CN108048481A - Application of the RLI1 albumen in adjusting and controlling rice leaf angle - Google Patents

Application of the RLI1 albumen in adjusting and controlling rice leaf angle Download PDF

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CN108048481A
CN108048481A CN201810015980.0A CN201810015980A CN108048481A CN 108048481 A CN108048481 A CN 108048481A CN 201810015980 A CN201810015980 A CN 201810015980A CN 108048481 A CN108048481 A CN 108048481A
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rli1
albumen
protein
plant
amino acid
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CN108048481B (en
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易可可
阮文渊
郭美娜
王俊敏
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Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses a kind of application of RLI1 albumen in adjusting and controlling rice leaf angle.The present invention provides the application of RLI1 albumen or its encoding gene in plant leaf blade angle is regulated and controled；The RLI1 albumen is as shown in SEQ ID No.1, or by SEQ ID No.1 by the substitution of amino acid residue and/or missing and/or addition and tool identical function, or have more than 80% homology with SEQ ID No.1 and have identical function or connect sequence label and tool identical function in the N-terminal and/or C-terminal of SEQ ID No.1.The present invention is overexpressed RLI1 by transgenosis, can increase blade angle, plant type is made to become loose.By being knocked out with CRISPER/Cas9 technologies to RLI1 genes in rice, discovery can promote rice plant compact, and upright blade is conducive to dense planting, have the population photosynthesis efficiency for improving rice, improve the potential value of yield.

Description

Application of the RLI1 albumen in adjusting and controlling rice leaf angle
Technical field
The invention belongs to biological technical fields, are related to a kind of application of RLI1 albumen in adjusting and controlling rice leaf angle.
Background technology
Green plants carries out photosynthesis by blade, and luminous energy is transformed into chemical energy, is the life of all animals on the earth Source is ordered, while is also the main matter of human society and the source of energy.Rice yield, only 5%~10% substance come From the nutriment of root absorption, and 90%~95% substance then comes from the photosynthetic product of crop leaf.Therefore, rice Form plays yield critical effect, and yield is improved with the improvement of plant type.Leaf morphology is the main of influence plant type Factor.From the four's or five of 20th century to present, rice in China cultivar is improved the breed from high stalk farm variety, high stalk to of short stem It improves the breed, short-stalked variety to hybrid rice, yield all has been improved.Although this twice output increased the reason for be not quite similar, But common ground is all related to the morphology of rice.Since the 1980s, in rice breeding field, successively there is multidigit Breeder proposes rice high yield theory Ideotype model, and is all referred to the breeding of leaf morphology.
Leaf angle refers to the bending degree between blade and the inclination angle of stem or blade and leaf sheath, blade angle and photosynthetic speed Rate has substantial connection, is one of important morphological characters of rice.It was found that blade angle and bending degree and light under the conditions of single leaf Closing rate has direct relation；And under the conditions of group, blade angle and bending degree to canopy light distribution, Canopy Apparent Photosynthetic Rate, Substance produces and yield all has a significant impact.Even if leaf area is identical with single leaf photosynthetic rate, the light of vertical flag leaf group --- light It is in unsaturated type to close effect curves, and is hung down loosely or group that blade angle is larger is in then saturation type.Under strong light, the former Population Light It is high to close rate, Dry Matter Production speed is fast.Therefore, rice leaf angle be influence yield Main Agronomic Characters, upright leaf Piece is remarkably improved population photosynthesis efficiency beneficial to plant dense planting, and then increases yield.
The content of the invention
The object of the present invention is to provide a kind of RLI1 albumen or the new applications of its encoding gene.
New application provided by the present invention is specially RLI1 albumen or its relevant biological material in regulation and control plant leaf blade angle In application.
Wherein, the RLI1 albumen is specially following any shown protein：
(A1) amino acid sequence is the protein of SEQ ID No.1；
(A2) by the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues substitution and/or Missing and/or addition and the protein with identical function；
(A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；
(A4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (A1)-(A3) Albumen；
The relevant biological material is that can express the nucleic acid molecules of the RLI1 albumen or containing the nucleic acid molecules Expression cassette, recombinant vector, recombinant bacterium or transgenic cell line.
