CN104086637A - Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof - Google Patents
Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof Download PDFInfo
- Publication number
- CN104086637A CN104086637A CN201410316861.0A CN201410316861A CN104086637A CN 104086637 A CN104086637 A CN 104086637A CN 201410316861 A CN201410316861 A CN 201410316861A CN 104086637 A CN104086637 A CN 104086637A
- Authority
- CN
- China
- Prior art keywords
- tobacco
- sequence
- seq
- ntpdr6
- expression vector
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention discloses tobacco strigolactones transport protein NtPDR6 and an interference expression vector and application thereof. The tobacco strigolactones transport protein NtPDR6 has an amino acid sequence shown in SEQ ID NO.4 or obtained through substituting, deleting or adding at least one amino acid on SEQ ID NO.4; and a nucleotide sequence for coding the protein is shown in SEQ ID NO.3 or is obtained through substituting, deleting or adding at least one nucleotide on SEQ ID NO.3. The protein has the effect of inhibiting lateral branch, axillary bud or lateral bud proliferation, the lateral branch growth of tobacco subjected to interference expression is obviously active, axillary bud or lateral bud proliferation is obvious, and the protein has significance for controlling the plant type of tobacco and improving the yield of tobacco.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to ester transfer protein NtPDR6 in tobacco witchweed, also relate to the gene of ester transfer protein NtPDR6 in encoding nicotiana witchweed and disturb carrier and the application of this genetic expression.
Background technology
Plant hormone is the secondary metabolites that is subject to the adjustable plant physiology reaction of a class of specific environment signal induction generation, and they play important regulating and controlling effect in the growth and development processes such as cell fission and elongation, tissue and Organ Differentiation, Florescence and seed set, maturation and aging, dormancy and sprouting.Common plant hormone has growth hormone (auxin), Plant hormones regulators,gibberellins ((gibberellin, GA), phytokinin (cytokinin, CTK), dormin (abscisic acid, ABA), ethene (ethyne, ETH), Whitfield's ointment (salicylic acid, SA), jasmonic (jasmonic acid, JA) and rape element sterol (brassinosteroid, BR).Wherein, growth hormone and phytokinin are two kinds of hormones of thinking that traditionally control plant branching is grown.But recent studies have found that, the hormone that also has another can regulating plant branch to grow, i.e. witchweed lactone (strigolactone, SL), belongs to sesquiterpenoids secondary metabolites.Witchweed lactone can suppress the branch of plant and lateral bud growth, and with growth hormone together with phytokinin regulating plant on the ground and the growth of bottom portion maintain plant type.
Natural witchweed lactone compound mainly contains 5 kinds: witchweed alcohol (strigol), 5-deoxidation witchweed alcohol (5-deoxystrigol), broomrape alcohol (orobanchol), Chinese sorghum lactone (sorgolactone) and black capsule alcohol (alectrol).The witchweed lactone of finding is the earliest witchweed alcohol, secretes, to stimulate pernicious Parasitic Weeds witchweed, the sprouting of broomrape seed from non-phytoparasite cotton root system.In corn, Chinese sorghum, tobacco and tomato, be all separated to subsequently witchweed lactone compound.Study and also show, witchweed lactone also can be used as stimulating factor and promotes AM hypha,hyphae branch, sets up the relation of symbiosis with plant.
Witchweed lactone has the function that suppresses plant branching, is found in the earliest the research of the mutant that branch is increased, and causes mutant plant branch increase reason to be the biosynthesis block of natural witchweed lactone.At present, in paddy rice, Arabidopis thaliana, pea, petunia, carried out comparatively deep research about the research of witchweed lactone route of synthesis regulating plant branch.Witchweed lactone is as a kind of novel hormone, and its regulation and control that plant branching is grown are and growth hormone and the interactional process of phytokinin.The induction of the signals such as witchweed lactone is can be hard to bear low-phosphorous at root, growth hormone is synthetic, participates in promoting that, AM mycelia branch, all the other will transport through xylem after the secretion of table section under root hair from bottom to top, participates in the inhibition of overground part side shoot.And growth hormone is after synthesize on the top of plant, to carry and participate in suppressing branch under polarity.And phytokinin to be polarity upwards transport, promote cell fission and participation to organize mitogenetic, be the second hormone of growth hormone regulation and control.Growth hormone can be brought into play the inhibition to branch by the level that promotes the synthetic of witchweed lactone or inhibition phytokinin.Witchweed lactone also can affect by retroactive effect the hormonal readiness of growth hormone.
