CN104195097B - Culture medium for promoting transgenic tobacco with early blossoming-promoting gene NtFT5 to root and method for obtaining short-period tobacco - Google Patents
Culture medium for promoting transgenic tobacco with early blossoming-promoting gene NtFT5 to root and method for obtaining short-period tobacco Download PDFInfo
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- CN104195097B CN104195097B CN201410449758.3A CN201410449758A CN104195097B CN 104195097 B CN104195097 B CN 104195097B CN 201410449758 A CN201410449758 A CN 201410449758A CN 104195097 B CN104195097 B CN 104195097B
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Abstract
The invention discloses a culture medium for promoting transgenic tobacco with an early blossoming-promoting gene NtFT5 to root and a method for obtaining short-period tobacco. The rooting culture medium is a 1/2MS solid culture medium which contains 0.1mg/L NAA with the pH value of 5.6-5.8. The culture medium can be used for promoting the transgenic tobacco with an early blossoming-promoting gene NtFT5 to root. The tobacco after rooting can normally blossom and set seeds and pass the early blossoming-promoting character to the next generation so as to successfully obtain a short-period tobacco offspring with stable inheritance after seeding, and the life cycle of the tobacco is shortened to 2.5 months, thereby satisfying the demand on gene function identification and tobacco breeding research. The culture medium has a good application prospect.
Description
Technical field
The invention belongs to field of plant tissue culture technique, it is related to promotion and turns Nicotiana tabacum L. and promote early blossoming gene NtFT5 Nicotiana tabacum L. take root
Culture medium, further relate to using this culture medium obtain cycle Nicotiana tabacum L. method.
Background technology
Blooming is the important economical character of one of crop production, and it is subject to autonomic signals approach, photoperiod, vernalization approach
And the signal pathway combined influence such as phytohormone gibberellin.In arabidopsiss, these signal pathways are all by PHOSPHATIDYL ETHANOLAMINE
Associated proteins family phosphatidylethanolamine-binding protein (PEBP) family member plays regulation and control
Effect.Three subfamilies of mutual antagonism in numerous plants, are entered to turn between PEBP family member, they are
FLOWERING LOCUS T (FT), TERMINAL FLOWER 1 (TFL1) and MOTHER OF FT AND TFL1 (MFT).FT
It is considered to be chiefly to facilitate the subfamily bloomed with MFT member.The equal energy of overexpression FT member FT, TSF and MFT member in arabidopsiss
Promote arabidopsiss early blossoming.
As a kind of model organism, it has simplest habit and most reliable conversion process to Nicotiana tabacum L..With Nicotiana tabacum L.
As model plant, study secondary metabolite biosynthesis pathway, plant and antibacterial interaction, plant and pest and disease damage interaction, dimension
The formation of tubing and flower development process and pattern are respectively provided with significant advantage and have defined study characteristics.In numerous plants
In, many identify with the genome research of cis-acting factors all with Nicotiana tabacum L. as recipient plant with regard to functional gene.However, Nicotiana tabacum L.
Breeding cycle length has become as the key constraints of tobacco bred cultivation.Research finds, arabidopsiss FT gene can shorten blooms
Time, NtFT5 also can promote Nicotiana tabacum L. early blossoming, but under existence conditionses, after this gene transformation, plant is most it was found that Nicotiana tabacum L. NtFT4
Pipe can early blossoming in culture medium, but flower generally withered quickly it is impossible to form seed it is impossible to take root it is impossible to obtain stable something lost
The short cycle tobacco seedling passing.Therefore, urgent need is a kind of is obtained in that stable heredity, the method for short cycle transgene tobacco Seedling, and
The transgene tobacco obtaining shortens flowering time, can take root and form seed.
Content of the invention
In view of this, an object of the present invention is to provide and promotes to turn what Nicotiana tabacum L. rush early blossoming gene NtFT5 Nicotiana tabacum L. took root
Culture medium；The second object of the present invention is to provide to obtain using described culture medium stablizes heredity, the method for short cycle Nicotiana tabacum L..
For achieving the above object, the present invention provides following technical scheme：
1st, promote to turn the culture medium that Nicotiana tabacum L. rush early blossoming gene NtFT5 Nicotiana tabacum L. takes root, described root media is containing 0.1mg/L
NAA, pH be 5.6～5.8 1/2MS solid medium, the MS culture medium that described 1/2MS halves for a great number of elements.
