CN108374015A

CN108374015A - A kind of application of gene Loc_Os01g12810

Info

Publication number: CN108374015A
Application number: CN201810241463.5A
Authority: CN
Inventors: 董景芳; 刘斌
Original assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Current assignee: Rice Research Institute of Guangdong Academy of Agricultural Sciences
Priority date: 2018-03-22
Filing date: 2018-03-22
Publication date: 2018-08-07

Abstract

The invention belongs to gene technology field more particularly to a kind of applications of gene Loc_Os01g12810.The gene Loc_Os01g12810 can be applied in rice chloroplast developmental regulation Mechanism Study, and the expression of the gene Loc_Os01g12810 is regulated and controled by gene means to carry out the research of rice chloroplast developmental regulation mechanism；SEQ ID NO in the nucleotide sequence of the gene Loc_Os01g12810 such as sequence table：Shown in 1；SEQ ID NO in the amino acid sequence of the gene Loc_Os01g12810 codings albumen such as sequence table：Shown in 2.First demonstration that paddy gene Loc_Os01g12810 is rice chloroplast development related gene.Loc_Os01g12810 is a PPR gene.Effect study of the Study on Molecular Mechanism and PPR family genes that the clone of the gene and biological function verification develop rice chloroplast in Development of Rice has important reference significance.

Description

A kind of application of gene Loc_Os01g12810

Technical field

The invention belongs to gene technology field more particularly to a kind of applications of gene Loc_Os01g12810.

Background technology

Rice is one of most important cereal crops in the world, and the whole world is more than the population of half all using rice as staple food, Therefore, Rice Production has a decisive role safely world food.However, in the past 10 years, with the increasing of world population Add, the reduction of cultivated area, crisis in food occurs in worldwide.

The dry matter 90%~95% of rice accumulation is directly or indirectly from photosynthesis.Important as plant is photosynthetic The development of organ, chloroplaset directly affects plant carbon fixed efficiency, and then influences the various growth and development dynamics of plant, understands The development mechanism of chloroplaset has very important theory significance for improving Rice Photosynthetic Efficiency, culture high-yield variety.

Chlorophyll is that photosynthetic important pigment is participated in plant chloroplast, and the significant changes of content can lead to leaf color Variation, so chlorophyll deficiency mutant is also referred to as leaf color mutant.Leaf color mutant be mainly shown as leaf color occur yellow, The symptoms such as albefaction, light green, zebra, striped.With functional genomics and the continuous development of bioinformatics research, leaf color is prominent The molecule mechanism of variant research Development of Chloroplasts is an effective research point of penetration.Most of albino mutant (albino Mutant) lack chlorophyll, cannot be normally carried out photosynthesis, when seedling grows by the nutrition of endosperm in seed, works as embryo Plant will be dead when breast exhausts, and seedling stage lethality occurs.The seedling stage lethal effect part of albino mutant limits albefaction Utility value of the mutant in theoretical research and practical application.And turn green mutant (albino green-revertible in vain Mutant), albefaction character is shown as in seedling stage, but gradually multiple green in the leaf tissue of development later stage chlorosis, normal synthesis leaf is green Element, mutant can normal growth and announcement.So it is considered as genetic breeding and research plant photosynthesis to turn green mutant in vain The good material of the physiology courses such as the mechanism of action and the function of related gene, photomorphogenesis.

