CN108841797A - The protein and its encoding gene of a kind of adjusting and controlling rice Chlorophyll synthesis and application - Google Patents

The protein and its encoding gene of a kind of adjusting and controlling rice Chlorophyll synthesis and application Download PDF

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CN108841797A
CN108841797A CN201810634708.0A CN201810634708A CN108841797A CN 108841797 A CN108841797 A CN 108841797A CN 201810634708 A CN201810634708 A CN 201810634708A CN 108841797 A CN108841797 A CN 108841797A
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圣忠华
胡培松
唐绍清
谢黎虹
焦桂爱
魏祥进
邵高能
胡时开
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China National Rice Research Institute
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Abstract

The invention discloses a kind of protein of adjusting and controlling rice Chlorophyll synthesis and its encoding gene and applications.The albumen is with SEQ ID NO:Amino acid sequence shown in 3.The encoding gene of the protein has SEQ ID NO:Nucleotide sequence shown in 1.Rice chlorophyll precursor synthesis associated protein encoding gene mutation can lead to rice plant blade and macula lutea line leaf occurs, and mutation is serious to even result in Albino Seedling death.The gene is knocked out using Crisp/cas9 gene editing technology in normal plant, normal green plant phenotypic mutation Albino Seedling can be made, it is dead after tri-leaf period.It is applied to the work such as genetic modification of plants, is important indicator, can be used as target gene applied to breeding of hybrid rice.It, on the one hand can quick and convenient detection filial generation F in seedling stage especially in the double-linear hybrid rice production of hybrid seeds1Purity.Simultaneously during planting hybrid rice, the sterile line etiolated seedling for carrying target gene can quickly be removed in seedling stage.

Description

The protein and its encoding gene of a kind of adjusting and controlling rice Chlorophyll synthesis and application
Technical field
The invention belongs to field of plant genetic project technology, and in particular to a kind of protein of adjusting and controlling rice Chlorophyll synthesis And its encoding gene and application.
Background technique
Rice is important cereal crops, and is the mode crop for studying higher plant.The yield of rice and its blade profile, Leaf function is directly related.The change of rice leaf color is due to the related gene of control Development of Chloroplasts and Chlorophyll synthesis approach hair Caused by having given birth to mutation, because photosynthetic efficiency is affected that the yield of rice is made to be influenced to different extents (Wang Ping Honor etc., 2006).
Currently, rice leaf color mutant is mainly by induced mutations and natural variation two types, it is many relevant with leaf color Gene has been cloned.Chlorophyll synthesis and Development of Chloroplasts relevant metabolic pathway are substantially bright in model plant arabidopsis Really, the enzyme gene for participating in these metabolic pathways can all cause leaf color to change in case of mutation.Mochizauk etc. utilizes methyl sulphur Acetoacetic ester (EMS) induced mutation, obtains mutantgun1,gun2,gun3,gun4,gun5, these mutant and Chlorophyll synthesis Approach is closely related（Mochizauki at al.,2001）.At least 15 kinds of enzymes are identified at present participates in ALA（5 ' glycyls Propionic acid）It is eventually converted into the process (Masuda at al., 2008) of chlorophyll a and chlorophyll b.Glutamyl-tRNA synthetase （GluRS）It is the initial enzyme for adjusting ALA and being formed（Eckhardt at al.,2004;Liu at al., 2007）,OsGluRSIt is prominent Change causes rice yellow leaf and phenotype of short stem (Liu at al., 2007).T-DNA insertionOsChlHIt will lead to the conjunction of chlorophyll biology It is obstructed and the subsequent plant phenomena of mortality (Zhang at al., 2006) at approach.Last in chlcrophyll biosynthesis Step, chlorophyll b are formed methyl oxidation on D ring by chlorophyll a, wherein chlorophyll a oxygenase（CAO）Play catalytic action (Espineda at al.,1999; Harper at al., 2004; Lee at al., 2005).From the full base of rice Because identifying two in groupCAOHomologous gene,OsCAO1WithOsCAO2。OsCAO1It is the leaf under chlorophyll b synthesis and illumination The key of green body development, andOsCAO2It may be to work in the dark.T-DNA mutant the study found thatOsCAO2It knocks out prominent There were significant differences with wild type leaf color for variant leaf color, andOsCAO1Knockout mutations body has light green leaf phenotype (Lee at al., 2005)。OsDVRYellowish green leaf is presented in gene mutation body, and chlorophyll levels reduce, and Development of Chloroplasts is stagnated, and the speed of growth is slow Slowly (Rodriguez at al., 2016).YGL1It is the important Chlorophyll synthesis enzyme gene being cloned.ygl1Mutation Body shows yellow green blade in Seedling Stage, and Chlorophyll synthesis amount is reduced, Development of Chloroplasts also postpone (Wu at al., 2007).Although many leaf color related genes have been cloned, during rice chlorophyll synthesis and Development of Chloroplasts still It is not accredited there are many gene, therefore it is green for illustrating plant chlorophyll synthesis and leaf further to excavate Rice Leaf form and aspect correlation gene Body development is still significant, while having theoretical direction effect to the rice breeding of high photosynthetic efficiency.