In the application, the regulation and control are embodied as：The RLI1 albumen or its encoding gene are in the plant Expression quantity and/or activity it is higher, the leaf angle of the plant is bigger；The RLI1 albumen or its encoding gene are in the plant Expression quantity and/or activity in object is lower, and the leaf angle of the plant is smaller.
The present invention also provides a kind of methods for cultivating the smaller plant of leaf angle.
The method provided by the present invention for cultivating the smaller plant of leaf angle, it may include make RLI1 albumen in recipient plant Expression quantity and/or activity reduce the step of.Wherein, the RLI1 albumen is any shown protein in (A1)-(A4) above.
Further, the present invention provides a kind of method for cultivating the smaller genetically modified plants of leaf angle, specifically may include Following steps：Inhibition expression is carried out to the encoding gene of RLI1 albumen in recipient plant, obtains genetically modified plants；The transgenosis Plant leaf angle smaller compared with the recipient plant.Wherein, the RLI1 albumen is any shown in (A1)-(A4) above Protein.
It is described previously cultivate the smaller plant of leaf angle method (or it is described cultivation the smaller transgenosis of leaf angle The method of plant) it is following it is any in application fall within protection scope of the present invention：
(1) plant variety suitable for dense planting is cultivated；
(2) photosynthetic efficiency of plant population is improved；
(3) plantation yield of the plant on unit area is improved.
The present invention also provides a kind of methods for the plant for cultivating leaf angle bigger.
The method of the plant provided by the present invention for cultivating leaf angle bigger, it may include make RLI1 albumen in recipient plant Expression quantity and/or activity improve the step of.Wherein, the RLI1 albumen is any shown protein in (A1)-(A4) above.
Further, the present invention provides a kind of method for the genetically modified plants for cultivating leaf angle bigger, specifically may include Following steps：The nucleic acid molecules of the RLI1 albumen can be expressed by being imported into recipient plant, obtain genetically modified plants；Described turn Gene plant leaf angle bigger compared with the recipient plant.Wherein, the RLI1 albumen is any in (A1)-(A4) above Shown protein.
In above-mentioned each application or method, the nucleic acid molecules that can express RLI1 albumen are the coding base of RLI1 albumen Cause, concretely following any DNA molecular：
(B1) DNA molecular shown in SEQ ID No.2；
(B2) DNA molecular limited under strict conditions with (B1) hybridizes and encodes the DNA molecular of the RLI1 albumen；
(B3) have more than 99%, more than 95%, more than 90%, 85% with the DNA sequence dna of any restriction in (B1)-(B2) Above or the DNA molecular of more than 80% homology and the coding RLI1 albumen.
Above-mentioned stringent condition can be with 6 × SSC, and the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
It is described " in recipient plant in the method for the previously described cultivation smaller genetically modified plants of leaf angle The encoding gene of RLI1 albumen carries out inhibition expression " it can be realized by any technological means that can realize this purpose, it is such as logical It crosses sequence specific nuclease (such as CRISPR/Cas9 nucleases) and specific cleavage is carried out to the encoding gene, so as to reduce it Expression in the recipient plant.
In the present invention, described " inhibition expression is carried out to the encoding gene of RLI1 albumen in recipient plant " particular by What CRISPER/Cas9 technologies were realized；To meet 5 '-N in DNA fragmentation shown in SEQ ID No.2X- NGG-3 ' or 5 '-CCN-NX- The segment of 3 ' series arrangements rule is target sequence；N represents any one of A, G, C and T, 14≤X≤30, and X as integer, NXTable Show X continuous deoxyribonucleotides.More specifically, in one particular embodiment of the present invention, the X is 19.Phase It answers, the target sequence is specially SEQ ID No.3.
It is described " to be led into recipient plant in the method for the genetically modified plants of previously described cultivation leaf angle bigger Enter the encoding gene of RLI1 albumen " it can be realized by any technological means that can realize this purpose.
In the present invention, described the encoding gene of RLI1 albumen " into recipient plant import " particular by it is described by The recombinant expression carrier realization of the encoding gene containing the RLI1 albumen is imported in body plant.