Witchweed lactone is brought into play its effect at root or overground part, relate to a series of transport processes from root to overground part, and this just needs the participation of translocator.Recently, having found in plant ester transfer protein PhPDR1 in first witchweed in petunia, is abc transport albumin A BCG subfamily member.Tobacco is as important farm crop, and tobacco leaf grows to the later stage, does not shift to reproductive organ for the nutrition in guarantee tobacco leaf, take the operation of " pinching ".But this operation will destroy the apical dominance of plant, cause side shoot mitogenetic obviously, if erase not in time lateral bud, can consume equally nutrition in blade, affect yield of tobacco.The transport molecule mechanism of research tobacco witchweed lactone by the branch of Molecular tools control plant, has important theory and realistic meaning to the study on regulation of tobacco plant type by contributing to.But, in existing tobacco research report, about the transhipment genes involved of tobacco witchweed lactone has no report.
Summary of the invention
In view of this, one of object of the present invention is to provide the ester transfer protein NtPDR6 in witchweed that grows tobacco, and two of object of the present invention is to provide the gene of ester transfer protein NtPDR6 in encoding nicotiana witchweed; Three of object of the present invention is to provide the transgenosis interference expression vector that disturbs ester transfer protein NtPDR6 expression in tobacco witchweed; Four of object of the present invention is to provide the construction process of above-mentioned interference expression vector; Five of object of the present invention is to provide the application of ester transfer protein NtPDR6 in tobacco witchweed; Six of object of the present invention is to provide the application of tobacco witchweed lactone transporter gene NtPDR6; Seven of object of the present invention is to provide the application of transgenosis interference expression vector.
For achieving the above object, the invention provides following technical scheme:
1, ester transfer protein NtPDR6 in tobacco witchweed, its aminoacid sequence as shown in SEQ ID NO.4 or shown in SEQ ID NO.4 aminoacid sequence be substituted, lack or add at least one amino acid and derive from the aminoacid sequence with ester transfer protein function in witchweed of tobacco.
2, the gene of ester transfer protein NtPDR6 in tobacco witchweed described in coding claim 1, its nucleotide sequence as shown in SEQ ID NO.3 or shown in SEQ ID NO.3 nucleotide sequence be substituted, lack or add at least one Nucleotide and derive from the nucleotide sequence with ester transfer protein function in witchweed of tobacco.
3, disturb the transgenosis interference expression vector that in described tobacco witchweed, ester transfer protein NtPDR6 expresses.
Preferably, described interference expression vector is to contain to disturb hairpin structure that shown in SEQ ID NO.3, sequence is expressed to be connected into pART27 plant expression vector through Not I restriction enzyme site to obtain.
Preferred, described hairpin structure contains just like forward sequence and the reverse sequence shown in SEQ ID NO.9.
4, the construction process of described interference expression vector, comprise the steps: taking sequence shown in SEQ ID NO.10 and SEQ ID NO.11 as primer, the root tissue cDNA of tobacco is that template is carried out pcr amplification acquisition forward sequence, forward sequence is connected into pHANNIBAL carrier by XhoI and KpnI restriction enzyme site, obtains the pHANNIBAL carrier that contains forward sequence; Then taking sequence shown in SEQ ID NO.12 and SEQ ID NO.13 as primer, the root tissue cDNA of tobacco is that template is carried out pcr amplification acquisition reverse sequence, the reverse sequence of acquisition is connected into the pHANNIBAL carrier that contains forward sequence by XbaI and HindIII, the pHANNIBAL carrier that acquisition contains forward sequence and reverse sequence, recycling Not I restriction enzyme site cuts hairpin structure sequence and is connected into through Not I enzyme from the pHANNIBAL carrier that contains forward sequence and reverse sequence and cuts, dephosphorylized pART27 plant expression vector, obtains interference expression vector.
5, the application of ester transfer protein NtPDR6 in inhibition tobacco side shoot, axillalry bud or lateral bud increase in described tobacco witchweed.
