CN106967160A - A kind of chlorophyll content of rice GAP-associated protein GAP OsWSL4 and its encoding gene and application - Google Patents
A kind of chlorophyll content of rice GAP-associated protein GAP OsWSL4 and its encoding gene and application Download PDFInfo
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- 235000019804 chlorophyll Nutrition 0.000 title claims abstract description 39
- 229930002875 chlorophylls Natural products 0.000 title claims abstract description 39
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 37
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- NSMUHPMZFPKNMZ-VBYMZDBQSA-M Chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 description 2
- 241000088885 Chlorops Species 0.000 description 2
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- 239000002253 acid Substances 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 229930002868 chlorophyll a Natural products 0.000 description 2
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- PCDQPRRSZKQHHS-XVFCMESISA-N ({[({[(2R,3S,4R,5R)-5-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}oxy)phosphonic acid Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-XVFCMESISA-N 0.000 description 1
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- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
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Classifications
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- 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
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of chlorophyll content of rice GAP-associated protein GAP OsWSL4 and its encoding gene and application.Disturb the DNA molecular shown in the present invention（Target gene is OsWSL4 genes）, obtainOsWSL4The transgenic rice plant of gene interference, transfer-gen plant shows as informal voucher line phenotype, chlorophyll content substantially reduce andOsWSL4Gene expression amount is lowered.First identified of the present invention and rice chloroplast content GAP-associated protein GAP OsWSL4, its encoding gene can cause the phenotype of paddy rice informal voucher line under conditions of function is lost or expression quantity declines, it was demonstrated that chlorophyll content of rice GAP-associated protein GAP OsWSL4 or its gene play a significant role in terms of chlorophyll content of rice is controlled.The present invention is not only that further Study On Rice chlorophyll content provides molecule mechanism basis, and provides for rice breeding new gene and breeding resources.
Description
Technical field
The present invention relates to gene engineering technology field, and in particular to chlorophyll content of rice GAP-associated protein GAP and its encoding gene
With application.
Background technology
Paddy rice solves the food problem of only about half of population in the world as one of important cereal crops.Normally
Photosynthesis is the premise for ensureing production, and chlorophyll content of rice related mutants are research photosynthesis, probe into photosynthetic utilization
Rate is to improve the ideal material of yield.Chloroplaset is semiautonomous organelle, possesses the genome of itself, and it is not only photosynthetic work
Place, is also the center of various metabolic activities and regulation and control, and there is complicated signal with nucleus and other organelles hands over
Stream.
PPR families are one of maximum families for finding in plant, there is 477 members in paddy rice, are one kind with 35 letters
And amino acid is the protein of recurring unit's continuous arrangement composition, these degeneracy amino acid repeated are referred to as PPR motifs.
This family is largely positioned in mitochondria or chloroplaset, influence plastid RNA montage, editor, processing and stability
Deng.Although coding PPR families largely exist in plant, the influence rice chloroplast gene RNA montage having now been found that
PPR albumen is but very limited.The PPR albumen for being positioned at chloroplaset found in paddy rice has OsPPR1, (Gothandam et
al., 2005)、YSA (Su et al., 2012)、OsV4 (Gong et al., 2014)、WSL (Tan et al.,
2014), ASL3 (Lin et al., 2015a) and OspTAC2 (Wang et al., 2016), wherein only WSL is reported
Road has participated in the RNA montage processes of chloroplast gene, and WSL is the albumen of a PSL subfamily, influences chloroplast generpl2
Montage（Tan et al., 2014）.Our WSL4 albumen has influence on chloroplaset II class introne genesatpF,ndhA, rpl2Withrps12RNA montage processes, this be for the first time report core coding P subfamilies PPR albumen in Rice Leaf
The RNA montages in many sites are participated in green body.
The content of the invention
It is an object of the invention to provide a kind of chlorophyll content of rice GAP-associated protein GAP OsWSL4 and its encoding gene and application.