4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase, it is highly conserved in higher plant, arabidopsis it is same Source gene (HDR) report is relatively more.HDR is isoprenoid, is played an important role in the growth and development of plant.Base The biosynthesis of this isoprenoid units is occurred by two kinds of different approaches：Mevalonic acid（MVA）Approach and 2- methyl D-are red Moss sugar alcohol -4- phosphoric acid（MEP）Approach.MEP access works in plastid and is related to providing monoterpene, isoprene, chlorophyll, class Carrotene, tocopherol, Japanese yew diene, the precursor of gibberellin and abscisic acid.In recent years, the enzyme that entire MEP approach is related to is all It is accredited in Escherichia coli, and has demonstrated the activity and its effect of corresponding enzyme extensively.HDR（Also referred to as IspH or LytB）It urges Change the final step of MEP approach and plays speed limit effect in approach.Therefore the mutation of the homologous genes such as HDR will lead to chlorophyll Resulting anomaly is embodied in plant leaf blade and then shows as yellow speckle leaf.
Summary of the invention
In view of the problems of the existing technology, the purpose of the present invention is to provide a kind of eggs of adjusting and controlling rice Chlorophyll synthesis The technical solution of white matter and its encoding gene and application.
The protein of a kind of adjusting and controlling rice Chlorophyll synthesis, it is characterised in that the protein has SEQ ID Amino acid sequence shown in No.3
（MATITTQLRSALLSPAASPSRRARRAPSSVRCDSSAASSLSASASLDADFDKKQFRHNLT
RSDNYNRKGFGHKKETLELMSQEYTSDVIKTLKENGNQHTWGPVTVKLAEAYGFCWGVERAVQIAYEARKQFP DDRIWLTNEIIHNPTVNKRLEDMGVQNIPVDAGIKDFDVVEQGDVVVLPAFGAAVEEMYTLNEKKVQIVDTTCPWVS KVWNMVEKHKKGDYTSIIHGKYSHEETVATASFAGTYIIVKNIAEASYVCDYILGGQLDGSSSTKEEFLEKFKNAVS PGFDPDVDLVKVGIANQTTMLKGETEEIGKLVEKTMMRRFGVENVNDHFIAFNTICDATQERQDAMYQLVKEKVDLI LVVGGWNSSNTSHLQEIGELSGIPSYWIDSEQRIGPGNKISYKLNHGELVEKENWLPEGPITIGVTSGASTPDKVVE DALQKVFEIKRQEVLQAA*）.
The protein, it is characterised in that the amino acid sequence further includes the amino shown in SEQ ID No.3 The homologous sequence of addition, substitution, insertion or deletion one or more amino acid or other species in acid sequence and the amino generated Acid sequence or derivative.
The gene of the protein of a kind of encoding regulator rice chlorophyll synthesis, it is characterised in that the gene has Nucleotide sequence shown in SEQ ID No.1.
A kind of gene of the protein of encoding regulator rice chlorophyll synthesis, it is characterised in that the nucleotide Sequence further includes addition, substitution, insertion or deletion one or more nucleotide in the nucleotide sequence shown in SEQ ID No.1 And mutant, allele or the derivative generated.
The transgenic cell line containing rice chlorophyll synthesis associated protein encoding gene.
The transgenosis recombinant bacterium containing rice chlorophyll synthesis associated protein encoding gene.
Described is cultivating turning for Chlorophyll synthesis changed condition containing rice chlorophyll synthesis associated protein encoding gene Application in gene plant.
Described plants containing rice chlorophyll synthesis associated protein encoding gene in the transgenosis for cultivating seedling leaf yellow Application in object.
Specific implementation step of the invention is as follows：
One, rice xantha mutantys-1Separation and genetic analysis
This research mutantys-1Early 17 are praised in super early rice kind（YK17）EMS mutagenesis mutant library, field table Type observation finds being mainly characterized by for the mutant：The mutant shows as yellow speckle leaf under cryogenic conditions in seedling stage, with The progress of development, the raising of temperature, the yellow speckle leaf phenotype fade away, until heading stage restores to close to wild type greenery table Type.Net Photosynthetic Rate,ys-1（27.65）Lower than YK17（33.96）.Chlorophyll measuring the result shows thatys-1It is mainly shown as that leaf is green Plain a compared with wild type there is a serious shortage of（Fig. 1）.Reciprocal cross is carried out with rice varieties D50, Dular and 02428 respectively using mutant, Obtained F1Offspring shows as normally afforesting, in its selfing F2Normal plant and mutant plant segregation ratio are close to 3 in group:1 （Table 1）, show that this character is controlled by a pair of of recessive nuclear gene.