There is the variation of leaf color change and temperature caused by some gene mutations closely related.As OsTSV encodes a pigment Body oxidoreducing enzyme, after mutation, rice under 20 DEG C of cryogenic conditions chlorophyll content in leaf blades reduction, Development of Chloroplasts defect, Seedling stage is lethal；But be not in Relevant phenotype under 30 DEG C of room temperature.The one chloroplaset positioning of OsTCD10 codings has 27 PPR The PPR albumen of motif, after mutation, at 20 DEG C, there is albefaction phenotype in chloroplaset deformity, and dead before 5 leaf phases； At 32 DEG C, phenotype is then normal.Ostcd5 encodes the monooxygenase of a conservative plastid positioning, and rice mutant Ostcd5 is 20 Long white leaf, the long greenery at 32 DEG C under DEG C low temperature.The Deg protease of OsTCM5 encoding chloroplasts positioning, mutant is at 32 DEG C Under growing environment, there is albefaction phenotype, chloroplaset defect, it is lethal before 5 leaf phases.Mutant is under 20 DEG C of growing environments, then phenotype Normally.TCD9 encodes a subunits of Cpn60 albumen, shows as albefaction phenotype when mutant is at 24 DEG C before 3 leaf phases, however 28 DEG C show as normal green.PPR albumen OsV4 containing 4 PPR motifs is under cryogenic to the influence of Development of Chloroplasts. The study found that under 20 DEG C of cryogenic conditions, OsV4 mutant whitens in 3 leaf phase front vanes, chloroplaset is abnormal, and four leaf stage gradually turns Green, under 32 DEG C of temperature conditions, mutant leaf color and Development of Chloroplasts are normal.The OsHsp70CP1 of chloroplaset positioning, in height It plays a significant role during Development of Chloroplasts under temperature；Serious yellow leaf at 40 DEG C of its corresponding mutant is normal at 27 DEG C.

Research is it has also been found that the two genes of ZN and SLAC7 are related with the Development of Chloroplasts under illumination condition at present.

With going deep into for research, it is found that some genes not only regulate and control the development of blade chloroplaset, while also regulating and controlling in glume The development of chloroplaset.Such as：The supermethylation of OsAK1 promoter regions can cause rice spire and the chloroplast structure of flower spike abnormal And albefaction.After OsWSP1 point mutation, the editor in the sites various kinds of cell device RNA is affected, the biology of chloroplast ribosome Synthesis and the montage of ndhA are destroyed, and chlorophyll content is reduced, Development of Chloroplasts is abnormal, and white stripes occurs in blade, takes out Ear period generates almost white immature inflorescence.OsValRS2 is bis- to be located in chloroplaset and mitochondria, Development of Chloroplasts with And it plays an important role in the biosynthetic process of chloroplast ribosome；In its mutant, rice seedling will appear leaf color albefaction Phenomenon, meanwhile, heading stage also will appear white inflorescence.

There is a Development of Chloroplasts related gene more than 60 to be reported in ncbi database, they belong to different genes man mostly Race, but wherein there are 8 to belong to PPR gene families.They be respectively OspTAC2, OsTCD10, OsV4, OsASL3, OsWSL, ysa、OsMPR25、OsPPR1.PPR family members are formed by 2-27 PPR motifs arranged in series in varying numbers, PPR (pentatricopeptide repeat) motif is the genome sequence to arabidopsis earliest in Small and Peeters in 2000 It is found and names when row progress bioinformatic analysis, each PPR motifs are made of 35 conserved amino acids.With multiple The completion of species genome sequencing and evolutionary analysis, research find PPR gene families have in different plant species 2 it is apparent Characteristic distributions：(1) it is difficult to find in prokaryotes, eucaryote is peculiar；(2) quantity is seldom in animal, quantity in higher plant Very much.Up to the present, 2 PPR genes are only found in drosophila, 5 in yeast, 6 in the mankind；However arabidopsis, rice, In the higher plants such as corn, sorghum, more than 400 PPR genes have been predicted.PPR albumen may participate in higher plant one A little specific biological functions.

To PPR protein biological functions research shows that PPR albumen is a kind of rna binding protein.Each PPR motifs are formed Antiparallel αhelix, multiple antiparallel α spiral arranged in series form the supercoil knot of crescent shape (crescent) Structure.Superhelix concave surface (the concave surface) imparts the binding function of PPR albumen and special RNA；Convex surface (the convex surface) is responsible for the interaction of PPR albumen and other albumen.So PPR albumen specifically identifies RNA sequence, And by and other albumen combination, the regulation and control of degradation, montage, editor, translation of complete paired rna etc., in post-transcriptional level The expression of controlling gene, to influence many aspects of growth and development of plants, fertility restorer, embryo such as cytoplasmic male sterility Formation, the formation of chloroplaset and the response etc. to abiotic stress.

