CN102180956A - Rice-tillering-related protein, and coding gene and application thereof - Google Patents
Rice-tillering-related protein, and coding gene and application thereof Download PDFInfo
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- CN102180956A CN102180956A CN2011101124987A CN201110112498A CN102180956A CN 102180956 A CN102180956 A CN 102180956A CN 2011101124987 A CN2011101124987 A CN 2011101124987A CN 201110112498 A CN201110112498 A CN 201110112498A CN 102180956 A CN102180956 A CN 102180956A
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Abstract
The invention discloses a rice-tillering-related protein, and a coding gene and application thereof. The protein provided by the invention is named TE and is (a) a protein composed of the amino acid sequence shown as a sequence 2 in a sequence list; or (b) a protein derived from the sequence 2, which is formed by enabling the amino acid sequence shown as the sequence 2 in the sequence list to be subjected to the substitution and/or deletion and/or addition of one or more amino acid residues and is related to the number of tillers of a plant. Experiment proves that by using the transgenic technology to overexpress the TE gene obtained by cloning, the transgenic rice with obviously reduced number of tillers is obtained, and the number of tillers is 1/4-1/2 of that of the wild type rice, thereby proving that the TE gene can effectively reduce the number of tillers of rice, can be used for adjusting the plant type of the plant and possibly forming a rational plant population structure, and can improve the yield and quality of crops in unit area of cultivated land.
Description
Technical field
The present invention relates to plant genetic engineering field, relate in particular to a kind of rice tillering associated protein and encoding gene thereof and application.
Background technology
Tillering is the important morphological feature of most grasses.The every joint of the cane of gramineous crop all has bud, and bud can be grown the formation branch, and the branch base portion forms adventive root, and this class branch is tillered exactly.The morphological specificity and the stem of tillering are basic identical, tiller can separate with stem and survival separately.Between paddy rice cane first segment meristem zone is arranged, the cell of this meristem zone divides and can extend between the envoy, regulates tillering node, makes the soil Shen rudiment of tillering in the different transplanting degree of depth.Rice seedlings enter tillering phase when growing to 4 blades.Since most of vegetative organ of paddy rice as blade, tiller and root system all is to form in this period, so be the important period in the rice growth process tillering phase.Spike number is the important determinative of rice yield, and the individual plant tiller number is the important factor of decision spike number, and crossing low or too high tiller number all can influence output.Therefore, ability for tillering is important economical character, is to influence the rice yield important factor.Past mainly concentrates on morphologic observation and Crop Physiology aspect to the research of tillering.The physiological research of rice cropping has preferably the relation of tillering quantity and envrionment conditions illustrates, comprise the adjusting that factors such as temperature, illumination, moisture, farming and mineral nutrition are grown to tillering, the influence of especially nitrogen nutrition level and temperature to tillering.It is generally acknowledged to tiller from genetic angle and be subjected to polygene combined adjusting.Present a plurality of genes that studies show that have participated in the tiller regulating of paddy rice, and this is comprising MOCl, LAX, the sequence regulatory factor in OsTBl and the MAX/RMS/D path.Wherein the generation of MOCl regulation and control tiller bud also promotes the growth of tiller bud always, and LAX, the regulatory factor in OsTBl and the MAX/RMS/D path is all regulated the growth of the tiller bud after the generation.
Paddy rice (Oryza sativa) mutant is the single-gene recessive mutation, meets Mendelian's mode genetic development.
Summary of the invention
An object of the present invention is to provide a kind of rice tillering associated protein and encoding gene thereof.
The protein that rice tillering branch provided by the invention is relevant, name is called TE, derives from paddy rice (Oryza sativa), is following (a) or (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the tiller number of plant by sequence 2 deutero-protein.
The replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
Sequence 2 in the sequence table is made up of individual 523 amino-acid residues.
The gene of encoding said proteins also belongs to protection scope of the present invention.
