CN101096676A - Rice Chlorophyll Synthase Mutant Gene and Its Application in Genetic Engineering - Google Patents

Rice Chlorophyll Synthase Mutant Gene and Its Application in Genetic Engineering Download PDF

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CN101096676A
CN101096676A CNA2007100235543A CN200710023554A CN101096676A CN 101096676 A CN101096676 A CN 101096676A CN A2007100235543 A CNA2007100235543 A CN A2007100235543A CN 200710023554 A CN200710023554 A CN 200710023554A CN 101096676 A CN101096676 A CN 101096676A
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gene
rice
osygl1
ygl1
chlorophyll
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CN100569949C (en
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万建民
吴自明
张欣
江玲
陈亮明
苏宁
程治军
刘世家
刘喜
何冰
王春明
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Nanjing Agricultural University
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Nanjing Agricultural University
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Abstract

本发明公开了一个水稻叶绿素合成酶突变基因Osygl1的核苷酸序列及基因工程应用，属于基因工程领域。Osygl1的cDNA序列SEQ ID NO.1及编码的氨基酸序列SEQ ID NO.2。基因OsYGL1为水稻中一个叶绿素合成酶基因，OsYGL1参与水稻的叶绿素a和叶绿素b的合成，mRNA表达分析表明该基因呈组成性表达。该基因的突变(Osygl1基因)导致水稻黄绿叶的表型。转基因实验证明了该基因的黄绿叶功能，利用本发明Osygl1突变基因控制的黄绿叶性状可以作为遗传标记用于农业生产中的品种鉴定以及水稻遗传育种。The invention discloses a nucleotide sequence of a rice chlorophyll synthase mutant gene Osygl1 and its application in genetic engineering, belonging to the field of genetic engineering. The cDNA sequence of Osygl1 is SEQ ID NO.1 and the encoded amino acid sequence is SEQ ID NO.2. Gene OsYGL1 is a chlorophyll synthase gene in rice. OsYGL1 is involved in the synthesis of chlorophyll a and chlorophyll b in rice. mRNA expression analysis shows that the gene is constitutively expressed. Mutations in this gene (Osygl1 gene) result in a yellow-green leaf phenotype in rice. The transgenic experiment proves the yellow-green leaf function of the gene, and the yellow-green leaf trait controlled by the Osygl1 mutant gene of the present invention can be used as a genetic marker for variety identification in agricultural production and rice genetic breeding.

Description

Rice chlorophyll synthase mutant gene and genetically engineered thereof are used
Technical field
New rice chlorophyll synthase mutant gene Osygl1 of the present invention and genetically engineered thereof are used, and belong to the genetically engineered field, relate to the gene that rice chlorophyll is synthetic and paddy rice leaf look is grown specifically.
Technical background
The leaf look is the general performance of various pigments in the chloroplast(id), and chlorophyll is preponderated in the normal leaf, is usually expressed as green.Leaf variegation is the higher and mutant character that is easy to identify of mutation frequency in the higher plant.Up to now, in nearly all higher plants such as paddy rice, wheat, barley, corn, soybean, cotton, tobacco, corn, Sunflower Receptacle, tomato, cucumber, potato, mulberry tree, all found leaf look mutant.Its mutator gene directly or indirectly influences photosynthetic pigments, and particularly chlorophyllous synthetic and degraded causes the content of various pigments and ratio to change, thereby causes the leaf chromatic variation.
Paddy rice (Oryza sativa L.) is as one of most important food crop of the China and even the world, and the raising of its output has crucial strategic importance to solving following global food problem.For a long time, traditional cross-breeding means are not obtaining gratifying effect aspect the raising light-use as yet.Leaf look mutant is the ideal material that the research light energy of rice utilizes.At present, though obtained some yellowish green pallette variants in paddy rice, great majority research only is confined to the Primary Location of gene.The mutator gene of furtheing investigate these yellowish green pallette variants has important use value for improvement light energy of rice utilising efficiency and cross breeding seed control of purity.
This research is by map based cloning and functional analysis to the yellowish green pallette variant of the paddy rice of natural variation ygl1 gene, disclosed the molecule mechanism that causes the yellowish green pallette sex change of paddy rice shape from molecular level, and systematic study this gene and chloroplast(id) form, the relation of photosynthetic efficiency and relevant photosynthetic index, proposed to utilize the Osygl1 gene to carry out the method for the yellowish green leaf genetic improvement of paddy rice.