6, the application of the gene of ester transfer protein NtPDR6 in inhibition tobacco side shoot, axillalry bud or lateral bud increase in described encoding nicotiana witchweed.
7, described interference expression vector is in the application promoting in tobacco side shoot, axillalry bud or lateral bud growth.
Beneficial effect of the present invention is: the present invention utilizes the primers sequence of ester transfer protein PhPDR1 in petunia witchweed, obtain the nucleotide sequence of tobacco witchweed lactone transporter gene through amplification, thereby obtain the aminoacid sequence of this genes encoding, this gene can improve the expression amount of NtPDR6 gene under scarce phosphorus, MeJA and NAA induction, but is subject to the impact of NaCl and ABA less; The invention also discloses the interference expression vector that disturbs NtPDR6 genetic expression, utilize the transgenic positive strain NtPDR6 gene expression amount obtaining after interference expression vector transformation of tobacco to reduce, from plant type transgenic positive strain branch showed increased, collateral generation is active, stem's axillalry bud and lateral bud hyperplasia are obvious, therefore can utilize this gene to suppress tobacco branch, suppress axillalry bud and lateral bud hyperplasia, contrary also can passing through disturbs the expression of this gene to promote tobacco branch, promote axillalry bud and lateral bud hyperplasia, the regulation and control of tobacco plant type are had great importance.
Brief description of the drawings
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing:
Fig. 1 is that in abduction delivering feature, ("+P " represents to cultivate in plant interpolation phosphoric nutritive medium NtPDR6 gene, and " P " represents to cultivate in scarce phosphorus nutrition liquid, " H
2o ", " ABA ", " MeJA ", " NAA " and " NaCl " be illustrated respectively in and in nutritive medium, add H
2lower cultivation processed in O, ABA, MeJA, NAA and NaCl induction).
Fig. 2 is that (35Sp is expressed as 35S promoter to NtPDR6 (RNAi)-pART27 transgenosis interference carrier schematic diagram, NtPDR6S is NtPDR6 forward fragment, NtPDR6R is the reverse fragment of NtPDR6, and intron is intron fragment, and OCS terminator is OCS termination signal; NPT II neomycin phosphotransferase gene, L and R represent respectively the border, left and right of T-DNA transgene carrier).
Fig. 3 is T
0for the qualification result of transgenic line, (" WT " represents wild-type tobacco, and 1,2,3,4,5 is the T that obtain after leaf dish transforms
0for transgenic line; A figure is the pcr amplification result of NptII gene in plant genome; B figure is the pcr amplification result of CaMV35S promotor in plant genome).
Fig. 4 is the gene expression results (" WT " represents wild-type tobacco, and L2 and L5 represent transgenosis interference positive plant) of transgenic positive seedling NtPDR6.
Fig. 5 is the phenotypic characteristic (A: left is wild-type tobacco, and the right side is transgenic positive tobacco of transgenic positive seedling; B figure shows the mitogenetic side shoot of transgenic positive plant, C represent stem of place near the ground axillalry bud and lateral bud hyperplasia phenotype obvious, the side shoot of arrow instruction hyperplasia).
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, for example, condition described in molecular cloning experiment guide (third edition, the work such as J. Pehanorm Brooker), or the condition of advising according to manufacturer.
Embodiment 1, clone tobacco NtPDR6 gene
According to the sequence of ester transfer protein PhPDR1 in petunia witchweed, the primer of design amplification tobacco witchweed lactone transporter gene (NtPDR6) coding region, concrete primer is as follows:
NtPDR6-fw：5'-atggagggtggtgaagacag-3'(SEQ?ID?NO.1)；
NtPDR6-rev：5'-atggagggtggtgaagacag-3'(SEQ?ID?NO.2)；
Then taking the root tissue cDNA of the large gold dollar of tobacco safflower as template, with SEQ ID NO.1 and SEQ ID NO.2, shown primer pair carries out pcr amplification respectively, and pcr amplification condition is: 94 DEG C of denaturation 4min.94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 240s, totally 30 circulations.72 DEG C are extended 10min, 4 DEG C of preservations eventually again.After the sequence order-checking that amplification is obtained, obtaining sequence as shown in SEQ ID NO.3, contain 4482bp Nucleotide, is a complete open reading frame, and the aminoacid sequence of its coding is as shown in SEQ ID NO.4.
