CN104818258A - Gossypium hirsutum glycosyltransferase GhUGT85O1, coding gene and applications thereof - Google Patents
Gossypium hirsutum glycosyltransferase GhUGT85O1, coding gene and applications thereof Download PDFInfo
- Publication number
- CN104818258A CN104818258A CN201510095549.8A CN201510095549A CN104818258A CN 104818258 A CN104818258 A CN 104818258A CN 201510095549 A CN201510095549 A CN 201510095549A CN 104818258 A CN104818258 A CN 104818258A
- Authority
- CN
- China
- Prior art keywords
- ghugt85o1
- gene
- glycosyltransferase
- cotton
- transgenic
- 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.)
- Granted
Links
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- 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
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8266—Abscission; Dehiscence; Senescence
-
- 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
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
-
- 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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
-
- 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
- C12N15/8291—Hormone-influenced development
- C12N15/8293—Abscisic acid [ABA]
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Endocrinology (AREA)
- Physiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to the field of genetic engineering, particularly to Gossypium hirsutum glycosyltransferase GhUGT85O1, a coding gene and applications thereof, wherein the amino acid sequence is represented by SEQ ID No.1. According to the present invention, the gene GhUGT85O1 in the cotton glycosyltransferase family 1 is cloned, and the expression pattern analysis and the transgene progeny characteristic research are performed, such that it is identified that the gene GhUGT85O1 is involved in the plant stress, flowering time regulating and aging process regulating so as to establish the foundation for the further function clarification.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to upland cotton glycosyltransferase GhUGT85O1 and encoding gene thereof and application.
Background technology
Glycosylation is a kind of modification mode of general Secondary Metabolism of Plant, relates to the adjustment of hormone homeostasis, the detoxification of xenobiontics, the various procedures such as the synthesis of secondary species and storage.In plant, this series of reaction is all by the specific enzyme-glycosyltransferase (GT) of a class-realize.GT catalysis glycan molecule is transferred to specific acceptor from the donor of activation, and their acceptor is diversified, can be carbohydrate, lipid, albumen, nucleic acid, microbiotic and other small molecules.According to the feature of gene product and sequence, GT has been divided into 94 different extended familys, and wherein the member that comprises of multigene family UGT is maximum, and the function of it and plant is the tightst.Identify the glucoside different more than 200 in grape, the highly plastic during synthesis of these materials makes plant can respond various environmental change in time.Identified altogether the gene of 120 coding UGT family proteins in Arabidopis thaliana, according to Phylogenetic Relationships, they be divide into three, the member of two sprigs contains sterol and lipid GT, and the 3rd bough then contains the PSPG die body of high conservative.The UGT comprising PSPG die body participates in the detoxification etc. of the synthesis of bioactive natural product, the adjustment of plant hormone, cell homeostasis and xenobiotic.
Although UGT family amino acid C terminal sequence is very conservative, contain PSPG structural domain, the variation of N terminal sequence is very large, and the function between member also has difference.UGT87A2 in Arabidopis thaliana take part in the adjustment of flowering time; In Arabidopis thaliana, process LAN UGT74D1 gene makes leaf rolling; UGT73B3 and UGT73B5 participates in the response that Arabidopis thaliana is coerced cause of disease; The ectopic expression Arabidopis thaliana UGT85A5 genes amplification salt resistance of plant in tobacco; UGT76B1 gene Delaying Leaf-Senescence in process LAN Arabidopis thaliana, and enhance the resistance of plant.
Cotton is a kind of important cash crop.Along with becoming increasingly conspicuous of ground contradiction striven by China's grain and cotton, promoting short season cotton kind becomes one of important channel ensureing grain security.But, precocious often along with the generation of early ageing.Blade carries out photosynthetic major organs, and cotton leaf early ageing has a strong impact on output (10% ?20%) and the cotton quality of cotton.
This research has been separated a UGT family gene from upland cotton, called after GhUGT85O1, shows that it is in great expression in maturation and ageing leaves to the analysis of its spatial and temporal expression pattern.Build plant over-express vector, adopt agriculture bacillus mediated inflorescence dip method transformation mode plant Arabidopsis thaliana, transgenic arabidopsis, than non-transgenic Arabidopis thaliana Blooming and aging, makes shorten than contrast the breeding time of transfer-gen plant.
