CN105254728B - Improve GAP-associated protein GAP TaMOR-D and its encoding gene and the application of root system of plant and yield - Google Patents
Improve GAP-associated protein GAP TaMOR-D and its encoding gene and the application of root system of plant and yield Download PDFInfo
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Abstract
The invention discloses GAP-associated protein GAP TaMOR-D and its encoding gene and the applications of improvement root system of plant and yield.Protein provided by the present invention is following protein a) or b): a) protein that the amino acid sequence shown in sequence 2 in sequence table forms；B) by the amino acid sequence of sequence 2 in sequence table by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein with the same function as derived from a).TaMOR albumen and its encoding gene provided by the present invention are of great significance at improvement root system of plant, increase yield and aspect resistant to lodging, will play a significant role in cultivating well developed root system, high yield and plant variety resistant to lodging.
Description
Technical field
The invention belongs to plant genetic engineering fields, more particularly to the GAP-associated protein GAP TaMOR- of improvement root system of plant and yield
D and its encoding gene and application.
Background technique
Food problem is the cardinal task concerning national security, but global warming and the variation of other environmental factors are led
The a large amount of underproduction of grain in global range are caused, food supply is reduced, upheaval is caused because of food shortage in some areas.Global people simultaneously
Mouth is continuously increased, and consumption demand adds bioenergy increase in demand, so that grain demand increases.So increases in grain production problem is complete
The huge challenge that ball agricultural faces.
Root system supports plant strain growth and the foundation of root system microenvironment etc. to play an important role in moisture and nutrient uptake.Root
System's improvement is increasingly a kind of effective means of crop improvement by approval.Tune of the growth and development of root system by series of genes
Section.The regulatory mechanism of root growth and development is specified, scientific basis will be provided because of engineering research and application for root system basis.Study table
It is bright, molecular genetics and genetic engineering research are combined, it will help Gene mining and crop improvement, to reach volume increase
Purpose.
Therefore, it using Modern Molecular Biotechnology, excavates Root morphology and builds up related gene, root is improved by genetic engineering
System, is of great significance to increases in grain production.
Summary of the invention
It is an object of the present invention to provide a kind of GAP-associated protein GAP TaMOR-D and its coding for improveing root system of plant and yield
Gene and application.
Protein provided by the invention is named as TaMOR albumen, derives from wheat (Triticum aestivum L.) product
Kind drought selects No. 10, is following protein a) or b):
A) protein that the amino acid sequence shown in sequence 2 in sequence table forms；
B) amino acid sequence of sequence 2 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues
And/or addition and the protein with the same function as derived from a).
For the ease of the purifying of protein shown in above-mentioned (a), can in by sequence table sequence 1 amino acid residue sequence
The upper label as shown in the table of amino terminal or carboxyl terminal connection of the protein of composition.
Table 1: the sequence of label
| 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 by will be in DNA sequence dna shown in sequence 4, sequence 5 and sequence 6 in sequence table
The codon of one or several amino acid residues is lacked, and/or carries out the missense mutation of one or several base-pairs.
The DNA molecular of code for said proteins also belongs to protection scope of the present invention.
Above-mentioned DNA molecular is following 1) -5) in any DNA molecular:
1) code area is DNA molecular shown in sequence 1 in sequence table；
2) code area is DNA molecular shown in sequence 1 14-739 in sequence table；
3) code area is DNA molecular shown in sequence 1 14-742 in sequence table；4) with 1) or 2) or 3) limit
DNA sequence dna at least has 70%, at least has with 75%, at least with 80%, at least with 85%, at least 90%, at least
With 95%, at least with 96%, at least with 97%, at least with 98% or at least with 99% homology and coding right
It is required that the DNA molecular of 1 protein；
1) or 2) or 3) 5) hybridize under strict conditions with the DNA sequence dna limited and encode the DNA molecular of above-mentioned protein.
Wherein, sequence 1 is made of 926 nucleotide, and 14-742 are ORF, egg shown in sequence 2 in polynucleotide
White matter.
Recombinant vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus containing above-mentioned DNA molecular also belong to
Protection scope of the present invention.
The recombinant vector can be recombinant expression carrier, can also be recombinant cloning vector.
The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture
Bacillus carrier and the carrier etc. that can be used for plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300,
PCUbi 1390, the derivative plant expression vector of pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.
The plant expression vector also may include 3 ' end untranslated regions of foreign gene, i.e., comprising polyadenylation signals and any other
Participate in the DNA fragmentation of mRNA processing or gene expression.The bootable polyadenylic acid of polyadenylation signals is added to mRNA precursor
3 ' end.It, can be plus any enhancing before its transcription initiation nucleotide when using the gene constructed recombinant expression carrier
Type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene
Ubiquitin promoter (pUbi), stress induced promoter rd29A etc., they can be used alone or open with other plants
Mover is used in combination；In addition, enhancer, including translation also can be used when using gene constructed recombinant expression carrier of the invention
Enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but
Must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control signal and starting are close
The source of numeral be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region
Domain or structural gene.For the ease of transgenic plant cells or plant are identified and screened, recombinant expression used can be carried
Body is processed, and can produce the enzyme of color change or the gene of luminophor, tool as the coding that can be expressed in plant is added
Resistant antibiotic marker or anti-chemical reagent marker gene etc..
