CN107312794B - Rose RrNUDX1 gene is changing the interim application of plant flowers - Google Patents
Rose RrNUDX1 gene is changing the interim application of plant flowers Download PDFInfo
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Abstract
The invention belongs to plant biotechnology fields, disclose rhodinyl because RrNUDX1 is changing the interim application of plant flowers.The over-express vector of present invention building RrNUDX1 gene, using agrobacterium-mediated transformation by pCAMBIA1304-RrNUDX over-express vector leading-in petunia, after plant blossom, we have found that, after transplanting 35 days, part turns RrNUDX1 gene plant and starts to bloom, and WT lines are then just bloomed after transplanting 60 days successively.Meanwhile the blade of transgenic plant obviously broadens, and it is whole leaf from oblong to class heart transformation, illustrate that being overexpressed RrNUDX1 gene has enhancing petunia growing way, the function of promoting it to bloom ahead of time.
Description
Technical field
The invention discloses rhodinyl because RrNUDX1 is changing the interim application of plant flowers, belong to Plant Biotechnology neck
Domain.
Background technique
Rose (Rosa rugosa Thunb.) is the traditional medicine food of important in the world natural perfume plant and China
Homologous flowers, citronellol, geraniol, nerol and its acetate esters derivative in monoterpenes compound are to form rose feature
The main component of fragrance.
Monoterpene is distributed in the secretory tissues such as the body of gland grease chamber of plant, usually mostly as terpenoid main species
With stronger fragrance and bioactivity, monoterpene plays a significant role plant life and human lives, in addition to they are being planted
Outside function in object defence, monoterpene also serves as fragrance and drug, while the main component of many rare plants essential oils is by monoterpene
Class compound composition, as Peppermint essential oil is mainly made of citral and carvol.In recent years, researcher is raw by separation monoterpene
Key gene in object route of synthesis selectively regulates and controls monoterpene metabolic pathway, to improve terpene by genetic engineering means
Class synthesizes the yield of precursor and downstream monoterpenes compound, achievees the purpose that improve scent and essential oil quality.
NUDX1 belongs to Nudix hydrolase gene family, is found in bacterium, virus, eucaryote, studies have shown that
NUDX1 gene is only one horse special type enzyme in NUDX family, and with 8-oxo- (d) GTP, (8- aoxidizes three phosphorus of deoxyguanosine to albumen
Sour enzyme), dNTP, NADH, Dihydroneopterin (dihydroneopterin) be substrate, can be effectively in scavenger-cell because of ROS oxygen
Change the nucleotide being damaged, reduce the accumulation of Cellular Oxidation damage, reduce cell mutation and programmed death, enhances to bad
The adaptive faculty of environment.
The cDNA full length sequence overall length 777bp of rose RrNUDX1 gene has initiation codon, complete open reading
Frame 453bp, terminator codon, 5' noncoding region 68bp, 3 ' noncoding region 244bp and poly (A) tail 12bp, encodes 151 altogether
A amino acid.The gene number of logging in is KX096710.1.But the application of RrNUDX1 gene not yet has been reported that.
It is the committed step of higher plant transition stage at flower, is the qualitative leap process that plant completes its life cycle, it
Common regulation by plant itself internal factor and outside environmental elements.It is always domestic and international to the exploration of flowering of plant mechanism
The emphasis focused extensively, and the hot spot of current plant developmental biology area research.Floral induction is plant from nutrient growth
In the first stage of transformation and plant reproductive starting to reproductive growth, it directly decides it at the time spent.In recent years
Come, people have found the floral induction process of plant by one in the research to model plant arabidopsis, rice, toad's-mouth etc.
