CN104788553A - Application of stress-tolerant related protein R10 to regulating plant adverse resistance - Google Patents
Application of stress-tolerant related protein R10 to regulating plant adverse resistance Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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Abstract
The invention discloses an application of a stress-tolerant related protein R10 to regulating plant adverse resistance. The stress-tolerant related protein R10 provided by the invention is (a), (b) or (c), wherein (a) is a protein with the amino acid sequence of SEQ ID NO.2; (b) is a fused protein obtained by connecting tags on N terminal and/or C terminal of the protein shown in SEQ ID NO.2; (c) is a protein with the stress-tolerant related protein function obtained by substituting and/or losing and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2. The experiment proves that the stress-tolerant related protein R10 provided by the invention is capable of regulating the plant stress resistance and increasing the plant drought resistance.
Description
Technical field
The present invention relates to the application of resistance relevant protein R10 in regulating plant resistance in biological technical field.
Background technology
A series of responsing reaction can be produced in plant materials, along with many Physiology and biochemistries and change developmentally under environment stress.Specify the reaction mechanism of plant to adverse circumstance, science argument will be provided for adversity gene engineering research and application.At present, the approach obtaining degeneration-resistant new crop varieties comprises and utilizes the conventional herd breeding technology of classical genetic method and utilize genetic engineering means to cultivate transgenosis new variety technology.Conventional herd breeding technology cycle is long, non-directional, and excellent proterties difficulty controls; By contrast, transgenic technology is quicker, can orderly improvement single traits.Therefore, utilize Protocols in Molecular Biology to transform farm crop, the anti-adversity ability of raising farm crop has become study hotspot in recent years.
Water resources shortage is grave danger that China's agriculture production faces.Paddy rice is most important food crop, is the staple food grain of China's population over half, and rice growing season water requirement is large, and be especially easily subject to the impact of drought and water shortage, arid has become affects one of maximum limiting factor of Rice Production.Under the form of the general shortage of current water resources, how to improve the defensive ability/resistance ability of paddy rice to arid and there is important production meaning.
Summary of the invention
Technical problem to be solved by this invention how to improve the resistance of plant.
For solving the problems of the technologies described above, the present invention provide firstly the application of resistance relevant protein in regulating plant resistance.
In the application of resistance relevant protein provided by the present invention in regulating plant resistance, described resistance relevant protein R10 is a) or b) or c):
A) aminoacid sequence is the protein shown in SEQ ID No.2;
The N end of the protein b) shown in SEQ ID No.2 is or/and C end connects the fused protein that label obtains;
C) by the protein relevant to resistance that the aminoacid sequence shown in SEQ ID No.2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
Wherein, SEQ ID No.2 is made up of 212 amino-acid residues.
In order to make the protein a) be convenient to purifying, label as shown in table 1 can be connected at the N-terminal of the protein shown in SEQ ID No.2 or C-terminal.
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 c) in protein R10, the replacement of one or several amino-acid residue described and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
Above-mentioned c) in protein R10 can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned c) in the encoding gene of protein R10 by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in SEQ ID No.1, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.
In the application of above-mentioned resistance relevant protein in regulating plant resistance, described plant is terrestrial plant.Described terrestrial plant can be dicotyledons and/or monocotyledons.Described monocotyledons can be grass, as paddy rice (Oryza sativa).
For solving the problems of the technologies described above, present invention also offers the biomaterial relevant to described R10.