In the present invention, the promoter for starting the genetic transcription in the recombinant expression carrier is specially 35S promoter.
More specifically, the recombinant expression carrier is at more grams of 1390 carrier of pCAMBIA1300-GFP and pCUBi
Long Weidianchu is inserted into the recombinant plasmid obtained after the gene.The multiple cloning sites specifically be respectively HindIII and SmaI,
BamHI and SpeI.
The expression cassette is by that can start the promoter of the gene expression, the gene and transcription terminator group
At.
Above-mentioned protein or the DNA molecular or the recombinant vector, expression cassette, transgenic cell line, recombinant bacterium or again
Application of the group virus in regulation root system of plant and/or yield also belongs to protection scope of the present invention:
Above-mentioned regulation root system of plant is to improve plant lateral roots, coronal radical amount and/or root dry weight；
Above-mentioned regulation plant products, which are embodied in, improves plant single-strain grain number and/or single plant yield.
Above-mentioned protein or the DNA molecular or the recombinant vector, expression cassette, transgenic cell line, recombinant bacterium or again
Group virus is also the scope of protection of the invention cultivating the application in high-improved root system and/or high yield genetically modified plants.
Above-mentioned protein is being also the scope of protection of the invention as the application in transcription factor.
In above-mentioned application, the plant is monocotyledon or dicotyledon；
Or the plant is monocotyledon, the monocotyledon is specially rice；
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis.
Another object of the present invention is to provide a kind of method for cultivating improvement root system and/or high yield genetically modified plants, packet
It includes following steps: above-mentioned DNA molecular being imported into purpose plant, obtains genetically modified plants；
The genetically modified plants have following 1) -8) at least one of phenotype:
1) the root system improvement of the genetically modified plants；
2) the genetically modified plants yield is higher than the purpose plant；
3) plant height of the genetically modified plants is greater than the purpose plant；
4) the ground dry weight of the genetically modified plants is greater than the purpose plant；
5) tiller number of the genetically modified plants is greater than the purpose plant；
6) the main spike length of the genetically modified plants is greater than the purpose plant；
7) Main stem diameter of the genetically modified plants is greater than the purpose plant；
8) the main fringe Primary branch number of the genetically modified plants is greater than the purpose plant.
In the above method, the root system improvement of the genetically modified plants is embodied at least one of following A-C:
A) the lateral root number of the genetically modified plants is greater than the purpose plant；
B) the coronal radical of the genetically modified plants is greater than the purpose plant；
C) root dry weight of the genetically modified plants is greater than the purpose plant；
The genetically modified plants yield is higher than the purpose plant and is embodied in following D and/or E:
D) single-strain grain number of the genetically modified plants is greater than the purpose plant；
E) single plant yield of the genetically modified plants is greater than the purpose plant；
The plant is monocotyledon or dicotyledon；
Or the plant is monocotyledon, the monocotyledon is specially rice；Embodiment is wild type water
Rice Kitaake.
Or the plant is dicotyledon, the monocotyledon is specially arabidopsis, and in embodiment is quasi- south
0 type of mustard Colombia (Clo-0).
The protein is also belonging to protection scope of the present invention as the application in transcription factor.
The experiment proves that being overexpressed TaMOR-D can effectively improve present invention finds new albumen TaMOR-D
Arabidopsis root system, and effectively improve the characters such as rice root, stalk and its yield, it was demonstrated that TaMOR-D and the root system of plant are raw
Length is related with single plant yield, provides basis to cultivate the genetically modified plants of high yield.
Detailed description of the invention
Fig. 1 is TaMOR-D subcellular localization schematic diagram；A, B is respectively that wheat protoplast and cigarette are observed under Confocal
The case where TaMOR-D-GFP is expressed in blade of grass piece.
Fig. 2 is the transcriptional activation activity testing result of TaMOR-D full length protein and its truncation；A is TaMOR truncation signal
Figure；B is that TaMOR truncation is connected to upgrowth situation of the yeast converted on BD carrier on defect culture medium.
Fig. 3 is relative expression quantity of the TaMOR in wheat different development stage different tissues；A and B is respectively real-time quantitative
The relative expression's situation of PCR and Semi quantitative PCR analysis TaMOR in different times different tissues.
Fig. 4 is from wheat seeds sprouting to the expression of Seedling Stage TaMOR；A is that wheat is sprouted from seed to Seedling Stage
The upgrowth situation of (1-13 days)；B is TaMOR 13 days expression variation tendencies after seed starts to sprout.
Fig. 5 TaMOR is expressed by growth hormone induction；A is the TaMOR expression under auxin (IAA) processing；B is in life
Long element and protein expression inhibitor cycloheximide (CHX) handle the expression of lower TaMOR.
Fig. 6 is to be overexpressed TaMOR arabidopsis phenotype；A is wild type (WT) and transgenic arabidopsis strain (Line1-3)
Plant forms；B is the analysis result of wild type and transgenic line lateral root number.
