CN103613649A - Paddy rice leaf color control gene OscpSRP54 and protein encoded by same - Google Patents
Paddy rice leaf color control gene OscpSRP54 and protein encoded by same Download PDFInfo
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Abstract
The invention discloses a cDNA sequence represented by the SEQ ID No.1 of a paddy rice leaf color control gene OscpSRP54 coding area and an amino acid sequence represented by SEQ ID No.2 of a protein encoded by the gene. The gene OscpSRP54 which controls the paddy rice leaf color is cloned from a paddy rice light green leaf mutant HM14 through a map-based cloning technology, and the gene OscpSRP54 is proved to be the gene that control the paddy rice leaf color through a functional complementation experiment. Through observation of chloroplast by using a transmission electron microscope, the fact that the OscpSRP54 gene has the function of modulating chloroplast growth and then further controlling the leaf color is proved. The OscpSRP54 gene can be used to modulate paddy rice chloroplast growth so as to improve the photosynthesis efficiency and increase the paddy rice output. The leaf color control gene provided by the invention can also be used as a tracing marker in a genetic transferred plant progeny or an indicating marker for distinguishing pure species from hybrid species in a hybrid seed production process, and can be applied to the fields of good species breeding and hybrid breeding.
Description
Technical field
The invention belongs to plant genetic engineering field, be specifically related to a kind of rice Os cpSRP54 gene that utilizes map-based cloning clone, can utilize this gene regulating rice chloroplast to grow, improve photosynthetic efficiency, increase rice yield.Leaf color control gene of the present invention also can be used as the cue mark of true hybrid in gene transformation plant offspring's tracking mark and hybrid seeding process.
Background technology
Paddy rice is important with China in the world food crop, and the whole world has 35Yi Ren， China to have more than 60% population to take rice as staple food.For guaranteeing grain security, China started super hybridization rice evolutionary operation(EVOP) in 1996.Leaf photosynthesis efficiency is the important factor that determines rice yield, and the photosynthetic normal development that efficiently depends on chlorophyllous synthetic and chloroplast(id), chloroplast(id) is photosynthesis of plant and be the place of many important compound metabolism still not, therefore significant to the research of the growth course of chloroplast(id).
About 100 protein of higher plant chloroplast gene group coding, and in rice chloroplast, contain nearly 4800 albumen, enzyme and polypeptide.Therefore, most chloroplast proteins, by nuclear gene encoding, are transported in chloroplast(id) after translation in tenuigenin.Leaf look mutant refers to that leaf development process middle period color table type obviously differs from a class mutant of normal wild type leaf look, and this class mutant often shows as chlorophyll content to be reduced, the slow or abortion of Development of Chloroplasts.The clone of these mutator genes and functional study are grown understanding rice chloroplast, and the rice material of cultivating high light efficiency has important Research Significance.
Rice Leaf chromatic variation has and is easy to differentiate, be not subject to the features such as such environmental effects as morphological markers, the economical characters such as output is affected to little leaf variegation and in Rice Production, have important using value.Leaf color marker, as improving one of effective way of hybrid rice seeds purity, is applied by breeding man in the practices of breeding.
Only have at present the Rice Leaf look mutant gene of minority identified, still have a large amount of Ye Sejiyin to have to be identified.
Summary of the invention
The object of the present invention is to provide a kind of new Rice Leaf color base because of, there is the function of Rice Leaf color control, can utilize this gene regulating rice chloroplast to grow, improve photosynthetic efficiency, increase rice yield.
A first aspect of the present invention, provides a kind of paddy rice cpSRP54 albumen of separation.
A protein for Rice Leaf color control gene OscpSRP54 coding, the aminoacid sequence shown in the aminoacid sequence of described protein and SEQ ID No.2 has at least 90% homology, and described protein has the function of Rice Leaf color control.
Further, described protein amino acid sequence is in the aminoacid sequence shown in SEQ ID NO.2, to add, replace, insert or delete the aminoacid sequence with identical function or the derivative that one or more amino acid generates.
