CN104561043B - Paddy rice B3 transcription factor family genes RAV2 application - Google Patents
Paddy rice B3 transcription factor family genes RAV2 application Download PDFInfo
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- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention discloses a kind of paddy rice B3 transcription factor family genes RAV2 application, belongs to plant genetic engineering field.The present invention is cloned into the RAV2 genes of control rice leaf intersection angle using the method for map based cloning, and the gene belongs to the RAV subfamilies of B3 class transcription factor families；Show that RAV2 is a key gene of control rice leaf intersection angle by genetic analysis and functional complementation experiment.Furthermore, the research to the gene contributes to people to understand the molecular mechanism of rice growth, and using modern genetic engineering method, design and context plays an important roll to improving grain yield.Meanwhile, using the functional characteristic of the gene, Molecular design breeding is carried out in paddy rice, the kind of the suitable plant type of rice of seed selection has very strong operability.Described RAV2 genes have such as SEQ ID NO:Nucleotide sequence shown in 1, its amino acid sequence such as SEQ ID NO encoded:Shown in 2.
Description
Technical field
The invention belongs to plant genetic engineering field, and in particular to a kind of paddy rice B3 transcription factor family genes RAV2's should
With.The present invention has obtained the gene RAV2 of a control rice leaf intersection angle by forward genetics method from paddy rice, and passes through
Transgenosis complementary assay demonstrates the function of RAV2 genes；The invention further relates to trained using the gene or the similar gene of its function
Educate the method with different Leaf angle paddy rice.The paddy rice B3 transcription factor family genes RAV2 of the present invention is related to plant type of rice,
It is significant to abundant plant type of rice resource.
Background technology
B3 class transcription factors are the distinctive class transcription factors of plant, are gained the name because it contains B1, B2 and B3 domain, can
It is divided into 5 subtribes：ABI3/VP1；HSI(High-level expression of sugar-inducible gene)；RAV
(Related to ABI3/VP1)；ARF(Auxin Response Factor)and REM(ReproductiveMeristem).The research of arabidopsis shows, their function be related to plant hormone synthesis and signal transduction, seed development,
Bloom regulation and control, biological and abiotic stress response etc., important regulating and controlling effect is played in the growth course of plant
(Romanel,E.A.C.,et al.,Evolution of the B3 DNA Binding Superfamily:New
Insights into REM Family Gene Diversification.PLoS ONE,2009,4(6):e5791)。
The B3 gene families of paddy rice have 91 members, and wherein RAV subfamilies have 12 genes (Romanel, E.A.C., et
al.,Evolution of the B3DNA Binding Superfamily:New Insights into REM Family
Gene Diversification.PLoS ONE,2009,4(6):e5791)。
Inventor analyzes the research conditions of 12 genes of paddy rice RAV subfamilies, and to the genomic locations where it
Compared with RAV2.As a result show, only RAVL1 (LOC_Os04g49230) gene positioned at the 4th chromosome has correlation
Functional study.Research shows that transcription factor RAVL1 (LOC_Os04g49230) participates in BR signal pathways and synthesis by adjusting
The expression of pathway gene, to maintain intracellular BR homeostasises (Je, B.I., et al., RAV-Like1Maintains
Brassinosteroid Homeostasis via the Coordinated Activation of BRI1and
Biosynthetic Genes in Rice.The Plant Cell Online,2010.22(6):p.1777-1791)。
RAVL1 Ds insertion and deletion mutant is mainly shown as that leaf color is dark green, the character such as semi-dwarf mutant.