Fig. 7 is to be overexpressed TaMOR rice phenotype；A is T0For plant phenotype lopsided in transgenic paddy rice strain.B-E is positive
It is frequently grown transgenic paddy rice strain and control comparison chart；B is Transgenic Rice Seedlings and control comparison chart；C is transgenic paddy rice
Maturity period plant and control comparison chart；D is Transgenic Rice Seedlings root system and control comparison chart；E is the transgenic paddy rice maturity period
Root system and control comparison chart；The main fringe of F transgenic paddy rice and control comparison chart.
Fig. 8 is to be overexpressed TaMOR rice phenotypic character statistical result；A is seedling stage transgenic line (Line1-3) and wild
The coronal radical comparison result of type (WT)；B-K is followed successively by maturity period transgenic paddy rice strain (Line1-3) and wild type (WT)
Character comparison result: root dry weight (B), ground dry weight (C), tiller number (D), plant height (E), main spike length (F), Main stem diameter (G), master
Fringe Primary branch number (H), mass of 1000 kernel (I), single-strain grain number (J) and single plant yield (K).
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Wheat (Triticum aestivum L.) kind drought selects No. 10: drought-enduring variety (is compiled from national genebank
Number: ZM009279).It is recorded in " Wang Zhenghang, Wu Xianshan, prosperous small equality wheat flag leaf chlorophyll content and Change of Chlorophyll Fluorescence Kinetics Parameters
With the grey relational grade analysis Acta Agronomica Sinica of yield, 02 phase in 2010 " text, the public can obtain from applicant, be only limitted to use
It is of the invention in repeating.
PCAMBIA1300-GFP carrier: being transformed as follows by the pCAMBIA1300 carrier of CAMBIA company and
Come: (1) DNA fragmentation shown in insetion sequence 3 (contains 2 between the restriction enzyme site BamHI and XbaI of pCAMBIA1300 carrier
CaMV 35S promoter) after obtain intermediate vector；(2) it is inserted into sequence table between the site KpnI and SacI of the intermediate vector
DNA fragmentation shown in sequence 4 (open reading frame of GFP), finally obtains the pCAMBIA1300-GFP carrier.
1390 carrier of pCUBi: it is recorded in " the degeneration-resistant functional analysis of paddy gene OsASIE1.Acta Agronomica Sinica, 2011 37
(10) a 1771-1778. " text, the public can obtain from applicant, only limit the use of in the duplication present invention.
Agrobacterium tumefaciems GV3101 (Agrobacterium tumefaciens StrainGV3101): Biovector
Science Lab, Inc product.
Agrobacterium tumefaciems EHA105 (Agrobacterium tumefaciens strain EHA105): Beijing Hua Yue is raw
Object Products.
0 type of arabidopsis Colombia (Clo-0): it is recorded in that " ringgit beautiful woman exogenous NO gas is to Arabidopsis callus respiratory intensity
And the influence Lanzhou University of mitochondrial complex Ⅰ albumen, Master's thesis in 2007 " text, the public can obtain from applicant,
It is only limitted to for repeating the present invention.
Wild rice Kitaake: it is recorded in " rice breeding of high photosynthetic efficiency progress.Biotechnological Advances, 2014 4
(3) a 153-157. " text, the public can obtain from applicant, only limit the use of in the duplication present invention.
Embodiment 1, TaMOR albumen and its encoding gene and its expression study
One, the acquisition of TaMOR albumen and its encoding gene
The seed that consistent wheat (Triticum aestivum L.) kind drought uniform in size is selected to No. 10, is placed in diameter
In 9cm culture dish, at 22 DEG C, 150 μm of olm of light intensity-2·s-1, the photoperiod is 12h illumination, 12h dark, relative humidity 70%
Under the conditions of, add deionized water culture.The long seedling leaves to a leaf wholeheartedly are taken, with liquid nitrogen flash freezer, -80 DEG C are saved backup.With
TRIZOL extracts total serum IgE, synthesizes the first chain cDNA (Invitrogen) with M-MLV reverse transcription reagent box, using this cDNA as template,
Primers F 1 and R1 carry out PCR amplification, obtain the pcr amplification product of 926bp.By sequencing, which has sequence in sequence table
Nucleotide shown in column 1, is TaMOR-D by unnamed gene shown in the PCR product, and the code area of the gene is 1 14- of sequence
742 nucleotide, the albumen of gene coding are named as TaMOR-D, and amino acid sequence is sequence 2 in sequence table.
Two, TaMOR-D protein expression is studied
A, the subcellular localization of TaMOR-D albumen
Recombinant vector pCAMBIA1300-TaMOR-D-GFP is that TaMOR-D shown in sequence 1 is inserted into pCAMBIA1300-
The carrier obtained between the Hind III and SmaI restriction enzyme site of GFP carrier.
Recombinant vector pCAMBIA1300-TaMOR-D-GFP is transferred in wheat protoplast and tobacco leaf respectively.
Wheat protoplast Transient Expression step:
1, the expression vector Plasmid DNA of preparation high concentration fusion target gene, concentration should reach 2 μ g/ μ l.
