CN102702337A - Rice blast disease-resisting protein, coding gene and application thereof - Google Patents
Rice blast disease-resisting protein, coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a rice blast disease-resisting protein, a coding gene and an application thereof. The protein provided by the invention is following (a) or (b): (a) a protein composed of amino acid sequences shown in the sequence 2 in the sequence table; (b) a protein obtained by substituting and/deleting and/adding one or more of amino acid residue for the amino acid sequence shown in the sequence 2 and deriving from the (a) related to plant blast disease resistance. The PID3-A4 protein and the coding gene provided by the invention can be used for cultivating a new blast disease-resisting plant variety, particularly, a variety showing resistance for blast disease caused by at least one of following Magnaporthe oryzae microspecies: JS2001-108-1, Zhong10-8-14, 97-27-2, 03-10-66-1, 07-31-1-2, 03-10-76-3, 10-25-1-1, 10-32-2-1, 03-11-37-1, 07-55-1-1, 10-47-9-2, 10-120-21-2, 07-21-1-1, 10-62-2-1, 07-26-22, 09-87-2-1, Y34, 99-26-2, CH706 and 99-20-2. The protein is beneficial to improve rice variety and has an important significance for enlarging crop planting range and improving crop yield.
Description
Technical field
The invention belongs to plant genetic engineering field, relate to a kind of rice anti-rice blast albumen and encoding sox and application.
Background technology
Plant can receive the invasion and attack of various pathogenic micro-organisms in growth and development process; Though plant does not possess the initiatively ability of " going after profits and advoiding disadvantages "; But in long-term and various pathogenic micro-organism coevolution processes, plant has formed perception of pathogenic micro-organism accurate signal and system of defense.The plant congenital immunity mainly comprises three aspect contents: the resistance and the systemic acquired resistance of basic resistance, disease-resistant gene mediation.Wherein the resistance of disease-resistant gene mediation is because the reaction times is fast, and disease resistance response is strong, and the allergy and the programmed cellization that usually are accompanied by pathogen infection point are dead, make it become an important component part of plant disease-resistant.Flor has proposed " gene pairs gene " theory when the heredity of research flax rust resistant gene; It specifically is expressed as the different microspecies of pathogenic bacteria and has different nontoxic genes; During nontoxic gene in the middle of the disease-resistant gene in the plant can the special recognition pathogen bacterium, just can be through signal conduction startup disease resistance response.Therefore to the structural analysis and the research of plant disease resistance genes product, be the basis of understanding plant disease-resistant mechanism.
Paddy rice is one of most important food crop in the world, and whole world population over half is main food with rice.Rice blast is one of topmost disease of paddy rice, and the annual paddy rice underproduction that causes because of rice blast in the whole world reaches 11%～30%, and the underproduction reaches 40%～50% when serious, even No kernels or seeds are gathered, as in a year of scarcity.Facts have proved that utilizing the rice varieties that contains blast resistant gene is one of effective way of reply rice blast harm.Up to the present; Localized blast resistant gene has more or less a hundred in paddy rice; That has cloned has 21; They are respectively Pib, Pi-ta, Pi9, Piz-t, Pi2, Pid2, Pi36, Pi37, Rbr2, Pik-m, Pi5, Pid3, pi21, Pit, Pb1, Pish, Pik-p, Pik, Pia, Pi54 and Pi25; Wherein except a gene Pid2 similar kinases of silk Threonine acceptor of coding and rich proline protein of recessive blast resistant gene pi21 coding, other 19 blast resistant genes all belong to Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) gene family.The NBS-LRR gene holds rich leucine to repeat (LRR) zone formation by N end conservative nucleotide binding site (NBS) and C, in the LRR zone, is made up of the incomplete LRR structure of dozens of (general 15～40).Although cloned 21 blast resistant genes at present, because the Pyricularia oryzae microspecies are numerous and speed of mutation is fast, so, clone more blast resistant gene and just seem particularly important with the harm of tackling rice blast.
The NBS-LRR genoid extensively exists in Plant Genome, and in paddy rice of having checked order and Arabidopis thaliana, the NBS-LRR genoid has all accounted for more than 1% of all encoding sox numbers.Blast resistant gene to having cloned is at present analyzed discovery, has 19 to belong to NBS-LRR genoid family in 21 genes, and we have reason to believe that the blast resistant gene overwhelming majority belongs to NBS-LRR genoid family in the paddy rice.The more important thing is in these 19 NBS-LRR genes has 12 all to be allelotrope, and wherein Pi37/Pish is positioned on the number one karyomit(e); Pib/Rbr2 is positioned on No. second karyomit(e); Piz-t/Pi2/Pi9 is positioned on No. six karyomit(e), and the not far position of distance is Pid3/Pi25; Pik/Pik-p/Pik-m is positioned on the ride on Bus No. 11 karyomit(e).We also have reason to believe so, in 100 blast resistant genes nearly that identify, much all should be homotopic each other.At present, it is generally acknowledged that the bigger LRR structural domain of variation has determined the specialization of this type of disease-resistant gene to pathogenic bacteria identification.
