CN104861051B - Plant development associated protein AtUBP15 and its encoding gene and application - Google Patents
Plant development associated protein AtUBP15 and its encoding gene and application Download PDFInfo
- Publication number
- CN104861051B CN104861051B CN201410064497.3A CN201410064497A CN104861051B CN 104861051 B CN104861051 B CN 104861051B CN 201410064497 A CN201410064497 A CN 201410064497A CN 104861051 B CN104861051 B CN 104861051B
- Authority
- CN
- China
- Prior art keywords
- sequence
- atubp15
- plant
- protein
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 101000841472 Arabidopsis thaliana Ubiquitin carboxyl-terminal hydrolase 15 Proteins 0.000 title claims abstract description 36
- 230000008121 plant development Effects 0.000 title abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 241000196324 Embryophyta Species 0.000 claims description 93
- 241001233957 eudicotyledons Species 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 8
- 241000209510 Liliopsida Species 0.000 claims description 6
- 108091006146 Channels Proteins 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 5
- 230000018109 developmental process Effects 0.000 abstract description 5
- 125000000539 amino acid group Chemical group 0.000 abstract description 3
- 238000003976 plant breeding Methods 0.000 abstract description 2
- 238000006467 substitution reaction Methods 0.000 abstract description 2
- 241000219194 Arabidopsis Species 0.000 description 27
- 239000013612 plasmid Substances 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 239000002689 soil Substances 0.000 description 7
- 238000003306 harvesting Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 4
- 241000219195 Arabidopsis thaliana Species 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000219198 Brassica Species 0.000 description 3
- 235000003351 Brassica cretica Nutrition 0.000 description 3
- 235000003343 Brassica rupestris Nutrition 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 3
- 241001070944 Mimosa Species 0.000 description 3
- 235000016462 Mimosa pudica Nutrition 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 235000010460 mustard Nutrition 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000009331 sowing Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 229910052902 vermiculite Inorganic materials 0.000 description 2
- 235000019354 vermiculite Nutrition 0.000 description 2
- 239000010455 vermiculite Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101710197650 Actin-7 Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- YCSMVPSDJIOXGN-UHFFFAOYSA-N CCCCCCCCCCCC[Na] Chemical compound CCCCCCCCCCCC[Na] YCSMVPSDJIOXGN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000710118 Maize chlorotic mottle virus Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 241000592344 Spermatophyta Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of plant development associated protein AtUBP15 and its encoding gene and application.Protein provided by the invention, AtUBP15 albumen is named as, is as follows（a）Or（b）Or（c）：（a）The protein being made up of the amino acid sequence shown in sequence in sequence table 1；（b）The protein being made up of the amino acid sequence shown in sequence in sequence table 2；（c）By substitution of the amino acid sequence of sequence 1 or sequence 2 by one or several amino acid residues and/or missing and/or addition and the protein as derived from sequence 1 or sequence 2 related to development of plants.The present invention also protects a kind of method for cultivating genetically modified plants, is that in the AtUBP15 channel genes purpose plant, will obtain genetically modified plants of the development better than the purpose plant.The present invention has substantial worth for plant breeding.
Description
Technical field
The present invention relates to a kind of plant development associated protein AtUBP15 and its encoding gene and application.
Background technology
Grain is the primary condition of human survival, as population quickly increases, cultivated area is persistently reduced, improves unit plane
Store up grain food yield naturally just into people solve food problem main path.
China is a country for very lacking soil, although area is not small, most of is unbroken mountains high mountain, height
Former desert, the Plain of livable preferably agriculture only accounts for the 12% of area, therefore raising per mu yield has king-sized importance to China.
Recently due to the application of bioengineering, improve per mu yield and show unprecedented huge chance.
Size of plant seed is highly important for crop yield.
The content of the invention
It is an object of the invention to provide a kind of plant development associated protein AtUBP15 and its encoding gene and application.
Protein provided by the invention, come from Columbia ecotype arabidopsis, be named as AtUBP15 albumen, be as
Under（a）Or（b）Or（c）：
（a）The protein being made up of the amino acid sequence shown in sequence in sequence table 1；
（b）The protein being made up of the amino acid sequence shown in sequence in sequence table 2；
（c）By substitution of the amino acid sequence of sequence 1 or sequence 2 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 or sequence 2 related to development of plants.