Leaf morphology is to influence the principal element of plant type, and posture is stretched in Leaf inclination influence plant space, is leaf morphology
Important component.Found by the research to leaf cornicult variant, most mutators have been involved in brassinosteroid
(BR) biosynthesis and signal transduction path, (is waited quietly including LC2, OsBRI1, OsLIC1, OsBAK1, BU1, ILI1 etc. slowly
Rice leaf morphogenesis molecular regulation Recent Advances in Mechanism Acta Agronomica Sinicas, 2013,39 (5):767-774).These genes lead to
The division and growth of pulvinus (junction of blade and leaf sheath) adaxial and its surface cell are overregulated, and then influences blade tilt.In addition,
ILA1 is by influenceing the gene of pulvinus mechanical tissue intensity and then change leaf angle, and the reaction nothing of the gene and brassinosteroid
Close (Ning J, Zhang B, Wang N, et al.Increased leaf angle1, a Raf-like MAPKKK that
interacts with a nuclear protein family,regulates mechanical tissue formation
in the lamina joint of rice.Plant Cell.2011,23(12):4334-4347).Another influence blade
Developed with leaf sheath junction and the gene TLD1 unrelated with brassinosteroid reaction also play a part of to adjust leaf angle size (Zhang,
et al.Altered architecture and enhanced drought tolerance in rice via the
down-regulation of indole-3-acetic acid by TLD1/OSGH3.13activation.Plant
Physiol,2009,151(4):1889-1901).Therefore, the regulation and control of leaf angle size are a complicated processes, largely
On developed and influenceed by pulvinus, plant endogenous plant hormone brassinosteroid plays critically important regulation and made again during this
With.
The regulation of plant form mechanism of action is fully understanded, it is necessary to identify and clone more related genes.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the invention be to provide a kind of paddy rice B3 transcriptions because
Sub-family gene RAV2 and its encoding proteins.The present invention spends in 11 offspring from wild rice and screens a mutant
Rav2, not only downgrade but also blade is sagging by plant for the mutant.Genetic analysis shows that this sports incomplete dominant lnheritance.Profit
Mutant candidate gene RAV2 (LOC_Os02g45850) is obtained with map based cloning strategy, the gene belongs to B3 class transcription factors
The RAV subfamilies of family.Therefore, purpose of the present invention gene OsRAV2 is located at the chromosome of paddy rice the 2nd, is a regulation paddy rice strain
The new gene of type.
It is another object of the present invention to provide above-mentioned paddy rice B3 transcription factor family genes RAV2 in regulation leaves of plants
Application in angle.Purpose of the present invention gene RAV2 belongs to B3 class transcription factor families, and corresponding mutant strain is high substantially to be become
It is short, and blade is sagging, and indication RAV2 plays an important roll in plant type of rice regulation and control.
Another object of the present invention is to provide above-mentioned paddy rice B3 transcription factor family genes RAV2 in plant breeding
Using.
Transgenosis plant is being cultivated it is still another object of the present invention to provide above-mentioned paddy rice B3 transcription factor family genes RAV2
Application in thing.
The purpose of the present invention is achieved through the following technical solutions：
The present invention utilizes paddy rice natural variation mutant, determines that the mutant candidate gene is located at by map based cloning strategy
The chromosome of paddy rice the 2nd, belongs to the RAV subfamilies of B3 transcription factor genes family, is named as RAV2, at present still not on the base
Because of the research of function aspects.
Based on described above, the present invention provides a kind of paddy rice B3 transcription factor RAV2, its amino acid sequence such as SEQ ID
NO:Shown in 2, or such as SEQ ID NO:Amino acid sequence shown in 2 by one or more amino acid substitutions, insertion, lack
That loses and obtain still has the analog of control rice leaf intersection angle function.
Above-mentioned paddy rice B3 transcription factors RAV2 is encoded by paddy gene RAV2, and the gene is following tri- kinds of nucleotides of A, B, C
One of sequence：
A sequence table SEQ ID NO:DNA sequence dna shown in 1；
B is encoded and A encoding proteins matter identical protein DNA sequences；
The analog still with control Leaf angle function that more than C A and B are inserted by base, lack or replaced and obtain.
Recombinant vector, transgenic cell line and recombinant bacterium containing above-mentioned rice leaf intersection angle related protein encoding gene also belong to
In protection scope of the present invention.
Above-mentioned rice leaf intersection angle gene RAV2 application, its method is to import the recombinant expression carrier containing RAV2 genes
Plant cell, obtains the transgenic paddy rice of changeable leaf angle；The transgenic paddy rice of above-mentioned changeable leaf angle can also be used as confession
Body parent, rice leaf intersection angle is improved by backcross breeding method, i.e., by the way that the paddy rice of the changeable leaf angle obtained is improved with intending
Rice varieties (recurrent parent) hybridization, then filial generation through repeatedly with recurrent parent hybridization with the water of seed selection changeable leaf angle
Rice.