At present; Rely on map based cloning to the clone of blast resistant gene is still main, whole genome sequence all arranged, for the map based cloning of paddy gene has brought huge convenience though long-grained nonglutinous rice 9311 and japonica rice Japan are fine; But, still also there are a lot of difficulties for the clone of blast resistant gene.Such as the rice blast resistance of each individual plant in the Fine Mapping colony is identified that not only workload is huge, and field rice blast incidence also is easy to affected by environmently, causes the proterties mistake of statistics, and can not accurately locate; In addition, from checked order 9311 with Japanese fine genome sequence, the NBS-LRR gene all is that cluster exists mostly, even can target gene be navigated to a certain section, is difficult to also judge that in the genes in cluster which is only real disease-resistant gene; Moreover the rice varieties genetic background of utilizing at present is narrow, and available blast resistant gene is few.Yet in wild-rice, but contain abundant disease-resistant gene resource, owing to present research to wild-rice also is in the starting stage, do not have meticulous genetic map and physical map, wherein blast resistant gene is obviously difficult especially to utilize map based cloning.Therefore; We can directly utilize the NBS-LRR type gene order information of having cloned; Clone it and rice blast is had the homologous gene in the rice varieties of high antiatherosclerotic effect, directly transform the rice varieties of susceptible rice blast, thereby filter out gene with better resistance at wild-rice or other.
Summary of the invention
The purpose of this invention is to provide a kind of rice anti-rice blast albumen and encoding sox and application.
Protein provided by the present invention, name is called PID3-A4, derives from common wild-rice (Oryza.rufipogon) A4, is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant blast resisting by (a) deutero-protein.
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can pass through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1, and/or carries out the missense mutation of one or several base pair.
Wherein, the sequence in the sequence table 2 is made up of 924 amino-acid residues.
For the ease of the proteic purifying of PID3-A4, label as shown in the table on proteinic N-terminal that can the amino acid residue sequence of sequence 2 is formed in by sequence table or C-terminal connect.
Table: the sequence of 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 (a) but in PID3-A4 albumen synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.The proteic encoding sox of PID3-A4 in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table.
The proteic nucleic acid molecule of said PID3-A4 of encoding also belongs to protection scope of the present invention.
Said nucleic acid molecule can be DNA, like cDNA, genomic dna or recombinant DNA; Said nucleic acid molecule also can be RNA, like mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, said nucleic acid molecule is specially coding said PID3-A4 proteic gene (name is called Pid3-A4), and said Pid3-A4 gene is following 1)-4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize that is limited and the proteic dna molecular of said PID3-A4 of encoding;
4) with 1) or 2) dna molecular that limited has 90% above homology and the proteic dna molecular of said PID3-A4 of encoding at least.
Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and 2 * SSC is used in hybridization then under 65 ° of C, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 1 is made up of 2775 Nucleotide, and whole sequence 1 is the encoding sequence of said Pid3-A4 gene, the protein (said PID3-A4 albumen) shown in the sequence 2 in the code sequence tabulation.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.
Said recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Said recombinant expression vector can be used existing plant expression vector construction.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment, like pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 or other plant expression vector of deriving.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant expression vector; Before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter; For example cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter Rd29A etc., they can use separately or be used in combination with other plant promoter; In addition; When using gene constructed recombinant 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 said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used recombinant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.Also can not add any selected marker, directly with Pyricularia oryzae inoculation screening transformed plant.
In one embodiment of the invention, the promotor of the said Pid3-A4 genetic transcription of startup is specially 35S promoter in the said recombinant expression vector.More concrete, said recombinant expression vector is for to insert the recombinant plasmid that said Pid3-A4 gene obtains at the MCS place of PZH01 carrier.Said MCS is specially Xba I and Kpn I.
Said PZH01 carrier is at Xiao, H., Wang, Y., Liu; D., Wang, W., Li, X.; Zhao, X., Xu, J., Zhai; W.and Zhu, L. (2003) Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference.Plant Mol.Biol.52 disclosed among 957 – 966., and the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
Said expression cassette is by the promotor that can start said Pid3-A4 genetic expression, said Pid3-A4 gene, and transcription termination sequence is formed.
Said PID3-A4 albumen, or said nucleic acid molecule, or said recombinant expression vector, expression cassette or reorganization bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:
A1) the regulation and control plant is to the resistance of rice blast;
A2) seed selection blast resisting plant variety.
In one embodiment of the invention, said regulation and control plant is specially the resistance of raising plant to rice blast to the resistance of rice blast.
The method of said seed selection blast resisting plant variety specifically can comprise the step that the plant that said PID3-A4 expressing quantity is higher hybridizes as the parent.
Another object of the present invention provides a kind of method of cultivating the blast resisting transgenic plant.
This method comprises the steps: the proteic gene of the said PID3-A4 of coding is imported in the purpose plant, obtains the transgenic plant of blast resisting, said transgenic plant expressing said gene; Said gene (being the Pid3-A4 gene) is following 1) to 4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize that is limited and the proteic dna molecular of said PID3-A4 of encoding;
4) with 1) or 2) dna molecular that limited has 90% above homology and the proteic dna molecular of said PID3-A4 of encoding at least.
Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and 2 * SSC is used in hybridization then under 65 ° of C, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Said Pid3-A4 gene specifically can import in the said purpose plant through above-mentioned arbitrary said recombinant expression vector, obtains said transgenic plant.Specifically can be through using conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated, particle gun with said recombinant expression vector transformed plant cells or tissue, and the plant transformed tissue cultivating become plant.The agriculture bacillus mediated biological method that waits is transformed in vegetable cell or the tissue.