Chlorophyll content of rice GAP-associated protein GAP provided by the present invention, entitled OsWSL4, from Oryza paddy rice
（Oryza sativa）Kind RX69 amino acid sequence, is following a）Or b）：
a）The protein of amino acid residue sequence composition as shown in SEQ ID NO. 1；
b）The substitution by one or several amino acid residues and/or missing by the amino acid residue sequence in SEQ ID NO. 1
And/or add and related to chlorophyll content of plant by a）Derivative protein.
Above-mentioned b）In one or several amino acid residues substitution and/or missing and/or be added to no more than 10 ammonia
The substitution and/or missing and/or addition of base acid residue.
Above-mentioned b）In protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
SEQ ID NO. 1 in above-mentioned sequence table are made up of 554 amino acid residues.
The DNA molecular for encoding the WSL4 albumen falls within protection scope of the present invention.
The DNA molecular is as follows（1）Or（2）Or（3）DNA molecular.
1）DNA molecular as shown in SEQ ID NO. 2；
2）Under strict conditions with 1）The DNA sequence dna hybridization of restriction and the DNA molecular related to chlorophyll content of plant；
3）With 1）Or 2）The DNA sequence dna of restriction at least have 70%, at least have 75%, at least have 80%, at least have 85%, extremely
Have 90% less, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least there is 99% homology
And the DNA molecular related to chlorophyll content of plant.
The stringent condition can be in 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS solution, it is miscellaneous at 65 DEG C
Hand over and wash film.
SEQ ID NO. 2 in above-mentioned sequence table are made up of 1665 nucleotides.
It is a further object to provide a kind of method for cultivating genetically modified plants, to suppress WSL4 in purpose plant
The expression of protein coding gene, obtains the genetically modified plants of chlorophyll content defect.
The genetically modified plants of chlorophyll content defect described above are embodied in the blade to form informal voucher line phenotype, Ye Lv
Cellulose content is abnormal.
Plant described above is specially dicotyledonous or monocotyledon, and the monocotyledon is specially further paddy rice.
Above-mentioned suppression is realized by being transferred to interference carrier in the purpose plant.Weight containing the encoding gene
Group carrier, recombinant bacterium or transgenic cell line belong to protection scope of the present invention.
The interference carrier is to insert specific DNA fragment 1 and specific DNA fragment 2 in binary expression vector, described
DNA fragmentation 1 is as shown in SEQ ID NO. 2 from the nucleotides of 5 ' end the 1162nd to 1493；The DNA fragmentation 2 and the DNA
The reverse complemental of fragment 1.
Above-mentioned expression vector is specially pCUbi1390- △ FAD2 carriers.
The primer pair for expanding the full length gene or its any fragment falls within the scope of protection of the invention.
Application of the albumen in terms of chlorophyll content is adjusted is also protection scope of the present invention.
The invention has the advantages that：
The experiment proves that, present invention clone obtainsWSL4Gene, missingWSL4There is informal voucher line phenotype in the plant of gene,
Chlorophyll content is reduced.Transgenosis complementation demonstrates the function of this gene, and Subcellular Localization has been also positioned in chloroplaset, it was demonstrated that
WSL4 albumen plays a significant role during early stage chlorophyll content.It therefore, it can the gene based on the application, initiative is different
The plant variety material of character, as the material of crop breeding, while the also mechanism for further research Chlorophyll synthesis process
And be used and provide the foundation.
Brief description of the drawings
Fig. 1 is pCUbi1390- △ FAD2 Vector maps.
Fig. 2 isOsWSL4Disturb the phenotype of plant.
Fig. 3 is the detection of interference plant gene transcription level and chlorophyll content measurement.