2, it takes wheat breed drought to select No. 10 leaf one heart stage wheat green seedlings blades, removes vane tip and base with blade
Remaining blade-section, is cut into the strip of 0.5-1mm by portion's tissue.
3, the blade cut is placed in 30ml enzymolysis liquid, under dark condition, is placed in shaking table, gentle agitation digests 3-
4h。
4, enzymolysis liquid is diluted with isometric W5 solution.
5, with W5 solution rinse stainless steel mesh screen (200 mesh) 2-3 times.
6, with the zymolyte after stainless steel mesh screen (200 mesh) filtering dilution, protoplast solution, 100 × g centrifugation are obtained
4min。
7, protoplast is softly resuspended with the 30ml W5 solution of ice bath, 100 × g is centrifuged 4min.
8, it is gently sucked out after supernatant and protoplast is resuspended again with 30ml W5 solution, and place it in 30min on ice；Together
When with microexamination protoplast number and state.
9,100 × g is centrifuged 4min, and protoplast is resuspended with suitable (20ml) MMg solution.
10,20 μ g plasmids are added in 5ml centrifuge tube.
11,1ml protoplast is slowly drawn, is added in the centrifuge tube containing plasmid.
12, the PEG4000 solution of 1ml is added, at room temperature, stands 20min.
13,3ml W5 solution is added, mixes gently, terminates reaction.
14,100 × g is centrifuged 4min, supernatant is gently sucked out, and protoplast is resuspended with 3ml W5 solution.
15, previous step is repeated, protoplast, 24 DEG C of illumination cultivation 16h are resuspended with 1ml W5 solution.
16, GFP expression is observed and recorded under Leika TCS-NT laser scanning co-focusing microscope.
The main buffer configuration of wheat protoplast Transient Expression:
(1) enzymolysis liquid
55 DEG C of water-bath 10min, are added following reagent after being cooled to room temperature:
10mM CaCl2 1ml 0.15M CaCl2
0.1%BSA 1ml 1.5%BSA
(2) 4000 solution of PEG
(3) W5 solution
(4) MMg solution
Mannitol (mannitol) 400mM
Magnesium chloride (MgCl2) 15mM
2-morpholine ethane sulfonic acid (MES) 4mM, pH5.7
Transformation of tobacco step:
1, the purpose plasmid thermal shock built is transformed into Agrobacterium GV3101, is inverted, 28 DEG C of culture 2-3d.
2, picking passes through the positive colony of bacterium colony PCR identification, is inoculated in 5ml YEB (Kan+Rif resistance) fluid nutrient medium,
28 DEG C of 200rpm overnight incubations.
3, OD value is detected when bacterium solution is in orange red, and draws bacterium solution according to following formula according to OD value.
Vconstruct=Vfinal × 0.5/OD, Vp19=Vfinal × 0.3/OD
4, the bacterium solution of carrier and p19 are mixed in a test tube according to the ratio calculated, 12000rpm is centrifuged 1min.
5, using Buffer resuspension thallus is infected, continue to cultivate 4h.
Buffer configuration method is as follows:
6, it is injected in the back side of tobacco (Nicotiana benthamiana) blade, the tobacco normal growth 3- injected
4d。
7, GFP luminous situation is observed with Confocal.
Subcellular localization result is as shown in Figure 1.The result shows that positioning of the TaMOR in wheat protoplast and tobacco cell
It is identical, all it is located on nucleus and cell membrane.
B, the transcriptional activation activity of TaMOR-D albumen
TaMOR-D albumen P1 truncate is 2 1-242 amino acids of sequence；
TaMOR-D albumen P2 truncate is 2 7-242 amino acids of sequence；
TaMOR-D albumen P3 truncate is 2 29-242 amino acids of sequence；
TaMOR-D albumen P4 truncate is 2 109-242 amino acids of sequence；
TaMOR-D albumen P5 truncate is 2 7-108 amino acids of sequence；
TaMOR-D albumen P6 truncate is 2 1-108 amino acids of sequence；
TaMOR-D albumen P7 truncate is 2 1-28 amino acids of sequence；
Above-mentioned each truncate encoding gene is inserted into pGBKT7 carrier (BD carrier) (Clontech Products, product
Catalog number (Cat.No.) is VT1638) EcoRI and BamHI restriction enzyme site between obtain recombinant vector (Fig. 2A).
Using Yeast system, the transcriptional activation activity and its activity of TaMOR are determined by two reporter genes HIS and LACZ
Region.(SD/-Trp-His) and three scarce culture mediums (SD/-Trp-His-Ade) are lacked (using the basis of general Jino company two
The scarce Trp-His-Ade-dropout configuration of culture medium Minimal SD Base, two scarce Trp-His-dropout and three) on, inspection
Can yeast be surveyed grow on auxotrophy culture medium.
As a result, it has been found that lacking on (SD/-Trp-His) and three scarce culture mediums (SD/-Trp-His-Ade) two, only contain P4
The yeast growth of segment is good, as shown in Figure 2 B.Show that TaMOR-D has transcriptional activation activity, C-terminal is the transcription of TaMOR-D
Active region.