Said rice blast is by at least a the causing in following Pyricularia oryzae (Magnaporthe oryzae) microspecies: JS2001-108-1, Zhong10-8-14,97-27-2,03-10-66-1,07-31-1-2,03-10-76-3,10-25-1-1,10-32-2-1,03-11-37-1,07-55-1-1,10-47-9-2,10-120-21-2,07-21-1-1,10-62-2-1,07-26-22,09-87-2-1, Y34,99-26-2, CH706 and 99-20-2.
Said plant is monocotyledons or dicotyledons; Said monocotyledons such as paddy rice.In one embodiment of the invention, be specially rice varieties TP309.
Increase total length or arbitrary segmental primer of said PID3-A4 gene to also belonging to protection scope of the present invention.
The experiment proof; PID3-A4 albumen provided by the present invention and encoding sox thereof can be used for cultivating the blast resisting new variety of plant, particularly to showing resistance by at least a rice blast that causes in following Pyricularia oryzae (Magnaporthe oryzae) microspecies: JS2001-108-1, Zhong10-8-14,97-27-2,03-10-66-1,07-31-1-2,03-10-76-3,10-25-1-1,10-32-2-1,03-11-37-1,07-55-1-1,10-47-9-2,10-120-21-2,07-21-1-1,10-62-2-1,07-26-22,09-87-2-1, Y34,99-26-2, CH706 and 99-20-2.This will help the improvement to rice varieties, and to enlarging the crop-planting scope, it is significant to improve crop yield.
Description of drawings
Fig. 1 is the pcr amplification product electrophoresis result of blast resistant gene Pid3-A4.Wherein, swimming lane 1 is the PCR product of Pid3-A4 gene; Swimming lane M is a dna molecular amount standard.
Fig. 2 is the plasmid map of recombinant expression vector PZH01-Pid3-A4.
Fig. 3 is T
0It is the Molecular Identification result that generation is changeed the strain of Pid3-A4 gene.Wherein, HPT is the PCR detected result to hygromycin gene; CAPS cuts detected result for the pcr amplification-BamH I enzyme to the Pid3-A4 gene.
Fig. 4 is T
0In generation, change that Pid3-A4 gene overexpression in the Pid3-A4 gene strain system is analyzed and to the resistant phenotype of Pyricularia oryzae zhong-10-8-14.
Fig. 5 is for changeing Pid3-A4 expression of gene in the Pid3-A4 gene strain system and to the analysis of dependency between the rice blast resistance.Wherein, Hpt is the PCR detected result to hygromycin gene; CAPS cuts detected result for the pcr amplification-BamH I enzyme to the Pid3-A4 gene.
Fig. 6 is for changeing the specificity analysis result of Pid3-A4 genetic expression in the Pid3-A4 gene strain system.Wherein, A is for connecing the bacterium group; B is the water receiving group.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The acquisition of embodiment 1, blast resistant gene Pid3-A4
Blast resisting homologous gene among blast resistant gene Pid3 sequence information separating clone common wild-rice (Oryza.rufipogon) A4 that the present embodiment utilization is cloned from the local cultivar of paddy rice ground paddy.
1, the acquisition of rice blast resistant gene Pid3 sequence information
Blast resistant gene Pdi3 clones from the local cultivar of paddy rice ground paddy and obtains, and it has resistance preferably to japonica rice district Pyricularia oryzae microspecies, and wherein being used for the discriminating microspecies of positional cloning is japonica rice district Pyricularia oryzae microspecies zhong-10-8-14.Obtain blast resistant gene Pid3 complete genome sequence (the 3010-5784 position of GenBank:FJ745364.1) the ground paddy from NCBI (http://www.ncbi.nlm.nih.gov); Pid3 full length gene 2775bp; Do not contain intron, the NBS-LRR albumen that contains one section conservative motif:RSLALSIEDVVD (78-89aa) of encode a N end non-TIR, non-CC.The NBS structural domain by the 158th to 466 amino acids codings, at 565 to 13 incomplete LRR are arranged between 924 amino acids.
2, utilize the homologous gene Pid3-A4 of Pid3 among the ground paddy Pid3 gene order design primer amplification wild-rice A4
1) design of primers
Pid3 gene order in the paddy of base area (the 3010-5784 position of GenBank:FJ745364.1); Design primer amplification wild-rice A4 (Shang; J.J.et al.; 2009Identification of a New Rice Blast Resistance Gene; Pid3, by Genomewide Comparison of Paired Nucleotide-Binding Site-Leucine-Rich Repeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes.Genetics182:1303-1311) homologous gene in.Institute's designed primer is following:
D3F:5 '-atggcggagggtgttgtgggctc-3 ' (sequence 1 preceding 23)
D3R:5 '-ttattgaatcctttctgcagcc-3 ' (back 22 reverse complementary sequence of sequence 1)
For the ease of follow-up the Pid3 homologous gene that is increased is connected into the test of expression vector, in this upstream and downstream primer, adds respectively and contain Xba I and the Kpn I restriction enzyme site sequence of protecting base, it is following to obtain primer:
Pid3F:5 '-TT
TCTAGAAtggcggagggtgttgtgggctc-3 ' (the underscore place is the recognition sequence of Xba I)
Pid3R:5 '-CT
GGTACCTtattgaatcctttctgcagcc-3 ' (the underscore place is the recognition sequence of Kpn I)
2) acquisition of blast resistant gene Pid3-A4
Blade genomic dna with wild-rice A4 is a template, carries out pcr amplification with primer Pid3F and Pid3R.
