CN108395473A - Carotenoid in Plants synthesis associated protein and its encoding gene and application - Google Patents
Carotenoid in Plants synthesis associated protein and its encoding gene and application Download PDFInfo
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- CN108395473A CN108395473A CN201810148402.4A CN201810148402A CN108395473A CN 108395473 A CN108395473 A CN 108395473A CN 201810148402 A CN201810148402 A CN 201810148402A CN 108395473 A CN108395473 A CN 108395473A
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Abstract
The invention discloses a kind of Carotenoid in Plants synthesis associated protein and its encoding gene and applications.It is following (a1) or (a2) the present invention provides a kind of protein:(a1) protein that amino acid sequence forms shown in sequence in sequence table 2;(a2) by amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or lack and or add and with the protein with the same function derived from sequence 2.The present inventor clones WF1 genes from M. truncatula, successfully builds plant complementing vector, converts M. truncatula white flower-mutant using agriculture bacillus mediated leaf disk method, compared with the control, the flower for being transferred to the white flower M. truncatula of WF1 genes becomes yellow.Present disclosure can be to study the gene to regulate and control carotenogenesis in plant and lay the first stone.
Description
Technical field
The present invention relates to a kind of Carotenoid in Plants synthesis associated protein and its encoding gene and applications.
Background technology
Pulse family (Leguminosae) plant is the third-largest section for being only second to composite family and orchid family, extensively there are about 700 18000 kinds of categories
It is general to be distributed in the whole world, it is the important sources of human foods and oil plant and herbage, green manure, medicinal material and timber etc., has important
Economy, ecology and biological value.But since most of legume genome is larger, genetic conversion system is still immature
Etc. factors, limit the research of legume functional genomics.M. truncatula (Medicagotruncatula) is annual
Herbaceous plant, grazing-type herbage are beans because growth period is short, ploidy is small, genome is small, self-pollination and the features such as fixed nitrogen
The idealized model plant of section's Plant genetics and genomics research.
Carotenoid is the general name of a kind of C40 terpenoid compounds and its derivative, be all photosynthetic organisms it is basic at
Point, it is widely present in animal, plant, algae, bacterium and fungi, but animal itself cannot synthesize carotenoid, it can only be from outer
It absorbs on boundary.Carotenoid is the precursor substance of vitamin A needed by human, plays anti-oxidant, the work(such as removing free radical in vivo
Can, and it is related to the colors such as the yellow in the flowers of plant, fruit to red.Carotenoid identified at present is tied by it
Structure difference is divided into hydrocarbon carotenes, in orange, red;Alcohol carotenoid is in yellow;Ketone and acid carotenoid take on a red color.
Invention content
The object of the present invention is to provide a kind of Carotenoid in Plants synthesis associated protein and its encoding gene and applications.
The present invention provides a kind of protein (being named as WF1 albumen), are obtained from M. truncatula, are following (a1) or (a2):
(a1) protein that amino acid sequence forms shown in sequence in sequence table 2;
(a2) by amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/
Or lack and or add and with the protein with the same function derived from sequence 2.
In order to make WF1 albumen in (a1) convenient for purifying and detection, can in by sequence table amino acid sequence shown in sequence 1
Arrange the amino terminal or the upper label as shown in Table 1 of carboxyl terminal connection of the protein of composition.
The sequence of 1 label of table
Label | Residue | Sequence |
Poly--Arg | 5-6 (being usually 5) | RRRRR |
Poly-His | 2-10 (being usually 6) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
WF1 albumen in above-mentioned (a2) can be artificial synthesized, also can first synthesize its encoding gene, then carries out biological expression and obtain
It arrives.The encoding gene of WF1 albumen in above-mentioned (a2) can be by will lack one in DNA sequence dna shown in sequence in sequence table 1
Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 ' end and/
Or 3 ' end connect the coded sequence of label shown in table 1 and obtain.
The gene (WF1 genes) for encoding the WF1 albumen also belongs to protection scope of the present invention.
The gene is any DNA molecular in following (b1)-(b4):
(b1) DNA molecular shown in sequence 1 in code area such as sequence table;
(b2) DNA molecular shown in the sequence 5 of sequence table;
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode albumen described in claim 1
The DNA molecular of matter;
(b4) from M. truncatula and homologous with 90% or more with the DNA sequence dna of (b1) or (b2) or (b3) restriction
Property and encode claim 1 described in protein DNA molecular.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is miscellaneous in DNA or RNA
It hands over and hybridizes at 65 DEG C in experiment and wash film.
Recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing the WF1 genes belong to the present invention
Protection domain.
The recombinant expression carrier concretely inserts sequence table in the KpnI restriction enzyme sites of pCAMBIA2300 plasmids
Sequence 3 shown in DNA fragmentation, and DNA fragmentation shown in the sequence 4 of insetion sequence table obtains in PstI restriction enzyme sites
Recombinant plasmid.
The expression vector of the recombinant expression carrier concretely double chain DNA molecule shown in the sequence 5 containing ordered list.
The present invention also protects the application of WF1 albumen or WF1 genes in regulating and controlling Carotenoid in Plants content.
The present invention also protects a kind of method (method 1) for cultivating genetically modified plants, includes the following steps:Inhibit purpose plant
The expression of middle WF1 genes obtains the genetically modified plants of carotenoid content reduction.
The present invention also protects a kind of method (method 2) reducing Carotenoid in Plants content, includes the following steps:Inhibit
The activity and/or expression quantity of WF1 albumen in purpose plant obtain the genetically modified plants of carotenoid content reduction.
The present invention also protects a kind of method (method 3) for cultivating genetically modified plants, includes the following steps:WF1 genes are led
Enter purpose plant, obtains the genetically modified plants of carotenoid content raising.
In the method, the WF1 genes can import purpose plant by recombinant expression carrier.The recombinant expression carries
Body can be conventional by Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated etc.
Biological method is transformed into plant cell or tissue.
The recombinant expression carrier concretely inserts sequence table in the KpnI restriction enzyme sites of pCAMBIA2300 plasmids
Sequence 3 shown in DNA fragmentation, and DNA fragmentation shown in the sequence 4 of insetion sequence table obtains in PstI restriction enzyme sites
Recombinant plasmid.
The expression vector of the recombinant expression carrier concretely double chain DNA molecule shown in the sequence 5 containing ordered list.
The present invention also protects a kind of method (method 4) that raising Carotenoid in Plants content improves, and includes the following steps:
The activity and/or expression quantity for improving WF1 albumen in purpose plant obtain the genetically modified plants of carotenoid content raising.
The present invention also protects the application of WF1 albumen or the method for WF1 genes or any description above in plant breeding.
The purpose of the breeding is to cultivate the low plant of carotenoid high plant (method 3 or method 4) or carotenoid
Object (method 1 or method 2).
Any description above purpose plant is dicotyledon or monocotyledon.The dicotyledon is planted for Rosales
Object, the Rosales plant are legume.The legume is clover.The clover is puncture vine lucerne
Mu.In method 1 or method 2, the purpose plant more specifically can be M. truncatula R108.In method 3 or method 4, the purpose
Plant more specifically can be wf1 mutant (The Nobel Foundation, number:NF4496).
Any description above carrotene concretely lutein.
Any description above plant is dicotyledon or monocotyledon.The dicotyledon is Rosales plant,
The Rosales plant is legume.The legume is clover.The clover is M. truncatula, more
Concretely M. truncatula R108.
The present inventor clones WF1 genes from M. truncatula, plant complementing vector is successfully built, using agriculture bar
The leaf disk method conversion M. truncatula white flower-mutant that bacterium mediates is transferred to the flower of the white flower M. truncatula of WF1 genes compared with the control
Become yellow.Present disclosure can be to study the gene to regulate and control carotenogenesis in plant and lay the first stone.