Summary of the invention
The object of this invention is to provide upland cotton glycosyltransferase GhUGT85O1.
Another object of the present invention is to provide the gene of the above-mentioned upland cotton glycosyltransferase GhUGT85O1 that encodes.
Another object of the present invention is to provide the application of above-mentioned upland cotton glycosyltransferase GhUGT85O1.
According to upland cotton glycosyltransferase GhUGT85O1 of the present invention, its aminoacid sequence is as shown in SEQ ID No.1:
MGSLETSKPHIVIVPFPAQGHVNPMMLLAKLLHSRGFFITFVNTEFNHRRLVRSKGPDFVKGLPDFQFETIPEGLTSSDRNATQDLPVLCDSIRKHCLAPFVDLLAKLNSSPQVPTVTCIISDGLMSFAIKAAEQLGIPEVQFWTASACSFMGYLHFSELVKRGIIPFQSETFLDEPIDWVPGMSNIRLRDFPSFVRANDPNDILFDYLGSEAQNCLKASAIIFNAFEEFEHEVLDAIAAKFPRIYTVGPLHLVARHLHADPSKSMNPSLWKEDTSCIEWLNEREPNSVVYVNYGSITVMSAKHLKEFAWGLANSKHPFLWIVRPDVVMGDSAILDLEFLKEIKERGLITSWCNQYEVLSHPSVGVFLTHCGWNSTVETISEGVPVVCWPFFADQQTNCRYACTHWGIGMEVDHDVKRENIEFLVKEMMEGEEGKKKKEKALGWKKKAEEAVEVGGSSYIDFDRFVKEALKHG。
According to upland cotton glycosyltransferase GhUGT85O1 encoding gene of the present invention, its nucleotide sequence is as shown in SEQ ID No.2:
ATGGGTTCACTTGAAACCAGTAAACCCCACATTGTAATCGTCCCATTTCCAGCACAAGGTCATGTTAACCCCATGATGCTACTTGCTAAGCTCTTACACTCTAGAGGCTTCTTCATAACCTTTGTTAACACTGAGTTCAACCATAGGCGTTTGGTCAGGTCCAAAGGCCCTGACTTTGTTAAAGGTCTGCCTGATTTCCAGTTCGAAACAATTCCGGAAGGGCTGACATCGTCCGATCGAAATGCAACACAGGATCTTCCAGTTCTGTGTGATTCGATACGAAAGCATTGCTTGGCACCATTCGTAGATCTACTAGCTAAGTTAAACTCCTCGCCCCAAGTGCCCACTGTTACTTGCATAATCTCTGATGGACTTATGAGCTTTGCTATTAAGGCTGCTGAACAACTTGGCATACCAGAAGTTCAGTTTTGGACTGCCTCAGCATGTAGTTTCATGGGATATCTTCACTTCAGTGAACTGGTTAAACGAGGCATTATTCCATTCCAAAGTGAAACATTTCTCGATGAACCTATTGACTGGGTCCCTGGAATGAGTAACATTCGCCTCAGAGATTTTCCAAGCTTCGTCAGAGCCAACGATCCGAATGACATTTTGTTTGATTATTTAGGATCCGAAGCTCAAAATTGCCTAAAAGCTTCAGCAATAATCTTCAACGCATTTGAAGAGTTCGAACATGAAGTGCTCGACGCCATCGCTGCCAAATTTCCTCGAATTTATACAGTAGGACCACTTCATTTGGTTGCCAGGCACCTACATGCCGATCCCTCCAAGTCAATGAACCCAAGCCTATGGAAGGAAGATACAAGCTGCATTGAATGGCTTAACGAAAGGGAACCCAATTCAGTTGTGTATGTGAACTATGGAAGCATTACTGTCATGTCGGCGAAGCATCTCAAAGAATTTGCATGGGGGTTGGCTAACTCTAAACACCCATTTTTATGGATCGTTAGACCAGATGTCGTGATGGGTGATTCTGCAATTCTGGATCTAGAGTTCCTGAAGGAGATTAAGGAAAGAGGGCTGATAAcAAGCTGGTGCAACCAATATGAGGTCCTTTCACATCCTTCAGTCGGTGTTTTTTTGACACACTGTGGGTGGAATTCTACCGTGGAAACCATATCAGAAGGTGTGCCAGTAGTTTGTTGGCCATTTTTTGCTGATCAGCAAACCAATTGTCGATATGCTTGCACTCATTGGGGCATTGGCATGGAGGTGGATCATGATGTGAAGCGAGAGAACATAGAGTTTTTAGTTAAGGAAATGATGGAAGGTGAGG AAGGAAAGAAAAAGAAAGAGAAGGCATTGGGATGGAAGAAGAAAGCGGAAGAAGCGGTTGAAGTTGGGGGATCATCTTATATTGATTTCGACAGATTTGTTAAGGAAGCTCTCAAACATGGTTAACTGGAAATACTATTGTCAGTGGGAAATTACTTAAATAATGAATGGGGATCATTCAATGTCTCTATTTCTGCTTTTTTTCCTTTGTAAGTGAATGCAGTCGTGTGAGG。