Reaction system (25 μ l): 10 * PCR damping fluid, 2.5 μ l; DNTP Mixture (2.5mM) 2.5 μ l; KOD enzyme (5U/ μ l) 0.2 μ l; Each 0.25 μ l of primer Pid3F and Pid3R (all 10mM); Template (DNA) 1 μ l; MgSO
4(25mM) 1.6 μ l; DMSO 1.6 μ l; DdH
2O is supplemented to 25 μ l.
Reaction conditions: 94 ℃ of preparatory sex change 4min of elder generation; 94 ℃ of sex change 30S then, 58 ℃ of annealing 30S, 68 ℃ are extended 3min, totally 30 circulations; Last 68 ℃ are extended 5min.
The PCR product is carried out 1% agarose gel electrophoresis detect, the result shows that pcr amplification obtains the purpose band (Fig. 1) that size is about 3000bp.Downcut this purpose band, after the purifying and recovering PCR product is connected on the pEASY-Blunt carrier (Beijing Quanshijin Biotechnology Co., Ltd), transformed into escherichia coli DH5a obtains transformant, transformant is shaken bacterium and extracts plasmid, the sample presentation order-checking.Order-checking shows that this PCR product contains the nucleotide sequence shown in the sequence 1 in the ordered list, and its sequence is specially TT
TCTAGA+ sequence 1+
GGTACCAG (underscore partly is respectively the recognition sequence of Xba I and Kpn I restriction enzyme site).Wherein, sequence 1 is made up of 2775 Nucleotide, and whole sequence 1 is a complete ORFs, the protein shown in the sequence 2 in the code sequence tabulation.Sequence 2 is made up of 924 amino-acid residues in the sequence table.With unnamed gene shown in the sequence 1 is Pid3-A4, with the protein called after PID3-A4 of this genes encoding.
3, the homologous gene Pid3-A4 sequence that obtains is analyzed
Utilize DNAMAN software over the ground in the paddy among Pid3 gene (the 3010-5784 position of GenBank:FJ745364.1) and the wild-rice A4 Pid3-A4 gene (sequence 1) sequence similarity analyze discovery; Pid3-A4 gene similarity in the ground paddy among Pid3 gene and the wild-rice A4 is very high; Reached 99.5%, had only 14 base differences altogether.Utilize online predictive genes software (http://linux1.softberry.com) that sequence 1 is carried out predictive genes, find that 1 of sequence contains an ORF, do not have intron to exist, this with ground paddy in Pid3 gene structure characteristic consistent.Utilize online software (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) analysis to find the aminoacid sequence (sequence 2) of Pid3-A4 genes encoding among the wild-rice A4; Its NBS-LRR albumen of also encoding; And the position of conservative region NBS is the same with the position of Pid3 in the ground paddy; All by the amino acid of 158-466 position coding, and only there are 9 amino acid whose differences in the two, and wherein 6 are positioned at the LRR zone; One is positioned at the NBS zone, and other two at N-terminal.
Acquisition and the functional study thereof of embodiment 2, commentaries on classics Pid3-A4 trans-genetic hybrid rice
One, changes the acquisition of Pid3-A4 trans-genetic hybrid rice
1, the structure of recombinant expression vector PZH01-pid3-A4
PCR product (the TT that embodiment 1 is obtained
TCTAGA + sequence 1+
GGTACCAG) cut the fragment that obtains and cut plant through same enzyme binary expression vector PZH01 (Xiao, H., Wang, Y. through Xba I and Kpn I enzyme; Liu, D., Wang, W., Li; X., Zhao, X., Xu, J.; Zhai, W.and Zhu, L. (2003) Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference.Plant Mol.Biol.52, the 957-966. public can be from Chinese Academy of Sciences heredity and the acquisition of developmental biology institute) carrier segments be connected; Connect product transformed into escherichia coli DH5a, obtain transformant, transformant is shaken bacterium and extracts plasmid, the sample presentation order-checking.To be illustrated between Xba I and the Kpn I restriction enzyme site of PZH01 carrier the recombinant plasmid called after PZH01-Pid3-A4 of the Pid3-A4 gene shown in the sequence 1 in the insertion sequence table through order-checking, this plasmid is recombinant expression vector, and its plasmid map is as shown in Figure 2.
2, change the acquisition of Pid3-A4 trans-genetic hybrid rice
The recombinant expression vector PZH01-pid3-A4 that step 1 is made up passes through electric shock transformation method (Hiei; Y.; S.Ohta; T.Komari and T.Kumashiro; 1994 Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant are J.6:271-282.) change Agrobacterium LBA4404 (Chen over to; X., et al., (2006) A B-lectin receptor kinase gene conferring rice blast resistance.Plant J.46:794 – 804. public can obtain with developmental biology institute from Chinese Academy of Sciences heredity) obtain transformant.The contrast that changes the PZH01 empty carrier over to is set simultaneously.With the Agrobacterium LBA4404 called after LBA4404/PZH01-pid3-A4 that changes recombinant expression vector PZH01-pid3-A4 over to; With the Agrobacterium LBA4404 called after LBA4404/PZH01 that changes the PZH01 empty carrier over to.
