CN106554397B - Protein OsGRF4-M and its relevant biological material from rice are regulating and controlling the application in plant organ size - Google Patents
Protein OsGRF4-M and its relevant biological material from rice are regulating and controlling the application in plant organ size Download PDFInfo
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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Abstract
The invention discloses a kind of application of protein OsGRF4-M from rice and its relevant biological material in regulation plant organ size.Protein provided by the invention is obtained from rice, is named as OsGRF4-M albumen, is following (a) or (b) or (c): (a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；(b) by the amino acid sequence of sequence 1 by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to the seed size of plant and/or seed length and/or seed grain weight；(c) by the amino acid sequence of sequence 1 by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to plant organ size.The present invention can be used for increasing plant seed production, for plant breeding, especially rice breeding, have substantial worth.
Description
Technical field
It is big in regulation plant organ that the present invention relates to the protein OsGRF4-M for deriving from rice and its relevant biological material
Application in small.
Background technique
Rice is one of cereal crops important in the world.With the reduction of cultivated area and the rapid growth of population, mention
High rice yield has a very important significance guarantee China's grain security.
Rice yield is determined by available tillering, grain number per spike and grain shape.Rice grain shape includes the length of seed, width
And thickness.Length and width by increasing rice grain can effectively improve rice yield.How research plant regulates and controls
The mechanism of organ size has become one of the Critical policies for improving crop yield.
Summary of the invention
The object of the present invention is to provide a kind of protein OsGRF4-M from rice and its relevant biological material to adjust
Control the application in plant organ size.
Protein provided by the invention is obtained from rice, is named as OsGRF4-M albumen, is following (a) or (b) or (c):
(a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(b) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and the protein as derived from sequence 1 relevant to the seed size of plant and/or seed length and/or seed grain weight；
(c) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add and the protein as derived from sequence 1 relevant to plant organ size.
In order to make protein in (a) convenient for purifying, amino acid sequence shown in sequence 1 can be formed in by sequence table
The amino terminal or carboxyl terminal of protein connect upper label as shown in Table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of protein in above-mentioned (b) can be one or several by will lack in DNA sequence dna shown in sequence 2 in sequence table
The codon of amino acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end and/or 3 ' ends
The coded sequence for connecting label shown in table 1 obtains.
The gene (OsGRF4-M gene) for encoding the OsGRF4-M albumen also belongs to protection scope of the present invention.
The OsGRF4-M gene concretely following DNA molecular 1) or 2) or 3) or 4) or 5):
1) DNA molecular shown in sequence 2 in sequence table；
2) hybridize and the seed size and/or seed length of coded plant with the DNA sequence dna 1) limited under strict conditions
And/or the DNA molecular of seed grain weight GAP-associated protein GAP；
1) or 2) 3) there is the seed size and/or kind of 90% or more homology and coded plant with the DNA sequence dna limited
The DNA molecular of sub- length and/or seed grain weight GAP-associated protein GAP；
4) hybridize under strict conditions with the DNA sequence dna 1) limited and the DNA of coded plant organ size GAP-associated protein GAP divides
Son；
1) or 2) 5) there is 90% or more homology and coded plant organ size GAP-associated protein GAP with the DNA sequence dna limited
DNA molecular.
Above-mentioned stringent condition can are as follows: and 50 DEG C, in 7%SDS, 0.5M Na3Hybridize in the mixed solution of PO4 and 1mM EDTA,
It is rinsed in 65 DEG C, 0.1 × SSC, 0.1%SDS.Above-mentioned stringent condition can also are as follows: in 6 × SSC, the solution of 0.5%SDS,
Hybridize at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, it is primary that 0.1%SDS respectively washes film.
Recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing the OsGRF4-M gene belong to
Protection scope of the present invention.The recombinant expression carrier can be the recombination for obtaining OsGRF4-M gene insertion expression vector
Plasmid.The expression vector concretely carrier pMDC43.The preparation method of the recombinant expression carrier is specific as follows: (1) will
The OsGRF4-M gene is connect with carrier pCR8/GW/TOPO, obtains recombinant plasmid TOPO-OsGRF4-M；(2) recombinant plasmid
TOPO-OsGRF4-M and carrier pMDC43 carries out LR recombining reaction, obtains the recombinant plasmid containing the OsGRF4-M gene, will
It is named as recombinant plasmid pMDC43-OsGRF4-M.