The present invention has cloned a GhUGT85O1 gene from No. 10, " CCRI, by the analysis of its gene structure, finds that it contains two exons and an intron.GhUGT85O1 gene is up-regulated expression in cotton ageing leaves, and the trend that raises is up to 200 times, and this illustrates that it has certain influence to leaf senile.By the further observation to transgenic arabidopsis, find that the chlorophyll content of transgenic line is starkly lower than wild type control, and the expression amount of several old and feeble marker gene is higher than wild-type in transgenic line, and this illustrates that this gene serves the effect promoting leaf senile.
Protein sequence total length 473 amino acid of GhUGT85O1 translation.Whether there is response to abiotic stress to study GhUGT85O1 gene, eight kinds of process having been carried out to blade, that is: Plant hormones regulators,gibberellins, heat, jasmonic, nitric oxide donors SNP, injury, Whitfield's ointment, dormin and PEG.Result shows, after treatment in 12 hours, this gene pairs ABA, JA and PEG have response.ABA process, after 4 hours, has raised 4 times when GhUGT85O1 expression amount is less than 0, and this trend is continued until 12 hours.JA process, after 8 hours, raised 15 times when GhUGT85O1 expression amount is less than 0, to 12 hours return to primitive age levels.PEG process is after 4 hours, and GhUGT85O1 has raised 3 times, and up to 40 times after 8 hours, then this rise trend slowly reduces, to 12 hours about 15 times.For other 5 kinds of processing modes, in 12 hours of sampling, the expression trend of this gene does not significantly change.In addition, the senescence process of L5, L7, L53 tri-transgenic lines comparatively wild-type in advance, from phenotype, when the most of yellow of blade of transgenic line, when presenting evidence for senescence, the leaf look of most of blade of wildtype Arabidopsis thaliana is also in deep green.By showing the mensuration of chlorophyll content: now the chlorophyll content of 3 transgenic lines is lower than wild-type, the chlorophyll content of L5 and L7 is about 70% of wild-type, the chlorophyll content of L53 is that the range of decrease of 56% of wild-type, three strains all reaches pole conspicuous level (P<0.01).
The present invention has cloned the gene in a marsh-mallow based transferase family 1, GhUGT85O1, by the analysis to its expression pattern, and the research of transgenic progeny characteristic, identify that it take part in Adversity-stressed Plant, the regulation and control of flowering time, and the regulation and control of senescence process, lay a good foundation for illustrating its function further.
Accompanying drawing explanation
Fig. 1 shows the spatial and temporal expression pattern analysis of GhUGT85O1 gene;
The expression change of GhUGT85O1 gene after Fig. 2 .ABA, JA and PEG process;
Fig. 3 show pBI121 ?the qualification of the positive strain of GhUGT85O1 transgenic arabidopsis;
Fig. 4 shows transgenic arabidopsis aging in advance (A), the expression of chlorophyll content (B) and senescence-associated gene (C ?G).