Method (Hiei through agrobacterium mediation converted paddy rice mature seed callus; Y.; S.Ohta; T.Komari and T.Kumashiro; 1994 Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant are J.6:271-282.) the reorganization Agrobacterium LBA4404/PZH01-pid3-A4 (or LBA4404/PZH01) of above-mentioned preparation is imported to rice varieties TP309 (Junjun Shang.; Et al.Identification of a New Rice Blast Resistance Gene, Pid3, by Genomewide Comparison of Paired Nucleotide-Binding Site-Leucine-RichRepeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes.Genetics; 2009; 182:1303-1311, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity), obtained 21 T
0In generation, change the commentaries on classics Pid3-A4 gene strain system of recombinant expression vector PZH01-pid3-A4 and the contrast strain that 8 strains change the PZH01 empty carrier over to over to.
3, Molecular Detection
(1) PCR to the Pid3-A4 gene detects
Utilize commentaries on classics Pid3-A4 gene strain system that the CAPS mark obtains above-mentioned steps 2 and the contrast strain that changes the PZH01 empty carrier over to carry out the Molecular Detection on the dna level, specific as follows:
The T that obtains with above-mentioned steps 2 respectively
0The genomic dna that generation changes Pid3-A4 gene strain system and changes the contrast strain of PZH01 empty carrier over to is a template, is that primer carries out pcr amplification with Pid3JF and Pid3JR.Not genetically modified TP309 paddy rice is set simultaneously as contrast.
Pid3JF:5 '-TACTACTCATGGAAGCTAGTTCTC-3 ' (the 2057-2080 position of sequence 1)
Pid3JR:5 '-ACGTCACAAATCATTCGCTC-3 ' (corresponding to the 2695-2714 position of sequence 1)
Not only can the increase fragment (the 2057-2714 position of sequence 1) of one section 658bp of external source Pid3-A4 gene of above-mentioned primer; The fragment (the 2071-2728 position of GenBank:FJ745366.1) of one section 658bp of the endogenous Pid3-tp309 gene of the TP309 paddy rice of can also increasing (homologous gene of Pid3); But owing to contain a BamH I restriction enzyme site (sequence 1 the 2204th) from the fragment of Pid3-A4 gene; And the Pid3-tp309 gene since here the difference of a base do not have this restriction enzyme site; So can cut through the PCR product being carried out enzyme, thereby detect among the TP309 whether successfully changed the Pid3-A4 gene over to restriction enzyme BamH I.Do not change the purpose band that the TP309 of Pid3-A4 gene obtains after pcr amplification-BamH I enzyme is cut over to, size is 658bp; And the TP309 that has successfully changed the Pid3-A4 gene over to obtains two purpose bands after pcr amplification-BamH I enzyme is cut, and size is respectively 510bp and 148bp.
6 T to above-mentioned steps 2 acquisitions
0In generation, changeed Pid3-A4 gene strain system (line1, line2, line3, line4, line7 and line9) and not genetically modified TP309 paddy rice carries out above-mentioned pcr amplification, cuts with BamH I enzyme then, and the result is as shown in Figure 3.As can be seen from the figure, 6 T
0In generation, changes Pid3-A4 gene strain system (line1, line2, line3, line4, line7 and line9) and after pcr amplification-BamH I enzyme is cut, obtains two purpose bands; And the purpose band that not genetically modified TP309 paddy rice obtains after pcr amplification-BamH I enzyme is cut, and stripe size conforms to expected results.This result shows 6 T
0In generation, changes Pid3-A4 gene strain system and is Pid3-A4 gene masculine plant.In addition, consistent to the qualification result of the contrast strain that changes the PZH01 empty carrier over to not genetically modified TP309 paddy rice.
(2) PCR to hygromycin gene detects
6 T that obtain with above-mentioned steps 2 respectively
0The genomic dna that generation changes Pid3-A4 gene strain system and changes the contrast strain of PZH01 empty carrier over to is a template, is that primer carries out pcr amplification with HYGF and HYGR.Not genetically modified TP309 paddy rice is set simultaneously as contrast.Do not change TP309 no purpose band behind pcr amplification of hygromycin gene over to; Be about 500bp and the TP309 that has successfully changed hygromycin gene over to obtains size behind pcr amplification) the purpose band.
HYGF：5′-GACGGTGTCGTCCATCACAGTTT-3′
HYGR：5′-ACTCACCGCGACGTCTGTCGAGAA-3′
6 T to above-mentioned steps 2 acquisitions
0In generation, changeed Pid3-A4 gene strain system (line1, line2, line3, line4, line7 and line9) and not genetically modified TP309 paddy rice carries out above-mentioned pcr amplification, and the result is as shown in Figure 3.As can be seen from the figure, 6 T
0In generation, changes Pid3-A4 gene strain system (line1, line2, line3, line4, line7 and line9) and behind pcr amplification, obtains the purpose band that size is about 500bp; And not genetically modified TP309 paddy rice no purpose band in back behind pcr amplification.This result shows 6 T
0In generation, changes Pid3-A4 gene strain system and is the hygromycin gene positive plant.In addition, it is also positive to change the qualification result of contrast strain of PZH01 empty carrier over to.