Embodiment 2, the NtPDR6 abduction delivering analysis under hormone and low-phosphorous processing
The tobacco seedling that grows to " grand cross " phase is transferred in deionized water from Nutrition Soil, recovers growth and after 3 days, lack phosphorus, hormone and high salt induction processing.
Lack phosphorus processing: the tobacco seedling that recovers growth is proceeded in the MS substratum of phosphorus element-containing not and cultivate, control group is put into normal MS substratum and cultivated, and gets seedling after two weeks in 1.5mL centrifuge tube, liquid nitrogen flash freezer ,-80 DEG C save backup.
HORMONE TREATMENT: the tobacco seedling that recovers growth is put into respectively to the nutritive medium that has added 50 μ M ABA, 100 μ M MeJA, 30 μ M NAA and cultivate, simultaneously with the nutritive medium of the water gagings such as interpolation in contrast, after processing 24h, get seedling in 1.5mL centrifuge tube, liquid nitrogen flash freezer ,-80 DEG C save backup.
The induction of high salt is processed: the tobacco seedling that recovers growth put into the nutritive medium that has added 20mM NaCl and cultivates, get seedling in 1.5mL centrifuge tube after processing 24h, and liquid nitrogen flash freezer ,-80 DEG C save backup.
Design detects the fluorescent quantitation primer of NtPDR6 gene, be specially: NtPDR6-q-fw:5'-tggaatgaaggacaggaggctaa-3'(SEQ ID NO.5), NtPDR6-q-rev:5'-gtccaaccatcatctcccctgtag-3'(SEQ ID NO.6); Taking tobacco GAPDH (NtGAPDH) gene as internal reference, concrete primer is NtGAPDH-q-fw:5'-tgggtgtcaacgagaaggaa-3'(SEQ ID NO.7 simultaneously); NtGAPDH-q-rev:5'-tctgggtggcagtaaggga-3'(SEQ ID NO.8).Then the material of preservation is used for extracting RNA, carries out reverse transcription and become cDNA, then above-mentioned fluorescent quantitation carries out fluorescent quantitation detection, and quantitative fluorescent PCR condition is 95 DEG C of denaturation 10s, 95 DEG C of sex change 5s, and 60 DEG C of annealing 30s, 39 circulations, then add up C
tvalue.By C
tvalue is by 2
-△ △ Ctcalculate NtPDR6 gene expression amount, result as shown in Figure 1.As shown in Figure 1, after scarce phosphorus, MeJA and NAA induction are processed, NtPDR6 expression amount increases substantially, and NtPDR6 expression amount changes little after NaCl and ABA induction processing.Show the expression amount that can improve NtPDR6 gene under phosphorus, MeJA and NAA induction lacking.
Embodiment 3, NtPDR6 gene interference carrier build
Be the primer (comprising forward fragment primer and reverse fragment primer) that 315bp blocks fragment (SEQ ID NO.9) according to the NtPDR6 gene order obtaining in N end design pair for amplification length, and design respectively restriction enzyme site according to the restriction enzyme site of pHANNIBAL intermediate carrier at the upstream and downstream of primer, and with the direction of insertion of restriction enzyme site control sequence, thereby obtain the interference sequence of RNAi interference carrier.