Summary of the invention
Technical problem
The objective of the invention is to disclose a new rice chlorophyll synthase mutant gene Osygl1 and genetically engineered thereof uses, this mutator gene Osygl1 can be used as goal gene and imports in the middle of the rice breed sterile line, to change the leaf look of blade, be used for the production of hybrid seeds of paddy rice two-line hybrid rice or genetic breeding.
Technical scheme
A kind of rice chlorophyll synthase mutant gene Osygl1, it is from paddy rice (Oryza sativa L.), length is 1131bp, 376 amino acid of corresponding encoded chlorophyll synthase, the mutational site that comprises the 198th amino acids sports Serine Ser (CCT) by proline(Pro) Pro (TCT), and complete sequence is seen SEQ ID NO.1.
With transgenic method this mutator gene Osygl1 is transferred in the light sensitive nuclear sterility based material, obtain to contain the Osygl1 gene, have the sterile line material of yellowish green leaf phenotype, can be used for the production of hybrid seeds of paddy rice two-line hybrid rice.
Beneficial effect
1, the invention discloses a kind of rice chlorophyll synthase mutant gene (Osygl1 gene) and application thereof.This gene can be used as goal gene and imports paddy rice from paddy rice (Oryza sativa L.), makes the blade yellow-green colour, or by selection cross, transformation yellow-green colour proterties obtains leaf look yellowish green rice varieties.Detect linked marker RM3838 (with the genetic distance 0.1cM of ygl1 gene in the yellowish green leaf look mutant), can be used for the yellowish green leaf marker gene of paddy rice cross breeding breeding molecular marker assisted selection ygl1.
2, the Osygl1 gene function that provides of the inventor is the chlorophyll synthetic final step reaction of involved in plant, and the mRNA expression analysis shows Osygl1 gene constructive expression in paddy rice.
3, Osygl1 gene of the present invention is from paddy rice, coded protein has the chlorophyll synthase activity, itself and higher plant and algae bio etc. have higher homology, be respectively 88.39%, 74.68% and 53.94% with avenaceous chlorophyll synthase ChlG, arabidopsis ' chlorophyll synthetic enzyme G4, Synechocystis sp.PCC 6803 chlorophyll synthase similaritys, be reduced to 20～30% with bacteriochlorophyll synthetic enzyme sequence similarity.Wherein motif (WAGHDF-197) exists only in the chlorophyll synthase of higher plant, lacks this motif in the bacterium, and yellowish green pallette becomes the end that gene ygl1 mutational site (Pro-198) is positioned at this motif.
4, utilize wild-type OsYGL1 gene to make up plant expression vector as goal gene, its conversion can make the rice varieties of yellow-green recover normal green.The invention provides the transgenosis application method of Osygl1 gene.
5, with transgenic method with the Osygl1 transgenosis in the light sensitive nuclear sterility based material, can obtain to contain the Osygl1 gene, have the sterile line material of yellowish green leaf phenotype, be used for the production of hybrid seeds of paddy rice two-line hybrid rice.
Embodiment
The molecular cloning of embodiment 1 rice chlorophyll synthase mutant gene
1, the location in mutator gene ygl1 site
Utilize yellowish green pallette variant 249 Huangs of paddy rice (ygl1) to train short by 64, USSR5, W002 and 02428 etc. with leaf look normal different rice varieties respectively and carry out positive and negative hybridization, the F that obtains 2Colony carries out genetic analysis, and the result shows that the yellowish green leaf phenotype of ygl1 controlled by recessive single-gene.From ygl1 mutant and the short 64 deutero-F of training 2In colony's (12,300 strain), chosen 2741 recessive individual plants of yellow leaf and be used for this mutant gene Fine Mapping.Summer in 2005, in 3 leaves, 1 heart stage evaluation phenotype, DNA was extracted in sampling simultaneously in the plantation of Institute of Crop