CN106434647B - By the promoter and its application of phytohormone Regulation - Google Patents
By the promoter and its application of phytohormone Regulation Download PDFInfo
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Abstract
The invention discloses the promoters and its application by phytohormone Regulation.The promoter provided by the present invention regulated and controled by brassinosteroid, it is following DNA fragmentations a) or b) or c): a) 1557-2266 nucleotide sequences of the 3 ' ends at least containing SEQ ID No.1 in ordered list, and extends since the 1557th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1, obtain any one DNA fragmentation that length is 710 to 2266bp；The DNA molecular has promoter function；B) nucleotide sequence and a) limited has 75% or 75% or more identity, and the DNA fragmentation with promoter function；C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.
Description
Technical field
The present invention relates in field of biotechnology by the promoter of phytohormone Regulation and its application.
Background technique
Promoter is one section of DNA sequence dna that gene can be made to be transcribed, and the RNA of transcription is by further translating and modifying
Form functional protein.Different according to the mode of promoter effect and function, can be roughly divided into 4 classes: constitutive promoter lures
Conductivity type promoter, tissue or stage of development specific promoter and synthesis type promoter.Foreign gene is in tissue or stage of development
Under the regulation of specific promoter, it can be expressed in certain specific histoorgans.
Auxin (auxin) is a kind of endogenous hormones containing unsaturated an aromatic ring and an acetic acid side chain, English
Literary abbreviation IAA.In addition to heteroauxin, chloro- IAA, 5- hydroxyl-IAA of 4-, methyl α-naphthyl acetate (NAA), indolebutyric acid etc. are class auxin.
Poland gardening scholar Xie Liansiji in 1872 studies tip of a root control root elongation zone growth；Later Darwin father and son is to grass
Plumule phototropism is studied.Nineteen twenty-eight Wen Te confirms that the tip of plumule produces certain substance really, can control
Plumule growth.1934, triumphant lattice et al. isolated this substance from some plants and have named it for heteroauxin, thus practise
Often using heteroauxin as the synonym of auxin on used.
Auxin plays an important role during the growth and development of plant, these effects can promote including auxin
The vascular tissues such as cell elongation and stem growth, induced synthesis layer and cell division, xylem and bast in tissue cultures
Differentiation and root generation and differentiation, mediate gravity and light tropic reaction, maintain apical dominance, Delaying Leaf-Senescence and fruit
It is mature, inhibit blade and fruit abscission, induction fruit setting and growth, increase distribution from assimilation activity to auxin source, promote male and female
Feminizing for different strain flower and blooming for bromelia.It is formed in addition, auxin has an effect on the plumular axis in early embryonic development, shadow
It rings former base development and coordinates the form generation of plant.
Brassinosteroid (Brassinolide, BR) is a kind of growth regulatory substance, since being found from 1970s, later
Many decades research shows that brassinosteroid is similar with the steroid hormones of animal, can promote cell extension and point
It splits, regulates and controls the response of the aging of plant, the development of pollen, the maturation of fruit and plant to various environmental signals.Brassinosteroid
Route of synthesis is illustrated with 1990s in arabidopsis and brassinosteroid deficiency, insensitive mutunt in other several crops
Discovery confirm with as other hormones known to us including auxin, the basic element of cell division including gibberellin to plant
The normal growth and development of object plays a key role.
With auxin as the basic element of cell division, BR not only takes part in simple cell elongation, but also regulates and controls complex biological
The proliferation and differentiation of cell during, and then affect the size of cell cycle and separate living tissue.The presence of BR ensure that leaf
The normal development of green body and normally blooming for plant.BR takes part in development and floral organ and the carpel margins point of anther and pollen
The formation of raw tissue.BR can by modification include superoxide dismutase, catalase, glutathione peroxidase,
The antioxidant of the antioxidases such as ascorbate peroxidase enzyme and non-enzymatic, thus facilitate superoxide radical, hydrogen peroxide with
And the removing of hydroxyl, reduce injury of the Oxdative stress to plant.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of promoters induced by plant hormone BR and IAA.
In order to solve the above technical problems, present invention firstly provides the DNA molecular of entitled Pbhlh7, DNA molecular tool
There is promoter function, can star destination gene expression.
DNA molecular provided by the present invention is following DNA fragmentations a) or b) or c):
A) 3 ' 1557-2266 nucleotide sequences of the end at least containing SEQ ID No.1 in ordered list, and from SEQ
The 1557th of ID No.1 starts, extends according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1, obtains
Any one DNA fragmentation that length is 710 to 2266bp；The DNA molecular has promoter function；
B) nucleotide sequence and a) limited has 75% or 75% or more identity, and the DNA with promoter function
Segment；
C) under strict conditions with the nucleotide sequence hybridization that a) or b) limits, and the DNA fragmentation with promoter function.
Wherein, SEQ ID No.1 is made of 2266 nucleotide.
In above-mentioned DNA molecular, the stringent condition is hybridized simultaneously at 68 DEG C in 2 × SSC, the solution of 0.1%SDS
It washes film 2 times.In the solution of each 5min 0.5 × SSC, 0.1%SDS, hybridizes at 68 DEG C and wash film 2 times.Each 15min.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90%, 95% or more identity.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated DNA molecular nucleotide sequence of the invention.Those have and separate with the present invention by manually modified
The nucleotide of the DNA molecular nucleotide sequence 75% or higher identity that arrive, as long as maintaining the promoter of expression target gene
Activity is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
Bright promoter nucleotide sequence has 75% or higher or 85% or higher or 90% or higher or 95% or higher same
The nucleotide sequence of property.Identity can with the naked eye or computer software is evaluated.It is two or more using computer software
Identity between sequence can be indicated with percentage (%), can be used to evaluate the identity between correlated series.
In above-mentioned DNA molecular, last nucleotide of the DNA molecular is the 2266th of SEQ ID No.1.
In above-mentioned DNA molecular, the DNA molecular is following A 1)-A3) in any DNA fragmentation:
A1) 783-2266 nucleotides sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list
Column, and to SEQ ID No.1's since the 783rd of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1
5 ' ends extend, and obtain any one DNA fragmentation that length is 1484 to 2266bp；
A2) 458-2266 nucleotides sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list
Column, and to SEQ ID No.1's since the 458th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1
5 ' ends extend, and obtain any one DNA fragmentation that length is 1809 to 2266bp；
A3) 358-2266 nucleotides sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list
Column, and to SEQ ID No.1's since the 358th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1
5 ' ends extend, and obtain any one DNA fragmentation that length is 1909 to 2266bp.
In above-mentioned DNA molecular, the DNA molecular is following 1) -5) in any one:
1) nucleotides sequence is classified as DNA molecular shown in the 358-2266 nucleotide of SEQ ID No.1 in sequence table；
2) nucleotides sequence is classified as DNA molecular shown in the 783-2266 nucleotide of SEQ ID No.1 in sequence table；
3) nucleotides sequence is classified as DNA molecular shown in the 1-2266 nucleotide of SEQ ID No.1 in sequence table；
4) nucleotides sequence is classified as DNA molecular shown in the 1557-2266 nucleotide of SEQ ID No.1 in sequence table；
5) nucleotides sequence is classified as DNA molecular shown in the 458-2266 nucleotide of SEQ ID No.1 in sequence table.
In order to solve the above technical problems, the present invention also provides the biomaterials for containing the DNA molecular.
Biomaterial provided by the present invention containing the DNA molecular is following B1) any one of to B19):
B1) contain the expression cassette of the DNA molecular；
B2) contain the recombinant vector of the DNA molecular；
B3) contain B1) recombinant vector of the expression cassette；
B4) contain the recombinant microorganism of the DNA molecular；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain the transgenic plant cells system of the DNA molecular；
B9) contain B1) the transgenic plant cells system of the expression cassette；
B10) contain B2) the transgenic plant cells system of the recombinant vector；
B11) contain B3) the transgenic plant cells system of the recombinant vector；
B12) contain the Transgenic plant tissue of the DNA molecular；
B13) contain B1) Transgenic plant tissue of the expression cassette；
B14) contain B2) Transgenic plant tissue of the recombinant vector；
B15) contain B3) Transgenic plant tissue of the recombinant vector；
B16) contain the genetically modified plants organ of the DNA molecular；
B17) contain B1) the genetically modified plants organ of the expression cassette；
B18) contain B2) the genetically modified plants organ of the recombinant vector；
B19) contain B3) the genetically modified plants organ of the recombinant vector.