In order that（a）Or（b）In protein be easy to purify, can in as sequence table sequence 1 or the amino shown in sequence 2
The amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of acid sequence composition.
The sequence of the label of table 1
| 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 |
It is above-mentioned（c）In protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned（c）In the encoding gene of protein can be by by the DNA sequence dna shown in sequence in sequence table 3, sequence 4 or sequence 5
The codon of one or several amino acid residues is lacked, and/or carries out the missense mutation of one or several base-pairs, and/or
The coded sequence that its 5 ' end and/or 3 ' ends connect the label shown in table 1 obtains.
Encode the gene of the AtUBP15 albumen（AtUBP15 genes）Fall within protection scope of the present invention.
The AtUBP15 genes are as follows（1）Or（2）Or（3）Or（4）DNA molecular：
（1）DNA molecular shown in the sequence 3 of sequence table；
（2）DNA molecular of the code area as shown in the sequence 4 of sequence table or the sequence 5 of sequence table；
（3）Under strict conditions with（1）Or（2）The DNA sequence dna hybridization of restriction and the DNA of coded plant development associated protein
Molecule；
（4）With（1）Or（2）The DNA sequence dna of restriction at least has more than 90% homogeneity and coded plant development associated protein
DNA molecular.
Above-mentioned stringent condition can be as follows：50 DEG C, in 7% lauryl sodium sulfate（SDS）、0.5M NaPO4And 1mM
Hybridize in EDTA mixed solution, rinsed in 50 DEG C, 2 × SSC, 0.1%SDS；Can also be：50 DEG C, in 7%SDS, 0.5M
NaPO4Hybridize with 1mM EDTA mixed solution, rinsed in 50 DEG C, 1 × SSC, 0.1%SDS；Can also be：50 DEG C, 7%
SDS、0.5M NaPO4Hybridize with 1mM EDTA mixed solution, rinsed in 50 DEG C, 0.5 × SSC, 0.1%SDS；May be used also
For：50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize with 1mM EDTA mixed solution, at 50 DEG C, 0.1 × SSC, 0.1%SDS
Middle rinsing；Can also be：50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize with 1mM EDTA mixed solution, at 65 DEG C, 0.1 ×
Rinsed in SSC, 0.1%SDS；Or：In 6 × SSC, 0.5%SDS solution, hybridize under 65oC, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Above-mentioned " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " can with the naked eye or computer is soft
Part is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage（%）Represent, it can
With for evaluating the homogeneity between correlated series.
Expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing the AtUBP15 genes belong to the present invention
Protection domain.
The recombinant expression carrier of the gene can be contained with existing expression vector establishment.The expression vector can also include
3 ' end untranslated regions of foreign gene, i.e., process or gene expression comprising polyadenylation signals and any other participations mRNA
DNA fragmentation.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Using described gene constructed heavy
During group expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotides, it
Can be used alone or be used in combination with other promoters；In addition, when using the gene constructed recombinant expression carrier of the present invention,
Enhancer, including translational enhancer or transcriptional enhancer also can be used, but must be identical with the reading frame of coded sequence, to ensure
The correct translation of whole sequence.The source of the translation control signal and initiation codon is extensive, can be it is natural,
It can be synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of being identified and being sieved
Choosing, can be processed to the recombinant expression carrier, and the enzyme of color change or the base of luminophor can be produced by such as adding coding
Cause, resistant antibiotic marker or anti-chemical reagent marker gene etc..
The recombinant vector concretely following recombinant plasmid：AtUBP15 genes insertion plasmid pMDC99 is obtained
Recombinant plasmid.
The present invention also protects a kind of method for cultivating genetically modified plants, is by the AtUBP15 channel genes purpose plant
In, obtain the genetically modified plants that developmental level is better than the purpose plant.The AtUBP15 genes can pass through the recombinant vector
Import the purpose plant.The AtUBP15 genes specifically can import the purpose plant by the recombinant plasmid.The side
In method, the recombinant vector can be by using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, electricity
Conventional biology methods conversion plant cell or the tissue such as lead, be agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.