4, the Pid3-A4 gene overexpression is analyzed
For further verifying the transgenic situation, 6 T that the present invention obtains above-mentioned steps 2 again
0In generation, changes the strain of Pid3-A4 gene and is the expression analysis of crossing that Pid3-A4 has been carried out in (line1, line2, line3, line4, line7 and line9) and the contrast strain that changes the PZH01 empty carrier over to, and is specific as follows:
6 T with above-mentioned steps 2 acquisitions
0The contrast strain that generation changes Pid3-A4 gene strain system and changes the PZH01 empty carrier over to is an experiment material, extracts the total RNA of its blade, and utilizes the DNAse I to handle and remove residual DNA, utilizes oligdT that its reverse transcription is cDNA.With this cDNA is template, is Pid3-A4 gene that changes over to and the endogenous Pid3-tp309 gene of TP309 paddy rice (homologous gene of Pid3) of 658bp with primer Pid3JF and Pid3JR amplification size.Be to detect confidential reference items simultaneously with Actin, the primer is ActinF and ActinR.Not genetically modified TP309 paddy rice is set simultaneously as contrast.With Actin confidential reference items; Carry out under the situation of half-quantitative detection; Obviously be better than not genetically modified TP309 paddy rice if the size that the amplification of strain to be detected system obtains is the brightness of the purpose band of 658bp, judge that then this strain to be detected system is for expressing the transgenic line of Pid3-A4 gene as contrast.
ActinF：5′-AGCAACTGGGATGATATGGA-3′
ActinR：5′-CAGGGCGATGTAGGAAAGC-3′
The result is as shown in Figure 4; 4 transgenic line (line1 therein; Line2; Line3 and line4) in detected Pid3-A4 expression of gene (brightness of purpose band is better than the not genetically modified TP309 paddy rice as contrast), and other 2 transgenic lines (line7 and line9) are consistent with the contrast strain detected result that changes the PZH01 empty carrier over to, all do not have to detect Pid3-A4 expression of gene (brightness of purpose band is consistent with the not genetically modified TP309 paddy rice that conduct contrasts).Analyze its reason, this possibly be because the on position of Pid3-A4 gene in transgenic line line7 and line9 causes the Pid3-A4 gene that changes over to not express.
Two, change of the resistance detection of Pid3-A4 trans-genetic hybrid rice to rice blast
1, to the Preliminary detection of rice blast resistance
To 6 T that obtain in the above-mentioned steps 1
0The blast resisting dientification of bacteria is carried out in the contrast strain that generation changes Pid3-A4 gene strain system and changes the PZH01 empty carrier over to; Used Pyricularia oryzae (Magnaporthe oryzae) microspecies are zhong-10-8-14 (Shang; J.J.; Et al.; 2009 Identification of a New Rice Blast Resistance Gene; Pid3, by Genomewide Comparison of Paired Nucleotide-Binding Site-Leucine-Rich Repeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes.Genetics 182:1303-1311, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity).Specific as follows:
Big Tanaka, with 6 T that obtain in the above-mentioned steps 1
0It is that (line1, line2, line3, line4, line7 and line9) and the contrast strain plant that changes the PZH01 empty carrier over to are transplanted that generation is changeed the strain of Pid3-A4 gene; All utilizing syringe to inject respectively in transplanting back about month (not before the booting) connects bacterium, is specially with sterilized water Pyricularia oryzae (Magnaporthe oryzae) microspecies zhong-10-8-14 is configured to 5 * 10
4The spore suspension of spore/milliliter overflows until suspension-s the injection of 1ml spore suspension from paddy rice cane base portion with common injector for medical purpose from rice stem masthead portion.One all " Invest, Then Investigate " incidences (judgment criteria is seen table 1).Be provided with simultaneously without genetically modified TP309 paddy rice as contrast.The experiment triplicate.
Table 1 rice plant incidence judgment criteria
| Resistance | Rank | Incidence |
| Anti-(R) | 0 | There is not any scab |
| Anti-(R) | 1 | Diameter is less than the brown some scab of 0.5mm |
| In anti-(mR) | 2 | Diameter is about the brown some scab of 0.5-1mm |
| Middle sense (mS) | 3 | The oval scab of diameter 1-3mm, edge brown, central pearl |
| Sense (S) | 4 | Typical fusiform scab, diameter 3mm or longer, scab have fusion or amixis slightly |
| Sense (S) | 5 | Typical fusiform scab, because scab merges, the blade first half is withered |
The result finds; Only detected 4 strains systems (line1, line2, line3 and line4) that Pid3-A4 expresses in 6 transgenic lines and shown as resistance (Fig. 4) Pyricularia oryzae zhong-10-8-14; And other two the strain systems (line7 and line9) that fail to detect the Pid3-A4 expression all show as susceptible with identical with the contrast strain that changes the PZH01 empty carrier over to without genetically modified TP309 paddy rice.