Extremely cumbersome netted approach control.Oneself confirms at least there is the tune that four major signalling pathways participate in Flowering transformation at present
Control process, it may be assumed that vernalization approach (Vernalization pathway), Photoperiod pathway (Photoperiod pathway), from
Main approach (Autonomous pathway) and gibberellin pathway (Gibberellin pathway).Wherein vernalization approach and light
Cycle pathways are response of the plant to temperature and Irradiance in external environment, and autonomous pathway and gibberellin pathway then by
The regulation of plant developmental state itself and Endogenous Hormones.Although four approach of regulation Flowering may independently be rung
The stimulation of various environmental signals is answered, but is mutually correlated with each other interaction between these approach again, composition one is numerous and complicated
Organic network system, to realize the precision control to flowering of plant.Signal from each approach finally comes together in several important
Integration factor of blooming, such as SOC1 (SUPPRESSOR OF OVEREXPRSSION OF CO1), FT (FLOWERING
LOCUS T), LFY (LEAFY), FLC (FLOWERING LOCUS C) etc. integrate these signals by them, in turn
Start the expression of downstream floral meristem characterizing gene, it is ensured that plant blooms in due course.
Currently, by the method for genetic engineering, flower color, plant type, polyphyll, male sterility and in terms of
Upgrading has achieved good progress, but is rarely reported in terms of early blossoming breeding.Using technological means of the invention, we
The petunia distinguished germ plasm material bloomed for nearly 30 days can be shifted to an earlier date by obtaining, this for using RrNUDX1 change the plant florescence provide
Important means.The present invention and related ends will further enrich that Flowering regulation is theoretical, for cultivation early blossoming new variety of plant,
It reduces production cost, improve the basis that breeding efficiency establishes initiative, there is important theory significance and more practical value.
Summary of the invention
The object of the present invention is to provide the applications of rose RrNUDX1 gene.
The invention discloses rose RrNUDX1 genes to change the interim application of plant flowers.
The invention also discloses RrNUDX1 genes to cultivate the purposes in early blossoming plant variety.
A kind of breeding method of early blossoming plant variety is the over-express vector for constructing RrNUDX1 gene, is situated between using Agrobacterium
Over-express vector is imported plant to be transformed by inducing defecation by enema and suppository, obtains early blossoming plant variety.
The present invention is by the over-express vector of building RrNUDX1 gene, using agrobacterium-mediated transformation by pCAMBIA1304-
RrNUDX over-express vector leading-in petunia, after plant blossom, it has been found that after transplanting 35 days, partially turn RrNUDX1 gene
Plant starts to bloom, and WT lines are then just bloomed after transplanting 60 days successively.Meanwhile the blade of transgenic plant obviously becomes
Width, it is whole leaf from oblong to class heart transformation, illustrate that being overexpressed RrNUDX1 gene has enhancing petunia growing way, promotees
The function of blooming ahead of time into it.
Using technological means of the invention, the petunia distinguished germ plasm material bloomed for nearly 30 days can be shifted to an earlier date by obtaining.This hair
Bright and related ends will further enrich that Flowering regulation is theoretical, for cultivate the new variety of plant of early blossoming, reduce production cost,
It improves breeding efficiency and establishes initiative basis, there is important theory significance and more practical value.
Detailed description of the invention
Fig. 1 petunia plant transformation process；A: blade pretreatment, B: infecting, C: it takes root, D: strong sprout, E: transplanting.
Fig. 2 PCR product gel detection, the PCR product of 2000 N:RrNUDX1 of M:Marker DL.
The single endonuclease digestion electrophoretogram of Fig. 3 recombinant plasmid pCAMBIA1304-RrNUDX1,
M1:Marker DL 15000M2:Marker DL 2000 A1-A2: recombinant plasmid single endonuclease digestion N: negative control.
Fig. 4 RrNUDX1-1 and RrNUDX1-2 sequence compares,
RrNUDX1-1: the target gene RrNUDX1-2: recombinant plasmid of clone.
Fig. 5 pCAMBIA1304-RrNUDX1 Agrobacterium-mediated Transformation daughter bacteria liquid PCR electrophoretogram
2000 P1-P3:pCAMBIA1304 of M:Marker DL is template N1:ddH2O is template N2: Agrobacterium EHA
Bacterium solution is that template N3:YEB fluid nutrient medium is that template A1-A7:pCAMBIA1304-RrNUDX1 Agrobacterium-mediated Transformation daughter bacteria liquid is
Template.
Fig. 6 petunia callus GUS coloration result
A:WT；B-D: turn RrNUDX1 gene callus.
Fig. 7 transgenic petunia plant DNA verifying
M:Marker DL2000；A1-A6: it is overexpressed resistant plant；P:pCAMBIA1304-RrNUDX1 plasmid；WT1-2:
Wild type.