Currently, had the report of part leaf color mutant in rice, but plant leaf color change is to be related to multiple regulation and control The complex biological of approach and metabolic process process, it is by inhereditary material and the collective effect of external environment.It needs exist for By identifying new gene come the abundant cognition with in-depth to leaf color change and Development of Chloroplasts mechanism.It is right in existing scientific research The research of rice chloroplast development is very few, therefore is badly in need of finding that a kind of gene grinds for the development mechanism of Study On Rice chloroplaset Study carefully.

Invention content

For above-mentioned prior art problem, one aspect of the present invention provides a kind of gene Loc_Os01g12810 and exists Application in rice chloroplast developmental regulation Mechanism Study regulates and controls the gene Loc_Os01g12810's by gene means Expression is to carry out the research of rice chloroplast developmental regulation mechanism；

SEQ ID NO in the nucleotide sequence of the gene Loc_Os01g12810 such as sequence table：Shown in 1；The gene SEQ ID NO in the amino acid sequence of Loc_Os01g12810 coding albumen such as sequence table：Shown in 2.It should be understood that, it is contemplated that it is close The degeneracy of numeral modifies to the nucleotide sequence of above-mentioned encoding gene under the premise of not changing amino acid sequence, In belonging to the scope of protection of the present invention.

Preferably, by the gene silent technology expression of the gene Loc_Os01g12810 is declined, The gene silent technology includes the following steps：

(1) a kind of recombinant plasmid is built；

(2) recombinant plasmid built in step (1) is imported in normal rice paddy seed, obtains the rice of transgenic line Seed.

It is furthermore preferred that the recombinant plasmid in the step (1) is obtained by importing interference genetic fragment into carrier, it is described SEQ ID NO in the nucleotide sequence of interference genetic fragment such as sequence table：Shown in 3.

It is furthermore preferred that take total mRNA in rice leaf, by total mRNA reverse transcriptions at cDNA after, by primer 1 to described CDNA obtains the interference genetic fragment after carrying out PCR amplification；The forward and reverse nucleotide sequence of the primer 1 is respectively such as sequence SEQ ID NO in list：4 and SEQ ID NO：Shown in 5.

Best, the carrier is pYLRNAi.5；The interference genetic fragment is first astern reversely cloned into pUbi respectively In the MCS1 and MCS2 in promoter downstream, to obtain the recombinant plasmid.

It is furthermore preferred that the recombinant plasmid built in the step (1) is imported by method for transformation in normal rice paddy seed, The expression of recombinant plasmid cryptiogene Loc_Os01g12810, obtains the rice paddy seed of transgenic line.

The second aspect of the present invention discloses the rice paddy seed of the transgenic line obtained in item application.

Third aspect of the present invention discloses the rice paddy seed of above-mentioned transgenic line in Rice Photosynthesis research Application.

The 4th aspect of the present invention discloses hybrid rice of the rice paddy seed in screening homozygosis of above-mentioned transgenic line PRODUCTION TRAITS in application.

Compared with prior art, the invention has the advantages that：

(1) first demonstration that paddy gene Loc_Os01g12810 is rice chloroplast development related gene. Loc_Os01g12810 is a PPR gene.What the clone of the gene and biological function verification developed rice chloroplast The Effect study of Study on Molecular Mechanism and PPR family genes in Development of Rice has important reference significance.

(2) the present invention provides the rice conversion loads that Loc_Os01g12810 gene expressions are interfered using pUbi promoters Body.The expression quantity of Loc_Os01g12810 can be greatly reduced after the carrier rice transformation, along with the reduction of expression quantity, conversion is planted The seedling leaf of strain and the chlorophyll content of heading stage glume are substantially reduced, and the phenotype of albefaction occur, and with the later stage Growth, albefaction can restore to green, and transfer-gen plant can also be normal solid.Therefore, the present invention passes through pUbi promoter low amounts The technology for expressing Loc_Os01g12810, obtain one it is new turn green mutant in vain, can be applied to rice chloroplast development and adjust The research of control mechanism, and lay the foundation with utilization for photosynthetic research.Due to turning for Loc_Os01g12810 low amounts expression Gene plant show as white turn it is green, have no effect on the normal solid of rice, therefore the homozygous hybridization water of screening can also be applied to In the production practices of rice.