In above-mentioned biomaterial, the expression cassette can be by the DNA molecular, the purpose base of DNA molecular starting expression
Cause and transcription terminator composition；The DNA molecular is connect with functional way with the target gene, and the purpose
Gene is connect with the transcription terminator.In one embodiment of the invention, the target gene is specially β-glucose
Thuja acid enzyme (GUS) gene.In another embodiment of the present invention, the target gene is specially Green Fluorescent Protein (GFP)
Gene.
In the recombinant vector, by the expression of DNA molecular starting target gene.
In one embodiment of the invention, the recombinant vector is specially the multiple cloning sites insertion in pIG46 carrier
The recombinant plasmid pIG46-Pbhlh7-Intron-GUS that the promoter obtains.The multiple cloning sites are specially in restricted
Enzyme cutting recognition site ClaI and PstI.The target gene is specially beta-glucuronidase (GUS) gene.
In another embodiment of the present invention, the recombinant vector is specially by pIG46-Pbhlh7-Intron-GUS
Described in promoter starting target gene replace with the recombinant plasmid pIG46- that Green Fluorescent Protein (GFP) gene obtains
Pbhlh7-GFP。
Above-mentioned expression cassette or recombinant vector can be by Agrobacterium_mediated methods, particle bombardment, pollen tube passage method, micro-
Conventional biology methods transformed animal organ or tissue or the cells such as injection method, electroporation and microbeam laser boring method, obtain
Transgenetic animal cell or tissue or organ.
In order to solve the above technical problems, the application the present invention also provides the DNA molecular as promoter.
In order to solve the above technical problems, the present invention also provides the reagent sets of following P1 or P2:
The reagent set of P1, regulating plant growth are made of the DNA molecular with plant hormone；
The reagent set of P2, regulating plant growth are made of the biomaterial and the plant hormone.
In above-mentioned reagent set, the plant hormone concretely brassinosteroid or IAA.The brassinosteroid can be
Brassinosteroid solution or brassinosteroid aqueous solution.The brassinosteroid solution is made of solute and solvent, described molten
Matter is brassinosteroid, and the solvent is W5 solution, and the concentration of brassinosteroid can be 0.1- in the brassinosteroid solution
10 μM, such as 1 μM and 0.5 μM.The W5 solution can be the solution being made of solute and solvent, and the solvent is water, the solute
And its concentration is respectively 0.154mol/L NaCl, 0.125mol/L CaCl2, 5mmol/L KCl and 2mmol/L 2- (N- morphine
Quinoline) ethanesulfonic acid (MES).
The brassinosteroid aqueous solution is the solution that brassinosteroid is added into water and obtains, the brassinosteroid water
The concentration of brassinosteroid can be 0.1-100 μM, such as 1 μM and 10 μM in solution.
The IAA can be IAA solution or IAA aqueous solution.The IAA solution is made of solute and solvent, the solute
For IAA, the solvent is W5 solution, and the concentration of oil IAA can be 0.1-10 μM, such as 0.5 μM, 1 μM and 5 μM in the IAA solution.
In above-mentioned reagent set, the DNA molecular can be the A3).The DNA molecular concretely it is described 1).
In order to solve the above technical problems, the present invention also provides following any applications:
M1, the DNA molecular start the application in destination gene expression in plant；
M2, the biomaterial start the application in destination gene expression in plant；
M3, the reagent set start the application in destination gene expression in plant.
In order to solve the above technical problems, the present invention also provides following any applications:
N1, the DNA molecular are cultivating the application in genetically modified plants；
N2, the biomaterial are cultivating the application in genetically modified plants；
N3, the reagent set are cultivating the application in genetically modified plants.
In the present invention, the plant can be terrestrial plant.The terrestrial plant concretely monocotyledon.The list
Leaf plant can be corn.The corn can be corn inbred line neat 319.
It is demonstrated experimentally that DNA molecular of the invention --- Pbhlh7 has promoter activity, can star the table of target gene
Reach: Pbhlh7 can star the expression of target gene GUS, contain pIG46-Pbhlh7-Intron-GUS (pIG46-Pbhlh7-
In Intron-GUS, Pbhlh7 is connected with target gene GUS) protoplast in gus protein opposite enzyme activity be containing
53.6 times of the opposite enzyme activity of gus protein in the protoplast of pIG46 zero load carrier；Pbhlh7 can also start target gene
The expression of GFP.
It is demonstrated experimentally that DNA molecular of the invention --- the promoter activity of Pbhlh7 by plant hormone induction, and
The promoter activity of Pbhlh7 changes with the variation of phytohormone concentration and induction time: 0.1 μM, 0.5 μM, 1 μM, 5 μM and
The enzyme activity of gus protein is respectively 0 μM of BR in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 10 μM of BR processing
5.36,6.81,3.69,0.93 and 1.28 times of the protoplast containing pIG46-Pbhlh7-GUS of processing；At 0.5 μM of BR
Reason 0 hour, 0.25 hour, 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours contains pIG46-Pbhlh7-Intron-
The enzyme activity of gus protein is 0 μM of BR each respective handling time to contain pIG46-Pbhlh7- respectively in the protoplast of GUS
1.03,3.34,4.51,2.80,1.47,1.32,0.76 times of the enzyme activity of gus protein in the protoplast of Intron-GUS.?
In stable conversion system, the GUS enzyme activity of the transgenic plant of 0.1 μM, 1 μM, 10 μM and 100 μM of BR processing is respectively 0 μM of BR
5.68,3.31,3.40 and 1.02 times of GUS enzyme activity in the transgenic plant of processing；0.1 μM of BR handle 0 hour, it is 0.25 small
When, 0.5 hour, 1 hour, 3 hours, the GUS enzyme activity in the transgenic plants of 6 hours and 12 hours be 0 μM of BR each corresponding respectively
Handle 1.04,1.26,1.70,5.03,3.28,1.14,0.86 times of the GUS enzyme activity in the transgenic plant of time.0.1μM,
Gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM, 1 μM, 5 μM and 10 μM of IAA processing
Opposite enzyme activity is respectively the enzyme of gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of IAA processing
Living 1.66,1.47,1.89,1.48,1.16 times.1 μM of IAA handle 0 hour, 0.1 hour, 0.25 hour, 0.5 hour, it is 1 small
When, 3 hours, in the protoplasts containing pIG46-Pbhlh7-Intron-GUS of 6 hours and 12 hours gus protein enzyme activity
It is gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of IAA each respective handling time respectively
0.96,1.61,1.75,1.42,1.75,1.55,1.32,0.93 times of enzyme activity.
It is demonstrated experimentally that DNA molecular of the invention --- the different truncated segments of Pbhlh7 also have promoter activity, and
The promoter activity of part truncated segment is also by the induction of plant hormone: what 0 μM of BR was handled contains pIG46-Pbhlh7-
Intron-GUS, pIG46-Pbhlh7-1484-Intron-GUS, pIG46-Pbhlh7-710-Intron-GUS and pIG46-
The enzyme activity of gus protein is respectively the unloaded containing pIG46 of 0 μM of BR processing in the protoplast of Pbhlh7-74-Intron-GUS
148.5,165.86,134.9,1.34 times of the enzyme activity of gus protein, illustrate the truncated segment of Pbhlh7 in the protoplast of body
(Pbhlh7-1484 and Pbhlh7-710) all has promoter activity.What 0.5 μM of BR was handled contains pIG46-Pbhlh7-
Intron-GUS、pIG46-Pbhlh7-1909-Intron-GUS、pIG46-Pbhlh7-1809-Intron-GUS、pIG46-
The enzyme activity of gus protein in the protoplast of Pbhlh7-1484-Intron-GUS and pIG46-Pbhlh7-710-Intron-GUS
5.96 containing the GUS enzyme activity in each corresponding recombinant vector protoplast of respectively 0 μM of BR processing, 5.20,1.21,1.41,
1.01 times, illustrate induction of the promoter activity by BR of the truncated segment (Pbhlh7-1909) of Pbhlh7.In stable conversion system
In, the GUS enzyme activity of the transgenic plant of 1 μM of IAA processing is 1.76 of the GUS enzyme activity in the transgenic plant of 0 μM of IAA processing
Times.
