CN105153289A - Protein for controlling color of rice leaves and coding gene and application of protein - Google Patents

Protein for controlling color of rice leaves and coding gene and application of protein Download PDF

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CN105153289A
CN105153289A CN201510728279.XA CN201510728279A CN105153289A CN 105153289 A CN105153289 A CN 105153289A CN 201510728279 A CN201510728279 A CN 201510728279A CN 105153289 A CN105153289 A CN 105153289A
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严长杰
郭旻
杨海莲
刘敏
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Yangzhou University
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Abstract

The invention belongs to the technical field of plant molecular biology and particularly relates to protein for controlling the color of rice leaves and a coding gene and application of the protein. The sequence of the rice ZL9 protein for controlling the color of the rice leaves is shown in SEQ ID No.2. The invention further discloses a gene of the rice ZL9 and an application of the gene in preparing a transgenic cell system and transgenic plant with the deepened color of the leaves and improved photosynthetic efficiency. The gene ZL9 is obtained through a map-based cloning strategy. The expression quantity of the gene is changed with a gene engineering method, and the new germ plasm of rice zebra leaves can be cultured and serve as the marker characteristic in crossbreeding; a new material with the chlorophyll content larger than that of a wild type material can be cultured, and photosynthetic efficiency is improved. Thus, the gene has high application value in the aspect of high-yield rice culture.

Description

A kind of control rice leaf color albumen and encoding gene and application
Technical field
The invention belongs to molecular biology of plants technical field, be specifically related to the albumen controlling rice leaf color and encoding gene thereof and application.
Background technology
Blade is the topmost photosynthesis organ of paddy rice.Leaf photosynthesis power directly can affect the height of rice yield.Zebra pallette variant (zebraleaf9, zl9) is screened in the mutant library that this research spends 11 to be background in japonica rice.Adopt map based cloning strategy, cloned ZL9 gene, obtained the encoding sequence of this gene.The clone of this gene and functional analysis are reported first in paddy rice, grow and chlorophyll metabolism regulatory mechanism and then be used to guide breeding and established important foundation for people understand rice chloroplast.
The annotation of gene LOC-Os09g28480 on NCBI website is: conservative hypothetical protein, by paddy rice full-length genome database search, shows that ZL9 gene is single copy gene.Gene order total length 4578bp, cDNA are 618bp, 205 amino acid of encoding.The homologous gene CYO1 of this gene in Arabidopis thaliana encodes a disulfide bond isomerase, and it has C4 type Zinc finger domain, and with intestinal bacteria DnaJ structural similitude, CYO1 protein localization is on Thylakoid membrane.CYO1 may be from GSH (glutathione in cotyledon Development of Chloroplasts process, gsh) receive electronics, and accelerating containing the PSI subunit (A1, A2 and psaK) of halfcystine and folding of PSII subunit (CP43 and CP47) by constantly open and close disulfide linkage, is that the thylakoid membrane biosynthesizing in cotyledon is necessary.
The homologous protein of ZL9 gene protein and jowar, corn, false bromegrass, barley, grape, castor-oil plant, cucumber, peach, soybean, tomato, Arabidopis thaliana, strawberry and clover is carried out amino acid alignment analysis, result shows, different this biological gene has very strong conservative property, and ZL9 and monocotyledons have higher homology.86.7%, 84.2%, 80.6% and 78.2% is respectively with the similarity of jowar, corn, false bromegrass, barley, lower with the similarity of dicotyledons, only there is 45%-54%, be respectively 51.5%, 53.9% and 46.1% with the similarity of Arabidopis thaliana, tomato, cucumber.The Phylogenetic tree analysis that DNAMAN software maximum likelihood number method is carried out also shows: ZL9 gene be both the Chinese sorghum of grass, corn has closer sibship than other biological.
Summary of the invention
The object of this invention is to provide a kind of paddy rice ZL9 albumen and encoding gene thereof and application.
Paddy rice ZL9 provided by the invention, derives from paddy rice, is following 1) or 2) protein:
1) protein be made up of the amino acid residue sequence of the SEQID № .2 in sequence table;
2) the SEQID № .2 amino acid residue sequence in sequence table had the protein of the identical activity of amino acid residue sequence of SEQID № .2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
In sequence table, sequence 2 is made up of 205 amino-acid residues.