The purpose plant is monocotyledon or dicotyledon.The dicotyledon concretely arabidopsis, such as Colombia
Arabidopsis thaliana ecotype." developmental level is excellent " can specifically be presented as that seed is big and/or seed weight is big and/or it is big to spend and/or
Blade is big and/or cell propagation is fast.
In the above method, AtUBP15 genes can be modified first as follows, then import purpose plant, to reach more preferable table
Up to effect：
1. modified and optimized according to being actually needed, so that gene efficient expression；For example, can be according to recipient plant institute partially
The codon of love, the codon optimization of DNA level is carried out while amino acid sequence is kept to meet plant-preference；Optimization
During, it is desirable that certain G/C content is kept in the DNA after optimization, to be best implemented with the high level expression of gene, its
Middle G/C content can be 35%, 45%, 50% or more than 60%；
2. the nucleotide sequence of the neighbouring initial methionine of modification, so that translation effectively starting；For example, using in plant
Known effective sequence is modified；
3. it is connected with the promoter of various plants expression, in favor of its expression in plant；The promoter may include
Composing type, induction type, sequential regulation, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter
Selection will need and change with expression time and space, and also depend on target kind；Such as the specificity of tissue or seed
Promoter is expressed, acceptor as needed is depending on what period of development；Although demonstrate many from dicotyledon
Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for
Expression in dicotyledon, the expression that monocotyledonous promoter is used in monocotyledon；
4. being connected with suitable transcription terminator, the expression efficiency of gene can also be improved；Such as from CaMV's
Tml, from rbcS E9；Any known available terminator to be worked in plant can enter with gene of the present invention
Row connection；
5. enhancer sequence is introduced, such as intron sequences（Such as from Adhl and bronzel）And viral leader sequence
（Such as from TMV, MCMV and AMV）.
The present invention also protects the AtUBP15 albumen, the AtUBP15 genes or the recombinant vector cultivating development water
Application in flat excellent genetically modified plants.The plant that sets out of the genetically modified plants is monocotyledon or dicotyledon.Institute
Dicotyledon concretely arabidopsis is stated, such as Columbia ecotype arabidopsis." developmental level is excellent " is embodied as
Seed is big and/or seed weight is big and/or spends big and/or blade big and/or cell propagation is fast.
The present invention has substantial worth for plant breeding.
Brief description of the drawings
Fig. 1 is the relative expression quantity of AtUBP15 genes
Fig. 2 compares for plant phenotype；Scale is respectively 0.5mm, 5cm and 1mm from top to bottom；It is horizontal that * represents P ＜ 0.01
Significant difference.
Fig. 3 is the apparent surface's projected area and relative weight of single seed.
Fig. 4 is the 5th true leaf of plant（Maturity period）Blade area and blade cell area.
Fig. 5 is second on the stem of plant（Maturity period）To the 5th flower（Maturity period）Single flower average petal
Area, average petal length, average petal width and average petal cell area.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Entry vector pDONR207：Purchased from Invitrogen companies, article No. 12213-013.
Plasmid pMDC99：Bibliography：Curtis MD,Grossniklaus U(2003)A gateway cloning
vector set for high-throughput functional analysis of genes in planta.Plant
Physiol.133:462-469。
Agrobacterium tumefaciems GV3101：Bibliography：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 Dev22,1331-1336.
Columbia ecotype arabidopsis（Col-0）：ABRC（Arabidopsis Biological Resource
Center）, seed numbering CS28166.
T1It is T for the plant that seed grows up to1For plant.T2It is T for the plant that seed grows up to2For plant.T3Grow up to for seed
Plant be T3For plant.
Inventor has found a protein from Columbia ecotype arabidopsis, as shown in the sequence 1 of sequence table or such as
Shown in the sequence 2 of sequence table（Two kinds of shear patterns）, it is named as AtUBP15 albumen.The gene of AtUBP15 albumen will be encoded
AtUBP15 genes are named as, its full-length genome is as shown in the sequence 3 of sequence table, the institute of sequence 4 of ORFs such as sequence table
Show or as shown in the sequence 5 of sequence table（Two kinds of shear patterns）.