1. mutant plant of table is from normal plant in different F2Separation in group
Two, map based cloningYS-1Gene
1. YS-1The molecule of gene positions：
In order to separateYS-1Gene, the present invention have initially set up the high F of big polymorphism2Target group, by tropic Japonica Kind D50 is male parent, mutantys-1For female parent, F is harvested2Single plant identifies offspring's genotype, to the F of acquisition2There is phenotype in group Isolated group chooses recessive macula lutea line leaf extremists therein and carries out the assignment of genes gene mapping, utilizes SSR molecular marker pairYS-1Position Point carries out Primary Location, by its Primary Location the 3rd chromosome it is long-armed on, and marked between RM15851 and two SSR of RM15880 Between note.Then by analyzing the BAC sequence between RM15851 and RM15880 two label, new Indel has been developed Label, willYS-1Finely positioning is between Indel label Indel3 and Indel4 within the scope of 122kb（Fig. 2）, pass through analysis This section open reading frame（ORF）Speculate candidate gene and gene sequencing analysis, finds mutational site.
2. YS-1The identification and functional analysis of gene
According to finely positioning as a result, within the scope of 122kb according to RiceGAAS (Rice Automat ed Systrm, http://ricegaas.dna.affrc.go.jp/) and TIGR（http://rice.plantbiology.msu.edu/）'s Prediction, discovery share 12 candidate genes in this section, we devise the sequencing primer of each gene, using the method for PCR Respectively fromys-1All candidate genes are amplified in mutant and wild type YK17 genome carries out sequencing analysis.It was found that wherein 1 In the DNA fragmentation of a gene, single base mutation is had occurred in the product of mutant amplification compared with wild-type variety, causes amino acid Mutation.The gene order of wild-type variety YK17 amplification is SEQ ID NO.1, is named asYS-1Gene.In order to confirm mutation Body surface type be byYS-1Caused by gene mutation, we have carried out transgenosis to mutant and have restored confirmatory experiment.Transgenosis is restored Verifying is mainly by wild typeYS-1Full length gene genome sequence clones and is building up to binary plant transgene carrier pUbi- At 1390 multiple cloning sites KpnI/SpeI, Overexpression vector is obtained.The Overexpression vector built is passed through into Agrobacterium The genetic transformation system untransformed mutants callus of mediation induces by kanamycin-resistant callus tissue and then is divided into transgenic seedling.It has converted outer SourceYS-1The mutant of gene（That is transgenic positive strain）Seedling Color is multiple green,YS-1Expression quantity obviously increases, and Fig. 4 is wild Type, mutant, gene knockout and T0 transgenosis are overexpressed the phenotype of plant.Transgenosis, which is overexpressed to restore to test, to be confirmed to be mutated Body surface type be byYS-1Caused by gene mutation, show that present invention obtains the transgenosis water for making mutant restore normal function Rice.
The present invention utilizes rice yellow line pallette variant, is cloned by map based cloning methodYS-1Gene, the gene are compiled Code Chlorophyll synthesis correlation 4- hydroxy-3-methyl but-2-ene base diphosphonic acid reductase.Confirmatory experiment mirror is overexpressed by transgenosis It is fixedYS-1The function of gene.Thus the present invention is adjustable the synthesis of rice chlorophyll, and then obtains seedling stage macula lutea line leaf phenotype. It is the gene further using laying the foundation.
Detailed description of the invention
Fig. 1 Fig. 1 macula lutea line leaf phenotype（A, B, C）With wild type YK17 and mutantys-1Seedling Stage blade（D） With heading stage sword-like leave（E）, chlorophyll a, content of chlorophyll b comparative analysis.A, macula lutea line leaf seedling stage variable rate technology；B is left, wild Early 17 seedling are praised in type；In, macula lutea line leaf mutant seedlings；The right side, the seedling after target gene is knocked out using Crisp/Cas9 technology Phase performance.
Fig. 2YS-1Finely positioning on the 3rd chromosome of rice.
Fig. 3 wild type YK17（It is left）, mutantys-1（In）, T0Transgenosis is overexpressed plant（It is right）Seedling stage phenotype.
Fig. 4 wild type YK17, mutantys-1, transgenosis overexpression plantYS-1Gene（LOC_OS03g52170） Relative expression quantity analysis.YK17 praises early 17 in wild type；ys-1, mutant；YS-1E , transgenosis overexpression plant.
Specific embodiment
In order to understand the present invention, the present invention is further illustrated with embodiment below, but do not limit the present invention.