Embodiment
Experiment material
Supply examination material to be short season cotton kind CCRI 10 (CCRI10, Gossypiumhirsutum), get the different tissues such as the cotyledon of cotton, root, stem, leaf, flower, terminal bud, ovule respectively.Full-bloom stage carries out listing mark to Leaves on main stem, and what flatten the same day is 0 day, samples, often get once every other day, until leaf senile from 15 days.Carry out in process test in the controlled environment chamber.The quick-frozen in liquid nitrogen of the sample got, then guarantor deposits in ?80 DEG C of refrigerators stand-by.
Embodiment 1 gene clone and conversion
One, the clone of GhUGT85O1 gene in cotton
Design Auele Specific Primer amplifies complete sequence from No. 10, " CCRI.PCR primer is: upstream primer 5 ’ ?ATGGGTTCACTTGAAACCAG ?3 '; Downstream primer 5 ’ ?CCTCACACGACTGCATTCAC ?3 '.
Carry out pcr amplification according to TAKARA LA Taq Hot Start Version 2.0 (DRR042) specification sheets, then reclaim object fragment and check order.
Two, the spatial and temporal expression pattern analysis of GhUGT85O1 gene
1, extract the different tissues of cotton variety " No. 10, CCRI " and the total serum IgE of Different Leaf Age blade, and reverse transcription is cDNA respectively.
10 times of volume dilution liquid of the cDNA 2, obtained with step 1, for template, carry out quantitative fluorescent PCR.
For detecting the primer of the expression amount of GhUGT85O1 gene be: upstream primer: 5 ’ ?GGCGAAGCATCTCAAAGAAT ?3 '; Downstream primer: 5 ’ ?CAGAATCACCCATCACGACA ?3 '.For detecting the primer pair of the expression amount of reference gene (actin) be: upstream primer: 5 ’ ?ATCCTCCGTCTTGACCTTG ?3 '; Downstream primer: 5 ’ ?TGTCCGTCAGGCAACTCAT ?3 '.
Quantitative fluorescent PCR reaction system: 10 μ l SYBR Premix Ex Tag, 0.8 μ l upstream primer, 0.8 μ l downstream primer, 2 μ l cDNA templates, 6 μ l ddH2O, 0.4 μ l ROX Reference Dye II, cumulative volume 20ul.
Quantitative fluorescent PCR response procedures: 95 DEG C of denaturation 1min; 95 DEG C of sex change 5s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 circulations; 72 DEG C extend 10min; At 72 DEG C, fluorescent signal is collected at 30s place.
Relative quantification Δ Δ Ct method is adopted to analyze.
GhUGT85O1 gene is shown in Fig. 1 at the blade of Different Leaf Age and the expression of other tissue.Result shows: the height of this gene expression amount comparatively young leaflet tablet (15 to 30 days) of (35 to 45 days) in ripe and ageing leaves; Between different tissues, GhUGT85O1 blade, cotyledon, spend in expression amount higher, and expression amount is lower in bud, ovule, stem, root and hypocotyl.
Whether there is response to abiotic stress to study GhUGT85O1 gene, eight kinds of process having been carried out to blade, that is: Plant hormones regulators,gibberellins, heat, jasmonic, nitric oxide donors SNP, injury, Whitfield's ointment, dormin and PEG.Result shows, after treatment in 12 hours, this gene pairs ABA, JA and PEG have response (Fig. 2).ABA process, after 4 hours, has raised 4 times when GhUGT85O1 expression amount is less than 0, and this trend is continued until 12 hours (A).JA process, after 8 hours, raised 15 times when GhUGT85O1 expression amount is less than 0, to 12 hours return to primitive age levels (B).PEG process was after 4 hours, and GhUGT85O1 has raised 3 times, and up to 40 times after 8 hours, then this rise trend slowly reduces, to 12 hours about 15 times (C).For other 5 kinds of processing modes, in 12 hours of sampling, the expression trend of this gene does not significantly change.