Fig. 8 wild type and turn RrNUDX1 gene petunia plant forms and florescence compares；
WT1-2: wild type petunia；RrNUDX11-3: the petunia of overexpression RrNUDX1 gene；
A: Reducing sugar after transplanting 35 days；B: transgenic plant shows bud after transplanting 35 days；C: wild type and turn base
Because plant leaf size compares；D: transgenic plant is bloomed ahead of time than wild type.
Specific embodiment
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention.
Biomaterial: pCAMBIA1304 expression vector (Biovector company, Beijing), EHA105 Agrobacterium (Hua Yue ocean
Biotechnology Co., Ltd, Beijing)
1 rose RrNUDX1 genetic transformation petunia and functional verification
1.1 vector plasmids and bacterial strain
PCAMBIA1304 expression vector and EHA105 Agrobacterium are that this laboratory saves.
The preparation of 1.2 main agents
(1) YEB fluid nutrient medium: 0.05g MgSO4·7H2O, 1g yeast extract, 5g peptone, 5g beef extract, 5g
Sucrose uses ddH2O dissolves and is settled to 1000mL, adjusts pH value to 7.0, saves for 4 DEG C after sterilizing.YEB solid medium: in YEB
Add 20g/L agar on the basis of fluid nutrient medium, dispenses and save after sterilizing.
(2) Kan: mother liquor 50mg/mL.0.5g kan is dissolved in 10mL sterilizing ddH2In O, 0.22 μm of membrane filtration is used
The packing of 1.5mL centrifuge tube, -20 DEG C of preservations.
(3) Rif: mother liquor 5mg/mL.0.05g Rif uses sterilizing ddH after being dissolved in a small amount of dehydrated alcohol2O constant volume to 10mL,
0.22 μm of membrane filtration is dispensed with 1.5mL centrifuge tube, -20 DEG C of preservations.
(4) 6-BA: mother liquor 0.1mg/mL.10mg 6-BA is dissolved with the NaOH solution of a small amount of 0.1moI/L, finally with going out
Bacterium ddH2O is settled to 100mL, and -20 DEG C are kept in dark place.
(5) IAA: mother liquor 0.1mg/mL.10mg IAA is dissolved with the NaOH solution of a small amount of 1mol/L, is settled to 100mL,
0.22 μm of membrane filtration, -20 DEG C are kept in dark place.
(6) As: 50 μm of ol/L of mother liquor.0.1962g As is dissolved in DMSO and is settled to 20ml, 0.22 μm of membrane filtration, and -20 DEG C
It is kept in dark place.
(7) MES: mother liquor 10mg/mL.0.1g MES is dissolved in 10mL sterilizing ddH2It dissolves, 0.22 μm of membrane filtration, uses in O
The packing of 1.5mL centrifuge tube, 4 DEG C of preservations.
(8) Cb: mother liquor 50mg/mL.0.5g Cb is dissolved in 10mL sterilizing ddH2It dissolves, 0.22 μm of membrane filtration, uses in O
The packing of 1.5mL centrifuge tube, -20 DEG C of preservations.
1.3 petunia genetic transformation minimal mediums
MS1: sucrose 30g/L+MS basis, pH5.8
MS2 (callus differentiation): MS1+6-BA 3.0mg/L+IAA 0.2mg/L, pH5.8
MS3 (is taken root): sucrose 30g/L+1/2MS, pH5.8
MS4 (infected liquid): MS+MES 1mg/L+As 20mol/L, pH5.8
MS5 (co-cultivation): MS2+As 20mol/L, pH5.8
1.4RrNUDX1 gene overexpression vector construction
3.4.1 the preliminary acquisition of target gene
3.4.4.1 the synthesis and design of primer
Separately designing upstream and downstream primer according to RrNUDX1 gene (KX096710.1) cDNA full length sequence, (primer institute is transregional
Domain includes complete CDs sequence), it is used for in-vitro separation target gene, primer information to be as follows:
The primer of the amplification RrNUDX1 gene open reading frame of table 1
3.4.4.2 the PCR amplification of target gene
(1) reaction system are as follows:
(2) reaction condition:
X: annealing temperature
(3) by PCR reaction solution on 1% Ago-Gel electrophoresis detection.