Concrete primer sequence is as follows:
Forward fragment primer:
NtPDR6-if-fw:5'-ccg
ctcgagtaactaaacttgatttggtggaaag-3'(SEQ ID NO.10), underscore represents XhoI restriction enzyme site;
NtPDR6-if-rew:5'-cgg
ggtaccctggtttgatgattccactgactt-3'(SEQ ID NO.11), underscore represents KpnI restriction enzyme site;
Oppositely fragment primer:
NtPDR6-ir-fw:5'-tgc
tctagataactaaacttgatttggtggaaag-3'(SEQ ID NO.12), underscore represents XbaI enzyme cutting site;
NtPDR6-ir-rew:5'-ccc
aagcttctggtttgatgattccactgactt-3'(SEQ ID NO.13), underscore represents HindIII restriction enzyme site;
Then respectively with SEQ ID NO.10 and SEQ ID NO.11, the sequence of SEQ ID NO.12 and SEQ ID NO.13 is primer, and in embodiment 1, synthetic cDNA is that template is carried out pcr amplification, and pcr amplification condition is: 94 DEG C of denaturation 4min.94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 240s, totally 30 circulations.72 DEG C are extended 10min eventually again, 4 DEG C of preservations, and amplified production is connected into respectively cloning vector pEASY-T1 after sepharose purifying, obtains respectively the pEASY-T1 carrier that contains forward fragment and the pEASY-T1 carrier that contains reverse fragment.After the pEASY-T1 carrier that contains forward fragment is cut by XhoI and KpnI enzyme, be connected in the pHANNIBAL carrier of cutting through same enzyme, must contain the pHANNIBAL carrier of forward fragment; Again by the pEASY-T1 carrier that contains reverse fragment by XbaI and HindIII double digestion, and be connected in the pHANNIBAL carrier that contains forward fragment of cutting through same enzyme, form intermediate carrier, called after NtPDR6 (RNAi)-pKANNIBAL.After being cut with Not I enzyme, intermediate carrier NtPDR6 (the RNAi)-pKANNIBAL building is connected in pART27 plant expression vector that cut through Not I enzyme, dephosphorylized, obtain transgenosis interference expression vector, called after NtPDR6 (RNAi)-pART27, its result as shown in Figure 2.
The Agrobacterium-mediated Transformation of embodiment 4, gene interference carrier and the acquisition of transgenic tobacco plant
(1) NtPDR6 (RNAi)-pART27 interference expression vector transforms Agrobacterium
From-80 DEG C of refrigerators, take out Agrobacterium LBA4404 competent cell, in freeze thawing on ice, in the time being about to thaw, add NtPDR6 (RNAi)-pART27 interference expression vector, flick and mix; Mixture is joined in the 2mm electricity revolving cup of precooling, be placed in 5 minutes on ice; Electroporation parameter is adjusted to: voltage 2.5kV; Electric capacity: 25 μ F; Resistance 200 Ω; Paper using is drawn the water droplet on electric revolving cup outer wall, and electric revolving cup is put into electric shock tank, and electric shock 5ms, then adds rapidly the YEB liquid nutrient medium of 800 μ L28 DEG C preheatings, and in 220rpm, 28 DEG C of vibration recoveries 3 hours; After recovery by bacterium liquid centrifugal 1min under 4500rpm condition, abandon the supernatant of half volume, on the YEB solid medium being evenly coated with after Eddy diffusion and contain Rif (100 μ g/mL), Str (50 μ g/mL) and Kan (50 μ g/mL), be inverted for 28 DEG C and cultivate about 2-3 days, until single bacterium colony forms; Picking list bacterium colony, after spreading cultivation, bacterium liquid is identified positive colony by PCR, obtains engineering bacteria.
(2) acquisition of transgene tobacco
Get tobacco aseptic seedling blade, with punch tool, blade be processed into the leaf dish of diameter 0.5cm size, blade back down, preculture 3 days on MS solid medium; Activate engineering bacteria, concrete grammar is simultaneously: the positive single bacterium colony of picking, the YEB substratum incubated overnight that contains Rif (100 μ g/mL), Str (50 μ g/mL) and Kan (50 μ g/mL) at 2mL; Get 1mL bacterium liquid, join 50mL containing in the YEB substratum of identical resistance, 28 DEG C, 200rmp is cultured to OD and reaches 0.6 left and right, then centrifugal 5min under 4000rpm condition, the MS liquid nutrient medium suspension thalline of use 20mL 30 minutes; Then pre-incubated blade is put into suspension bacteria liquid, infect 10 minutes, blot the unnecessary bacterium liquid of blade with aseptic filter paper, dark cultivation 3 days on the solid medium of MS+6-BA (2mg/L)+NAA (0.5mg/L); Then with the sterile water wash leaf dish that contains 400mg/L cephamycin (Cef), aseptic filter paper sucks unnecessary liquid, again leaf dish is forwarded in the MS solid screening culture medium that contains 6-BA (2mg/L), NAA (0.5mg/L), Cef (200mg/L) and Kan (50mg/L) to 28 DEG C of illumination cultivation; In the time that indefinite bud grows to 0.5cm, transfer on the MS solid medium containing Cef (200mg/L) and Kan (50mg/L) and take root, obtain T
0for transgenic line.