For the ease of the purifying of ZL9, the aminoterminal of the protein that can form at the amino acid residue sequence by sequence 2 or carboxyl terminal connect label as shown in table 1.
The sequence of table 1 label
Label Residue Sequence
Poly-Arg 5-6 (being generally 5) RRRRR
Poly-His 2-10 (being generally 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
Above-mentioned 2) ZL9 in can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned 2) encoding gene of the ZL9 in is by lacking the codon of one or several amino-acid residue in the DNA sequence dna that sequence in sequence table 1 shown, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.
The gene of above-mentioned paddy rice ZL9 of encoding also belongs to protection scope of the present invention.
Described paddy rice ZL9 albumen cDNA gene can be following 1) or 2) or 3) or 4) DNA molecular:
1) DNA sequence dna of SEQ ID № .1;
2) polynucleotide of SEQID № .2 protein sequence in polynucleotide;
3) DNA sequence dna limited with SEQ ID № .1 has more than 90% homology, and coding identical function protein DNA sequence;
4) nucleotide sequence that the DNA sequence dna that can limit with sequence in sequence table 1 is under strict conditions hybridized.
Sequence 1 in sequence table is by 618 based compositions, and its open reading frame (ORF) is from 5 ' end 1-618 position Nucleotide.
Above-mentioned stringent condition can be at 0.1 × SSPE (or 0.1 × SSC), in the solution of 0.1%SDS, hybridizes and wash film under 65 DEG C of conditions.
Recombinant vectors containing above arbitrary described gene also belongs to protection scope of the present invention, as recombinant expression vector.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.
Described plant expression vector comprises double base agrobacterium vector (as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.
When using described gene constructed recombinant plant expression vector, any one enhancement type promotor, constitutive promoter or inducible promoter can be added before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin), stress induced promoter Rd29A etc. of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can be used alone or are combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.
For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. of colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.
Expression cassette containing above arbitrary described gene (ZL9), transgenic cell line and recombinant bacterium all belong to protection scope of the present invention.
The amplification total length of said gene or the primer pair of arbitrary fragment also belong within protection scope of the present invention.
Any one in described albumen, described gene, described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium all can be applicable to the paddy rice cultivating " zebra leaf " phenotype.
The carrier utilizing any one can guide foreign gene to express in plant, by the gene transfered plant cell of encoding said proteins, can obtain leaf look and deepen, the transgenic cell line that photosynthetic efficiency improves and transfer-gen plant.The plant tissue of conversion by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, conventional biology methods transformed plant cells or the tissue such as agriculture bacillus mediated, and is cultivated into plant by the expression vector carrying described gene.
The present invention screens a paddy rice zebra pallette variant zl9 in mutant library, this mutant material source in spend 11, transplant under hindering root condition, the 2-3 newly grown opens blade and is interrupted chlorosis, namely horizontal " zebra-stripe "; When not transplanting, mutant does not have mutant phenotype to occur.The present invention utilizes mutant zl9 and Nanjing 11 to hybridize F 2in generation, by ZL9 gene Primary Location on Chromosome 9; Further expansion colony, navigates to a LOC-Os09g2848 gene.It is a conservative hypothetical protein, NCBI website is explained without other, by paddy rice full-length genome database search, shows that ZL9 gene is single copy gene.After gene clone, not only to the growth of understanding rice chloroplast and chlorophyll metabolism regulatory mechanism, there is important theory value, meanwhile, also can explore and directly be applied to rice breeding.Present invention obtains paddy rice ZL9 gene, utilize gene engineering method to change the expression amount of this gene, both may be used for cultivating paddy rice zebra leaf new germ plasm, as mark property in cross-breeding; The novel material of chlorophyll content higher than wild-type can be cultivated again, improve photosynthetic efficiency.
Accompanying drawing explanation
The phenotype of Fig. 1 zebra pallette variant.Mutant phenotype in seedling stage is normal, transplant rear blade interruption and turn white, and total number of mutant also reduces to some extent.
Fig. 2 gene clone.The gene of control zl9 mutant character may just in the 172kb fragment of disappearance.
The expression analysis of Fig. 3 candidate gene.The expression amount of gene LOCOs09g28480 first reduces, after slowly raise again, present regular change.