1.4.4.3PCR the recycling and purifying of product
Referring to the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit of TaKaRa company
Ver.4.0 kit carries out.
1.4.4.4PCR the connection, conversion, expansion culture of product
The connection of target fragment, the pEASY for converting reference Trans companyTM- T5Zero Cloning Kit kit explanation
Book carries out.
1.4.4.5 the extraction and detection of Plasmid DNA
With reference to the method for the part 2.2.4.3, the raw work in Shanghai is entrusted to carry out objective gene sequence sequencing, by sequencing result and
The RrNUDX1 gene order of login carries out homologous comparison.
1.4.4.6RrNUDX1 the clone of gene and sequence are analyzed
The RNA extracted using rose variety ' Tang Hong ' petal, reverse transcription are template at cDNA, pass through product after PCR amplification
Electrophoretogram such as Fig. 2, it can be seen that amplified fragments are close with desired value between 500-750bp.Recovery purifying amplified fragments, insertion
pEASYTM- T5Zero Cloning Vector is transferred to Escherichia coli screening positive clone on the LB plate of additional 1 ‰ Km.It surveys
Sequence is the results show that the objective gene sequence cloned is 540bp or so, containing initiation codon, terminator codon, coding 151
Amino acid is compared with the RrNUDX1 gene similitude cloned up to 100%, thus it is speculated that amino acid sequence similarity is up to 100%.
1.4.2 the acquisition of target gene (containing restriction enzyme site)
1.4.2.1 the design and synthesis of primer
It is required according to In-Fusion kit specification, on binding purpose gene and binary expression vector pCAMBIA1304
Restriction enzyme site, designed for over-express vector building PCR primer, Primer and its oligonucleotide sequence are such as
Under:
RrNUDX1 single endonuclease digestion primer:
RrNUDX1-F:5’-GGACTCTTGACCATGGTTATGGGAAACGAGACAGTTGTAG-3’(Nco I)(SEQID
NO.3)RrNUDX1-R:5’-GTCAGATCTACCATGGTCATGTTGGAAAAGGGTTAAATC-3’(Nco I)(SEQID
NO.4) 1.4.2.2 contains the PCR amplification of restriction enzyme site target gene
The cloning vector plasmids containing RrNUDX1 coding sequence obtained using 2.1.3.5 are protected as template using height
True enzyme carries out pcr amplification reaction, and NcoI restriction enzyme site is introduced the gene coding region RrNUDX1 both ends.
(1) reaction system are as follows:
(2) reaction condition:
(3) product is detected with 1% agarose gel electrophoresis, cuts the blob of viscose containing target fragment and recycling.
1.4.2.3 binary expression vector pCAMBIA1304 digestion and product recycling
(1) the over-express vector pCAMBIA1304 endonuclease reaction system of RrNUDX1 gene is connected are as follows:
(2) reaction condition: 37 DEG C of 3h；
(3) product is detected with 1% agarose gel electrophoresis.
1.4.2.4 the connection of target fragment and expression vector
The over-express vector of the target gene and acquisition that will acquire is with 1:3 Hybrid connections.
(1) coupled reaction system are as follows:
(2) PCR reaction condition: 50 DEG C of 15min
1.4.3 connection conversion
The connection of target fragment, the pEASY for converting reference Trans companyTM- T5Zero Cloning Kit kit explanation
Book carries out.
The identification and detection of 1.5 recombinant plasmids
1.5.1 digestion identification and detection
Select the single colonie on LB solid culture ware, be respectively placed in the sterilizing 10mL of the LB of liquid containing 3mL (containing 1 ‰ Kan) from
In heart pipe, after 37 DEG C of 200rpm shaken overnight cultures, the DNA of pCAMBIA1304-RrNUDX1 recombinant plasmid is extracted.