The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture Bacillus carrier and carrier available for plant micropellet bombardment etc., as pCAMBIA-1300-221, pGreen0029, PCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative Plant expression vector.The plant expression vector can also include 3 ' end untranslated regions of foreign gene, i.e., comprising polyadenylic acid Signal and any other DNA fragmentation for participating in mRNA processing or gene expression.The bootable polyadenylic acid of polyadenylation signals It is added to 3 ' ends of mRNA precursor.During using the gene constructed recombinant expression carrier, it can add before its transcription initiation nucleotide Upper any one is enhanced, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S Promoter, ubiquitin gene Ubiquitin promoters (pUbi), stress induced promoter rd29A etc., they can be used alone or It is used in combination with other plant promoters；In addition, when using the gene constructed recombinant expression carrier of the present invention, it also can be used and increase Hadron, including translational enhancer or transcriptional enhancer, these enhancer regions can be that ATG initiation codon or neighboring region rise Beginning codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of entire sequence.The translation control The source of signal and initiation codon is extensive, can be natural or synthesis.Translation initiation region can come From transcription initiation region or structural gene.It, can be to institute for the ease of transgenic plant cells or plant are identified and screened It is processed with recombinant expression carrier, the enzyme or light emitting compound of color change can be generated as added in the coding that can be expressed in plant The gene of object, resistant antibiotic marker or anti-chemical reagent marker gene etc..Also any selectivity mark can be not added with Remember gene, transformed plant is directly screened with adverse circumstance.
In the present invention, the promoter for starting the encoding gene transcription of the RLI1 albumen in the recombinant vector is flower coconut palm Cauliflower mosaic virus (CAMV) 35S promoter.
More specifically, the recombinant expression carrier is that the encoding gene of the RLI1 albumen is inserted into pF3PZPY122 Recombinant plasmid at the multiple cloning sites of carrier obtained by (XbaI and BamHI).
In the above-mentioned methods, by the recombinant expression carrier for the encoding gene for carrying the RLI1 albumen or it is used for The gene editing instrument used when " carrying out inhibition expression to the encoding gene of RLI1 albumen in recipient plant " imports the receptor Plant, concretely：By using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture The conventional biology methods such as bacillus mediation conversion plant cell or tissue, and the plant tissue of conversion is cultivated into plant.
In above application or method, the plant can be dicotyledon or monocotyledon.
Further, the monocotyledon can be grass.
More specifically, the grass is rice.In one embodiment of the invention, the rice is specially Rice varieties Nipponbare (Nipponbare).
In the present invention, the leaf angle be specially fall a leaf (L1), fall two leaves (L2) and/or the leaf of three leaves (L3) Piece angle.
The present invention is overexpressed RLI1 by transgenosis, can increase blade angle, plant type is made to become loose.Pass through utilization CRISPER/Cas9 technologies knock out RLI1 genes in rice, and discovery can promote rice plant compact, upright blade, favorably In dense planting, there is the population photosynthesis efficiency for improving rice, improve the potential value of yield.
Description of the drawings
Fig. 1 can improve rice leaf orthostatic to knock out RLI1 genes.A：Wild type (WT), mutant rli1 and turn zero load The potting phenotypic analysis of body plant (NC, negative control)；Rli1-1 and rli1-2 is two different independent water Rice RLI1 knock out mutants bodies；NC-1 and NC-2 is two transgenic lines for turning empty carrier；B：The sequencing of mutant rli1 Analysis；C：Potted plant experiment wild type, mutant rli1 and three leaves (L3) the leaf angle size analysis for turning empty carrier plant.It is aobvious Write sex differernce analysis * P<0.05.
Fig. 2 can increase rice leaf intersection angle for overexpression RLI1 genes.A：RLI1 genes overexpress potting phenotypic analysis； OE-3, OE-7 and OE-10 are three different independent Transgenic Rice strains；NC-3 and NC-4 is turn empty carrier two turns Gene strain.B：RLI1 genes overexpress the Molecular Identification of strain.C：OE-3, OE-7 and OE-10 are in basin for Transgenic Rice strain Under the conditions of cultivation, fall a leaf (L1), fall two leaves (L2), fall three leaves (L3) leaf angle size.Significant difference analysis * * P< 0.01。
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
PYLsgRNA-OsU3 plasmids and pYLCRISPR/Cas9P35S-H plasmids：It is obtained from Liu Yaoguang professors laboratory.Note It is loaded in " Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B.et al (2015) A robustCRISPR/Cas9system for convenient high‐efficiency multiplex genome Editing inmonocot and dicot plants.Mol.Plant, 8,1274-1284. " text, the public can be from applicant Place obtains, and can only be used to repetition present invention experiment and uses.