For the Pid3-A4 gene further confirming to change over to and disease-resistant dependency, to T
0The T that 1 strain system (line1) of disease-resistant performance was arranged in generation
1In generation, separates plant (16 strain) and carries out Molecular Detection and blast resisting (Pyricularia oryzae zhong-10-8-14) character analysis, and wherein, blast resisting character analysis step is with the above, and the Molecular Detection step is seen (1) and (2) in the step 13.Not genetically modified TP309 paddy rice is set simultaneously as the blast resisting negative control, wild-rice A4 is set as the blast resisting positive control.The experiment triplicate.
The result is as shown in Figure 5, and changeing the strain of Pid3-A4 gene is the T of line1
1Whether blast resisting proterties and Pid3-A4 expression of gene that generation is separated each plant in the plant are one to one.The result shows that this instance isolating Pid3-A4 gene from common wild-rice A4 is a blast resistant gene that function is arranged.
2, the Pid3-A4 gene expression characteristics is analyzed
Utilize RT-PCR that Pid3-A4 expression of gene pattern is analyzed, specific as follows:
Big Tanaka, with syringe the wild-rice A4 of resisting rice blast bacteria zhong-10-8-14 is injected and to connect bacterium, be specially with sterilized water Pyricularia oryzae (Magnaporthe oryzae) microspecies zhong-10-8-14 is configured to 5 * 10
4The spore suspension of spore/milliliter overflows until suspension-s the injection of 1ml spore suspension from paddy rice cane base portion with common injector for medical purpose from rice stem masthead portion.The water receiving contrast is set simultaneously.Different time point (0h, 3h, 6h, 12h, 24h, 48h and 72h) is gathered leaf sheath and blade and is extracted its total RNA after connecing bacterium or water receiving, utilizes the DNAse I to handle and removes residual DNA, utilizes oligdT that its reverse transcription is cDNA.With this cDNA is template, carries out pcr amplification with primer Pid3JF and Pid3JR, and is the detection confidential reference items with Actin, and the primer is ActinF and ActinR.
The result is as shown in Figure 6, connects in bacterium group and the water receiving control group expression amount basically identical of Pid3-A4 gene on each time point.This result shows that the Pid3-A4 gene shows as constitutive expression in wild-rice A4, and not induced by Pyricularia oryzae microspecies zhong-10-8-14.
3, the mensuration of the anti-spectrum of Pid3-A4 gene pairs Pyricularia oryzae
Although Pid3-A4 gene and Pid3 gene difference on sequence are very little; But LRR structural domain that it is generally acknowledged the NBS-LRR genoid has determined the specific recognition of these type of gene pairs Pyricularia oryzae microspecies; And Pid3-A4 gene and Pid3 gene have 6 amino acid whose differences in the LRR zone; Therefore the present invention is to having identical transgene receptor TP309; And all the Pid3-A4 transfer-gen plant T2 under the control of CAMV35S promotor for the line1 strain is and Pid3 transfer-gen plant (Shang; J.J.; Et al.; 2009, Identification of a New Rice Blast Resistance Gene, Pid3; By Genomewide Comparison of Paired Nucleotide-Binding Site-Leucine-Rich Repeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes.Genetics 182:1303-1311; The public can obtain with developmental biology institute from the Chinese Academy of Sciences is hereditary) carried out the anti-spectrum mensuration of Pyricularia oryzae, totally 30 of used Pyricularia oryzae microspecies, wherein 12 are anti-12 Pyricularia oryzae microspecies (numbering 19-30 in the table 2) (Shang that compose of the original Pid3 of mensuration; J.J.; Et al., 2009Identification of a New Rice Blast Resistance Gene, Pid3; By Genomewide Comparison of Paired Nucleotide-Binding Site-Leucine-Rich Repeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes.Genetics 182:1303-1311; The public can obtain with developmental biology institute from Chinese Academy of Sciences heredity), other 18 be the Pyricularia oryzae microspecies (numbering 1-18 the table 2) of collecting from the multiple zone of Sichuan Province's Pyricularia oryzae (reference: Zhang Xuemei. the anti-pest property evaluation of rice in Sichuan province main breed with in the anti-pest sex-controlled inheritance of extensive 99-14 analyze [D]. Sichuan Agricultural University, 2007. white jade connects. the Sichuan Pyricularia oryzae is to the toxic research of hybridisation rice [D]. Sichuan Agricultural University; 2008. provided in the collection of area, Sichuan by the Peng Yunliang researcher of Institute of Plant Protection of academy of agricultural sciences, Sichuan Province, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity).
Respectively above-mentioned 30 Pyricularia oryzae microspecies (shown in the table 2) are inoculated into Pid3-A4 transfer-gen plant T2 and carry out the anti-spectrum of rice blast for line1 strain system with the Pid3 transfer-gen plant and measure, concrete inoculation method is following: with sterilized water different microspecies are disposed 5 * 10 respectively
4The spore suspension of spore/milliliter overflows until suspension-s the injection of 1ml spore suspension from paddy rice cane base portion with common injector for medical purpose from rice stem masthead portion, each bacterium connects three strains, and every strain connects three and tillers.One all " Invest, Then Investigate " incidences (judgment criteria is seen table 1).Be provided with simultaneously without genetically modified TP309 paddy rice and the paddy rice that changes the PZH01 empty carrier over to as contrast.The experiment triplicate.