Single endonuclease digestion identification is carried out to pCAMBIA1304-RrNUDX1, reaction condition is 37 DEG C of 3h, with 1% agarose
Detected through gel electrophoresis product.For digestion pCAMBIA1304 as negative control, electrophoresis detection result is as shown in Figure 3 simultaneously.It can see
Out, negative control N shows that the band for being about 12000bp, single endonuclease digestion A1-A2 cut out an about band of 500bp, with
RrNUDX1 open reading frame is in the same size.It is therefore shown that target gene is tentatively inserted into expression vector pCAMBIA1304.
Send the positive recombinant plasmid after digestion verification to sequencing, sequencing result analyzes (Fig. 4) with DNAMAN, and has cloned
As a result it is compared, discovery similitude is 100%, no base mutation, thus it is speculated that amino acid sequence is identical, it was demonstrated that target gene
RrNUDX1 is successfully connected into expression vector pCAMBIA1304, and over-express vector pCAMBIA1304-RrNUDX1 is constructed successfully.And
Correct recombinant plasmid will be sequenced to save in -20 DEG C.
1.5.2 recombinant plasmid imports Agrobacterium EHA105 and screening and identification
1.5.2.1 prepared by Agrobacterium EHA105 competence
Test method carries out the preparation of Agrobacterium EHA105 competence referring to the method for this laboratory Wang Jia (2015)
1.5.2.2 frozen-thawed method converts Agrobacterium
(1) Agrobacterium EHA105 competent cell is slowly melted on trash ice, 5 μ L recombinant plasmids are added and mixed, set
In 30min on ice, 1min liquid nitrogen flash freezer, 37 DEG C of heat shock 5min, ice bath 2min；
(2) 800 μ L or so are added is free of antibiotic liquid YEB, 120-160rpm, 28 DEG C, and 4-5h is containing 40mg/L
Rif and 50mg/L Kan solid YEB on be coated with 300 μ L, 28 DEG C of dark culture 2d or so.
3.5.2.3 the screening and identification of Agrobacterium-mediated Transformation
(1) single colonie on picking YEB plate is inoculated in containing 3mL (Kan of Rif and 50mg/L containing 40mg/L)
In the sterilizing 10mL centrifuge tube of liquid YEB, 28 DEG C of 200rpm dark cultures；
(2) bacterium solution PCR detects positive transformant；
(3) product is detected with 1% agarose gel electrophoresis.
1.5.2.4 Agrobacterium-mediated Transformation saves
25% sterile glycerol is added by being accredited as in the Agrobacterium bacterium solution of positive transformant through bacterium solution PCR, is used after mixing
The packing of 1.5mL sterile centrifugation tube, -70 DEG C of preservations.
1.5.2.5 result
It will test correct recombinant plasmid (pCAMBIA1304-RrNUDX1) and Agrobacterium imported using frozen-thawed method
EHA105 competence screens positive transformant on the YEB solid medium for being attached with 1 ‰ Km and 1 ‰ Rif, after bacterium solution is made
Bacterium solution PCR verifying is carried out using it as template, while being directed to one group of positive control (P) of template-setup, three groups of negative controls (N1, N2,
N3), to guarantee the accuracy of result.Product shows (Fig. 5) that it is 500bp or so that A1-A7, which has amplified length, through electrophoresis detection
Target fragment.
The optimization of 1.6 petunia genetic conversion systems
1.6.1 the acquisition of vegetable material
1.6.1.1 the acquisition of aseptic seedling
Petunia seeds are placed in 75% alcohol and impregnate 5min, aseptic water washing 3 times, then with 12.5% hypochlorous acid
10min is impregnated in sodium solution, aseptic water washing 3 times, is inoculated on 1/2MS culture medium and is cultivated.
1.6.1.2 tissue culture transplantation of seedlings
It is transplanted when tissue-cultured seedling grows to five leaves wholeheartedly, bottle cap is opened hardening one week or so in room temperature, is then moved
It plants in artificial greenhouse climate box, light dark cycles 16h/8h, intensity of illumination 200umol.m-2s-1, temperature is 25 DEG C/23 DEG C,
Relative humidity is 70%.
