery fertility, so with this ORF as the Rf1 candidate gene, by following its function of functional complementation experimental verification.2. the paddy rice candidate recovers the conversion of gene and the fertility detection of transformant
TAC is cloned comprise the Rf1 candidate gene in insertion of A24 a 7.3kb fragment subclone to plant conversion carrier pCAMBIA1300, import Agrobacterium EHA105.Paddy rice is wrapped bench-type cytoplasmic male sterile line WYJ8A mature seed evoked callus on inducing culture.The EHA105 that contains the goal gene conversion carrier was cultivated 1 day for 28 ℃ at the YM nutrient agar, be collected in the MB liquid nutrient medium of the Syringylethanone that contains 100 μ mol/L.The rice callus tissue was immersed bacterium liquid 20 minutes, blot and transfer to the MB nutrient agar behind the bacterium liquid and cultivate.Shift callus to the culture medium culturing that contains the 50mg/L Totomycin, per 14 days subcultures once, subculture 2 times.Behind the resistance screening, change regeneration culture medium over to and differentiate the conversion seedling, obtained the transformant of strain more than 50.
Rf1m has carried out the PCR detection to transformant with the SNP mark.3 primers are used in this experiment, and wherein primer Rf1m-1 is: GTGGAACCCATCATCTTTAAC-3 ', primer Rf1m-2 is: GCAATAATGGAGCTGTAAA-3 ', primer Rf1m-3R is: GGAATGACCGAGCTCG-3 '.Rf1m-3R is the special nested primer of allelotrope OsRf1 for recovering.The PCR step is: (1) the 1st PCR is with primer Rf1m-1 and 33 circulations of primer Rf1m-2 amplification, each circulation be 94 ℃ 30 seconds, 60 ℃ 60 seconds, 72 ℃ 60 seconds; (2) getting 1 μ l PCR product is template, carries out the 2nd PCR reaction (25 μ l) with primer Rf1m-3R and Rf1m-2,10 circulations of increasing, each circulation be 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds.
Detected result proof target gene fragment has imported these transformant (Fig. 2).Pollen potassiumiodide dyeing microscopic examination is got in the transformant heading when blooming, find that fertile flower powder rate reaches about 50%-80%, and the pollen germination that the pollen germination experiment can be observed transformant goes out pollen tube (Fig. 3), and the transformant selfing can be solid.And the fertile flower powder rate of the sterile line of contrast is zero, and pollen can not be sprouted, and selfing can not be solid.This experiment is the explanation of functional complementation result of experiment, the candidate who is transformed into sterile line recovers the pollen fertility that expression of gene has been recovered sterile line, the biological function that has proved this gene has confirmed that for recovering the fertility of cytoplasmic male sterility in plants this gene is male sterile and recovers gene.It is OsRf1 (Oriza sativa Rf1) that the present invention recovers unnamed gene with this cytoplasmic male sterility that obtains from the Rf1 site clone of rice genome, and the allelotrope of the non-recovery of paddy rice system is Osrf1.3.OsRf1 Gene Sequence Analysis
By Screening of Rice cDNA library and reverse transcription PCR (RT-PCR) amplification, cDNA clone and the order-checking of OsRf1 have been obtained.Sequence shown in the sequence 1 is the genomic DNA fragment that has comprised the OsRf1 gene in the sequence table, and it comprises the sequence of promoter region, opening code-reading frame and 3 ' end non-translational region.The opening code-reading frame of OsRf1 has 1521 Nucleotide, is a gene that does not have intron, the protein polypeptide (Fig. 4, sequence 2) that coding is made up of 506 amino acid.The order-checking comparative analysis finds that the nucleotide sequence of the Osrf1 that OsRf1 and a plurality of non-recovery are has only the difference of 3 bases, wherein the variation of 2 bases causes the change of 2 amino-acid residues, promptly the Gly (G) the 156th of sequence 2 becomes Ser (S), and the 412nd Asn (N) becomes Ser (S).
The database comparative analysis does not find that the gene of OsRf1 and known organism function has tangible homology.The T type cytoplasmic male sterility of OsRf1 and corn is recovered gene Rf2 (acetaldehyde dehydrogenase gene) does not have homology on nucleotide sequence and amino acid polypeptide sequence.The fertility restorer gene Rf-PPR592 of OsRf1 and petunia lists detection less than homology at nucleotides sequence, 29% the homology of only having an appointment in the subregion on the amino acid polypeptide sequence.Therefore OsRf1 is that a new cytoplasmic male sterility in plants recovers gene.