Preferably, described root media is 1/2MS+0.1mg/L NAA+30g/L sucrose+7.5g/L agar, adjusts pH
To 5.8.
2nd, the method utilizing described culture medium to obtain short cycle Nicotiana tabacum L., comprises the steps：
A. with SEQ ID NO.1 and sequence shown in SEQ ID NO.2 as primer, Nicotiana tabacum L. cDNA enters performing PCR amplification for template,
Obtain Nicotiana tabacum L. and promote early blossoming gene NtFT5, the Nicotiana tabacum L. that amplification is obtained promotees early blossoming gene NtFT5 and is connected into pCXSN carrier Xcm I enzyme action
Site, obtains NtFT5-pCXSN recombiant plasmid；By gained NtFT5-pCXSN recombinant plasmid transformed Agrobacterium, NtFT5- must be contained
The engineering bacteria of pCXSN recombiant plasmid；
B. step A gained engineering bacteria is adopted leaf disk method transformation of tobacco, in 6-BA containing 1mg/L, 0.1mg/L NAA30g/L
Sucrose, 8g/L agar, pH be 5.8 MS culture medium on co-culture 3 days, then in 1mg/L 6-BA, 0.1mg/L NAA,
250mg/L Carbenicillin, 20mg/L hygromycin, 30g/L sucrose, 8g/L agar, pH be 5.8 MS culture medium on induction of resistance
Seedling, then utilize PCR screen the plant containing hygromycin gene, obtain T0 generation turn NtFT5 genetic tobacco；
C. by step B gained T0 generation turn NtFT5 genetic tobacco root culture in described culture medium, transplant after taking root to
Cultivate in culture matrix to blooming, setting seeds, after the seed of acquisition is sprouted, obtain short cycle Nicotiana tabacum L..
Preferably, in step A, the condition of described PCR amplification is 95 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 54.0 DEG C are moved back
Fiery 45s, 72 DEG C of extension 40s, circulate 35 times；Extend 10min after last 72 DEG C.
Preferably, in step A, described Agrobacterium is LBA4404.
The beneficial effects of the present invention is：The present invention turns rush early blossoming gene FLOWERING LOCUS T (FT) for Nicotiana tabacum L.
Afterwards it is difficult to knot of taking root, cannot obtain stable heredity short cycle tobacco seedlings offspring situation, in root media and cultural method
On carried out systematic study and optimization, gained culture medium rooting rate is high, and test tube seedling transplanting survival rate is high, can produce fertility kind
Son, successfully obtains the short cycle tobacco seedlings offspring of stable heredity, and the life cycle turning NtFT5 gene tobacco seedlings foreshortens to 2.5 months, energy
Enough meet Functional identification of genes and the needs of tobacco breeding research, have a good application prospect.
Brief description
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：
Fig. 1 is NtFT5-pCXSN recombiant plasmid schematic diagram.
Fig. 2 is to turn NtFT5 genetic tobacco resistance Seedling.
Fig. 3 is to turn situation of taking root after NtFT5 genetic tobacco is cultivated in D3 culture medium.
Fig. 4 turns to transplant after NtFT5 genetic tobacco is taken root successfully blooms to peat cultivation matrix (Pin Shi).
Fig. 5 is to grow fine in NtFT5 genetic tobacco T1 generation.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted concrete in embodiment
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. writes)
Described in condition, or according to the condition proposed by manufacturer.
Embodiment 1, Nicotiana tabacum L. promote early blossoming gene (FLOWERING LOCUS T) NtFT5 transgene carrier and build
Nicotiana tabacum L. NtFT5 gene code is expanded according to (FH969747.1) sequential design in Genbank Nicotiana tabacum L. GSS data base
The primer in area, forward primer NtFT5-F is：5’-atgccaagagaacgtgaacc-3’(SEQ ID NO.1)；Downstream primer
NtFT5-R is：5’-tcaatcggcagaccttctac-3’(SEQ ID NO.2)；Then with tobacco leaf cDNA as template, with
SEQ ID NO.1 and sequence shown in SEQ ID NO.2 are primer, enter performing PCR amplification.PCR amplification system is 25 μ L, each group split
Long-pending as follows：CDNA 1.0 μ L, NtFT5-F 1.0 μ L, NtFT5-R 1.0 μ L,Green Master Mix enzyme 12.5 μ
L, ddH2O 9.5 μ L, then enters performing PCR amplification by following program：95 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 54.0 DEG C of annealing
45s, 72 DEG C of extension 40s, circulate 35 times；72 DEG C of extension 10min.
