CN102477091B - Rice male sterile protein and coding gene and application thereof - Google Patents
Rice male sterile protein and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a rice male sterile protein and a coding gene and application thereof. The protein is one of 1) a protein formed by amino acid residue sequences of the SEQ ID No.2 in a sequence table or 2) a protein which is formed after one or more amino acid residues in the amino acid residue sequences of the SEQ ID No.2 in the sequence table are substituted and/or deleted and/or added and has the same activities as the amino acid residue sequences of the SEQ ID No.2. The rice male sterile protein gene can be used for breeding male sterile rice, and makes contributions for removing male hybrid rice.
Description
Technical field
The present invention relates to male sterility of rice albumen and encoding gene thereof and application.
Background technology
Paddy rice is one of topmost food crop of the mankind, the population of half is approximately arranged in the world take rice as main food.The China rice accounts for global 23% as area, rice yield accounts for global 37% left and right.The rice growing area of China accounts for 30% of national food crop sown area, and ultimate production accounts for more than 42% of food crop output, yield per unit on average exceeds 45.7% than whole food crop, and one of them important reason is exactly heterotic widespread use.Hybrid vigour is a kind of universal phenomenon of organic sphere, refers to two parent's hybridization that heredity is different, and first generation of hybrid the Characters has stronger vitality, growth potential, resistance, adaptability and yielding ability than parents.Hybrid rice shows significant hybrid vigour on many proterties, general performance is at aspects such as nutritional advantages, Reproductive heterosis, resistance advantage and quality heterosis.Applying of hybrid rice, for the increases in grain production in China and even the world make a great contribution.
The basis that rice heterosis can effectively utilize is male sterility of rice.Plants male sterility is can not produce the phenomenon with normal function pollen in the plant sexual reproduction process, and this phenomenon ubiquity in higher plant is effectively to utilize hybrid vigour, improves prerequisite and the basis of crop yield.Forming angle from genotype divides, the male sterile of paddy rice is divided into: nuclear male sterility (Genie Male Sterility, GMS) and nucleo-cytoplasmic interreaction male sterility (that is cytoplasmic male sterility, Cytoplasmic Male Sterility, CMS).Nuclear male sterility is controlled by the nucleus sterile gene, without positive and negative friendship hereditary effect.Aobvious recessive relation according between sterile gene and corresponding educated gene, can be divided into recessive cytoblast sterile and dominant genic male sterile again, and the male nuclear sterile of most plants belongs to recessive cytoblast sterile, accounts for 88%.The dominant genic male sterile paddy rice, for educating and sterile separating than being 1: 1,, not only without restorer but also without maintenance line, can not be directly used in production because of its F1 usually; Single-gene recessive cytoblast sterile, dual-gene recessive cytoblast sterile and polygene recessive cytoblast sterile, these a few class sterile lines, because be difficult to find corresponding maintenance line on producing, can not be realized three series mating, have limited its application in heterosis utilization.So far comparatively successfully applying recessive cytoblast sterile is that rice photo-thermo-sensitive core is sterile.Find that there is the responsive male sterility line of light (temperature) in paddy rice, be divided into temperature sensitive type, Photosensitive and warm light interaction type.
Summary of the invention
The purpose of this invention is to provide a kind of male sterility of rice albumen and encoding gene and application.
Male sterility of rice albumen provided by the invention (MSPL), derive from paddy rice, is following 1) or 2) protein:
1) protein that is formed by the amino acid residue sequence of the SEQ ID № .2 in sequence table;
2) with replacement and/or disappearance and/or the interpolation of one or several amino-acid residue of the process of the SEQ ID № .2 amino acid residue sequence in sequence table and the protein with identical activity of amino acid residue sequence of SEQ ID № .2.
In sequence table, sequence 2 is comprised of 127 amino-acid residues.
For the ease of the purifying of MSPL, label as shown in table 1 on the aminoterminal of the protein that can form at the amino acid residue sequence by sequence 2 or carboxyl terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (b) but in the MSPL synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of MSPL in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the DNA sequence dna shown in sequence 1 or 3 in sequence table, and/or carry out the missense mutation of one or several base pair, and/or at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in table 1, obtain.
The gene (MSPL) of above-mentioned male sterility of rice albumen (MSPL) of encoding also belongs to protection scope of the present invention.