Fig. 4 RNA interferes transfer-gen plant phenotype.The leaf morphology of transfer-gen plant is similar to mutant, and the blade newly grown is zebra leaf.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is mass percentage
Biological material source designed by the present invention is as follows:
Spend 11 in japonica rice, long-grained nonglutinous rice Nanjing 11: research of agricultural science institute of Jiangsu Province
Carrier pCAMBIA1301: open material, this carrier is purchased from Takara company
Carrier p-MD18-T: open material, this carrier is purchased from Takara company
Carrier p1022: open material, this carrier is purchased from Takara company
Bacillus coli DH 5 alpha: open material, this carrier is purchased from Generay company
Agrobacterium EHA105: open material, this carrier is purchased from Takara company
Embodiment 1: the phenotype of mutant zl9 and genetic analysis
1. the phenotype analytical of mutant zl9
Obtain a paddy rice leaf color mutant by radiation, this mutant is compared with WT lines, and mutant phenotype in seedling stage is identical, after transplanting, several the blades interruptions newly grown are turned white, and show as " zebra-stripe ", and leaf sheath also turns white, the leaf morphology grown again is afterwards normal.After heading, zebra leaf hickie starts to turn green gradually, and the hickie of the mutant zebra leaf at heading stage has not had tillering phase obvious, and the hickie of the mutant zebra leaf being in the milk early stage turns green completely.To the ripening stage, except the leaf sheath of zl9 mutant turns white and setting percentage is on the low side, mutant is substantially identical with the phenotype of wild-type.Except leaf look changes, the Other Main Agronomic Characters of mutant: plant height, examples explain, total grain number, Primary branch number, Secondary branch number, setting percentage all significantly or extremely significantly reduce; And panicle number per hill, spike length, flat grain number and thousand seed weight are all without significant difference.When the examples explain of mutant extremely significantly reduces, the reduction of total grain digital display work is due to spike length indifference, Primary branch digital display work reduces and Secondary branch number extremely significantly reduces and causes.Visible, economic yield and the biological yield of mutant decline all to some extent.As can be seen here, although zl9 mutant under normal operation, only have 2-3 to open often being off color of blade, show interruption chlorosis, it but creates larger impact (Fig. 1) to the growth of plant.
2. the genetic analysis of mutant zl9
With mutant zl9 for female parent, 11 preparing hybrids are spent to combine respectively with rice variety Nanjing 11 and japonica rice variety.Its hybrid F 1all show as leaf look normal.F 1by selfing, obtain corresponding F 2colony, all isolates normal leaf and zebra leaf plant two type, occurs there are no intermediate type individuality.For carrying out the F of genetic analysis 2totally 536 strains of (spending 11 in zl9/) colony, wherein identical with mutation type surface have 129 strains, χ 2=0.6944< χ 2 0.05,1=3.84, meet the theoretical segregation ratio (3:1) of Mendelian individual gene.Another one F 2totally 400 strains of (zl9/ Nanjing 11) colony, wherein identical with mutation type surface have 87 strains, carries out χ 2test, χ 2=2.0833< χ 2 0.05,1=3.84, its ratio also meets Mendelian theoretical segregation ratio (3:1).As can be seen here, this mutant character controls by single recessive nuclear gene.
