CN108048481B - 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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CN108048481B
CN108048481B CN201810015980.0A CN201810015980A CN108048481B CN 108048481 B CN108048481 B CN 108048481B CN 201810015980 A CN201810015980 A CN 201810015980A CN 108048481 B CN108048481 B CN 108048481B
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rli1
plant
albumen
rice
seq
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CN108048481A (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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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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 regulation plant leaf blade angle；The RLI1 albumen is as shown in SEQ ID No.1, or substitution and/or deletion and/or addition and tool identical function by SEQ ID No.1 by amino acid residue, or have 80% or more homology and tool identical function with SEQ ID No.1, 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 knocking out with CRISPER/Cas9 technology to RLI1 gene 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 field of biotechnology, it is related to a kind of application of RLI1 albumen in adjusting and controlling rice leaf angle.
Background technique
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 being also main matter and the source of energy of human society.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 critical effect to yield, 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 of 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 or blade and leaf sheath of stem, blade angle and photosynthetic speed Rate has substantial connection, is important one of the 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 production and yield all have a significant impact.Even if leaf area is identical with single leaf photosynthetic rate, the light of vertical flag leaf group --- light Closing effect curves is in unsaturated type, and is hung down loosely or the biggish group of blade angle is then in 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 is the Main Agronomic Characters for influencing yield, upright leaf Piece is conducive to plant dense planting and is remarkably improved population photosynthesis efficiency, and then increases yield.
Summary 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, specially RLI1 albumen or its relevant biological material are in regulation 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 amino acid sequence shown in SEQ ID No.1 by one or several amino acid residues substitution and/or Deletion and/or addition and protein with the same function；
(A3) with (A1)-(A2) in it is any defined by amino acid sequence have 99% or more, 95% or more, 90% with Above, 85% or more or 80% or more homology and protein with the same function；
(A4) fusion obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (A1)-(A3) Albumen；
The relevant biological material is that can express the nucleic acid molecules of the RLI1 albumen, or contain the nucleic acid molecules Expression cassette, recombinant vector, recombinant bacterium or transgenic cell line.
In the application, the regulation embodies are as follows: 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 shown protein any 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 compared with the recipient plant is smaller.Wherein, the RLI1 albumen is any shown in (A1)-(A4) above Protein.
Method (or the smaller transgenosis of the cultivation leaf angle described previously for cultivating the smaller plant of leaf angle The method of plant) it is following it is any in application also belong to protection scope of the present invention:
(1) plant variety for being 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 of plant that cultivation leaf angle is bigger.
The method provided by the present invention for cultivating the bigger plant of leaf angle, it may include make RLI1 albumen in recipient plant Expression quantity and/or activity improve the step of.Wherein, the RLI1 albumen is shown protein any in (A1)-(A4) above.
Further, the present invention provides a kind of method of genetically modified plants that cultivation leaf angle is bigger, specifically may include Following steps: the nucleic acid molecules that can express the RLI1 albumen are imported into recipient plant, obtain genetically modified plants；Described turn Gene plant leaf angle compared with the recipient plant is bigger.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) hybridize under strict conditions with (B1) DNA molecular limited and encode the DNA molecular of the RLI1 albumen；
(B3) there is 99% or more, 95% or more, 90% or more, 85% with the DNA sequence dna of any restriction in (B1)-(B2) Above or the DNA molecular of 80% or more homology and the coding RLI1 albumen.
Above-mentioned stringent condition can for 6 × SSC, the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
It is described " in recipient plant in the previously described method for cultivating the 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 be realized this purpose, it is such as logical It crosses sequence specific nuclease (such as CRISPR/Cas9 nuclease) and specific cleavage is carried out to the encoding gene, 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 technology was 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 indicates that any one of A, G, C and T, 14≤X≤30, and X are 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 bigger genetically modified plants of previously described cultivation leaf angle Enter the encoding gene of RLI1 albumen " it can be realized by any technological means that can be realized 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 the carrier etc. that can be used for plant micropellet bombardment, as pCAMBIA-1300-221, pGreen0029, PCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivatives Plant expression vector.The plant expression vector also may include 3 ' end untranslated regions of foreign gene, that is, include 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.When 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 promoter (pUbi), stress induced promoter rd29A etc., they can be used alone or It is used in combination with other plant promoters；In addition, also can be used and increase when using gene constructed recombinant expression carrier of the invention Hadron, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region rises Beginning codon etc., but must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control The source of signal and initiation codon be it is extensive, can be natural, be also possible to 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, can produce the enzyme or light emitting compound of color change as the coding that can be expressed in plant is added The gene of object, resistant antibiotic marker or anti-chemical reagent marker gene etc..Any selectivity mark can also be not added Remember gene, transformed plant is directly screened with adverse circumstance.