Embodiment 1, the transfer-gen plant excellent by importing AtUBP15 genes cultivation developmental level
First, the preparation of recombinant plasmid
1st, the genomic DNA of Columbia ecotype arabidopsis is extracted.
2nd, the genomic DNA extracted using step 1 is entered as template using the AtUBP15-F and AtUBP15-R primer pairs formed
Performing PCR expands, and obtains pcr amplification product.
AtUBP15-F:5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTCAAATCCACAAAAAATCCGA；
AtUBP15-R:5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAAGCTCTGCTTAGTGAACC。
Underscore mark attB1 sites in AtUBP15-F.Underscore mark attB2 sites in AtUBP15-R.
BP reactions occur for the pcr amplification product and entry vector pDONR207 that the 3, step 2 obtains, and obtain containing ordered list
The recombinant plasmid of DNA molecular shown in sequence 2（It is named as recombinant plasmid pDNOR207-AtUBP15）.
4th, LR reactions occur for recombinant plasmid pDNOR207-AtUBP15 and plasmid pMDC99, obtain the sequence containing ordered list
The recombinant plasmid of DNA molecular shown in 2（It is named as recombinant plasmid pMDC99-AtUBP15）.
2nd, the acquisition of genetically modified plants
1st, recombinant plasmid pMDC99-AtUBP15 is imported into Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium.
2nd, the seed of Columbia ecotype arabidopsis is taken, 1/2MS solid mediums, 4 DEG C of vernalization 3 are laid in after sterilization
My god, in normal condition（22 DEG C/18 DEG C, 16h illumination/8h is dark, 75% humidity）Lower culture 8 days, is then transplanted to soil property culture medium
（Turfy soil：Vermiculite=1：2；Volume ratio）And cultivated 3 weeks in greenhouse, seedling starts bolting and bloomed.
3rd, with conversion buffer solution（Sucrose containing 5g/100ml, 0.05g/100ml MES, 0.03% volume ratio Silwet
L-77 1/2MS fluid nutrient mediums, pH5.7）The recombinational agrobacterium that step 1 obtains is resuspended, obtains OD600nm=0.3-0.5 bacterium is hanged
Liquid.
4th, the plant for taking step 2 to obtain, the bacteria suspension obtained with suction pipe aspiration step 3, careful drops on titbit, repeats
Drop 3-5 times, then covers plant with plastic sheeting and carries out moisture-heat preservation, removes film after 2 days by plant normal growth, harvest kind
Son, as T1For seed.
5th, by T1For seed sowing in the 1/2MS solid mediums of the hygromycin containing 50mg/L, normal culture（Can normal growth
Plant be resistant plant, the plant that can only grow the just not regrowth of tiny cotyledon is sensitive plant）, collect resistant plant
Seed, as T2For seed.
6th, by T2For seed sowing in the 1/2MS solid mediums of the hygromycin containing 50mg/L, normal culture（Can normal growth
Plant be resistant plant, the plant that can only grow the just not regrowth of tiny cotyledon is sensitive plant）, collect resistant plant
Seed, as T3For seed.
7th, by the T of sampling3For seed sowing in the 1/2MS solid mediums of the hygromycin containing 50mg/L, normal culture（Can be just
The plant being frequently grown is resistant plant, and the plant that can only grow the just not regrowth of tiny cotyledon is sensitive plant）.
For a certain T2For plant, if the T of its sampling Detection3It is positive plant for plant, the T2It is for plant
Homozygous transfer-gen plant, the plant and its self progeny are a homozygous transgenic line.
3rd, the acquisition of empty carrier plant is turned
Replace recombinant plasmid pMDC99-AtUBP15 to carry out step 2 with plasmid pMDC99, obtain turning empty carrier plant.
4th, Molecular Identification
Take Columbia ecotype arabidopsis, 3 homozygous transgenic lines（OE1 strains, OE2 strains and OE3 strains）
T3 for plant, extraction total serum IgE and reverse transcription be cDNA, the primer pair identification formed using qUBP15-F and qUBP15-R
AtUBP15 genes, the primer pair formed using ACTIN7F and ACTIN7R identify ACTIN7 genes（Reference gene）, calculate
The relative expression quantity of AtUBP15 genes, is as a result shown in Fig. 1（The average value of 3 repetition plant）.
qUBP15-F：5’-GGAGACGTTCCTCCGCTTTATATGC-3’；
qUBP15-R：5’-TCCTCTTTGAGGACGTGGATACGAT-3’.