Description of the drawings
Fig. 1 is that M. truncatula R108 wild types flower and M. truncatula wf1 white flower-mutants spend phenotypic map.
Fig. 2 is that R108 flowers and wf1 spend middle carotenoid content and composition measurement.
Fig. 3 is that WF1 is analyzed with white flower phenotype linkage relationship.
Fig. 4 is wf1 mutant complementations transfer-gen plant pattern phenotypic analysis and wf1 mutant complementation transfer-gen plant molecules
Detection.
Fig. 5 is the expression analysis of wf1 mutant complementation transfer-gen plant carotenogenesis related genes.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
M. truncatula R108 (wild type):The Nobel Foundation.
Wf1 mutant:The Nobel Foundation, number:NF4496.
Wf1-2 mutant:The Nobel Foundation, number:NF10625.
Wf1-3 mutant:The Nobel Foundation, number:NF10301.
PCAMBIA2300 plasmids:The Wuhan bio tech ltd Zhong Liyuan, article No.:P0283.
Agrobacterium AGL1:Biomed, article No.:BC302-01.
YEP solid mediums:Peptone 10g, yeast extract 10g, sodium chloride 5g, agar powder 10g, distilled water are settled to
1L。
YEP fluid nutrient mediums:Peptone 10g, yeast extract 10g, sodium chloride 5g, distilled water are settled to 1L.
Callus induced fluid culture medium (PH 5.8):A great number of elements mother liquor 100mL, micro- mother liquor 1mL, organic element
Mother liquor 1mL, mother liquid of iron salt 140mg, inositol 100mg, sucrose 30g, auxin 4mg, basic element of cell division 0.5mg, cephalo 200mg,
Ticarcillin/Clavulanate Acid 250mg, cremart 2mg, are settled to 1L.
Callus induces solid medium (PH 5.8):A great number of elements mother liquor 100mL, micro- mother liquor 1mL, organic element
Mother liquor 1mL, mother liquid of iron salt 140mg, inositol 100mg, sucrose 30g, auxin 4mg, basic element of cell division 0.5mg, cephalo 200mg,
Ticarcillin/Clavulanate Acid 250mg, cremart 2mg, Phytagel 3.2g, are settled to 1L.
Differential medium (PH 5.8):A great number of elements mother liquor 100mL, micro- mother liquor 1mL, organic element mother liquor 1mL,
Mother liquid of iron salt 140mg, inositol 100mg, sucrose 20g, cephalo 200mg, Ticarcillin/Clavulanate Acid 250mg, cremart 2mg, Phytagel
3.2g is settled to 1L.
Root media (PH 5.8):Murashige&Skoog Basal Medium with Vitamins 2.215g/
L (companies:PhytoTechnology Laboratories, article No.:16B0519138A)
Mother liquid of iron salt:Disodium ethylene diamine tetraacetate 37.3mg, green vitriol 27.8mg.
A great number of elements mother liquor:Bitter salt 1.85g, potassium nitrate 28.3g, ammonium sulfate 4.63g, CALCIUM CHLORIDE DIHYDRATE
1.66g, potassium dihydrogen phosphate 4g, distilled water are settled to 1L.
Micro- mother liquor:Manganous sulfate monohydrate 1g, boric acid 500mg, Zinc vitriol 100mg, potassium iodide 100mg,
Two molybdic acid hydrates receive 10mg, Salzburg vitriol 20mg, cobalt chloride hexahydrate 10mg, and distilled water is settled to 1L.
Organic element mother liquor:Niacin 500mg, thiamine hydrochloride 500mg, puridoxine hydrochloride 500mg, distilled water are settled to
1L。
The discovery of embodiment 1, WF1 and its encoding gene WF1
One, mutant acquisition and phenotypic analysis
By screening M. truncatula Tnt1 insertional mutagenesis libraries, obtains a pattern and white is become by the yellow of wild type
Extreme abnormal sudden change body, be named as white flower1 (wf1).
The flower Phenotypic Observation the result is shown in Figure 1 of wf1 mutant and M. truncatula R108 (wild type).In Fig. 1, A:Wild type
R108 spends front;B:Wild type R108 star-spangled banner valves;C:The ala and fossil fragments of wild type R108 flower fusions;D:Wild type R108 flowers
Diadelphous stamen;E:Wf1 spends front;F:Wf1 star-spangled banner valves;G:The ala and fossil fragments of wf1 flower fusions;H:Wf1 spends diadelphous stamen.It is wild
The ala closure bone flap for spending front, star-spangled banner valve, flower to merge of raw type R108 is yellow;Wf1 spends front, star-spangled banner valve, flower fusion
Ala and fossil fragments be white.
Two, pigments composition measures
Plant to be measured:M. truncatula R108 (wild type) and wf1 mutant.
The pigment composition in plant to be measured is analyzed, steps are as follows:
1, mortar and medicine spoon are cleaned, pour into appropriate absolute ethyl alcohol and lights calcination, room temperature cools down 1h, is used before being put into sample
Liquid nitrogen freezes 2-3 times.
2, take growth conditions consistent, wide-open plant petal about 20mg to be measured (about 8-9, removal petiole, sepal with
Stamen), it is put into liquid nitrogen rapidly after precise weight and freezes.
3, after completing step 2, petal is ground in the mortar that step 1 is handled well, after grinding, 100 μ l6% is added
(w/v) KOH/methanol solution, after vortex 10s mixings, the lower 60 DEG C of heating 1h of dark condition.
4, after completing step 3,100 μ l Tris-HCl buffer (50mM, pH7.5, NaCl containing 1M) is added, run up and down
8-10 times, 10min is placed after mixing well on ice.
5, after completing step 4,400 μ L chloroforms is added, 10min is placed on ice after the mixing that turns upside down.
6, after completing step 5,4 DEG C, 3000g centrifugation 5min take lower liquid.
7, upper layer remaining liq is extracted again according to step 5,6, the lower liquid that 2 times are obtained mixes, nitrogen
It is dissolved with 100 μ L ethyl acetate after drying, pigment composition is detected by HPLC.
Standard items:Lutein Lutein (Carotenature, NO.0133);
Chromatographic column:5 μm of C of YMC companies3020 × 4.6mmC of column (250 × 4.6mm) series connection30Column;
Sample size:10μL;
Flow velocity:1.0mL·min-1;
Column temperature:25℃;
Detection wavelength:450nm;
Mobile phase:A:Methanol;B:Water/methanol (volume ratio 1/4 contains 0.2% ammonium acetate);C:Tertiary methyl butyl ether
Gradient elution:95%A, 5%B isocratic elution 12 minutes, 80%A, 5%B, 15%C elution 12 minutes, subsequent 30%
A, 5%B, 65%C are eluted 30 minutes.
The results are shown in Figure 2.The result shows that it is lutein that M. truncatula R108 (wild type), which spends middle primary pigments,
(Lutein), the lutein content during wf1 mutant is spent is far below wild type.
Three, the confirmation of WF1 and white flower phenotype linkage relationship and its acquisition of encoding gene WF1
1, according to M. truncatula mutant database (https://medicago-mutant.noble.org/mutant/)
The flanking sequence of the Tnt1 retrotransponsons insertion points of offer, designs corresponding primer WF1-Insertion-6-F and WF1-
Insertion-6-R is combined with the primer of Tnt1 (LTR6 or LTR31) respectively, and screening is isolated with wf1 mutation type surfaces
Tnt1 insertion points, preliminary latch target gene.