It is demonstrated experimentally that DNA molecular of the invention --- Pbhlh7 and its different segments have promoter activity, and this
The induction of the DNA molecular of invention --- Pbhlh7 and its Partial Fragment by plant hormone can use DNA of the invention points
Son --- Pbhlh7 and its Partial Fragment cultivate genetically modified plants, such as being induced by plant hormone (such as brassinosteroid or IAA)
Genetically modified plants.
Detailed description of the invention
Fig. 1 is the building process of recombinant vector pIG46-Pbhlh7-Intron-GUS.
Fig. 2 is the building process of recombinant vector pIG46-Pbhlh7-GFP.
Fig. 3 is the functional verification result of Pbhlh7.Wherein, A is to contain pIG46-Pbhlh7-Intron-GUS after cultivating
With the opposite enzyme activity of gus protein in the protoplast of pIG46, Pbhlh7:GUS indicates to contain pIG46-Pbhlh7- after cultivating
The protoplast of Intron-GUS, pIG46 indicate the protoplast containing pIG46 after culture；B is containing after cultivating
GFP expression in the protoplast of pIG46-Pbhlh7-GFP and pIG46, a are to contain pIG46-Pbhlh7- after cultivating
The protoplast Green fluorescin luminous situation of GFP, b be white light under culture after contain pIG46-Pbhlh7-GFP's
Protoplast, c be culture after the protoplast Chloroplast containing pIG46-Pbhlh7-GFP autofluorescence, d a, b and
The superposition of c.
Fig. 4 is brassinosteroid in instantaneous conversion system to the inducing action of Pbhlh7 promoter activity.Wherein, A is not
The opposite enzyme activity of gus protein, NT indicate 0 μM in the protoplast containing pIG46-Pbhlh7-GUS handled with the BR of concentration
The opposite enzyme activity of gus protein in the protoplast containing pIG46-Pbhlh7-GUS of BR processing；B is the processing of brassinosteroid
Influence of the time to Pbhlh7 promoter activity.
Fig. 5 is brassinosteroid in stable conversion system to the inducing action of Pbhlh7 promoter activity.Wherein, A is not
The enzyme activity of gus protein, NT indicate the transgenic plant gus protein of 0 μM of BR processing in the transgenic plant handled with the BR of concentration
Enzyme activity；B is influence of the processing time of brassinosteroid to Pbhlh7 promoter activity.
Fig. 6 is the promoter activity and brassinosteroid pair of the different truncated segments of Pbhlh7 in instantaneous conversion system
The inducing action of the different truncated segment promoter activities of Pbhlh7.Wherein, A is the promoter of the different truncated segments of Pbhlh7
Activity, NT indicate the opposite enzyme activity of gus protein in the protoplast containing pIG46 of 0.5 μM of BR processing；B is brassinosteroid
To the inducing action of the different truncated segment promoter activities of Pbhlh7.
Fig. 7 is auxin in instantaneous conversion system to the inducing action of Pbhlh7 promoter activity.Wherein, A is different dense
The opposite enzyme activity of gus protein, NT indicate 0 in the protoplast containing pIG46-Pbhlh7-Intron-GUS of the IAA processing of degree
The opposite enzyme activity of gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of μM BR processing；B is IAA's
Handle influence of the time to Pbhlh7 promoter activity.
Fig. 8 is IAA in stable conversion system to the testing result of Pbhlh7 promoter activity inducing action.Wherein,
Control is the GUS enzyme activity in the transgenic plant of 0 μM of IAA processing.
Fig. 9 is the building process of pCAMBIA3301-Pbhlh7-Intron-GUS.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
(Wang Yanli etc., corn inbred line neat 319 is in Genetic improvement for corn inbred line neat 319 in following embodiments
2013 (8) are communicated using, agricultural science and technology) public can obtain from applicant, and which only attaches most importance to the correlation of duplicate invention
Used in experiment, it not can be used as other purposes and use.
Carrier pIG46 (Ono A et al., The rab16B promoter of rice in following embodiments
contains two distinct abscisic acid responsive elements,Plant physiology,
1996Oct；112 (2): 483-491.) public can obtain from applicant, the biomaterial only attach most importance to duplicate invention correlation it is real
Used in testing, it not can be used as other purposes and use.
Carrier pCAMBIA3301 (Liu et al., Identification and in following embodiments
characterization of promoters specifically and strongly expressed in maize
Embryos, Plant Biotechnology Journal (2014) 12, pp.1286-1296) public can obtain from applicant
, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.
Luciferase Assay System with Reporter Lysis Buffer reagent in following embodiments
Box is Promega Products, article No. E4030.
Enzymolysis liquid in following embodiments is that cellulase (Cellulase R10), macerozyme are added into deionized water
(Macerozyme R10), mannitol, KCl, CaCl2The solution obtained with BSA, in enzymolysis liquid, cellulase (Cellulase
R10), macerozyme (Macerozyme R10), mannitol, KCl, CaCl2Concentration with BSA be respectively 0.15g/10ml,
0.04g/10ml, 0.4mol/L, 2mM, 10mM and 0.01g/10ml.
W5 Solutions Solution in following embodiments is the solution being made of solute and solvent, wherein solvent is water, solute and
Its concentration is respectively 0.154mol/L NaCl, 0.125mol/L CaCl2, 5mmol/L KCl and 2mmol/L 2- (N- morphine
Quinoline) ethanesulfonic acid (MES).
Embodiment 1, the clone of Pbhlh7 promoter and functional verification
1, the building of the clone of Pbhlh7 promoter and recombinant vector
To extract the obtained genomic DNA of neat 319 blade of corn inbred line as template, with primers F: 5 '-ATCGATGTTCGCTTTCATGTAATCGTTGTGC-3 ' (being the identification sequence of ClaI at underscore) and primer R:5 '-CTGCAGTGAAAGAGAGAAAGGC-3 ' (being the identification sequence of PstI at underscore) carries out PCR amplification.The reaction of PCR amplification
System are as follows:
The reaction system PCR amplification program of PCR amplification are as follows: 94 DEG C of 2min；94 DEG C of 20s, 56 DEG C of 20s, 72 DEG C of 70s, 33
Circulation；72℃5min.
Pcr amplification product and carrier pIG46 are subjected to double digestion with ClaI and PstI respectively, then carry out digestion products
Connection, picking monoclonal is sequenced after screening, and will show after being sequenced containing DNA fragmentation shown in SEQ ID No.1 in ordered list
(Pbhlh7) recombinant vector is named as pIG46-Pbhlh7-Intron-GUS, the building of pIG46-Pbhlh7-Intron-GUS
Process is as shown in Figure 1.SEQ ID No.1 is made of 2266 nucleotide, is the nucleotide sequence of promoter Pbhlh7.
DNA fragmentation shown in SEQ ID No.2 and pIG46-Pbhlh7-Intron-GUS are used into PstI and EcoRI respectively
Double digestion is carried out, is then attached digestion products, picking monoclonal is sequenced after screening, and will show after being sequenced containing orderly
(DNA fragmentation shown in SEQ ID No.2 contains the encoding gene and terminator of GFP to DNA fragmentation shown in SEQ ID No.2 in list
Tnos recombinant vector) is named as pIG46-Pbhlh7-GFP, and the building process of pIG46-Pbhlh7-GFP is as shown in Figure 2.
Wherein, 1-6 of SEQ ID No.2 are the identification sequence of PstI, and 987-992 of SEQ ID No.2 are
The identification sequence of EcoRI, 7-723 of SEQ ID No.2 are the encoding gene of GFP, the 734-986 of SEQ ID No.2
Position is the sequence of terminator Tnos.
2, the Function Identification of Pbhlh7 promoter
The rough leaf that the corn inbred line of two leaves wholeheartedly neat 319 is grown under dark condition is chosen, it will be in the true leaf
The part portion 5-6cm is cut into the filament that width is 1mm or so along the direction perpendicular to vein, and filament is placed in 10ml enzymolysis liquid, is kept away
Light digests 5 hours, and enzymolysis process remains 40r/min sustained oscillation, and corn mesophyll protoplast is obtained after enzymatic hydrolysis.
