CN104818258B - Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application - Google Patents
Upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, and in particular to upland cotton glycosyl transferase GhUGT85O1 and its encoding gene and application, its amino acid sequence is as shown in SEQ ID No.1.The present invention has cloned the gene in a cotton glycosyl transferase family 1, GhUGT85O1, pass through 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, laid a good foundation to further elucidate its function.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to upland cotton glycosyl transferase GhUGT85O1 and its encoding gene
And application.
Background technology
Glycosylation is a kind of modification mode of universal Secondary Metabolism of Plant, is related to the regulation of hormone homeostasis, heteroplasia
The various procedures such as the detoxication of material, the synthesis and storage of secondary species.The a series of reaction of this in plant is all by one kind
Specific enzyme-glycosyl transferase (GT)-come what is realized.GT catalysis glycan molecules are transferred to specific acceptor from the donor of activation
On, their acceptor is diversified, can be carbohydrate, lipid, albumen, nucleic acid, antibiotic and other small molecules.Root
According to gene outcome and the feature of sequence, GT is divided into 94 different extended familys, the member that wherein multigene family UGT is included
At most, it is most close with the function of plant.The glucoside different more than 200, height when these materials synthesize are identified in grape
Degree plasticity enables plant to timely respond to various environmental changes.120 coding UGT families have been identified in arabidopsis altogether
The gene of albumen, three being classified into according to Phylogenetic Relationships, the member of two sprigs contains sterol and lipid GT,
3rd bough then contains highly conserved PSPG die bodys.The synthesis of UGT participation bioactive natural products comprising PSPG die bodys,
Detoxication of the regulation of plant hormone, cell homeostasis and xenobiotic etc..
Although UGT families amino acid C-terminal sequence is very conservative, PSPG domains are contained, N-terminal sequence variations are very
Greatly, the function between member also has difference.UGT87A2 in arabidopsis take part in the regulation of flowering time；In arabidopsis
Being overexpressed UGT74D1 genes makes leaf rolling；UGT73B3 and UGT73B5 participates in the response that arabidopsis is coerced cause of disease；In tobacco
The middle ectopic expression arabidopsis UGT85A5 genes amplifications salt-resistance of plant；It is overexpressed UGT76B1 genes in arabidopsis and delays leaf
Piece aging, and enhance the resistance of plant.
Cotton is a kind of important industrial crops.As China's grain and cotton strives becoming increasingly conspicuous for ground contradiction, short season cotton product are promoted
Kind turns into one of important channel for ensureing grain security.However, the precocious generation for being usually associated with early ageing.Blade is that progress is photosynthetic
The major organs of effect, cotton leaf early ageing have a strong impact on the yield (10%-20%) and cotton quality of cotton.
This research has separated a UGT family gene from upland cotton, is named as GhUGT85O1, to its spatial and temporal expression mould
The analysis shows of formula its in the great expression in ripe and ageing leaves.Plant over-express vector is built, use is agriculture bacillus mediated
Inflorescence dip method transformation mode plant Arabidopsis thaliana, transgenic arabidopsis makes than non-transgenic arabidopsis Blooming and aging
The breeding time for obtaining transfer-gen plant shortens than control.
The content of the invention
It is an object of the invention to provide upland cotton glycosyl transferase GhUGT85O1.
Another object of the present invention is to provide the gene for encoding above-mentioned upland cotton glycosyl transferase GhUGT85O1.
Another object of the present invention is to provide above-mentioned upland cotton glycosyl transferase GhUGT85O1 application.