Embodiment 1：YL-1The clone of gene
A) rice material
Rice（Oryza sativaL）Mutantys-1(yellow leaves 1), original wild type material is super early rice Early 17 are praised in kind（YK17）.
B) genetic analysis and target group
It is determined using positive and negative hybridizationys-1For cryptic mutant.It chooses mutant and D50 is hybridized, F1Generation selfing, single plant are received Kind plantation F2Group selects 1500 recessive individuals from the group for have separation（Seedling macula lutea line leaf）As target group.? Every plant of four leaf stage takes 0.2 gram or so of tender leaf, carries out the assignment of genes gene mapping for extracting total DNA.
c)YS-1The Primary Location and finely positioning of gene
The base for the assignment of genes gene mapping is extracted from rice leaf using the rapid extracting method of rice minim DNA
The method of group DNA, the DNA extracting is SDS method (Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19-21).It takes about 100mg rice leaf is put into 2ml centrifuge tube after shredding, steel ball is added after liquid nitrogen frozen, crushes on sample grinding machine, then mentions DNA, the DNA of acquisition is taken to be precipitated and dissolved in 400 μ L ultrapure waters, each PCR reacts 1 μ LDNA sample.?YS-1Gene In Primary Location, using by 30 F with mutant phenotype2Extremists carry out ssr analysis.First according to the japonica rice of announcement and The molecular genetic linkage map of long-grained nonglutinous rice creation chooses approaches uniformity and is distributed in the progress PCR amplification of the SSR primer on each chromosome（Reaction System is as follows）.Pass through 8% polyacrylamide gel（Gel configuration method is as follows）Electrophoretic separation, passes through the polymorphic of test strip Property, by the galianconism of gene Primary Location to the 3rd chromosome, and between two SSR marker of RM15851 and RM15880.
PCR reaction system：
8% polyacrylamide gel formula：
Polyacrylamide gel colour developing formula of liquid：
Note：Formaldehyde is now to add before use, other three are prepared in advance by corresponding amount.
Then by analyzing the BAC sequence between RM15851 and RM15880 two label, new Indel has been developed Label, finally by F2Target group is expanded to 1500 progress finely positionings, willYS-1It is accurately located at Indel label Indel3 Between Indel4 within the scope of 122kb,（Fig. 2）, by analyzing this section open reading frame（ORF）Speculate candidate gene simultaneously Gene sequencing analysis, finds mutational site.
Indel labeled primer sequence：
Indel3-F TCGAAAGTTGGCGAACAG(SEQ ID NO.6)
Indel3-R AGGGCAACATACTGGCTCT (SEQ ID NO.7)
Indel4-F CTAGAATTGCCCAGATAC(SEQ ID NO.8)
Indel4-R ACTTCATCCGTTTTACAA (SEQ ID NO.9)。
D) predictive genes and comparative analysis
According to finely positioning as a result, within the scope of 122kb according to RiceGAAS (Rice Automat ed Systrm, http://ricegaas.dna.affrc.go.jp/) and TIGR（http://rice.plantbiology.msu.edu/）'s Prediction, discovery share 12 candidate genes in this section, we devise the sequencing primer of each gene, using the method for PCR Respectively fromys-1All candidate genes are amplified in mutant and wild-type variety genome carries out sequencing analysis.It was found that wherein 1 A gene (LOC-Os03g52170) DNA fragmentation in, mutant amplification product single alkali has occurred compared with wild-type variety Base is mutated, and A has been mutated into T at the 165th base of the 2nd exon（Base A is mutated into T）, corresponding amino acid is by asparagus fern Amide is mutated into isoleucine.The gene order of wild-type variety amplification is SEQ ID NO.1, is named asYS-1Gene is compiled The nucleotides sequence that the protein sequencing of code obtains is classified as SEQ ID NO.2.
Embodiment 2：Transgenic experiments
1. vector construction
Design a pair is completely covered entirelyYS-1The primer of gene ORF, and separately design on primer restriction enzyme site KpnI and The site SpeI, PCR amplification wild type gene group DNA, electrophoresis detection gel extraction, recovery product KpnI and SpeI digestion, even It is connected on the pUbi-1390 carrier of same digestion, there is no base mutations for sequencing confirmation, and the carrier structure figure after building is The carrier built is transferred to Agrobacterium by the method to shock by electricity by pUbi1390-YS-1（A grobacterium tumefaciens）In bacterial strain.
Amplification ORF sequence primer sequence be：
OG2-KpnI:5’- TTACTTCTGCACTAGGTACCATGGCGACCATCACCACGC -3’ (SEQ ID NO.4)
OG2-SpeI: 5’- TAGCGTTAACACTAGTCGCGGCCTGCAAAACTTCC -3’ (SEQ ID NO.5)
2. genetic transformation：
（1）The selection of transformation receptor
It willys-1Plumule is cut after induced medium induces 2 weeks, continues culture 1 by seed mature embryo evoked callus Select the receptor that eugonic callus is used as conversion in week.