The acquisition of embodiment 2:ZL9 albumen and encoding gene ZL9 thereof
Utilize 236 SSR marker to carry out polymorphism analysis to mutant zl9 and Nanjing 11, that wherein discloses two parent's polymorphisms is marked with 53, applies these 53 marks and hybridizes to zl9 and Nanjing 11 F produced 2in colony, the microcommunity of 10 strain typical case zebra leaf individual plant compositions makes linkage analysis, found that the SSR marker RM410 on Chromosome 9 shows obvious association between two ponds.Utilize further on Chromosome 9 and between two parents, show polymorphic mark to F 222 strain zebra leaf mutant plants of colony are analyzed, and result shows ZL9 gene and RM410 close linkage, determines that this gene is positioned on Chromosome 9.In order to further Fine Mapping ZL9 gene, we have developed 52 STS marks on the both sides of RM410, these marks are carried out polymorphism analysis to parent, and have 38 and can expand single slice, wherein 11 show polymorphism between parent.And then utilize these 11 to mark F 2in (zl9/ Nanjing 11) colony, totally 161 strain zebra pallettes become individual plant and carry out genotype detection and linkage analysis, pac clone AP5419 detects that 11 restructuring exchange in strain to AP6057 above expectation gene ZL9 position and be then gradually reduced to 3 exchange strains, below is that 3 exchange strains reduce to 2 exchange individual plants from AP5555 to AP5399, on AP5676 and AP5399, 161 strains all do not find to exchange strain, wherein estimating that the exchange strain above and below ZL9 position is individual different, illustrate that they lay respectively at goal gene both sides, as can be seen from the figure recon number successively decreases from both sides to centre, mark Y9-10, Y9-11 and Y9-24, Y9-28, RM410 is closer from target gene, thus ZL9 is positioned in the section of about 548kb between Y9-40 and Y9-28.In order to reduce between positioning area further, we expand target group.At F 2in (zl9/ Nanjing 11) colony, in units of individual plant, totally 50 strains of the random results normal individual plant of phenotype, next year continues plantation and forms F 3for colony.Wherein, 30 F 3(zl9/ Nanjing 11) strain isolates recessive individual plant, individual plant totally 513 strains of zebra leaf trait phenotypes.Meanwhile, we are also intensive STS mark, this interval has been developed again 20 STS marks, and these marks are carried out polymorphism analysis to parent, and have 18 and can expand single slice, wherein 10 show polymorphism between parent, utilize these 10 marks to F 3in (zl9/ Nanjing 11) strain, totally 513 strain zebra pallettes become individual plant and carry out gene type assay, mark Y9-28 detects that 16 restructuring exchange strain, mark Y9-25, Y9-24, Y9-10 be then reduced to 5 exchange strains, then toward central marker Y9-33 and Y9-32 reduce to only have 2 exchange strains.And mark Y9-11, Y9-62, Y9-61 and Y9-41 and all do not find to exchange in 513 strains thus to be positioned at strain ZL9 in the section of about 272kb between Y9-40 and Y9-32.
Curiously, we find all individual plants of mark Y9-44, Y9-46, Y9-57, Y9-49 and H64 in colony (the recessive individual plant of 513 strains), and all can not increase any PCR band.We guess that this interval of mutant may lack, and disappearance interval has on earth does not muchly still know.Between mark Y9-41 and Y9-44 and intensive mark between H64 and Y9-61, with in spend 11 and zl9 for template, carry out pcr amplification.If mark with in spend 11 to do template can to amplify band, and be that template amplification does not go out band with zl9, then the chromosome segment of this mark correspondence has disappearance; If with in spend 11 and zl9 do template and can amplify band, then the chromosome segment of this mark correspondence does not lack.Respectively with drawing before the nearest mark in distance deletion fragment two ends, after draw the new mark of composition one, using in spend 11 and zl9 as template, carry out pcr amplification.Finally, we find to form a new mark with drawing x9-11r after drawing x9-2f and mark x9-11 before mark x9-2, using in spend 11 and zl9 as template, carry out pcr amplification, the specific band that zl9 can amplify.Reclaim this specific band PCR primer, deliver to biotech firm and check order, sequencing result shows: the 17th, 946,595-18,118 of the Chromosome 9 of mutant, 259 bases (NCBI website) 171,665bp have lacked totally.Total on this deletion fragment: 19 functional genes, 11 expressing proteins, 2 retrotransponsonses.According to particular location and the size of deletion fragment, we devise specific marker xs9-1 (F:TTCCCTTAGTGGTTTTG (SEQIDNo.3), R:ATCTCTAACCCCTTCAA (SEQIDNo.4), respectively with in spend 11 and zl9 for template, carry out pcr amplification, only have with zl9 when being template, the band of 772bp can be amplified.Meanwhile, we also analyze the recessive individual plant in target group with mark xs9-1, and we find that all recessive individual plants can amplify the specific band of this 772bp.As can be seen here, the gene of control zl9 mutant character may just in the 172kb fragment of disappearance.Involved portion markings and primer are in table 2.