In the application of the relevant biomaterial of provided by the present invention and described R10 in regulating plant resistance, the biomaterial relevant to described R10 is following A 1) to A20) in any one:
A1) to encode the nucleic acid molecule of described R10;
A2) containing A1) expression cassette of described nucleic acid molecule;
A3) containing A1) recombinant vectors of described nucleic acid molecule;
A4) containing A2) recombinant vectors of described expression cassette;
A5) containing A1) recombinant microorganism of described nucleic acid molecule;
A6) containing A2) recombinant microorganism of described expression cassette;
A7) containing A3) recombinant microorganism of described recombinant vectors;
A8) containing A4) recombinant microorganism of described recombinant vectors;
A9) containing A1) the transgenic plant cells system of described nucleic acid molecule;
A10) containing A2) the transgenic plant cells system of described expression cassette;
A11) containing A3) the transgenic plant cells system of described recombinant vectors;
A12) containing A4) the transgenic plant cells system of described recombinant vectors;
A13) containing A1) Transgenic plant tissue of described nucleic acid molecule;
A14) containing A2) Transgenic plant tissue of described expression cassette;
A15) containing A3) Transgenic plant tissue of described recombinant vectors;
A16) containing A4) Transgenic plant tissue of described recombinant vectors;
A17) containing A1) the transgenic plant organ of described nucleic acid molecule;
A18) containing A2) the transgenic plant organ of described expression cassette;
A19) containing A3) the transgenic plant organ of described recombinant vectors;
A20) containing A4) the transgenic plant organ of described recombinant vectors.
In the application of the relevant biomaterial of above-mentioned and described R10 in regulating plant resistance, A1) described nucleic acid molecule is following a1) a2) or a3) shown in gene:
1) nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.1;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of resistance relevant protein R10 described in coding claim 1 or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of the R10 of resistance relevant protein described in coding claim 1 or genomic DNA molecule.
Wherein, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.
Wherein, SEQ ID No.1 is made up of 639 Nucleotide, the aminoacid sequence shown in coding SEQ ID No.2.
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of coding R10 of the present invention.Those are through manually modified, have and be separated the nucleotide sequence 75% of the R10 obtained or the Nucleotide of higher identity with the present invention, as long as coding R10 and have R10 function is all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention.
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid.The nucleotide sequence that " identity " comprises the protein formed with the aminoacid sequence shown in the SEQ of coding ID No.2 of the present invention has 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.
In above-mentioned biomaterial, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C; Or, in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, hybridize under 65 DEG C of conditions and wash film.
More than above-mentioned 75% or 75% identity, can be the identity of more than 80%, 85%, 90% or 95%.
In above-mentioned biomaterial, the expression cassette (R10 expression casette) of the nucleic acid molecule containing coding R10 B2), refer to the DNA that can express R10 in host cell, this DNA not only can comprise the promotor starting R10 genetic transcription, also can comprise the terminator stopping R10 genetic transcription.Further, described expression cassette also can comprise enhancer sequence.Promotor used in the present invention includes but not limited to: constitutive promoter, the promotor that tissue, organ and growth are special, and inducible promoter.The example of promotor includes but not limited to: the constitutive promoter 35S of cauliflower mosaic virus: from the wound-inducible promoter of tomato, leucine aminopeptidase (" LAP ", the people such as Chao (1999) Plant Physiol 120:979-992); From tobacco chemical inducible promoter, pathogeny be correlated with 1 (PR1) (by Whitfield's ointment and BTH (diazosulfide-7-carbothioic acid S-methyl ester) induction); Tomato proteinase inhibitor II promotor (PIN2) or LAP promotor (all available methyl jasmonate induction); Heat-shock promoters (United States Patent (USP) 5,187,267); Tetracycline inducible promoter (United States Patent (USP) 5,057,422); Seed specific promoters, as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), the special promotor of seed storage protein matter (such as, the promotor (people (1985) EMBO such as Beachy J.4:3047-3053) of phaseollin, napin, oleosin and soybean beta conglycin).They can be used alone or are combined with other plant promoter.All reference cited herein all quote in full.Suitable transcription terminator includes but not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, such as: the people (I such as Odell
985) Nature 313:810; The people such as Rosenberg (1987) Gene, 56:125; The people such as Guerineau (1991) Mol.Gen.Genet, 262:141; Proudfoot (1991) Cell, 64:671; The people Genes Dev. such as Sanfacon, 5:141; The people such as Mogen (1990) Plant Cell, 2:1261; The people such as Munroe (1990) Gene, 91:151; The people such as Ballad (1989) Nucleic Acids Res.17:7891; The people such as Joshi (1987) Nucleic AcidRes., 15:9627).