（2）Genetic transformation
Using agrobcterium-mediated transformation (Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T‐DNA. The Plant Journal 6:271-282), by pUbi-1390 empty carrier and pUbi1390-YS-1The EHA105 bacterial strain of carrier infects rice Callus is cultivated on the screening and culturing medium containing 120mg/L G418 after co-culturing 3 days under the conditions of dark, 25 DEG C.Screening is anti- Property callus containing cultivating 10 days or so on the pre- differential medium of 120mg/L.The callus broken up in advance is gone on differential medium It is cultivated under illumination condition.Obtain within one month or so resistant transgenic plant.Identification and continuous observation, discovery are carried out to plant Turn the transgenic plant phenotype of empty carrier withys-1Compared to not changing, i.e. seedling stage macula lutea line leaf, and pUbi1390-YS-1It carries The positive transgenic plant of body and wild type performance are consistent, show as green, that is, restoreys-1Mutant phenotype, see Fig. 4.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>China Paddy Rice Inst
<120>The protein and its encoding gene of a kind of adjusting and controlling rice Chlorophyll synthesis and application
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3896
<212> DNA
<213>Rice (Oryza sativa)
<400> 1
gtggggagat cccaactccg aatccgacgc acccacaccc atccactcca gagctccccg 60
cggcacttgg caccaccacc accaccaccg gcatccggcg gcgcgggcgg tgatggcgac 120
catcaccacg cagctccggt cggccctcct ctcgccggcg gcctcgccga gccgccgtgc 180
caggcgcgcc ccgtcgtccg tgcggtgcga ctcctcggca gcgtcgtccc tgtccgcctc 240
cgcgtcgctc gacgccgact tcgacaagaa gcagttccgc cacaacctca cccgcagcga 300
caactacaac cgcaagggct tcggccacaa gaaggagacg ctcgagctca tgagccagga 360
gtacaccagt atgatttcct ctcctttccc tttctttttt tttctcttct tgattggggg 420
ctcaccggcg gcggcgtgtt cttggtctca ggcgacgtga tcaagacgct caaggagaac 480
gggaaccagc acacgtgggg ccccgtgacg gtgaagctgg cggaggcgta cggcttctgc 540
tggggcgtcg agcgcgccgt gcagatcgcc tacgaggcgc gcaagcagtt ccccgacgat 600
cgcatctggc tcaccaacga gatcatccac aaccccaccg tcaacaaggt gattatgaaa 660
tcctccccta tccatttcgt ttgattcagg cgtcgactcg tctccatggg gatgaatttt 720
gtggtttttc cccttcgtga atgcccgcct ccttccacga actgagtttg ttgcgtatga 780
ggggatagat tagcgtctgc gtgtgcgtgg taaaatgatc tatttggtgc aaatgagatt 840
tggagtaaaa tcctaccaat ttctagttga gaaagtgttc acagcgaaga gtggccacca 900
gattctgcgt tttagacttt tataccccct tgattaccca attaactagt ttctttttat 960
ctgctgctta agagtattca atcatctgtg cttcgcatga caacggactc tggaattgga 1020
agtgacttga attcgactcc tgctattgtg tttgcagcga ttggaggaca tgggcgtcca 1080
aaacattcct gttgatgcag gtattaagga tttcgatgtt gtcgagcaag gggatgttgt 1140
cgtattgcct gcatttggag cggccgtgga ggagatgtac acgctgaatg agaagaaggt 1200
gcagatagtc gatacaacgt gcccttgggt ttcaaaggtg ggtttccctt ttgaagatca 1260
cctgatcctt tctatgtcgc tctgagatta tcagaaatca aaggtatatc tggttgatga 1320
actccactac tatatatagg tgtggaacat ggttgaaaag cacaagaagg gtgactacac 1380
ttctattatt catggaaaat actcccatga agaaactgtt gccactgctt cctttgcagg 1440
gacatacatc attgttaaga acatagcaga ggtatgtttg atgcatttaa ttttttccta 1500
ttgcattgac ttccatgttg aatacattat ttggctcaaa taacaaaata ttatcaattg 1560
taacaggcta gctatgtatg tgattatata cttggtggcc aacttgatgg ttctagctcc 1620
acaaaagagg agttccttga ggtaaagcct ttcttgaatc atcttttttt ttttgccctg 1680
ttatgttgga aagaaatgcc aatgtctaat cgaatgtttt atcaacaact gcagaaattc 1740