C, expression of the TaMOR-D gene in different tissues
No. 10 growth different times are selected to take the different tissues position to be in wheat (Triticum aestivum L.) kind drought
Sample, through liquid nitrogen frozen, -80 DEG C are saved backup.Including germination period (plumule, foundation and root), Seedling Stage (leaf, foundation and root) and
Heading stage (root, leaf, leaf sheath, section, internode and fringe).The total serum IgE of above-mentioned Liquid nitrogen storage sample is extracted, respectively with TRIZOL with M-
MLV reverse transcription reagent box synthesizes the first chain cDNA, using real-time quantitative with semi-quantitative method detection TaMOR in different times difference
Differential expression in tissue.Use the Tubulin gene of constitutive expression as internal reference, the primer sequence of design is as follows:
The primer sequence for detecting gene TaMOR-D expression is as follows:
TaMOR-RT-F:5 '-GTCTTTGCGCCCTACTTCTG-3 ' (80-99 of sequence 1)；
TaMOR-RT-R:5 '-TCATGACCTGCTGCTGGAG-3 ' (281-299 reverse complemental sequences of sequence 1
Column)；
The primer sequence for detecting gene Tubulin expression is as follows:
Tubulin-RF:5 '-GAGGCCTCGTGTGGTCGCTTTGT-3 '；
Tubulin-RR:5 '-GCCCAGTTGTTACCCGCACCAGA-3 '.
Real-time quantitative and sxemiquantitative experimental result are as shown in figure 3, position, that is, root that TaMOR-D occurs in adventitious root and lateral root
Expression quantity is apparently higher than other tissues on base and root.
D, expression of the TaMOR gene from wheat seeds sprouting to seedling
Until the 13rd day since wheat seeds sprouting, intercepting wheat foundation respectively is sample, through liquid nitrogen frozen, -80 DEG C
It saves backup.The total serum IgE of above-mentioned Liquid nitrogen storage sample is extracted, respectively with TRIZOL with M-MLV reverse transcription reagent box synthesis first
Chain cDNA detects TaMOR from the expression sprouted to the 13rd day Seedling Stage using real-time quantitative method.Detection primer is the same as detection
The experiment of TaMOR tissue expression.
U-shaped trend is presented as shown in figure 4, expressing variation from sprouting Seedling Stage TaMOR in real-time quantitative experimental result, i.e., and the
1 day expression quantity is higher, declines within the 2nd day, the one 4-5 days minimum, begins to ramp up within the 6th day, reaches highest within the 13rd day.
E, the expression of TaMOR gene pairs auxin processing
Wheat seed water planting 5 day, seedling is moved on in 1 μM of heteroauxin (IAA) solution and is handled, respectively 0h, 1h, 3h,
6h, 12h, the foundation tissue for intercepting seedling for 24 hours are sample, and through liquid nitrogen frozen, -80 DEG C are saved backup.Take the 8th day small after sprouting
Wheat seedling, which is first dipped into 50 μM of cycloheximides (CHX), carries out 24 hours pre-processings, then immerses water, 50 μM of CHX, 1 respectively
μM IAA, 50 μM of CHX and 1 μM of IAA mixed solution in handle 3h.The foundation tissue for intercepting seedling is sample, cold through liquid nitrogen
Freeze, -80 DEG C save backup.The total serum IgE of above-mentioned Liquid nitrogen storage sample is extracted, respectively with TRIZOL with M-MLV reverse transcription reagent box
The first chain cDNA is synthesized, gene TaMOR is detected to the response condition of processing using the method for semiquantitive PCR.Detection primer is the same as inspection
Survey the experiment of TaMOR tissue expression.
Semiquantitive PCR testing result is as shown in figure 5, TaMOR reaches high by auxin processing up-regulated expression, in processing 3h
Peak.And growth hormone induction TaMOR expression is not influenced by protein inhibitor CHX.
The functional verification of embodiment 2, TaMOR-D gene
One, TaMOR-D gene is improving the application in arabidopsis lateral root number
1, the amplification of TaMOR-D gene
According to the primers of TaMOR-D gene order 1 to (F1 and R1), 5 ' end of primer introduces respectively
HindIII and SmaI digestion recognition site.Select No. 10 cDNA as template using wheat breed drought, PCR amplification TaMOR-D gene；
Pcr amplification product is subjected to 1% agarose gel electrophoresis, the band of recovery purifying 900bp or so.
F1:5 '-CCCAAGCTTATGACGGGACTTGGGTCG-3 ' (underscore part is the restriction enzyme site of HindIII,
Sequence afterwards is 14-31 of sequence 1 in sequence table)；
R1:5 '-TCCCCCGGGCGAGCGATTTAGGTACGCAT-3 ' (underscore part is the restriction enzyme site of SmaI,
Sequence afterwards is 720-739 reverse complementary sequences of sequence 1 in sequence table).