ACTIN7F：5’-ATCCTTCCTGATATCGAC-3’；
ACTIN7R：5’-GAGAAGATGACTCAGATC-3’.
5th, phenotypic evaluation
Take the seed of Columbia ecotype arabidopsis（30）, 3 homozygous transgenic lines（OE1 strains, OE2 strains
System and OE3 strains）T3For seed（Each strain 30）, turn the T of empty carrier plant3For seed（30）, carry out respectively as follows
Operation：Seed is taken, is laid in 1/2MS solid mediums after sterilization, 4 DEG C of vernalization 3 days, then in normal condition（22 DEG C/18 DEG C,
16h illumination/8h is dark, 75% humidity）Lower culture 8 days, is then transplanted to soil property culture medium（Turfy soil：Vermiculite=1：2；Volume ratio）
And cultivated in greenhouse, harvest the early stage seed of natural maturity on plant stem（The usually seed of the 2nd to the 10th silique）.
The photo of the seed of part harvesting is shown in Fig. 2A.Kind of the seed of transfer-gen plant than Columbia ecotype arabidopsis
It is sub big, and significant difference.It is consistent with the seed size of Columbia ecotype arabidopsis to turn the seed of empty carrier plant, without significantly
Difference.For the seed of harvest, apparent surface's projected area of the single seed of each strain（By Columbia ecotype
Surface projection's area of the seed of arabidopsis is as 100%）And relative weight（By the seed of Columbia ecotype arabidopsis
Weight is as 100%）See Fig. 3.The measuring method of surface projection's area of seed：Seed is kept flat（Cotyledon respectively has a piece of up and down）
In in plane, front shooting photo, surface projection of the obtained seed projection i.e. as seed, its area is the surface of seed
Projected area；Each strain from 500 seeds of 30 plant harvests to measuring, results averaged.The weight of seed
Measuring method：Each strain from 1500 seeds of 30 plant harvests to weighing, results averaged.
Plant photo after being cultivated 28 days in soil property culture medium is shown in Fig. 2 B.The mature leaf of transfer-gen plant is than brother rival
The mature leaf of sub- Arabidopsis thaliana ecotype is big, and significant difference.The blade and Columbia ecotype for turning empty carrier plant intend south
The leaf blade size of mustard is consistent, no significant difference.The 5th true leaf of plant（Maturity period）Relative vane area（Colombia is given birth to
5th true leaf of state type arabidopsis（Maturity period）Area as 100%）With relative vane cell area（Colombia is given birth to
The blade cell area of state type arabidopsis is as 100%）See Fig. 4（Each strain is surveyed to the 5th true leaf of 30 plant
Amount, results averaged）.The measuring method of blade cell area：Blade is taken, first in transparent liquid（80g chloraldurates, 30ml water
Mixed with 10ml glycerine）Middle soaking at room temperature 3 days, then tabletting and under DIC microscopes shoot cell photo, with ImageJ softwares
Build measurement cell area（The cell area at cell area and base portion 1/4 at every crop leaf measuring top 1/4, both add and
Average again）.As a result show, the blade area of the 5th true leaf of transfer-gen plant is noticeably greater than Columbia ecotype plan
Southern mustard, the blade area for turning the 5th true leaf of empty carrier plant are not significantly different with Columbia ecotype arabidopsis.Knot
Fruit shows, transfer-gen plant, Columbia ecotype arabidopsis and the blade cell area that turns empty carrier plant do not have significance difference
It is different, therefore it may be speculated that the difference of blade area is as caused by number of cells difference, i.e. overexpression AtUBP15 genes promote
The cell propagation of plant.