LRT31-F(5’-3’):CTCCTCTCGGGGTCGTGGTT
LTR6-R(5’-3’):GCTACCAACCAAACCAAGTCAA
WF1-Insertion-6-F(5’-3’):GGTGTTGCATGGACAGAA
WF1-Insertion-6-R(5’-3’):GCCCATGCCTAGTTGTTA
2, extract the total serum IgE of M. truncatula R108 (wild type) wide-open chrysanthemum, reverse transcription at cDNA as template,
It is expanded using primer pair WF1-F/WF1-R, obtains pcr amplification product.It is sequenced, obtains the code area sequence of target gene
Row, as shown in sequence 1 in sequence table, protein shown in the sequence 2 of polynucleotide.Egg shown in sequence 2 by sequence table
White matter is named as WF1, is made of 230 amino acid residues.The encoding gene of WF1 is named as WF1 genes.
3, further reversed to screen M. truncatula Tnt1 retrotransponsons insertional mutagenesis libraries, and obtain other two WF1
Tnt1 insertion mutation bodies wf1-2 and wf1-3.Then to M. truncatula R108 (wild type), wf1 mutant, wf1-2 and wf1-3
Mutant carries out genome and transcriptional level analysis, and is analysed in depth to the phenotype of completely open flower.
4, the genomic DNA of extraction M. truncatula R108 (wild type) and three mutant is template, according to M. truncatula
Mutant database (https://medicago-mutant.noble.org/mutant/) provide Tnt1 retrotransponsons be inserted into
Site sequence design primer analyzes the insertion situation of Tnt1 retrotransponsons in wf1-1, wf1-2 and wf1-3 respectively, the results show that
Tnt1 is inserted respectively on second exon and third exon in wf1-1 and wf1-2, leads to Huang of the pattern by wild type
Discoloration is at white, and Tnt1 is inserted into the 3 ' areas UTR of its genome in wf1-3 mutant, leads to yellow of the pattern by wild type
Become light yellow (Fig. 3 A).WF1 gene expression amount situations in three mutant are further analyzed on transcriptional level, extraction is wild respectively
The total serum IgE of raw type R108 and three mutant, reverse transcription at cDNA be template, with WF1 CDS amplimers WF1-F/WF1-R
The primer pair amplifies WF1 genes of composition, and in the primer pair amplifies M. truncatula formed with primer MtActin-F/MtActin-R
Join gene M tActin, carry out RT-PCR, amplified production the results show that compared with wild type in wf1-1 WF1 genes purpose piece
Duan Bian little, the sequencing fragment result show its second Exon deletion, lead to WF1 gene 130bp base deletions, to make this
Gene, which generates frameshift mutation, leads to afunction (Fig. 3 B).Through sequencing, WF1 gene mistakes splicing (scheming C) in wf1-1 mutant.
Respectively extract wild type R108 and three mutant total serum IgE, reverse transcription at cDNA be template, with WF1 special primers qWF1-
The primer pair amplifies WF1 genes of F/qWF1-R compositions, and the primer pair amplifies formed with primer qMtActin-F/qMtActin-R
M. truncatula reference gene MtActin carries out Real-time PCR, shows that the expression of WF1 genes in three mutant is equal
Significantly lower than wild type (Fig. 3 D), further demonstrating the missing of WF1 genes causes M. truncatula pattern to become white by yellow
Color illustrates that WF1 is played an important role in the regulation and control of M. truncatula pattern.Fig. 3 E:Wild type R108 spends front;Fig. 3 F:Wf1 is spent
Front;Fig. 3 G:Wf1-2 spends front;Fig. 3 H:Wf1-3 spends front.The front of spending of wild type R108 is yellow;Wf1 spend front and
It is white that wf1-2, which spends front, and it is light yellow that wf1-3, which spends front,.
WF1-F(5’-3’):CAAAAAAGCAGGCTTCATGGGTGGTGTTGCATGGAC
WF1-R(5’-3’):CAAGAAAGCTGGGTCCTAAGAAAAACCATTATATAGATCC
MtActin-F:TCTTACTCTCAAGTACCCCATTGAGC
MtActin-R:GTGGGAGTGCATAACCCTCATAGATT
The functional verification of embodiment 2, WF1 genes
In order to further verify effects of the WF1 in carotenogenesis regulation and control, by carrying out WF1 in wf1 mutant
Genome complementation experiment carries out functional verification.
One, the structure of complementary expression vector
DNA fragmentation shown in the sequence 3 of sequence table is inserted in the KpnI restriction enzyme sites of pCAMBIA2300 plasmids, and
And the DNA fragmentation shown in the sequence 4 of insetion sequence table in PstI restriction enzyme sites, obtain recombinant plasmid pWF1pro::WF1
GDNA-1.5KASC (sequence verification).
The sequence 3 of sequence table is WF1 upstream region of gene 3K promoters section and WF1 gene gDNA sections, the sequence 4 of sequence table
For the terminators WF1 downstream of gene 1.5K section.The sequence 5 of sequence table is WF1 upstream region of gene 3K promoter section-WF1 genes
The complete sequence of the gDNA sections-terminators carrier intermediate segment-WF1 downstream of gene 1.5K section.
Two, the acquisition of complemented mutant body plant
1, the recombinant plasmid pWF1pro for preparing step 1::WF1 gDNA-1.5KASC convert Agrobacterium AGL1, obtain
Recombinant bacterium AGL1/pWF1pro::WF1 gDNA-1.5KASC.
2, the recombinant bacterium AGL1/pWF1pro for obtaining step 1::WF1 gDNA-1.5KASC are inoculated in containing 50mg/mL
Rifampin antibiotic and 50mg/mL card that antibiotic YEP solid mediums on, 28 DEG C culture to single bacterium colony is grown, choose
Take single bacterium to drop down onto in YEP fluid nutrient mediums, 28 DEG C, 200rpm shaken cultivations stay overnight.
3, it after completing step 2, takes 500 μ L bacterium solutions to be inoculated in 5mLYEP fluid nutrient mediums, adds 5 μ L 100mg/mL
Acetosyringone, 28 DEG C, 200rpm shaken cultivations to OD600nm=0.8,3800rpm centrifuge bacterium solution 15min and collect thalline.
4, the thalline that step 3 obtains is resuspended using the SH3a fluid nutrient mediums of the acetosyringone containing 100mg/L, adjusts bacterium
Liquid OD600nm=0.2, it obtains infecting liquid.
5, growth 4 weeks or so wf1 mutant, first compound leaf is taken, about 10s is washed with 75% ethyl alcohol, then with 5% sodium hypochlorite
5min is sterilized, is at least washed with sterile water 5 times in super-clean bench, be put into that step 4 obtains after cut-off blade infects in liquid, infects
15min。
6, after completing step 5, the blade after infecting is transferred on SH3a solid mediums, culture surrounding is to there is white
Embryo callus (replaces a subculture in two weeks).
7, after completing step 6, white embryo callus is transferred on differential medium, culture surrounding is green to differentiating
Color embryoid (replaces a subculture in two weeks).
8, after completing step 7, green embryoid is transferred on root media, a subculture is replaced within two weeks, takes root
It is moved in vermiculite after coming into leaves, until seedling, obtains T0 for complemented mutant body plant.
T0 generation selfings obtain T1 generations, and T1 generation selfings obtain T2 generations.
9, recombinant plasmid pWF1pro is substituted using pCAMBIA2300 plasmids::WF1 gDNA-1.5KASC, according to step 1-
8 are operated, and obtain turning empty carrier plant (pCAMBIA2300-GFP-HA/wf1).