The recombinant expression carrier containing RAV2 genes can use existing plant expression vector construction；The plant expression
Carrier include double base agrobacterium vector and the carrier available for plant micropellet bombardment etc., such as pCAMBIA1300, pCAMBIA2300,
PCUbi1390 etc.；Recombinant expression carrier containing RAV2 genes can be mediated by particle gun, microinjection, protoplast, pollen
The various physics such as tube passage, agriculture bacillus mediated, chemistry and biological method are imported into host plant cell or tissue；The place
Main plant includes but is not limited to paddy rice, wheat, corn, sorghum, millet, sugarcane, cotton, tomato, clover and napier grass；It is imported into
Host plant is preferably paddy rice.
When building the recombinant expression carrier containing RAV2 genes, it can be added before the transcription initiation nucleotides of RAV2 genes
Any enhanced, composing type, tissue specificity or inducible promoter, such as 35S promoter, Ubi promoters.
For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out
Reused after transformation, including add reporter gene, antibiotics resistance gene or other anti-chemical reagent marker gene such as GUS, GFP
Deng.
By map based cloning method, RAV2 genes have been cloned in separation to the present invention from paddy rice first, one water of the gene code
Rice B3 transcription factors.Mutation type surface is analyzed and complementation analysis shows that RAV2 genes play important work in terms of rice leaf intersection angle is controlled
With.On the other hand, the research to the gene contributes to people to understand the molecular mechanism of rice growth, utilizes modern genetic work
Cheng Fangfa, design and context plays an important roll to improving grain yield.Meanwhile, using the functional characteristic of the gene, in paddy rice
Molecular design breeding is carried out, the kind of the suitable plant type of rice of seed selection has very strong operability.
Brief description of the drawings
Fig. 1 is the phenotype of paddy rice rav2 mutant；WT be wild type in spend 11；H is mutation heterozygote；M is mutant homozygous
Body.
Fig. 2 is the physical map of paddy gene RAV2 map based cloning.
Fig. 3 is the epigenetic variation site schematic diagram of paddy rice RAV2 genes；Grey rectangular segment show for RAV2
The promoter region of gene (Os02g45850) there occurs the region of DNA demethylations.
Fig. 4 is the expression analysis figure of paddy rice RAV2 genes；WT be wild type in spend 11；H is mutation heterozygote；M is mutation
Homozygote.
Fig. 5 is paddy rice RAV2 gene overexpression transfer-gen plant phenotypes；WT be wild type in spend 11；OE is overexpression
Transgenic line.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Rice varieties are to spend 11 (the open rice varieties used) in wild type japonica rice in embodiment；PCUbi1390 is normal
Plant expression vector, Hyg containing hygromycin gene, the Ubiquitin promoters and kermes for starting exogenous gene expression
The terminator of alkali synthase gene；PMD19-T carriers and DNA Ligation Kit Ver.2.0 kits are raw purchased from Dalian treasured
Thing Engineering Co., Ltd；Agrobacterium EHA105 bacterial strains are conventional bacterial strains, and there are preservation in most Molecular Biology Labs；DH5α
Competent cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd.Primer used in embodiment is by Invitrogen companies
Synthesis, and be sequenced.
Described plant expression vector pCUbi1390 and Agrobacterium EHA105 bacterial strains, in " Chinese invention patent
201110410931.5, rice extensin OsPEX1 and its application " disclosed in.