Science, Chinese Academy of Agricultural Science solarium.From F 2Choose 11 yellowish green leaf individual plants and 34 greenery individual plants in the colony at random, this microcommunity is carried out pcr amplification with candidate's mark, and the linkage relationship in post analysis candidate mark and mutational site.SSR labeled primer RM516, RM164 (RM series primer is public, sees website www.gramene.org) that discovery is positioned in the middle part of the 5th karyomit(e) are chain with mutator gene.Analyze F with RM516 and RM164 2Colony, the mutator gene Primary Location between SSR mark RM516 and RM164, with RM516, RM164 respectively at a distance of 4.8cM and 13.1cM.Choose the SSR labeled primer between RM516 and RM164, the parent is carried out polymorphism analysis.The result has only SSR mark RM3838 and RM5454 (www.gramene.org) to show polymorphic between the parent.Utilize these two marks to whole F 2Colony analyzes, and the result shows that the two and mutational site are respectively at a distance of 0.0cM and 8.8cM.The 23 pairs of SSR primers that further utilized the rice genome design data are numbered y1～y23.The result has only y1 (SEQ ID NO.3,4), y5 (SEQID NO.5,6) and y22 (SEQ ID NO.7,8) to detect polymorphism between the parent, and wherein, mark y1 is between RM516 and RM3838, and mark y5 and y22 are between RM3838 and RM5454.With these several polymorphism marks to F 2Analyze for 2741 recessive individual plants of colony, find mutator gene (called after ygl1, yellow greenleaf1) between y1 and RM3838, apart from y1, the genetic distance of RM3838 and y22 is respectively 0.2cM, 0.1cM and 0.7cM.Subsequently according to Japan's warm and fine 9311 genomic sequence informations (www.ncbi.nlm.nih.gov), between SSR mark y1 and RM3838,26 pairs of CAPS marks have been designed, through polymorphism analysis, find out 7 pairs and had polymorphic CAPS mark, mark P25 (SEQ ID NO.19 wherein, 20) and P26 (SEQ ID NO.21,22) be divided into from, other mark P20 (SEQ ID NO.15,16) with the ygll gene locus, P5 (SEQ ID NO.9,10), P23 (SEQ ID NO.17,18), P8 (SEQ ID NO.11,12) and P11 (SEQ ID NO.13,14) reorganization exchange individual plant number are respectively 8,8,1,5 and 5.Therefore, between the mark P23 and P8 on the BAC AC136221, physical distance is 11kb to the ygl1 gene locus by Fine Mapping.
The molecule marker on table 1 PCR-based basis
Labeling pattern The mark title Primer sequence Amplification segment size (bp) Restriction enzyme
SSR mark CAPS mark y1 y5 y22 P5 P8 P11 P20 P23 P25 5′GCGGTTGAAGGCGTCGTA3′ 5′AGGGTGCTGAGTCACAATAGGT3′ 5′CCCCAAACTAATTTCCTCCT3′ 5′ACATCTGTAACCAATCCTCCC3 5′CTGCCCTTGAATAATGACG3′ 5′GCGACTGATCGGTACTCCT3′ 5′GGCTAGTTATGGGTTAGAGGGTA3 5′TCCCTTTTCAAATCACACGA3′ 5′CGTGCAGGTTGTGTGACTCT3′ 5′GTTACAGAGCCAGCCAGGAG3 5′TTTGATCGCCGTCATGTTTA3′ 5′CCGGTGGTGAAGTCGTAGAT3′ 5′GCAGTTATTGGAAGTCAGC3′ 5′ATTACATCTACGTGCAAAGTC 3′ 5′CAACCACCTCAAGCTCTTT3′ 5′ATATTTCCTTCCCTACCCA3′ 5′GGAATTAGTGCCACCAAAC3′ 5′GGGAATCAACAAGAAACGA3′ 116 100 177 709 922 934 685 197 834 XbaI TaqI HapII Nsil TaqI StyI
P26 5′CCTCATTTTCCTTGGAGCAG3′ 5′TCTTCGCACCTAAGGTCACA3′ 740 MfeI
Utilize genetic analysis and forecasting software (www.softberry.com) that predictive genes is carried out in this 11kb interval, find only to have two open reading frame (Open reading frame, ORFs), one is chlorophyll synthase gene (Chlorophyll synthetase), and we call YGL1 (GenBank accession number: EF432576) to it; Another is em gene (Embryogenic abscisi acid-inducible gene).
2, the clone of mutator gene ygl1
With the method for RT-PCR, primer is SEQ ID NO.23, SEQ ID NO.24; Wild-type OsYGL1 and mutant Osygl1 goal gene regional code district cDNA are increased out, directly reclaim, the repetition measurement preface of laying equal stress on compares with the DNAstar analysis software then.The RT-PCR amplification program is as follows: 94 ℃ of 2min, and 94 ℃ of 40s, 59 ℃ of 40s, 72 ℃ of 1min, totally 35 circulations, last 72 ℃ are extended 7min, 4 ℃ of Pause.