2, the building of recombinant vector
1. recycling digestion products with the PCR product of restriction enzyme HindIII and SmaI digestion step 1 recovery purifying；
2. recycling carrier framework with restriction enzyme HindIII and SmaI digestion carrier pCAMBIA1300-GFP；
3. the digestion products of step 1. are connected with the carrier framework of step 2.；
4. by the electroporated e.colistraindh5α of the connection product of step 3., 37 DEG C of overnight incubations, positive gram of picking
It is grand to be sequenced.Sequencing result shows to have obtained recombinant vector pCAMBIA1300-TaMOR-D-GFP, for by sequence 1 the
The carrier that the HindIII and SmaI restriction enzyme site of 14-739 nucleotides inserted pCAMBIA1300-GFP obtains.
2, the acquisition of transgenic arabidopsis
1) 2 μ l correct recombinant plasmid pCAMBIA1300-TaMOR-D-GFP is constructed to be added to 20 μ l Agrobacterium tumefaciems
In GV3101 competent cell, 28 DEG C of thermal shock conversions.
2) Agrobacterium of conversion is subjected to bacterium colony PCR identification, will identifies correct single colonie, access 5ml YEB liquid training
Support in base (kanamycins containing 50mg/l, 50mg/l rifampin), 28 DEG C, 250rpm shaken cultivation it is overnight.
3) the 5ml bacterium solution of step 2 is gone into 250ml YEB fluid nutrient medium (kanamycins containing 50mg/l, 50mg/l benefit good fortune
It is flat) in, 28 DEG C, 250rpm shaken cultivation about 14h (bacterium solution OD600Reach 0.8-1.0).
4) Agrobacterium infection buffer is configured.Agrobacterium infection buffer: 1/2MS 100ml adds 5g sucrose, 80 μ l
Silwet-77。
5) thalline were collected by centrifugation, and thallus is resuspended with Agrobacterium infection buffer.
6) arabidopsis Colombia 0 type (Clo-0) plant just to have bloomed is chosen, inflorescence is immersed in Agrobacterium infection buffering
About 30sec in liquid puts on freshness protection package moisturizing, and dark culturing for 24 hours, then removes freshness protection package, normal to cultivate, and harvest seed is T0
Generation.T0Generation selfing obtains T1For seed.
7) by T1It after seed disinfection, is laid on MS culture medium (hygromycin containing 50mg/L), 4 DEG C of vernalization 2d, 22 DEG C,
16h illumination/8h dark culturing 14d, selects the transgenic Arabidopsis plants of hygromycin, and transgenic line breeding plus generation obtain
To multiple T3Turn TaMOR-D arabidopsis strain for homozygosis (it is homozygous line that all offsprings, which all have the parental generation of hygromycin resistance).
3, the Molecular Identification of transgenic arabidopsis
Extract T3The generation homozygous genomic DNA for turning TaMOR-D arabidopsis strain, carries out PCR amplification with above-mentioned F1 and R1, obtains
It is positive T to 926bp3Turn TaMOR-D arabidopsis strain, after testing, T for homozygosis3Turn TaMOR-D arabidopsis strain for homozygosis
All positives.
Empty carrier pCAMBIA1300-GFP is imported in 0 type of arabidopsis Colombia (Clo-0) using same method,
Obtain T3Turn empty carrier arabidopsis for homozygosis.
4, the phenotypic evaluation of transgenic arabidopsis
1), the culture of experimental material
Take 0 type of arabidopsis Colombia (Clo-0) (wild type control, WT), T3Turn TaMOR-D arabidopsis strain for homozygosis
(Line1, Line2 and Line3) and T3Turn the seed of empty carrier arabidopsis (Vector) for homozygosis, first with added with 0.01% (v/
V) 10% (v/v) NaClO solution disinfection of Triton X-100 handles 15min, then clear with aqua sterilisa in superclean bench
It washes 8 times.It is seeded into addition 3.0% (w/v) sucrose again, the MS culture medium of 0.8% (w/v) agar powder carries out sprouting culture.
It sprouts culture first through 4 DEG C of processing 2d, then moves on to 22 DEG C, vertically cultivates 8d in the incubator of 12h illumination.
3 plants of each strain, experiment is repeated 3 times, and results are averaged.
2), the statistical analysis of arabidopsis lateral root number
Arabidopsis thaliana Seedlings root system is scanned using EXPSON EXTRESSION 10000XL scanner (EXPSON, JAPAN),
Root system scan image is analyzed using winRHIZO software.
Seedlings root and its statistical result are as shown in fig. 6, T3Turn TaMOR-D arabidopsis strain (Line1, Line2 for homozygosis
And Line3) lateral root number be significantly higher than wildtype Arabidopsis thaliana.
0 type of arabidopsis Colombia (Clo-0) and T3Turn empty carrier arabidopsis without significant difference for homozygosis.
The above results illustrate that the lateral root number of arabidopsis can be effectively increased by being overexpressed TaMOR-D.
Two, TaMOR-D gene significantly improves rice root and improves yield
1, the amplification of TaMOR-D gene
According to the primers of TaMOR-D gene to (F2 and R2), 5 ' end of primer introduces BamHI and SpeI respectively
Digestion recognition site.Select No. 10 cDNA as template using wheat breed drought, PCR amplification TaMOR-D gene；By pcr amplification product
Carry out 1% agarose gel electrophoresis, the band of recovery purifying 900bp or so.