According to the upland cotton glycosyl transferase GhUGT85O1 of the present invention, its amino acid sequence is as shown in SEQ ID No.1：
MGSLETSKPHIVIVPFPAQGHVNPMMLLAKLLHSRGFFITFVNTEFNHRRLVRSKGPDFVKGLPDFQFETIPEGLTS
SDRNATQDLPVLCDSIRKHCLAPFVDLLAKLNSSPQVPTVTCIISDGLMSFAIKAAEQLGIPEVQFWTASACSFMGY
LHFSELVKRGIIPFQSETFLDEPIDWVPGMSNIRLRDFPSFVRANDPNDILFDYLGSEAQNCLKASAIIFNAFEEFE
HEVLDAIAAKFPRIYTVGPLHLVARHLHADPSKSMNPSLWKEDTSCIEWLNEREPNSVVYVNYGSITVMSAKHLKEF
AWGLANSKHPFLWIVRPDVVMGDSAILDLEFLKEIKERGLITSWCNQYEVLSHPSVGVFLTHCGWNSTVETISEGVP
VVCWPFFADQQTNCRYACTHWGIGMEVDHDVKRENIEFLVKEMMEGEEGKKKKEKALGWKKKAEEAVEVGGSSYIDF
DRFVKEALKHG。
According to the upland cotton glycosyl transferase GhUGT85O1 encoding genes of the present invention, its nucleotide sequence such as SEQ ID
Shown in No.2：
ATGGGTTCACTTGAAACCAGTAAACCCCACATTGTAATCGTCCCATTTCCAGCACAAGGTCATGTTAACCCCATGAT
GCTACTTGCTAAGCTCTTACACTCTAGAGGCTTCTTCATAACCTTTGTTAACACTGAGTTCAACCATAGGCGTTTGG
TCAGGTCCAAAGGCCCTGACTTTGTTAAAGGTCTGCCTGATTTCCAGTTCGAAACAATTCCGGAAGGGCTGACATCG
TCCGATCGAAATGCAACACAGGATCTTCCAGTTCTGTGTGATTCGATACGAAAGCATTGCTTGGCACCATTCGTAGA
TCTACTAGCTAAGTTAAACTCCTCGCCCCAAGTGCCCACTGTTACTTGCATAATCTCTGATGGACTTATGAGCTTTG
CTATTAAGGCTGCTGAACAACTTGGCATACCAGAAGTTCAGTTTTGGACTGCCTCAGCATGTAGTTTCATGGGATAT
CTTCACTTCAGTGAACTGGTTAAACGAGGCATTATTCCATTCCAAAGTGAAACATTTCTCGATGAACCTATTGACTG
GGTCCCTGGAATGAGTAACATTCGCCTCAGAGATTTTCCAAGCTTCGTCAGAGCCAACGATCCGAATGACATTTTGT
TTGATTATTTAGGATCCGAAGCTCAAAATTGCCTAAAAGCTTCAGCAATAATCTTCAACGCATTTGAAGAGTTCGAA
CATGAAGTGCTCGACGCCATCGCTGCCAAATTTCCTCGAATTTATACAGTAGGACCACTTCATTTGGTTGCCAGGCA
CCTACATGCCGATCCCTCCAAGTCAATGAACCCAAGCCTATGGAAGGAAGATACAAGCTGCATTGAATGGCTTAACG
AAAGGGAACCCAATTCAGTTGTGTATGTGAACTATGGAAGCATTACTGTCATGTCGGCGAAGCATCTCAAAGAATTT
GCATGGGGGTTGGCTAACTCTAAACACCCATTTTTATGGATCGTTAGACCAGATGTCGTGATGGGTGATTCTGCAAT
TCTGGATCTAGAGTTCCTGAAGGAGATTAAGGAAAGAGGGCTGATAAcAAGCTGGTGCAACCAATATGAGGTCCTTT
CACATCCTTCAGTCGGTGTTTTTTTGACACACTGTGGGTGGAATTCTACCGTGGAAACCATATCAGAAGGTGTGCCA
GTAGTTTGTTGGCCATTTTTTGCTGATCAGCAAACCAATTGTCGATATGCTTGCACTCATTGGGGCATTGGCATGGA
GGTGGATCATGATGTGAAGCGAGAGAACATAGAGTTTTTAGTTAAGGAAATGATGGAAGGTGAGG
AAGGAAAGAAAAAGAAAGAGAAGGCATTGGGATGGAAGAAGAAAGCGGAAGAAGCGGTTGAAGTTGGGGGATCATCT
TATATTGATTTCGACAGATTTGTTAAGGAAGCTCTCAAACATGGTTAACTGGAAATACTATTGTCAGTGGGAAATTA
CTTAAATAATGAATGGGGATCATTCAATGTCTCTATTTCTGCTTTTTTTCCTTTGTAAGTGAATGCAGTCGTGTGAG
G。
The present invention has cloned a GhUGT85O1 gene from " CCRI 10, passes through point of its gene structure
Analysis, it is found that it contains two extrons and an introne.GhUGT85O1 genes up-regulated expression in cotton ageing leaves, on
Tune trend is up to 200 times, and this illustrates that it has certain influence on leaf senile.By being further looked to transgenic arabidopsis,
It was found that the chlorophyll content of transgenic line is significantly lower than wild type control, and the expression quantity of several aging marker gene is turning
It is higher than wild type in gene strain, this illustrates that the gene serves the effect for promoting leaf senile.