aaaaatgctg tttctccagg atttgatcct gatgttgact tagtgaaagt tggcattgcc 1800
aatcaaacaa caatgcttaa aggagaaact gaagaaattg gttcgtatgc tagcagatgc 1860
taattgccat tagcttcatg cctgttagtt tgtgttatta tatacagtac aatttggttt 1920
taaatatcaa atttgactat aatgctgtat aactaacagg gaagcttgtt gagaagacaa 1980
tgatgcgtag gtttggagtt gagaatgtca atgaccactt cattgccttc aacactatct 2040
gtgatgccac acaggtaaac tttgttgtac tattaagtca tttatggctc cattgtaatc 2100
tgcataagta accttgttcg agcatattcc tacaaacttt gtggtgtttt tggatacaat 2160
cagatcatag tctatgtttg gttcatatat tggtgacacc cttccccttc atatatcagg 2220
aaagacaaga tgcaatgtac cagctagtga aggagaaagt tgatcttatt ctggttgttg 2280
gaggatggaa ctccagtaac acctctcatt tgcaagaaat tggagaactc agtggaattc 2340
cgtcttactg gattgacagt gaacaaagga ttggacctgg aaacaagatc agctacaagt 2400
taaatgtatg ttgtaatact catatctcat caagaatatt cttttttctt caaaaaaaaa 2460
tgttagtgaa tattatgaaa gaatagtgta gatttttttt tggcaaccaa taagcagaat 2520
tctgacaaaa taagcagaaa catcaattga aagtatagtc ttatcactga aatctgaatc 2580
attttaacag catggcgagc ttgtcgagaa agagaactgg ttaccagaag gccctatcac 2640
aatcggagtt acttctggcg cctcaactcc ggacaaggta atacggctaa cctggattat 2700
gtttttcttt tgtacgcagc ctgcttgtct tctgcacata tttctgcaca gtggtcactg 2760
gataaaacta aaaacgcata actttccttc ttatgcaggt tgttgaggat gcgttacaga 2820
aggtgtttga gatcaagcgc caggaagttt tgcaggccgc gtaaattttc atcaggtttg 2880
gtcacggtag ctgatggctt ggtgaaggtt aatagaacca gtgcacaagc gatgtctgta 2940
cagtagtgaa gaatgctacg cttttaggag cgcatactac ggcatgtgct gagatttacg 3000
tagtatgttg ggggcaaatg ttgtaatcaa ttatcacacg aagaacagtt gctgtatcgg 3060
ctgtcataac tttctgaccc cctcaaataa cgttgcagaa gtatctttct tcggtataaa 3120
tatcacccaa tttgttagct agtactattt gatagagctc aaatgcagga ctccctgtat 3180
atatatggca agtacaagaa atgcagggtt ccctatgtat aacattaagt tcagaaccaa 3240
tcttgatatg ccacatatgt tgttataaaa ttccatttct ctctgcaact ttcatactgt 3300
agtagtattg ttcttcagct tttgaacaaa aaaaaataat cagtagtagc agctgttagc 3360
tcacgaccat tctgtgtgtg tgtgtgtcgc ctcactccca tgacttgaca cactgggagt 3420
gcggcggcca cctgagcacc ggcgacgcgc cgtcgccgtc gaggcgccgc cgcgccgcgg 3480
cgaggacggc cgcgaggtcg gcgaccttgg cgcgcatggt ccggcagcag ttggcgtgca 3540
cggtggcgac ctcgcgggcg tcgcggctgt cctggcagaa gccgctgaag cgcgccgtgt 3600
cgaggacgcg ggcgcggacg ccgaggcggc ggagcgcgcc gcggcgcttc atgtcgttga 3660
gcacgtcctg ctccttcatt cccgccgagc ggtcccgcgc cgcgtgccac tcgtcgaaca 3720
gcgccgccgt gcggttgttg gaggccacga agaagaagcc ggtgttgagc tcgttgccgg 3780
cgatgtcgcc ggggcggccg ttgaactggt cggagctgat caggaggtcc tcctcctccg 3840
ccctgtactc cagcctcgac aacgggttcc tcagccacat cacatccaaa tccttg 3896
<210> 2
<211> 1380
<212> DNA
<213>Rice (Oryza sativa)
<400> 2
atggcgacca tcaccacgca gctccggtcg gccctcctct cgccggcggc ctcgccgagc 60
cgccgtgcca ggcgcgcccc gtcgtccgtg cggtgcgact cctcggcagc gtcgtccctg 120
tccgcctccg cgtcgctcga cgccgacttc gacaagaagc agttccgcca caacctcacc 180
cgcagcgaca actacaaccg caagggcttc ggccacaaga aggagacgct cgagctcatg 240
agccaggagt acaccagcga cgtgatcaag acgctcaagg agaacgggaa ccagcacacg 300
tggggccccg tgacggtgaa gctggcggag gcgtacggct tctgctgggg cgtcgagcgc 360
gccgtgcaga tcgcctacga ggcgcgcaag cagttccccg acgatcgcat ctggctcacc 420
aacgagatca tccacaaccc caccgtcaac aagcgattgg aggacatggg cgtccaaaac 480
attcctgttg atgcaggtat taaggatttc gatgttgtcg agcaagggga tgttgtcgta 540
ttgcctgcat ttggagcggc cgtggaggag atgtacacgc tgaatgagaa gaaggtgcag 600
atagtcgata caacgtgccc ttgggtttca aaggtgtgga acatggttga aaagcacaag 660
aagggtgact acacttctat tattcatgga aaatactccc atgaagaaac tgttgccact 720