Bibliography (Yoo SD, Cho YH, Sheen J.Arabidopsis mesophyll protoplasts:a
versatile cell system for transient gene expression analysis.Nature
Protocols, 2007,2:1565-1572) PEG in induces mesophyll protoplast transient transformation methods, induces the PEG
Mesophyll protoplast transient transformation methods carry out change below: 1. after washing away enzymolysis liquid, receipts are softly resuspended with W5 solution
The protoplast collected, places 40min on ice, to achieve the purpose that thoroughly to settle protoplast；2. completing protoplast sedimentation
Afterwards, 50g is centrifuged 1min, and excessively high centrifugal force is avoided to cause iuntercellular pressure excessive, reduces the injury to protoplast；3. being added
The PEG-Ga of 220 μ l 30%2+After solution, room temperature induces 10min, respectively by pIG46, the pIG46-Pbhlh7- of step 1
Intron-GUS and pIG46-Pbhlh7-GFP is imported in corn mesophyll protoplast, respectively obtains the plasm containing pIG46
Body, the protoplast containing pIG46-Pbhlh7-Intron-GUS and the protoplast containing pIG46-Pbhlh7-GFP.
By the protoplast containing pIG46, the protoplast containing pIG46-Pbhlh7-Intron-GUS and contain
The protoplast of pIG46-Pbhlh7-GFP is incubated overnight in W5 solution 16 hours under dim light respectively respectively obtain culture after
The protoplast containing pIG46, after the protoplast containing pIG46-Pbhlh7-Intron-GUS after culture and culture
Protoplast containing pIG46-Pbhlh7-GFP.
Measurement of the GUS with respect to enzyme activity: Luciferase Assay System with Reporter Lysis is utilized
Buffer kit measures the GUS and LUC in the protoplast containing pIG46-Pbhlh7-Intron-GUS after culture respectively
Enzyme activity contains pIG46-Pbhlh7-Intron-GUS's after being cultivated using the LUC enzyme activity of constitutive expression as internal reference
The opposite enzyme activity (A in Fig. 3) of gus protein in protoplast.
According to the method described above, the protoplast containing pIG46-Pbhlh7-Intron-GUS after culture is replaced with into training
The protoplast containing pIG46 after supporting, other steps are constant, GUS in the protoplast containing pIG46 after being cultivated
The opposite enzyme activity (A in Fig. 3) of albumen.
The results show that culture after the protoplast containing pIG46-Pbhlh7-Intron-GUS in gus protein it is opposite
Enzyme activity is 53.6 times of opposite enzyme activity of gus protein in the protoplast containing pIG46 after culture, shows Pbhlh7 with opening
Promoter activity can drive the expression of gus gene.
Under laser confocal microscope, containing after the protoplast containing pIG46 and culture after observation culture
The expression of GFP gene in the protoplast of pIG46-Pbhlh7-GFP, as a result as shown by B in fig. 3.The results show that training
Visible green fluorescence in the nucleus of the protoplast containing pIG46-Pbhlh7-GFP, chloroplaset, cytoplasmic matrix after supporting,
Show that Pbhlh7 has promoter activity, the expression of GFP gene can be driven.
Embodiment 2, Pbhlh7 promoter activity by brassinosteroid induction
Experiment is repeated four times, and repeating experiment every time, specific step is as follows:
1, induction of the brassinosteroid to Pbhlh7 promoter activity in instantaneous conversion system
1.1 the brassinosteroid of various concentration is to the inducing action of Pbhlh7 promoter activity
By the protoplast containing pIG46-Pbhlh7-GUS of 1 step 2 of embodiment in 0.5 μM of BR solution under dim light
Culture 1 in (0.5 μM of BR solution is the solution that the BR concentration that addition brassinosteroid (BR) obtains into W5 solution is 0.5 μM)
Hour, obtain the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of BR processing.
According to the method described above, 0 μM, 0.1 μM, 1 μM, 5 μM and 10 μM is replaced with respectively by 0.5 μM, other steps are constant,
Obtain the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of BR processing, 0.1 μM of BR processing contains
The original containing pIG46-Pbhlh7-Intron-GUS that the protoplast of pIG46-Pbhlh7-Intron-GUS, 1 μM of BR are handled
What the protoplast containing pIG46-Pbhlh7-Intron-GUS and 10 μM of BR that raw plastid, 5 μM of BR are handled were handled contains
The protoplast of pIG46-Pbhlh7-Intron-GUS.
Measuring method according to GUS in 1 step 2 of embodiment with respect to enzyme activity measures containing for above-mentioned 0 μM of BR processing respectively
What the protoplast of pIG46-Pbhlh7-Intron-GUS, 0.1 μM of BR were handled contains pIG46-Pbhlh7-Intron-GUS's
Protoplast, the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of BR processing, 1 μM of BR processing contain
The original containing pIG46-Pbhlh7-Intron-GUS that the protoplast of pIG46-Pbhlh7-Intron-GUS, 5 μM of BR are handled
The opposite enzyme activity of gus protein in raw plastid and the protoplast containing pIG46-Pbhlh7-Intron-GUS of 10 μM of BR processing
(A in Fig. 4), by the opposite enzyme of gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of BR processing
Work is defined as 1.
The results show that by gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of BR processing
Opposite enzyme activity be set to 1 (A in Fig. 4), the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.1 μM of BR processing,
What the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of BR processing, 1 μM of BR were handled contains pIG46-
The protoplast containing pIG46-Pbhlh7-Intron-GUS that the protoplast of Pbhlh7-Intron-GUS, 5 μM of BR are handled
The opposite enzyme activity of gus protein is respectively in the protoplast containing pIG46-Pbhlh7-Intron-GUS handled with 10 μM of BR
5.36,6.81,3.69,0.93,1.28 (A in Fig. 4).Show that brassinosteroid can induce the promoter activity of Pbhlh7, and
And the promoter activity of Pbhlh7 changes with the concentration of brassinosteroid, the Pbhlh7 when concentration of brassinosteroid is 0.5 μM
Promoter activity highest.
1.2 brassinosteroids handle the time to the inducing action of Pbhlh7 promoter activity
By the protoplast containing pIG46-Pbhlh7-Intron-GUS of 1 step 2 of embodiment in step under dim light
It is cultivated 1 hour in 1.1 0.5 μM of BR solution, obtains 0.5 μM of BR and handle 1 hour to contain pIG46-Pbhlh7-Intron-
The protoplast of GUS；By 0 μM of BR solution (i.e. W5 solution) as compareing, obtain 0 μM of BR and handle 1 hour to contain pIG46-
The protoplast of Pbhlh7-Intron-GUS.
According to the method described above, 1 hour is replaced with 0 hour, 0.25 hour, 0.5 hour, 3 hours, 6 hours and 12 respectively
Hour, other steps are constant, obtain 0.5 μM with 0 μM of BR processing 0 hour containing pIG46-Pbhlh7-Intron-GUS's
Protoplast, 0.5 μM with 0 μM of BR handle 0.25 hour the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5
μM with 0 μM of BR handle 0.5 hour the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM at 0 μM of BR
3 hours protoplasts containing pIG46-Pbhlh7-Intron-GUS of reason, 0.5 μM contained with 0 μM of BR processing 6 hours
The protoplast of pIG46-Pbhlh7-Intron-GUS, 0.5 μM handled 12 hours with 0 μM of BR and contain pIG46-Pbhlh7-
The protoplast of Intron-GUS.
Measuring method according to GUS in 1 step 2 of embodiment with respect to enzyme activity, measures GUS egg in above-mentioned each protoplast respectively
White opposite enzyme activity (B in Fig. 4).