With the OsRf1 gene is probe Screening of Rice cDNA library and sequencing analysis, and the comparative analysis of binding data library searching finds to have at least in the rice genome other 7 genes and OsRf1 height homology.In these homologous genes, have only the function of OsRf1 to be determined, all the other homogenic Unknown Function by the present invention.Database retrieval finds that the genome of a kind of plant Arabidopis thaliana (Arabidopsis thaliana) also has the amino acid polypeptide sequence of a plurality of Unknown Function and OsRf1 amino acids coding peptide sequence that in various degree homology is arranged.Utilize the NCBI website to analyze demonstration, the OsRf1 amino acid sequence coded has 7 PPR to repeat (Fig. 4), therefore OsRf1 belongs to a newcomer of the super gene family that contains the PPR functional domain, is one of a few gene of clear and definite function in this gene family.4.OsRf1 expression of gene specificity analysis
With RT-PCR the tissue and the space-time specificity of this genetic expression are analyzed.RT-PCR uses 4 primers of SNP mark Rf1m, promptly except embodiment 2 described 3 primers, also has a sterile line or the special nested primer Rf1m-3B:GGAATGACCGAGCTCA-3 ' of maintenance line allelotrope Osrf1.The PCR step is: (1) the 1st PCR is with primer Rf1m-1 and 33 circulations of primer Rf1m-2 amplification, each circulation be 94 ℃ 30 seconds, 60 ℃ 60 seconds, 72 ℃ 60 seconds; (2) getting 0.3 μ l PCR product is template, prepares 2 the 2nd PCR reactions (25 μ l), is 1 reaction with primer Rf1m-3R and Rf1m-2 promptly, be other 1 reaction with Rf1m-3B and Rf1m-2,10 circulations of increasing, each circulation be 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 60 seconds.
Experimental result shows, can amplify specific fragment from the young fringe of the sterile line transformant of recovering system and sterile line and importing OsRf1 gene thereof and the RNA reverse transcription template of leaf texture, illustrate that OsRf1 and allelotrope Osrf1 thereof have expression at rice young panicle and leaf texture, promptly belong to the gene (Fig. 6) of constitutive expression.5. transform the application that the OsRf1 gene produces new restorer line and hybridization
OsRf1 gene subclone to plant conversion carrier pCAMBIA1300, is imported Agrobacterium EHA105, be used for rice transformation bag bench-type cytoplasmic male sterile line WYJ8A, obtained the transformant of strain more than 50.T1 with transformant is hybridized for homozygous plants and another rice sterile line KFA, has produced hybrid F
1Seed.These F
1Plant grows fine, and fertility normal (Fig. 7), and explanation can produce new recovery system with transforming the OsRf1 gene, is used for cenospecies production.6.OsRf1 the application of gene order in the molecular marker assisted selection breeding
Utilization the invention provides recovery gene order information can produce molecule marker, is used to identify to have the plant that the range gene type is Rf1Rf1, Rf1rf1, rf1rf1 on the Rf1 site, can use in the molecular marker assisted selection breeding.Figure 8 shows that with SNP mark Rf1m parental rice and F
2Genotype for plant identifies that the PCR reaction conditions is identical with embodiment's 4.The result shows that the marker gene type of all plant and pollen fertility phenotype are in full accord.