After treating that seedling takes root on the resistance culture base that contains kantlex, clip blade, identifies positive transgenosis strain at DNA level.Concrete grammar is that the amplimer of CaMV35S promoter sequence and Npt II gene on design transgenosis interference carrier, is specially:
35S-fw：5'-gaagggtcttgcgaaggata-3'(SEQ?ID?NO.14)；
35S-rev：5'-ttgtaaaacgacggccagtg-3'(SEQ?ID?NO.15)；
Npt?II-fw:5'-ataccgtaaagcacgaggaag-3'(SEQ?ID?NO.16)；
Npt?II-rev:5'-ctgaagcgggaagggact-3'(SEQ?ID?NO.17)；
, then with T
0be that template is carried out pcr amplification for transgenic line DNA, pcr amplification condition is: 94 DEG C of denaturation 4min.94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 45s, totally 25 circulations; 72 DEG C are extended 10min, 4 DEG C of preservations eventually again.Amplified production is through agarose gel electrophoresis, and result as shown in Figure 3.Result shows, No. 2 and No. 5 transgenic lines have goal gene amplification, show No. 2 and No. 5 positive strains of transgenic line, distinguish called after L2 and L5.
Get the RNA that L2 and L5 transgenic positive strain are extracted tobacco root, after the synthetic cDNA of reversion, analyze NtPDR6 gene expression amount by qRT-PCR, extract wild-type tobacco in contrast simultaneously, detection method is identical with embodiment 2, and its detected result as shown in Figure 4.Result demonstration, in L2 and L5 transgenic positive strain, NtPDR6 gene expression amount is starkly lower than the NtPDR6 gene expression amount of wild-type tobacco, shows that in transgenic positive strain, NtPDR6 gene expression amount is obviously lowered.
Observe the phenotypic characteristic of L2 and L5 transgenic positive seedling: respectively wild-type and transgenic positive strain are transplanted to basin, hot-house culture about 5 weeks, then observes the phenotypic characteristic of transgenic positive strain and wild-type tobacco, and result as shown in Figure 5.Result demonstration, transgenic positive seedling is compared with wild-type tobacco, and L5 strain is obvious multi-branched phenotype, and lobe numbers increases.After showing to disturb NtPDR6 genetic expression, the mitogenetic collateral generation of plant enlivens, and stem of place near the ground axillalry bud and lateral bud hyperplasia phenotype are obvious.On the contrary, the expression of NtPDR6 gene has the hyperplasia that suppresses plant axillalry bud and lateral bud, therefore can be by controlling the hyperplasia of NtPDR6 genetic expression control plant axillalry bud and lateral bud.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.
Claims (9)
1. ester transfer protein NtPDR6 in tobacco witchweed, it is characterized in that: aminoacid sequence as shown in SEQ ID NO.4 or shown in SEQ ID NO.4 aminoacid sequence be substituted, lack or add at least one amino acid, and derive from the aminoacid sequence with ester transfer protein function in witchweed of tobacco.
2. the gene of the interior ester transfer protein NtPDR6 of tobacco witchweed described in coding claim 1, it is characterized in that: nucleotide sequence as shown in SEQ ID NO.3 or shown in SEQ ID NO.3 nucleotide sequence be substituted, lack or add at least one Nucleotide, and derive from the nucleotide sequence with ester transfer protein function in witchweed of tobacco.
3. the transgenosis interference expression vector that described in interference claim 1, in tobacco witchweed, ester transfer protein NtPDR6 expresses.
4. interference expression vector according to claim 3, is characterized in that: described interference expression vector is to contain to disturb hairpin structure that shown in SEQ ID NO.3, sequence is expressed to be connected into pART27 plant expression vector through Not I restriction enzyme site to obtain.
5. interference expression vector according to claim 4, is characterized in that: described hairpin structure contains just like forward sequence and the reverse sequence shown in SEQ ID NO.9.