The primer pair of the overall length or its any segment that expand the OsGRF4-M gene also belongs to protection scope of the present invention.
The present invention also protects a kind of method for cultivating genetically modified plants, is by the OsGRF4-M channel genes purpose plant
In, it obtains seed and/or seed grain weight and/or seed length is greater than the genetically modified plants of the purpose plant.The OsGRF4-
M gene specifically can import the purpose plant by the recombinant expression carrier.In the method, the recombinant expression carrier can
It is conventional by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus etc.
Biological method converts plant cell or tissue, and the plant tissue of conversion is cultivated into plant.The OsGRF4-M gene tool
Body can import the purpose plant by recombinant plasmid pMDC43-OsGRF4-M.The purpose plant is monocotyledon or double
Cotyledon plant.The monocotyledon can be gramineae plant, and No. 11 are concretely spent in rice, such as rice varieties.
The present invention also protects the OsGRF4-M albumen, the OsGRF4-M gene, the recombinant expression carrier, expression
Box, transgenic cell line or recombinant bacterium or the method, cultivating, seed is big and/or seed length is big and/or seed grain is great
Plant in application.The plant is monocotyledon or dicotyledon.The monocotyledon can be gramineae plant,
No. 11 are spent in concretely rice, such as rice varieties.
The present invention also protects the OsGRF4-M albumen in the seed size and/or seed length and/or kind of regulation plant
Application in seed weight.The plant is monocotyledon or dicotyledon.The monocotyledon can be gramineae plant,
No. 11 are spent in concretely rice, such as rice varieties.
Any description above seed is alternatively referred to as seed.
Any description above grain heavy concretely mass of 1000 kernel or 100-grain weight.
The present inventor has cloned OsGRF4-M gene from huge Large grain rice, it is found that its coded sequence spends 11 in
It is different compared to there is several places, OsGRF4-M channel genes are spent in 11 into wild-type variety, the middle seed for spending 11 can be made aobvious
It writes and increases.The present invention can be used for increasing plant seed production, for plant breeding, especially rice breeding, have substantial worth.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of carrier pCR8/GW/TOPO.
Fig. 2 is the structural schematic diagram of carrier pMDC43.
Fig. 3 is the partial results of PCR identification.
Fig. 4 is the result of OsGRF4-M gene expression amount analysis.
Fig. 5 is seed photo.
Fig. 6 is the result of seed length.
Specific embodiment
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.
Carrier pCR8/GW/TOPO:Invitrogen.The structural schematic diagram of carrier pCR8/GW/TOPO is shown in Fig. 1.
Refer to the bibliography of " carrier pMDC43 ": Curtis MD, Grossniklaus U (2003) A gateway
cloning vector set for high-throughput functional analysis of genes in
planta.Plant Physiol.133:462-469)；The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research
, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.The knot of carrier pMDC43
Structure schematic diagram is shown in Fig. 2.
Refer to the bibliography of " Agrobacterium tumefaciems GV3101 ": Li, Y., Zheng, L., Corke, F., Smith, C., and
Bevan,M.W.(2008)Control of final seed and organ size by the DA1gene family in
Arabidopsis thaliana.Genes Dev 22,1331-1336；The public can be from Chinese Academy of Sciences's heredity and development biology
It learns research institute to obtain, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.
Rice varieties " in spend No. 11 " (11 are spent in abbreviation): Zhu Xudong, Chen Hongqi, Luo Da, Zhang Jianjun, Fang Hongmin, Min Shao
Pattern；The separation and identification of 11 radiomutants are spent in rice；Rice in China science, 2003,17 (3): 205-210..
Induced medium: the organic+2,4-D 2.5mg/L+proline of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
500mg/L+glutamine 500mg/L+CH 300mg/L+ sucrose 30g/L+Gelrite 2.6mg/L, pH 5.8.
Subculture medium: the organic+2,4-D 2.0mg/L+proline of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
500mg/L+glutamine 500mg/L+CH 300mg/L+ sucrose 30g/L+Gelrite 2.6mg/L, pH 5.8.
Co-culture medium (solid): the organic+2,4-D 2.0mg/L of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
+ CH 500mg/L+ inositol 2000mg/L+AS 100 μM+sucrose 30g/L+Gelrite 2.6mg/L, pH 5.5.