＜110〉＜120〉＜160〉2＜210〉1＜211〉3453＜212〉DNA＜213〉 ( Orisa sativa L. )＜220〉＜221〉CDS＜222〉 ( 1460 ) ... ( 2980 )＜400〉1ggcgagctcg ctggtggccg tgatgatgca ggcgctcgcc caccgcgcca gctacctgtg 60ggtgctcgac gaggacgacg actgccgcct cgccgggatc gtcacgttcg ccgacgtgct 120cacggtgttc cgcgagcagc tgcagtgaag gccgctgccg cggcgacgcc aagaaagagt 180tctttttttt ttttctgttg cggcgatcga tcgatggatc gttgtgcatg cctgcatcct 240tgttctgaaa acattttgga gtgtttattt tgaagcgttt tgaactctga actctgaagt 300atttgtctct gccttggcct ggtgttgatg aacaaactga gactttttgg atatgccttc 360caaataaata agcatttgag ttttcatgct gtagtgaaaa attctaaatt gtgattcagt 420ttagaatagt tgaggacatg ttttgtgaga aacaagtgat caaagtggaa tattttaaaa 480agaaactaaa acacgacgga gatgcagtaa tttggattga tgagtaggga taggaagcag 540agagatcgaa gagtgacatt ctcgttggca tcatttttct ctactattat aaaaattgaa 600aatattttta ctaatatttt cgtacgtcat ccgggtacga gacgattttt aaattcgttt 660gcttttagaa atacatatcc gtatttgagt cggtttttaa cattgttcac ttttggcaat 720acagaaagaa tcatataaga aatctgttta aaaaaaactc gcatgctaac ttaaacgatc 780ggacttctaa ctgcagctca tgattttata aaaatatata tatccaagcg aactccatag 840tgaattttat tttaactaaa ccatataata ataacaagat taaaatagac tttacccatt 900gtaatgcacg ggcatttttt ctagtctatg aaaaaattga gaaaattttg caagtgaaat 960attattatgc cacattagca acaaagaaag ttggaaatat ttgcagtata aaaaaaacaa 1020gtaaaaacat gccactcgta gctagaaaaa aaaagtaact taaggaatat aaaaacatgc 1080ttcccatata gaaaaaaaaa gagtaacccc aaatcaagta atactcgttg aagcaagatg 1140tatgtgaaac cgaaaaaaga aaaaaaaaag aacaaataag acgctgatct gattgatctc 1200ttctcgtcgt cttcttcccg cgcctttcgc gaagccaact gctcgaacgg acgcacccgt 1260gcagccgcgc tcgtcggccg tcgccgtcga ggccgcccct ccgccgccgc cagcagctga 1320tccgttcgcc attccttcgc gcgctggtta gttccacctt cctttgcctc tggttcctag 1380tttcctagta aaccacgggg tgatttattc agcgctgccg caggtttcca cctttccttg 1440cccgtccgtc aggccggaca tggcacgccg cgtcgctgcc cgcgcccgcg cccgcgccgg 1500cggcgtcccg cgctcggagg gtacgatcca agaccgagca cgcgttggga gcggtggcgc 1560cgaggacgca ctcgacgtgt tcgacgaatt gctccggcga ggcatcggcg ccccgatccg 1620cagcttgaac ggcgctctcg ccgacgtcgc gcgcgacaac cccgcggccg ctgtgtcccg 1680cttcaaccgc atggcacgag ctggtgccag catggtaact cccaccgtgc acacctatgg 1740catcctcatc ggctgctgct gcagtgcggg ccgcttagac ctcggtttcg cggccttggg 1800ccatgtcgtt aagaagggat tcagagtgga acccatcatc tttaatcctc tgctcaaggg 1860cctctgtgca gacaagagga cggacgacgc aatggacata gtgctccgcg gaatgaccga 1920gctcggctgc gtgccaaatg tcttctccca caccattatt ctcaagggac tctgtcatga 1980gaacagaagc caagaagctc tcgagctgct ccacatgatg gctgatgatg gaggaggctg 2040cttacctaat gttgtgtcat acagcaccgt catcgatggc ctcttgaaag gaggggatcc 2100ggacaaagcc tacgctacat accgtgaaat gcttgaccgg aggattttgc caaatgttgt 2160gatttacagc tccattattg ctgccctatg caagggtcaa gcaatggaca aggccatgga 2220ggtacacgat aggatggtta agaatggagt tacacccaat