F2:5 '-CGGGATCC(underscore part is the restriction enzyme site of BamHI to ATGACGGGACTTGGGTCG-3 ', thereafter
Sequence be 14-31 of sequence 1 in sequence table)；
R2:5 '-GGACTAGTTTACGAGCGATTTAGGTACGC-3 ' (underscore part is the restriction enzyme site of SpeI,
Sequence afterwards is 722-742 reverse complementary sequences of sequence 1 in sequence table).
2, the building of recombinant vector
1. recycling digestion products with the PCR product of restriction enzyme BamHI and SpeI digestion step 1 recovery purifying；
2. recycling carrier framework with restriction enzyme BamHI and SpeI digestion carrier pCUbi1390；
3. the digestion products of step 1. are connected with the carrier framework of step 2.；
4. by the electroporated e.colistraindh5α of the connection product of step 3., 37 DEG C of overnight incubations, positive gram of picking
It is grand to be sequenced.Sequencing result shows to have obtained recombinant vector pCUbi1390-TaMOR-D, for by sequence 1 14-742
The carrier that the BamHI and SpeI restriction enzyme site of nucleotides inserted pCUbi1390 obtains.
2, the acquisition of transgenic paddy rice
Using rice varieties Kitaake as receptor, pass through the mature embryo of agrobacterium mediation converted method rice transformation Kitaake
Callus is taken root, strong sprout process by the screening and differentiation of resistant calli, finally obtains 21 T0In generation, turns TaMOR-D
Rice strain.
3, the Molecular Identification of transgenic arabidopsis
Extract T0In generation, turns the genomic DNA of TaMOR-D rice strain, carries out PCR amplification with above-mentioned F2 and R2, obtains
926bp is positive T0In generation, turns TaMOR-D rice strain, after testing, T0In generation, turns all positives of TaMOR-D rice strain.
Using same method by empty carrier pCUbi1390 Introduced into Rice kind Kitaake, T is obtained3Homozygous turn of sky of generation
Carrier rice.
4, transgenic paddy rice phenotypic evaluation
Fetch water rice varieties Kitaake (wild type control, WT), T3Generation turn TaMOR-D rice strain (Line1, Line2 and
) and T Line33Turn the seed of empty carrier rice (vector) for homozygosis, sowing carries out potting outside scenery.Each strain sowing
15 seeds, experiment are repeated 3 times, and results are averaged.
Respectively in seedling stage (after planting the 20th day) and maturity period (after planting the 70th day) to transgenic line and wild type into
Row phenotypic evaluation.Seedling stage is mainly for statistical analysis to the coronal radical of plant root.Maturity period to root dry weight, ground dry weight, point
Tiller number, plant height, main spike length, Main stem diameter, main fringe Primary branch number, mass of 1000 kernel, single plant yield and single-strain grain number carry out statistical
Analysis.Statistical result such as table 2.
2 seedling stage of table and maturity period phenotype statistical result
| Character | WT | Vector | Line1 | Line2 | Line3 |
| Coronal radical | 21.3±3.2B | - | 31±1.0A | 30.0±2.6A | 31.0±1.0A |
| Root dry weight | 1.73±0.1B | 1.71±0.09B | 2.10±0.18A | 2.07±0.15A | 1.99±0.09A |
| Ground dry weight | 29.57±1.08B | 29.62±1.05B | 32.46±1.19A | 32.30±1.54A | 31.68±1.32A |
| Tiller number | 9.4±1.17 | 9.3±0.90 | 9.4±1.30 | 9.2±1.12 | 9.5±0.87 |
| Plant height | 60.6±1.45B | 60.4±1.59B | 62.5±1.37A | 62.4±1.75A | 62.4±1.43A |
| Main spike length | 11.5±0.53B | 11.6±0.52B | 14.1±0.78A | 14.0±0.71A | 13.9±0.78A |
| Main stem diameter | 4.66±0.27B | 4.77±0.24B | 5.88±0.34A | 5.24±0.46A | 5.62±0.29A |
| Main fringe Primary branch number | 6.4±0.53B | 6.4±0.75B | 11.7±0.86A | 11.1±0.78A | 10.0±0.71A |
| Mass of 1000 kernel | 21.14±0.21A | 21.32±0.25A | 19.26±0.97B | 19.31±0.24B | 19.58±0.32B |
| Single plant yield | 10.37±0.67B | 10.28±0.50B | 11.92±0.75A | 11.83±0.80A | 11.78±1.20A |
| Single-strain grain number | 493.6±21.7B | 485.4±26.9B | 650.9±11.4A | 630.7±26.5A | 634.9±51.6A |
Annotation: A, B indicates that there are significant differences in 0.01 level.
T0In generation, has 16 to turn TaMOR-D rice to show profuse coronal, but aerial growth developmental deformity,
Seed (Fig. 7 A) cannot be finally obtained, remaining 5 strain growth is normal.
T3For seedling stage phenotypic evaluation as shown in fig. 7, Fig. 7 B seedling stage plant entirety growing way comparison diagram, Fig. 7 D are seedling stage root table
Type comparison diagram.Coronal radical statistical result is as shown in table 2, shows to be significantly more than control strain in transgenic plant seedling stage coronal radical
System.