Co-culture medium (liquid): the organic+2,4-D 2.0mg/L of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
+ CH 100 μM+sucrose of 500mg/L+ inositol 2000mg/L+AS 30g/L, pH 5.5.
Screening and culturing medium: the organic+2,4-D 2.0mg/L+proline of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
500mg/L+glutamine 500mg/L+CH 300mg/L+ sucrose 30g/L+Gelrite 2.6mg/L+cef 250mg/L+
Hyg 50mg/L, pH 5.8.
Differential medium: the organic+NAA 0.1mg/L+KT 4mg/L+ of N6 a great number of elements+MS-Fe salt+B5 microelement+B5
Proline 500mg/L+glutamine 500mg/L+CH 300mg/L+ sucrose 30g/L+Gelrite 2.6mg/L+cef
250mg/L+Hyg 50mg/L, pH 5.8.
Root media: 1/2N6 a great number of elements+MS-Fe salt+B5 microelement+0.8% (quality of sucrose 30g/L+Agar
Percentage composition), pH 5.8.
The discovery of embodiment 1, OsGRF4-M albumen and its encoding gene
1, it extracts the total serum IgE of huge Large grain rice and reverse transcription is cDNA.
2, the cDNA obtained using step 1 carries out PCR expansion using the primer pair that OsGRF4-S and OsGRF4-A is formed as template
Increase.
OsGRF4-S:5'-ATGACGATGCCGTATGCCTC-3'；
OsGRF4-A:5'-TCAGTCACCATTAGTTGATCG-3’。
3, the pcr amplification product for obtaining step 2 carries out agarose gel electrophoresis and recycles the band of about 1185bp.
Open reading frame shown in sequence 2 in pcr amplification product with sequence table, the open reading frame polynucleotide
Sequence 1 shown in protein.
Protein shown in sequence 1 by sequence table is named as OsGRF4-M albumen, will encode the base of OsGRF4-M albumen
Because being named as OsGRF4-M gene.The middle sequence 3 spent in 11 with such as sequence table of the highest protein of OsGRF4-M albumen homology
Shown (being named as OsGRF4 albumen, existing sequence), coded sequence (is named as shown in the sequence 4 of sequence table
OsGRF4 gene, existing sequence).
The acquisition and identification of embodiment 2, genetically modified plants
One, the preparation of recombinant plasmid
1, double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, the double chain DNA molecule obtained using step 1 is template, the primer pair formed using OsGRF4-S and OsGRF4-A into
Row PCR amplification, obtains pcr amplification product.
OsGRF4-S:5'-ATGACGATGCCGTATGCCTC-3'；
OsGRF4-A:5'-TCAGTCACCATTAGTTGATCG-3’。
3, the pcr amplification product for obtaining step 2 carries out agarose gel electrophoresis and recycles the segment of about 1185bp.
4, the segment that step 3 recycles is connect with carrier pCR8/GW/TOPO, obtains recombinant plasmid TOPO-OsGRF4-M.
According to sequencing result, structure is carried out to recombinant plasmid TOPO-OsGRF4-M and is described as follows: carrier pCR8/GW/TOPO's
OsGRF4-M gene shown in the sequence 2 of sequence table is inserted between attL1 and attL2.
5, recombinant plasmid TOPO-OsGRF4-M and carrier pMDC43 carries out LR recombining reaction, obtains the sequence containing ordered list
The recombinant plasmid of OsGRF4-M gene shown in column 2, is named as recombinant plasmid pMDC43-OsGRF4-M.It is tied according to sequencing
Fruit carries out structure to recombinant plasmid pMDC43-OsGRF4-M and is described as follows: will be between the attR1 and attR1 of carrier pMDC43
Small fragment replaces for the small fragment between the attL1 and attL2 of recombinant plasmid TOPO-OsGRF4-M.
The recombination matter of OsGRF4 gene shown in the sequence 4 containing ordered list is prepared referring to the method for step 1 to 5
Grain, is named as recombinant plasmid pMDC43-OsGRF4.
Two, turn the acquisition of OsGRF4-M gene plant
1, recombinant plasmid pMDC43-OsGRF4-M is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium.
2,11 will be spent in the conversion of recombinational agrobacterium that step 1 obtains, concrete operations are as follows:
(1) Fiber differentiation of Mature Embryos of Rice callus
1. removing and spending 11 mature seeds in shell, carries out surface sterilizing and (first impregnate 1-2min with 70% ethyl alcohol, then use
0.1% mercuric chloride impregnates 10min), then seed is placed on suck dry moisture on aseptic filter paper by then aseptic water washing 3-4 times.