Table 2
Be 272Kb between the positioning area of mutator gene, except the 172Kb of disappearance, also there is 100Kb, the interval of this 100Kb there are 10 functional genes, we check order to the possible candidate gene of conjecture, comprising: cytochrome P450 gene LOC_Os09g28390, RNA identify motif LOC_Os09g28810, serine carboxypeptidase homologous gene LOC_Os09g28830 and LOC_Os09g28840, and sequencing result and the fine sequence alignment of Japan announced of mutant, do not find differences.
The albumen homology sequence alignment of the gene that the deletion fragment between positioning area comprises and correspondence carries out at NCBI website (http://www.ncbi.nlm.nih.gov/), the prediction of proteins encoded Subcellular Localization is carried out at website TargetP1.1Server (http://www.cbs.dtu.dk/services/TargetP/), and the homologous gene in Arabidopis thaliana and functional annotation carry out at TAIR website (http://www.arabidopsis.org/).Wherein there is the product Subcellular Localization of 5 genes on chloroplast(id).These 5 genes are LOC_Os09g28480, LOC_Os09g28540, LOC_Os09g28550, LOC_Os09g28580 and LOC_Os09g28620 respectively.And these genes other position does not on chromosome have redundancy, be the gene of single copy in genome.Therefore, we assign these 5 genes as candidate gene (Fig. 2).
We will spend after the dry seeds of 11 soaks 2 days in mutant zl9 and corresponding wild-type, be divided into two, a part zl9 and in spend 11 to send out seedling in dark conditions, another part is placed on illumination condition in intelligent artificial climate culturing room and issues seedling, and temperature is all arranged to 30 DEG C.After 3-4 days, all grow incomplete leaf, the mutant zl9 under dark condition and middlely spend 11 to be all yellow.Under illumination condition, mutant zl9 and middlely spend 11 to be all green.Then, we cultivate under again the etiolated seedling sent under dark condition being forwarded to illumination condition, after for some time, spend the incomplete leaf of 11 to turn green by Huang, and the incomplete leaf of mutant zl9 are still yellow in wild-type.Thus, we infer: when etiolated seedling proceeds to illumination condition, and the demand of certain gene increases, Huang Ye just can be made to be transformed into normal green, and wild-type is normal, can turn green, and this transgenation of mutant, afunction, incomplete leaf can not turn green by yellow.
During we 0h, 1h, 3h, 6h, 12h, 7d respectively after dark proceeds to illumination cultivation, get in wild-type the incomplete leaf spending 11, and during 7d, get the first leaf, also have the incomplete leaf under illumination condition and the first leaf, proceed to immediately in liquid nitrogen, at-70 DEG C of Refrigerator stores, extract RNA.In order to determine the candidate gene of ZL9 by the expression of gene, we have carried out Semiquatitative RT-PCR assay expression analysis to above-mentioned 5 candidate genes.Found that: the expression amount of gene LOC_Os09g28480 first reduces, after slowly raise again, present regular change.We have done fluorescent quantitation expression analysis to LOC-Os09g28480 gene again, and the result of its result and semi-quantitative expressed analysis matches.Therefore, we are defined as LOC_Os09g28480 gene the candidate gene (Fig. 3) of Zl9 mutator gene.
Embodiment 3: zebra phyllopodium because of RNA interference analysis experiment
The structure of 1.RNA interference vector pRNAiZL9
Submit the cDNA sequence of ZL9 gene to ncbi database (http://www.ncbi.nlm.nih.gov/), carry out homologous sequence comparison with Blastn, and carry out sequence homology comparison with DNAMAN software.According to RNA interference vector design of primers principle, choose the fragment of the long 133bp of ZL9 gene cDNA sequence the preceding paragraph as RNA interference fragment.Software primer5.0 is adopted to design the primer of cloning RNA interference fragment; and according to the restriction enzyme site that fragment forward and reverse on intermediate carrier p1022 is inserted; add BamHI site and SpeI site respectively at 5 ' end of upstream and downstream primer respectively, and add protection base in restriction enzyme site upstream.