6. the construction process of interference expression vector described in claim 3-5 any one, it is characterized in that, comprise the steps: taking sequence shown in SEQ ID NO.10 and SEQ ID NO.11 as primer, the root tissue cDNA of tobacco is that template is carried out pcr amplification acquisition forward sequence, forward sequence is connected into pHANNIBAL carrier by XhoI and KpnI restriction enzyme site, obtains the pHANNIBAL carrier that contains forward sequence; Then taking sequence shown in SEQ ID NO.12 and SEQ ID NO.13 as primer, the root tissue cDNA of tobacco is that template is carried out pcr amplification acquisition reverse sequence, the reverse sequence of acquisition is connected into the pHANNIBAL carrier that contains forward sequence by XbaI and HindIII, the pHANNIBAL carrier that acquisition contains forward sequence and reverse sequence, recycling Not I restriction enzyme site cuts hairpin structure sequence and is connected into through Not I enzyme from the pHANNIBAL carrier that contains forward sequence and reverse sequence and cuts, dephosphorylized pART27 plant expression vector, obtains interference expression vector.
7. the application of ester transfer protein NtPDR6 in inhibition tobacco side shoot, axillalry bud or lateral bud increase in tobacco witchweed described in claim 1.
8. the application of the gene of ester transfer protein NtPDR6 in inhibition tobacco side shoot, axillalry bud or lateral bud increase in encoding nicotiana witchweed described in claim 2.
9. the application of interference expression vector in promotion tobacco side shoot, axillalry bud or lateral bud increase described in claim 3-5 any one.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410316861.0A CN104086637B (en) | 2014-07-04 | 2014-07-04 | Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410316861.0A CN104086637B (en) | 2014-07-04 | 2014-07-04 | Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104086637A true CN104086637A (en) | 2014-10-08 |
| CN104086637B CN104086637B (en) | 2017-04-19 |
Family
ID=51634421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410316861.0A Active CN104086637B (en) | 2014-07-04 | 2014-07-04 | Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104086637B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017156535A1 (en) * | 2016-03-11 | 2017-09-14 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
| CN108473544A (en) * | 2016-01-15 | 2018-08-31 | 英美烟草(投资)有限公司 | Modify the method laterally sprouted |
| WO2018168015A1 (en) * | 2017-03-16 | 2018-09-20 | 日本たばこ産業株式会社 | Tobacco plant and production method thereof |
| CN109628475A (en) * | 2019-01-22 | 2019-04-16 | 中国农业科学院郑州果树研究所 | Brassinosteroid synthesizes purposes of the gene PaCYP724B1 in regulation plant branching |
| CN110156883A (en) * | 2019-05-17 | 2019-08-23 | 中国烟草总公司郑州烟草研究院 | Tobacco SLs signal transducer NtDAD2 and its encoding gene, recombinant expression carrier, gene editing carrier and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008152091A2 (en) * | 2007-06-15 | 2008-12-18 | Bayer Cropscience Sa | Pesticidal composition comprising a strigolactone derivative and an insecticide compound |
-
2014
- 2014-07-04 CN CN201410316861.0A patent/CN104086637B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008152091A2 (en) * | 2007-06-15 | 2008-12-18 | Bayer Cropscience Sa | Pesticidal composition comprising a strigolactone derivative and an insecticide compound |
Non-Patent Citations (3)
| Title |
|---|
| TOBIAS KRETZSCHMAR: "A petunia ABC protein controls strigolactone-dependentsymbiotic signalling and branching", 《NATURE》 * |
| 牛维环 等: "植物类萜代谢及其在烟草中的研究进展", 《中国烟草科学》 * |
| 谢小东 等: "植物ABC和MATE转运蛋白与次生代谢物跨膜转运", 《植物生理学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108473544A (en) * | 2016-01-15 | 2018-08-31 | 英美烟草(投资)有限公司 | Modify the method laterally sprouted |
| WO2017156535A1 (en) * | 2016-03-11 | 2017-09-14 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
| CN109715810A (en) * | 2016-03-11 | 2019-05-03 | 奥驰亚客户服务有限公司 | For producing the composition and method of tobacco plant and product that cigarette wooden fork reduces or eliminates |
| JP2019512247A (en) * | 2016-03-11 | 2019-05-16 | アルトリア クライアント サーヴィシーズ リミテッド ライアビリティ カンパニー | Compositions and methods for producing tobacco plants and tobacco products with reduced or eliminated suckers |