Described male sterility of rice albumen cDNA gene can be following 1) or 2) or 3) or 4) DNA molecular:
1) DNA sequence dna of SEQ ID № .1 in sequence table;
2) polynucleotide of SEQ ID № .2 protein sequence in the code sequence list;
3) with sequence table in the DNA sequence dna that limits of SEQ ID № .1 have 90% above homology, and the identical function protein DNA sequence of encoding;
The nucleotide sequence of the DNA sequence dna hybridization that 4) can limit with sequence in sequence table 2 under stringent condition.
Sequence 1 in sequence table is by 384 based compositions, and its open reading frame (ORF) is from 5 ' end 1-384 position Nucleotide.
Above-mentioned stringent condition can be at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridizes and washes film under 65 ℃ of conditions.
The recombinant vectors that contains above arbitrary described gene also belongs to protection scope of the present invention, as recombinant expression vector.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.
Described plant expression vector comprises double base agrobacterium vector (as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for carrier of plant micropellet bombardment etc.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of as the Agrobacterium crown-gall nodule, inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean, storing protein gene) 3 ' end to transcribe all has similar functions.
While using described gene constructed recombinant plant expression vector, can add any enhancement type promotor, constitutive promoter or inducible promoter before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, stress induced promoter Rd29A etc., they can use separately or with other plant promoter, be combined with; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, as add the coding that can express in plant can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.), have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc., from the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
The expression cassette, transgenic cell line and the recombinant bacterium that contain above arbitrary described gene (MSPL) all belong to protection scope of the present invention.
Within the total length of amplification said gene or the primer pair of arbitrary fragment also belong to protection scope of the present invention.
Any one in described albumen, described gene, described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium all can be applicable to cultivate the male sterile transgenic paddy rice.
Utilize any carrier that can guide foreign gene to express in plant,, with the gene transfered plant cell of encoding said proteins, can obtain transgenic cell line and the transfer-gen plant of anti-salt, drought tolerance enhancing.Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that will transform is cultivated into plant.
The present invention obtains the abiogenous male-sterile mutation body of Xian 3037 in a long-grained nonglutinous rice, and mspl (microsporocyteless) does not produce pollen mother cell in flower pesticide after this mutant heading, cause without any pollen, but the megagamete growth is unaffected.This mutant phenotype is controlled by a recessive nuclear gene.By building indica-japonica hybrid colony, utilize the method for map based cloning, obtained to control the gene of this proterties.Sequence comparison shows that, glutaredoxin of this genes encoding, and there is no at present this genoid sudden change in paddy rice affects the report that pollen mother cell is grown, and illustrates that this is a new gene of controlling nuclear male sterility.The acquisition of this new mutant body, have important using value to the formation developmental mechanism of understanding paddy pollen.After novel gene cloning, can explore and use it for rice breeding, enrich the genetic diversity of existing rice breed.Male sterility of rice protein gene of the present invention, can be used for cultivating male sterile rice, for removing male hybrid rice from, contributes.
Description of drawings
Fig. 1 be 3037 with the flower pesticide of mutant mspl; A and B are respectively the flower pesticide of contrast 3037 (left sides) and mspl (right side); C and D are respectively the crosscut of the mature anther of contrast 3037 (C) and mspl (D).
Fig. 2 is the location of MSPL gene and the structure of complementary expression vector
The A.MSPL gene locus is positioned between molecule marker S8 and S9 on No. 6 the short arm of a chromosome of paddy rice.
The structure of B.MSPL gene.Black represents exon, and this gene only has an exon.What arrow indicated is the insertion point of external source fragment.
C. the structure of complementary expression vector.PCMSPL, complementary expression vector, comprise MSPL upstream region of gene 3870bp and downstream 1306bp; PCMSPLC, complementary expression vector contrast, compare the part promoter region that has lacked the MSPL gene, whole coding region and 3 ' control region with pMSPL.
Fig. 3 be 3037 with the flower pesticide of MSPL gene RNA interference of transgene plant.
Fig. 4 is wild-type salt rice No. 8 and pMSPLRNAi plant MSPL gene expression amount.
Embodiment
Experimental technique in following embodiment, if no special instructions, be ordinary method.