2. complete step 1. after, seed is placed on induced medium plate, 26 DEG C dark culture 10-15 days.
3. peeling the callus that mature embryo scultellum is grown after completing step 2., being placed on subculture medium plate and carry out
(squamous subculture is primary every two weeks for squamous subculture；26 DEG C of dark cultures), select squamous subculture 5-7 days, the callus of pale yellow
It carries out step (3).
(2) bacteria suspension is prepared
The recombinational agrobacterium that step 1 is obtained is suspended in liquid co-culture medium, and adjustment bacteria concentration is OD600nm=
Then 0.3-0.5 is added acetosyringone and makes its concentration 100mM.
(3) callus that step (1) obtains is placed in the bacteria suspension that step (2) obtains, is placed at room temperature for 20min not
When shake, then take out callus and be placed on aseptic filter paper to suck extra bacterium solution, callus is then transferred to paving
On the co-culture medium plate for having one layer of aseptic filter paper, 26 DEG C dark culture 2-3 days.
(4) after completing step (3), callus is placed on screening and culturing medium plate simultaneously 26 DEG C dark culture 14 days, then
Callus is transferred to 26 DEG C dark culture 14 days on new screening and culturing medium plate.Most of callus is 10 days after screening
Then left and right browning is regrowed out milky resistant calli at the edge of browning tissue.
(5) after completing step (4), the resistant calli of milk yellow densification is selected, is placed on differential medium plate, first
It dark culture 3 days, is then transferred under 15h/d illumination condition and cultivates.Culture has green point to occur for 15-25 days or so, cultivates 30-40 days
Seedling is further differentiated afterwards.
(6) it in step (5), when seedling bud is about 2cm, transfers them on root media and cultivates two weeks, selection is high
The seedling of about 10cm, well developed root system wash away culture medium, transplanting to field, and as T0 generation intends turning OsGRF4-M gene plant.
It obtains 35 plants of T0 and turns OsGRF4-M gene plant for quasi-.
3, turn the identification of OsGRF4-M gene plant
(1) 35 plants of T0 generation quasi- OsGRF4-M gene plant that turns that step 2 obtains is subjected to PCR identification.
Extract the genomic DNA of the fresh blade of plant and as template, the primer pair formed with K4S-F and K4S-R into
Row PCR amplification, PCR product with HindIII carries out digestion, and (OsGRF4-M gene can be cut, and OsGRF4 gene cannot be cut
It cuts).
K4S-F:5 '-CCCTTTCAAGCTATTCTAAGC-3 '；
K4S-R:5 '-TTCCCAAAGAACGAAAGTGG-3 '.
Partial detection is shown in Fig. 3.Turn OsGRF4-M gene plant because containing OsGRF4-M gene and OsGRF4 simultaneously
Gene has two bands after digestion.In spend 11 to contain only OsGRF4 gene, cannot be digested, therefore only one single
Band.In Fig. 1, swimming lane 1-17 is to intend in part T0 generation turning OsGRF4-M gene plant, and swimming lane 18 spends 11 in.
In 35 plants of T0 generations, intend turning in OsGRF4-M gene plant, and 28 plants are to turn OsGRF4-M gene plant.
(2) in T0 generation, is turned into OsGRF4-M gene plant and carries out the analysis of OsGRF4-M gene expression amount.
It extracts the total serum IgE of the fresh blade of plant and reverse transcription is cDNA, then pass through real-time PCR
(Lightcycler 480, ROCHE) detects the expression quantity of OsGRF4-M gene in plant.Fluorescent dye is Lightcycler
480SYBR Green I Master(ROCHE).Internal reference is ACTIN1.For detect the primer pair of OsGRF4-M gene by
K4RT-F and K4RT-R composition.Primer for detecting ACTIN1 is made of ACTIN1F and ACTIN1R.
K4RT-F:5 '-ATTCCAAGTACTGCGAGCGC-3 '；
K4RT-R:5 '-GGCGACCAGCTGCGTTT-3 '.
ACTIN1F:5 '-TGCTATGTACGTCGCCATCCAG-3 '；
ACTIN1R:5 '-AATGAGTAACCACGCTCCGTCA-3 '.