Described gene is the dna molecular of following (1) or (2) or (3):
(1) dna molecular shown in the sequence 1 in the sequence table;
(2) dna molecular of the tiller number associated protein of the dna sequence dna hybridization that under stringent condition, limits and coding and plant with (1);
(3) dna sequence dna that limits with (1) has 70% at least, has 75% at least, has 80% at least, has 85% at least, has 90% at least, has 95% at least, has 96% at least, has 97% at least, has 98% or the dna molecular that has the tiller number associated protein of 99% homology and coding and plant at least at least.
Described stringent condition is at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Sequence 1 in the described sequence table is made up of 1572 Nucleotide.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain described gene also are the scope of protection of the invention.
Described recombinant vectors is following 1) or 2):
1) be that described gene is inserted the recombinant vectors that the SmaI recognition site place of pCambia1305.1 carrier obtains;
2) be that described gene is inserted the recombinant vectors that obtains between the KpnI of pCUbil390 carrier and SpeI recognition site.
Increase described full length gene or its any segmental primer to also being the scope of protection of the invention.
Another object of the present invention provides a kind of method of cultivating transgenic plant.
Method provided by the invention is that described gene is imported in the purpose plant, obtains transgenic plant; The individual plant tiller number of described transgenic plant is less than described purpose plant.
Described gene imports in the described purpose plant by described recombinant vectors.
Described purpose plant is dicotyledonous or monocotyledons, and described monocotyledons is specially paddy rice, and described paddy rice is mutant cl.
Of the present invention experimental results show that, the present invention clone obtains the TE gene, by transgenic technology overexpression TE gene, the transgenic paddy rice that acquisition is tillered and obviously reduced, tiller number is the 1/4-1/2 of the wild-type sisters system of mutant, proof TE gene can effectively reduce the rice tillering number, and using TE gene adjustment plant plant type and forming rational plant population structure becomes possibility, can improve output and the quality of farm crop in the unit surface arable land.
Description of drawings
Fig. 1 is the phenotype of function complementation experiment T1 for transgenic paddy rice
Fig. 2 is the tiller number of overexpression TE transgenic paddy rice
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Paddy rice (Oryza sativa ssp.Japonica) Japan is fine, the wild-type sisters of paddy rice (Oryza sativa) mutant cl, mutant cl are and binary expression vector pCubil390 all puts down in writing in following article: the Fine Mapping of paddy rice leaf twist gene CL is perfect with " training short 64S/93-11 " chromosome segment substitution line molecule marker, the author is public outstanding, graduated 2008, deliver in June, 2009, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Agrobacterium tumefaciens EHA105 (Agrobacterium tumefaciens EHA105) is documented in NewAgrobacterium helper plasmids for gene transfer to plants.Hood, Elizabeth E; Gelvin, Stanton B; Melchers, Leo S; Hoekema, Andre.Transgenic Research, 2 (4): p.208-218-218 (1993). the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
The cultivation condition of rice material: rice paddy seed was soaked in water 3 days, sows on the seedbed then.The rice seedling of 4 leaf phases is transplanted in the paddy field, divides the individual plant rice transplanting.
The acquisition of embodiment 1, TE gene
Extract the fine genomic dna of paddy rice (Oryza sativa ssp.Japonica) Japan, carry out pcr amplification with a pair of primer pGR-3F and pGR-4R, obtain the PCR product and send to order-checking, the result has the Nucleotide shown in the sequence 1 in the sequence table for this PCR product, the gene of this PCR product is TE, and the coding region of this gene is a sequence 1 in the sequence table; The albumen called after TE of this genes encoding, this proteic aminoacid sequence are the sequence 2 in the sequence table.
PGR-3F (5 ' end adds phosphate group): 5 '-TGAATCTTACTCTTCTATATATTGCCTCAC-3 '
PGR-4R (5 ' end adds phosphate group): 5 '-GGATCCCATAACCTTATTTATTTTTCTAGT-3 '
Also but artificial synthesized sequence 1, is primer with pGR-3F and pGR-4R, and amplification obtains the PCR product.