Sequential analysis is found, the coding region cDNA that amplifies wild-type OsYGL1 gene (is called for short wild-type YGL1 gene, GenBank accession number: EF432576) (be called for short mutator gene ygl1 with the coding region cDNA that amplifies mutant Osygl1 gene, SEQ ID NO.1), compare, find on the 8th exon, to have single base difference between the two, become cytosine(Cyt) (C), cause proline(Pro) (Pro-198) to Serine (Ser) sudden change by chest acyl pyrimidine (T-592).
Proline(Pro) is a kind of imino-acid, and its side chain is the heterocycle structure that nitrogen-atoms and α carbon are connected to form.Although its imino-can form peptide bond with other amino acid, side chain has limited rotating freely of α carbon, so proline(Pro) exists in the proteinic secondary structure element βZhuan Jiao of being everlasting; Serine is the uncharged polare Aminosaeren of side chain, and its side chain can form hydrogen bond with other polar group.If proline(Pro) is mutated into Serine in the chlorophyll synthase, the activity of enzyme may be affected.
3, the sequence information and the specificity analysis of mutator gene ygl1, wild-type YGL1 gene
The cDNA coding region, coding region of wild-type YGL1 gene is 1131bp, 376 the amino acid whose 41kDa albumen of encoding, the N end has 47 amino acid whose leader sequences, be the plastid membranin, this albumen is a new chlorophyll synthase, catalysis chlorophyllide (Chlorophyllide) and phytol tetra-sodium (Phytyl-PP) or two yak base tetra-sodium (Geranylgeranyl-PP) phytolization generate chlorophyll a.By search rice genome database, the result shows that YGL1 is a single copy gene, and by 15 exons, 14 introns are formed.YGL1 and higher plant and algae bio etc. have higher homology.Be respectively 88.39%, 74.68% and 53.94% with avenaceous chlorophyll synthase ChlG, arabidopsis ' chlorophyll synthetic enzyme G4, Synechocystis sp. PCC 6803 chlorophyll synthase similaritys, be reduced to 20～30% with bacteriochlorophyll synthetic enzyme sequence similarity.In addition, sequential analysis finds that a motif (WAGHDF-197) exists only in the middle of the chlorophyll synthase, and the bacteriochlorophyll synthetic enzyme lacks.
Single base mutation takes place in mutator gene ygl1 on the 8th exon, other sequence information and characteristic are identical with wild-type YGL1 gene.
4, mutator gene ygl1, YGL1 expression of gene are analyzed
Use quantitative RT-PCR method, compared the expression level of mutator gene ygl1 and wild type gene YGL1, the result shows that mutator gene ygl1 and YGL1 gene are all expressed, and do not have difference between mutant and wild-type in root, stem, leaf and fringe.The YGL1 expression of gene does not have difference yet in half-light treatment condition and the different development stage, intravital mutator gene ygl1 of mutant plant and wild-type plant body.These results show ygl1 gene and the YGL1 genome expression characterization that becomes second nature, and single base mutation does not influence the ygl1 gene and expresses in the ygl1 intravital stable state of suddenling change.
5, proline(Pro) changes to Serine and causes the active decline of chlorophyll synthase
Utilize external prokaryotic expression recombinant protein, use two kinds of substrate GGPP and PhyPP, recombinase is carried out the esterification activity analysis.The result shows: compare (establishing recombinase YGL1 catalytic substrate Chlide a esterification GGPP and PhyPP activity value is 1) with the wild-type recombinase, the activity of mutant recombinase ygl1 catalytic substrate Chlide a esterification substrate GGPP and PhyPP all descends, what be respectively wild-type recombinase esterification activity is 35.22% and 21.75%, shows that the change of ygl1 single amino acids influences the esterification activity of enzyme.
The transgenosis application test of embodiment 2 chlorophyll synthase gene YGL1 and the functional verification of chlorophyll synthase mutant gene ygl1
(1) contains the structure of YGL1 coding region cDNA expression vector
(the biological Lu Tiegang researcher of institute of the Chinese Academy of Agricultural Sciences provides with Pst I and Spe I (public) double digestion hygromycin resistance expression vector pCUbi1390; The Chinese Academy of Agricultural Sciences's Ph D dissertation, Peng Hao, 2005), reclaim carrier segments, standby; (the joint primer sequence is SEQ ID NO.25 to add Pst I/Spe I joint with the PCR mediation respectively at YGL1 gene cDNA sequence two ends, 26) insert the pMD18-T carrier, after order-checking is confirmed, enzyme is cut, electrophoresis and reclaim the purpose fragment, and connects and enter above-mentioned expression vector and obtain the YGL1 gene recombined vector.