The results show that 0 μM of BR respectively to be handled to the plasm containing pIG46-Pbhlh7-Intron-GUS of time respectively
The opposite enzyme activity of gus protein is defined as 1,0.5 μM of BR and handles 0 hour to contain pIG46-Pbhlh7-Intron-GUS's in body
Protoplast, 0.5 μM of BR handle 0.25 hour the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM of BR
The protoplast containing pIG46-Pbhlh7-Intron-GUS, the 0.5 μM of BR of processing 0.5 hour, which is handled 1 hour, to be contained
The protoplast of pIG46-Pbhlh7-Intron-GUS, 0.5 μM of BR, which are handled 3 hours, contains pIG46-Pbhlh7-Intron-
The protoplast of GUS, 0.5 μM of BR handle 6 hours the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM
The opposite enzyme activity of gus protein is relative to 0 in the protoplast containing pIG46-Pbhlh7-Intron-GUS of BR processing 12 hours
The opposite enzyme activity of gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of μM BR each respective handling time
Respectively 1.03,3.34,4.51,2.80,1.47,1.32,0.76 (B in Fig. 4).Show that brassinosteroid can induce
The promoter activity of Pbhlh7, and the promoter activity of Pbhlh7 is with the processing time change of brassinosteroid, in rape element
The promoter activity highest of Pbhlh7 when ester is handled 0.5 hour.
2, induction of the brassinosteroid to Pbhlh7 promoter activity in stable conversion system
Pcr amplification product and carrier pCAMBIA3301 that 1 step 1 of embodiment obtains are carried out with EcoRI and NcoI respectively
Then digestion products are attached by double digestion, picking monoclonal is sequenced after screening, and will show to contain ordered list after being sequenced
The recombinant vector of DNA fragmentation (Pbhlh7) shown in middle SEQ ID No.1 is named as pCAMBIA3301-Pbhlh7-Intron-
The building process of GUS, pCAMBIA3301-Pbhlh7-Intron-GUS are as shown in Figure 9.SEQ ID No.1 is by 2266 nucleosides
Acid composition is the nucleotide sequence of promoter Pbhlh7.
According to document (Frame BR et al., Agrobacterium tumefaciens-Medicated
Transformation of Maize Embryos Using a Standard Binary Vector system,Plant
Physiology,2002May；129 (1): 13-22.) in corn genetic transforming method utilize pCAMBIA3301-Pbhlh7-
Intron-GUS maize transformation self-mating system neat 319, obtains transgenic plant.
With identification BAR resistance transgenic plant, the specific method is as follows: take the seedling leaves of transgenic plant in 2ml from
In heart pipe, about 400 μ l deionized waters are added and with Glass rod grinding obtain blade lapping liquid, then (test strips are by test strips
AGDIA Products, article No. STX14200/0012) it is inserted into blade lapping liquid, when 2 purple bands of appearance in test strips
When be positive transgenic plant, be negative transgenic plant, i.e. nontransgenic plants when 1 band.
Inducing action of the brassinosteroid of 2.1 various concentrations to Pbhlh7 promoter activity
Above-mentioned positive transgenic plant, 10 plants of each concentration are handled with the brassinosteroid of various concentration, experiment repeats three
Secondary, repeating experiment every time, specific step is as follows:
With 0.1 μM of the BR solution (preparation method of 0.1 μM of BR solution are as follows: weigh 48 milligrams of brassinosteroid with less
It is settled to 10ml after amount alcohol dissolution, the mother liquor that brassinosteroid concentration is 10mmol/L is obtained, mother liquor is diluted with water
100000 times, obtain 0.1 μM of BR solution) sprinkling two leaves wholeheartedly when positive transgenic plant, sprinkling 1 hour after obtain
The transgenic plant of 0.1 μM of BR processing, utilizes Luciferase Assay System with Reporter Lysis
GUS enzyme activity (A in Fig. 5) in the transgenic plant of 0.1 μM of BR of Buffer kit measurement processing.
According to the method described above, 0 μM, 1 μM, 10 μM and 100 μM is replaced with respectively by 0.1 μM, other steps are constant, respectively
Obtain the GUS enzyme activity (A in Fig. 5) in the transgenic plant of 0 μM, 1 μM, 10 μM and 100 μM of BR processing.
GUS enzyme activity determination in the transgenic plant of above-mentioned 0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM of BR processing is in phase
With what is measured under total protein concentration.Being measured as total protein concentration utilizes BCA determination of protein concentration kit (Beijing Suo Laibaoke
Skill Co., Ltd product, article No. PC0020) carry out.
The results show that by 0 μM of BR processing transgenic plant in GUS enzyme activity be defined as 1,0.1 μM, 1 μM, 10 μM and
The GUS of the transgenic plant of 100 μM of BR processing is respectively 5.68,3.31,3.40 and 1.02 with respect to enzyme activity, shows that BR can be lured
The promoter activity of Pbhlh7 in transgenic corns is led, the promoter activity of Pbhlh7 changes with BR concentration and changed, wherein 0.1
μM induction Pbhlh7 promoter activity highest.
2.2 brassinosteroids handle the time to the inducing action of Pbhlh7 promoter activity
Positive transgenic plant when with two leaf of BR spray solution of 0.1 μM in above-mentioned steps 2.1 wholeheartedly, in sprinkling 1
The transgenic plant that 0.1 μM of BR handles 1 hour is obtained when hour, utilizes Luciferase Assay System with
Reporter Lysis Buffer kit measurement obtains the GUS enzyme activity in the transgenic plant of 0.1 μM of BR processing 1 hour,
Totally 10 repetitions；By 0 μM of BR solution (i.e. water) as control, the GUS in the transgenic plant of 0 μM of BR processing 1 hour is obtained
Enzyme activity, totally 10 repetitions.
According to the method described above, 1 hour is replaced with 0 hour, 0.25 hour, 0.5 hour, 3 hours, 6 hours and 12 respectively
Hour, other steps are constant, respectively obtain GUS enzyme activity in 0.1 μM of transgenic plant with 0 μM of BR processing 0 hour, 0.1
μM handled with 0 μM of BR GUS enzyme activity in 0.25 hour transgenic plant, 0.1 μM turn base within BR processing 0.5 hour with 0 μM
Because of the GUS enzyme activity in plant, the GUS enzyme activity in 0.1 μM of transgenic plant with 0 μM of BR processing 3 hours, 0.1 μM and 0 μM of BR
GUS enzyme activity, 0.1 μM of GUS enzyme handled with 0 μM of BR in 12 hours transgenic plants in the transgenic plant of processing 6 hours
GUS enzyme activity (B in Fig. 6) in transgenic plant living.
What above-mentioned each GUS enzyme activity determination measured under identical total protein concentration.Being measured as total protein concentration utilizes BCA
What determination of protein concentration kit (Beijing Suo Laibao Science and Technology Ltd product, article No. PC0020) carried out.
The results show that GUS enzyme activity in the transgenic plant of 0 μM of BR processing different time is defined as 1,0.1 μM respectively
BR handles 0 hour transgenic plant, 0.1 μM of BR handles 0.25 hour transgenic plant, 0.1 μM of BR is handled 0.5 hour
Transgenic plant, 0.1 μM of BR handles 1 hour transgenic plant, 0.1 μM of BR handles 3 hours transgenic plants, 0.1 μ
The transgenic plant of M BR processing 6 hours, 0.1 μM of BR handle the GUS enzyme activity in 12 hours transgenic plants relative to 0 μM
The opposite enzyme activity of GUS enzyme activity in the transgenic plant of BR each respective handling time is respectively 1.04,1.26,1.70,5.03,
3.28,1.14,0.86 (B in Fig. 5).Show in transgenic corns, brassinosteroid can induce the promoter of Pbhlh7 living
Property, and the promoter activity of Pbhlh7 is with the processing time change of brassinosteroid；In transgenic corns, brassinosteroid
The promoter activity highest of Pbhlh7 when handling 1 hour.
Embodiment 3, Pbhlh7 different truncated segments promoter activity and brassinosteroid to its inducing action
1, the building of the recombinant vector containing Pbhlh7 promoter difference truncation segment
Pbhlh7 is truncated at the 5 ' ends of Pbhlh7,5 DNA fragmentations of acquisition are respectively Pbhlh7-1909 (SEQ
DNA fragmentation shown in 358-2266 of ID No.1), Pbhlh7-1809 is (shown in 458-2266 of SEQ ID No.1
DNA fragmentation), Pbhlh7-1484 (DNA fragmentation shown in 783-2266 of SEQ ID No.1), Pbhlh7-710 (SEQ
DNA fragmentation shown in 1557-2266 of ID No.1), Pbhlh7-74 is (shown in 2195-2266 of SEQ ID No.1
DNA fragmentation).