Fig. 4 is the complementary plant phenotype of transgenosis and chlorophyll content measurement, wherein a, wild type（WT）,wsl4Mutant,
Complementary plant（wsl4/WSL4）Phenotype during 3 leaf phase；B, wild type（WT）,wsl4Mutant, complementary plant（wsl4/WSL4）
Measuring chlorophyll content, average value is from three independent experimental datas.Chl a, chlorophyll a；Chl b, chlorophyll b；FW,
Fresh weight.
Fig. 5 is the Subcellular Localization of the albumen wsl4 after WSL4 albumen and mutation, wherein a, WSL4-GFP and wsl4-
Transient expressions of the GFP in rice protoplast；B, WSL4 and wsl4 (GFP) and chloroplaset nucleoid Marker PEND
（CFP）Common location.GFP, WSL4 and wsl4 GFP fluorescence signals, CFP, chloroplaset nucleoid Marker PEND CFP fluorescence letter
Number, Auto, chlorophyll autofluorescence, Merged, GFP, CFP and Auto fluorescence merges image.Bars, 5 μm.
Fig. 6 is the montage that WSL4 albumen participates in chloroplaset II type introne genes, and left side is Gene Name, and right side is to cut
Connect（S）With no montage（U）Transcript；RNA is extracted from wild type and mutant under the conditions of L30/D25（White portion）
L3-3, L3-3 represents the 3rd leaf of 3 leaf phases..
Embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment provided is only for more preferable
Illustrate the present invention, without limit the present invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, biochemical reagents used etc., unless otherwise specified, are commercially obtained in following embodiments.
Paddy rice in following embodiments（Oryza sativa ssp.Japonica）(also referred to as wild rice, simple by RX69
Claiming WT) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, and the biomaterial is only attached most importance to the correlation of duplicate invention
Experiment is used, can not be used as other purposes.
Agrobacterium is that Agrobacterium tumefaciems EHA105 (Agrobacterium tumefaciens EHA105) is recorded in New
Agrobacterium helper plasmids for gene transfer to plants.Hood,Elizabeth E;
Gelvin,Stanton B;Melchers,Leo S;Hoekema,Andre. Transgenic research,2(4):
p.208-218(1993).The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Embodiment 1,OsWSL4The acquisition of gene
According to ncbi database, design primer PPR-CDS-F and PPR-CDS-R
PPR-CDS-F: ATGGCCGCGCCCGCGCCCAC
PPR-CDS-R: TCAATCAAGCAGTCTAATGG
With the total extracts kit of RNAprep pure plants（Tiangeng biochemical technology Co., Ltd）, extract paddy rice RX69 10 days
The rice seedling total serum IgE of left and right, reversion obtains cDNA.As template, expanded with primer PPR-CDS-F and PPR-CDS-R
Increase, the obtained corresponding unnamed gene of PCR primer isOsWSL4, the code area of the gene is the nucleosides shown in SEQ ID NO. 2
Acid；The albumen of the gene code is named as OsWSL4, and amino acid sequence is sequence shown in SEQ ID NO. 1.
Embodiment 2,OsWSL4Rna interference vector structure
1、 OsWSL4The acquisition of gene interference fragment
（1）Using cDNA in example 1 as template, expanded with WSL4-RNAi-SacI-InF and WSL4-RNAi-SacI-InR primers
Increase, obtain fragment 1.
WSL4-RNAi-SacI-InF：CTAGGTACCAGGCCTGAGCTCGTTGAATTGCTTCATCAA
WSL4-RNAi-SacI-InR：GACGTAGGGGCGATAGAGCTTAAGCGAAAAGATCAATG
（2）Using cDNA in example 1 as template, with WSL4-RNAi- BamHI-InF and WSL4-RNAi- BamHI-InR primers
Expanded, obtain fragment 2.