Main agents in embodiment are：Restriction enzyme, Taq enzyme, T4Ligase, RNase A, M-MLV reverse transcriptions
Enzyme etc. is purchased from biotech firms such as TAKARA (Dalian), Promega, NEB, Toyobo；DNTPs is purchased from Genestar companies；
The small extraction reagent kit of plasmid and Ago-Gel QIAquick Gel Extraction Kit are purchased from Beijing TIANGEN companies；TRLzol RNA extracts reagents
Box is purchased from Invitrogen companies；MS culture mediums, agar powder, agarose, ampicillin (Amp), kanamycins (Kan),
The antibiotic and DEPC, LB Medium etc. such as gentamicin sulphate (Gen), rifampin (Rif) are purchased from Sigma companies；Embodiment
Used in other chemical reagent be that import or domestic analysis are pure.
The phenotypic characteristic of the paddy rice rav2 mutant of embodiment 1 and genetic analysis
Spent in the wild type japonica rice variety of plantation in 11 offspring, screen plant dwarfing and blade is sagging
Plant, is named as paddy rice rav2 mutant.Compared with spending 11 in wild type, the mutation heterozygote shows as plant dwarfing, blade
The character such as sagging, and rav2 mutant homozygous body Relevant phenotypes are more serious, and it is completely sterile (Fig. 1).
To verify the hereditary capacity of mutant, point 3 strains of the sagging mutant of single-strain planting leaf (mutation heterozygote).Phenotype
Observation shows that the offspring from the sagging mutant individual plant of 3 leaves there occurs under wild type, leaf sagging (heterozygote) and serious leaf
The separation of vertical (homozygote), totally 537 plants of 3 strains, its segregation ratio meets 1:2:1(127:273:137；χ2=0.52<χ2 0.05,2
=5.99).Result above shows that mutant phenotype is in incomplete dominant lnheritance, and rav2 is mutated for incomplete dominance.
The map based cloning and candidate gene approach of the mutator of embodiment 2
The F built using rav2 and the magnificent round-grained rice Xian 74 of rice variety (the open rice varieties used, commercially available)2Colony (rav2
F is obtained with magnificent round-grained rice Xian 74 hybridization1, F1Selfing obtains F2Colony) to RAV2 carry out the assignment of genes gene mapping, find its with the 2nd chromosome
Mark RM3512 and RM318 is chain, and genetic distance is respectively 1.6cM and 3.7cM.By developing Indel between being marked two
(insertion-deletion) mark, further the paddy rice RAV2 assignments of genes gene mapping Indel mark CID4178-26 and
Between CID4255-3, the physical distance between 2 marks is 18.5kb.It is according to the physical map of the chromosome of paddy rice the 2nd and finely fixed
The result of position, the present invention constructs the physical map (Fig. 2) of covering OsRAV2 genes.
On this basis, the present invention utilizes rice genome annotations database RGAP (http://
Rice.plantbiology.msu.edu/ the paddy rice dyeing between mark CID4178-26 and mark CID4255-3) is analyzed
Body section, finds only have 1 candidate gene in the section, the corresponding site that the gene is annotated in RGAP paddy genes storehouse is LOC_
Os02g45850.Further analysis shows, candidate gene encodes a B3 class transcription factor.
The epigenetic variation of embodiment 3 causes the overexpression of RAV2 genes
In order to find mutational site, the present invention designs primer according to Public Rice Genome Sequence Data, expands wild type and prominent
Become homozygotic purpose section, sequencing analysis show, without the change for finding DNA sequence dna in 18.5kb positioning is interval.Cause
This, thus it is speculated that purpose section may have occurred epigenetic variation.Therefore, the present invention is analyzed by bisulfite sequencing
Methylate modification variation situation.As a result show, the promoter region of OsRAV2 genes there occurs DNA demethylation (Fig. 3).DNA
Methylation detecting method is summarized as follows：
It is all that methyl does not occur with bisulf iotate-treated wild type (11 are spent in wild type) and mutant gene group DNA
The cytimidine of change is converted into uracil, and the cytimidine methylated is constant；Then redesign primer enters performing PCR amplification, by mesh
Product purification after carry out TA clones, each clone's picking positive colony sequencing, finally by the sequence measured and original series ratio
It is right, statistics methylation sites and quantity, and analyze wild type (11 are spent in wild type) and mutant methylation differential.