Embodiment 2, the mutant phenotype of mutant of having changeed the complementation of TE paddy rice
1, the acquisition of recombinant expression vector
(this carrier all can be to CambiaLabs laboratory obtainable post-free for research purposes researchist to cut binary expression vector pCambia1305.1 with the SmaI enzyme.CambiaLabs，G301，2George?Street?Brisbane，QLD?4000，Australia。), dephosphorylation is not more contained the pCambia1305.1 carrier framework of the flat end of 5 ' phosphate group; The PCR product that will obtain with primer pGR-3F and pGR-4R amplification (the PCR product that obtains by embodiment 1) purifying, purified product is connected with the pCambia1305.1 carrier framework of above-mentioned acquisition, and the connection product that obtains changes in the intestinal bacteria, obtains transformant.Extract the plasmid of transformant, send to order-checking, the carrier that the SmaI recognition site place that the result inserts pCambia1305.1 for this plasmid for the sequence 1 that will comprise in the sequence table obtains, this plasmid called after pGTE.
2, change the acquisition of TE paddy rice
The plasmid pGTE of above-mentioned acquisition is changed among the agrobacterium tumefaciens EHA105 (Agrobacteriumtumefaciens EHA105) by the method for heat shock, obtain the bacterium of recombinating, extract the plasmid of reorganization bacterium, send to order-checking, the result will contain the reorganization bacterium called after EHA105/pGTE of this plasmid for this plasmid is pGTE.
(it is short that plant height becomes for the increase of tillering, boot leaf serious distortion with paddy rice (Oryza sativa) mutant cl.) mature seed shelling sterilization, be inoculated in the substratum of callus induction.After cultivating for 3 weeks, grow callus from scultellum, it is vigorous to select growth, and color is pale yellow, and more open embryo callus subculture is as the acceptor that transforms.
The EHA105/pGTE of above-mentioned acquisition is infected embryo callus subculture, cultivate after 3 days for 25 ℃, containing screening kanamycin-resistant callus tissue and transfer-gen plant on the selection substratum of 50mg/L Totomycin at the dark place.With hygromycin resistance plant hardening in the cool, be transplanted to the paddy field after several days, obtain T
0In generation, changeed TE paddy rice (te).
With Hyg-F (5 '-AAGAAGATGTTGGCGACCTCGTATT-3 ') and Hyg-R (5 '-GAAGTGCTTGACATTGGGGAGTTTA-3 ') is primer, to T
0In generation, changes TE paddy rice (te) and carries out pcr amplification, obtains the purpose fragment of 475bp size, illustrates to have changed the TE gene over to, obtains the positive T of 5 strains altogether
0In generation, changeed TE paddy rice (te).
From positive T
0In generation, changes TE paddy rice (te) and goes up the results seed, and sowing obtains T
1In generation, changeed TE paddy rice (te).
T
1In generation, changeed the wild-type sisters system of TE paddy rice (te), cl mutant and mutant cl relatively, observes phenotype, the result as shown in Figure 1, wherein, pGTE is T
1In generation, changeed TE paddy rice (te); WT is the wild-type sisters system of cl mutant; Te is mutant cl, as seen from the figure, and T
1In generation, change TE paddy rice (te) too much the tillering of mutant returned to the WT level, and the plant height that change is short and the boot leaf isophenous of distortion return to the WT level.