WSL4-RNAi- BamHI -InF：TCTTAGAATTCCCGGGGATCCGTTGAATTGCTTCATCAA
WSL4-RNAi- BamHI -InR：CGTTACGTAGTCGACGGATCCTAAGCGAAAAGATCAAT
2、OsWSL4Gene RNA interference carrier（Recombinant vector pCUbi1390- △ FAD2-OsWSL4）Structure
(1) restriction enzyme is usedSacI digestion expression vector pCUbi1390- △ FAD2, obtain linear expression vector, and reclaiming should
Linear fragment.Fragment 1 is recombinated onto the linear expression vector using the method for homologous recombination directed cloning（Specific method is referred to
Clontech infusion kit specifications）, obtain recombinant vector pCUbi1390- △ FAD2-sense-OsWSL4。
（2）To recombinant vector pCUbi1390- △ FAD2-sense-OsWSL4It is sequenced, obtains correctly plasmid, then
Use restriction enzymeBamHICorrectly recombinant vector pCUbi1390- △ FAD2-sense- are sequenced in digestionOsWSL4, obtain
Linear carrier, reclaims the linear carrier.Fragment 2 is recombinated onto the linear carrier using the method for homologous recombination directed cloning
（Specific method refers to clontech infusion kit specifications）, obtain recombinant vector pCUbi1390- △ FAD2-OsWSL4。
（3）To recombinant vector pCUbi1390- △ FAD2-OsWSL4It is sequenced, as a result shows the recombinant vector
pCUbi1390-△FAD2- OsWSL4BeSacI restriction enzyme site forward directions insert fragment 1,BamHIRestriction enzyme site is inserted
Fragment 2, that is, successfully construct, and is named as pCUbi1390- △ FAD2-OsWSL4。
Embodiment 3, RNA disturb the acquisition and identification of plant
First, RNA disturbs the acquisition of plant
1st, by recombinant vector pCUbi1390- △ FAD2-OsWSL4It is transferred in Agrobacterium tumefaciems EHA105 and is contained with heat shock method
The Agrobacterium tumefaciems EHA105 of recombinant vector, is named as EHA105/pCUbi1390- △ FAD2-OsWSL4。
2nd, recombinant vector EHA105/pCUbi1390- △ FAD2- will be containedOsWSL4Agrobacterium infect wild rice
RX69 embryo callus.By co-culturing, subculture, a series of processes of differentiation, when plant to be planted is highly about 15cm on the ground,
Abroach experience and tempering 1 day, then wash culture medium, transplant to greenhouse production, as T0For plant.T0Harvest and plant for plant selfing
Son is simultaneously planted, the T stablized1For plant.
2nd, the PCR identifications of RNAi interference of transgene plant
By the T of above-mentioned acquisition1For seedling and the seedling of receptor parent paddy rice RX69 plant（Referred to as WT）Genomic DNA, and
Using primer 1390-F（5’-TGCCTTCATACGCT ATTTATTTGC-3’）With primers F AD2-R（5’-
TGCCTTCATACGCTATTTATTTGC-3’）Enter the positive seedling of performing PCR Molecular Detection identification, obtain containing fragment 1PCR products
Plant is positive seedling, takes three plants of positive seedlings, is respectively designated as RNAi-1, RNAi-2, RNAi-3 plant.