By pcr amplification product according to Transgen company EasyPure Quick Gel Extraction Kit explanation
Book carries out reclaiming and purification, obtains NtFT5 amplified production after purification.
By pCXSN carrier, (pCXSN carrier is disclosed in Chen, S., et al., A versatile zero background
T-vector system for gene cloning and functional genomics.Plant Physiol,
2009.150(3):P.1111-1121. plasmid ccdB containing suicide gene, through Xcm I enzyme action produce end T, can with A end
Genes of interest passes through TA Strategies For The Cloning, obtains the over-express vector containing genes of interest) carry out enzyme action with the Xcm I of NEB company,
Enzyme action system is as follows：PCXSN plasmid 1.4 μ g, Xcm I 1.0 μ L, NEB buffer 25.0 μ L, ddH2O adds to 50 μ L, then
Enzyme action 3 hours under the conditions of 37 DEG C.After enzyme action, pCXSN carrier linear fragment is reclaimed, then with the purification reclaiming
NtFT5 amplified production is 6 in molar ratio:1～7:1 connection, linked system is as follows：
Condition of contact：Connect 3 hours under the conditions of 25 DEG C.
Connection product is added -80 DEG C to take out the DH5 α competence just melted, ice bath 30min, then 42 DEG C of heat shock 45s,
300 μ L LB culture medium are added, in 37 DEG C of 200rpm shaking table cultures 1 hour after ice bath 2min；On abandoning after bacterium solution being centrifuged after culture
Clearly, thalline is applied in the LB resistant panel containing 50mg/L Kan, in 37 DEG C of constant temperature carton upside down overnight incubation.
Next day, choose single bacterium colony to 20 μ L ddH2In O, using bacterium colony PCR, positive colony is identified.PCR primer is：
NtFT5-F：5′-atgccaagagaacgtgaacc-3′(SEQ ID NO.1)；
NOS-R：5′-taaatgtataattgcgggactc-3′(SEQ ID NO.3)；
PCR system is as follows：Template 4.0 μ L, NtFT5-F 0.5 μ L, NOS-R 0.5 μ L,Green Master
Mix enzyme 5 μ L；
PCR response procedures are：95 DEG C of denaturations 10min；94 DEG C of degeneration 30s, 52.0 DEG C of annealing 45s, 72 DEG C of extension 50s,
Circulation 28 times；Last 72 DEG C of extension 10min.
Because NOS-R primer is apart from genes of interest insertion point downstream 200bp, therefore, when amplified fragments PCR primer length
Than NtFT5 genetic fragment about 200bp when, then regard as preliminary screening to the positive positive colony connecting.Preliminary screening is arrived
Positive bacterium solution, take 4.0 μ L to 6mL LB (containing Kan) fluid medium incubated overnight, then take 500 μ L bacterium solution send Hua Da survey
Sequence.500 μ L permanent bacterium to be saved, 5mL bacterium solution is collected by centrifugation precipitation and is stored in -20 DEG C.Sequencing result shows, the nucleotide of NtFT5
Sequence is as shown in SEQ ID NO.4, and NtFT5 genetic fragment has been connected into pCXSN carrier, obtains NtFT5-pCXSN recombiant plasmid, knot
Structure is as shown in Figure 1.