Three, the phenotype of complemented mutant body plant
Plant to be measured:M. truncatula R108 (wild type), wf1 mutant, complemented mutant body plant (F2 generations) (pWF1:WF1
GDNA-GFP-HA/wf1), turn empty carrier plant (pCAMBIA2300-GFP-HA/wf1).
Observe the wide-open colored phenotype of plant to be measured.As a result as illustrated in figs. 4 a-4d.The result shows that complemented mutant body
The pattern of plant is become the yellow of similar wild type by the white of wf1 mutant.The pattern of control vector plant is to be mutated with wf1
The identical white of body.It is white to turn empty carrier plant pattern still, and excluding carrier influences pattern.
Four, complemented mutant body plant carotenogenic gene expression analysis
Plant to be measured:M. truncatula R108 (wild type), wf1 mutant, empty carrier transformed plant (F2 generations)
(pCAMBIA2300/wf1-7), complemented mutant body plant (F2 generations) (pWF1:WF1 gDNA-GFP-HA/wf1-1).
1, the wide-open colored total serum IgE of plant to be measured is extracted, and is reversed to cDNA.
2, the cDNA obtained using step 1 is template, using qRT-PCR detection wild types R108, wf1 mutant, wf1-2 and
Wf1-3 mutant spends the expression of middle carotenogenesis related gene, the following (MtActin of gene and its detection primer of detection
For reference gene):
qMtActin-F(5’-3’):TCAATGTGCCTGCCATGTATGT
qMtActin-R(5’-3’):ACTCACACCGTCACCAGAATCC
qMtWF1-F(5’-3’):CTAGAAGCTGATCAAGATAGGTCAC
qMtWF1-R(5’-3’):CTTGGCTCCTTCTCTTCACTG
qMtPSY-F(5’-3’):ATGACTCCTGAAAGGCGAAG
qMtPSY-R(5’-3’):TGATTCCCACCTATCCATTGC
qMtPDS-F(5’-3’):TTCTACCTTTCGTGCTTCTCC
qMtPDS-R(5’-3’):TTTCCACCTAGAACGTCTCTTG
qMtZDS-F(5’-3’):CCTCCCGTTTTACTAAGACTCG
qMtZDS-R(5’-3’):CTCGATAATGTTCAGGCTCCG
qMtCRTISO-F(5’-3’):CTCTCCCTTTCTAACCACACC
qMtCRTISO-R(5’-3’):TTTCACCGCCACCCTTTT
qMtLYCE-F(5’-3’):AGCATGTTTGGAAGGATACCG
qMtLYCE-R(5’-3’):GAGCCAAGATATGAGACACCTG
qMtLYCB-F(5’-3’):TGTGGATCTTGTCGTTGTCG
qMtLYCB-R(5’-3’):CCTCAAATTCATCCACCCAAAC
qMtECH-F(5’-3’):GGCTGTTCAAATTGGTGCTG
qMtECH-R(5’-3’):TCCACAGCTCCATTACCTTTC
qMtBCHa-F(5’-3’):TTTGCTCTATCAGTGGGTGC
qMtBCHa-R(5’-3’):CACAAGGAAGCATGCCAAAG
qMtBCHb-F(5’-3’):TCACTTACCTAGTTGCAGCTG
qMtBCHb-R(5’-3’):GCCAAACATTTCAGACCAAGG
qMtVDE-F(5’-3’):ACCAGAGCCTTCCATTGTG
qMtVDE-R(5’-3’):CCTTCTCCAAATCCTTCTCCAC
qMtZEP-F(5’-3’):AGTGGTCTTGGATAATGGTCAG
qMtZEP-R(5’-3’):CCCGAGTATGTAGCTTCTGTTG
qMtNXS-F(5’-3’):ACCAAACTTCTCTTTACTGTTCTGT
qMtNXS-R(5’-3’):AGATTGAAGGAGAAATGATTGTGTG
Each strain detects 3 plants.
Testing result (in the testing result of each gene, is followed successively by M. truncatula R108, wf1 from left to right as shown in Figure 5
Mutant, pCAMBIA2300/wf1-1 and pWF1:WF1 gDNA-GFP-HA/wf1-1).Wf1 mutant complementation transfer-gen plants
The expression quantity of middle carotenogenesis related gene is spent to be increased with respect to wf1 mutant, and expression quantity is significantly more than wild type R108
The testing result of the expression quantity of middle carotenogenesis related gene, control vector plant is identical as wf1 mutant, it was demonstrated that WF1
Gene can effectively improve the expression of carotenogenesis related gene, to improve carotenoid output.
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>Carotenoid in Plants synthesis associated protein and its encoding gene and application
<160> 5
<210> 1
<211> 690
<212> DNA
<213>M. truncatula (Medicago truncatula)
<400> 1
atgggtggtg ttgcatggac agaagaagaa gatcacttgc ttaagaaatg catacaacaa 60
tatggtgaag gaaagtggca tagggttcca ctattggctg gtctaaacag atgcagaaag 120
agttgtaggc taagatggtt gaactatcta cgtcctaaca taaagagagg aaattttgct 180
gaggaggaag tggaaatgat tgtcaaactc cacaaattat taggcaacag gtggtccctg 240
attgcaggaa ggctaccagg aaggacagca aatgacgtga aaaactattg gaactgtcat 300
ctaagcaaaa aactaaatgc tctagaagct gatcaagata ggtcacaatc atccaaagat 360
gttcaaatca ttaggccaca ggcaagaaac attggttcaa gctcagtgaa gagaaggagc 420
caaggagagt caccaactga ccaagttcta gttcaacaag agagtgacat gacaacattt 480
gatgctgatg gaaagaatca tatgcttgaa tcacaacaag acatgatggt gttttcatgc 540
ttggaccaac aaggtatggt tggtgagttt ccaatggatt ttcaattaga aggatttgaa 600
gctatggtaa gtggaggaga gggtagtagt agccaatgga attgggagga tttgctctta 660
gatatggatc tatataatgg tttttcttag 690
<210> 2
<211> 229
<212> PRT
<213>M. truncatula (Medicago truncatula)
<400> 2
Met Gly Gly Val Ala Trp Thr Glu Glu Glu Asp His Leu Leu Lys Lys
1 5 10 15
Cys Ile Gln Gln Tyr Gly Glu Gly Lys Trp His Arg Val Pro Leu Leu
20 25 30
Ala Gly Leu Asn Arg Cys Arg Lys Ser Cys Arg Leu Arg Trp Leu Asn
35 40 45
Tyr Leu Arg Pro Asn Ile Lys Arg Gly Asn Phe Ala Glu Glu Glu Val
50 55 60
Glu Met Ile Val Lys Leu His Lys Leu Leu Gly Asn Arg Trp Ser Leu
65 70 75 80
Ile Ala Gly Arg Leu Pro Gly Arg Thr Ala Asn Asp Val Lys Asn Tyr
85 90 95
Trp Asn Cys His Leu Ser Lys Lys Leu Asn Ala Leu Glu Ala Asp Gln
100 105 110
Asp Arg Ser Gln Ser Ser Lys Asp Val Gln Ile Ile Arg Pro Gln Ala
115 120 125
Arg Asn Ile Gly Ser Ser Ser Val Lys Arg Arg Ser Gln Gly Glu Ser
130 135 140
Pro Thr Asp Gln Val Leu Val Gln Gln Glu Ser Asp Met Thr Thr Phe
145 150 155 160
Asp Ala Asp Gly Lys Asn His Met Leu Glu Ser Gln Gln Asp Met Met
165 170 175
Val Tyr Ser Cys Leu Asp Gln Gln Gly Met Val Gly Glu Phe Pro Met
180 185 190
Asp Phe Gln Leu Glu Gly Phe Glu Ala Met Val Ser Gly Gly Glu Gly
195 200 205
Ser Ser Ser Gln Trp Asn Trp Glu Asp Leu Leu Leu Asp Met Asp Leu
210 215 220
Tyr Asn Gly Phe Ser
225
<210> 3
<211> 6095