Available existing expression vector establishment contains the recombinant vectors of described R10 expression casette.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as rouge alkali synthetase gene Nos), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.When using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene is (as given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to herbicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene given methotrexate resistance, give EPSPS gene to glyphosate) or chemical resistance reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.
In the application in regulating plant resistance of the biomaterial of above-mentioned and described R10, described carrier can be plasmid, glutinous grain, phage or virus vector.
In the application in regulating plant resistance of the biomaterial of above-mentioned and described R10, described microorganism can be yeast, bacterium, algae or fungi, as Agrobacterium.
In the application in regulating plant resistance of the biomaterial of above-mentioned and described R10, described transgenic plant cells system, Transgenic plant tissue and transgenic plant organ all do not comprise reproductive material.
In an embodiment of the invention, the encoding gene (i.e. DNA molecular shown in SEQ ID No.1) of R10 is imported in Agrobacterium EHA105 by the recombinant vectors of the expression cassette of the encoding gene containing R10.Described recombinant vectors is by after carrier pCUbi1390 PstI single endonuclease digestion, and insert the recombinant vectors pCUbi1390-R10 that the DNA molecular shown in SEQ ID No.1 obtains, pCUbi1390-R10 expresses R10 albumen.
The application of biomaterial in cultivation resistance transgenic plant of above-mentioned resistance relevant protein R10 or described R10 also belongs to protection scope of the present invention.
The biomaterial of the application in regulating plant resistance of the biomaterial of above-mentioned and described R10 and resistance relevant protein R10 or described R10 is being cultivated in the application in resistance transgenic plant, and described plant is terrestrial plant.Described terrestrial plant can be dicotyledons and/or monocotyledons.Described monocotyledons can be grass, as paddy rice (Oryza sativa).
For solving the problems of the technologies described above, present invention also offers a kind of method of cultivating resistance transgenic plant.
A kind of method of cultivating resistance transgenic plant provided by the present invention, comprises the encoding gene importing described R10 in recipient plant and obtains the step of resistance higher than the resistance transgenic plant of described recipient plant.
In the method for above-mentioned cultivation resistance transgenic plant, the encoding sequence of the encoding gene of described R10 is following 1) or 2) or 3) shown in gene:
1) nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.1;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of the described resistance relevant protein R10 that encodes or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of the R10 of described resistance relevant protein of encoding or genomic DNA molecule.
In an embodiment of the present invention, the encoding gene (i.e. DNA molecular shown in SEQ ID No.1) of described R10 is imported in described recipient plant by the R10 gene recombinant vectors containing R10 expression casette.
In aforesaid method, wherein said R10 gene can first be modified as follows, then imports in recipient plant, to reach better expression effect:
1) carry out according to actual needs modifying and optimizing, to make gene efficient expression; Such as, the codon can had a preference for according to recipient plant, changes its codon to meet plant-preference while the aminoacid sequence keeping R10 gene of the present invention; In optimizing process, keep certain GC content in the encoding sequence after preferably making optimization, to realize the high level expression of quiding gene in plant best, wherein GC content can be 35%, more than 45%, more than 50% or more than about 60%;
2) gene order of contiguous initial methionine is modified, to make translation effectively initial; Such as, effective sequence known in plant is utilized to modify;
3) be connected with the promotor of various expression of plants, be beneficial to its expression in plant; Described promotor can comprise composing type, induction type, sequential adjustment, Growth adjustment, Chemical Regulation, tissue preferably and tissue-specific promoter; The selection of promotor will change along with expression time and space requirement, and depend on target species; The such as specific expressing promoter of tissue or organ, acceptor in what period of growing is determined as required; Although it is operational for demonstrating the many promotors deriving from dicotyledons in monocotyledons, vice versa, but ideally, select dicot promoters for the expression in dicotyledons, monocotyledonous promotor is used for the expression in monocotyledons;
4) with the Transcription Termination sub-connection be applicable to, the expression efficiency of gene of the present invention can also be improved; Such as derive from the tml of CaMV, derive from the E9 of rbcS; Any known available terminator worked in plant can be connected with gene of the present invention;
5) enhancer sequence is introduced, as intron sequences (such as deriving from Adhl and bronzel) and viral leader sequence (such as deriving from TMV, MCMV and AMV).