Percentage composition in following embodiment, if no special instructions, be the quality percentage composition
The phenotype of embodiment 1, rice male sterility mutant mspl and genetic analysis
1, the phenotype analytical of rice male sterility mutant mspl
The natural mutant of the middle Xian 3037 (WT, available from Agricultural College Affiliated to Yangzhou Univ.) that this laboratory of rice male sterility mutant mspl is found in field.The mspl mutant vegetative growth phase with compare and there is no notable difference with contrast 3037, after heading, mutant just shows typical male sterile phenotype, shows as flower pesticide and is white in color, the pollen that sheds can not ftracture.The semithin section result shows in this mutant flower pesticide without any pollen, and the size shape that distributing in pollen sac is similar parenchyma cell all, and some pollen sac central authorities are lignifying even, is similar to fascicular tissue differentiation (Fig. 1).
We authorize the mspl mutant pollen of wild-type, find that it can be normally solid, and this cenospecies can normal germination and growth, its plant at aspects such as survival rate, the reproductive growths of nourishing and growing all with wild-type plant no significant difference, show that the mspl mutant is female to educate.
2, the genetic analysis of rice male sterility mutant mspl
Fine (the Chinese Academy of Agricultural Sciences's crop science institute Rice Germplasm Resources center preservation of rice male sterility mutant mspl and round-grained rice type wild-type material (Japonica) Japan, storehouse numbering I1A13071), single two-way cross obtains F1 generation, F1 produces F2 generation for selfing, F2 is carried out phenotypic evaluation for plant, the warm and fine mspl of Japan is respectively as positive and negative contrast, and F2 is as shown in table 2 for the phenotypic evaluation result of plant, shows that this proterties of male sterility of rice meets the Dominant gene genetic development.In table 2, the strain number that normal strain number refers to have Japanese fine phenotype, the strain number that male sterile mspl strain number refers to have the mspl phenotype.
The genetic analysis of table 1 rice male sterility mutant mspl
| Combination | Normal strain number | Mspl strain number | Total strain number | Separate ratio |
| Mspl/ Japan is fine | 306 | 94 | 400 | 3.25∶1 |
The acquisition of embodiment 2, MSPL and encoding gene MSPL thereof
1. the genomic gene of map based cloning MSPL
In order to clone the MSPL gene, we will with the male-sterile mutation body mspl that isozygotys and Japan fine hybridization, the F of acquisition
1Obtain F for selfing
2Colony, carry out the Primary Location of MSPL gene to 236 F2 recessive individual (having the F2 of male sterile phenotype for individuality) wherein.Apply STS (Sequence-Tagged Site) molecule marker, utilize the method for PCR, we find that STS mark S1, S2, S3 and the S4 and the mutational site that are positioned on the 7th karyomit(e) have obvious linksystem in position.Exchange individual plant between mutational site and S2, most also exchange between mutational site and S1, and the exchange individual plant between mutational site and S3, and the overwhelming majority is included in exchange individual plant between mutational site and S4.Therefore exchange individual plant between simultaneous mutation site and S3 is different from the exchange individual plant between mutational site and S2, infers the zone that mutator gene may be between mark S2 and mark S3.On this basis, we further enlarge the fine cross combination of mutant mspl and Japan, have obtained to comprise the F of 1020 plant mutant individual plants
2Segregating population is used for the MSPL Fine Mapping.The rice genome sequence that reference has been completed (
Http:// www.tigr.org/tdb/e2k1/osa1/With http://btn.genomics.org.cn), the sequence of japonica rice and long-grained nonglutinous rice is compared, utilize sequence difference to develop 5 new STS molecule markers (table 3).The MSPL Fine Mapping is between BAC clone AP003704 mark S8 and S9 the most at last, and the physical distance between these two marks is about 13kb.Utilize rice genome annotation database RiceGAAS (
Http:// ricegaas.dna.affrc.go.jp/rgadb) the analysis showed that, have 4 genes in the 13kb zone, we carry out determined dna sequence with these 4 genes, mutant and wild-type sequence have been compared, find all consistent with wild-type of the sequence of three genes in mutant wherein, the gene that only has a coding glutaredoxin, its ORF district can not be by pcr amplification out, and we determine the external source fragment is arranged after the 223bp of this gene ORF insertion the method by TAIL-PCR (thermal asymmetric interlaced PCR).Therefore, we are defined as target gene with the glutaredoxin gene, called after MSPL.In Fig. 2, C is the MSPL gene structure, is labeled as the mutational site of gene in figure.