3rd, RNAi interference of transgene plantOsWSL4The identification of gene expression dose
RNAi-1 plant, RNAi-2 plant, RNAi-3 plant and receptor parent paddy rice RX69 plant are extracted respectively（Referred to as WT）
The RNA of blade, sets internal reference as Ubiquitin, using internal control primer UBI-F and UBI-R, andOsWSL4Gene specific is determined
Measure primer WSL4-RT-F and WSL4-RT- R and carry out quantitative fluorescent PCR reaction.As a result show（Fig. 1）, with wild type control phase
Than in RNAi interference of transgene plantOsWSL4Gene expression dose is remarkably decreased.Above-mentioned primer is as follows：
UBI-F：5’-GCTCCGTGGCGGTATCAT -3’
UBI-R：5’-CGGCAGTTGACAGCCCTAG-3’
WSL4-RT-F：5’- GCCCTCCAAATGTGGTAACT -3’
WSL4-RT-R: 5’- GCCATCGCTTTATCCATCTT -3’
4th, the phenotypic evaluation of RNAi interference of transgene plant
Respectively by RNAi-1 plant, RNAi-2 plant, RNAi-3 plant and receptor parent paddy rice RX69 plant（Referred to as WT）Kind
It is implanted in incubator（30 °C of illumination 12h, 25 °C of dark 12h circulations, humidity 60%）, in tri-leaf period to be grown to, observation leaf color phenotype is
It is no variant.Observe result such as Fig. 2, compared with receptor parent paddy rice RX69 plant, RNAi-1 plant, RNAi-2 plant with
There is the phenotype of informal voucher line in RNAi-3 plant,OsWSL4Gene expression dose and chlorophyll content are substantially reduced（Fig. 3）, so that
Demonstrating OsWSL4 influences the content of chlorophyll, disturbs its gene, blade occurs in that the phenotype of informal voucher line and leaf pigment is obvious
Reduction.
Embodiment 4, the structure of transgenosis complementing vector and identification
1st, the structure of complementing vector
According to ncbi database, design primer PPR-G-F and PPR-G-R
PPR-G-F: CCATGATTACGAATTCATGGCGTATCCTCCTATCGTTGC
PPR-G-R: TACCGAGCTCGAATTCGCCGACTTCCGACCGAACAT
Extract paddy rice RX69 DNA.As template, PrimeSTAR HS DNA are used with primer PPR-G-F and PPR-G-F
Polymerase（TakaRa）Expanded, obtain the fragment of a 5.7 kb sizes, include 2.2 kb upstream promoter region sequence,WSL4Complete code area and the kb of downstream 2 termination region sequence.Product is run after glue reclaim, In-Fusion (Clontech)
Homologous recombination is connected into pCAMBIA1305 carriers, obtains complementing vector pCAMBIA1305-GWSL4.
2nd, the identification of transgenosis complementing vector
Complementing vector pCAMBIA1305-GWSL4 is transferred towsl4In mutant callus, by co-culturing, subculture, the one of differentiation is
Row process, when plant to be planted is highly about 15cm on the ground, then experience and tempering 1 day of abroaching wash culture medium, transplant to greenhouse and plant
Training, as T0For plant.T0Seed is harvested for plant selfing and is planted, the T stablized1For plant.By the T of above-mentioned acquisition1Generation
Seedling, mutantwsl4The genomic DNA of the seedling of plant（Negative control）, complementing vector pCAMBIA1305-GWSL4 plasmids
（Positive control）And using primer GWSL4-F（5'TAGGCACCCCAGGCTTTACACT 3'）With primer GWSL4-R（5'
GTAACGTAGGACAAAACC3'）Enter the positive seedling of performing PCR Molecular Detection identification, can amplify 692bp fragments for positive seedling,
It is named aswsl4/WSL4Plant.6 transgenic positive strains are obtained altogether, and transgenic positive strain has recovered the table of wild type
Type, while chlorophyll a and chlorophyll b have all recovered the level of wild type,WSL4The expression of gene also almost maintains an equal level with wild type
（Fig. 4）.Result above showswsl4The phenotype of mutant informal voucher line is due toOsWSL4（LOC_Os02g35750）Mutation is caused
's.