DNA fragmentation between the ClaI of pIG46-Pbhlh7-Intron-GUS and PstI identification sequence is replaced with into SEQ ID
DNA fragmentation (Pbhlh7-1909) shown in 358-2266 of No.1, other nucleotide are constant, obtain recombinant vector
PIG46-Pbhlh7-1909-Intron-GUS, pIG46-Pbhlh7-1909-Intron-GUS and pIG46-Pbhlh7-
The difference of Intron-GUS is only that: pIG46-Pbhlh7-1909-Intron-GUS is by pIG46-Pbhlh7-Intron-
The recombinant vector that DNA fragmentation shown in 1-357 of SEQ ID No.1 is deleted in GUS.
DNA fragmentation between the ClaI of pIG46-Pbhlh7-Intron-GUS and PstI identification sequence is replaced with into SEQ ID
DNA fragmentation (Pbhlh7-1809) shown in 458-2266 of No.1, other nucleotide are constant, obtain recombinant vector
PIG46-Pbhlh7-1809-Intron-GUS, pIG46-Pbhlh7-1809-Intron-GUS and pIG46-Pbhlh7-
The difference of Intron-GUS is only that: pIG46-Pbhlh7-1809-Intron-GUS is by pIG46-Pbhlh7-Intron-
The recombinant vector that DNA fragmentation shown in 1-457 of SEQ ID No.1 is deleted in GUS.
DNA fragmentation between the ClaI of pIG46-Pbhlh7-Intron-GUS and PstI identification sequence is replaced with into SEQ ID
DNA fragmentation (Pbhlh7-1484) shown in 783-2266 of No.1, other nucleotide are constant, obtain recombinant vector
PIG46-Pbhlh7-1484-Intron-GUS, pIG46-Pbhlh7-1484-Intron-GUS and pIG46-Pbhlh7-
The difference of Intron-GUS is only that: pIG46-Pbhlh7-1484-Intron-GUS is by pIG46-Pbhlh7-Intron-
The recombinant vector that DNA fragmentation shown in 1-782 of SEQ ID No.1 is deleted in GUS.
DNA fragmentation between the ClaI of pIG46-Pbhlh7-Intron-GUS and PstI identification sequence is replaced with into SEQ ID
DNA fragmentation (Pbhlh7-710) shown in 1557-2266 of No.1, other nucleotide are constant, obtain recombinant vector
PIG46-Pbhlh7-710-Intron-GUS, pIG46-Pbhlh7-710-Intron-GUS and pIG46-Pbhlh7-Intron-
The difference of GUS is only that: pIG46-Pbhlh7-710-Intron-GUS is by SEQ in pIG46-Pbhlh7-Intron-GUS
The recombinant vector that DNA fragmentation shown in 1-1556 of ID No.1 is deleted.
DNA fragmentation between the ClaI of pIG46-Pbhlh7-Intron-GUS and PstI identification sequence is replaced with into SEQ ID
DNA fragmentation (Pbhlh7-74) shown in 2195-2266 of No.1, other nucleotide are constant, obtain recombinant vector
PIG46-Pbhlh7-74-Intron-GUS, pIG46-Pbhlh7-74-Intron-GUS and pIG46-Pbhlh7-Intron-
The difference of GUS is only that: pIG46-Pbhlh7-74-Intron-GUS is by SEQ ID in pIG46-Pbhlh7-Intron-GUS
The recombinant vector that DNA fragmentation shown in 1-2194 of No.1 is deleted.
According to the method for 1 step 2 of embodiment, respectively by pIG46, pIG46-Pbhlh7-1909-Intron-GUS,
pIG46-Pbhlh7-1809-Intron-GUS、pIG46-Pbhlh7-1484-Intron-GUS、pIG46-Pbhlh7-710-
Intron-GUS and pIG46-Pbhlh7-74-Intron-GUS is imported in the corn mesophyll protoplast of 1 step 2 of embodiment, point
The protoplast containing pIG46 is not obtained, the protoplast containing pIG46-Pbhlh7-1909-Intron-GUS, is contained
The protoplast of pIG46-Pbhlh7-1809-Intron-GUS contains the primary of pIG46-Pbhlh7-1484-Intron-GUS
Plastid, the protoplast containing pIG46-Pbhlh7-710-GUS and contain the primary of pIG46-Pbhlh7-74-Intron-GUS
Plastid.
2, inducing action of the brassinosteroid to the different truncated segment promoter activities of Pbhlh7
In triplicate, repeating experiment every time, specific step is as follows for experiment:
By the protoplast containing pIG46-Pbhlh7-Intron-GUS of 1 step 2 of embodiment at 0.5 μM under dim light
BR solution (0.5 μM of BR solution is the solution that the BR concentration that addition BR is obtained into W5 solution is 0.5 μM) middle culture 1 hour molten
Liquid obtains the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of BR processing.
According to the method described above, by the protoplast containing pIG46-Pbhlh7-Intron-GUS replace with respectively containing
The protoplast of pIG46, contains pIG46-Pbhlh7- at the protoplast containing pIG46-Pbhlh7-1909-Intron-GUS
The protoplast of 1809-Intron-GUS, contains the protoplast containing pIG46-Pbhlh7-1484-Intron-GUS
The protoplast of pIG46-Pbhlh7-710-Intron-GUS and plasm containing pIG46-Pbhlh7-74-Intron-GUS
Body, other steps are constant, respectively obtain the protoplast containing pIG46 of 0.5 μM of BR processing, 0.5 μM of BR processing contains
There are the protoplast of pIG46-Pbhlh7-1909-Intron-GUS, 0.5 μM of BR processing to contain pIG46-Pbhlh7-1809-
The plasm containing pIG46-Pbhlh7-1484-Intron-GUS that the protoplast of Intron-GUS, 0.5 μM of BR are handled
What the protoplast containing pIG46-Pbhlh7-710-Intron-GUS and 0.5 μM of BR that body, 0.5 μM of BR are handled were handled contains
There is the protoplast of pIG46-Pbhlh7-74-Intron-GUS.
According to the method described above, 0.5 μM of BR solution is replaced with into 0 μM of BR solution, other steps are constant, obtain 0 μM of BR
The protoplast containing pIG46-Pbhlh7-Intron-GUS of processing.
According to the method described above, 0.5 μM of BR solution is replaced with into 0 μM of BR solution, and pIG46-Pbhlh7- will be contained
The protoplast of Intron-GUS replaces with the protoplast containing pIG46 respectively, contains pIG46-Pbhlh7-1909-
The protoplast of Intron-GUS, contains pIG46- at the protoplast containing pIG46-Pbhlh7-1809-Intron-GUS
The protoplast of Pbhlh7-1484-Intron-GUS, the protoplast containing pIG46-Pbhlh7-710-Intron-GUS and
Protoplast containing pIG46-Pbhlh7-74-Intron-GUS, other steps are constant, respectively obtain 0 μM of BR processing
The protoplast containing pIG46-Pbhlh7-1909-Intron-GUS, 0 μ that protoplast containing pIG46,0 μM of BR are handled
What the protoplast containing pIG46-Pbhlh7-1809-Intron-GUS of M BR processing, 0 μM of BR were handled contains pIG46-
What the protoplast of Pbhlh7-1484-Intron-GUS, 0 μM of BR were handled contains pIG46-Pbhlh7-710-Intron-GUS
Protoplast and 0 μM of BR processing the protoplast containing pIG46-Pbhlh7-74-Intron-GUS.
Measuring method according to GUS in 1 step 2 of embodiment with respect to enzyme activity measures containing for 0.5 μM of BR processing respectively
The protoplast containing pIG46, the 0.5 μM of BR that the protoplast of pIG46-Pbhlh7-Intron-GUS, 0.5 μM of BR are handled
What the protoplast containing pIG46-Pbhlh7-1909-Intron-GUS of processing, 0.5 μM of BR were handled contains pIG46-
What the protoplast of Pbhlh7-1809-Intron-GUS, 0.5 μM of BR were handled contains pIG46-Pbhlh7-1484-Intron-
The protoplast of GUS, the protoplast containing pIG46-Pbhlh7-710-Intron-GUS of 0.5 μM of BR processing, 0.5 μM
What the protoplast containing pIG46-Pbhlh7-74-Intron-GUS of BR processing, 0 μM of BR were handled contains pIG46-
The protoplast of Pbhlh7-Intron-GUS, the protoplast containing pIG46 of 0 μM of BR processing, 0 μM of BR processing contain
What the protoplast of pIG46-Pbhlh7-1909-Intron-GUS, 0 μM of BR were handled contains pIG46-Pbhlh7-1809-
The protoplast of Intron-GUS, the protoplast containing pIG46-Pbhlh7-1484-Intron-GUS of 0 μM of BR processing, 0
The protoplast containing pIG46-Pbhlh7-710-Intron-GUS of μM BR processing and 0 μM of BR processing contain pIG46-
The opposite enzyme activity of gus protein in the protoplast of Pbhlh7-74-Intron-GUS.