Three, transgenic arabidopsis
1, the structure of PBI121-GhUGT85O1 plant expression vector
With CCRI No. 10 cDNA for template, the primer upstream with corresponding restriction enzyme site: 5 ’ ?CACGGGGGAC
tCTAGAaTGGGTTCACTTGAAACCAG ?3 ' (underscore is XbaI enzyme cutting site); Downstream: 5 ’ ?GATCGGGGAAATTC
gAGCTCcCTCACACGACTGCATTCAC ?3 ' underscore be SacI restriction enzyme site), amplification obtain the PCR primer comprising GhUGT85O1 gene coding region.
Plant expression vector pBI121 is cut through XbaI and SacI enzyme, obtains carrier framework;
Above-mentioned carrier framework is connected with above-mentioned purpose gene fragment.
2, the acquisition of GhUGT85O1 Arabidopis thaliana is turned
By plasmid PBI121 ?GhUGT85O1 proceed in Agrobacterium LBA4404 competent cell, obtain recombinant bacterium.By PCR detect containing plasmid PBI121 ?GhUGT85O1 recombinant bacterium called after LBA4404/PBI121 ?GhUGT85O1.The pod of the Hua Hejie pollinated by the wild-type Arabidopsis plants of blooming cuts off with scissors, thaliana flower is immersed to be equipped with in the small beaker of permeabilization buffer and contaminates 50s, then the Arabidopsis plant contaminated is placed dark culturing in large plastic tank, continue to cultivate in Arabidopis thaliana culturing room after 24h; After fruit pod maturation, mix sowing, obtain the Arabidopis thaliana seed that T0 generation turns GhUGT85O1.Cultivated on the MS substratum of added with antibiotic kantlex after sterilization vernalization for seed by T0, negative transgenosis strain can not normal growth, and screening obtains positive T0 generation and turns GhUGT85O1 Arabidopis thaliana.
3, the Molecular Identification of transgenic progeny
Extract the genomic dna that positive T0 generation turns GhUGT85O1 Arabidopis thaliana, with forward primer 5 ’ ?GACGCACAATCCCACTATCC ?3 ' (mating with one section of nucleotide sequence of 35S promoter on carrier) and reverse primer 5 ’ ?CCTCACACGACTGCATTCAC ?3 ' carry out pcr amplification, with wildtype Arabidopsis thaliana (WT) for contrast for primer.
1% agarose electrophoresis detects, and as shown in Figure 3, L2, L5, L7, L53 can detect object fragment to result, are defined as positive T0 for transgenic line; The genomic dna extracting wild-type (WT) Arabidopis thaliana in contrast, with same primer, does not obtain object fragment.Positive T0 generation is turned GhUGT85O1 Arabidopis thaliana individual plant sowing, sowing, and screening, until T3 is for above homozygous lines.
Embodiment 2GhUGT85O1 functional study
One, turn GhUGT85O1 Arabidopis thaliana to bloom in advance
Turning GhUGT85O1 Arabidopis thaliana, wildtype Arabidopsis thaliana (WT) sowing by being numbered isozygotying of L2, L5, L7, L53, cultivating under long-day conditions (16h illumination/8h is dark).
Turn empty carrier Arabidopis thaliana consistent with wildtype Arabidopsis thaliana phenotype.Add up 4 the isozygoty flowering time, the lotus throne number of sheets and the stem leaf numbers that turn GhUGT85O1 strain and wildtype Arabidopsis thaliana, in triplicate, each strain is that reference carries out significance analysis with wild-type, and statistic analysis result is in table 1 in experiment.Except L2, other 3 strains (L5, L7, L53) extremely significantly promote Arabidopis thaliana early blossoming (P<0.01), and the quantity of lotus throne leaf also extremely significantly reduces (P<0.01), the change of stem leaf quantity is compared with wild-type, L53 significantly reduces (P<0.05), and other 3 strains are all not obvious.
Table 1 transgenic arabidopsis flowering time, lotus throne leaf and stem leaf number statistical
Note: WT: wildtype Arabidopsis thaliana; L2: transgenic line 2; L5: transgenic line 5; L7: transgenic line 7; L53: transgenic line 53.* represent significant difference (P<0.05), * * represents pole significant difference (P<0.01).