The Subcellular Localization of embodiment 5, WSL4 albumen
In order to understand the Subcellular Localization situation of WSL4 albumen, we use ChloroP (http first://www.cbs.dtu.dk/
) and TargetP services/ChloroP/（http://www.cbs.dtu.dk/services/TargetP/）To WSL4 eggs
It is predicted in vain, it is found that the N-terminal of WSL4 albumen has chloroplast transit peptides（CTP）, addwsl4The table of chlorophyll deficiency
Type, we guess that WSL4 may be positioned in chloroplaset.Whether the albumen after true positioning and mutation in order to determine WSL4 changes
Become positioning, we using wild type and mutants cDNA as template, amplify coding respectivelyWSL4Full length sequence andwsl4After mutation
Sequence（Remove terminator）, it is connected on carrier pAN580, is respectively designated as pAN580-WSL4-GFP and pAN580-wsl4-
GFP is simultaneously transformed into rice protoplast, and 28 DEG C of dark culturings are taken pictures after 16 hours with laser confocal microscope, are as a result shown
Show, WSL4-GFP and wsl4-GFP all with chlorophyll autofluorescence（For chloroplaset Marker）Common location（Fig. 5 a）, further
It was found that WSL4 and wsl4 and chloroplaset nucleoid Marker PEND（CFP）Common location（Fig. 5 b）, summary result illustrates WSL4
Albumen is positioned in chloroplaset and does not change positioning with the albumen after chloroplaset nucleoid common location, and mutation, also fixed
In chloroplaset nucleoid.
Embodiment 6, RNA montages analysis
Rice chloroplast introne has two types, respectively I types and II types.I types only include genetrnL, II types includeatpF、ndhA、ndhB、petB、petD、rpl2、rpl16、rps12、rps16、trnA、trnG、trnK、trnI、trnVWithycf3, whereinrps12、ycf3There are two intrones (Michel et al., 1989; de Longevialle., 2010).
We have detected the chloroplast gene of 17 II type intrones（atpF、ndhA、ndhB、petB、petD、rpl2、rpl16、rps12-1、rps16、trnA、trnG、trnK、trnI、trnV、ycf3-1、ycf3-2）With the chloroplaset of a Group I Introns
GenetrnL, the CDS of all splice site genes in wild type and mutant is amplified with PCR method, Ago-Gel is used
The situation of electrophoresis detection transcript is simultaneously taken pictures, and finds II type intronesatpF、ndhA、rpl2、rps12-1Wild type in gene
Had differences with mutant transcript（Fig. 6）.So OsWSL4 may beatpF、ndhA、rpl2、rps12-1It is green Deng four leaves
Played an important role in the montage of body II class introne genes.
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>A kind of chlorophyll content of rice GAP-associated protein GAP OsWSL4 and its encoding gene and application
<130> 1
<160> 2
<210> 1
<211> 554
<212>Amino acid
<400> 1
MAAPAPTASPPPPPAMSGLLSFASSRPYPPLPAPRPAAAAPRPRLRIAGSAAAAPNAVSHRASSSFSSGDRLR
SLVRRGELDEALRLVGSARRPDAGTCAALIKKLSASGRTAEARRVLAACGPDVMAYNAMVAGYCGAGQLDAARRLVA