Research shows that DNA demethylations normally result in the overexpression of gene.So, mutant candidate gene RAV2
Again what kind of change can occur for expressionTherefore, the present invention 1 pair of gene specific primer of design utilizes semi-quantitative RT-PCR analysis open country
The expression of RAV2 genes, as a result shows to be mutated heterozygosis in raw type (11 are spent in wild type), mutation heterozygote, mutant homozygous body
The expression of RAV2 genes is apparently higher than wild type in body, mutant homozygous body, and mutant homozygous body is higher than mutation heterozygote (figure
4).There is dosage effect in this explanation mutation type surface and RAV2 expression.The extraction of plant total serum IgE and RT-PCR reaction bar
Part and program, referring in particular to " Chinese invention patent 201110410931.5, rice extensin OsPEX1 and its application ".
Primer used in RT-PCR is：
Sense primer：5′-GTGGTGGAGAAGGAGCACAT-3′；
Anti-sense primer：5′-GGCTGCTGTTCCAGTAGGAG-3′.
Embodiment 4 has complementary functions checking
In order to further verify RAV2 gene functions, the RAV2 overexpressions that the present invention constructs the driving of Ubi promoters are carried
Body pCUbi1390-U-RAV2, is mediated by Agrobacterium (EHA105), and 11 are spent in conversion wild rice, is turned by PCR detections
Gene masculine plant.As a result show, RAV2 overexpression transfer-gen plant phenotypes are similar with mutant (Fig. 5).This is also further
Illustrate, can Effective Regulation rice leaf intersection angle size by adjusting RAV2 gene expressions.The present embodiment is involved the steps of：
1st, PCR expands RAV2 total length ORFs (ORF), is connected with plasmid pCUbi1390, builds overexpression matter
Grain Ubi::RAV2；Overexpression plasmid Ubi::RAV2 converts bacillus coli DH 5 alpha, and transformant is identified by bacterium colony PCR, digestion,
And after sequence verification, then by plasmid convert Agrobacterium (EHA105).
2nd, by the overexpression plasmid Ubi of structure::RAV2 is mediated by Agrobacterium (EHA105), and rice transformation kind is wild
11, PCR is spent to detect transgenic positive plant in raw type；Observation and the change of analysis plant phenotype.
Specific experiment process is as follows：
(1) target gene overexpression plasmid Ubi::RAV2 structure
According to the sequence of RAV2 genes, for the multiple cloning sites of pCUbi1390 plasmids, 1 pair of primer of design (contains BglII
With SpeI restriction enzyme sites)：
Sense primer RAV-1BXF (underscore is BglII restriction enzyme sites)
5′–AGATCTTCTCGAGATGGAGTTCACTACAAGCAG -3 ',
Anti-sense primer RAV-1239RNS (underscore is SpeI restriction enzyme sites)
5′–ACTAGTCTAGCTAGCCAGATCGAGATCCAAGGCCG–3′。
Using sense primer RAV-1BXF and anti-sense primer RAV-1239RNS amplification RAV2 genes, by the PCR primer of acquisition
Carry out after recovery purifying, be cloned on pMD19-T carriers, screening positive clone simultaneously carries out digestion identification, filter out containing positive gram
Grand plasmid pMD19-T-RAV2 positive bacteria, send handsome company to carry out gene sequencing, contains double enzyme site it is determined that obtaining
RAV2 genes.
Obtained after RAV2 genes are cut from pMD19-T-RAV2 cloned plasmids with BglII and SpeI restriction enzymes
Double digestion after RAV2 gene DNA fragments, the expression vector reconnected to after two kinds of enzymes double zyme cuttings of BamHI and SpeI
On pCUbi1390, recombinant plasmid pCUbi1390-RAV2 is obtained, and recombinant plasmid pCUbi1390-RAV2 is transferred to Escherichia coli
DH5 α competent cells.The connection of expression vector pCUbi1390 after RAV2 genes and double digestion DNA Ligation Kit
Ver.2.0 kits.