(2) agrobacterium mediation converted
With agrobacterium strains EHA105 is mediation, the importing of YGL1 gene recombined vector is had the yellow leaf round-grained rice of ygl1 allelotrope acceptor material strain ygl2, and (this strain is to be hybridized for No. 8 with military fortune round-grained rice by yellowish green pallette variant ygl1, backcross through 3 generations then, the yellow leaf japonica rice strain with yellowish green leaf feature that the selfing of 5 generations obtains contains mutator gene ygl1 through Molecular Identification):
(1) 28 ℃ of cultivation contains the Agrobacterium 16hr of the YGL1 that recombinates, collects thalline, and be diluted in the N6 liquid nutrient medium that contains 100 μ mol/L to concentration be OD 600≈ 0.5, obtains bacterium liquid;
(2) will be cultured to one month paddy rice mature embryo embryo callus and above-mentioned bacterium liquid mixed infection 30min, filter paper changes in the common culture medium (the N6 solid is culture medium altogether, and Sigma company buys) after blotting bacterium liquid, cultivates altogether 3 days for 24 ℃;
(3) above-mentioned callus is seeded on the N6 solid screening culture medium that contains 150mg/L Totomycin (purchase of Sigma company) and screened 16 days for the first time;
(4) the healthy callus of picking changes programmed screening on the N6 solid screening culture medium of 200mg/L Totomycin over to, and per 15 days subcultures once;
(5) the picking kanamycin-resistant callus tissue changes on the division culture medium that contains the 150mg/L Totomycin and breaks up;
(6) the reuse water rice plants of seedling differentiation is the transfer-gen plant of the chlorophyll synthase gene YGL1 that is obtained.
(3) transfer-gen plant Molecular Identification and ygl1 gene function checking
The transfer-gen plant of chlorophyll synthase gene YGL1 is through PCR Molecular Detection, Southern blot analysis, T 1Analyze for separating experiment and recovery phenotype plant chlorophyll content, confirmed that chlorophyll synthase gene YGL1 transforms successfully.Its phenotype is the green (the YGL1 gene is a dominant gene) after the yellow-green colour (the ygl1 gene is a recessive gene) before the transgenosis changes transgenosis into.The yellowish green leaf proterties of having verified the yellow leaf round-grained rice strain that transgenosis is preceding is by the ygl1 Gene Handling.
Those skilled in the art know, and the transgenosis application method of ygl1 mutator gene is used with the transgenosis of wild-type YGL1.With transgenic method this mutator gene is transferred in the light sensitive nuclear sterility based material, obtained to contain the Osygl1 gene, have the sterile line material of yellowish green leaf phenotype, can be used for the production of hybrid seeds of paddy rice two-line hybrid rice.