Described R10 gene recombinant vectors is by using Ti-plasmids, plant virus carrying agent, directly delivered DNA, microinjection, the standard biologic technological methods such as electroporation import vegetable cell (Weissbach, 1998, Method forPlant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geisersonand Corey, 1998, Plant Molecular Biology (2nd Edition)).
In aforesaid method, described transgenic plant are interpreted as the first-generation transgenic plant not only comprising and obtained by described R10 gene transformation object plant, also comprise its filial generation.For transgenic plant, this gene can be bred in these species, also with traditional breeding method, this transgenosis can be entered other kind of same species, particularly including in commercial variety.Described transgenic plant comprise seed, callus, whole plant and cell.
In the present invention, described resistance can be drought resistance.Described drought resistance specifically can be the drought resistance in seedling stage.The drought resistance in described seedling stage specifically can be presented as compared with recipient plant, and plant survival rate is greater than described recipient plant.
Experiment proves, resistance relevant protein R10 provided by the invention and encoding gene thereof can improve the drought resistance of plant: before Osmotic treatment, wild rice, T
1in generation, turns empty carrier paddy rice and T
1the growth conditions that generation turns R10 trans-genetic hybrid rice strain OE1-OE6 is good; T after Osmotic treatment
1it is less that in generation, turns R10 trans-genetic hybrid rice strain OE1-OE6 dehydration, wild rice and T
1the dehydration that generation turns empty carrier paddy rice is more; T after rehydration
1the seedling that generation turns empty carrier paddy rice is all dead after Osmotic treatment, and T
1generation turns R10 trans-genetic hybrid rice strain OE1-OE6 has a certain proportion of survival, T
1the survival rate that generation turns R10 trans-genetic hybrid rice strain OE3 is 85.0%, and the survival rate of wild rice is 18.3%, wild rice (WT) and T after rehydration
1for the survival rate no significant difference turning empty carrier paddy rice.Result shows, resistance relevant protein R10 of the present invention and encoding gene thereof can be utilized to improve the resistance of plant.
Accompanying drawing explanation
Fig. 1 is pCUbi1390 carrier structure schematic diagram.
Fig. 2 is the expression of results that quantitative fluorescent PCR qualification turns R10 gene in R10 trans-genetic hybrid rice plant.
WT is wild rice; OE1 is for turning R10 trans-genetic hybrid rice strain OE1; OE2 is for turning R10 trans-genetic hybrid rice strain OE2; OE3 is for turning R10 trans-genetic hybrid rice strain OE3; OE4 is for turning R10 trans-genetic hybrid rice strain OE4; OE5 is for turning R10 trans-genetic hybrid rice strain OE5; OE6 is for turning R10 trans-genetic hybrid rice strain OE6.
Fig. 3 is wild rice before and after Osmotic treatment and after rehydration and T
1for the growth conditions turning R10 trans-genetic hybrid rice strain OE3.
WT is wild rice; OE3 is for turning R10 trans-genetic hybrid rice strain OE3.
Fig. 4 is wild rice (WT) after rehydration and T
1for the survival rate statistics turning R10 trans-genetic hybrid rice strain OE3.
WT is wild rice; OE3 is for turning R10 trans-genetic hybrid rice strain OE3.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Paddy rice (Oryza sativa) japonica.cv.Nipponbare (J Exp Bot.2015Jan in following embodiment; 66 (1): 271-81.) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.Paddy rice (Oryza sativa) japonica.cv.Nipponbare is hereinafter referred to as wild rice.
Agrobacterium tumefaciens EHA105 (Agrobacterium tumefaciens EHA105) (Plant Cell.2014Jan in following embodiment; 26 (1): 410-25.). the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.
Expression vector pCUbi1390 (J Exp Bot.2015Jan in following embodiment; 66 (1): 271-81.) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application's experiment.