PF3PZPY122 carriers：It is obtained from Deng Xingwang professors laboratory.It is recorded in " Feng S, et al.The COP9signalosome interacts physically with SCF COI1and modulates jasmonate responses.PlantCell.2003；15(5):1083-1094 " text, the public can obtain at applicant, can only be used to weight Duplicate invention experiment uses.
Embodiment 1, inhibit RLI1 genes expression can rice leaf angle become smaller
Involved RLI1 genes come from rice (Oryza sativa L.), the sequence of RLI1 genes in the present embodiment As shown in SEQ ID No.2, the RLI1 albumen shown in SEQ ID No.1 is encoded.The present embodiment will use CRISPER/Cas9 skills Art knocks out rice RLI1 genes, and then studies its influence to rice plant leaf angle.It is specific as follows：
First, the selection of the target site of rice RLI1 genes
A chain in the target double-strand knocked out using CRISPER/Cas9 technologies has such as lower structure：5’-CCN-NX- N in 3 ', PAM (NGG) represents any one of A, T, C and G, and the N in Nx represents any one of A, T, C and G, x=19. The target sequence of RLI1 genes is as follows, and the base with underscore is PAM (prototype intervening sequence adjoins motif).
RLI1：5’-CCTGACGATCTACCACATCAAG-3’(SEQ ID No.3)；
After the knockout carrier rice transformation, under the mediation of sgRNA, Cas9 albumen is cut in target sequence region, forms DNA Double-strand break triggers the in vivo self-inflicted injury repair mechanism of machine, and mutation (this can be introduced during the cell spontaneous reparation notch Place's " mutation " refers to that broad sense is mutated, and including being inserted into, lacking, the forms such as narrow sense mutation, most in these mutation is gene Functionally inactive is mutated).
2nd, the structure of knockout carrier is recombinated
1st, with restriction enzyme BsaI digestion pYLsgRNA-OsU3 plasmids, the carrier framework of about 3kb is recycled, is named as pYLsgRNA-OsU3-BasI。
2nd, according to the target site RLI1 sequences of design, synthesis carries the primer of cohesive end (underscore part) as follows：
RLI1-1F：5’-gccaCTTGATGTGGTAGATCGTC-3’；
RLI1-1R：5’-aaacGACGATCTACCACATCAAG-3’。
3rd, RLI1-1F and RLI1-1R are annealed, the double-stranded DNA for being formed with cohesive end is named as RLI1, by itself and Glue recovery product pYLsgRNA-OsU3-BasI connections in step 1, using connection product as template with primer Uctcg-B1 ' and GRcggt-BL carries out PCR and obtains the segment of about 600bp, carries out digestion with BsaI after recycling, is named as pYLsgRNA-OsU3- RLI1-BasI。
Uctcg-B1’：5’-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3’；
gRcggt-BL:5’-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3’。
4th, with restriction enzyme BsaI digestions pYLCRISPR/Cas9P35S- H plasmids recycle the carrier framework of about 16kb, It is named as pYLCRISPR/Cas9P35S-H-BsaI。
5th, the carrier framework for obtaining the pYLsgRNA-OsU3-RLI1-BasI segments that step 3 obtains with step 4 pYLCRISPR/Cas9P35S- H-BsaI is attached, and the recombinant plasmid after sequence verification is correct is named as pYLCRISPR/ Cas9-U3-RLI1。
3rd, rice transformation
The recombinant plasmid pYLCRISPR/Cas9-U3-RLI1 of step 2 structure is imported by agriculture bacillus mediated method Rice varieties Nipponbare.Using Nipponbare callus as transformation receptor, complete regenerated plant is obtained by tissue cultures after conversion. Experiment sets be transferred to pYLCRISPR/Cas9P into rice varieties Nipponbare simultaneously35SThe control of-H empty carriers is (referred to as unloaded right According to).
Obtain the transfer-gen plant of RLI1 gene lacks functionalities, i.e., the homozygous plants that RLI1 sites are undergone mutation.Extraction turns Gene plant genomic DNA carries out genomic DNA of the conversion containing hygromycin with the special primer containing target site RLI1 PCR amplification, PCR product sequence verification.