Public kind involved in the present invention is as follows:
Yellowish green pallette variant 249 Huangs of paddy rice (ygl1) (public kind, Gong Hongbing, Chen Liangming, Diao Li equality (2001) rice chlorophyll b reduces the genetic analysis and the correlation properties thereof of mutant. Scientia Agricultura Sinica, 34:686-689) training short by 64 (public kind, Luo Xiaohe, the seed selection of Yuan Longping (2000) paddy rice light affinity system. hybrid rice, 2000,15:1-3)
USSR5 (public kind, Jiang Ling, Xu Junfeng, the precocial genetic analysis of Wei Xiangjin etc. (2007) rice varieties USSR5. Acta Genetica Sinica 2007,34 (1): 46-55, the Chinese Academy of Agricultural Sciences's germplasm resource bank externally provides) W002 (public kind, Zhang Jianyong, universal unit, the stability analysis of Xiao Yinghui etc. (2004) rice varieties food flavor quality trait. Scientia Agricultura Sinica 2004,37 (6): this laboratory of 788-794 externally provides) 02428 (public kind, Li Hebiao (1989) paddy rice light affinity resource---02428. China seed industry 1989,4:4)
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉rice chlorophyll synthase OsYGL1 gene and transgenic engineering thereof are used
＜130〉specification sheets
<140>00
<141>2007-06-05
<160>26
<170>PatentIn version 3.1
<210>1
<211>1131
<212>DNA
＜213〉Oryza sativa (paddy rice)
<220>
<221>Osygl1 gene
<222>(1)..(1131)
<223>
<400>1
atggccacct cccacctcct cgccgccgcc tcctccaccg ccgcctcctc cgccaccttc 60
cgccctcccc tcctcagcct ccgctccccg ccgccctctt ctctccgcct caaccgcagg 120
cgccacttcc aggtggtccg cgcggccgag accgacaaag agacgaaggc caatgcgccc 180
gagaaagctc ccgcgggagg ctccagcttc aaccagctgc tcggcatcaa gggcgccaag 240
caagagaacg acatatggaa gattcgcctt caacttacta agccagtgac atggcctccg 300
cttgtatggg gagtgctttg tggagcagct gcctctggaa atttccactg gacagttgaa 360
gatgttgcaa aatctattgt ctgcatgata atgtctggtc catgtcttac aggatacaca 420
cagacaatta acgactggta tgatcgagat atagatgcga taaatgaacc ttaccgtcct 480
attccttcag gcgctatatc agaaaatgag gtcattactc aaatttgggc actgttgtta 540
gcagggcttg gcctgggtgc tctgttagat gtatgggcag gacatgattt ttctattatt 600
ttttatcttg ctgttggtgg gtccttgctt tcttacatat attcagctcc acctctgaag 660
ctcaagcaga atggatggat tggaaatttt gctcttggtg cgagctacat tggcttgccc 720
tggtgggctg gccaggcatt atttggaacc cttactcctg atattgttgt cctgacttct 780
ttgtatagca tagctgggct agggattgct attgtaaatg actttaagag tgttgaggga 840
gatagagctc tggggcttca gtcactcccg gttgcttttg gtatggaaac tgcaaagtgg 900
atatgtgtag gagcgattga cataactcaa ttatccgttg caggctacct tttcagttcc 960
ggcaagcctt attatgccct ggcactgcta ggactcacaa ttcctcaggt ggtctttcag 1020
ttccaatact tcctcaagga ccctgtgaag tatgacgtca aataccaggc aagcgcgcaa 1080
ccgttcttcg tcttgggcct tctggtgacc gcccttgcaa ccagccactg a 1131
<210>2
<211>376
<212>PRT
＜213〉Oryza sativa (paddy rice)
<220>
＜221〉the proteinic aminoacid sequence of OsYGL1
<222>(1)..(376)
<223>
<400>2
Met Ala Thr Ser His Leu Leu Ala Ala Ala Ser Ser Thr Ala Ala Ser
1 5 10 15
Ser Ala Thr Phe Arg Pro Pro Leu Leu Ser Leu Arg Ser Pro Pro Pro
20 25 30
Ser Ser Leu Arg Leu Asn Arg Arg Arg His Phe Gln Val Val Arg Ala
35 40 45
Ala Glu Thr Asp Lys Glu Thr Lys Ala Asn Ala Pro Glu Lys Ala Pro
50 55 60
Ala Gly Gly Ser Ser Phe Asn Gln Leu Leu Gly Ile Lys Gly Ala Lys
65 70 75 80
Gln Glu Asn Asp Ile Trp Lys Ile Arg Leu Gln Leu Thr Lys Pro Val
85 90 95
Thr Trp Pro Pro Leu Val Trp Gly Val Leu Cys Gly Ala Ala Ala Ser
100 105 110
Gly Asn Phe His Trp Thr Val Glu Asp Val Ala Lys Ser Ile Val Cys
115 120 125
Met Ile Met Ser Gly Pro Cys Leu Thr Gly Tyr Thr Gln Thr Ile Asn
130 135 140
Asp Trp Tyr Asp Arg Asp Ile Asp Ala Ile Asn Glu Pro Tyr Arg Pro
145 150 155 160
Ile Pro Ser Gly Ala Ile Ser Glu Asn Glu Val Ile Thr Gln Ile Trp