T3As shown in Figure 7 and Figure 8 for maturity period phenotypic evaluation, Fig. 7 C indicates that control and transgenic line tie up to the whole of maturity period
Body comparison, the root system of Fig. 7 E expression control and transgenic line maturity period, Fig. 7 F indicate control and transgenosis maturity period strain
Main fringe.In Fig. 8, A-K is respectively coronal radical, root dry weight, ground dry weight, the tiller number, plant height, master compareed with transgenic paddy rice
Spike length, Main stem diameter, main fringe Primary branch number, mass of 1000 kernel, single-strain grain number, single plant yield comparing result.T3In generation, turns TaMOR-D water
The plant height of rice, main spike length degree and Primary branch number are significantly higher than wild type.The increase of plant height is as caused by main spike length degree increase.
The transgenic line underground and aboveground dry weight in maturity period is significantly higher than wild type.The mass of 1000 kernel of transgenic line is substantially less than wild
Type, but single-strain grain number and grain yield are significantly higher than wild type.In addition, the stalk diameter of transgenic line is noticeably greater than wild
Type.The experimental results showed that rice root can significantly be improved and increase yield by being overexpressed TaMOR-D, while big root system and sturdy stem
Stalk can also enhance the anti-volt of plant.
Claims (12)
1. a kind of protein, the protein that the amino acid sequence shown in sequence 2 in sequence table forms.
2. encoding the DNA molecular of protein described in claim 1.
3. DNA molecular according to claim 2, it is characterised in that: the DNA molecular is that code area is sequence in sequence table
1 DNA molecular shown in 14-739.
4. recombinant vector, expression cassette, recombinant bacterium or recombinant virus containing DNA molecular described in Claims 2 or 3.
5. DNA molecular described in protein or Claims 2 or 3 described in claim 1 or recombinant vector as claimed in claim 4,
The application of expression cassette, recombinant bacterium or recombinant virus in regulation root system of plant and/or yield.
6. DNA molecular described in protein or Claims 2 or 3 described in claim 1 or recombinant vector as claimed in claim 4,
Expression cassette, recombinant bacterium or recombinant virus are cultivating the application in root system improvement and/or high yield genetically modified plants；
The genetically modified plants of the root system improvement are at least one of following A-C:
A) the lateral root number of the genetically modified plants is greater than purpose plant；
B) the coronal radical of the genetically modified plants is greater than purpose plant；
C) root dry weight of the genetically modified plants is greater than purpose plant.
7. application according to claim 5 or 6, it is characterised in that: the plant is monocotyledon or dicotyledon.
8. application according to claim 7, it is characterised in that: the plant is monocotyledon or dicotyledon；Institute
Stating monocotyledon is rice；The dicotyledon is arabidopsis.
9. protein described in claim 1 is as the application in transcription factor.
10. a kind of method for cultivating improvement root system and/or high yield genetically modified plants, include the following steps: claim 2 or
3 DNA moleculars import purpose plant, obtain genetically modified plants；
The genetically modified plants have following 1) -8) at least one of phenotype:
1) the root system improvement of the genetically modified plants；
The root system improvement of the genetically modified plants is embodied at least one of following A-C:
A) the lateral root number of the genetically modified plants is greater than the purpose plant；
B) the coronal radical of the genetically modified plants is greater than the purpose plant；
C) root dry weight of the genetically modified plants is greater than the purpose plant；
2) the genetically modified plants yield is higher than the purpose plant；
3) plant height of the genetically modified plants is greater than the purpose plant；
4) the ground dry weight of the genetically modified plants is greater than the purpose plant；
5) tiller number of the genetically modified plants is greater than the purpose plant；
6) the main spike length of the genetically modified plants is greater than the purpose plant；
7) Main stem diameter of the genetically modified plants is greater than the purpose plant；
8) the main fringe Primary branch number of the genetically modified plants is greater than the purpose plant.
11. according to the method described in claim 10, it is characterized by:
The genetically modified plants yield is higher than the purpose plant and is embodied in following D and/or E:
D) single-strain grain number of the genetically modified plants is greater than the purpose plant；
E) single plant yield of the genetically modified plants is greater than the purpose plant；
The plant is monocotyledon or dicotyledon.
12. according to the method for claim 11, it is characterised in that:
The monocotyledon is rice；
The dicotyledon is arabidopsis.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006006956A2 (en) * | 2004-02-02 | 2006-01-19 | E.I. Dupont De Nemours And Company | Altering root structure during plant development |
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006006956A2 (en) * | 2004-02-02 | 2006-01-19 | E.I. Dupont De Nemours And Company | Altering root structure during plant development |
Non-Patent Citations (2)
| Title |
|---|
| Overexpression of wheat gene TaMOR improves root system architecture and grain yield in Oryza sativa;Bo Li等;《Journal of Experimental Botany》;20160526;第67卷(第14期);第4155-4167页 * |
| The maize (Zea mays L.)RTCS gene encodes a LOB domain protein that is a key regulator of embryonic seminal and post-embryonic shoot-borne root initiation;Graziana Taramino等;《The Plant Journal》;20070531;第50卷(第4期);第649-659页 * |
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