The concrete operation step of said process is：1) by the expression vector pCUbi1390 and double digestion after above-mentioned double digestion
RAV2 gene DNA fragments afterwards mix and are prepared into the DNA solution (expression vector after double digestion that volume is 10 μ L
PCUbi1390 and the RAV2 gene DNA fragments after double digestion mole ratio is：0.1pmol:0.3pmol)；2) to above-mentioned DNA
Isometric Solution Ι (DNA Ligation Kit Ver.2.0 kits are carried) are added in solution, are fully mixed；3)16
DEG C reaction is stayed overnight；4) reaction solution is directly added into Bacterial Transformation, i.e., 20 μ L reaction solution is added to 100 μ L competent cell
In, 42 DEG C of heat shock methods convert (reference《Molecular Cloning:A Laboratory guide》(third edition) (in translate version) Huang Peitang etc. is translated, scientific publication
Society, in September, 2002 is published).Positive colony bacterium colony is selected, and by the Preliminary Identification of round pcr progress positive colony, then extract
The plasmid of positive colony carries out double digestion identification, determines that recombinant plasmid pCUbi1390-RAV2 is successfully constructed.
(2) genetic transformation
By the above-mentioned recombinant plasmid pCUbi1390-RAV2 conversion Agrobacteriums EHA105 successfully constructed.Comprise the following steps that：
1 μ g or so recombinant plasmid pCUbi1390-RAV2 is taken to be added to 100mL EHA105 competent cells (self-control, reference《Molecule
Cloning experimentation guide》(third edition) (in translate version) Huang Peitang etc. is translated, Science Press, and in September, 2002 is published) in, after mixing, ice
Bathe 30min, -70 DEG C of placement 10min.Again in 42 DEG C of water-bath 1min, then ice bath 2min, adds 800mL YM fluid nutrient mediums
(pH=7.0, containing KH2PO40.5g/L, NaCl 0.2g/L, MgSO4·7H2O 0.2g/L, mannitol (Mannitol) 10g/L,
Glu (L-Glutamine) 2g/L, yeast extract (Yeast extract) 0.3g/L, agar (Agar) 15g/L) 28 DEG C,
175rpm is coated on the YM flat boards containing 50 μ g/mL kanamycins (Kanamycin) after shaking training 3hr.28 DEG C of cultures are single to formation is arrived
Bacterium colony.The identification of positive colony is carried out by round pcr, positive colony bacterium colony is selected, YM fluid nutrient mediums are inoculated in (same
On), -80 DEG C of preservations produce the Agrobacterium EHA105 strains of the pCUbi1390-RAV2 containing recombinant plasmid, standby.
The Agrobacterium EHA105 strains of the pCUbi1390-RAV2 containing recombinant plasmid are transformed into spent in wild type 11 callus,
By preculture, dip-dye, co-culture, screening with hygromycin resistance callus, break up, take root, acclimatization and transplantses are obtained to crop field
Transfer-gen plant.
Rice transformation is comprised the following steps that：
1) rice paddy seed is shelled, NB culture mediums is inoculated in after sterilization, 25 DEG C of light culture 4d remove the budding of rataria length
Afterwards, rataria is inoculated in fresh NB culture mediums again, light culture 4d, 25 DEG C.Using callus as converting material, by callus group
Preculture is knitted in NB culture mediums 4d, 25 DEG C, light culture.NB culture mediums are that Rice Callus is induced and subculture medium, formula
For：PH=5.8, N6A great number of elements, 2,4-D 2mg/L, Glu (L-Glutamine) 500mg/L, B5Trace element, enzyme
Solve casein 300mg/L, sucrose 30g/L, B5Vitamin, proline (L-proline) 500mg/L, agar (Agar) 8g/L.
2) by the Agrobacterium EHA105 strains (being stored in -80 DEG C) of the pCUbi1390-RAV2 containing recombinant plasmid, flat board is first drawn
It is activated, then the bacterium for taking the flat board to grow is coated on fresh flat board, 28 DEG C of light culture 2d collect Agrobacterium and are suspended in MBAS agricultures
Bacillus suspension medium, utilizes constant-temperature table (28 DEG C, 100rpm) shaken cultivation to OD600About 0.6, for converting.MBAS agriculture bars
Bacterium suspension medium formula is：MS a great number of elements, 2,4-D 2mg/L, sucrose 30g/L, B5It is micro, casein hydrolysis 500mg/L,
Acetosyringone (AS) 100 μm of ol/L, B5It is organic, inositol 2g/L, pH=5.2.