Because this gene has and does not have complete cDNA sequence at the fine cDNA database of KOME (http://cdna01.dna.affrc.go.jp/cDNA) paddy rice Japan, and rice genome annotation database RiceGAAS predicts that there is 579bp in this gene ORF district.We have carried out 5 ' RACE (5 ' Rapid Amplification of cDNA Ends) PCR and 3 ' RACE (3 to this gene, RapidAmplification of cDNA Ends) PCR, find that the genome total length of this gene from the initiator codon to the terminator codon is 384bp, there is no intron, 127 amino acid of encoding altogether.The complete ORF sequence of this gene is the sequence shown in sequence 1 in sequence table.5 ' RACE and 3 ' RACE adopt the PCR cDNA of Clonetech company synthetic agent box (K1052-1) to operate to specifications.
The STS mark of the new initiative of this research of table 3
2.MSPL the acquisition of full length gene ORF
The total RNA of the fine blade of paddy rice Japan extracts and adopts the RNA of Bioteke company extraction test kit (Bioteke, RP1201) to operate to specifications., take Oligo (dt)-18 as primer, carry out reverse transcription take the total RNA that is extracted as template and synthesize the first chain cDNA.Take this cDNA as template, primer 1 (5 '-AGGATGAGGATGCAGGTGGTGG-3 ') and primer 2 (5 '-AACCGATTCAACAGCACAC-3 '), carry out pcr amplification reaction, reaction conditions is as follows:
Reaction volume 50 μ l, wherein contain: template (cDNA) 5 μ l (5ng), primer forward primer, each 0.2 μ M of reverse primer final concentration, each 200 μ M of dNTP final concentration, Taq archaeal dna polymerase 2.5U, 10 * Taq DNA polymerase buffer liquid, 5 μ l, complement to 50 μ l volumes with distilled water.
Temperature of reaction, time: 94 ℃, sex change 5 minutes; Then 94 ℃ of sex change are 30 seconds, 58 ℃ of annealing 30 seconds, and 72 ℃ were extended 30 seconds, 30 circulations of increasing; Finally 72 ℃ of downward-extensions 5 minutes.
amplified production is 3 ' 3 ' the outstanding cohesive end fragment of base A to be arranged, reclaim test kit (Biomed with Biomed glue, 28706) carry out purifying by product description, then with 3 ' the linear pBM19-T carrier (Biomed that base T arranged, A1360) at room temperature lower connection is 20 minutes, use the 2mm pole cup, 2500V transforms bacillus coli DH 5 alpha, conversion product is grown containing on the LB plate culture medium of penbritin, selected clone, extract plasmid, use AbI PRISM3700DNA analyser (Perkin-Elmer/Applied Biosystem) order-checking, the ORF of the cDNA of the MSPL of acquisition as shown in sequence in sequence table 1, its total length 384bp, the aminoacid sequence of its coding is seen sequence 2 in sequence table.
The complementation test of embodiment 3, male-sterile mutation body MSPL phenotype
1, the structure of complementary carrier pCMSPL and complementary control vector pCMSPLC
Utilize the EcoRI enzyme to cut BAC OsJNBa0057C01 (available from Shanghai country of Chinese Academy of Sciences cara gene, numbering OsJNBa0057C01), acquisition includes the DNA fragmentation (5360bp) of the full length sequence of 3870 bases of initiator codon ATG upstream of MSPL and 1306 bases after terminator codon TGA, be cloned into pCAMBIA1300 (DingGuo, MCV033) EcoRI site, namely be built into complementary expression vector pCMSPL.The complementary carrier pCMSPL that builds is cut with the SacI enzyme, remove part promoter region, whole coding region and the 3 ' control region of MSPL gene, keep the part promoter region of 5 ' end, namely be built into complementary control vector pCMSPLC (C in Fig. 2).
2, acquisition and the phenotypic evaluation thereof of pCMSPL and pCMSPLC transformation plant
Two carrier pCMSPL and pCMSPLC change over to respectively in Agrobacterium (AgroBacterium tumefaciens) strain EHA105 (inferior Pingning, Beijing is biological) by the method for electric shock, utilize the mediated method of Agrobacterium respectively pCMSPL and pCMSPLC to be changed in the embryo of selfing F2 for seed of male-sterile mutation body mspl and Japanese fine hybrid Population.The concrete grammar that transforms is to cut out after this F2 is sterilizing for the rataria of seed, is inoculated in the substratum of evoked callus.After cultivating for 1 week, select growth vigorous, color is pale yellow, and more open embryo callus, as the acceptor that transforms.Infect respectively Rice Callus with the EHA105 bacterial strain that contains pCMSPL and pCMSPLC plasmid, after 25 ℃, dark place cultivates 3 days, containing screening resistant calli and transfer-gen plant on the selection substratum of 50mg/L Totomycin.