Description of the drawings

Fig. 1 is the carrier figure of pYLRNAi.5；

Fig. 2 is that Transgenic Rice strain compares cylindricality relative to the expression quantity of the gene Loc_Os01g12810 of wild system Figure；

Fig. 3 is that Transgenic Rice strain blade the photo of part albefaction occurs；

Fig. 4 is the photo that Transgenic Rice strain blade is gradually brought to normal green；

Fig. 5 is the photo of Transgenic Rice strain and wild system's reproductive stage.

Specific implementation mode

The following examples are further illustrations of the invention, rather than limiting the invention.In the following example not It is usually conventional means well-known to those skilled in the art to indicate specific experiment condition and method, used technological means.

The structure of 1 recombinant plasmid of embodiment

The present embodiment construction recombination plasmid, is as follows：

The seedling leaves position of water intaking rice varieties Lijiang xintuanheigu (LTH), with TriZol Reagent (Invitrogen Company, article No. are：15596026) blade total serum IgE is extracted, it is total using agarose gel electrophoresis and UV spectrophotometer measuring The purity and amount of RNA, takes the total serum IgE of 1 μ g to do starting reverse transcription reaction, and used reverse transcriptase is PrimeScript The step of (TAKARA companies), reverse transcription reaction the operation instruction with reference to the reverse transcriptase.Using reverse transcription product as template, use Primer 1 carries out PCR amplification, and the forward and reverse nucleotide sequence of the primer 1 is respectively such as SEQ ID NO in sequence table：4 Hes SEQ ID NO：Shown in 5.Polymerase used in PCR is KOD FX (Toyobo companies).Reaction system is 50uL, according to KOD FX Specification prepare PCR reaction systems.Reaction condition is：94℃5min；94 DEG C of 30sec, 60 DEG C of 30sec, 68 DEG C of 90sec, 35 A cycle；68℃10min.PCR amplification obtains the segment of about 452bp.

After recycling the segment using agarose gel electrophoresis, with BamHI and HindIII to segment and pYLRNAi.5 carriers (as shown in Figure 1) carries out double digestion respectively, and target fragment and carrier segments is separately recovered after digestion, and with T4 ligases, (NEB is public Department) 16 DEG C of connections 16 hours, linked system is：T4 ligases 1uL, 10Xbuffer 1uL, Loc_Os01g12810 genetic fragment 6ul (200ng), pYLRNAi.5 carriers 2uL (50ng).1uL connection products are taken, are transformed into bacillus coli DH 5 alpha with electric shocking method, Converted product is coated with the LB solid mediums of kalamycin resistance.37 DEG C of overnight incubations choose 10 monoclonals and extract plasmid, Digestion is identified, two positive colonies is selected to carry out sequencing detection (sequencing primer is amplimer).To be transferred to the sun of the first segment Property grain is template, carries out the PCR amplification of the second segment using universal primer 1 and universal primer 2, the universal primer 1 and logical With the nucleotide sequence of primer 2 respectively such as SEQ ID NO in sequence table：6 and SEQ ID NO：Shown in 7.Polymerase used in PCR For KOD FX (Toyobo companies).Reaction system is 50uL, and PCR reaction systems are prepared according to the specification of KOD FX.React item Part is：94℃5min；94 DEG C of 30sec, 60 DEG C of 30sec, 68 DEG C of 90sec, 35 cycles；68℃10min.PCR amplification obtains about After recycling the segment using agarose gel electrophoresis, to segment and first has been connected into MluI and PstI for the second segment of 500bp The pYLRNAi.5 carriers of segment carry out double digestion respectively, and target fragment and carrier segments is separately recovered after digestion, with T4 ligases 16 DEG C of (NEB companies) connection 16 hours, linked system are：T4 ligases 1uL, 10Xbuffer 1uL, Loc_Os01g12810 base Second segment 6ul (200ng) of cause, has been connected into the pYLRNAi.5 carriers 2uL (50ng) of the first segment.1uL connection products are taken, It is transformed into bacillus coli DH 5 alpha with electric shocking method, converted product is coated with the LB solid mediums of kalamycin resistance.37 DEG C of cultures Overnight, 10 monoclonals are chosen and extract plasmid, the identification of BamHI single endonuclease digestions selects positive colony to carry out sequencing detection, thus structure Build recombinant plasmid.