Preferably, the aminoacid sequence of described protein is as shown in SEQ ID NO.2.
In a second aspect of the present invention, provide a kind of polynucleotide of the above-mentioned protein of coding of separation.
Nucleotide sequence shown in the nucleotide sequence of described gene and SEQ ID NO.1 has at least 90% homology.
The nucleotide sequence of described gene is included in the nucleotide sequence of the mutant, allelotrope or the derivative that add, replace, insert or delete one or more Nucleotide in the nucleotide sequence shown in SEQ ID NO.1 and generate.
Preferably, the nucleotide sequence of described gene is as shown in SEQ ID NO.1.
The present invention also provides plasmid or plant expression vector or the host cell that contains said gene sequence.
Described plant expression vector is PcpSRP54.
Described host cell is Bacillus coli cells, agrobatcerium cell or vegetable cell.Host cell contains above-mentioned carrier, or in genome, is integrated with described polynucleotide.
In a fifth aspect of the present invention, a kind of Rice Leaf look thin out method that makes is provided, and the method comprises that the method by RNAi, Anti-senseRNA, gene knockout or gene inactivation reduces expression or the activity with the polypeptide of aminoacid sequence shown in SEQ ID NO:2.
The present invention clones the gene OscpSRP54 that controls Rice Leaf look from paddy rice pale green leaf mutant HM14 by map-based cloning, function complementation experiment proof OscpSRP54 is the gene of controlling Rice Leaf look.Chloroplast(id) transmission electron microscope observing proof the present invention clone's gene has regulation and control Development of Chloroplasts, and then controls the function of leaf look.Can utilize this gene regulating rice chloroplast to grow, improve photosynthetic efficiency, increase rice yield.Leaf color control gene of the present invention also can be used as the cue mark of true hybrid in gene transformation plant offspring's tracking mark and hybrid seeding process, is applied to stock breeding and cross-breeding.
Accompanying drawing explanation
Fig. 1 is Rice Leaf look mutation type surface; Wherein, 1: wild-type IR64; 2: mutant HM14.
Fig. 2 is
oscpSRP54the Fine Mapping of gene on paddy rice the 11st karyomit(e).
Fig. 3 is expression vector PcpSRP54 collection of illustrative plates.
Fig. 4 is that function complementation experiment T0 is for the phenotype schematic diagram of transgenic paddy rice; Wherein, 1:IR64; 2:HM14; 3:HM14-OscpSRP54.
Fig. 5 is the RT-PCR checking electrophorogram of transfer-gen plant; Wherein, M:Marker; 1:IR64; 2:HM14; 3-7: transfer-gen plant.
Fig. 6 is the expression electrophorogram of OscpSRP4 gene; Wherein, M:Marker; 1: leaf; 2: leaf sheath; 3: stem; 4: root; 5: young fringe.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further.
embodiment 1: the separation of mutant and genetic analysis
By the screening to the paddy rice of EMS mutagenesis (IR64) mutant library, separation has obtained a Rice Leaf look mutant HM14, and its phenotype energy genetic stability all shows as light green (as shown in Figure 1) within whole breeding time.Utilize mutant HM14 and Moroberekan hybridization to obtain F1, F1 plant has normal leaf color table type, the wild-type of F2 in generation and the separated separation ratio than meeting the single recessive mutation of Mendelian of mutation type surface, illustrate that the pale green leaf phenotype of mutant HM14 is controlled by single recessive gene.
embodiment 2: the Fine Mapping of mutator gene
According to the molecular genetic linkage map of the japonica rice of having announced and long-grained nonglutinous rice, choose the approximate SSR primer being uniformly distributed on each karyomit(e) and carry out polymorphic detection and Primary Location analysis, using the individual plant that there is mutation type surface in the F2 colony of mutant HM14 and Moroberekan hybridization acquisition as target group, identify that altogether 1008 strains have the F2 individuality of mutation type surface, 72 strains are wherein as first target group.Just positioning result shows that mutator gene is between the 11st the short arm of a chromosome RM1812 and RM26092.