During being cultivated in soil property culture medium, the photo in second colored maturity period on the stem of plant is shown in figure
2C.The maturation flower of transfer-gen plant is spent greatly than the maturation of Columbia ecotype arabidopsis, and significant difference.Turn empty carrier plant
Ripe flower and the maturation of Columbia ecotype arabidopsis spend in the same size, no significant difference.It is single on the stem of plant
Colored relative petal area（Using the petal area of the single flower of Columbia ecotype arabidopsis as 100%）, relative petal grows
Degree（Using the petal length of the single flower of Columbia ecotype arabidopsis as 100%）, relative petal width（By Colombia
The petal width of the single flower of Arabidopsis thaliana ecotype is as 100%）With relative petal cell area（Columbia ecotype is intended
The petal cell area of southern mustard is as 100%）See Fig. 5（Each strain to second on the stem of 30 plant to the 5th into
Ripe flower measures, results averaged）.The measuring method of petal cell area：Petal is taken, first the soaking at room temperature in transparent liquid
1 day, then tabletting and the shooting cell photo under DIC microscopes, cell area was measured with ImageJ softwares（Every petal detection
The cell area of the widest part）.As a result show, petal area, petal length and the petal width of transfer-gen plant are noticeably greater than
Columbia ecotype arabidopsis, petal area, petal length and the petal width for turning empty carrier plant are given birth to Colombia
State type arabidopsis is not significantly different.As a result show, transfer-gen plant, Columbia ecotype arabidopsis and turn empty carrier plant
Petal cell area be not significantly different, therefore it may be speculated that the difference of petal area is as caused by number of cells difference,
It is overexpressed the cell propagation that AtUBP15 genes promote plant.
Result above shows, with Columbia ecotype arabidopsis（Wild type）And turn empty carrier plant and compare, turn base
Because the seed, flower, blade of plant significantly become big, and enhanced cellular proliferation is to cause organ to become the reason for big.
Claims (6)
1. a kind of method for cultivating genetically modified plants, it is by the channel genes purpose plant for encoding AtUBP15 albumen, is sent out
Educate the horizontal genetically modified plants for being better than the purpose plant；" developmental level is excellent " is presented as that seed is big and/or seed weight
It is big and/or spend big and/or blade big and/or cell propagation is fast；
The AtUBP15 albumen is following (a) or (b)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 2.
2. the method as described in claim 1, it is characterised in that：The gene of the coding AtUBP15 albumen be following (1) or
(2) DNA molecular：
(1) DNA molecular shown in the sequence 3 of sequence table；
(2) DNA molecular of the code area as shown in the sequence 4 of sequence table or the sequence 5 of sequence table.
3. the method as described in claim 1, it is characterised in that：The purpose plant is monocotyledon or dicotyledon.
The recombinant vector of 4.AtUBP15 albumen, the gene for encoding AtUBP15 albumen or the gene containing coding AtUBP15 albumen
Application in the excellent genetically modified plants of developmental level are cultivated；" developmental level is excellent " is presented as that seed is big and/or plants
Sub- weight is big and/or spends big and/or blade big and/or cell propagation is fast；
The AtUBP15 albumen is following (a) or (b)：
(a) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(b) protein being made up of the amino acid sequence shown in sequence in sequence table 2.
5. application as claimed in claim 4, it is characterised in that：The gene of the coding AtUBP15 albumen be following (1) or
(2) DNA molecular：
(1) DNA molecular shown in the sequence 3 of sequence table；
(2) DNA molecular of the code area as shown in the sequence 4 of sequence table or the sequence 5 of sequence table.