Embodiment 1, the transgenic paddy rice utilizing resistance relevant protein gene cultivation resistance to strengthen
1, the structure of recombinant vectors and recombinational agrobacterium
The total cDNA obtained with wild rice 2 weeks seedling extraction RNA reverse transcription is for template, increase with primer 5 '-TCTGCACTAGGTACCTGCAGATGGAGCAGCAGGAGGAAGTG-3 ' and 5 '-ATGGATCCGTCGACCTGCAGCTATTCCGCCGCCGCCGGTGGAG-3 ', obtain the PCR primer of 679bp.Through order-checking, this PCR primer has the Nucleotide shown in SEQ ID No.1.
Reclaim above-mentioned PCR primer, by clontech infusion kit (takara Products) homologous recombination on the restriction enzyme site PstI of binary expression vector pCUbi1390, the connection product obtained proceeds in intestinal bacteria, obtains transformant.The plasmid extracting transformant sends to order-checking, and the carrier of this plasmid for the binary expression vector pCUbi1390 that recombinated to by the nucleotide homology shown in SEQ ID No.1 obtains, by its called after pCUbi1390-R10, is process LAN recombinant vectors.PCUbi1390-R10 expresses the R10 albumen shown in SEQ ID No.2.
Recombinant vectors pCUbi1390-R10 is imported in agrobacterium tumefaciens EHA105, obtains the recombinational agrobacterium EHA105/pCUbi1390-R10 containing recombinant vectors pCUbi1390-R10.
Empty carrier plasmid pCUbi1390 is proceeded to agrobacterium tumefaciens EHA105, obtains the recombinational agrobacterium EHA105/pCUbi1390 containing plasmid pCUbi1390.
2, the acquisition of R10 trans-genetic hybrid rice is turned
By wild rice seed shelling sterilizing, paddy rice such as transformed wild type such as EHA105/pCUbi1390-R10 such as recombinational agrobacterium such as the method (Hiei, Y.et al.Plant J, 1994,6 (2): 271 – 282) of Hiei etc. is then adopted to obtain T
0in generation, turns R10 trans-genetic hybrid rice plant.T
0generation turn R10 trans-genetic hybrid rice plant sowing after sowing obtain T
1in generation, turns R10 trans-genetic hybrid rice strain, gets 6 T being numbered 0E1-OE6 respectively
1in generation, turns R10 trans-genetic hybrid rice strain and carries out following experiment.。
According to the method described above, recombinational agrobacterium EHA105/pCUbi1390-R10 is replaced with recombinational agrobacterium EHA105/pCUbi1390, other steps are all identical, obtain T
1for the plant turning empty carrier paddy rice.
3, quantitative fluorescent PCR qualification turns the expression of R10 gene in R10 trans-genetic hybrid rice plant
In triplicate, each concrete steps repeated are as follows in experiment:
The RNA turning the leaf tissue of R10 trans-genetic hybrid rice strain OE1 growing 30 days is extracted in paddy field with plant total serum IgE rapid extraction test kit (Tian Gen biochemical technology company limited), obtain the RNA turning R10 trans-genetic hybrid rice strain OE1, be called for short the RNA of strain OE1.
According to the method described above, R10 trans-genetic hybrid rice strain OE1 will be turned replace with respectively and turn R10 trans-genetic hybrid rice strain OE2, turn R10 trans-genetic hybrid rice strain OE3, turn R10 trans-genetic hybrid rice strain OE4, turn R10 trans-genetic hybrid rice strain OE5 and turn R10 trans-genetic hybrid rice strain OE6, other step is all constant, obtains the RNA of strain OE2, RNA, the RNA of strain OE4 of strain OE3, the RNA of the RNA of strain OE5 and strain OE6 respectively.
Be extracted in paddy field the RNA of the leaf tissue of the wild rice growing 30 days, obtain contrasting RNA.
Be extracted in paddy field the RNA turning the leaf tissue of empty carrier paddy rice growing 30 days, obtain unloaded contrast RNA.
Quantitative analysis is carried out to the expression amount of the R10 encoding gene in above-mentioned RNA, identify that the primer of R10 gene is 5 '-TCTTCGGCTTCACCATCACCG-3 ' and 5 '-GATGTAGGACTCGAGGACGAG-3 ', internal reference is UBI-F:5 '-TGAAGACCCTGACTGGGAAG-3 ' and UBI-R:5 '-CACGGTTCAACAACATCCAG-3 '.