3) by above-mentioned steps 1) vegetable material immersion step 2 after preculture) Agrobacterium bacterium solution 15min is obtained, afterwards
Bacterium solution is abandoned, material is placed on aseptic filter paper and blots surface moisture.
4) co-culture base planar surface in MBAS and put an aseptic filter paper, the material of wipe dry is placed in aseptic filter paper
On, (can put the material of 40 pieces of 2mm sizes per ware culture medium) half opens wide ware lid, about 30min is blown in superclean bench, afterwards
Seal ware, 25 DEG C of light culture 3d.The formula that MBAS co-cultures base is added on the basis of MBAS Agrobacterium suspension medium formulas
Agar (Agar) 8g/L.
5) material is gone into NBCCH screening and culturing mediums, 25 DEG C of light culture 15d.
6) material subculture is in NBCCH screening and culturing mediums, 25 DEG C of light culture 15d.5), 6) in NBCCH screening and culturing mediums one
Cause, be formulated and be：NB culture mediums (with the NB culture mediums in step 1)), carbenicillin (Carbenicilline) 250mg/L, head
Spore rhzomorph (Cefazollin) 250mg/L, hygromycin (Hm) 50mg/L.
7) callus of picking fresh color is inoculated in the pre- differential mediums of PRCCH, 25 DEG C, 12h/d, illumination about 12d,
Obtain milky or the nodositas kanamycin-resistant callus tissue with green point.The pre- differential medium formulas of PRCCH are：PH=5.8, N6It is basic into
Point, proline (L-proline) 500mg/L, carbenicillin (Carbenicilline) 250mg/L, abscisic acid (ABA) 5mg/
L, Glu (L-Glutamine) 500mg/L, cynnematin (Cefazollin) 250mg/L, 6-benzyl aminopurine (6-
BA) 6mg/L, sucrose 30g/L, hygromycin B (Hygromycin B) 50mg/L, methyl α-naphthyl acetate (NAA) 2mg/L, agar (Agar) 8g/
L。
8) by step 7) milky that is obtained or the nodositas kanamycin-resistant callus tissue access RCCH differential mediums with green point, 25
DEG C, 12h/d, illumination about 12d obtain the green budlet of resistance.RCCH differential mediums：N6Largely, 6-benzyl aminopurine (6-
BA) 3mg/L, methyl α-naphthyl acetate (NAA) 1mg/L, MS is micro, sorbierite (Sorbitol) 18g/L, agar (Agar) 8g/L, B5It is organic,
Sucrose 20g/L, pH=5.8；Cynnematin (Cefazollin) 250mg/L, carbenicillin (Carbenicilline)
250mg/L, hygromycin (Hm) 50mg/L.
9) by step 8) the green budlet of resistance that is obtained is inoculated in RTCCH root medias, 25 DEG C, 12h/d, illumination
About 20d, obtains resistant plant.The formula of RTCCH root medias is：N6Culture medium, methyl α-naphthyl acetate (NAA) 1mg/L, sucrose 20g/
L, agar (Agar) 8g/L, pH=5.8；Cynnematin (Cefazollin) 250mg/L, carbenicillin
(Carbenicilline) 250mg/L, hygromycin (Hm) 50mg/L.
10) by step 9) the resistant plant length that is obtained moved to 5~more than 8cm, when root perfects it is outdoor potted plant.
Wherein：The disinfectant program of Mature seed of rice is：
Seed is shelled, about 30s is soaked with 70% ethanol；Ethanol is removed, 0.2%HgCl, 20min is added；Go 0.2%
HgCl, adds 1%NaClO, 10min；Remove NaClO, with it is sterile washing 5 wipe dries after be inoculated in NB medium cultures.AS：
Acetosyringone, Sigma companies；Hm：Hygromycin, Roche companies are on sale.