gcttcctttg cagggacata catcattgtt aagaacatag cagaggctag ctatgtatgt 780
gattatatac ttggtggcca acttgatggt tctagctcca caaaagagga gttccttgag 840
aaattcaaaa atgctgtttc tccaggattt gatcctgatg ttgacttagt gaaagttggc 900
attgccaatc aaacaacaat gcttaaagga gaaactgaag aaattgggaa gcttgttgag 960
aagacaatga tgcgtaggtt tggagttgag aatgtcaatg accacttcat tgccttcaac 1020
actatctgtg atgccacaca ggaaagacaa gatgcaatgt accagctagt gaaggagaaa 1080
gttgatctta ttctggttgt tggaggatgg aactccagta acacctctca tttgcaagaa 1140
attggagaac tcagtggaat tccgtcttac tggattgaca gtgaacaaag gattggacct 1200
ggaaacaaga tcagctacaa gttaaatcat ggcgagcttg tcgagaaaga gaactggtta 1260
ccagaaggcc ctatcacaat cggagttact tctggcgcct caactccgga caaggttgtt 1320
gaggatgcgt tacagaaggt gtttgagatc aagcgccagg aagttttgca ggccgcgtaa 1380
<210> 3
<211> 459
<212> PRT
<213>Rice (Oryza sativa)
<400> 3
Met Ala Thr Ile Thr Thr Gln Leu Arg Ser Ala Leu Leu Ser Pro Ala
1 5 10 15
Ala Ser Pro Ser Arg Arg Ala Arg Arg Ala Pro Ser Ser Val Arg Cys
20 25 30
Asp Ser Ser Ala Ala Ser Ser Leu Ser Ala Ser Ala Ser Leu Asp Ala
35 40 45
Asp Phe Asp Lys Lys Gln Phe Arg His Asn Leu Thr Arg Ser Asp Asn
50 55 60
Tyr Asn Arg Lys Gly Phe Gly His Lys Lys Glu Thr Leu Glu Leu Met
65 70 75 80
Ser Gln Glu Tyr Thr Ser Asp Val Ile Lys Thr Leu Lys Glu Asn Gly
85 90 95
Asn Gln His Thr Trp Gly Pro Val Thr Val Lys Leu Ala Glu Ala Tyr
100 105 110
Gly Phe Cys Trp Gly Val Glu Arg Ala Val Gln Ile Ala Tyr Glu Ala
115 120 125
Arg Lys Gln Phe Pro Asp Asp Arg Ile Trp Leu Thr Asn Glu Ile Ile
130 135 140
His Asn Pro Thr Val Asn Lys Arg Leu Glu Asp Met Gly Val Gln Asn
145 150 155 160
Ile Pro Val Asp Ala Gly Ile Lys Asp Phe Asp Val Val Glu Gln Gly
165 170 175
Asp Val Val Val Leu Pro Ala Phe Gly Ala Ala Val Glu Glu Met Tyr
180 185 190
Thr Leu Asn Glu Lys Lys Val Gln Ile Val Asp Thr Thr Cys Pro Trp
195 200 205
Val Ser Lys Val Trp Asn Met Val Glu Lys His Lys Lys Gly Asp Tyr
210 215 220
Thr Ser Ile Ile His Gly Lys Tyr Ser His Glu Glu Thr Val Ala Thr
225 230 235 240
Ala Ser Phe Ala Gly Thr Tyr Ile Ile Val Lys Asn Ile Ala Glu Ala
245 250 255
Ser Tyr Val Cys Asp Tyr Ile Leu Gly Gly Gln Leu Asp Gly Ser Ser
260 265 270
Ser Thr Lys Glu Glu Phe Leu Glu Lys Phe Lys Asn Ala Val Ser Pro
275 280 285
Gly Phe Asp Pro Asp Val Asp Leu Val Lys Val Gly Ile Ala Asn Gln
290 295 300
Thr Thr Met Leu Lys Gly Glu Thr Glu Glu Ile Gly Lys Leu Val Glu
305 310 315 320
Lys Thr Met Met Arg Arg Phe Gly Val Glu Asn Val Asn Asp His Phe
325 330 335
Ile Ala Phe Asn Thr Ile Cys Asp Ala Thr Gln Glu Arg Gln Asp Ala
340 345 350
Met Tyr Gln Leu Val Lys Glu Lys Val Asp Leu Ile Leu Val Val Gly
355 360 365
Gly Trp Asn Ser Ser Asn Thr Ser His Leu Gln Glu Ile Gly Glu Leu
370 375 380
Ser Gly Ile Pro Ser Tyr Trp Ile Asp Ser Glu Gln Arg Ile Gly Pro
385 390 395 400
Gly Asn Lys Ile Ser Tyr Lys Leu Asn His Gly Glu Leu Val Glu Lys
405 410 415
Glu Asn Trp Leu Pro Glu Gly Pro Ile Thr Ile Gly Val Thr Ser Gly
420 425 430
Ala Ser Thr Pro Asp Lys Val Val Glu Asp Ala Leu Gln Lys Val Phe
435 440 445
Glu Ile Lys Arg Gln Glu Val Leu Gln Ala Ala
450 455
<210> 4
<211> 39
<212> DNA
<213>Primer (primer)
<400> 4
ttacttctgc actaggtacc atggcgacca tcaccacgc 39
<210> 5
<211> 35
<212> DNA
<213>Primer (primer)
<400> 5
tagcgttaac actagtcgcg gcctgcaaaa cttcc 35
<210> 6
<211> 18
<212> DNA
<213>Primer (primer)
<400> 6
tcgaaagttg gcgaacag 18
<210> 7
<211> 19
<212> DNA
<213>Primer (primer)
<400> 7
agggcaacat actggctct 19
<210> 8
<211> 18
<212> DNA
<213>Primer (primer)