Screening obtains between RM1812 and RM26092 primer RM26076, RM26079 and the RM26085 of polymorphism between parent, and analyzes the mutated individual in 1008 GeF2 colonies with these 5 pairs of primers, utilizes the information architecture physical map obtaining.Mutator gene Fine Mapping is within the scope of 40.7kb between the 11st karyomit(e) BAC clone AC116949 and the upper RM26076 of AC138196 and RM26079 mark (as shown in Figure 2) the most at last.
embodiment 3: predictive genes and sequence alignment analysis
According to the result of Fine Mapping, 8 of this interval predicted genes are analyzed, find a white cpSRP54 gene of coding chloroplast(id) signal identifying particle protein 8.In order-checking discovery mutant HM14, in this gene First Intron there is sudden change in last base, may affect normal montage, therefore this gene is decided to be to candidate gene called after OscpSRP54.
Utilize RT-PCR technology, with primer pair SEQ ID No.3(TCGCTCATCGGCAATGGA) and SEQ ID No.4(TTCCACGGCATTCTTGGTTATT) amplification cDNA, compare the clip size of OscpSRP54 gene in wild-type and mutant HM14, this experiment confirmation is because base mutation has caused the reservation of First Intron in mutant, and its amplified fragments than the large 119bp(of the wild-type of normal montage as shown in Figure 5).
the checking of embodiment 4:OscpSRP54 functional complementation
The complementary carrier (Fig. 3) that structure comprises OscpSRP54 full length gene coding region (SEQ ID NO.1) and self promotor, is transformed into Agrobacterium EHA105, with agrobacterium-mediated transformation, transforms mutant HM14.Transfer-gen plant shows as normal leaf color table type, by order-checking, find that it is bimodal in mutational site, RT-PCR checking simultaneously confirms to contain the fragment (Fig. 5) of normal montage in transfer-gen plant, and experimental result shows that complementary carrier can recover the mutant phenotype (Fig. 4) of HM14 completely.PcpSRP54 as shown in Figure 3, this carrier can be expressed above-mentioned nucleotide sequence coded polypeptide or its homology analogue.
the expression of embodiment 5:OscpSRP54 gene
Utilize RT-PCR technology, compared the expression of OscpSRP54 gene in each tissue, the expression that can obviously observe OscpSRP54 gene blade, leaf sheath from Fig. 6 is apparently higher than the expression in root and fringe, and the expression of OscpSRP54 in stem, do not detected.
The foregoing is only several embodiments of the present invention, it should be pointed out that the content disclosed by the invention of all distortion can directly derive or associate from to(for) those of ordinary skill in the art, all should think protection scope of the present invention.
Sequence table
<110> China Paddy Rice Inst
The protein of <120> Rice Leaf color control gene OscpSRP54 and coding thereof
<130> 2013
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 747
<212> DNA
<213> Oryza sativa
<400> 1
atggaggcca cggcgctcac gctctcctcc cgccccgccg ccgccagccg ctcgccggcg 60
aagggaacct tcgcctccct ccacccccgc cgccgcttct ccgcgcacgc tgtccacctc 120
cgcgcggccc agagcgcctc cctgcgcgcc ccctccccgg gggcgaggag gagacggagg 180
agggggagcg ggctggtggt gcgggcggag atgttcgggc agctcaccac cggcctggag 240
tccgcctgga acaagctgcg tggcgtcgat gtactaacaa aggagaatat cgttgagcca 300
atgcgggata taagaagagc acttctggaa gctgatgtta gtttgcctgt agtgagaaga 360
tttgtttcgt ctataagtga aaaggctctg ggttctgatc ttataagagg agtccgaccg 420