The hygromycin resistance plant is practiced seedling in the cool, be transplanted to paddy field after 7 days.Because F2 is for the recessive homozygous individual of existing mspl in seed, the individuality that carries wild-type MSPL gene is arranged again,, so we identify the genotype of transfer-gen plant, pick out the transfer-gen plant that comes from the recessive homozyous seed of mspl, then observe the phenotype recovery situation of these transfer-gen plants.Result shows that it is the wild-type phenotype that complementary carrier pCMSPL can recover the male sterile phenotype, and complementary control vector pCMSPLC can not recover the male sterile phenotype.Function complementation experiment shows that MSPL controls male-sterile mutation body mspl phenotype
The acquisition of embodiment 4, MSPL gene RNA interference of transgene plant
The detailed construction process of MSPL gene RNA interference plasmid pMSPLRNAi:
To carry out the Japanese fine genomic dna of PCR reaction amplification with primer pair primer 3 (5 '-ACCGTATCGTGGTGGAAGG-3 ') and primer 4 (5 '-ACGAGGGTGCCATTGATGTG-3 '), reaction conditions is as follows:
Reaction volume 50 μ l, wherein contain: template (cDNA) 5 μ l (5ng), primer forward primer, each 0.2 μ M of reverse primer final concentration, each 200 μ M of dNTP final concentration, Taq archaeal dna polymerase 2.5U, 10 * TaqDNA polymerase buffer, 5 μ l, complement to 50 μ l volumes with distilled water.
Temperature of reaction, time: 94 ℃, sex change 5 minutes; Then 94 ℃ of sex change are 30 seconds, 58 ℃ of annealing 30 seconds, and 72 ℃ were extended 30 seconds, 30 circulations of increasing; Finally 72 ℃ of downward-extensions 8 minutes.
amplified production is 3 ' 3 ' the outstanding cohesive end fragment of base A to be arranged, reclaim test kit (Biomed with Biomed glue, 28706) carry out purifying by product description, then be connected into the 3 ' linear pMD19-T (Takara that base T arranged, D101) obtain carrier pMD19-MSPL in carrier, obtain fragment 1 and fragment 2 with Sal I and BamHI and Pst I and Xba I double digestion carrier pMD19-MSPL respectively, obtain fragment 3 with Bgl II and Xba I double digestion carrier pUCCRNAi (available from Chinese Academy of Sciences's heredity, growing institute), with fragment 1, 2, 3 are connected between the Pst I and Sal I recognition site of over-express vector pCam13OX simultaneously, namely be built into RNAi interference carrier pMSPLRNAi.The construction process of pCam13OX is as follows:
Use primer pair 35S-F:(5 '-AAGCTTCCCAGATTAGCCTTTTCAAT-3 ') and 35S-R:(5 '-CTGCAGTCCCCCGTGTTCTCTCCAA-3 ') PCR amplification plasmid pBI 121 (DingGuo, MCV032) obtain the approximately composing type CaMV35S promoter fragment of 850bp, then this fragment is connected into carrier pCAMBIA1300 (DingGuo with Pst I and Hind III double digestion, between MCV033) Pst I and HindIII recognition site, form intermediate carrier pCam13OXM.With EcoR I and Sac I double digestion plasmid pBI121, the fragment that obtains is connected between the EcoR I and Sac I recognition site of intermediate carrier pCam13OXM, finally obtain over-express vector pCam13OX.
Method by electric shock changes carrier pMSPLRNAi in Agrobacterium (AgroBacterium tumefaciens) strain EHA105 over to, utilize the mediated method of Agrobacterium that it is changed in round-grained rice type wild-type material salt rice No. 8 (paddy rice Data centre of country of China Paddy Rice Inst preservation, accession designation number Soviet Union examines rice 200307).Method for transformation is with embodiment 2.