473 amino acid of protein sequence total length of GhUGT85O1 translations.In order to whether study GhUGT85O1 genes to non-
Biotic has response, eight kinds of processing has been carried out to blade, i.e.,：Gibberellin, heat, jasmonic, nitric oxide donors SNP, injury,
Salicylic acid, abscisic acid and PEG.As a result show, after treatment in 12 hours, the gene pairs ABA, JA and PEG have response.
After ABA is handled 4 hours, GhUGT85O1 expression quantity raised 4 times than 0 hour, and this trend is continued until 12 hours.At JA
Reason is after 8 hours, and GhUGT85O1 expression quantity raised 15 times than 0 hour, to 12 hours return to primitive age levels.PEG is handled 4 hours
Afterwards, 40 times are up to after GhUGT85O1 has raised 3 times, 8 hours, then this up-regulation trend slowly reduced, by 12 hours about 15
Times.For other 5 kinds of processing modes, in 12 hours of sampling, the expression trend of the gene does not change significantly.This
Outside, the senescence process of tri- transgenic lines of L5, L7, L53 shifts to an earlier date compared with wild type, from phenotype, when the leaf of transgenic line
The most of yellows of piece, when evidence for senescence is presented, the leaf color of most of blade of wildtype Arabidopsis thaliana is also dark green.By to leaf
The measure of chlorophyll contents shows：Now the chlorophyll content of 3 transgenic lines is less than the chlorophyll content of wild type, L5 and L7
About 70%, L53 of wild type chlorophyll content is the 56% of wild type, and the range of decrease of three strains has reached extremely notable water
Flat (P<0.01).
The present invention has cloned the gene in a cotton glycosyl transferase family 1, GhUGT85O1, by expressing mould to it
The analysis of formula, 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, laid a good foundation to further elucidate its function.
Brief description of the drawings
Fig. 1 shows the spatial and temporal expression profile analysis of GhUGT85O1 genes；
The expression change of GhUGT85O1 genes after Fig. 2 .ABA, JA and PEG are handled；
Fig. 3 shows the identification of pBI121-GhUGT85O1 transgenic arabidopsis positive strains；
Fig. 4 shows that transgenic arabidopsis aging shifts to an earlier date (A), the expression (C- of chlorophyll content (B) and aging related genes
G)。
Embodiment
Experiment material
Material to be tested is short season cotton kind CCRI 10 (CCRI10, Gossypiumhirsutum), takes the son of cotton respectively
The different tissues such as leaf, root, stem, leaf, flower, terminal bud, ovule.Full-bloom stage carries out listing mark to Leaves on main stem, flatten the same day for 0
My god, sampled since 15 days, per taking every other day once, until leaf senile.Processing experiment middle progress in the controlled environment chamber.Taken
Sample is quick-frozen in liquid nitrogen, is then stored in stand-by in -80 DEG C of refrigerators.
The gene cloning of embodiment 1 and conversion
First, in cotton GhUGT85O1 genes clone
Design specific primer amplifies complete sequence from " CCRI 10.PCR primer is：Sense primer
5’‐ATGGGTTCACTTGAAACCAG‐3’；- the CCTCACACGACTGCATTCAC-3 ' of anti-sense primer 5 '.
Performing PCR amplification is entered according to (DRR042) specifications of TAKARA LA Taq Hot Start Version 2.0, then
Recovery purpose fragment is simultaneously sequenced.
2nd, the spatial and temporal expression profile analysis of GhUGT85O1 genes
1st, the different tissues of cotton variety " CCRI 10 " and the total serum IgE of different leaf age blades are extracted, and are inverted respectively
Record as cDNA.
2nd, the cDNA obtained using step 1 10 times of volume dilution liquid carry out quantitative fluorescent PCR as template.
The primer of expression quantity for detecting GhUGT85O1 genes is：Sense primer:5’‐
GGCGAAGCATCTCAAAGAAT‐3’；Anti-sense primer:5’‐CAGAATCACCCATCACGACA‐3’.For detecting reference gene
(actin) primer pair of expression quantity is：Sense primer:5’‐ATCCTCCGTCTTGACCTTG‐3’；Anti-sense primer:5’‐
TGTCCGTCAGGCAACTCAT‐3’。
Quantitative fluorescent PCR reaction system：10 μ l SYBR Premix Ex Tag, 0.8 μ l sense primers, 0.8 μ l downstreams are drawn
Thing, 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 pre-degeneration 1min；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 72 DEG C of extensions
30s, 40 circulations；72 DEG C of extension 10min；At 72 DEG C, fluorescence signal is collected at 30s.
Analyzed using relative quantification Δ Δ Ct methods.