6. application as claimed in claim 4, it is characterised in that：The plant that sets out of the genetically modified plants for monocotyledon or
Dicotyledon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410064497.3A CN104861051B (en) | 2014-02-25 | 2014-02-25 | Plant development associated protein AtUBP15 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410064497.3A CN104861051B (en) | 2014-02-25 | 2014-02-25 | Plant development associated protein AtUBP15 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104861051A CN104861051A (en) | 2015-08-26 |
| CN104861051B true CN104861051B (en) | 2018-01-09 |
Family
ID=53907277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410064497.3A Expired - Fee Related CN104861051B (en) | 2014-02-25 | 2014-02-25 | Plant development associated protein AtUBP15 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104861051B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365370B (en) * | 2017-08-31 | 2019-12-31 | 中国农业科学院蔬菜花卉研究所 | Protein related to plant fruit development and coding gene and application thereof |
| CN109679965B (en) * | 2018-09-28 | 2020-08-04 | 中国林业科学研究院林业研究所 | Gene for regulating and controlling leaf type development of poplar and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757486A (en) * | 2011-04-27 | 2012-10-31 | 中国农业大学 | Plant development related protein GA20ox, and encoding gene and application thereof |
| CN103224555A (en) * | 2013-05-27 | 2013-07-31 | 中国农业科学院棉花研究所 | Plant-development-related protein GhSOC1, and coding gene and application thereof |
-
2014
- 2014-02-25 CN CN201410064497.3A patent/CN104861051B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757486A (en) * | 2011-04-27 | 2012-10-31 | 中国农业大学 | Plant development related protein GA20ox, and encoding gene and application thereof |
| CN103224555A (en) * | 2013-05-27 | 2013-07-31 | 中国农业科学院棉花研究所 | Plant-development-related protein GhSOC1, and coding gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| NP_001185019.1;Theologis A等;《Genbank》;20130605;第1页 * |
| NP_564014.1;Theologis A等;《Genbank》;20130605;第1页 * |
| Ubiquitin C-terminal hydrolases 1 and 2 affect shoot architecture in Arabidopsis;Peizhen yang等;《The plant Journal》;20071231;第51卷;第441-457页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104861051A (en) | 2015-08-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105753953B (en) | Disease-resistant wheat albumen and encoding gene and its application in regulation disease resistance of plant | |
| CN110724183B (en) | Application of GmXTH91 protein in regulation and control of plant stress resistance and plant height | |
| CN103695438A (en) | Arabidopsis MYB family transcription factor AtMYB17 gene as well as coding sequence and application thereof | |
| CN110295183A (en) | A method of citrus is improved to canker resistance based on CsPrx25 overexpression | |
| CN104725495A (en) | Cotton GhWRKY51 transcription factor, and coding gene and application thereof | |
| CN109971766A (en) | A kind of and plant stress tolerance-associated protein PwRBP1 and its encoding gene and application | |
| CN117535311A (en) | Upland cotton GhCRP21 gene and encoding protein and application thereof | |
| CN104861051B (en) | Plant development associated protein AtUBP15 and its encoding gene and application | |
| CN106367433B (en) | Plant is improved to the method and its application of gibberellin inhibitor sensitiveness | |
| CN105820220B (en) | The application of resistance relevant protein and its encoding gene in regulation plant alkali resistance | |
| CN110218247A (en) | Two kinds of interactions between protein collaborations of PwRBP1 and PwNAC1 improve plant stress tolerance and its application | |
| CN103172717B (en) | Plant low potassium stress resistant related protein GmWRKY50 as well as encoding gene and application thereof | |
| CN102732553B (en) | Improve the gene engineering method and material of plant products | |
| Yara et al. | Production of transgenic japonica rice (Oryza sativa) cultivar, Taichung 65, by the Agrobacterium-mediated method | |
| CN110627887B (en) | Application of SlTLFP8 protein and related biological material thereof in regulation and control of tomato drought resistance | |
| CN103172718B (en) | Plant low nitrogen stress resistant related protein GmDUF-CBS and encoding gene and application thereof | |
| CN106397558A (en) | Application of protein and encoding gene of protein in regulation of verticillium wilt resistance of plants | |
| CN106480069B (en) | Cucumber CsERF025 gene and its promote the straight developmental application of cucumber fruits | |
| CN105732785B (en) | Application of protein GhDHN1 in regulation and control of plant stress resistance | |
| CN104805100A (en) | Application of paddy rice gene OsS[mu]BP-2 in delaying plant leaf senescence | |
| CN103819548B (en) | Heat Resistance of Plant associated protein TaOPR3 and encoding gene thereof and application | |
| CN104610438A (en) | Cotton stress response associated protein GhGeBP and coding gene and application thereof | |
| CN104673803A (en) | Application of gene methylation in gene expression regulation | |
| CN113249394B (en) | Application of receptor-like kinase gene MdMRLK2 in improving plant water utilization efficiency | |
| CN103834624B (en) | The cold-resistant associated protein GST of plant and encoding gene thereof are applied with it |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180109 Termination date: 20210225 |