Experimental result is shown in Fig. 2.The expression amount turn R10 trans-genetic hybrid rice strain OE1, turn R10 trans-genetic hybrid rice strain OE2, turn R10 trans-genetic hybrid rice strain OE3, turn R10 trans-genetic hybrid rice strain OE4, turning R10 trans-genetic hybrid rice strain OE5 and turn R10 gene in R10 trans-genetic hybrid rice strain OE6 is respectively 18.5 times, 15.6 times, 25.8 times, 30.5 times, 23.7 times and 29.0 times of the expression amount of R10 gene in wild rice; In wild rice R10 gene expression amount and turn R10 gene in empty carrier paddy rice expression amount without significant difference.Result shows, T
1in generation, turns the expression all having R10 gene in R10 trans-genetic hybrid rice strain OE1, OE2, OE3, OE4, OE5 and OE6.
4, the resistance qualification of R10 trans-genetic hybrid rice plant is turned
By wild rice (WT), T
1in generation, turns empty carrier paddy rice and T
1the seed that generation turns R10 trans-genetic hybrid rice strain OE3 is planted in (Nutrition Soil and vermiculite volume ratio are 1:1) in the basin that Nutrition Soil and vermiculite are housed respectively, at 25 DEG C, cultivate under 12h illumination, obtain 3 weeks seedling respectively, above-mentioned 3 weeks seedling are carried out Osmotic treatment then rehydration.Be specially 3 weeks and water sufficient water before seedling Osmotic treatment, do not water any liquid (comprising nutritive medium and water) afterwards always, rewater after 20 days, rehydration is observed rice plant phenotype and adds up rice plant survival rate for 4 days afterwards.Experiment in triplicate, repeats 30 strains at every turn.
According to above-mentioned steps, by T
1the seed that generation turns R10 trans-genetic hybrid rice strain OE3 replaces with T respectively
1for the seed, the T that turn R10 trans-genetic hybrid rice strain OE1
1for the seed, the T that turn R10 trans-genetic hybrid rice strain OE2
1for the seed, the T that turn R10 trans-genetic hybrid rice strain OE4
1for the seed and the T that turn R10 trans-genetic hybrid rice strain OE5
1for the seed turning R10 trans-genetic hybrid rice strain OE6, other step is all identical, obtains each rice plant phenotype and adds up rice plant survival rate.
Result shows, before Osmotic treatment, and wild rice, T
1in generation, turns empty carrier paddy rice and T
1in generation, turns R10 trans-genetic hybrid rice strain OE1, T
1in generation, turns R10 trans-genetic hybrid rice strain OE2, T
1in generation, turns R10 trans-genetic hybrid rice strain OE3, T
1in generation, turns R10 trans-genetic hybrid rice strain OE4, T
1in generation, turns R10 trans-genetic hybrid rice strain OE5 and T
1the growth conditions that generation turns R10 trans-genetic hybrid rice strain OE6 is good; T after Osmotic treatment
1it is less that in generation, turns R10 trans-genetic hybrid rice strain OE1-OE6 dehydration, wild rice and T
1the dehydration that generation turns empty carrier paddy rice is more; T after rehydration
1the seedling that generation turns empty carrier paddy rice is all dead after Osmotic treatment, and T
1generation turns R10 trans-genetic hybrid rice strain OE1-OE6 a certain proportion of survival.Wild rice (WT) before and after Osmotic treatment and after rehydration and T
1in generation, turns the growth conditions of R10 trans-genetic hybrid rice strain OE3 as shown in Figure 3.Wild rice (WT), T after above-mentioned rehydration
1in generation, turns empty carrier paddy rice and T
1the survival rate statistics that generation turns R10 trans-genetic hybrid rice strain OE3 shows (Fig. 4), T
1the survival rate that generation turns R10 trans-genetic hybrid rice strain OE3 is 85.0%, and the survival rate of wild rice is 18.3%, wild rice (WT) and T after rehydration
1for the survival rate no significant difference turning empty carrier paddy rice.