GhUGT85O1 genes are shown in Fig. 1 in the blade of different leaf ages and the expression of other tissues.As a result show：Should
Gene in ripe and ageing leaves (35 to 45 days) expression quantity compared with young leaflet tablet (15 to 30 days) height；In different tissues
Between, GhUGT85O1 in blade, cotyledon, spend in expression quantity it is higher, and the expression quantity in bud, ovule, stem, root and hypocotyl
It is relatively low.
In order to study whether GhUGT85O1 genes have response to abiotic stress, eight kinds of processing are carried out to blade, i.e.,：
Gibberellin, heat, jasmonic, nitric oxide donors SNP, injury, salicylic acid, abscisic acid and PEG.As a result show, after treatment
In 12 hours, the gene pairs ABA, JA and PEG have response (Fig. 2).After ABA is handled 4 hours, GhUGT85O1 expression quantity is smaller than 0
When raised 4 times, this trend is continued until 12 hours (A).After JA is handled 8 hours, GhUGT85O1 expression quantity was than 0 hour
15 times are raised, to 12 hours return to primitive age levels (B).After PEG is handled 4 hours, after GhUGT85O1 has raised 3 times, 8 hours
Up to 40 times, then this up-regulation trend slowly reduce, to 12 hours about 15 times (C).For other 5 kinds of processing modes,
In 12 hours of sampling, the expression trend of the gene does not change significantly.
3rd, transgenic arabidopsis
1st, the structure of PBI121-GhUGT85O1 plant expression vectors
Using No. 10 cDNA of CCRI as template, with the primer upstream with corresponding restriction enzyme site:5’‐
CACGGGGGACTCTAGAATGGGTTCACTTGAAACCAG-3 ' (underscore is XbaI enzyme cutting site)；Downstream:5’‐
GATCGGGGAAATTCGAGCTCCCTCACACGACTGCATTCAC-3 ' underscores are SacI restriction enzyme sites), amplification is included
The PCR primer of GhUGT85O1 gene coding regions.
Plant expression vector pBI121 is passed through into XbaI and SacI digestions, obtains carrier framework；
Above-mentioned carrier framework is connected with above-mentioned purpose genetic fragment.
2nd, the acquisition of GhUGT85O1 arabidopsis is turned
Plasmid PBI121-GhUGT85O1 is transferred in Agrobacterium LBA4404 competent cell, obtains recombinant bacterium.By PCR
Recombinant bacterium of the detection containing plasmid PBI121-GhUGT85O1 is named as LBA4404/PBI121-GhUGT85O1.The open country that will be bloomed
The pod for the Hua Hejie that raw type Arabidopsis plant has been pollinated is cut off with scissors, and thaliana flower is immersed into the small burning equipped with permeabilization buffer
50s is contaminated in cup, the Arabidopsis plant contaminated then is placed into dark culturing in big Plastic Drum, 24h is after arabidopsis culture
Continue to cultivate in room；Sowing is mixed after Fruit pod maturation, obtains T0 for the arabidopsis seed for turning GhUGT85O1.T0 is passed through for seed
Cultivated after sterilization vernalization on the MS culture mediums of added with antibiotic kanamycins, negative transgenic line is unable to normal growth, screens
Turn GhUGT85O1 arabidopsis to positive T0 generations.
3rd, the Molecular Identification of transgenic progeny
Extract the genomic DNA that positive T0 generation turns GhUGT85O1 arabidopsis, with forward primer 5 '-
GACGCACAATCCCACTATCC-3 ' (being matched with one section of nucleotide sequence of 35S promoter on carrier) and reverse primer 5 '-
CCTCACACGACTGCATTCAC-3 ' is that primer enters performing PCR amplification, is control with wildtype Arabidopsis thaliana (WT).
1% agarose electrophoresis detects, and as a result as shown in figure 3, L2, L5, L7, L53 can detect purpose fragment, is defined as sun
T0 is for transgenic line for property；The genomic DNA of wild type (WT) arabidopsis is extracted as control, with same primer, is not obtained
Purpose fragment.In positive T0 generations, are turned into GhUGT85O1 arabidopsis individual plants sowing, are sowed, screening, until T3 is for above homozygous strain
System.
Embodiment 2GhUGT85O1 functional studies
First, turn GhUGT85O1 arabidopsis to bloom in advance
The homozygosis that numbering is L2, L5, L7, L53 is turned into GhUGT85O1 arabidopsis, wildtype Arabidopsis thaliana (WT) sowing, in length
Sunshine condition is cultivated under (16h illumination/8h is dark).