Embodiment 2

The recombinant plasmid obtained in embodiment 1 is transferred to by normal round-grained rice using the Agrobacterium EHA105 genetic transforming methods mediated In seed in rice product.Convert primary (T₀Generation) pass through PCR and fluorescence quantitative PCR detection, detection target gene Loc_ Os01g12810 is control with wild tying fruit in the expression quantity of rna level.Quantitative fluorescent PCR identifies Loc_Os01g12810 Gene expression effect in transfer-gen plant, steps are as follows：

The tri-leaf period seedling leaves that the difference for taking PCR detections positive turns transgenic line carry out Total RNAs extraction, the examination of use Agent is that (Invitrogen companies, article No. are TriZol Reagent：15596026), according to the specification of reagent the step of It carries out, the purity and amount of agarose gel electrophoresis and UV spectrophotometer measuring total serum IgE is used in combination, the total serum IgE of 1 μ g is taken to originate Reverse transcription reaction, used reverse transcriptase are PrimeScript (Takara companies), anti-with reference to this step of reverse transcription reaction The operation instruction of transcriptase.Using reverse transcription product as template, the expression feelings of Loc_Os01g12810 genes are detected using primer 2 Condition, the forward and reverse nucleotide sequence of the primer 2 is respectively such as SEQ ID NO in sequence table：8 and SEQ ID NO：Shown in 9. The expression of rice EF1a genes is detected as internal reference, reality using EF1aF the and EF1aR primer pairs of rice housekeeping gene EF1a genes It tests result and sees Fig. 2.As a result 8 transgenic lines are proved relative to wild system, the expression quantity of Loc_Os01g12810 all lowers 2-6 It differs again.

Embodiment 3

8 T in embodiment 2₀Seed for transgenic line and wild system ZH11 seeds, put in 49 DEG C of incubators The suspend mode that 72h breaks seed is set, seed is soaked for 24 hours under room temperature, and seed 48h is placed at 32 DEG C, the sowing extremely dress after seed sprouting There is in the vinyl disc of soil (3 biology of design repeat), grown under plastic foil covering, Loc_Os01g12810 interference turns Gene plant shows as aerial part albefaction (see Fig. 3).In the grown under normal conditions of same temperature condition, Loc_ Os01g12810 interference of transgene plant show as the part albinism of blade, are transferred in normal habitat, albefaction journey Degree can slowly weaken, and be gradually brought to normal green (see Fig. 4), and when growing to heading stage, tassel also lacks chlorophyll, presents Albinism, but compared with wild system, the normal solid (see Fig. 5, wherein left side is wild of interference of transgene plant is not influenced It is ZH11, right side is transgenosis pearl system).

The results show that in same growing environment, the seedling that wild system ZH11 seeds are grown is normal green, and 8 A T₀The rice seedlings of white turn of green phenotype are then all isolated for the seed of transgenic line.Illustrate that Loc_Os01g12810 is participated in The variation for regulating and controlling leaf color, participates in the morphogenesis of chloroplaset.It is also hybrid rice not influence the normal solid green phenotype that turns in vain Breeding provides the theoretical foundation of high frequency zone.

Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

<110>Inst. of Rice, Guangdong Academy of Agricultural Sciences

<120>A kind of application of gene Loc_Os01g12810

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1911

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

atggtagaga acggtgtgca gctcacattg acctccactg tcaagaccaa tggcaagata 60

agaatcaatg gagaagtgct aaacggatca cacctcaaag agaagcacga agctggcagc 120

aatggcgccc tgcacccatc aaacgggcaa gcaaagcagc cgccgtcgcc gccgcagaag 180

cagaagcaga agctgctctg cacgacgtgc ggcaaggggc acacgtgcca ggcggtgatc 240

gcccggacgc ggcagatgcg cgccatgatc gacgcgagga ggccgcacca ggcgcactcg 300

gcgttccgcc acctcgtcga cgacggccac cgcccgtcgc tggtgacgta caccacgctg 360

ctcgcggcgc tgacgagcca gcgggcgttc gacaccatcc cgtggctgct cgccgaggtg 420

gaggacgccg gcctccgccc ggactccatc ttcttcaacg cgctcatcaa cgccctcgtc 480

gaggcccggc ggatgggcga ggcgacgaac accttcctca ggatgggcca ctccggctgc 540

cgccccacgg cgagcacgtt caacacgctg atcaagggct acggcatagc cggccgcccc 600

gaggagtcgc agcgggtgtt cgacatgatg gcctccggcg gcgccggcgg cgaggcggcg 660

gtcaggccga acctgacgac gtacaacatc ctcgtcaagg cgtggtgcga cgccgggagg 720

ctggaggagg cgtggcgggt ggtggcgagg atgcgcgcgt cgggcgccga cccggacgtc 780

gtcacgtaca acaccctcgc cagcgcgtac gccaagaacg acgagacgtg gcgcgccgag 840

gagctcgtcg tggagatggc gcagcaggca gggctccgca ccagcgagcg cacctggggc 900

atcatcgtcg gcggctactg ccgcgagggc cgcctcggcg aggcgctccg ctgcgtccgc 960

cagatgaagg actccggcgt gctccccaac gtcatcgtct tcaacaccct gctcaagggc 1020

ttcctcgacg ccaacgacat ggccgccgtc gacgacgttc ttgggctgat ggagcagttc 1080

gggatcaagc cggacatcgt gacctacagc caccagctga acgcgctgag ctccatgggg 1140

cacatggcca agtgcatgaa ggtgttcgac aaaatgatcg aggccgggat cgagccggac 1200

ccgcaggtgt acagcatcct cgccaagggc tacgtgcgcg cgcagcagcc ggagaaggcg 1260

gaggagctcc tgcgccagat gggccgcctc ggcgtccgcc ccaacgtcgt caccttcacc 1320

accgtcatca gcgggtggtg cagcgtcgcc gacatgggca acgccgtgag ggtctacgcc 1380

gccatgcgcg acgccggcgt gcgcccgaac ctcaggacgt tcgagaccct catctggggc 1440

tacagcgagc tgaagcagcc atggaaggcc gaggaggtgc tccagatgat gcaggacgcc 1500

ggcgtccgcc ccaagcagac cacctactgc ctcgtcgccg acgcgtggaa ggccgtcggc 1560

ctcgtcgaga acgccaaccg cgcgctcggc tcctcctcct cctccggcga cctcctcgac 1620

gccgacgacg acgaggagcc ctacttcccg gacaaccatg gcgacgacaa gctgcagagc 1680

ttcgagagaa ccaatggcca tgccaagagt gacgcgtcac gatccatgca ggtgacaaga 1740

gcttccatga gcttgaagac ggcgaggtcg tcgtcgccgt cgctgtcgct gctccggcga 1800

tcgtgccggc ttccggtgag atccacgtgg ctttgcagga agcagcttca gatgcagtgt 1860

ggagtgtatg gccagagcat cagctcactg aagatggtgt tcctcagtta g 1911

<210> 2

<211> 636

<212> PRT

<213>Artificial sequence (Artificial Sequence)

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Met Val Gly Ala Gly Val Gly Leu Thr Leu Thr Ser Thr Val Leu Thr