B in Fig. 1 is shown in the sequencing result of Mutants homozygous, obtained mutant rli1-1 contains 5bp in RLI1 genes The missing of base, the final reading frame for changing RLI1 genes.Mutant rli1-2 lacking containing 2bp bases in RLI1 genes It loses, the final reading frame for changing RLI1 genes.
4th, the phenotypic evaluation of rice mutant
The rice mutant rli1-1 and rli1-2 obtained with step 3 and the wild rice plant without transgenosis (WT) and it is transferred to pYLCRISPR/Cas9P35SThe rice plant of-H plasmid empty carriers is experiment material.By each experiment material seedling After carry out potted plant experiment.In the Nutrition Soil of 10 -day-old of rice shoot insertion 10kg, plantation carries out statistical after 45 days to leaf angle Analysis.
The results are shown in Figure 1.As it can be seen that compared with wild rice plant (WT), rice mutant rli1-1 and rli1-2 Leaf angle significantly become smaller, entire plant is compacter, upright blade, this would be more advantageous in dense planting, have improve rice Population photosynthesis efficiency, improve the potential value of yield.In addition, the leaf angle of unloaded control and wild rice plant (WT) It is basically identical, no difference of science of statistics.
Embodiment 2, RLI1 gene overexpressions can rice leaf angle become larger
Involved RLI1 genes come from rice (Oryza sativa L.), the sequence of RLI1 genes in the present embodiment As shown in SEQ ID No.2, the RLI1 albumen shown in SEQ ID No.1 is encoded.The present embodiment will to RLI1 genes in rice into Row is overexpressed, and then studies its influence to rice plant leaf angle.It is specific as follows：
First, the structure of over-express vector
In order to obtain the rice plant of RLI1 gene overexpressions, inventor constructs drives RLI1 genes with 35S promoter Expression vector (35S:RLI1), converted for rice plant.Both ends as template, are used using RLI1 genes shown in SEQ ID No.2 The primer RLI1-2F and RLI1-2R for being respectively provided with restriction enzyme site XbaI and BamHI clone RLI1 gene orders, will clone Sequence be connected to the XbaI/BamHI restriction enzyme sites after pF3PZPY122 carrier 35S promoters.The carrier inscribe built Enzyme XbaI and BamHI are detected, and can cut out the target fragment of about 1050bp, show that the carrier of structure is correct.
RLI1-2F：5’-CGCTCTAGAATGTTGCAAGATATCATGAAC-3 ' (is XbaI enzyme cutting site at underscore Identify sequence)；
RLI1-2R：5’-CGCGGATCCGCAGCACTTGCACTCCATTG-3 ' (is BamHI restriction enzyme sites at underscore Identify sequence).
Over-express vector 35S:The structure of RLI1 is described as：By two restriction enzyme XbaI of pF3PZPY122 plasmids Segment (about 20bp) between the identification sequence of BamHI replaces with the restructuring matter of gained after DNA fragmentation shown in SEQ ID No.2 Grain.
2nd, rice transformation
Using the callus of rice Nipponbare maturation embryonal induction as receptor, callus is transferred to the Agrobacterium EHA105 methods mediated, Induce differentiation into seedling.Three plants are randomly selected from the transgenic paddy rice obtained, is denoted as OE-3, OE-7 and OE-10 respectively.
Experiment sets the control for being transferred to pF3PZPY122 empty carriers into rice varieties Nipponbare (referred to as unloaded right simultaneously According to).
3rd, to the Molecular Identification of transgenic paddy rice
Real-time PCR are to transgenic paddy rice OE-3, OE-7 and OE-10 that step 2 obtains further to be confirmed to turn base Because whether the expression of the RLI1 genes in rice significantly improves.Concrete operations are as follows：
Use the RNAeasy Plant Mini Kit (article No.s of Qiagen companies：74903) wild type and step 2 are extracted The total serum IgE of transgenic paddy rice OE-3, OE-7 and OE-10 of acquisition and unloaded control rice, take the total serum IgE of 1 μ g first to use 37 DEG C of DNase digestion 30min, then in 20 μ l systems using TOYOBO RT kit to specifications reverse transcription into cDNA； CDNA is expanded with the SYBR Premix Ex Taq kit of TaKaRa, with Bio-Rad CFX96real-time PCR detection systems System detects the amplification amount of cDNA in real time.For expanding the primer sequence of RLI1 genes as 5 '-CGAACCAGTCCCAAATCCCA-3 ' With 5 '-CGTCGAGCTGAACACGCAGT-3 ', for expand the primer sequence of internal reference Actin cDNA for 5 '- CAACACCCCTGCTATGTACG-3 ' and 5 '-CATCACCAGAGTCCAACACAA-3 '.