Embodiment 3, TE cross the expression paddy rice and have reduced tillering of acceptor material mutant
1, the acquisition of recombinant expression vector
Cut binary expression vector pCubil390 with KpnI and SpeI enzyme, do not contained segmental pCubil390 carrier framework between KpnI and the SpeI; Fine with paddy rice (Oryza sativa ssp.Japonica) Japan is template, the PCR product purification that obtains with primer RT-2LF (5 '-ATCCCCAAATCTCTCGCCCCCACCCA-3 ') and RT-2LR (5 '-TCGCTCCTACAAAGCCAATGAATAAA-3 ') amplification, purified product is cut with KpnI and SpeI enzyme, enzyme is connected with the pCubil390 carrier framework of above-mentioned acquisition after cutting product purification, the connection product that obtains changes in the intestinal bacteria, obtains transformant.Extract the plasmid of transformant, send to order-checking, the result is the carrier of this plasmid for obtaining between the KpnI of the 1 insertion pCubia1390 of the sequence in the sequence table and SpeI, this plasmid called after pUbi::TE.
2, TE crosses the acquisition of expressing paddy rice
PUbi::TE is changed among the agrobacterium tumefaciens EHA105 (Agrebacteriumtumefaciens EHA105) by the method for heat shock, obtain the bacterium of recombinating, extract the plasmid of reorganization bacterium, send to order-checking, the result will contain the reorganization bacterium called after EHA105/pUbi::TE of this plasmid for this plasmid is pUbi::TE.
Mature seed shelling sterilization with the mutant cl of paddy rice (Oryza sativa) is inoculated in the substratum of callus induction.After cultivating for 3 weeks, grow callus from scultellum, it is vigorous to select growth, and color is pale yellow, and more open embryo callus subculture is as the acceptor that transforms.
The EHA105/pUbi::TE of above-mentioned acquisition is infected embryo callus subculture, cultivate after 3 days for 25 ℃, containing screening kanamycin-resistant callus tissue and transfer-gen plant on the selection substratum of 50mg/L Totomycin at the dark place.With hygromycin resistance plant hardening in the cool, be transplanted to the paddy field after several days, obtain T
0Cross the expression paddy rice for TE.
With Pcubi-f (5 '-TGCCTTCATACGCTATTTATTTGC-3 ') and RT-2LR is primer, to T
0Cross the expression paddy rice for TE and carry out pcr amplification, obtain 2100bp purpose fragment, illustrate to have changed the TE gene over to, obtain the positive T of 16 strains altogether
0Cross the expression paddy rice for TE.
Adopt and use the same method, empty carrier pCubil390 is changed among the mutant cl of paddy rice (Oryza sativa), obtain T
0In generation, changeed the empty carrier paddy rice, extracts genomic dna, is primer with Pcubi-f and RT-2LR, carries out pcr amplification, do not obtain the purpose fragment, and the empty carrier that is that changes over to is described.
3, TE crosses the functional study of expressing paddy rice
Choose and be numbered 17,22,65 positive T
2For TE cross expression rice strain in May plantation in the paddy field, growth under field conditions (factors) is with mutant cl (te), the T of wild-type sisters system (WT) and the paddy rice (Oryza sativa) of mutant cl
1In generation, changes the empty carrier paddy rice and is contrast, and each strain system chooses 15 strains, results averaged.
The statistics individual plant tiller number, the result as shown in Figure 2, as seen from the figure,
Be numbered 17 T
2It is 3.7 that generation is changeed TE paddy rice (OE17) individual plant tiller number;
Be numbered 22 T
2It is 6.0 that generation is changeed TE paddy rice (OE22) individual plant tiller number;
Be numbered 65 T
2It is 6.5 that generation is changeed TE paddy rice (OE65) individual plant tiller number;
WT individual plant tiller number is 12.5;
Te individual plant tiller number is 31.9;
T
2The result that generation is changeed empty carrier paddy rice and te does not have significant difference.