1 5 10 15

Ala Gly Leu Ile Ala Ile Ala Gly Gly Val Leu Ala Gly Ser His Leu

20 25 30

Leu Gly Leu His Gly Ala Gly Ser Ala Gly Ala Leu His Pro Ser Ala

35 40 45

Gly Gly Ala Leu Gly Pro Pro Ser Pro Pro Gly Leu Gly Leu Gly Leu

50 55 60

Leu Leu Cys Thr Thr Cys Gly Leu Gly His Thr Cys Gly Ala Val Ile

65 70 75 80

Ala Ala Thr Ala Gly Met Ala Ala Met Ile Ala Ala Ala Ala Pro His

85 90 95

Gly Ala His Ser Ala Pro Ala His Leu Val Ala Ala Gly His Ala Pro

100 105 110

Ser Leu Val Thr Thr Thr Thr Leu Leu Ala Ala Leu Thr Ser Gly Ala

115 120 125

Ala Pro Ala Thr Ile Pro Thr Leu Leu Ala Gly Val Gly Ala Ala Gly

130 135 140

Leu Ala Pro Ala Ser Ile Pro Pro Ala Ala Leu Ile Ala Ala Leu Val

145 150 155 160

Gly Ala Ala Ala Met Gly Gly Ala Thr Ala Thr Pro Leu Ala Met Gly

165 170 175

His Ser Gly Cys Ala Pro Thr Ala Ser Thr Pro Ala Thr Leu Ile Leu

180 185 190

Gly Thr Gly Ile Ala Gly Ala Pro Gly Gly Ser Gly Ala Val Pro Ala

195 200 205

Met Met Ala Ser Gly Gly Ala Gly Gly Gly Ala Ala Val Ala Pro Ala

210 215 220

Leu Thr Thr Thr Ala Ile Leu Val Leu Ala Thr Cys Ala Ala Gly Ala

225 230 235 240

Leu Gly Gly Ala Thr Ala Val Val Ala Ala Met Ala Ala Ser Gly Ala

245 250 255

Ala Pro Ala Val Val Thr Thr Ala Thr Leu Ala Ser Ala Thr Ala Leu

260 265 270

Ala Ala Gly Thr Thr Ala Ala Gly Gly Leu Val Val Gly Met Ala Gly

275 280 285

Gly Ala Gly Leu Ala Thr Ser Gly Ala Thr Thr Gly Ile Ile Val Gly

290 295 300

Gly Thr Cys Ala Gly Gly Ala Leu Gly Gly Ala Leu Ala Cys Val Ala

305 310 315 320

Gly Met Leu Ala Ser Gly Val Leu Pro Ala Val Ile Val Pro Ala Thr

325 330 335

Leu Leu Leu Gly Pro Leu Ala Ala Ala Ala Met Ala Ala Val Ala Ala

340 345 350

Val Leu Gly Leu Met Gly Gly Pro Gly Ile Leu Pro Ala Ile Val Thr

355 360 365

Thr Ser His Gly Leu Ala Ala Leu Ser Ser Met Gly His Met Ala Leu

370 375 380

Cys Met Leu Val Pro Ala Leu Met Ile Gly Ala Gly Ile Gly Pro Ala

385 390 395 400

Pro Gly Val Thr Ser Ile Leu Ala Leu Gly Thr Val Ala Ala Gly Gly

405 410 415

Pro Gly Leu Ala Gly Gly Leu Leu Ala Gly Met Gly Ala Leu Gly Val

420 425 430

Ala Pro Ala Val Val Thr Pro Thr Thr Val Ile Ser Gly Thr Cys Ser

435 440 445

Val Ala Ala Met Gly Ala Ala Val Ala Val Thr Ala Ala Met Ala Ala

450 455 460

Ala Gly Val Ala Pro Ala Leu Ala Thr Pro Gly Thr Leu Ile Thr Gly

465 470 475 480

Thr Ser Gly Leu Leu Gly Pro Thr Leu Ala Gly Gly Val Leu Gly Met

485 490 495

Met Gly Ala Ala Gly Val Ala Pro Leu Gly Thr Thr Thr Cys Leu Val

500 505 510

Ala Ala Ala Thr Leu Ala Val Gly Leu Val Gly Ala Ala Ala Ala Ala

515 520 525

Leu Gly Ser Ser Ser Ser Ser Gly Ala Leu Leu Ala Ala Ala Ala Ala

530 535 540

Gly Gly Pro Thr Pro Pro Ala Ala His Gly Ala Ala Leu Leu Gly Ser

545 550 555 560

Pro Gly Ala Thr Ala Gly His Ala Leu Ser Ala Ala Ser Ala Ser Met

565 570 575

Gly Val Thr Ala Ala Ser Met Ser Leu Leu Thr Ala Ala Ser Ser Ser

580 585 590

Pro Ser Leu Ser Leu Leu Ala Ala Ser Cys Ala Leu Pro Val Ala Ser

595 600 605

Thr Thr Leu Cys Ala Leu Gly Leu Gly Met Gly Cys Gly Val Thr Gly

610 615 620

Gly Ser Ile Ser Ser Leu Leu Met Val Pro Leu Ser

625 630 635

<210> 3

<211> 452

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

ggtgtgcagc tcacattgac ctccactgtc aagaccaatg gcaagataag aatcaatgga 60

gaagtgctaa acggatcaca cctcaaagag aagcacgaag ctggcagcaa tggcgccctg 120

cacccatcaa acgggcaagc aaagcagccg ccgtcgccgc cgcagaagca gaagcagaag 180

ctgctctgca cgacgtgcgg caaggggcac acgtgccagg cggtgatcgc ccggacgcgg 240

cagatgcgcg ccatgatcga cgcgaggagg ccgcaccagg cgcactcggc gttccgccac 300