Result shows, the transfer-gen plant that obtains vegetative growth phase with compare for No. 8 and there is no notable difference with wild-type material salt rice, after heading, transfer-gen plant just shows the typical male sterile phenotype as the mspl mutant, shows as flower pesticide and is white in color, and the pollen that sheds can not ftracture.The semithin section result shows in this transfer-gen plant flower pesticide without any pollen, is also all similar parenchyma cells of size shape that distributing in pollen sac, illustrate that the MSPL gene carries out RNA Recombinant Interferon α-2b acquisition male sterile plants (Fig. 3).In Fig. 3, A and B are respectively the flower pesticide of salt rice No. 8 (left side) and transfer-gen plant (right side); C and D are respectively the crosscut of salt rice No. 8 (left side) and the mature anther of transfer-gen plant (right side).
The detection by quantitative of pMSPLRNAi plant MSPL expression amount:
Get the 2cm children fringe of transfer-gen plant and wild-type material salt rice No. 8, extract total RNA, adopt the RNA of Bioteke company to extract test kit (Bioteke, RP1201) and operate to specifications., take Oligo (dt)-18 as primer, carry out reverse transcription take the total RNA that is extracted as template and synthesize the first chain cDNA.Compare the expression amount of MSPL gene in leaf cDNA at fine heading stage of transfer-gen plant and wild-type material Japan by semi-qPCR, with the expression amount of paddy rice Ubiquitin gene, do reference.Amplification MSPL gene primer pair primer 5 (5 '-TCCATCCATCGATCCCTACC-3 ') and primer 6 (5 '-GCATTTCAAACTCATCGTCG-3 '), amplification Ubiquitin gene primer pair primer 7 (5 '-CAAGATGATCTGCCGCAAATGC-3 ') and primer 8 (5 '-TTTAACCAGTCCATGAACCCG-3 ').
The pcr amplification reaction condition is as follows:
Reaction volume 20 μ l, wherein contain: template (cDNA) 1 μ l (25ng), forward primer, each 0.2 μ M of reverse primer final concentration, each 200 μ M of dNTP final concentration, Taq archaeal dna polymerase 1U, 10 * Taq DNA polymerase buffer liquid, 2 μ l, complement to 20 μ l volumes with distilled water.
Temperature of reaction, time: 94 ℃, sex change 5 minutes; Then 94 ℃ of sex change are 25 seconds, 58 ℃ of annealing 25 seconds, and 72 ℃ were extended 25 seconds, several circulations of increasing; Finally 72 ℃ of downward-extensions 5 minutes.
Amplification cycles number: 36 circulations of MSPL gene; 25 circulations of Ubiquitin gene.
Result shows does reference with the expression amount of paddy rice Ubiquitin gene, and in the pMSPLRNAi plant, the expression of MSPL gene has obvious decline (seeing Fig. 4), has really reached the effect of disturbing.Fig. 4 wild-type and pMSPLRNAi plant MSPL gene expression amount; Upper is the expression amount of MSPL gene, lower to contrasting the expression amount of Ubiquitin (UBQ) gene; A left side is the 2cm children fringe cDNA of wild-type material salt rice No. 8 (WT), and the right side is the 2cm children fringe cDNA of transfer-gen plant pMSPLRNAi.
Claims (7)
1. albumen, the protein that is formed by the amino acid residue sequence of the SEQ ID № .2 in sequence table.
2. the encoding gene of the described albumen of claim 1.
3. encoding gene according to claim 2, the nucleotide sequence of its cDNA gene is one of following nucleotide sequences:
1) DNA sequence dna of SEQ ID № .1 in sequence table;
2) polynucleotide of SEQ ID № .2 protein sequence in the code sequence list.
4. the recombinant expression vector that contains claim 2 or 3 described genes.
5. the recombinant bacterial strain that contains claim 2 or 3 described genes.
6. the recombinant virus that contains claim 2 or 3 described genes.
7. albumen claimed in claim 1 or its encoding gene application in cultivating male sterile transgenic paddy rice.
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| CN1678745A (en) * | 2002-07-05 | 2005-10-05 | 日本烟草产业株式会社 | The rice restorer gene to the rice BT type cytoplasmic male sterility |
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| CN1678745A (en) * | 2002-07-05 | 2005-10-05 | 日本烟草产业株式会社 | The rice restorer gene to the rice BT type cytoplasmic male sterility |
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