Experimental result is shown in Fig. 5, wherein, WT be wild type in spend 11 plant.OE is gene RAV2 Overexpression vector
PCUbi1390-RAV2, which is transformed into wild type, spends 11 to obtain transfer-gen plant, and in mutation type surface, plant leaf is substantially sagging.
11 callus will be spent in Overexpression vector pCUbi1390-RAV2 importing wild types, 28 plants of transformation seedlings are obtained altogether, wherein 17 plants
Positive strain, is mutation type surface；11 plants of negative strains are wild type phenotype.In order to detect the genetic stability of transfer-gen plant, with
Machine chooses 10 parts of T0Transgenic seed, field planting T1For plant, the plant of wherein normal phenotype is transgene negative strain, and
What the sagging phenotype of blade was presented in Leaf angle increase is transgenic positive strain.Above result of study shows, RAV2 mutant it is prominent
Become phenotype, be strictly because the overexpression mutation of RAV2 genes is caused.
In summary, the present invention draws a conclusion：The DNA demethylations of target gene promoter region cause the mistake of RAV2 genes
It is the reason for mutation type surface makes a variation to measure expression, and mutation type surface and RAV2 expression have dosage effect.It is indicated above,
RAV2 is the transcription factor with critical function, has important adjustment effect to rice leaf form.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (7)
1. applications of the paddy rice B3 transcription factor family genes RAV2 in regulation rice leaf intersection angle, it is characterised in that：Described B3
Transcription factor family gene RAV2, its amino acid sequence such as SEQ ID No：Shown in 2.
2. application according to claim 1, it is characterised in that：B3 transcription factor family genes RAV2 is in rice breeding
Using.
3. application according to claim 1, it is characterised in that：B3 transcription factor family genes RAV2 is cultivating transgenosis water
Application in rice.
4. application according to claim 1, it is characterised in that：Recombinant expression carrier containing RAV2 genes is imported into plant
Cell, obtains the transgenic paddy rice of changeable leaf angle；Or it is used as donor parent by the use of the transgenic paddy rice of above-mentioned changeable leaf angle
This, rice leaf intersection angle, the i.e. water by the way that the paddy rice of the changeable leaf angle obtained is improved with plan are improved by backcross breeding method
Rice varieties hybridize, and then filial generation is through repeatedly with recurrent parent hybridization with the paddy rice of seed selection changeable leaf angle；
The described recombinant expression carrier containing RAV2 genes is to use existing plant expression vector construction.
5. the application according to any one of Claims 1 to 4, it is characterised in that：Described B3 transcription factor family genes
RAV2, its nucleotide sequence is：
Sequence table SEQ ID NO:DNA sequence dna shown in 1.
6. application according to claim 4, it is characterised in that：The described recombinant expression carrier containing RAV2 genes is in structure
When building, plus any enhanced, composing type, tissue specificity or induction type before the transcription initiation nucleotides of RAV2 genes
Promoter.
7. application according to claim 4, it is characterised in that：Described plant expression vector is first changed using preceding
Make, including it is GUS or GFP to add reporter gene a kind of in reporter gene and antibiotics resistance gene, described.
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| CN112063626B (en) * | 2019-06-10 | 2022-07-15 | 中国农业大学 | Corn gene ZmRAVL1 and functional site and application thereof |
| CN111004817B (en) * | 2019-12-30 | 2022-03-22 | 北京市农林科学院 | Agrobacterium-mediated rice genetic transformation method |
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Non-Patent Citations (4)
| Title |
|---|
| Byoung Il Je等.RAV-Like1 maintains brassinosteroid homeostasis via the coordinated activation of BRI1 and biosynthetic genes in rice.《The Plant Cell》.2010,第22卷(第6期),第1788页右栏第2段、附图1和3. * |
| Elisson A. C. Romanel等.Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification.《Plos One》.2009,第4卷(第6期),第2页右栏第5段、Table S4. * |
| Xiangqian Zhang等.Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice.《Plant Physiology》.2015,第169卷(第3期),第2118-2128页. * |
| 水稻叶片形态建成分子调控机制研究进展;徐静等;《作物学报》;20130219;第39卷(第5期);第767-774页 * |
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