The promoter activity of the different truncated segments of 2.1Pbhlh7
The opposite enzyme activity of gus protein in the protoplast containing pIG46 of 0 μM of BR processing is defined as 1, as the result is shown
(A in Fig. 6), what the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of BR processing, 0 μM of BR were handled contains
What the protoplast of pIG46-Pbhlh7-1484-Intron-GUS, 0 μM of BR were handled contains pIG46-Pbhlh7-710-
In the protoplast of Intron-GUS and the protoplast containing pIG46-Pbhlh7-74-Intron-GUS of 0 μM of BR processing
The opposite enzyme activity of gus protein is respectively 148.5,165.86,134.9,1.34.Show Pbhlh7, Pbhlh7-1484 and
Pbhlh7-710 all has promoter activity, and Pbhlh7-74 does not have promoter activity.
Inducing action of 2.2 brassinosteroids to the different truncated segment promoter activities of Pbhlh7
The opposite enzyme activity containing GUS in each recombinant vector protoplast by 0 μM of BR processing is defined as 1 respectively, as a result shows
Show (B in Fig. 6), the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of BR processing, 0.5 μM of BR are handled
What the protoplast containing pIG46-Pbhlh7-1909-Intron-GUS, 0.5 μM of BR were handled contains pIG46-Pbhlh7-
The original containing pIG46-Pbhlh7-1484-Intron-GUS that the protoplast of 1809-Intron-GUS, 0.5 μM of BR are handled
Gus protein in the protoplast bhlh7 containing pIG46-Pbhlh7-710-Intron-GUS that raw plastid, 0.5 μM of BR are handled
Opposite enzyme activity divide relative to 0 μM of BR opposite enzyme activity containing the GUS enzyme activity in each corresponding recombinant vector protoplast handled
It Wei 5.96,5.20,1.21,1.41,1.01.Show that brassinosteroid can induce the starting of Pbhlh7 and Pbhlh7-1909
Sub- activity, brassinosteroid not can induce the promoter activity of Pbhlh7-1809, Pbhlh7-1484 and Pbhlh7-710.
Embodiment 4, IAA are to the inducing action of Pbhlh7 promoter activity
In triplicate, repeating experiment every time, specific step is as follows for experiment:
1, IAA can induce the promoter activity of Pbhlh7
By the protoplast containing pIG46-Pbhlh7-Intron-GUS of 1 step 2 of embodiment at 0.5 μM under dim light
Culture in IAA solution (0.5 μM of IAA solution is the solution that the IAA concentration that addition IAA is obtained into W5 Solutions Solution is 0.5 μM)
1 hour solution obtains the protoplast containing pIG46-Pbhlh7-GUS of 0.5 μM of IAA processing.
According to the method described above, 0 μM, 0.1 μM, 1 μM, 5 μM and 10 μM is replaced with respectively by 0.5 μM, other steps are constant,
Obtain the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of IAA processing, 0.1 μM of IAA processing contains
What the protoplast of pIG46-Pbhlh7-Intron-GUS, 1 μM of IAA were handled contains pIG46-Pbhlh7-Intron-GUS's
What the protoplast containing pIG46-Pbhlh7-Intron-GUS and 10 μM of IAA that protoplast, 5 μM of IAA are handled were handled contains
There is the protoplast of pIG46-Pbhlh7-Intron-GUS.
Measuring method according to GUS in 1 step 2 of embodiment with respect to enzyme activity measures containing for 0.5 μM of IAA processing respectively
What the protoplast of pIG46-Pbhlh7-Intron-GUS, 0 μM of IAA were handled contains pIG46-Pbhlh7-Intron-GUS's
Protoplast, the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.1 μM of IAA processing, 1 μM of IAA processing contain
There are the protoplast of pIG46-Pbhlh7-Intron-GUS, 5 μM of IAA processing to contain pIG46-Pbhlh7-Intron-GUS
Protoplast and 10 μM of IAA processing the protoplast containing pIG46-Pbhlh7-Intron-GUS in gus protein phase
To enzyme activity.
By the opposite enzyme of gus protein in the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0 μM of IAA processing
Work is defined as 1, as the result is shown (A in Fig. 7), the plasm containing pIG46-Pbhlh7-Intron-GUS of 0.1 μM of IAA processing
Body, the protoplast containing pIG46-Pbhlh7-Intron-GUS of 0.5 μM of IAA processing, 1 μM of IAA processing contain
What the protoplast of pIG46-Pbhlh7-Intron-GUS, 5 μM of IAA were handled contains pIG46-Pbhlh7-Intron-GUS's
Gus protein is opposite in protoplast and the protoplast containing pIG46-Pbhlh7-Intron-GUS of 10 μM of IAA processing
Enzyme activity is respectively 1.66,1.47,1.89,1.48,1.16.Show that IAA can induce the promoter activity of Pbhlh7, and
The promoter activity of Pbhlh7 changes with the concentration of IAA, the promoter activity highest of Pbhlh7 when the concentration of IAA is 1 μM.
2. auxin handles the time to the inducing action of Pbhlh7 promoter activity
By the protoplast containing pIG46-Pbhlh7-Intron-GUS of 1 step 2 of embodiment in step 1 under dim light
0.5 μM of IAA solution in cultivate 1 hour, obtain 0.5 μM of IAA and handle 1 hour to contain pIG46-Pbhlh7-Intron-
The protoplast of GUS；By 0 μM of IAA solution (i.e. W5 solution) as compareing, obtain 0 μM of IAA and handle 1 hour to contain
The protoplast of pIG46-Pbhlh7-Intron-GUS.
According to the method described above, 0 hour, 0.1 hour, 0.25 hour, 0.5 hour, 3 hours, 6 were replaced with respectively by 1 hour
Hour and 12 hours, other steps are constant, obtain 0.5 μM and contain pIG46-Pbhlh7- with 0 μM of IAA processing 0 hour
The protoplast of Intron-GUS, 0.5 μM handled 0.1 hour with 0 μM of IAA and contain pIG46-Pbhlh7-Intron-GUS's
Protoplast, 0.5 μM with 0 μM of IAA handle 0.25 hour the protoplast containing pIG46-Pbhlh7-Intron-GUS,
0.5 μM of protoplast containing pIG46-Pbhlh7-Intron-GUS that 0.5 hour is handled with 0 μM of IAA, 0.5 μM and 0 μM
IAA handle 3 hours the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM with 0 μM of IAA processing 6 hours
Protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM handled 12 hours with 0 μM of IAA and contain pIG46-
The protoplast of Pbhlh7-Intron-GUS.
Measuring method according to GUS in 1 step 2 of embodiment with respect to enzyme activity, measures GUS egg in above-mentioned each protoplast respectively
White opposite enzyme activity (B in Fig. 7).
The results show that 0 μM of IAA respectively to be handled to the plasm containing pIG46-Pbhlh7-Intron-GUS of time respectively
The opposite enzyme activity of gus protein is defined as 1,0.5 μM of IAA and handles 0 hour to contain pIG46-Pbhlh7-Intron-GUS in body
Protoplast, 0.5 μM of IAA handle 0.1 hour the protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM
IAA handles 0.25 hour protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM of IAA is handled 0.5 hour
Protoplast containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM of IAA, which are handled 1 hour, contains pIG46-Pbhlh7-
The protoplast of Intron-GUS, 0.5 μM of IAA handle 3 hours plasms containing pIG46-Pbhlh7-Intron-GUS
Body, 0.5 μM of IAA handle 6 hours protoplasts containing pIG46-Pbhlh7-Intron-GUS, 0.5 μM of IAA handles 12
The opposite enzyme activity of gus protein is each relative to 0 μM of IAA in the protoplast containing pIG46-Pbhlh7-Intron-GUS of hour
The opposite enzyme activity of gus protein is respectively in the protoplast containing pIG46-Pbhlh7-Intron-GUS of respective handling time
0.96,1.61,1.75,1.42,1.75,1.55,1.32,0.93 (B in Fig. 7).Show that auxin can induce opening for Pbhlh7
Promoter activity, and the promoter activity of Pbhlh7 is with the processing time change of auxin, auxin are handled 0.25 hour and 1 small
When after Pbhlh7 promoter activity highest.