EMPVEPDAYTYNTLIRGLCGRGRTANALAVLDEMLRRRCVPDVVTYTILLEATCKRSGYKQAMKLLDEMRDKGCTPD
IVTYNVVVNGICQEGRVDDAIEFLKNLPSYGCEPNTVSYNIVLKGLCTAERWEDAEELMGEMGQKGCPPNVVTFNML
ISFLCRKGLVEPALEVLEQIPKYGCTPNSLSYNPLLHAFCKQKKMDKAMAFLDLMVSRGCYPDIVSYNTLLTALCRS
GEVDVAVELLHQLKDKGCAPVLISYNTVIDGLTKAGKTKEALELLNEMVSKGLQPDIITYSTIAAGLCREDRIEDAI
RAFGKVQDMGIRPNTVLYNAIILGLCKRRETHSAIDLFAYMIGNGCMPNESTYTILIEGLAYEGLIKEARDLLDELC
SRGVVRKSLINKGAIRLLD
<210> 2
<211> 1665
<212> DNA
<400> 2
ATGGCCGCGC CCGCGCCCAC AGCTTCGCCG CCGCCGCCGC CCGCCATGTC CGGCCTCCTC 60
TCCTTCGCCT CGTCCCGCCC CTACCCTCCC CTCCCCGCGC CCAGACCCGC CGCCGCCGCC 120
CCCAGGCCGC GCCTCCGCAT AGCGGGTTCC GCCGCCGCGG CCCCCAACGC CGTGTCCCAC 180
CGGGCATCCT CCTCCTTCTC CTCCGGAGAC CGCCTCCGCT CGCTCGTCCG CCGCGGGGAG 240
CTCGACGAGG CGCTCCGCCT CGTCGGGTCC GCGCGGAGGC CCGACGCGGG CACCTGCGCC 300
GCGCTCATCA AGAAGCTCAG CGCGTCGGGG CGCACCGCGG AGGCCCGCCG CGTGCTGGCC 360
GCGTGCGGCC CCGACGTCAT GGCGTACAAC GCCATGGTGG CCGGGTACTG CGGCGCGGGG 420
CAGCTCGACG CCGCGCGGCG GCTCGTCGCG GAGATGCCCG TGGAGCCCGA CGCCTACACC 480
TACAACACGC TCATCCGAGG CCTCTGCGGA CGCGGCCGGA CCGCGAACGC GCTGGCGGTG 540
CTCGACGAAA TGCTCCGCCG GCGATGTGTG CCGGACGTGG TCACCTACAC CATCCTCCTC 600
GAGGCCACGT GCAAGAGGAG CGGGTACAAG CAGGCCATGA AGCTTCTCGA CGAGATGCGA 660
GATAAGGGGT GCACCCCGGA CATTGTCACC TACAACGTCG TTGTCAATGG AATCTGCCAA 720
GAAGGACGGG TTGACGATGC CATTGAGTTC TTGAAGAACC TCCCGTCATA TGGCTGTGAA 780
CCAAACACAG TTAGCTATAA CATTGTGTTG AAGGGCCTGT GTACCGCGGA GCGATGGGAG 840
GATGCCGAGG AGCTCATGGG CGAGATGGGC CAGAAGGGTT GCCCTCCAAA TGTGGTAACT 900
TTTAACATGC TCATCAGTTT CTTATGCCGG AAAGGATTGG TTGAGCCAGC ACTGGAAGTC 960
CTTGAGCAGA TCCCCAAGTA CGGTTGCACA CCTAATTCGT TGAGTTACAA CCCGCTTCTC 1020
CACGCATTCT GCAAGCAAAA AAAGATGGAT AAAGCGATGG CGTTTTTGGA TTTGATGGTG 1080
TCCAGAGGCT GTTATCCAGA CATCGTTTCA TACAACACTC TACTTACTGC ACTCTGCCGC 1140
AGTGGTGAGG TTGACGTCGC TGTTGAATTG CTTCATCAAC TCAAGGACAA GGGCTGCGCT 1200
CCTGTCTTGA TAAGTTATAA CACGGTCATT GATGGGCTTA CGAAGGCTGG GAAGACAAAG 1260
GAAGCACTAG AGCTGCTCAA TGAGATGGTC AGCAAAGGGC TCCAACCAGA TATTATCACA 1320
TACTCGACAA TAGCTGCTGG TCTTTGTAGA GAAGATAGAA TTGAAGATGC AATTAGGGCA 1380
TTTGGTAAAG TGCAAGATAT GGGCATAAGG CCCAACACTG TGTTGTACAA TGCGATAATT 1440
CTTGGGCTTT GCAAAAGGCG TGAAACACAC AGTGCCATTG ATCTTTTCGC TTACATGATA 1500
GGGAATGGCT GCATGCCGAA TGAATCAACT TACACCATAT TGATTGAAGG CTTGGCTTAT 1560
GAGGGATTGA TCAAGGAGGC ACGGGATTTG CTGGATGAAT TATGTTCGAG AGGTGTTGTA 1620
AGGAAGAGCT TGATAAATAA GGGAGCCATT AGACTGCTTG ATTGA 1665
Claims (10)
1. a kind of chlorophyll content of rice GAP-associated protein GAP, it is characterised in that it is：
a）The protein that the amino acid residue sequence as shown in SEQ ID NO. 1 is constituted；Or
b）The substitution by one or several amino acid residues and/or missing by the amino acid residue sequence in SEQ ID NO. 1
And/or addition obtained by derivative protein, its still have a) shown in protein activity.