gaacagcaat tggtgaagat tgtgcatgat gaacttgtaa aactgatggg tggagaagta 480
tcagatctgg tatttgcaaa aagtggcccc acagtaatct tactggcagg cctgcaaggt 540
gttgggaaga ctactgtttg cgcaaagctt gcattttacc tgaagaaatt gatacttatg 600
ccagagttag gtgacaggga aagagttgta tgctggtcgc tgcagatgtt tacaggcctg 660
cagccattga tcaactcact atacttggtg aacaggtcgg tgtaccagtt tactcagaag 720
gaacagaagc caaacctgca caaataa 747
<210> 2
<211> 248
<212> PRT
<213> Oryza sativa
<400> 2
Met Glu Ala Thr Ala Leu Thr Leu Ser Ser Arg Pro Ala Ala Ala Ser
1 5 10 15
Arg Ser Pro Ala Lys Gly Thr Phe Ala Ser Leu His Pro Arg Arg Arg
20 25 30
Phe Ser Ala His Ala Val His Leu Arg Ala Ala Gln Ser Ala Ser Leu
35 40 45
Arg Ala Pro Ser Pro Gly Ala Arg Arg Arg Arg Arg Arg Gly Ser Gly
50 55 60
Leu Val Val Arg Ala Glu Met Phe Gly Gln Leu Thr Thr Gly Leu Glu
65 70 75 80
Ser Ala Trp Asn Lys Leu Arg Gly Val Asp Val Leu Thr Lys Glu Asn
85 90 95
Ile Val Glu Pro Met Arg Asp Ile Arg Arg Ala Leu Leu Glu Ala Asp
100 105 110
Val Ser Leu Pro Val Val Arg Arg Phe Val Ser Ser Ile Ser Glu Lys
115 120 125
Ala Leu Gly Ser Asp Leu Ile Arg Gly Val Arg Pro Glu Gln Gln Leu
130 135 140
Val Lys Ile Val His Asp Glu Leu Val Lys Leu Met Gly Gly Glu Val
145 150 155 160
Ser Asp Leu Val Phe Ala Lys Ser Gly Pro Thr Val Ile Leu Leu Ala
165 170 175
Gly Leu Gln Gly Val Gly Lys Thr Thr Val Cys Ala Lys Leu Ala Phe
180 185 190
Tyr Leu Lys Lys Leu Ile Leu Met Pro Glu Leu Gly Asp Arg Glu Arg
195 200 205
Val Val Cys Trp Ser Leu Gln Met Phe Thr Gly Leu Gln Pro Leu Ile
210 215 220
Asn Ser Leu Tyr Leu Val Asn Arg Ser Val Tyr Gln Phe Thr Gln Lys
225 230 235 240
Glu Gln Lys Pro Asn Leu His Lys
245
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<400> 3
tcgctcatcg gcaatgga 18
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
ttccacggca ttcttggtta tt 22
Claims (11)
1. the protein that Rice Leaf color control gene OscpSRP54 encodes, the aminoacid sequence shown in the aminoacid sequence of described protein and SEQ ID No.2 has at least 90% homology, and described protein has the function of Rice Leaf color control.
2. protein according to claim 1, it is characterized in that, described protein amino acid sequence is in the aminoacid sequence shown in SEQ ID NO.2, to add, replace, insert or delete the aminoacid sequence with identical function or the derivative that one or more amino acid generates.
3. protein according to claim 1, is characterized in that, the aminoacid sequence of described protein is as shown in SEQ ID NO.2.
4. the gene of protein described in the claim 1 of encoding, 2 or 3.
5. gene according to claim 4, is characterized in that, the nucleotide sequence shown in the nucleotide sequence of described gene and SEQ ID NO.1 has at least 90% homology.
6. gene according to claim 5, it is characterized in that, the nucleotide sequence of described gene is included in the nucleotide sequence of the mutant, allelotrope or the derivative that add, replace, insert or delete one or more Nucleotide in the nucleotide sequence shown in SEQ ID NO.1 and generate.
7. gene according to claim 4, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO.1.
8. plasmid or plant expression vector or a host cell that contains gene order described in the arbitrary claim of claim 4-7.
9. plant expression vector according to claim 8, is characterized in that, described plant expression vector is PcpSRP54.
10. host cell according to claim 8, is characterized in that, described host cell is Bacillus coli cells, agrobatcerium cell or vegetable cell.