3, IAA is in stable conversion system to the inducing action of Pbhlh7 promoter activity
In triplicate, repeating experiment every time, specific step is as follows for experiment:
With the positive transgenic plant of 2 step 2 of IAA Processing Example of various concentration, 10 plants of each concentration, experiment is repeated
Three times, specific step is as follows for repetition experiment every time:
With 1 μM of the IAA solution (preparation method of 1 μM of IAA solution are as follows: weigh 17.5 milligrams of heteroauxin on a small quantity
It is settled to 10ml after alcohol dissolution, the mother liquor that heteroauxin concentration is 10mmol/L is obtained, which is diluted with water 10000
Times, obtain 1 μM of IAA solution) sprinkling two leaves wholeheartedly when positive transgenic plant, sprinkling 1 hour after obtain 1 μM of IAA
The transgenic plant of processing utilizes Luciferase Assay System with Reporter Lysis Buffer kit
GUS enzyme activity (Fig. 8) in the transgenic plant of the IAA processing of 1 μM of measurement.
According to the method described above, 0 μM is replaced with by 1 μM, other steps are constant, and the transgenosis for obtaining 0 μM of IAA processing is planted
GUS enzyme activity (Fig. 8) in strain.
What above-mentioned each GUS enzyme activity determination measured under identical total protein concentration.Being measured as total protein concentration utilizes BCA
What determination of protein concentration kit (Beijing Suo Laibao Science and Technology Ltd product, article No. PC0020) carried out.
The results show that the GUS enzyme activity in the transgenic plant of 0 μM of IAA processing is defined as turning for 1,1 μM of IAA processing
The GUS of gene plant is 1.76 with respect to enzyme activity, shows that IAA can induce the promoter activity of Pbhlh7 in transgenic corns.
Claims (10)
1.DNA molecule is 1557-2266 nucleotide sequences of the 3 ' ends at least containing SEQ ID No.1 in ordered list, and
Prolong since the 1557th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1
It is long, obtain any one DNA fragmentation that length is 710 to 2266bp；The DNA molecular has promoter function.
2. DNA molecular according to claim 1, it is characterised in that: last nucleotide of the DNA molecular is SEQ
The 2266th of ID No.1.
3. DNA molecular according to claim 1 or 2, it is characterised in that: the DNA molecular is following A 1)-A3) in appoint
A kind of DNA fragmentation:
A1) 783-2266 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list, and
And prolong since the 783rd of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1
It is long, obtain any one DNA fragmentation that length is 1484 to 2266bp；
A2) 458-2266 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list, and
And prolong since the 458th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1
It is long, obtain any one DNA fragmentation that length is 1809 to 2266bp；
A3) 358-2266 nucleotide sequences of the 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list, and
And prolong since the 358th of SEQ ID No.1, according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1
It is long, obtain any one DNA fragmentation that length is 1909 to 2266bp.
4. DNA molecular according to claim 1 to 3, it is characterised in that: the DNA molecular is following 1) -5) in
Any one:
1) nucleotides sequence is classified as DNA molecular shown in the 358-2266 nucleotide of SEQ ID No.1 in sequence table；
2) nucleotides sequence is classified as DNA molecular shown in the 783-2266 nucleotide of SEQ ID No.1 in sequence table；
3) nucleotides sequence is classified as DNA molecular shown in the 1-2266 nucleotide of SEQ ID No.1 in sequence table；
4) nucleotides sequence is classified as DNA molecular shown in the 1557-2266 nucleotide of SEQ ID No.1 in sequence table；
5) nucleotides sequence is classified as DNA molecular shown in the 458-2266 nucleotide of SEQ ID No.1 in sequence table.
5. it is following B1 the biomaterial containing the DNA molecular any in claim 1-4) any one of to B7):
B1 the expression cassette) containing the DNA molecular any in claim 1-4；
B2 the recombinant vector) containing the DNA molecular any in claim 1-4；
B3) contain B1) recombinant vector of the expression cassette；
B4 the recombinant microorganism) containing the DNA molecular any in claim 1-4；
B5) contain B1) recombinant microorganism of the expression cassette；
B6) contain B2) recombinant microorganism of the recombinant vector；
B7) contain B3) recombinant microorganism of the recombinant vector.
6. application of any DNA molecular as promoter in claim 1-4.
7. the reagent set of regulating plant growth, is made of DNA molecular and plant hormone；The DNA molecular is nucleotide sequence
3 ' 358-2266 nucleotide sequences of the end at least containing SEQ ID No.1 in ordered list, and from SEQ ID No.1's
358th starts, extends according to the nucleotide sequence of SEQ ID No.1 to the 5 ' ends of SEQ ID No.1, and obtaining length is 1909
To any one DNA fragmentation of 2266bp.
8. the reagent set of regulating plant growth, the biomaterial described in claim 5 is formed with plant hormone；Claim 5
The DNA molecular in the biomaterial is the of 3 ' ends of nucleotide sequence at least containing SEQ ID No.1 in ordered list
358-2266 nucleotide sequences, and since the 358th of SEQ ID No.1, according to the nucleotides sequence of SEQ ID No.1
It arranges and extends to the 5 ' ends of SEQ ID No.1, obtain any one DNA fragmentation that length is 1909 to 2266bp.
9. following any applications:
Any DNA molecular starts the application in destination gene expression in plant in M1, claim 1-4；
Biomaterial described in M2, claim 5 starts the application in destination gene expression in plant；
M3, claim 7 or 8 reagent set start the application in destination gene expression in plant.
10. following any applications:
Any DNA molecular is cultivating the application in genetically modified plants in N1, claim 1-5；
Biomaterial described in N2, claim 5 is cultivating the application in genetically modified plants；
Reagent set described in N3, claim 8 is cultivating the application in genetically modified plants.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1213405A (en) * | 1996-02-14 | 1999-04-07 | 以色列国农业部 | Method for induction of genetic parthenocarpy in plants |
| CN1310760A (en) * | 1998-05-14 | 2001-08-29 | 德卡尔博遗传学公司 | Methods and compositions for expression of transgenes in plants |
| EP1301609A2 (en) * | 2000-07-13 | 2003-04-16 | Pioneer Hi-Bred International, Inc. | Zmaxig1-specific polynucleotides and methods of use |
| EP1570064A2 (en) * | 2002-11-06 | 2005-09-07 | Pioneer Hi-Bred International Inc. | Auxin-repressed, dormancy-associated promoter and uses thereof |
| CN102757966A (en) * | 2012-07-26 | 2012-10-31 | 中国热带农业科学院橡胶研究所 | Promoter and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7750207B2 (en) * | 2004-09-01 | 2010-07-06 | Monsanto Technology Llc | Zea mays ribulose bisphosphate carboxylase activase promoter |
-
2015
- 2015-08-11 CN CN201510489416.9A patent/CN106434647B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1213405A (en) * | 1996-02-14 | 1999-04-07 | 以色列国农业部 | Method for induction of genetic parthenocarpy in plants |
| CN1310760A (en) * | 1998-05-14 | 2001-08-29 | 德卡尔博遗传学公司 | Methods and compositions for expression of transgenes in plants |
| EP1301609A2 (en) * | 2000-07-13 | 2003-04-16 | Pioneer Hi-Bred International, Inc. | Zmaxig1-specific polynucleotides and methods of use |
| EP1570064A2 (en) * | 2002-11-06 | 2005-09-07 | Pioneer Hi-Bred International Inc. | Auxin-repressed, dormancy-associated promoter and uses thereof |
| CN102757966A (en) * | 2012-07-26 | 2012-10-31 | 中国热带农业科学院橡胶研究所 | Promoter and application thereof |
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