Result shows T
1the drought resistance that generation turns R10 trans-genetic hybrid rice significantly improves.
Claims (10)
1. the application of resistance relevant protein R10 in regulating plant resistance; Described resistance relevant protein R10 is a) or b) or c):
A) aminoacid sequence is the protein shown in SEQ ID No.2;
The N end of the protein b) shown in SEQ ID No.2 is or/and C end connects the fused protein that label obtains;
C) by the protein relevant to resistance that the aminoacid sequence shown in SEQ ID No.2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. the application of the biomaterial relevant to resistance relevant protein R10 described in claim 1 in regulating plant resistance;
The described biomaterial relevant to resistance relevant protein R10 described in claim 1 is following A 1) to A20) in any one:
A1) nucleic acid molecule of resistance relevant protein R10 described in coding claim 1;
A2) containing A1) expression cassette of described nucleic acid molecule;
A3) containing A1) recombinant vectors of described nucleic acid molecule;
A4) containing A2) recombinant vectors of described expression cassette;
A5) containing A1) recombinant microorganism of described nucleic acid molecule;
A6) containing A2) recombinant microorganism of described expression cassette;
A7) containing A3) recombinant microorganism of described recombinant vectors;
A8) containing A4) recombinant microorganism of described recombinant vectors;
A9) containing A1) the transgenic plant cells system of described nucleic acid molecule;
A10) containing A2) the transgenic plant cells system of described expression cassette;
A11) containing A3) the transgenic plant cells system of described recombinant vectors;
A12) containing A4) the transgenic plant cells system of described recombinant vectors;
A13) containing A1) Transgenic plant tissue of described nucleic acid molecule;
A14) containing A2) Transgenic plant tissue of described expression cassette;
A15) containing A3) Transgenic plant tissue of described recombinant vectors;
A16) containing A4) Transgenic plant tissue of described recombinant vectors;
A17) containing A1) the transgenic plant organ of described nucleic acid molecule;
A18) containing A2) the transgenic plant organ of described expression cassette;
A19) containing A3) the transgenic plant organ of described recombinant vectors;
A20) containing A4) the transgenic plant organ of described recombinant vectors.
3. application according to claim 2, is characterized in that: A1) described nucleic acid molecule is following 1) or 2) or 3) shown in gene:
1) nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.1;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of resistance relevant protein R10 described in coding claim 1 or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of the R10 of resistance relevant protein described in coding claim 1 or genomic DNA molecule.
4. the application in resistance transgenic plant cultivated by resistance relevant protein R10 according to claim 1 or biomaterial according to claim 2.
5., according to the arbitrary described application of claim 1-4, it is characterized in that: described plant is monocotyledons or dicotyledons.
6., according to the arbitrary described application of claim 1-5, it is characterized in that: described resistance is drought resistance.
7. cultivate a method for resistance transgenic plant, comprise the encoding gene importing resistance relevant protein R10 described in claim 1 in recipient plant and obtain the step of resistance higher than the resistance transgenic plant of described recipient plant.
8. method according to claim 7, is characterized in that: the encoding gene of resistance relevant protein R10 described in claim 1 is following 1) or 2) or 3) shown in gene:
1) nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.1;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of resistance relevant protein R10 described in coding claim 1 or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of the R10 of resistance relevant protein described in coding claim 1 or genomic DNA molecule.
9. the method according to claim 7 or 8, is characterized in that: described recipient plant is monocotyledons or dicotyledons.
10., according to described method arbitrary in claim 7-9, it is characterized in that: described resistance is drought resistance.
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Non-Patent Citations (3)
| Title |
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| HYUNMI KIM 等: "Overexpression of PYL5 in rice enhances drought tolerance,inhibits growth,and modulates gene expression", 《JOURNAL OF EXPERIMENTAL BOTANY》 * |
| KIKUCHI,S.等: "Oryza sativa Japonica Group cDNA clone:006-310-C11，full insert sequence", 《GENBANK》 * |
| MIGUEL GONZALEZ-GUZMAN 等: "Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist puinabactin，and the capability to enhance plant drought resistance", 《JOURNAL OF EXPERIMENTAL BOTANY》 * |
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