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
actcttttac tacaattgca agttattata tttcaggaac aaaatattct aaagattgag 60
tcttggagca aaagaaagga gaattggagc tgattcgtgc caaaaatata aaagatctta 120
tacaaaaata tatggtgcaa ggattgagga aaaatatatg aatatctcat acaaaaatat 180
catgtgcaaa gaatttgaag atcttgtaca aatctataca agaggcgcgc cccactttag 240
tccttttgct gtttttgctt tccagacaag ctacctatta gtttatttct gacctaaaag 300
cctttggttt cttgtggaac attctagtga tgtattcctt aggttttaag tcgttataga 360
ctataaatag agtagctagc tatcataaca aatcatcttt tgttctctga aataataagt 420
gacaattgtt ttttttaata aaagttcatc tttattttct ttattttatt ttatgttctt 480
tttcttcttc ttcttcttct tcttttctat ggctacaatg aacgttagtg agtagactct 540
tcttgttttg ggattgttgg ataagtctaa tgacataatc ttaatcaaac caagccttaa 600
attctaattt atgtaaccct aatttctaat ctaaattcac cgttttaaca tgacttaaca 660
attatcaaac tgtagaagcg aaagtggagg gttagtaatt gttaattcat catctcaaat 720
atcaattcat aaaacgaaag tggagttttg atattcgaac aagtgaattt agacaaggat 780
tgcgaaacat gaaaatagtc tagcaaacct tgagacatat aaagtttcga acttcaagga 840
ttctaatagc aatcaaccat gaaaataggc gtgattgcta gaggaacaca ctcaatgaga 900
ccgaaagaag atgtgatagg ctaaagagaa acttgtcttt agaaattaag tctaacataa 960
ggttctaggt ttaatggttt ggtttgtgaa gggtttgtcg ccatgacgga ccaataatca 1020
caaggctctt ttatttttat tattttctta attaaaaatc caaacttttt aacctttgaa 1080
actttcatct aatcaatatt aaaagttaat tgaattcata actccctgtc ggaacgatac 1140
tctttttact acttcggtag aaccgtgcac ttgcggtttt atcccatcaa aagccatgat 1200
agtggatcct ttttccataa tggtggtaag gagatcaaga gagatggggt gataaattaa 1260
tagaagaacg gttgaatcaa gaaccgtcca catttcaaaa ctagcgtcaa atggaagtag 1320
gcttctcttt tccgggttga tgaatgatat gattgataac cttgttgaca ctagcatgaa 1380
tctcaaacaa ttcagtccac acggcatagt gatccttctc catttctagc acaaaagcaa 1440
tgtttttttt ttttttttaa tttttttttt tatattggtc actacaaggg ccgagtgaaa 1500
atcgggtttt gccgaattgg aggtcacgac agcgatggag gacacgaaag tgaacgacaa 1560
catggcaggg gaggaaggca tggtgtgacg agagagagag agagagagag agagagagag 1620
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 1680
agagagagag agctgctggc acggtggtta gtgcggcaaa ggaagacact tgatcgacca 1740
tgggcggctc aagagaatag gcagcggcgg ccttgggtga agaaaatggc ggcggttgca 1800
aggaagaaag gggtaggtta cgtgggaaat tagactcgtg ataccataaa ggaaatcaat 1860
tcgtgaattt cattgataat gataagagta tatatatagt tacaataggt agagtcaaat 1920
ctaggagagt aattagaaaa taatggagag taattacaaa gtaataacta acataattac 1980
taacataatt acataaggtc aaacattcta tcatgaaact tcccaacaca cccggctcac 2040
acttagagct gaaaatccga agcatgacca gagtcattca ataatgggcg gtccaacgag 2100
tcttagaagg ttctaataaa atcttagaat taggattgaa cctaatccaa tcttagaaaa 2160
ccgatttgtg agacgatgag gacccaccga ttatagctaa tacttttaaa ttactcccta 2220
aatttaagat agaaagaaga aagctaattt ttttttagga ctacgtaatt tgttgctgcg 2280
cgatgcaatg cattagttgc gcctactatg tcctgacatt acaaagcggt tttaatagtg 2340
atttacaatc acactgcaat tgtagcctga tgcgattggc accaccacaa ccacaatatt 2400
gggaccgtat caggtgatac ggtccccaat ttgaaacatt gattggggct acaattgcag 2460
ttgtttagtg tattttaaaa tatggttgat atttagttaa aggggagtaa aaggcggaga 2520
ggattcacgt ccttgttcga taactaactt tatgcattct gaaagacttc actgttggca 2580
ttattgttat tctaagaaaa ctctcctaat cttaatgaga tgacaaatga cagaaaaata 2640
cagatgggat aggtttggct ttgaatagga aataatacaa gcttctaatg tatgatgaga 2700
tccaatttct ttgagatgat gaggaccacc attggatact tatgatgtct atttaaaaac 2760
atagagcttg gtatcagagt tttagaaaga gcaatggacc atttactatg aattttgaag 2820
catacgacat agtatattac tctgccttgc tggctagttt ggcacatctt acttcaagaa 2880
acatgtgtgt gtgtaggtgt gtatgtgttt atcaccacca cacatatata taaaagatgc 2940
aggtgtgggt cgaatatgag ggtgaaaaag tagtagtggt tgaagagttt cataagagat 3000
gggtggtgtt gcatggacag aagaagaaga tcacttgctt aagaaatgca tacaacaata 3060
tggtgaagga aagtggcata gggttccact attggctggt aaaagttaat caattatttt 3120
tcctttttct tgatatttct atttgtcttc tctaaaacgg atatttaatt acttctttga 3180
agtatgtcat ctgaggttcg atctctcaca aatatgtatg gaagaacatt tttttattga 3240
aaaatcaatc gtcgacctct taaaacggat catattattt tgagagattg gtccatactc 3300
ttgactagga aaaccttggg ttcactatta aaatgaaatc atcatgaaat gaccgactct 3360
tgaagtatgt catctgaggt tccatctctg ataaatatgt atggaagaac atttttttat 3420
tgaaaatcaa ctgtcgacct cttaaaacgg atccgttatt ttgagagatt ggtccatact 3480
cttcttgact agaaaaacct tgggtttact attaaaatga aatcatcatg aaatgactga 3540
cttgttcaat ctttgcaatt ggttcttaca caattgaacc aacatactta cttagttgat 3600
tatttttctt ttttcatcaa acattggttt gttgaaggtc taaacagatg cagaaagagt 3660
tgtaggctaa gatggttgaa ctatctacgt cctaacataa agagaggaaa ttttgctgag 3720
gaggaagtgg aaatgattgt caaactccac aaattattag gcaacaggta aactttgatc 3780
tttaagactt tcctttggta ttttctaatg tgttctaagt tctaacaact aggcatgggc 3840
catgatagcc ttatgcagtt tttccaaagg tgttaaagct cattagtgaa gagtgtgcat 3900
agtaaaaagt tagtgttcaa gtatagtaaa tgatgttctc gctgaaaaaa agtatataat 3960
ataggatgtg taatattcat tcaatttgaa ccaattgatt tggttttggt gtgtttagat 4020
ttttagcaag taagatgttt ggattcactt tttgagctta tctataaata aagcatttat 4080
atttgttttg atctctaatt ttttttaaaa atgcactttt tatagatcca taacgaccaa 4140
tccttgcgtg aattgcaagg atccaataag tcctacattg attttctctg aggtgtcaat 4200
tggacaaata tatctatagt gactaggatg gatatcctgc atccgaaaac tagcagttcg 4260
tcccgtcaat cctccaggac ccaaataact caattttctc ccatgaacta tttgggtcaa 4320
tggattggtt cgatctaaaa cttgagataa tgggtgtaat ctaaaaaagg attcataagt 4380
ggttgctaat ggagttgaaa tcactaaatt ttgaggaatt cgtatcaata tatctaattg 4440
ctttatatat agttcctcaa attatatttt ctaaaccaac tagagccaat ccaaattcat 4500
cttttaatag atcactatca gtgggagtta tatgaaaaaa acttaagata tgtccatatg 4560