Two, GhUGT85O1 transgenic arabidopsis promotes old and feeble
By the observation to transgenic arabidopsis offspring, comparatively wild-type is in advance (Fig. 4) for the senescence process finding L5, L7, L53 tri-transgenic lines.From phenotype, when the most of yellow of blade of transgenic line, when presenting evidence for senescence, the leaf look of most of blade of wildtype Arabidopsis thaliana is also in deep green (A).By showing (B) the mensuration of chlorophyll content: now the chlorophyll content of 3 transgenic lines is lower than wild-type, the chlorophyll content of L5 and L7 is about 70% of wild-type, the chlorophyll content of L53 is that the range of decrease of 56% of wild-type, three strains all reaches pole conspicuous level (P<0.01).
To show shown fluorescent quantitation primer, take AtUBQ1 as reference gene, their expression amount has been carried out detecting (C ?G).Result shows: the expression amount of 6 genes such as AtNAP, AtORE1, AtPR1, AtSAG12, AtSAG13 and AtWRKY6 in transgenic line is higher than wildtype Arabidopsis thaliana.
The fluorescent quantitation primer of table 2 senescence-associated gene
GhUGT85O1 gene is up-regulated expression in cotton ageing leaves, and the trend that raises is up to 200 times, and this illustrates that it has certain influence to leaf senile.By the further observation to transgenic arabidopsis, find that the chlorophyll content of transgenic line is starkly lower than wild type control, and the expression amount of several old and feeble marker gene is higher than wild-type in transgenic line, and this illustrates that this gene serves the effect promoting leaf senile.The flowering time of the process LAN Arabidopis thaliana strain of the GhUGT85O1 gene of cloning in cotton is significantly early than wild-type, and the lotus throne number of sheets is less than wild-type, and this is that the relation of UGT family gene and flowering of plant provides a new evidence.Therefore, by the analysis to its expression pattern, and the research of transgenic progeny characteristic, identify that it take part in Adversity-stressed Plant, the regulation and control of flowering time, and the regulation and control of senescence process, lay a good foundation for illustrating its function further.
Claims (5)
1. upland cotton glycosyltransferase GhUGT85O1, is characterized in that, its aminoacid sequence is as shown in SEQ ID No.1.
2. upland cotton glycosyltransferase gene GhUGT85O1, is characterized in that, encode upland cotton glycosyltransferase GhUGT85O1 according to claim 1.
3. upland cotton glycosyltransferase gene GhUGT85O1, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.2.
4. comprise the plant expression vector of upland cotton glycosyltransferase gene GhUGT85O1 described in claim 2.
5. the application of upland cotton glycosyltransferase GhUGT85O1 described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510095549.8A CN104818258B (en) | 2015-03-04 | 2015-03-04 | Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510095549.8A CN104818258B (en) | 2015-03-04 | 2015-03-04 | Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104818258A true CN104818258A (en) | 2015-08-05 |
| CN104818258B CN104818258B (en) | 2018-01-30 |
Family
ID=53728739
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510095549.8A Expired - Fee Related CN104818258B (en) | 2015-03-04 | 2015-03-04 | Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104818258B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755007A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC1a and its application |
| CN106755010A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC4 and its application |
| CN106755008A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC6b and its application |
| CN112941044A (en) * | 2019-12-11 | 2021-06-11 | 中国科学院分子植物科学卓越创新中心 | Novel gene for synergistically controlling plant yield and stress resistance traits and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008155139A2 (en) * | 2007-06-21 | 2008-12-24 | Vib Vzw | Methods and means for the production of plants with improved stress resistance |
| CN103952414A (en) * | 2014-05-22 | 2014-07-30 | 中南民族大学 | Tobacco glycosyltransferase inducible promoter and application thereof |
-
2015
- 2015-03-04 CN CN201510095549.8A patent/CN104818258B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008155139A2 (en) * | 2007-06-21 | 2008-12-24 | Vib Vzw | Methods and means for the production of plants with improved stress resistance |