As a result the expression quantity of RLI1 genes is all significantly higher than open country in B such as in Fig. 2, transgenic paddy rice OE-3, OE-7 and OE-10 Raw type (WT) plant illustrates that the expression quantity of RLI1 genes in transgenic paddy rice OE-3, OE-7 and OE-10 that step 2 obtains is certain It significantly improves.And as the expression quantity of RLI1 genes and wild rice plant (WT) basic one in the rice plant of zero load control It causes, no difference of science of statistics.
4th, the phenotypic evaluation of transgenic paddy rice
Transgenic paddy rice OE-3, OE-7 and OE-10 for being obtained with step 2 and the wild rice without transgenosis are planted Strain (WT) and the rice plant for being transferred to pF3PZPY122 empty carriers are experiment material.Each experiment material is subjected to potted plant experiment. Transgenic seedling of the greenhouse rice nursery after 10 days is moved into the bucket equipped with Nutrition Soil (10kg soil/bucket).Plantation culture is united after 45 days Meter analysis leaf angle size.
As a result as shown in C in A in Fig. 2 and Fig. 2.As it can be seen that compared with wild rice plant (WT), transgenic paddy rice OE- 3rd, the leaf angle of OE-8 and OE-11 significantly becomes larger, and becoming for entire plant is loose.In addition, the leaf angle of unloaded control with Wild rice plant (WT) is basically identical, no difference of science of statistics.
<110>INST OF AGRICULTURAL RESOURCES
<120>Application of the RLI1 albumen in adjusting and controlling rice leaf angle
<130> GNCLN180084
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 350
<212> PRT
<213>Rice（Oryza sativa L.）
<400> 1
Met Leu Gln Asp Ile Met Asn Thr Lys Lys Ile Lys Leu His Asp Cys
1 5 10 15
His Phe Gly Ser Pro Leu Cys Asp Pro Ser Pro Ala Pro His Leu Leu
20 25 30
Ser Ser Ala Ala Ala Ala Gly Leu Ser Phe His Pro Gly Leu Val Ser
35 40 45
Ser Ala Ala Gln His Gln Gln His Gly Ala Gly Gly Trp Leu His Glu
50 55 60
Glu Tyr Tyr Ala Pro Arg Ser Ser Pro Pro Ser Ser Leu Leu Ala Gln
65 70 75 80
Thr Cys Val Gly Ser Asn Ala Thr Ala Phe Tyr Ala Ala Glu Asn Leu
85 90 95
Pro Gln Phe Asp Phe Pro Ala Leu Gly Thr Ala Ala Ala Ala Ala Ala
100 105 110
Lys Ala Pro Phe Arg Ser Ser Glu Ser Glu Leu Tyr Arg Pro Val Asp
115 120 125
Pro Leu Leu Leu Arg Ala Asp His Ser Val Arg Thr Tyr Tyr Val Arg
130 135 140
Pro Gln Lys Arg Asp Ser Gly Glu Arg Thr Pro Leu Pro Pro Pro Ser
145 150 155 160
Gln Gln Gln His Gln Asp Arg Ile His Gly Leu Phe Ala Gly Ala Pro
165 170 175
Thr Thr Arg Leu Leu Ser Gly Glu Pro Lys Ile His Ser Phe Pro Pro
180 185 190
Gln Val Ala Ala Lys Pro Ile Leu Pro Ala Met Asp Ala Pro Ser Leu
195 200 205
Gln Asn Gln Met Glu Asn Gln Leu Thr Arg Asn Cys Ile Gly Ala Ala
210 215 220
Thr Pro Val Thr Pro Thr Gly Asn Leu Ala Gly Ser Gly Ala Pro Ser
225 230 235 240
Lys Thr Arg Ile Arg Trp Thr Gln Asp Leu His Glu Arg Phe Val Asp
245 250 255
Cys Val Asn Gln Leu Gly Gly Ala Asp Lys Ala Thr Pro Lys Gly Ile
260 265 270
Leu Lys Leu Met Asn Ser Asp Gly Leu Thr Ile Tyr His Ile Lys Ser
275 280 285
His Leu Gln Lys Tyr Arg Ile Ala Lys Tyr Met Pro Ala Ser Ser Glu
290 295 300
Gly Lys Gln Leu Glu Lys Arg Ala Thr Gly Asn Asp Met Gln Asn Leu
305 310 315 320