ttgttttctg tttattttca aaaactatct ataataactt acaaaaacaa cttacagctc 4620
atatgaaaac atttttcatt tttttacaga taaaaatagt ttattttgta aaaaaaaaaa 4680
aaaatagttt attcataagc attgatataa taagtgctta attaagttgt ttatctaaac 4740
attactcaaa tgattttagt tattgacccc tttagagtcc gcttattata gttttattgt 4800
gaaaaaaaaa aaaaattcta aacaaagttt tatcacttat aaaagttttt cccatgtgtt 4860
acaattttct tttgaaaaag agaatccgaa aaatatctaa aaattgcttc aagaaactgt 4920
gatgaataat ttctccgaaa aatcattttt atatgtggtt gttttaaggg ttaaatatgt 4980
ttttggtccc tataaatata tttacttttt gttttagtcc ctctaaattt ttctttcaac 5040
tattagtccc tataaaattt tcaatcacta cttttggtcc ctatttttaa gttaattttt 5100
gtatttttta atgaaattgt gtgaaaatgt gtagaatatt gtaaaaatct ttcttagaaa 5160
aaaagttaga tttttttaac aaaacataaa ttaaatatta atttttaacc ataaaaaata 5220
taaaaattca tttttaattc atgttttgtt aaaaaataga attttctttt gcgagagatt 5280
cttataatat tatgattttt actgcaaaat tttattgaaa atatgaattc tacatatgag 5340
tttactttaa agtagggacc aaaaatgaag atggaaaacc tttgggggac taaaagtcga 5400
agaaattttt tagagggact aaaacgaaaa gttgacatat ttatagggac caaaaacata 5460
tttagccatt gttttaaaaa ggtaaattta attaggatga acaaacacaa actagcttca 5520
acttttaatt tttttttaaa aatttctaaa aattaatctt taaaattttc aaaatcacct 5580
ttttgcaatt agtaacaaat ggaccctaag ctatttgcta caattttttt ttcttcaggt 5640
ggtccctgat tgcaggaagg ctaccaggaa ggacagcaaa tgacgtgaaa aactattgga 5700
actgtcatct aagcaaaaaa ctaaatgctc tagaagctga tcaagatagg tcacaatcat 5760
ccaaagatgt tcaaatcatt aggccacagg caagaaacat tggttcaagc tcagtgaaga 5820
gaaggagcca aggagagtca ccaactgacc aagttctagt tcaacaagag agtgacatga 5880
caacatttga tgctgatgga aagaatcata tgcttgaatc acaacaagac atgatggtgt 5940
tttcatgctt ggaccaacaa ggtatggttg gtgagtttcc aatggatttt caattagaag 6000
gatttgaagc tatggtaagt ggaggagagg gtagtagtag ccaatggaat tgggaggatt 6060
tgctcttaga tatggatcta tataatggtt tttct 6095
<210> 4
<211> 1503
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
tagattattg ttccttattg ccaataggga agacaatgta gtctctatac atgggttgtg 60
tgtcaatttc aaagttaaat gttatccaag gaaatggtgg cttaatcgat gtattttgta 120
aatcgaagta gttgttgttt aaataaacca ataaagtcgg tcttgtgaga catagttagc 180
cctaaaactg gttagtaata tggaaatagt tttgtagctt tttaaaccct attactatat 240
atctagtaga cgatttgttt ttttttaata gtatcctagt tgataataaa aaggaatatc 300
ctcctacata agagaggcag ataaatattc ctttatgttc tattggttga tcttagtgtc 360
ataaaaaaat tagctaccaa tgttaataaa gtttttaaat tttgattatg gtaacgattg 420
ttctgcaatc cttgatattg tagaaaatta tagttaaata caatcacaac tatctaaatt 480
atagtcgtaa acctttgttt aaaatttaag tcattatatt tccatctctt tcattccttc 540
agaatttgag gaatgaaaat acaaattttt tcttagatta tcctagtatg acatgctatt 600
agcttctttc ttcgtctccc ttctcccttc ttccttcatt ctttttaccc tccacactta 660
ttgggggaaa ttctctttta ttgcaatcct ttattctagg tgaatctttt gagttgtcaa 720
tgattatttg tgacttttat aagagttatt tgtactttta cgtttctttg atatgtggct 780
catatagaaa aatagaaatg ccaaacagct cttgataagg catgaacagt taaatcctac 840
cccttcacca caaaagaaga aagtcaaatc ctgccccacc tctgtttctg tttgagtatc 900
acctgtcttc atgatatcaa ttaaatattg gtgtttcttt gtgacaaaat atccattttt 960
ttagcttaac tagtttgttt ggtcgttttt ttaattttag ccctatggtt ttttaatagt 1020
gtgattttga ccttcgtact tttaaacatg cgattttgcc ctctctagtt aaccctattt 1080
ttagtttcac aaccctctag ctctgaagcc catctacttt aaaatggatg acgtattgta 1140
ttacatgtgt atcctgcacc gatacctata tgatacttcc tgatacatat tagaagagta 1200
tctgatgatt atattttatt ttatttttta aaacaattat ctgatactat tcagatacat 1260
ctggaatacg agggataagc ggtagaaaac cgatatgtgc gggctacctg atgtttttat 1320
tatatttgat acatatgcaa ttgactaagt aatgatgttt tctatttagg atatatatca 1380
tatatgtaat tgattaattg tcgatttatt tattatcaat attacttaca tttatatctc 1440
catcgttgcc gtcgtctgtt ttgtgtctct ttatttgttt gagaacgatg ctattgttga 1500
ttg 1503
<210> 5
<211> 8417
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
actcttttac tacaattgca agttattata tttcaggaac aaaatattct aaagattgag 60
tcttggagca aaagaaagga gaattggagc tgattcgtgc caaaaatata aaagatctta 120
tacaaaaata tatggtgcaa ggattgagga aaaatatatg aatatctcat acaaaaatat 180
catgtgcaaa gaatttgaag atcttgtaca aatctataca agaggcgcgc cccactttag 240
tccttttgct gtttttgctt tccagacaag ctacctatta gtttatttct gacctaaaag 300
cctttggttt cttgtggaac attctagtga tgtattcctt aggttttaag tcgttataga 360
ctataaatag agtagctagc tatcataaca aatcatcttt tgttctctga aataataagt 420
gacaattgtt ttttttaata aaagttcatc tttattttct ttattttatt ttatgttctt 480
tttcttcttc ttcttcttct tcttttctat ggctacaatg aacgttagtg agtagactct 540
tcttgttttg ggattgttgg ataagtctaa tgacataatc ttaatcaaac caagccttaa 600
attctaattt atgtaaccct aatttctaat ctaaattcac cgttttaaca tgacttaaca 660
attatcaaac tgtagaagcg aaagtggagg gttagtaatt gttaattcat catctcaaat 720
atcaattcat aaaacgaaag tggagttttg atattcgaac aagtgaattt agacaaggat 780
tgcgaaacat gaaaatagtc tagcaaacct tgagacatat aaagtttcga acttcaagga 840
ttctaatagc aatcaaccat gaaaataggc gtgattgcta gaggaacaca ctcaatgaga 900
ccgaaagaag atgtgatagg ctaaagagaa acttgtcttt agaaattaag tctaacataa 960
ggttctaggt ttaatggttt ggtttgtgaa gggtttgtcg ccatgacgga ccaataatca 1020
caaggctctt ttatttttat tattttctta attaaaaatc caaacttttt aacctttgaa 1080
actttcatct aatcaatatt aaaagttaat tgaattcata actccctgtc ggaacgatac 1140
tctttttact acttcggtag aaccgtgcac ttgcggtttt atcccatcaa aagccatgat 1200
agtggatcct ttttccataa tggtggtaag gagatcaaga gagatggggt gataaattaa 1260
tagaagaacg gttgaatcaa gaaccgtcca catttcaaaa ctagcgtcaa atggaagtag 1320
gcttctcttt tccgggttga tgaatgatat gattgataac cttgttgaca ctagcatgaa 1380
tctcaaacaa ttcagtccac acggcatagt gatccttctc catttctagc acaaaagcaa 1440