Embodiment 2, the engineering bacteria containing NtFT5-pCXSN recombiant plasmid for the preparation
By the Agrobacterium LBA4404 competence of -80 DEG C of NtFT5-pCXSN recombinant plasmid transformed taking-up, specific as follows：Will
100～200ng NtFT5-pCXSN recombiant plasmid adds in competence, mixes, after ice bath 30min, liquid nitrogen bath 1min, Ran Houyu
Heat shock 5min under the conditions of 37 DEG C, adds 800 μ L YEB culture medium, cultivates 4 to 6h in 28 DEG C of 200rpm；After culture, 800 μ are removed in centrifugation
L of supernatant, remaining bacterium solution is spread evenly across (Str and 40mg/ of the Kan containing 100mg/L, 20mg/L in YEB solid plate after suspending
The Rif of L), 28 DEG C of light culture 36, to 48h, obtain the engineering bacteria containing NtFT5-pCXSN recombiant plasmid.
Embodiment 3, turn the acquisition of NtFT5 genetic tobacco
(1) leaf disk method genetic transformation Nicotiana tabacum L.
Take the engineering bacteria bacterium solution containing NtFT5-pCXSN recombiant plasmid, add in 50mL YEB fluid medium (containing Kan,
Rif, Str), 28 DEG C, under the conditions of 200rpm light culture to exponential phase；The converting material growth selection Flos Carthami of 2 months about is big
(Aseptic seedling culture base is gold dollar aseptic seedling：MS+30g/L sucrose+8g/L Agar, pH 5.8), conversion will grow into 2 months in first 3 days
Left and right aseptic seedling, avoids edge and master pulse, blade card punch is processed into the leaf dish of diameter 0.5cm, is inoculated in containing 1mg/L
6-BA, 0.1mg/L NAA, 30g/L sucrose and 8g/L Agar, pH be 5.8 MS culture medium in 28 DEG C of precultures 3 days.Then
The engineering bacteria containing NtFT5-pCXSN recombiant plasmid growing to exponential phase is centrifuged 15 minutes in 4000rpm, abandons supernatant,
Collects thalline, thalline is resuspended with 30mL MS fluid medium.Blade aseptic nipper is transferred in resuspended bacterium solution, infects
10min, aseptic filter paper blots bacterium solution, and blade is transferred to 6-BA containing 1mg/L, 0.1mg/L NAA, 30g/L sucrose and 8g/L
Agar, pH be 5.8 MS culture medium under the conditions of 28 DEG C light culture 3 days.
(2) screening of transfer-gen plant
By the blade of the light culture aseptic water washing containing 500mg/L Carbenicillin 4 times, transfer to 6- containing 1mg/L
BA, 0.1mg/L NAA, 250mg/L Carbenicillin, 20mg/L hygromycin, 30g/L sucrose, 8g/L Agar, pH are 5.8 MS
Screening and culturing in culture medium, every two weeks subculture once, obtain the resistance Seedling that breaks up out, result is as shown in Figure 2.Then will break up
Resistance Seedling amplification Hpt genescreen positive transgenic plant out, the primer of amplification Hpt gene is as follows：
Hpt-F：5′-ctgctccatacaagccaaccac-3′(SEQ ID NO.5)；
Hpt-R：5′-gaaaaagcctgaactcaccgc-3′(SEQ ID NO.6).
In 25 plants of T0 generations of acquisition, turn NtFT genetic tobacco altogether after testing.
(3) root culture of transfer-gen plant
For ensureing transgenic positive plant energy smoothly subculture sowing, transgenic positive plant is moved to D1, D2, D3 and D4 tetra-
Plant in root media and carry out root culture, count situation of taking root, result is as shown in table 1.
Root media D1：1/2MS+30g/L sucrose+7.5g/L Agar, pH 5.8；
Root media D2：1/2MS+250mg/L Carbenicillin+20mg/L hygromycin+30g/L sucrose+7.5g/
LAgar, pH 5.8；
Root media D3：1/2MS+0.1mg/L NAA+30g/L sucrose+7.5g/L Agar, pH 5.8；
Root media D4：1/2MS+0.1mg/L NAA+250mg/L Carbenicillin+20mg/L hygromycin+30g/L sugarcane
Sugar+7.5g/L Agar, pH 5.8.