tgcttcacgt atactagtct 2280tgtgcatgga ttttgctctt cagggcagtt gacagaggct attaaatttc tagaaaagat 2340gtgcagcaat ggtgttgaac caaatgttgt tacttatagc tcgtttatgg actatctctg 2400caagaacgga agatgcacag aagctagaaa gatttttgat tctatggtca agaggggcct 2460aaagcctgat attactacct acagtagctt acttcatggg tatgctatcg aaggagctct 2520tgttgagatg catggtctct ttgatttgat ggtacaaagt gatatgcaac ccgatcatta 2580tgtcttcaac acactaatat atgcatccgc caagcaagga aaagtagatg aggccatgct 2640tgtatttagc aaaatgaggc agcaaggatt gaaacctaat tgtgttacgt ataacacttt 2700gattaatggc tactgtaaaa ttactaggat ggagaatgct ttagcacttt tccaagagat 2760ggtgagcaat ggtgttagtc ctaattttat cacatataac ataatgctgc aaggtttatt 2820tcgtacagga agaactgcta ctgcaaaaga attctatgta cagattatca aaagtggcaa 2880aaaagatctt atagaacagg ggttgctaga agaattggat gatctatttc tttcaatgga 2940ggacaatgac tgtagtactg tgtcgactcc tgcatgctaa attgcagagt tacaaagtta 3000cttcaaaaag gggaagtagg cactaggcag tgctggggtt tacatgtcca aaattgatca 3060aaataacctc cccgttgaag cttcgactgt tgatttgctg attttgctga tttcaagcag 3120gaaattcaat caaacatacg gaatttctcc ctgaaaagtt ttgctctgaa gtgaaatcca 3180tcctgtttga aatggcctac agttatgcag ttgtaactgg ctattgttgg ccttctcact 3240gaattagtaa tacaaacaag aaattctgga ccactctatg tttggggatc acctttatct 3300gttacatgtg gattgctagg tacaaactgc cctttttttt gtcacactat atctgattcc 3360cttcgaagaa aaattcccag atgatattat agttctctaa ttttgctact ttttgtagct 3420ttgttagaac taatttatta tctgctaaac ttg 3453＜210〉2＜211〉506＜212〉PRT＜213〉 ( Orysa sativa L. )＜220〉＜221〉DOMAIN＜222〉 ( 149 ) ... ( 443 )＜400〉2Met Ala Arg Arg Val Ala Ala Arg Ala Arg Ala Arg Ala Gly Gly1 5 10 15Val Pro Arg Ser Glu Gly Thr Ile Gln Asp Arg Ala Arg Val Gly
20 25 30Ser?Gly?Gly?Ala?Glu?Asp?Ala?Leu?Asp?Val?Phe?Asp?Glu?Leu?Leu
35 40 45Arg?Arg?Gly?Ile?Gly?Ala?Pro?Ile?Arg?Ser?Leu?Asn?Gly?Ala?Leu
50 55 60Ala?Asp?Val?Ala?Arg?Asp?Asn?Pro?Ala?Ala?Ala?Val?Ser?Arg?Phe
65 70 75Asn?Arg?Met?Ala?Arg?Ala?Gly?Ala?Ser?Met?Val?Thr?Pro?Thr?Val
80 85 90His?Thr?Tyr?Gly?Ile?Leu?Ile?Gly?Cys?Cys?Cys?Ser?Ala?Gly?Arg
95 100 105Leu?Asp?Leu?Gly?Phe?Ala?Ala?Leu?Gly?His?Val?Val?Lys?Lys?Gly
110 115 120Phe?Arg?Val?Glu?Pro?Ile?Ile?Phe?Asn?Pro?Leu?Leu?Lys?Gly?Leu
125 130 135Cys?Ala?Asp?Lys?Arg?Thr?Asp?Asp?Ala?Met?Asp?Ile?Val?Leu?Arg
140 145 150Gly?Met?Thr?Glu?Leu?Gly?Cys?Val?Pro?Asn?Val?Phe?Ser?His?Thr
155 160 165Ile?Ile?Leu?Lys?Gly?Leu?Cys?His?Glu?Asn?Arg?Ser?Gln?Glu?Ala
170 175 180Leu?Glu?Leu?Leu?His?Met?Met?Ala?Asp?Asp?Gly?Gly?Gly?Cys?Leu
185 190 195Pro?Asn?Val?Val?Ser?Tyr?Ser?Thr?Val?Ile?Asp?Gly?Leu?Leu?Lys
200 205 210Gly?Gly?Asp?Pro?Asp?Lys?Ala?Tyr?Ala?Thr?Tyr?Arg?Glu?Met?Leu
215 220 225Asp?Arg?Arg?Ile?Leu?Pro?Asn?Val?Val?Ile?Tyr?Ser?Ser?Ile?Ile
230 235 240Ala?Ala?Leu?Cys?Lys?Gly?Gln?Ala?Met?Asp?Lys?Ala?Met?Glu?Val
245 250 255His?Asp?Arg?Met?Val?Lys?Asn?Gly?Val?Thr?Pro?Asn?Cys?Phe?Thr