165 170 175
Ala Leu Leu Leu Ala Gly Leu Gly Leu Gly Ala Leu Leu Asp Val Trp
180 185 190
Ala Gly His Asp Phe Ser Ile Ile Phe Tyr Leu Ala Val Gly Gly Ser
195 200 205
Leu Leu Ser Tyr Ile Tyr Ser Ala Pro Pro Leu Lys Leu Lys Gln Asn
210 215 220
Gly Trp Ile Gly Asn Phe Ala Leu Gly Ala Ser Tyr Ile Gly Leu Pro
225 230 235 240
Trp Trp Ala Gly Gln Ala Leu Phe Gly Thr Leu Thr Pro Asp Ile Val
245 250 255
Val Leu Thr Ser Leu Tyr Ser Ile Ala Gly Leu Gly Ile Ala Ile Val
230 265 270
Asn Asp Phe Lys Ser Val Glu Gly Asp Arg Ala Leu Gly Leu Gln Ser
275 280 285
Leu Pro Val Ala Phe Gly Met Glu Thr Ala Lys Trp Ile Cys Val Gly
290 295 300
Ala Ile Asp Ile Thr Gln Leu Ser Val Ala Gly Tyr Leu Phe Ser Ser
305 310 315 320
Gly Lys Pro Tyr Tyr Ala Leu Ala Leu Leu Gly Leu Thr Ile Pro Gln
325 330 335
Val Val Phe Gln Phe Gln Tyr Phe Leu Lys Asp Pro Val Lys Tyr Asp
340 345 350
Val Lys Tyr Gln Ala Ser Ala Gln Pro Phe Phe Val Leu Gly Leu Leu
355 360 365
Val Thr Ala Leu Ala Thr Ser His
370 375
<210>3
<211>18
<212>DNA
＜213〉synthetic
<220>
＜221〉y1 forward primer
<222>(1)..(18)
<223>
<400>3
gcggttgaag gcgtcgta 18
<210>4
<211>22
<212>PRT
＜213〉synthetic
<220>
＜221〉y1 reverse primer
<222>(1)..(22)
<223>
<400>4
Ala Gly Gly Gly Thr Gly Cys Thr Gly Ala Gly Thr Cys Ala Cys Ala
1 5 10 15
Ala Thr Ala Gly Gly Thr
20
<210>5
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉y5 forward primer
<222>(1)..(20)
<223>
<400>5
ccccaaacta atttcctcct 20
<210>6
<211>21
<212>DNA
＜213〉synthetic
<220>
＜221〉y5 reverse primer
<222>(1)..(21)
<223>
<400>6
acatctgtaa ccaatcctcc c 21
<210>7
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉y22 forward primer
<222>(1)..(19)
<223>
<400>7
ctgcccttga ataatgacg 19
<210>8
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉y22 reverse primer
<222>(1)..(19)
<223>
<400>8
gcgactgatc ggtactcct 19
<210>9
<211>23
<212>DNA
＜213〉synthetic
<220>
＜221〉P5 forward primer
<222>(1)..(23)
<223>
<400>9
ggctagttat gggttagagg gta 23
<210>10
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P5 reverse primer
<222>(1)..(20)
<223>
<400>10
tcccttttca aatcacacga 20
<210>11
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P8 forward primer
<222>(1)..(20)
<223>
<400>11
cgtgcaggtt gtgtgactct 20
<210>12
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P8 reverse primer
<222>(1)..(20)
<223>
<400>12
gttacagagc cagccaggag 20
<210>13
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P11 forward primer
<222>(1)..(20)
<223>
<400>13
tttgatcgcc gtcatgttta 20
<210>14
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P11 reverse primer
<222>(1)..(20)
<223>
<400>14
ccggtggtga agtcgtagat 20
<210>15
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉P20 forward primer
<222>(1)..(19)
<223>
<400>15
gcagttattg gaagtcagc 19
<210>16
<211>21
<212>DNA
＜213〉synthetic
<220>
＜221〉P20 reverse primer
<222>(1)..(21)
<223>
<400>16
attacatcta cgtgcaaagt c 21
<210>17
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉P23 forward primer
<222>(1)..(19)
<223>
<400>17
caaccacctc aagctcttt 19
<210>18
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉P23 reverse primer
<222>(1)..(19)
<223>
<400>18
atatttcctt ccctaccca 19
<210>19
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉P25 forward primer
<222>(1)..(19)
<223>
<400>19
ggaattagtg ccaccaaac 19
<210>20
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉P25 reverse primer
<222>(1)..(19)
<223>
<400>20
gggaatcaac aagaaacga 19
<210>21
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P26 forward primer
<222>(1)..(20)
<223>
<400>21
cctcattttc cttggagcag 20
<210>22
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉P26 reverse primer
<222>(1)..(20)
<223>
<400>22