Table 1, positive transgenic plant take root statistical result
Result shows, can take root in tetra- kinds of root medias of transgenic positive Seedling D3 and D4, but in D4 root culture
Rooting rate in base is low, less than 10%.In addition, T0For transgenic positive Seedling in D1, although on 3 kinds of root medias of D2 and D4
Alabastrum can be normally formed, bloom, but because the florescence is shorter, be difficult to set seeds.And the T cultivating on D3 root media0In generation, turns
Gene masculine Seedling not only rooting rate height (Fig. 3), transplants and can also successfully bloom to culture matrix (Fig. 4) and set seeds, and obtain
The seed obtaining is through after planting sprouting (Fig. 5), and growth cycle foreshortens to 2.5 months.The studies above shows, the present invention passes through excellent
Change prescription of rooting medium, transgenic positive plant can be induced to take root, receive T0After seed for transgenic positive plant, can cross and obtain
The short cycle tobacco lines of heredity must be stablized, the method is that the genetic breeding of Nicotiana tabacum L. provides the foundation.
In above-described embodiment, MS culture medium is the training that Murashige and Skoog in 1962 is culture tobacco cell designs
Foster base, is the conventional culture medium in plant tissue culture；1/2MS culture medium is the culture that a great number of elements halves in MS culture medium
Base.Root media D4, be inventor be directed to Nicotiana tabacum L. turn rush early blossoming gene FLOWERING LOCUS T (FT) optimize acquisition afterwards
Culture medium prescription.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and various changes are made to it, without departing from claims of the present invention limited range in details.
Claims (5)
1. promote to turn Nicotiana tabacum L. rush early blossoming geneNtFT5The root media of Nicotiana tabacum L. turns Nicotiana tabacum L. in induction and promotees early blossoming geneNtFT5Cigarette
Grass take root in application it is characterised in that：Described root media is NAA, pH containing 0.1 mg/L for 5.6 ~ 5.8 1/2
MS solid medium, the MS culture medium that described 1/2 MS halves for a great number of elements.
2. according to claim 1 application it is characterised in that：Described root media is 1/2 MS+0.1 mg/L
NAA+30g/L sucrose+7.5 g/L agar, adjusts pH to 5.8.
3. turn Nicotiana tabacum L. using promotion and promote early blossoming geneNtFT5The method that the root media of Nicotiana tabacum L. obtains short cycle Nicotiana tabacum L., it is special
Levy and be, comprise the steps：
A. with SEQ ID NO.1 and sequence shown in SEQ ID NO.2 as primer, Nicotiana tabacum L. cDNA enters performing PCR amplification for template, obtains
Nicotiana tabacum L. promotees early blossoming geneNtFT5, the Nicotiana tabacum L. of amplification acquisition is promoted early blossoming geneNtFT5It is connected into pCXSN carrierXcm Enzyme action position
At point, obtain NtFT5-pCXSN recombiant plasmid；By gained NtFT5-pCXSN recombinant plasmid transformed Agrobacterium, NtFT5- must be contained
The engineering bacteria of pCXSN recombiant plasmid；
B. step A gained engineering bacteria is adopted leaf disk method transformation of tobacco, containing 1 mg/L 6-BA, 0.1 mg/L NAA, 30g/L
Sucrose, 8 g/L agar, pH be 5.8 MS culture medium on co-culture 3 days, then in 1 mg/L 6-BA, 0.1 mg/L NAA,
250 mg/L Carbenicillins, 20 mg/L hygromycin, 30g/L sucrose, 8 g/L agar, pH be 5.8 MS culture medium on lure
Impedance Seedling, then utilizes PCR to screen the plant containing hygromycin gene, obtains T0 generation turnNtFT5Genetic tobacco；
C. in step B gained T0 generation, is turnedNtFT5Genetic tobacco root culture on root media, transplants after taking root and plants to peat
Cultivate to blooming, setting seeds in training substrate, after the seed of acquisition is sprouted, obtain short cycle Nicotiana tabacum L.；
Described root media is NAA, pH containing 0.1 mg/L 1/2 MS solid medium for 5.6 ~ 5.8, described 1/2
The MS culture medium that MS halves for a great number of elements.
4. method according to claim 3 it is characterised in that：In step A, the condition of described PCR amplification is 95 DEG C of pre- changes
Property 3 min；94 DEG C of degeneration 30 s, 54.0 DEG C of annealing 45 s, 72 DEG C of extension 40 s, circulate 35 times；Extend 10 after last 72 DEG C
min.
5. method according to claim 3 it is characterised in that：In step A, described Agrobacterium is LBA4404.
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