260 265 270Tyr?Thr?Ser?Leu?Val?His?Gly?Phe?Cys?Ser?Ser?Gly?Gln?Leu?Thr
275 280 285Glu?Ala?Ile?Lys?Phe?Leu?Glu?Lys?Met?Cys?Ser?Asn?Gly?Val?Glu
290 295 300Pro?Asn?Val?Val?Thr?Tyr?Ser?Ser?Phe?Met?Asp?Tyr?Leu?Cys?Lys
305 310 315Asn?Gly?Arg?Cys?Thr?Glu?Ala?Arg?Lys?Ile?Phe?Asp?Ser?Met?Val
320 325 330Lys?Arg?Gly?Leu?Lys?Pro?Asp?Ile?Thr?Thr?Tyr?Ser?Ser?Leu?Leu
335 340 345His?Gly?Tyr?Ala?Ile?Glu?Gly?Ala?Leu?Val?Glu?Met?His?Gly?Leu
350 355 360Phe?Asp?Leu?Met?Val?Gln?Ser?Asp?Met?Gln?Pro?Asp?His?Tyr?Val
365 370 375Phe?Asn?Thr?Leu?Ile?Tyr?Ala?Ser?Ala?Lys?Gln?Gly?Lys?Val?Asp
380 385 390Glu?Ala?Met?Leu?Val?Phe?Ser?Lys?Met?Arg?Gln?Gln?Gly?Leu?Lys
395 400 405Pro?Asn?Cys?Val?Thr?Tyr?Asn?Thr?Leu?Ile?Asn?Gly?Tyr?Cys?Lys
410 415 420Ile?Thr?Arg?Met?Glu?Asn?Ala?Leu?Ala?Leu?Phe?Gln?Glu?Met?Val
425 430 435Ser?Asn?Gly?Val?Ser?Pro?Asn?Phe?Ile?Thr?Tyr?Asn?Ile?Met?Leu
440 445 450Gln?Gly?Leu?Phe?Arg?Thr?Gly?Arg?Thr?Ala?Thr?Ala?Lys?Glu?Phe
455 460 465Tyr?Val?Gln?Ile?Ile?Lys?Ser?Gly?Lys?Lys?Asp?Leu?Ile?Glu?Gln
470 475 480Gly?Leu?Leu?Glu?Glu?Leu?Asp?Asp?Leu?Phe?Leu?Ser?Met?Glu?Asp
485 490 495Asn?Asp?Cys?Ser?Thr?Val?Ser?Thr?Pro?Ala?Cys.
500 505
Claims (7)
1 one kinds of isolated cytoplasmic male sterility in plants recover gene, it is characterized in that:
(1) coding is a kind of has cytoplasmic male sterility in plants and recovers active amino acid polypeptide sequence, and this polypeptide has the sequence shown in the sequence 2 in the sequence table; Or coding tool same function by sequence 2 polypeptides derived sequences, or its conservative property variation polypeptide, or its active fragments;
(2) has the nucleotide sequence of sequence 1 in the sequence table; Or with the 1460-2980 bit base sequence of sequence 1 in section more than continuous 210 bases homology more than 85% and coding and the proteinic nucleotide sequence of sequence 2 same functions are arranged.
2. according to the said gene of claim 1, it is characterized in that coded amino acid polypeptide sequence contains the functional domain of being made up of 35 PPR of amino-acid residue repeating unit.
3. according to the said gene of claim 1, it is characterized in that the 1-1459 bit base in the sequence 1 is the sequence that contains promoter regulation element and 5 ' end non-translational region, the 1460-2980 bit base is an opening code-reading frame, and the 2981-3453 bit base is the sequence of 3 ' end non-translational region.
4. according to the said gene of claim 1, it is characterized in that sequence 1 for containing the sequence of the cytoplasmic male sterility recovery gene OsRf1 that derives from paddy rice, sequence 2 is the amino acid polypeptide sequence of the 1460-2980 bit base sequence encoding in the sequence 1.