tgtttttttt ttttttttaa tttttttttt tatattggtc actacaaggg ccgagtgaaa 1500
atcgggtttt gccgaattgg aggtcacgac agcgatggag gacacgaaag tgaacgacaa 1560
catggcaggg gaggaaggca tggtgtgacg agagagagag agagagagag agagagagag 1620
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 1680
agagagagag agctgctggc acggtggtta gtgcggcaaa ggaagacact tgatcgacca 1740
tgggcggctc aagagaatag gcagcggcgg ccttgggtga agaaaatggc ggcggttgca 1800
aggaagaaag gggtaggtta cgtgggaaat tagactcgtg ataccataaa ggaaatcaat 1860
tcgtgaattt cattgataat gataagagta tatatatagt tacaataggt agagtcaaat 1920
ctaggagagt aattagaaaa taatggagag taattacaaa gtaataacta acataattac 1980
taacataatt acataaggtc aaacattcta tcatgaaact tcccaacaca cccggctcac 2040
acttagagct gaaaatccga agcatgacca gagtcattca ataatgggcg gtccaacgag 2100
tcttagaagg ttctaataaa atcttagaat taggattgaa cctaatccaa tcttagaaaa 2160
ccgatttgtg agacgatgag gacccaccga ttatagctaa tacttttaaa ttactcccta 2220
aatttaagat agaaagaaga aagctaattt ttttttagga ctacgtaatt tgttgctgcg 2280
cgatgcaatg cattagttgc gcctactatg tcctgacatt acaaagcggt tttaatagtg 2340
atttacaatc acactgcaat tgtagcctga tgcgattggc accaccacaa ccacaatatt 2400
gggaccgtat caggtgatac ggtccccaat ttgaaacatt gattggggct acaattgcag 2460
ttgtttagtg tattttaaaa tatggttgat atttagttaa aggggagtaa aaggcggaga 2520
ggattcacgt ccttgttcga taactaactt tatgcattct gaaagacttc actgttggca 2580
ttattgttat tctaagaaaa ctctcctaat cttaatgaga tgacaaatga cagaaaaata 2640
cagatgggat aggtttggct ttgaatagga aataatacaa gcttctaatg tatgatgaga 2700
tccaatttct ttgagatgat gaggaccacc attggatact tatgatgtct atttaaaaac 2760
atagagcttg gtatcagagt tttagaaaga gcaatggacc atttactatg aattttgaag 2820
catacgacat agtatattac tctgccttgc tggctagttt ggcacatctt acttcaagaa 2880
acatgtgtgt gtgtaggtgt gtatgtgttt atcaccacca cacatatata taaaagatgc 2940
aggtgtgggt cgaatatgag ggtgaaaaag tagtagtggt tgaagagttt cataagagat 3000
gggtggtgtt gcatggacag aagaagaaga tcacttgctt aagaaatgca tacaacaata 3060
tggtgaagga aagtggcata gggttccact attggctggt aaaagttaat caattatttt 3120
tcctttttct tgatatttct atttgtcttc tctaaaacgg atatttaatt acttctttga 3180
agtatgtcat ctgaggttcg atctctcaca aatatgtatg gaagaacatt tttttattga 3240
aaaatcaatc gtcgacctct taaaacggat catattattt tgagagattg gtccatactc 3300
ttgactagga aaaccttggg ttcactatta aaatgaaatc atcatgaaat gaccgactct 3360
tgaagtatgt catctgaggt tccatctctg ataaatatgt atggaagaac atttttttat 3420
tgaaaatcaa ctgtcgacct cttaaaacgg atccgttatt ttgagagatt ggtccatact 3480
cttcttgact agaaaaacct tgggtttact attaaaatga aatcatcatg aaatgactga 3540
cttgttcaat ctttgcaatt ggttcttaca caattgaacc aacatactta cttagttgat 3600
tatttttctt ttttcatcaa acattggttt gttgaaggtc taaacagatg cagaaagagt 3660
tgtaggctaa gatggttgaa ctatctacgt cctaacataa agagaggaaa ttttgctgag 3720
gaggaagtgg aaatgattgt caaactccac aaattattag gcaacaggta aactttgatc 3780
tttaagactt tcctttggta ttttctaatg tgttctaagt tctaacaact aggcatgggc 3840
catgatagcc ttatgcagtt tttccaaagg tgttaaagct cattagtgaa gagtgtgcat 3900
agtaaaaagt tagtgttcaa gtatagtaaa tgatgttctc gctgaaaaaa agtatataat 3960
ataggatgtg taatattcat tcaatttgaa ccaattgatt tggttttggt gtgtttagat 4020
ttttagcaag taagatgttt ggattcactt tttgagctta tctataaata aagcatttat 4080
atttgttttg atctctaatt ttttttaaaa atgcactttt tatagatcca taacgaccaa 4140
tccttgcgtg aattgcaagg atccaataag tcctacattg attttctctg aggtgtcaat 4200
tggacaaata tatctatagt gactaggatg gatatcctgc atccgaaaac tagcagttcg 4260
tcccgtcaat cctccaggac ccaaataact caattttctc ccatgaacta tttgggtcaa 4320
tggattggtt cgatctaaaa cttgagataa tgggtgtaat ctaaaaaagg attcataagt 4380
ggttgctaat ggagttgaaa tcactaaatt ttgaggaatt cgtatcaata tatctaattg 4440
ctttatatat agttcctcaa attatatttt ctaaaccaac tagagccaat ccaaattcat 4500
cttttaatag atcactatca gtgggagtta tatgaaaaaa acttaagata tgtccatatg 4560
ttgttttctg tttattttca aaaactatct ataataactt acaaaaacaa cttacagctc 4620
atatgaaaac atttttcatt tttttacaga taaaaatagt ttattttgta aaaaaaaaaa 4680
aaaatagttt attcataagc attgatataa taagtgctta attaagttgt ttatctaaac 4740
attactcaaa tgattttagt tattgacccc tttagagtcc gcttattata gttttattgt 4800
gaaaaaaaaa aaaaattcta aacaaagttt tatcacttat aaaagttttt cccatgtgtt 4860
acaattttct tttgaaaaag agaatccgaa aaatatctaa aaattgcttc aagaaactgt 4920
gatgaataat ttctccgaaa aatcattttt atatgtggtt gttttaaggg ttaaatatgt 4980
ttttggtccc tataaatata tttacttttt gttttagtcc ctctaaattt ttctttcaac 5040
tattagtccc tataaaattt tcaatcacta cttttggtcc ctatttttaa gttaattttt 5100
gtatttttta atgaaattgt gtgaaaatgt gtagaatatt gtaaaaatct ttcttagaaa 5160
aaaagttaga tttttttaac aaaacataaa ttaaatatta atttttaacc ataaaaaata 5220
taaaaattca tttttaattc atgttttgtt aaaaaataga attttctttt gcgagagatt 5280
cttataatat tatgattttt actgcaaaat tttattgaaa atatgaattc tacatatgag 5340
tttactttaa agtagggacc aaaaatgaag atggaaaacc tttgggggac taaaagtcga 5400
agaaattttt tagagggact aaaacgaaaa gttgacatat ttatagggac caaaaacata 5460
tttagccatt gttttaaaaa ggtaaattta attaggatga acaaacacaa actagcttca 5520
acttttaatt tttttttaaa aatttctaaa aattaatctt taaaattttc aaaatcacct 5580
ttttgcaatt agtaacaaat ggaccctaag ctatttgcta caattttttt ttcttcaggt 5640
ggtccctgat tgcaggaagg ctaccaggaa ggacagcaaa tgacgtgaaa aactattgga 5700
actgtcatct aagcaaaaaa ctaaatgctc tagaagctga tcaagatagg tcacaatcat 5760
ccaaagatgt tcaaatcatt aggccacagg caagaaacat tggttcaagc tcagtgaaga 5820
gaaggagcca aggagagtca ccaactgacc aagttctagt tcaacaagag agtgacatga 5880
caacatttga tgctgatgga aagaatcata tgcttgaatc acaacaagac atgatggtgt 5940
tttcatgctt ggaccaacaa ggtatggttg gtgagtttcc aatggatttt caattagaag 6000