It is consistent with wildtype Arabidopsis thaliana phenotype to turn empty carrier arabidopsis.4 homozygosis of statistics turn GhUGT85O1 strains and wild
Flowering time, the lotus throne number of sheets and the stem leaf number of type arabidopsis, in triplicate, each strain is using wild type as reference for experiment
Significance analysis is carried out, statistic analysis result is shown in Table 1.In addition to L2, other 3 strain (L5, L7, L53) poles remarkably promote plan south
Mustard early blossoming (P<0.01), and the quantity of lotus throne leaf also pole substantially reduces (P<0.01), the change of stem leaf quantity and wild type
Compare, L53 substantially reduces (P<0.05), other 3 equal unobvious of strain.
The transgenic arabidopsis flowering time of table 1, 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.* significant difference (P is represented<0.05), * * represent pole significant difference (P<0.01).
2nd, GhUGT85O1 transgenic arabidopsis promotes aging
By the observation to transgenic arabidopsis offspring, find the senescence process of tri- transgenic lines of L5, L7, L53 compared with
Wild type shifts to an earlier date (Fig. 4).From phenotype, when the most of yellows of the blade of transgenic line, when evidence for senescence is presented, wild type
The leaf color of most of blade of arabidopsis is also dark green (A).By showing (B) the measure of chlorophyll content：Now 3 turn
The chlorophyll content of gene strain is less than wild type, the chlorophyll that L5 and L7 chlorophyll content are about 70%, L53 of wild type
Content is the 56% of wild type, and the range of decrease of three strains has reached the pole level of signifiance (P<0.01).
With fluorescent quantitation primer shown in table, using AtUBQ1 as reference gene, (C- is detected to their expression quantity
G).As a result show：6 genes such as AtNAP, AtORE1, AtPR1, AtSAG12, AtSAG13 and AtWRKY6 are in transgenic line
Expression quantity in system is higher than wildtype Arabidopsis thaliana.
The fluorescent quantitation primer of the aging related genes of table 2
GhUGT85O1 genes up-regulated expression in cotton ageing leaves, up-regulation trend are up to 200 times, and this illustrates it to leaf
Piece aging has certain influence.By being further looked to transgenic arabidopsis, it is found that the chlorophyll content of transgenic line is bright
It is aobvious to be less than wild type control, and the expression quantity of several aging marker gene is higher than wild type, this explanation in transgenic line
The gene serves the effect for promoting leaf senile.The overexpression arabidopsis strain for the GhUGT85O1 genes cloned in cotton
Flowering time significantly earlier than wild type, and the lotus throne number of sheets is less than wild type, and this is the pass of UGT family genes and flowering of plant
System provides a new evidence.Therefore, the analysis to its expression pattern, and the research of transgenic progeny characteristic, mirror are passed through
Determine it and take part in Adversity-stressed Plant, the regulation and control of flowering time, and the regulation and control of senescence process, established to further elucidate its function
Basis.
Claims (5)
1. upland cotton glycosyl transferase GhUGT85O1, it is characterised in that its amino acid sequence is as shown in SEQ ID No.1.
2. upland cotton glycosyltransferase gene GhUGT85O1, it is characterised in that the upland cotton glycosyl described in coding claim 1
Transferase GhUGT85O1.
3. upland cotton glycosyltransferase gene GhUGT85O1, it is characterised in that its nucleotide sequence is as shown in SEQ ID No.2.
4. include the plant expression vector of upland cotton glycosyltransferase gene GhUGT85O1 described in claim 2.
5. upland cotton glycosyl transferase GhUGT85O1 described in claim 1 Adversity-stressed Plant, flowering time regulation and control and decline
Application in terms of the regulation and control of old process.
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| 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 |
| 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 |
| 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 |
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| Title |
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| Characterization and Expression Analysis of Two Cotton Genes Encoding Putative UDP-Glycosyltransferases;F.-J. Tai et al.;《Molecular Biology》;20081231;第42卷(第1期);第50页第1段 * |
| The Arabidopsis Glucosyltransferase UGT76B1 Conjugates Isoleucic Acid and Modulates Plant Defense and Senescence;Veronica von Saint Paul et al.;《The Plant Cell》;20111130;第23卷;第4124–4145页 * |
| UGT87A2, an Arabidopsis glycosyltransferase, regulates flowering time via FLOWERING LOCUS C;Bo Wang et al.;《New Phytologist》;20121231;第194卷;第666–675页 * |
| 植物UDP-糖基转移酶生化特性和功能研究进展;姬向楠等;《食品科学》;20131231;第34卷(第9期);第316-323页 * |
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