5. contain the full sequence of the said gene of claim 1 or the carrier of partial sequence.
6. according to the application of the said gene of claim 1, it is characterized in that: with said gene or contain carrier rice transformation or other plant of said gene, cultivate the transgenic restorer line that to express said gene; With transgenic restorer line and cytoplasmic male sterile line hybridization, produce cenospecies.
7. according to the application of the said gene order of claim 1, it is characterized in that: produce specific molecular mark probe according to said gene order, identify the male sterile restoring line of paddy rice or other plant or recover plant or non-recovery system or non-recovery plant with this label probe, carry out the molecular marker assisted selection breeding.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN102726285A (en) * | 2010-07-27 | 2012-10-17 | 上海交通大学 | Preparation method, seed propageation method and application of seeds of rice male sterile line |
| CN104789648A (en) * | 2014-12-25 | 2015-07-22 | 中国种子集团有限公司 | Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof |
| CN106754964A (en) * | 2017-01-17 | 2017-05-31 | 华中农业大学 | The clone of cabbage type rape nap cytoplasmic male sterility Restore genes Rfn and its application |
| CN108130328A (en) * | 2017-11-28 | 2018-06-08 | 上海交通大学 | The application of male sterility gene OsDPW3 and the method for rice fertility restorer |
| CN110241251A (en) * | 2019-07-26 | 2019-09-17 | 中国农业科学院棉花研究所 | The InDel label of cotton cells matter fertility restorer gene Rf1, Rf2 are identified simultaneously |
| CN110268973A (en) * | 2019-04-19 | 2019-09-24 | 湖南杂交水稻研究中心 | The production method of hybrid rice seed |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981833A (en) * | 1994-11-29 | 1999-11-09 | Iowa State University Research Foundation, Inc. | Nuclear restorer genes for hybrid seed production |
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| 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 |
| CN102726285A (en) * | 2010-07-27 | 2012-10-17 | 上海交通大学 | Preparation method, seed propageation method and application of seeds of rice male sterile line |
| CN102726285B (en) * | 2010-07-27 | 2014-04-16 | 上海交通大学 | Preparation method, seed propagation method and two-line hybrid seed propagation method based on osms4 mutant |
| CN104789648B (en) * | 2014-12-25 | 2017-12-01 | 中国种子集团有限公司 | Identify molecular labeling and its application of the section haplotypes of rice CMS restoring genes Rf 1 |
| CN104789648A (en) * | 2014-12-25 | 2015-07-22 | 中国种子集团有限公司 | Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof |
| CN106754964B (en) * | 2017-01-17 | 2019-11-15 | 华中农业大学 | The clone of cabbage type rape nap cytoplasmic male sterility restoring gene Rfn and its application |
| CN106754964A (en) * | 2017-01-17 | 2017-05-31 | 华中农业大学 | The clone of cabbage type rape nap cytoplasmic male sterility Restore genes Rfn and its application |
| CN108130328A (en) * | 2017-11-28 | 2018-06-08 | 上海交通大学 | The application of male sterility gene OsDPW3 and the method for rice fertility restorer |
| CN108130328B (en) * | 2017-11-28 | 2021-10-29 | 上海交通大学 | Application of male sterility gene OsDPW3 and rice fertility restoration method |
| CN110268973A (en) * | 2019-04-19 | 2019-09-24 | 湖南杂交水稻研究中心 | The production method of hybrid rice seed |
| CN110268973B (en) * | 2019-04-19 | 2022-05-20 | 湖南杂交水稻研究中心 | Production method of rice hybrid |
| CN110241251A (en) * | 2019-07-26 | 2019-09-17 | 中国农业科学院棉花研究所 | The InDel label of cotton cells matter fertility restorer gene Rf1, Rf2 are identified simultaneously |
| CN110241251B (en) * | 2019-07-26 | 2022-08-16 | 中国农业科学院棉花研究所 | InDel markers for simultaneously identifying cytoplasmic male sterility restorer genes Rf1 and Rf2 of cotton |
| WO2023016097A1 (en) * | 2021-08-09 | 2023-02-16 | 华中农业大学 | Oryza sativa cytoplasmic male sterility restorer gene osrf19 and application thereof |
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