2. the encoding gene of chlorophyll content of rice GAP-associated protein GAP as claimed in claim 1.
3. the encoding gene of chlorophyll content of rice GAP-associated protein GAP as claimed in claim 2, it is as follows（1）Or（2）DNA
Molecule：
DNA molecular as shown in SEQ ID NO. 2；
Under strict conditions with 1）The DNA sequence dna hybridization of restriction and the DNA molecular related to chlorophyll content of plant；
The stringent condition can be in 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS solution, to hybridize simultaneously at 65 DEG C
Wash film.
4. recombinant vector, recombinant bacterium or transgenic cell line containing encoding gene as claimed in claim 2 or claim 3.
5. recombinant vector as claimed in claim 3, it is interference carrier, or plant expression vector.
6. a kind of method for cultivating genetically modified plants, to suppress in expression or overexpression purpose plant as in Claims 2 or 3
Encoding gene, obtain chlorophyll content change genetically modified plants.
7. method as claimed in claim 5, it is characterised in that：The suppression expression causes genetically modified plants chlorophyll content to lack
Fall into by importing interference carrier in genetically modified plants, its phenotype is the blade of informal voucher line phenotype；Gene is described in overexpression
Realized by being transferred to plant expression vector.
8. the method as described in claim 5 or 6, it is characterised in that：The plant is specially dicotyledonous or monocotyledon, institute
It is specially further paddy rice to state monocotyledon.
9. the abnormal method of the chlorophyll of a kind of plant identification, it is characterised in that it comprises the steps：
(1) selection phenotype has the plant sample of informal voucher line；
(2) its chloroplast DNA is extracted；
(3) related gene fragment is obtained by Molecular Detection such as PCR amplifications, and be sequenced, to judge that its rice chlorophyll contains
Measure related protein encoding gene and whether there is defect, to judge that its chlorophyll is abnormal whether caused by the gene mutation.
10. the abnormal method of the chlorophyll of plant identification as claimed in claim 8, it is characterised in that its primer is to being：
PPR-CDS-F: ATGGCCGCGCCCGCGCCCAC
PPR-CDS-R: TCAATCAAGCAGTCTAATGG。
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| CN104593395A (en) * | 2014-12-26 | 2015-05-06 | 中国水稻研究所 | Gene YWL1 for controlling rice leaf color at low temperature and application of gene YWL1 |
| CN105566463A (en) * | 2014-10-13 | 2016-05-11 | 中国科学院植物研究所 | Chlorophyll synthesis related protein, coding gene and application thereof |
| CN105820221A (en) * | 2015-12-14 | 2016-08-03 | 中国水稻研究所 | Rice leaf color regulatory protein and coding gene thereof, and application thereof |
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| CN105566463A (en) * | 2014-10-13 | 2016-05-11 | 中国科学院植物研究所 | Chlorophyll synthesis related protein, coding gene and application thereof |
| CN104593395A (en) * | 2014-12-26 | 2015-05-06 | 中国水稻研究所 | Gene YWL1 for controlling rice leaf color at low temperature and application of gene YWL1 |
| CN105820221A (en) * | 2015-12-14 | 2016-08-03 | 中国水稻研究所 | Rice leaf color regulatory protein and coding gene thereof, and application thereof |
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| 佚名: "GENEBANK登录号：AK068847.1", 《GENEBANK》 * |
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