1.6.2 the determination of the selection of petunia genetic conversion system pressure and bacteriostatic agent concentration
1.6.2.1 the determination of hygromycin selection pressure
Over-express vector pCAMBIA1304-RrDXR, pCAMBIA1304-RrNUDX1 of this test building carry hygromycin
(Hyg) resistant gene, it is thus determined that Hgy is the selection antibiotic of the petunia genetic transformation of mediated by agriculture bacillus.In ultra-clean work
On platform, eugonic petunia aseptic seedling is chosen, blade is cut to 1cm2Leaf dish, be respectively placed in Hyg concentration be 1,3,5,
6,7,9,10, on the culture medium MS1 of 15mg/L, each gradient handles 30 pieces of leaf tissues, cultivates 30 days, replaces 1 time within every 15 days
Culture medium is repeated 3 times.
From table 2 it can be seen that the concentration of Hyg is higher, bigger, callus induction and bud differentiation are influenced on the differentiation of explant
Critical value is 7mg/L, the critical concentration 6mg/L taken root, thus select culture when, respectively using respective concentration Hyg into
Row screening, it is ensured that the raising of positive rate.
The influence that 2 difference Hyg concentration of table breaks up petunia each period
1.6.2.2 the determination of carboxylic benzyl concentration
This test uses bacteriostatic agent of the carboxylic benzyl as genetic transformation, and on superclean bench, selection is eugonic short to be led
Blade is cut to 1cm by ox aseptic seedling2Leaf dish, be respectively placed in Cb concentration be 100,200,300,400,500,600,700mg/L
Culture medium MS1 on, each gradient handles 30 pieces of leaf tissues, cultivates 30 days, every 15 days 1 subcultures of replacement are repeated 3 times.
From table 3 it can be seen that carboxylic benzyl does not have apparent inhibiting effect to the growth of explant, it is right when concentration is 500mg/L
Callus and bud break up to have to be influenced by a small margin, when concentration is 500mg/L, is influenced by a small margin on taking root for having, therefore three kinds of Selective agar mediums
Bacteriostatic agent concentration select 500mg/L.
The influence that 3 difference Cb concentration of table breaks up petunia each period
1.6.2.3 the culture of Agrobacterium infected liquid
(1) agrobacterium strains containing target gene in -70 DEG C are taken out, on the YEB screening and culturing medium containing Km and Rif
Draw plate, 28 DEG C dark culture 2-3 days；
(2) single colonie is inoculated in YEB+Kan+Rif fluid nutrient medium, culture OD is 0.8-1.3；
(3) bacterium solution PCR is verified, and determines the correctness of target gene in bacterium solution；
(4) by the bacterium solution of identification, 50mL is added and is shaken 1-2 days without antibiotic liquid YEB in 28 DEG C of 200rpm；
(5) by the bacterium solution of re-activation after 4 DEG C of 5000rpm are centrifuged 12-15min, thallus is collected, then with being ready in advance
Infected liquid (MS4) be resuspended dilution bacterium solution OD be 0.3-0.6 it is spare.
1.7 the genetic transformation of petunia
1.7.1 the pretreatment of blade
It chooses eugonic petunia blade and is cut into 1cm on superclean bench2Small cube, the blade outside of belly to
Under, it is placed in dark culture 2 days on MS solid medium.
1.7.2 infecting
Co-culturing surface tiling aseptic filter paper piece first；The material of preparatory culture 2 days is put into and is ready for by second step
Infected liquid in infect 5-8min；Third step takes out the blade after infecting, and the bacterium solution on surface is blotted with aseptic filter paper, is placed in total training
It supports and is cultivated 3 days on base (MS5).
1.7.3 break up screening and culturing
Leaf dish after co-culturing 3 days is taken out, with the bacterium solution on aseptic filter paper blotting material surface；It is transferred to screening and culturing medium
Middle Fiber differentiation callus is placed in plant tissue culture room culture, and every 15d changes a subculture, until growing regrowth.
1.7.4 it takes root screening and culturing
When regrowth length that the callus of screening and culturing differentiates to 2-3cm or so, replacement is cut to screening of taking root
In culture medium, the callus for not breaking up budding needs to cut off the part of browning flavescence, is transferred to new differentiation screening and culturing
On base, (Fig. 1) can be taken root within positive plant 2-3 weeks.