Primer sequence is as follows:
ZL-RNAi-BamHI-f:5′-CGGGATCCGCGACGTGGAGATCGAGGATT-3′(SEQIDNo.5)
ZL-RNAi-SpeI-r:5′-GACTAGTCATGGCACGCATTTGCAGATG-3′(SEQIDNo.6)
With the cDNA of this gene for template, Standard PCR is carried out with above-mentioned primer, and after the gel electrophoresis of 1%, utilized by the product obtained gel recovery test kit to reclaim this object fragment, and be cloned into (called after ZL9-pMD18-T) on pMD18-T carrier, adopt dideoxy nucleotide chain cessation method ABI3730 type automatic dna sequencer on check order, sequence and ncbi database (TheNationalCenterforBiotechnologyInformation) after order-checking or the middle sequence alignment analysis of rice genome annotations database (RiceGenomeAnnotation), after the confirmation of its series formation is errorless, for the structure of back carrier.Then, double digestion (BamHI and SpeI) ZL9-pMD18-T and p1022 respectively, digestion products is after electrophoresis, reclaim the carrier p1022 of interference fragment and incision, then two products are connected with T4 ligase enzyme, and transformation of E. coli DH5 α (the carrier called after L-p1022 now formed).After choosing positive monoclonal qualification, the plasmid identified of double digestion (BglII and XbaI) again, and the object fragment again obtained with double digestion (BamHI and SpeI) ZL9-pMD18-T is connected, connect product conversion bacillus coli DH 5 alpha (the carrier called after L-p1022-R now formed).The positive colony of qualification is connected with the carrier pCAMBIA1301 cut through same enzyme after BamHI with SacI double digestion again, connects product conversion bacillus coli DH 5 alpha, the positive colony pRNAiZL9 obtained.
2. the acquisition of transgenic line and phenotypic evaluation
Plasmid pRNAiZL9 is imported Agrobacterium EHA105 by thermal shock, transforms in japonica rice wild-type variety and spend 11 ratarias.T 0for the transfer-gen plant obtaining leaf color anomaly.The leaf morphology of transfer-gen plant is similar to mutant, and the blade newly grown is zebra leaf (Fig. 4).

Claims (3)

1. controlling a paddy rice ZL9 albumen for rice leaf color, it is characterized in that, is following 1) or 2) protein:
1) protein be made up of the amino acid residue sequence of SEQID № .2;
2) SEQID № .2 amino acid residue sequence had the protein of the identical activity of amino acid residue sequence of SEQID № .2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. a gene of paddy rice ZL9, is characterized in that, is the cDNA gene of paddy rice ZL9 albumen described in claim 1, is following 1) or 2) or 3) or 4) DNA molecular:
1) DNA sequence dna of SEQID № .1;
2) polynucleotide of coding SEQID № .2 protein sequence;
3) DNA sequence dna limited with SEQID № .1 has more than 90% homology, and coding identical function protein DNA sequence;
4) nucleotide sequence that the DNA sequence dna that can limit with SEQID № .1 is under strict conditions hybridized.
3. gene described in claim 2 is preparing the application in the transgenic cell line and transfer-gen plant that leaf look is deepened, photosynthetic efficiency improves.
CN201510728279.XA 2015-10-30 2015-10-30 Protein for controlling color of rice leaves and coding gene and application of protein Pending CN105153289A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060123505A1 (en) * 2002-05-30 2006-06-08 National Institute Of Agrobiological Sciences Full-length plant cDNA and uses thereof
JP2014171451A (en) * 2013-03-11 2014-09-22 Hiroshima Univ Rice transformant and method of making the same
CN104087603A (en) * 2014-07-07 2014-10-08 西南大学 Rice zebra-leaf mutant gene ZEBRA15 as well as protein encoded by same and application of gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060123505A1 (en) * 2002-05-30 2006-06-08 National Institute Of Agrobiological Sciences Full-length plant cDNA and uses thereof
JP2014171451A (en) * 2013-03-11 2014-09-22 Hiroshima Univ Rice transformant and method of making the same
CN104087603A (en) * 2014-07-07 2014-10-08 西南大学 Rice zebra-leaf mutant gene ZEBRA15 as well as protein encoded by same and application of gene

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
何瑞锋 等: "水稻"斑马叶"叶绿素含量及几种酶活性的变化", 《武汉大学学报（理学版）》 *
刘胜 等: "一个水稻"斑马叶"叶色突变体基因zebra leaf2(zl2)的图位克隆", 《中国水稻科学》 *

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