tcttcgcacc taaggtcaca 20
<210>23
<211>19
<212>DNA
＜213〉synthetic
<220>
＜221〉forward primer of RT-PCR amplification OsYGL1 and Osygl1 coding region cDNA
<222>(1)..(19)
<223>
<400>23
cagtctccaa tggccacct 19
<210>24
<211>20
<212>DNA
＜213〉synthetic
<220>
＜221〉reverse primer of RT-PCR amplification OsYGL1 and Osygl1 coding region cDNA
<222>(1)..(20)
<223>
<400>24
tgctttcatc agtggc tggt 20
<210>25
<211>27
<212>DNA
＜213〉synthetic
<220>
＜221〉joint forward primer
<222>(1)..(27)
<223>
<400>25
aactgcagag tctccaatgg ccacctc 27
<210>26
<211>28
<212>DNA
＜213〉synthetic
<220>
＜221〉joint reverse primer
<222>(1)..(28)
<223>
<400>26
ggactagtgc tttcatcagt ggctggtt 28

Claims (2)

1. rice chlorophyll synthase mutant gene Osygl1, it is from paddy rice (Oryza sativa), length is 1131bp, 376 amino acid of corresponding encoded chlorophyll synthase, the mutational site that comprises the 198th amino acids of rice chlorophyll synthase gene OsYGL1 sports Serine Ser (TCT) by proline(Pro) Pro (CCT), and complete sequence is seen SEQ ID NO.1.
2. the genetically engineered of the described a kind of rice chlorophyll synthase mutant gene Osygl1 of claim 1 is used.
CNB2007100235543A 2007-06-08 2007-06-08 Rice Chlorophyll Synthase Mutant Gene and Its Application in Genetic Engineering Expired - Fee Related CN100569949C (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831439A (en) * 2010-05-19 2010-09-15 重庆大学 Rice mutator gene associated with high chlorophyll content and recombinant plant overexpression vector thereof
CN102993284A (en) * 2011-09-14 2013-03-27 中国科学院植物研究所 New use of synechocystis protein in paddy rice property improvement
CN103966178A (en) * 2014-05-28 2014-08-06 江苏省农业科学院 Rice yellow-green leaf related protein as well as encoding gene and application of rice yellow-green leaf related protein
CN104839015A (en) * 2015-06-10 2015-08-19 浙江新安化工集团股份有限公司 Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation
CN105420256A (en) * 2015-12-31 2016-03-23 西南大学 Rice yellow-green leaf mutation gene YGL8, protein coded by rice yellow-green leaf mutation gene YGL8, and application of rice yellow-green leaf mutation gene YGL8
CN115820895A (en) * 2022-07-27 2023-03-21 湖南农业大学 Molecular Markers Closely Linked to Maize Chlorophyll Content and Its Application

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831439A (en) * 2010-05-19 2010-09-15 重庆大学 Rice mutator gene associated with high chlorophyll content and recombinant plant overexpression vector thereof
CN102993284A (en) * 2011-09-14 2013-03-27 中国科学院植物研究所 New use of synechocystis protein in paddy rice property improvement
CN103966178A (en) * 2014-05-28 2014-08-06 江苏省农业科学院 Rice yellow-green leaf related protein as well as encoding gene and application of rice yellow-green leaf related protein
CN103966178B (en) * 2014-05-28 2016-04-13 江苏省农业科学院 A kind of yellowish green leaf related protein of paddy rice and encoding gene thereof and application
CN104839015A (en) * 2015-06-10 2015-08-19 浙江新安化工集团股份有限公司 Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation
CN104839015B (en) * 2015-06-10 2017-04-26 浙江新安化工集团股份有限公司 Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation
CN105420256A (en) * 2015-12-31 2016-03-23 西南大学 Rice yellow-green leaf mutation gene YGL8, protein coded by rice yellow-green leaf mutation gene YGL8, and application of rice yellow-green leaf mutation gene YGL8
CN115820895A (en) * 2022-07-27 2023-03-21 湖南农业大学 Molecular Markers Closely Linked to Maize Chlorophyll Content and Its Application
CN115820895B (en) * 2022-07-27 2024-07-02 湖南农业大学 Molecular markers tightly linked to chlorophyll content in maize and their application

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