The detection of 1.8 transgenic petunias
Resistant plant is detected using GUS decoration method and PCR method
1.8.1 petunia callus GUS is dyed
GUS dyeing is carried out to the normal petunia callus of growthform under hygromycin selection, the results show that wild type
In yellow-white, no GUS substrate active turns to have dyed different degrees of blue (Fig. 6) in the callus of RrNUDX1 gene, shows to sieve
Select marker gene GUS normal expression.
1.8.2 turning the petunia plant DNA verifying of RrNUDX1 gene
It selects 6 single plants at random in the petunia plant for turning RrNUDX1 gene of acquisition, extracts leaf DNA and carry out PCR
Amplification, it is 500bp or so that 5 plants, which can amplify single band and size, in 6 plants as the result is shown, with the control plasmid product being transferred to
Size is similar, and wild type cannot amplify band (Fig. 7), it can be seen that RrNUDX1 gene has been integrated into petunia gene
In group.
1.9 petunia transgenic plant Phenotypic Observations
By wild type and turn the petunia plantlet of transplant of RrNUDX1 gene in artificial greenhouse climate box, same culture conditions
Under cultivated, form, flowering time, flower size, pattern and the florescence of observational record transgenosis and WT lines blade
Variation (Fig. 8) in terms of isophenous records the time (number of days, d) required from be colonized to blooming.As a result it was found that transplanting 35
After it, partially turns RrNUDX1 gene plant and start to bloom, and WT lines are then just bloomed after transplanting 60 days successively.Meanwhile
The blade of transgenic plant obviously broadens, and entirety is leaf to change (Fig. 8, table 4) from oblong to class heart.
Wild type and rotaring gene plant blade form compare (mm) after table 4 is transplanted 35 days
2 conclusions
In summary result, it has been found that compared with wild type, turns RrNUDX1 gene petunia growing way and be remarkably reinforced, and
Florescence significantly shifts to an earlier date nearly 30 days, illustrates that being overexpressed RrNUDX1 gene has the change plant florescence, promotes the function bloomed ahead of time
Can, next this has established breakthrough basis using the florescence of the other plants of RrNUDX1 gene alteration for us.
SEQUENCE LISTING
<110>Yangzhou University
<120>rose RrNUDX1 gene is changing the interim application of plant flowers
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
atgggaaacg agacagttgt ag 22
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
tcatgttgga aaagggttaa atc 23
<210> 3
<211> 40
<212> DNA
<213>artificial sequence
<400> 3
ggactcttga ccatggttat gggaaacgag acagttgtag 40
<210> 4
<211> 39
<212> DNA
<213>artificial sequence
<400> 4
gtcagatcta ccatggtcat gttggaaaag ggttaaatc 39
Claims (1)
1. rose RrNUDX1Gene is changing the interim application of petunia, the roseRrNUDX1The accession number of gene is
KX096710.1。
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| CN113278639B (en) * | 2021-05-25 | 2023-06-06 | 云南中烟工业有限责任公司 | Tobacco NUDIX hydrolase gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268080A (en) * | 2010-06-02 | 2011-12-07 | 中国农业科学院作物科学研究所 | Plant blossom related protein GmFTLa, coding gene thereof and application thereof |
| CN105777884A (en) * | 2016-04-19 | 2016-07-20 | 贵州大学 | Plant-disease-resistant related protein NHRGP and encoding gene and application thereof |
| CN107267551A (en) * | 2017-07-27 | 2017-10-20 | 扬州大学 | Application of the rose RrNUDX1 genes in the enhancing plant fragrance of a flower |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268080A (en) * | 2010-06-02 | 2011-12-07 | 中国农业科学院作物科学研究所 | Plant blossom related protein GmFTLa, coding gene thereof and application thereof |
| CN105777884A (en) * | 2016-04-19 | 2016-07-20 | 贵州大学 | Plant-disease-resistant related protein NHRGP and encoding gene and application thereof |
| CN107267551A (en) * | 2017-07-27 | 2017-10-20 | 扬州大学 | Application of the rose RrNUDX1 genes in the enhancing plant fragrance of a flower |
Non-Patent Citations (1)
| Title |
|---|
| Biosynthesis of monoterpene scent compounds in roses;Jean-Louis Magnard等;《Science》;20150703;第349卷(第6243期);第83页左栏最后一段 * |
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