<400> 8
ctagaattgc ccagatac 18
<210> 9
<211> 18
<212> DNA
<213>Primer (primer)
<400> 9
acttcatccg ttttacaa 18

Claims (8)

1. a kind of protein of adjusting and controlling rice Chlorophyll synthesis, it is characterised in that the protein has shown in SEQ ID No.3 Amino acid sequence.
2. protein according to claim 1, it is characterised in that the amino acid sequence further includes in SEQ ID No.3 Shown in amino acid sequence the homologous sequence of addition, substitution, insertion or deletion one or more amino acid or other species and The amino acid sequence or derivative of generation.
3. a kind of gene of protein described in coding claim 1, it is characterised in that the gene has shown in SEQ ID No.1 Nucleotide sequence.
4. gene according to claim 3, it is characterised in that the nucleotide sequence further includes in SEQ ID No.1 institute Addition, substitution, insertion or deletion one or more nucleotide in the nucleotide sequence shown and generate mutant, allele or Derivative.
5. the transgenic cell line containing rice chlorophyll synthesis associated protein encoding gene described in claim 3 or 4.
6. the transgenosis recombinant bacterium containing rice chlorophyll synthesis associated protein encoding gene described in claim 3 or 4.
7. cultivating Chlorophyll synthesis shape containing rice chlorophyll synthesis associated protein encoding gene described in claim 3 or 4 Application in the genetically modified plants of condition variation.
8. cultivating seedling leaf yellow containing rice chlorophyll synthesis associated protein encoding gene described in claim 3 or 4 Genetically modified plants in application.
CN201810634708.0A 2018-06-20 2018-06-20 The protein and its encoding gene of a kind of adjusting and controlling rice Chlorophyll synthesis and application Pending CN108841797A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110964730A (en) * 2019-12-11 2020-04-07 浙江大学 Application of rice leaf whitening trait gene OsLCD1 in regulation and control of rice leaf color trait
CN111575298A (en) * 2020-06-08 2020-08-25 淮阴师范学院 Rice leaf albino gene OsHMBPP, protein and application
CN112778407A (en) * 2021-02-02 2021-05-11 四川农业大学 Maize seedling yellow-white leaf gene and coding protein and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047486A2 (en) * 2003-11-12 2005-05-26 E.I. Dupont De Nemours And Company Biological production of tetradehydrolycopene
CN103290027A (en) * 2013-04-27 2013-09-11 中国水稻研究所 Protein for regulating and controlling chloroplast growth and gene and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005047486A2 (en) * 2003-11-12 2005-05-26 E.I. Dupont De Nemours And Company Biological production of tetradehydrolycopene
CN103290027A (en) * 2013-04-27 2013-09-11 中国水稻研究所 Protein for regulating and controlling chloroplast growth and gene and application thereof

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Title
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110964730A (en) * 2019-12-11 2020-04-07 浙江大学 Application of rice leaf whitening trait gene OsLCD1 in regulation and control of rice leaf color trait
CN111575298A (en) * 2020-06-08 2020-08-25 淮阴师范学院 Rice leaf albino gene OsHMBPP, protein and application
CN112778407A (en) * 2021-02-02 2021-05-11 四川农业大学 Maize seedling yellow-white leaf gene and coding protein and application thereof

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