gatttgaagc tatggtaagt ggaggagagg gtagtagtag ccaatggaat tgggaggatt 6060
tgctcttaga tatggatcta tataatggtt tttctcgggg atctgtcgac ggtggcggag 6120
ggggtggcat gggtaaagga gaacttttca ctggagttgt cccaattctt gttgaattag 6180
atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat 6240
acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt ccctggccaa 6300
cacttgtcac tactttctct tatggtgttc aatgcttttc aagataccca gatcatatga 6360
agcggcacga cttcttcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct 6420
tcttcaagga cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc 6480
tcgtcaacag gatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc 6540
acaagttgga atacaactac aactcccaca acgtatacat catggcagac aaacaaaaga 6600
atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc gttcaactag 6660
cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc 6720
attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg 6780
tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaggt 6840
ctatcgtcga cggtagctac ccttatgacg tccccgacta cgctggctat ccctacgatg 6900
tgcctgatta tgcctagatt attgttcctt attgccaata gggaagacaa tgtagtctct 6960
atacatgggt tgtgtgtcaa tttcaaagtt aaatgttatc caaggaaatg gtggcttaat 7020
cgatgtattt tgtaaatcga agtagttgtt gtttaaataa accaataaag tcggtcttgt 7080
gagacatagt tagccctaaa actggttagt aatatggaaa tagttttgta gctttttaaa 7140
ccctattact atatatctag tagacgattt gttttttttt aatagtatcc tagttgataa 7200
taaaaaggaa tatcctccta cataagagag gcagataaat attcctttat gttctattgg 7260
ttgatcttag tgtcataaaa aaattagcta ccaatgttaa taaagttttt aaattttgat 7320
tatggtaacg attgttctgc aatccttgat attgtagaaa attatagtta aatacaatca 7380
caactatcta aattatagtc gtaaaccttt gtttaaaatt taagtcatta tatttccatc 7440
tctttcattc cttcagaatt tgaggaatga aaatacaaat tttttcttag attatcctag 7500
tatgacatgc tattagcttc tttcttcgtc tcccttctcc cttcttcctt cattcttttt 7560
accctccaca cttattgggg gaaattctct tttattgcaa tcctttattc taggtgaatc 7620
ttttgagttg tcaatgatta tttgtgactt ttataagagt tatttgtact tttacgtttc 7680
tttgatatgt ggctcatata gaaaaataga aatgccaaac agctcttgat aaggcatgaa 7740
cagttaaatc ctaccccttc accacaaaag aagaaagtca aatcctgccc cacctctgtt 7800
tctgtttgag tatcacctgt cttcatgata tcaattaaat attggtgttt ctttgtgaca 7860
aaatatccat ttttttagct taactagttt gtttggtcgt ttttttaatt ttagccctat 7920
ggttttttaa tagtgtgatt ttgaccttcg tacttttaaa catgcgattt tgccctctct 7980
agttaaccct atttttagtt tcacaaccct ctagctctga agcccatcta ctttaaaatg 8040
gatgacgtat tgtattacat gtgtatcctg caccgatacc tatatgatac ttcctgatac 8100
atattagaag agtatctgat gattatattt tattttattt tttaaaacaa ttatctgata 8160
ctattcagat acatctggaa tacgagggat aagcggtaga aaaccgatat gtgcgggcta 8220
cctgatgttt ttattatatt tgatacatat gcaattgact aagtaatgat gttttctatt 8280
taggatatat atcatatatg taattgatta attgtcgatt tatttattat caatattact 8340
tacatttata tctccatcgt tgccgtcgtc tgttttgtgt ctctttattt gtttgagaac 8400
gatgctattg ttgattg 8417
Claims (10)
1. a kind of protein is following (a1) or (a2):
(a1) protein that amino acid sequence forms shown in sequence in sequence table 2;
(a2) amino acid sequence shown in sequence in sequence table 2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the protein with the same function derived from sequence 2.
2. encoding the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that:The gene is any DNA in following (b1)-(b4)
Molecule:
(b1) DNA molecular shown in sequence 1 in code area such as sequence table;
(b2) DNA molecular shown in the sequence 5 of sequence table;
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode protein described in claim 1
DNA molecular;
(b4) derive from M. truncatula and with (b1) or (b2) or (b3) limit DNA sequence dna have 90% or more homology and
Encode the DNA molecular of protein described in claim 1.
4. the recombinant expression carrier, expression cassette, transgenic cell line containing gene described in Claims 2 or 3 or recombinant bacterium.
5. protein described in claim 1, or, gene described in Claims 2 or 3, in regulating and controlling Carotenoid in Plants content
Using.
6. a kind of method for cultivating genetically modified plants, includes the following steps:Inhibit base described in Claims 2 or 3 in purpose plant
The expression of cause obtains the genetically modified plants of carotenoid content reduction.
7. a kind of method reducing Carotenoid in Plants content, includes the following steps:Inhibit claim 1 institute in purpose plant
The activity and/or expression quantity for stating protein obtain the genetically modified plants of carotenoid content reduction.
8. a kind of method for cultivating genetically modified plants, includes the following steps:Channel genes purpose described in Claims 2 or 3 is planted
Object obtains the genetically modified plants of carotenoid content raising.
9. a kind of method for improving Carotenoid in Plants content and improving, includes the following steps:Improve claim in purpose plant
The activity and/or expression quantity of 1 protein obtain the genetically modified plants of carotenoid content raising.
10. protein described in claim 1, or, gene described in Claims 2 or 3, or, any sides claim 6-9
Application of the method in plant breeding.
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CN116970053B (en) * | 2023-09-22 | 2024-01-30 | 南京农业大学三亚研究院 | Plant carotenoid synthesis related protein DcAPRR2, and coding gene and application thereof |
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