In the present invention, start the promoter of the encoding gene transcription of the RLI1 albumen in the recombinant vector as 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 (XbaI and BamHI) resulting recombinant plasmid at the multiple cloning sites of carrier.
In the above-mentioned methods, it by the recombinant expression carrier for carrying the encoding gene of the RLI1 albumen or is used for The gene editing tool of " carrying out inhibition expression to the encoding gene of RLI1 albumen in recipient plant " Shi Caiyong imports the receptor Plant, concretely: by using Ti-plasmids, Ri plasmid, 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-mentioned application or method, the plant can be dicotyledon or monocotyledon.
Further, the monocotyledon can be gramineae plant.
More specifically, the gramineae plant is rice.In one embodiment of the invention, the rice is specially Rice varieties OryzasativaLcv.Nipponbare (Nipponbare).
In the present invention, the leaf angle is specially to 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 technology knocks out RLI1 gene 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.
Detailed description of the invention
Fig. 1 is to knock out RLI1 gene to improve rice leaf orthostatic.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 body；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 of falling for turning empty carrier plant are analyzed.It is aobvious It writes sex differernce and analyzes P < 0.05 *.
Fig. 2 is that overexpression RLI1 gene can increase rice leaf intersection angle.A:RLI1 gene overexpresses potting phenotypic analysis； OE-3, OE-7 and OE-10 are three different independent Transgenic Rice strains；NC-3 and NC-4 is two turns for turning empty carrier Gene strain.The Molecular Identification of B:RLI1 gene overexpression strain.C: OE-3, OE-7 and OE-10 are in basin for Transgenic Rice strain Under the conditions of cultivation, 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 plasmid and pYLCRISPR/Cas9P35S-H plasmid: it is obtained from Liu Yaoguang professor 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 carrier: it is obtained from Deng Xingwang professor 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 from applicant, can only be used to weight Duplicate invention experiment uses.
Embodiment 1, inhibit RLI1 gene expression can rice leaf angle become smaller
RLI1 gene involved in this embodiment is from rice (Oryza sativa L.), the sequence of RLI1 gene As shown in SEQ ID No.2, RLI1 albumen shown in SEQ ID No.1 is encoded.The present embodiment will use CRISPER/Cas9 skill Art knocks out rice RLI1 gene, and then studies its influence to rice plant leaf angle.It is specific as follows:
One, the selection of the target site of rice RLI1 gene
The chain in target double-strand knocked out using CRISPER/Cas9 technology is had the following structure: 5 '-CCN-NX- N in 3 ', PAM (NGG) indicates any one of A, T, C and G, and the N in Nx indicates any one of A, T, C and G, x=19. The target sequence of RLI1 gene 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 intracorporal 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, including the forms such as insertion, missing, narrow sense mutation, and most in these mutation is gene Functionally inactive mutation).
Two, the building of knockout carrier is recombinated
1, with restriction enzyme BsaI digestion pYLsgRNA-OsU3 plasmid, the carrier framework of about 3kb is recycled, is named as pYLsgRNA-OsU3-BasI。
2, according to the target site RLI1 sequence of design, synthesis has the primer of cohesive end (underscore part) as follows:
RLI1-1F:5 '-gccaCTTGATGTGGTAGATCGTC-3'；
RLI1-1R:5 '-aaacGACGATCTACCACATCAAG-3’。
3, 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 connection 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’。
4, with restriction enzyme BsaI digestion pYLCRISPR/Cas9P35S- H plasmid recycles the carrier framework of about 16kb, It is named as pYLCRISPR/Cas9P35S-H-BsaI。
5, by the pYLsgRNA-OsU3-RLI1-BasI segment that step 3 obtains and the carrier framework that step 4 obtains pYLCRISPR/Cas9P35S- H-BsaI is attached, and the recombinant plasmid after sequence verification is correct is named as pYLCRISPR/ Cas9-U3-RLI1。
Three, rice transformation
The recombinant plasmid pYLCRISPR/Cas9-U3-RLI1 of step 2 building is imported by the method for mediated by agriculture bacillus Rice varieties OryzasativaLcv.Nipponbare.Using OryzasativaLcv.Nipponbare callus as transformation receptor, complete regenerated plant is obtained by tissue cultures after conversion. It tests while being arranged and be transferred to pYLCRISPR/Cas9P into rice varieties OryzasativaLcv.Nipponbare35SThe control of-H empty carrier is (referred to as unloaded right According to).