Asp Pro Lys Thr Tyr Leu Ser Phe Ser Leu Ser Ala Ser Ser Asn Ser
325 330 335
Phe Ala Asn Gln Ser Gln Ile Pro Met Glu Cys Lys Cys Cys
340 345 350
<210> 2
<211> 1053
<212> DNA
<213>Rice（Oryza sativa L.）
<400> 2
atgttgcaag atatcatgaa caccaagaag attaagctgc acgactgcca cttcggctcg 60
ccgctatgtg acccttcgcc ggcgccgcac ctgctgagct ccgccgccgc cgccgggctg 120
tcgttccacc cggggctcgt gagctcggcg gcgcagcacc agcagcacgg cgcgggcggc 180
tggctgcacg aggagtacta cgcgccgagg tcgtcgccgc cgtcgtcgct tctcgcgcag 240
acctgcgtcg gctccaacgc gaccgcgttc tacgccgccg agaacctgcc gcagttcgac 300
ttcccagctc tcggtacggc ggcggcggcg gcggccaagg cgccgttccg gtcgtcggag 360
agcgagctgt accggccggt cgacccgctg ctcctccgtg cggaccactc ggtgaggacg 420
tactacgtcc ggccgcagaa gcgggattcc ggcgagagga caccattgcc gccgccgtcg 480
cagcaacagc atcaggacag aatccacggg ctcttcgccg gcgctcccac cactcggctt 540
ctcagcggcg aacccaaaat ccactcgttt ccacctcaag tggcggcgaa gccgattctg 600
ccggcgatgg atgcgccgag cctgcagaac cagatggaga atcagctgac aaggaactgc 660
atcggcgcgg caactccggt gacccccacc ggaaacctcg ccggatcagg tgcgccgagc 720
aagacgcgga tcaggtggac gcaggacctg cacgagcggt tcgtcgactg cgtcaatcag 780
ctcggcggcg cagacaaggc aactccgaag gggattctga agctgatgaa ttcggatggc 840
ctgacgatct accacatcaa gagccatctc cagaaatatc gcatagcgaa gtacatgcca 900
gcgtcatctg aagggaagca actggagaaa agagcaacag gaaatgacat gcagaatctg 960
gaccccaaaa cgtatctctc tttctctctg tctgctagta gtaatagctt tgcgaaccag 1020
tcccaaatcc caatggagtg caagtgctgc tag 1053
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
cctgacgatc taccacatca ag 22

Claims (10)

  1. The application of 1.RLI1 albumen or its relevant biological material in plant leaf blade angle is regulated and controled；
    The RLI1 albumen is following any shown protein：
    (A1) amino acid sequence is the protein of SEQ ID No.1；
    (A2) by substitution of the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing And/or it adds and there is the protein of identical function；
    (A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；
    (A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3)；
    The relevant biological material is the nucleic acid molecules that can express the RLI1 albumen or the expression containing the nucleic acid molecules Box, recombinant vector, recombinant bacterium or transgenic cell line.
  2. 2. application according to claim 1, it is characterised in that：The RLI1 albumen or its encoding gene are in the plant Expression quantity and/or activity it is higher, the leaf angle of the plant is bigger；The RLI1 albumen or its encoding gene are in the plant Expression quantity and/or activity in object is lower, and the leaf angle of the plant is smaller.
  3. 3. a kind of method for cultivating the smaller plant of leaf angle, including make in recipient plant the expression quantity of RLI1 albumen and/or The step of activity reduces；
    The RLI1 albumen is following any shown protein：
    (A1) amino acid sequence is the protein of SEQ ID No.1；
    (A2) by substitution of the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing And/or it adds and there is the protein of identical function；
    (A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；
    (A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3).