ctcgtcgacg acggccaccg cccgtcgctg gtgacgtaca ccacgctgct cgcggcgctg 360

acgagccagc gggcgttcga caccatcccg tggctgctcg ccgaggtgga ggacgccggc 420

ctccgcccgg actccatctt cttcaacgcg ct 452

<210> 4

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

aaaggatccg gtgtgcagct cacattgac 29

<210> 5

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

aaaaagctta gcgcgttgaa gaagatgga 29

<210> 6

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

caccctgacg cgtggtgtta cttctgaaga gg 32

<210> 7

<211> 33

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

actagaactg cagcctcaga tctaccatgg tcg 33

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

acgttcttgg gctgatggag 20

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

gatcccggcc tcgatcattt 20

Claims

1. a kind of application of gene Loc_Os01g12810, which is characterized in that the gene Loc_Os01g12810 can be applied to In rice chloroplast developmental regulation Mechanism Study, the expression water of the gene Loc_Os01g12810 is regulated and controled by gene means It puts down to carry out the research of rice chloroplast developmental regulation mechanism；

SEQ ID NO in the nucleotide sequence of the gene Loc_Os01g12810 such as sequence table：Shown in 1；The gene Loc_ SEQ ID NO in the amino acid sequence of Os01g12810 coding albumen such as sequence table：Shown in 2.

2. application according to claim 1, it is characterised in that the gene means are gene silent technology, pass through the base Because of silent technology so that the expression of the gene Loc_Os01g12810 declines.

3. application according to claim 2, which is characterized in that the gene silent technology includes the following steps：

(1) a kind of recombinant plasmid is built；

(2) recombinant plasmid built in step (1) is imported in normal rice paddy seed, obtains the rice seed of transgenic line Son.

4. application according to claim 3, which is characterized in that the recombinant plasmid in the step (1) passes through into carrier It imports interference genetic fragment to obtain, SEQ ID NO in the nucleotide sequence of the interference genetic fragment such as sequence table：Shown in 3.

5. application according to claim 4, which is characterized in that total mRNA in rice leaf is taken, by total mRNA reverse transcriptions After cDNA, the interference genetic fragment is obtained after carrying out PCR amplification to the cDNA by primer 1；The forward direction of the primer 1 With reverse nucleotide sequence respectively such as SEQ ID NO in sequence table：4 and SEQ ID NO：Shown in 5.

6. application according to claim 4, which is characterized in that the carrier is pYLRNAi.5；The interference genetic fragment It is first astern reversely cloned into respectively in the MCS1 and MCS2 in pUbi promoters downstream, to obtain the recombinant plasmid.

7. application according to claim 3, which is characterized in that the recombinant plasmid built in the step (1) passes through conversion Method imports in normal rice paddy seed, obtains the rice paddy seed of transgenic line.

8. the rice paddy seed of the transgenic line obtained in being applied according to claim 3-7 any one.

9. application of the rice paddy seed of transgenic line according to claim 8 in Rice Photosynthesis research.

10. the production according to the rice paddy seed of the transgenic line described in claim 8 in the homozygous hybrid rice of screening is ground Application in studying carefully.