Obtain the transgenic plant of RLI1 gene lacks functionality, the i.e. homozygous plants of the site RLI1 mutation.It extracts and turns Gene plant genomic DNA is carried out with genomic DNA of the primer pair conversion containing hygromycin 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 gene The missing of base, the final reading frame for changing RLI1 gene.Mutant rli1-2 lacking containing 2bp base in RLI1 gene It loses, the final reading frame for changing RLI1 gene.
Four, the phenotypic evaluation of rice mutant
With rice mutant rli1-1 and rli1-2 that step 3 obtains, and the wild rice plant without transgenosis (WT) and it is transferred to pYLCRISPR/Cas9P35SThe rice plant of-H plasmid empty carrier is experimental material.By each experimental material seedling After carry out potted plant experiment.In the Nutrition Soil of 10 -day-old of rice shoot insertion 10kg, statistical is carried out to leaf angle after plantation 45 days Analysis.
As a result as 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 more compact, upright blade, this would be more advantageous in dense planting, has and improves rice Population photosynthesis efficiency, improve the potential value of yield.In addition, the leaf angle and wild rice plant (WT) of unloaded control It is almost the same, no difference of science of statistics.
Embodiment 2, RLI1 gene overexpression can rice leaf angle become larger
RLI1 gene involved in this embodiment is from rice (Oryza sativa L.), the sequence of RLI1 gene As shown in SEQ ID No.2, RLI1 albumen shown in SEQ ID No.1 is encoded.The present embodiment will to RLI1 gene in rice into Row is overexpressed, and then studies its influence to rice plant leaf angle.It is specific as follows:
One, the building of over-express vector
In order to obtain the rice plant of RLI1 gene overexpression, inventor, which constructs, drives RLI1 gene with 35S promoter Expression vector (35S:RLI1), for rice plant convert.Using RLI1 gene shown in SEQ ID No.2 as template, both ends are used The primer RLI1-2F and RLI1-2R for being respectively provided with restriction enzyme site XbaI and BamHI clone RLI1 gene order, will clone Sequence be connected to the XbaI/BamHI restriction enzyme site after pF3PZPY122 carrier 35S promoter.The carrier inscribe built Enzyme XbaI and BamHI are detected, and the target fragment of about 1050bp can be cut out, and show that the carrier of building 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 site at underscore Identify sequence).
The structure of over-express vector 35S:RLI1 describes are as follows: by two restriction enzyme XbaI of pF3PZPY122 plasmid Segment (about 20bp) between the identification sequence of BamHI replaces with resulting recombination matter after DNA fragmentation shown in SEQ ID No.2 Grain.
Two, rice transformation
Using the callus of rice OryzasativaLcv.Nipponbare maturation embryonal induction as receptor, it is transferred to callus with the method that Agrobacterium EHA105 is mediated, Induce differentiation into seedling.Three plants are randomly selected from transgenic paddy rice obtained, are denoted as OE-3, OE-7 and OE-10 respectively.
Experiment simultaneously be arranged be transferred into rice varieties OryzasativaLcv.Nipponbare pF3PZPY122 empty carrier control (referred to as zero load it is right According to).
Three, to the Molecular Identification of transgenic paddy rice
Being Real-time PCR to transgenic paddy rice OE-3, OE-7 and OE-10 that step 2 obtains further confirms to turn base Because whether the expression of the RLI1 gene in rice significantly improves.Concrete operations are as follows:
Use the RNAeasy Plant Mini Kit (article No.: 74903) extraction wild type and step 2 of Qiagen company Transgenic paddy rice OE-3, OE-7 and OE-10 of acquisition, and the total serum IgE of unloaded control rice, take the total serum IgE of 1 μ g first to use 37 DEG C of digestion 30min of DNase, then in 20 μ l systems using TOYOBO RT kit to specifications reverse transcription at cDNA； CDNA is expanded with the SYBR Premix Ex Taq kit of TaKaRa, with Bio-Rad CFX96real-time PCR detection system The amplification amount of system real-time detection cDNA.Primer sequence for expanding RLI1 gene is 5 '-CGAACCAGTCCCAAATCCCA-3 ' It is 5 '-for expanding the primer sequence of internal reference Actin cDNA with 5 '-CGTCGAGCTGAACACGCAGT-3 ' CAACACCCCTGCTATGTACG-3 ' and 5 '-CATCACCAGAGTCCAACACAA-3 '.
As a result the expression quantity of RLI1 gene 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 gene in transgenic paddy rice OE-3, OE-7 and OE-10 of step 2 acquisition is certain It significantly improves.And expression quantity and wild rice plant (WT) basic one as RLI1 gene in the rice plant of zero load control It causes, no difference of science of statistics.