The relative expression quantity of OsGRF4-M gene is shown in Fig. 4 (1 spends 11,2 in turn OsGRF4-M gene plant).
Three, turn the acquisition of OsGRF4 gene plant
It replaces recombinant plasmid pMDC43-OsGRF4-M to carry out step 2 with recombinant plasmid pMDC43-OsGRF4, is turned
OsGRF4 gene plant.
Four, phenotypic evaluation
In T0 generation, turns OsGRF4-M gene plant and grows under the natural conditions of field, collects seed after mature, observes and take pictures,
(in 28 plants of T0 generations, turn OsGRF4-M gene plant to measurement seed length, as a result the length of 100 seeds of every plant of random measurement is made even
Mean value), (in 28 plants of T0 generations, turn OsGRF4-M gene plant to statistics mass of 1000 kernel, and the grain of 100 seeds of every plant of random measurement is weighed and converted
For mass of 1000 kernel, results are averaged).
In T0 generation, turns OsGRF4 gene plant and grows under the natural conditions of field, collects seed after mature, observes and take pictures, and surveys
(in 10 plants of T0 generations, turn OsGRF4 gene plant to amount seed length, as a result the length of 100 seeds of every plant of random measurement is averaged
Value), (in 10 plants of T0 generations, turn OsGRF4 gene plant to statistics mass of 1000 kernel, and the grain weight of 100 seeds of every plant of random measurement is simultaneously scaled thousand
Grain weight, results are averaged).
In spend 11 to grow under the natural conditions of field, collect seed after mature, observe and take pictures, measure seed length (10
11 are spent in strain, the length of 100 seeds of every plant of random measurement, results are averaged), and statistics mass of 1000 kernel (spend 11 in 10 plants, every plant
The grain of 100 seeds of random measurement weighs and is scaled mass of 1000 kernel, and results are averaged).
Above-mentioned each plant carries out parallel test, i.e., cultivates under conditions of identical.Using body formula mirror (LEICA
S8APO, Germany) it observes and takes pictures (LEICA DFC420, Germany).Seed length is measured using Image J1.41 software.It utilizes
EXCEL is for statistical analysis.
Photo is shown in Fig. 5, two, the left side be it is middle spend 11 seed, two, the right is the kind for turning OsGRF4-M gene plant in T0 generation
Son, scale 1mm.
The result of seed length is shown in Fig. 6,1 for it is middle spend 11 seed, 2 turn the seed of OsGRF4-M gene plant for T0 generation.
The result of seed length and mass of 1000 kernel the results are shown in Table 2 (mean+SDs).
The phenotype statistical result of 2 genetically modified plants of table
| It is middle spend 11 seed | In T0 generation, turns the seed of OsGRF4-M gene plant | In T0 generation, turns the seed of OsGRF4 gene plant | |
| Seed length (mm) | 6.72±0.03 | 9.94±0.09** | 7.81±0.04** |
| Mass of 1000 kernel (g) | 25.61±0.17 | 34.56±0.08** | 28.92±0.32** |
Note: it is significant that * * represents 0.01 level difference of P <.
Compared with spending 11 in, the seed length and mass of 1000 kernel for turning OsGRF4-M gene plant are dramatically increased.With turn
OsGRF4 gene plant is compared, and the seed length and mass of 1000 kernel for turning OsGRF4-M gene plant dramatically increase.
The above results show that the organ size of OsGRF4-M gene and plant is positively correlated, and OsGRF4-M gene is transferred to mesh
Plant in express after, the organ size of plant can be increased.
Claims (7)
1. a kind of protein, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.
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 DNA molecular shown in sequence 2 in sequence table.
4. recombinant expression carrier, expression cassette or recombinant bacterium containing gene described in Claims 2 or 3.
5. a kind of method for cultivating genetically modified plants, is planted in channel genes purpose plant described in Claims 2 or 3
Son and/or seed grain weight and/or seed length are greater than the genetically modified plants of the purpose plant；The purpose plant is rice.
6. protein described in claim 1, or, gene described in Claims 2 or 3, or, recombinant expression as claimed in claim 4
Carrier, expression cassette or recombinant bacterium, or, method described in claim 5, seed is big and/or seed length is big and/or kind cultivating
Application in the great plant of seed；The plant is rice.
7. the answering in the seed size and/or seed length and/or seed grain weight of regulation plant of protein described in claim 1
With；The plant is rice.
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