  4. 4. a kind of method for cultivating the smaller genetically modified plants of leaf angle, includes the following steps：To RLI1 eggs in recipient plant White encoding gene carries out inhibition expression, obtains genetically modified plants；Genetically modified plants blade compared with the recipient plant Angle smaller；
    The RLI1 albumen is following any shown protein：
    (A1) amino acid sequence is the protein of SEQ ID No.1；
    (A2) by substitution of the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing And/or it adds and there is the protein of identical function；
    (A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；
    (A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3).
  5. 5. method described in claim 3 or 4 it is following it is any in application：
    (1) plant variety suitable for dense planting is cultivated；
    (2) photosynthetic efficiency of plant population is improved；
    (3) plantation yield of the plant on unit area is improved.
  6. 6. a kind of method for the plant for cultivating leaf angle bigger, including make in recipient plant the expression quantity of RLI1 albumen and/or The step of activity improves；
    The RLI1 albumen is following any shown protein：
    (A1) amino acid sequence is the protein of SEQ ID No.1；
    (A2) by substitution of the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing And/or it adds and there is the protein of identical function；
    (A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；
    (A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3).
  7. 7. a kind of method for the genetically modified plants for cultivating leaf angle bigger, includes the following steps：Energy is imported into recipient plant The nucleic acid molecules of RLI1 albumen are enough expressed, obtain genetically modified plants；Genetically modified plants blade compared with the recipient plant Angle bigger；
    The RLI1 albumen is following any shown protein：
    (A1) amino acid sequence is the protein of SEQ ID No.1；
    (A2) by substitution of the amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues and/or missing And/or it adds and there is the protein of identical function；
    (A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；
    (A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3).
  8. 8. according to any application or method in claim 1-7, it is characterised in that：It is described to express the RLI1 eggs White nucleic acid molecules are the encoding gene of RLI1 albumen, are following any DNA moleculars：
    (B1) DNA molecular shown in SEQ ID No.2；
    (B2) DNA molecular limited under strict conditions with (B1) hybridizes and encodes the DNA molecular of the RLI1 albumen；
    (B3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (B1)-(B2) Or more than 80% homology and the coding RLI1 albumen DNA molecular.
  9. 9. the method according to claim 4 or 7, it is characterised in that：" to RLI1 in recipient plant described in claim 4 The encoding gene of albumen carries out inhibition expression " it is realized by CRISPER/Cas9 technologies；With DNA pieces shown in SEQID No.2 Meet 5 '-N in sectionX- NGG-3 ' or 5 '-CCN-NXThe segment of -3 ' series arrangements rule is target sequence；N is represented in A, G, C and T It is any, 14≤X≤30, and X be integer, NXRepresent X continuous deoxyribonucleotides；
    " nucleic acid molecules of the RLI1 albumen can be expressed by being imported into recipient plant " described in claim 7 is by institute State the recombinant expression carrier realization of encoding gene of the importing containing the RLI1 albumen in recipient plant.
  10. 10. according to any application or method in claim 1-9, it is characterised in that：The plant is dicotyledon Or monocotyledon；
    Specifically, the monocotyledon is grass；
    More specifically, the grass is rice.
CN201810015980.0A 2018-01-08 2018-01-08 Application of the RLI1 albumen in adjusting and controlling rice leaf angle Expired - Fee Related CN108048481B (en)

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CN110129358A (en) * 2019-05-17 2019-08-16 中国科学院植物研究所 The application of rice Os 01g32730 gene
CN112552383A (en) * 2020-12-07 2021-03-26 中国科学院遗传与发育生物学研究所 Application of transcription factor HINGE1 in regulation and control of plant nitrogen-phosphorus homeostasis
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Publication number Priority date Publication date Assignee Title
CN110129358A (en) * 2019-05-17 2019-08-16 中国科学院植物研究所 The application of rice Os 01g32730 gene
CN110129358B (en) * 2019-05-17 2021-12-17 中国科学院植物研究所 Application of rice Os01g32730 gene
CN112552383A (en) * 2020-12-07 2021-03-26 中国科学院遗传与发育生物学研究所 Application of transcription factor HINGE1 in regulation and control of plant nitrogen-phosphorus homeostasis
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CN112680474A (en) * 2021-01-19 2021-04-20 中国农业科学院作物科学研究所 Fluorescent-labeled CRISPR/SpCas9 system-mediated gene replacement system and application thereof in plants

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