11. 1 kinds make Rice Leaf look thin out method, it is characterized in that, described method comprises that the method by RNAi, Anti-senseRNA, gene knockout or gene inactivation reduces expression or the activity with the polypeptide of aminoacid sequence shown in SEQ ID NO:2.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820221A (en) * | 2015-12-14 | 2016-08-03 | 中国水稻研究所 | Rice leaf color regulatory protein and coding gene thereof, and application thereof |
| CN106011156A (en) * | 2016-07-06 | 2016-10-12 | 宁波大学 | Controlling gene OsERV1 for rice root development and leaf color formation and coded protein |
| CN106234210A (en) * | 2016-09-18 | 2016-12-21 | 四川农业大学 | Kytoplasm is utilized to control the method that leaf color mutant improves hybrid rice seeds purity |
| CN110423758A (en) * | 2019-06-30 | 2019-11-08 | 南京大学 | A kind of rice WSCP gene mutation body OsWSCP1 and its application |
| CN111705078A (en) * | 2020-06-19 | 2020-09-25 | 华南农业大学 | Application of CSL1 gene in regulation and control of rice chloroplast development |
| CN113999855A (en) * | 2021-10-29 | 2022-02-01 | 浙江省农业科学院 | Rice leaf color regulation gene mOsFC2 and application thereof |
| CN114574500A (en) * | 2022-03-22 | 2022-06-03 | 中国农业科学院深圳农业基因组研究所 | Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP |
| CN114836440A (en) * | 2022-06-06 | 2022-08-02 | 西南大学 | Rice leaf color regulation gene AF1 and application thereof |
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2013
- 2013-10-30 CN CN201310527040.7A patent/CN103613649A/en active Pending
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| TANAKA, T.等: "NCBI GenBank ID:NM_001189430", 《NCBI GENBANK》 * |
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| 魏彦林: "水稻淡绿叶突变体HM14的鉴定和基因定位", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820221A (en) * | 2015-12-14 | 2016-08-03 | 中国水稻研究所 | Rice leaf color regulatory protein and coding gene thereof, and application thereof |
| CN105820221B (en) * | 2015-12-14 | 2019-03-29 | 中国水稻研究所 | A kind of Rice Leaf color modulin and its encoding gene and application |
| CN106011156A (en) * | 2016-07-06 | 2016-10-12 | 宁波大学 | Controlling gene OsERV1 for rice root development and leaf color formation and coded protein |
| CN106011156B (en) * | 2016-07-06 | 2020-02-21 | 宁波大学 | Control gene OsERV1 for rice root development and leaf color formation and coded protein |
| CN106234210A (en) * | 2016-09-18 | 2016-12-21 | 四川农业大学 | Kytoplasm is utilized to control the method that leaf color mutant improves hybrid rice seeds purity |
| CN110423758A (en) * | 2019-06-30 | 2019-11-08 | 南京大学 | A kind of rice WSCP gene mutation body OsWSCP1 and its application |
| CN111705078A (en) * | 2020-06-19 | 2020-09-25 | 华南农业大学 | Application of CSL1 gene in regulation and control of rice chloroplast development |
| CN113999855A (en) * | 2021-10-29 | 2022-02-01 | 浙江省农业科学院 | Rice leaf color regulation gene mOsFC2 and application thereof |
| CN113999855B (en) * | 2021-10-29 | 2023-06-09 | 浙江省农业科学院 | Rice leaf tone control gene mOsFC2 and application thereof |
| CN114574500A (en) * | 2022-03-22 | 2022-06-03 | 中国农业科学院深圳农业基因组研究所 | Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP |
| CN114574500B (en) * | 2022-03-22 | 2022-11-22 | 中国农业科学院深圳农业基因组研究所 | Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP |
| CN114836440A (en) * | 2022-06-06 | 2022-08-02 | 西南大学 | Rice leaf color regulation gene AF1 and application thereof |
| CN114836440B (en) * | 2022-06-06 | 2023-04-07 | 西南大学 | Rice leaf color regulation gene AF1 and application thereof |
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Application publication date: 20140305 |