As can be seen from the figure, compare T with the wild-type sisters systems (WT) of mutant and the mutant cl (te) of paddy rice (Oryza sativa)
2Crossing the individual plant tiller number of expressing paddy rice for TE obviously reduces, only be 1/4-1/2 (the too many words of tillering of paddy rice of the wild-type sisters system of mutant, tillering that part is not eared can be wasted nutrition, reduces the nutrition supply of other spikes of rice of tillering that can ear, thereby influences the yield and quality of spike of rice.The TE gene can produce on tillering of suiting in the control paddy rice effect.)。
Claims (9)
1. protein is following (a) or (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the tiller number of plant by sequence 2 deutero-protein.
2. coding claim 1 described proteic gene.
3. gene as claimed in claim 2 is characterized in that: described gene is the dna molecular of following (1) or (2) or (3):
(1) dna molecular shown in the sequence 1 in the sequence table;
(2) dna molecular of the tiller number associated protein of the dna sequence dna hybridization that under stringent condition, limits and coding and plant with (1);
(3) dna sequence dna that limits with (1) has 70% at least, has 75% at least, has 80% at least, has 85% at least, has 90% at least, has 95% at least, has 96% at least, has 97% at least, has 98% or the dna molecular that has the tiller number associated protein of 99% homology and coding and plant at least at least.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.
5. recombinant vectors as claimed in claim 4 is characterized in that:
Described recombinant vectors is following 1) or 2):
1) be that claim 2 or 3 described genes are inserted the recombinant vectors that the SmaI recognition site place of pCambia1305.1 carriers obtains;
2) be to ask the recombinant vectors that obtains with the KpnI and the SpeI recognition site of claim 2 or 3 described genes insertion pCUbil390 carriers.
6. amplification claim 2 or 3 described full length genes or its any segmental primer are right.
7. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains transgenic plant; The individual plant tiller number of described transgenic plant is less than described purpose plant.
8. method as claimed in claim 7 is characterized in that: claim 2 or 3 described genes import in the described purpose plant by claim 4 or 5 described recombinant vectorss.
9. as claim 7 or 8 described methods, it is characterized in that: described purpose plant is dicotyledonous or monocotyledons.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584973A (en) * | 2012-03-12 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Rice plant type related protein LPA1 and coding gene and application thereof |
| CN102690341A (en) * | 2012-06-05 | 2012-09-26 | 中国科学院植物研究所 | Plant tillering related protein and coding gene thereof |
| CN112980873A (en) * | 2021-03-12 | 2021-06-18 | 中国农业科学院作物科学研究所 | Protein related to plant type and coding gene and application thereof |
| CN117304288A (en) * | 2023-09-05 | 2023-12-29 | 三峡大学 | Rice tillering angle related protein OsITAND and encoding gene and application thereof |
-
2011
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Non-Patent Citations (2)
| Title |
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| MATSUMOTO, T. ET AL: "NM_001055339.1", 《NCBI DATABASE》 * |
| WING, R. A. ET AL: "AAN74839.1", 《NCBI DATABASE》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584973A (en) * | 2012-03-12 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Rice plant type related protein LPA1 and coding gene and application thereof |
| CN102584973B (en) * | 2012-03-12 | 2013-09-25 | 中国科学院遗传与发育生物学研究所 | Rice plant type related protein LPA1 and coding gene and application thereof |
| CN102690341A (en) * | 2012-06-05 | 2012-09-26 | 中国科学院植物研究所 | Plant tillering related protein and coding gene thereof |
| CN112980873A (en) * | 2021-03-12 | 2021-06-18 | 中国农业科学院作物科学研究所 | Protein related to plant type and coding gene and application thereof |
| CN112980873B (en) * | 2021-03-12 | 2022-05-03 | 中国农业科学院作物科学研究所 | Protein related to plant type and coding gene and application thereof |
| CN117304288A (en) * | 2023-09-05 | 2023-12-29 | 三峡大学 | Rice tillering angle related protein OsITAND and encoding gene and application thereof |
| CN117304288B (en) * | 2023-09-05 | 2024-04-19 | 三峡大学 | Rice tillering angle related protein OsITAND, coding gene and application thereof |
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