| WO2008155139A3 (en) * | 2007-06-21 | 2009-04-23 | Vib Vzw | Methods and means for the production of plants with improved stress resistance |
| CN103952414A (en) * | 2014-05-22 | 2014-07-30 | 中南民族大学 | Tobacco glycosyltransferase inducible promoter and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| BO WANG ET AL.: "UGT87A2, an Arabidopsis glycosyltransferase, regulates flowering time via FLOWERING LOCUS C", 《NEW PHYTOLOGIST》 * |
| F.-J. TAI ET AL.: "Characterization and Expression Analysis of Two Cotton Genes Encoding Putative UDP-Glycosyltransferases", 《MOLECULAR BIOLOGY》 * |
| VERONICA VON SAINT PAUL ET AL.: "The Arabidopsis Glucosyltransferase UGT76B1 Conjugates Isoleucic Acid and Modulates Plant Defense and Senescence", 《THE PLANT CELL》 * |
| 姬向楠等: "植物UDP-糖基转移酶生化特性和功能研究进展", 《食品科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755007A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC1a and its application |
| CN106755010A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC4 and its application |
| CN106755008A (en) * | 2016-12-23 | 2017-05-31 | 山东大学 | A kind of upland cotton non-specificity phospholipase C gene GhNPC6b and its application |
| CN112941044A (en) * | 2019-12-11 | 2021-06-11 | 中国科学院分子植物科学卓越创新中心 | Novel gene for synergistically controlling plant yield and stress resistance traits and application thereof |
| CN112941044B (en) * | 2019-12-11 | 2024-03-26 | 中国科学院分子植物科学卓越创新中心 | Novel gene for cooperatively controlling plant yield and stress resistance and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104818258B (en) | 2018-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103695438B (en) | Arabidopsis MYB family transcription factor AtMYB17 gene, coded sequence and application thereof | |
| KR100647767B1 (en) | A Novel STAY-GREEN Gene and Method for Preparing Stay-green Transgenic Plants | |
| US9546377B2 (en) | Methods for the identification of genes involved in abiotic stress tolerance in plants | |
| CN102766618B (en) | Rice OsICL protein and coding gene thereof, and application of the two | |
| CN104818258B (en) | Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application | |
| Zhao et al. | Downregulation of SL-ZH13 transcription factor gene expression decreases drought tolerance of tomato | |
| CN110964740B (en) | Preparation method and application of tobacco with high flavonol content | |
| CN111303257B (en) | Positive regulation gene AtMIF1 for oil content of plant seeds and application thereof | |
| CN102477435A (en) | Method for improving plant drought resistance using Poncirus trifoliata transcription factor gene PtrABF | |
| CN114907465B (en) | OsLEA9 protein related to cold tolerance of rice in booting stage, related biological material and application thereof | |
| CN105624172B (en) | The genetic engineering application of paddy rice zinc finger protein gene ZFP214 | |
| US20170298378A1 (en) | Genes to provide environmental stress tolerance and the use thereof | |
| CN107904238A (en) | Thick boisiana high salt, drought-inducible promoter IpLEA PRO and its application | |
| CN111534539B (en) | SiMYB4 protein related to plant stress resistance and related biological material and application thereof | |
| CN114703199A (en) | Plant drought resistance related gene TaCML46 and application | |
| CN103525825B (en) | The clone of the resistance to manganese poisoning important gene ShMDH1 of one kind of plant and application thereof | |
| CN108018286A (en) | Initiative, detection and the application of corn transformation event ZM8-143 | |
| Gho et al. | Tissue-specific enhancement of OsRNS1 with root-preferred expression is required for the increase of crop yield | |
| JP2016103994A5 (en) | ||
| CN103102402A (en) | Soybean transcription active protein GmPHD5, and coding gene and application thereof | |
| CN116121298B (en) | Application of inhibiting expression of HSRP1 gene in improving heat resistance of plants | |
| CN104004076B (en) | A kind of apocarya MADS-box class transcription factor CiAG and encoding gene thereof and application | |
| CN103626856A (en) | Transcription factor AtGT4, coding gene thereof and applications | |
| CN114181946B (en) | Gene and promoter related to low nitrogen stress resistance of plants and application of gene and promoter | |
| CN115948454B (en) | Application of grape VvDREB2c gene in improving plant heat resistance |
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: 20180130 Termination date: 20210304 |