The result is as shown in table 2, without genetically modified TP309 paddy rice and all the Pyricularia oryzae microspecies equal non-resistant of the paddy rice that changes the PZH01 empty carrier over to detecting; Pid3-A4 transfer-gen plant institute resisting rice blast bacteria microspecies are more than the Pid3 transfer-gen plant; The resistance that mainly is presented as the Pyricularia oryzae microspecies (numbering 1-18 in the table 2) that Sichuan is collected is stronger; Be specially the Pid3-A4 transfer-gen plant Pyricularia oryzae microspecies 10-25-1-1,03-11-37-1,10-120-21-2 and 10-62-2-1 are shown resistance, and the Pid3 transfer-gen plant does not show resistance.Therefore, the Pid3-A4 gene is worth in the blast resisting breeding in Sichuan, having better utilised.
Table 2Pid3-A4 transfer-gen plant is to the anti-spectrum of Pyricularia oryzae
Claims (10)
1. protein is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant blast resisting by (a) deutero-protein.
2. coding claim 1 said proteinic nucleic acid molecule.
3. nucleic acid molecule according to claim 2 is characterized in that: said nucleic acid molecule is coding claim 1 said proteinic gene, and said gene is following 1)-4) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the sequence 1 in the sequence table;
2) dna molecular shown in the sequence 1 in the sequence table;
3) under stringent condition with 1) or 2) dna molecule hybridize and the said protein DNA molecule of coding claim 1 that are limited;
4) with 1) or 2) dna molecular that limited has 90% above homology and the said protein DNA molecule of claim 1 of encoding at least.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said nucleic acid molecule.
5. recombinant vectors according to claim 4 is characterized in that: said recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5 is characterized in that: the promotor that starts said genetic transcription in the said recombinant expression vector is a 35S promoter.
7. described protein of claim 1, or claim 2 or 3 described nucleic acid molecule, or claim 4 or 5 or 6 described recombinant expression vectors, expression cassette or reorganization bacterium are at following a1) or a2) in application:
A1) the regulation and control plant is to the resistance of rice blast;
A2) seed selection blast resisting plant variety.
8. a method of cultivating the blast resisting transgenic plant comprises the steps: the said proteinic gene of coding claim 1 is imported the purpose plant, obtains the transgenic plant of blast resisting.
9. application according to claim 7; Or the described method of claim 8, it is characterized in that: said rice blast is by at least a the causing in following Pyricularia oryzae (Magnaporthe oryzae) microspecies: JS2001-108-1, Zhong10-8-14,97-27-2,03-10-66-1,07-31-1-2,03-10-76-3,10-25-1-1,10-32-2-1,03-11-37-1,07-55-1-1,10-47-9-2,10-120-21-2,07-21-1-1,10-62-2-1,07-26-22,09-87-2-1, Y34,99-26-2, CH706 and 99-20-2.
10. according to arbitrary described application or method among the claim 7-9, it is characterized in that: said plant is monocotyledons or dicotyledons.
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| CN108913809A (en) * | 2018-09-25 | 2018-11-30 | 湖南杂交水稻研究中心 | InDel molecular marked compound, detection method and the application of blast resistant gene Pid3-A4 |
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| CN110922463A (en) * | 2019-12-20 | 2020-03-27 | 中国农业大学 | Rice disease-resistant gene RGA5-HMA5 and application thereof in rice breeding |
| CN111534536A (en) * | 2019-02-02 | 2020-08-14 | 湖南杂交水稻研究中心 | Method for improving rice blast resistance of rice and related biological material thereof |
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| CN104072596A (en) * | 2014-07-03 | 2014-10-01 | 中国科学院遗传与发育生物学研究所 | Rice blast resisting protein of rice, coding gene and application thereof |
| CN108913809A (en) * | 2018-09-25 | 2018-11-30 | 湖南杂交水稻研究中心 | InDel molecular marked compound, detection method and the application of blast resistant gene Pid3-A4 |
| CN108913809B (en) * | 2018-09-25 | 2020-12-25 | 湖南杂交水稻研究中心 | InDel molecular marker of rice blast resistant gene Pid3-A4, detection method and application |
| CN111534536A (en) * | 2019-02-02 | 2020-08-14 | 湖南杂交水稻研究中心 | Method for improving rice blast resistance of rice and related biological material thereof |
| CN110922462A (en) * | 2019-12-20 | 2020-03-27 | 中国农业大学 | Artificially modified rice disease-resistant gene RGA5-HMA2 |
| CN110922463A (en) * | 2019-12-20 | 2020-03-27 | 中国农业大学 | Rice disease-resistant gene RGA5-HMA5 and application thereof in rice breeding |
| CN112391407A (en) * | 2020-11-19 | 2021-02-23 | 贵州省水稻研究所 | Breeding method for improving rice blast resistance |
| CN113528703A (en) * | 2021-08-23 | 2021-10-22 | 华美瑞康(北京)国际生物技术研究院有限公司 | Development and application of KASP molecular marker of rice blast resistance gene Pid3-A4 |
| CN117947094A (en) * | 2024-03-26 | 2024-04-30 | 云南农业大学 | Method for improving rice blast resistance by Pi-Pprs42 gene and application |
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