Four, the phenotypic evaluation of transgenic paddy rice
It is planted with transgenic paddy rice OE-3, OE-7 and OE-10 that step 2 obtains, and the wild rice without transgenosis Strain (WT) and the rice plant for being transferred to pF3PZPY122 empty carrier are experimental material.Each experimental material is subjected to potted plant experiment.? Transgenic seedling after greenhouse rice nursery 10 days moves 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 and Fig. 2 in Fig. 2.As it can be seen that compared with wild rice plant (WT), transgenic paddy rice OE- 3, 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 almost the same, 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
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His Phe Gly Ser Pro Leu Cys Asp Pro Ser Pro Ala Pro His Leu Leu
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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
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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
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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
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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 (24)

  1. The application of 1.RLI1 albumen or its relevant biological material in regulation plant leaf blade angle；
    The RLI1 albumen is the protein that amino acid sequence is SEQ ID No.1；
    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. application according to claim 1 or 2, it is characterised in that: the encoding gene of the RLI1 albumen is SEQ ID DNA molecular shown in No.2.
  4. 4. application according to claim 1 or 2, it is characterised in that: the plant is dicotyledon or monocotyledon.
  5. 5. application according to claim 4, it is characterised in that: the monocotyledon is gramineae plant.
  6. 6. application according to claim 5, it is characterised in that: the gramineae plant is rice.
  7. 7. 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 the protein that amino acid sequence is SEQ ID No.1.
  8. 8. a kind of method for cultivating the smaller genetically modified plants of leaf angle, includes the following steps: to RLI1 egg in recipient plant White encoding gene carries out inhibition expression, obtains genetically modified plants；Genetically modified plants blade compared with the recipient plant Angle is smaller；
    The RLI1 albumen is the protein that amino acid sequence is SEQ ID No.1.
  9. 9. according to the method described in claim 8, it is characterized by: the encoding gene of the RLI1 albumen is SEQ ID No.2 Shown in DNA molecular.
  10. 10. according to the method described in claim 8, it is characterized by: described " to the encoding gene of RLI1 albumen in recipient plant Carry out inhibition expression " it is to be realized by CRISPER/Cas9 technology；To meet 5 '-in DNA fragmentation shown in SEQ ID No.2 NX- NGG-3 ' or 5 '-CCN-NXThe segment of -3 ' series arrangements rule is target sequence；Any one of N expression A, G, C and T, 14≤ X≤30, and X is integer, NXIndicate X continuous deoxyribonucleotides.
  11. 11. method according to claim 7 or 8, it is characterised in that: the plant is that dicotyledon or unifacial leaf are planted Object.
  12. 12. according to the method for claim 11, it is characterised in that: the monocotyledon is gramineae plant.
  13. 13. according to the method for claim 12, it is characterised in that: the gramineae plant is rice.
  14. 14. method described in claim 7 or 8 it is following it is any in application:
    (1) plant variety for being 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.
  15. 15. application according to claim 14, it is characterised in that: the plant is dicotyledon or monocotyledon.
  16. 16. application according to claim 15, it is characterised in that: the monocotyledon is gramineae plant.
  17. 17. application according to claim 16, it is characterised in that: the gramineae plant is rice.
  18. 18. a kind of method for cultivating the bigger plant of leaf angle, including make in recipient plant the expression quantity of RLI1 albumen and/or The step of activity improves；
    The RLI1 albumen is the protein that amino acid sequence is SEQ ID No.1.
  19. 19. a kind of method for cultivating the bigger genetically modified plants of leaf angle, includes the following steps: to import energy into recipient plant The nucleic acid molecules for enough expressing RLI1 albumen, obtain genetically modified plants；Genetically modified plants blade compared with the recipient plant Angle is bigger；
    The RLI1 albumen is the protein that amino acid sequence is SEQ ID No.1.
  20. 20. according to the method for claim 19, it is characterised in that: the nucleic acid molecules that the RLI1 albumen can be expressed It is DNA molecular shown in SEQ ID No.2 for the encoding gene of RLI1 albumen.
  21. 21. according to the method for claim 19, it is characterised in that: described " to be imported into recipient plant described in expressing The nucleic acid molecules of RLI1 albumen " are the recombinations by importing the encoding gene containing the RLI1 albumen into the recipient plant What expression vector was realized.
  22. 22. method described in 8 or 19 according to claim 1, it is characterised in that: the plant is that dicotyledon or unifacial leaf are planted Object.
  23. 23. according to the method for claim 22, it is characterised in that: the monocotyledon is gramineae plant.
  24. 24. according to the method for claim 23, it is characterised in that: the gramineae plant 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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CN110129358B (en) * 2019-05-17 2021-12-17 中国科学院植物研究所 Application of rice Os01g32730 gene
CN112552383B (en) * 2020-12-07 2022-03-22 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
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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