| JP7015243B2 (en) | 2016-03-11 | 2022-02-02 | アルトリア クライアント サーヴィシーズ リミテッド ライアビリティ カンパニー | Compositions and Methods for Producing Tobacco Plants and Tobacco Products with Reduced or Lost Sucking Branches |
| JP2022058708A (en) * | 2016-03-11 | 2022-04-12 | アルトリア クライアント サーヴィシーズ リミテッド ライアビリティ カンパニー | Compositions and methods for producing tobacco plants and tobacco products having reduced or eliminated suckers |
| US11807860B2 (en) | 2016-03-11 | 2023-11-07 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
| JP7459150B2 (en) | 2016-03-11 | 2024-04-01 | アルトリア クライアント サーヴィシーズ リミテッド ライアビリティ カンパニー | Compositions and methods for producing tobacco plants and tobacco products with reduced or absent suckers |
| WO2018168015A1 (en) * | 2017-03-16 | 2018-09-20 | 日本たばこ産業株式会社 | Tobacco plant and production method thereof |
| CN109628475A (en) * | 2019-01-22 | 2019-04-16 | 中国农业科学院郑州果树研究所 | Brassinosteroid synthesizes purposes of the gene PaCYP724B1 in regulation plant branching |
| CN109628475B (en) * | 2019-01-22 | 2022-06-21 | 中国农业科学院郑州果树研究所 | Application of brassinolide synthetic gene PaCYP724B1 in regulation and control of plant branches |
| CN110156883A (en) * | 2019-05-17 | 2019-08-23 | 中国烟草总公司郑州烟草研究院 | Tobacco SLs signal transducer NtDAD2 and its encoding gene, recombinant expression carrier, gene editing carrier and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104086637B (en) | 2017-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102154289B (en) | Corn drought inducible gene promoters and activity analysis thereof | |
| CN104086637A (en) | Tobacco strigolactones transport protein NtPDR6 and interference expression vector and application thereof | |
| WO2022135246A1 (en) | R gene for controlling matching of soybean-rhizobium, protein and use thereof | |
| Cui et al. | A stable Agrobacterium rhizogenes-mediated transformation of cotton (Gossypium hirsutum L.) and plant regeneration from transformed hairy root via embryogenesis | |
| CN105647940B (en) | The method and its application of OsGRF6 gene raising rice yield | |
| CN107881172A (en) | A kind of adverse circumstance inducible promoter, adverse circumstance inducible promoter plant expression vector and induction target gene expression | |
| CN104004073B (en) | Derive from disease resistance associated protein TaCPK7-R of Semen Tritici aestivi and relevant biological material thereof and application | |
| CN107058324B (en) | Rice root specific expression promoter POsRO4 and corresponding rice cultivation method | |
| CN102174517B (en) | Shepherd's-purse cold-acclimation COR15a gene promoter and application thereof in improving cold resistance of plants | |
| CN113024645B (en) | Application of wheat transcription factor WRKY70 gene in regulation and control of plant growth and development | |
| CN112048507B (en) | Cloning and application of miRNA for enhancing rice blast resistance | |
| CN1884545B (en) | Method for cultivating Atropa belladonna L of high tropane analog alkaloid yield using gene engineering technology and Atropa belladonna L | |
| CN103695422B (en) | Paddy rice root tip specific expression promoter Pro-Os04g24469 and application thereof | |
| CN103031303B (en) | Identification and applications of plant pulvinus specific expression promoter ProCol1 | |
| CN115807010B (en) | Honeysuckle leaf glandular hair-growing gene and application thereof | |
| CN104450708A (en) | Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE | |
| CN113025621B (en) | Application of CIPK14 gene in improving drought resistance of pigeon pea | |
| CN103397048A (en) | Method for cultivation of transgenic wheat resisting take-all and sharp eyespot and related biological materials thereof | |
| WO2022213453A1 (en) | Use of aluminum ion receptor alr1 gene or protein for regulating aluminum resistance of plant | |
| CN110229824B (en) | Salt mustard TsHKT1; cloning and application of 3 promoter | |
| CN110863006B (en) | Method for improving tillering and regeneration of rice | |
| CN110295192B (en) | Bivalent RNAi expression vector for constructing TYLCV and ToCV by Gateway technology and application thereof | |
| KR101825960B1 (en) | Root―specific promoter derived from Oryza sativa and use thereof | |
| CN117683809A (en) | Yield-related protein OsSOE, biological material and application thereof | |
| CN105950620B (en) | It is a kind of it is comprehensive lower cotton lycopene cyclase gene expression vector and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |