CN104560986A - Soybean-derived root-specific promoter GmPRP2p-852 and application thereof - Google Patents
Soybean-derived root-specific promoter GmPRP2p-852 and application thereof Download PDFInfo
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Abstract
The invention discloses a soybean-derived root-specific promoter GmPRP2p-852 and its application. The promoter provided by the invention is at least one of the following DNA fragments: a) any one DNA fragment which at least contains the 211-1062nt sequences in the Sequence 2 in the sequence table and extends from the 211nt of the Sequence 2 to the 5' end of the Sequence 2 according to nucleotide sequence of the Sequence 2 to the length of 852-1062bp; b) a DNA fragment which is more than 90% homologous with a nucleotide sequence defined by a) and has a promoter function; and c) a DNA fragment which is hybridized with a nucleotide sequence defined by a) or b) under stringent conditions and has a promoter function. The promoter provided by the invention can effectively initiate specific expression of target genes in root. The invention provides a means for molecular improvement of plant's root. The soybean-derived root-specific promoter GmPRP2p-852 is of certain significance for researches on stress resistance of plants and has a wide range of application and market prospect in the field of agriculture.
Description
Technical field
The present invention relates to a kind of the root-specific promoter GmPRP2p-852 and the application thereof that derive from soybean.
Background technology
Promotor is the integral part of gene, and the initial sum that controlling gene is expressed expresses degree.In transgenic breeding, the accuracy controlling of foreign gene in plant materials is mainly realized by suitable promotor.At present, widely used in genetically engineered is constitutive promoter, but be constant and lasting because constitutive promoter orders about the expression of goal gene in plant tissue, be not subject to the restriction of space-time and extraneous factor, intracellular matter and energy can be consumed excessively, produce a large amount of heterologous protein or meta-bolites, destroy the original physiological metabolism balance of plant, cause plant-growth abnormal even dead.Compared with constitutive promoter, its foreign gene of driving of root-specific promoter is expressed in recipient plant root, overcome constitutive promoter owing to continuing, start the non-specific expression of foreign gene efficiently and the consuming excessively of the cellular material that causes.Along with the raising of transgenic plant safety standards, comprise all commercialization transgenic crops, requirement must to the expression activity of promotor used, potential restructuring ability, carry out detailed research to surrounding enviroment and safety effects.
Soybean is that dual-purpose crop raised by a kind of important grain and oil, is mostly in arid, the region such as saline and alkaline and high and cold in China soybean producing region, and the soybean varieties resistance of reverse of application is strong, and the abiotic and biotic stress such as serious drought and waterlogging disease and pest coerces remarkably influenced soybean yields.What use in genetically engineered soybean at present is all CaMV35S promotor, root-specific promoter is applied in soybean transgene, for soybean transgene research provides more abundant promoter element, simultaneously also for cultivating drought-enduring, salt tolerant, the genetically engineered soybean new variety such as cold-resistant provide a kind of means.
Summary of the invention
The object of this invention is to provide a kind of DNA molecular with promoter function.
The DNA molecular with promoter function provided by the present invention, derives from pulse family Glycine the cultivated soybean (Glycine max(L.) Merr.) Williams82, be following a)-c) in arbitrary DNA fragmentation:
A) at least containing the 211-1062 position nucleotide sequence of sequence 2 in ordered list, and extend according to the 5 ' end of the nucleotide sequence of sequence 2 to sequence 2 from the 211st of sequence 2, obtain any one DNA fragmentation that length is 852 to 1062bp;
B) nucleotide sequence and a) limited has more than 90% homology, and has the DNA fragmentation of promoter function;
C) under strict conditions with nucleotide sequence hybridization a) or b) limited, and there is the DNA fragmentation of promoter function.
Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
In the present invention, there is the DNA molecular of promoter function for the DNA molecular shown in the 211-1062 position Nucleotide of sequence in sequence table 2, called after GmPRP2p-852 described in.
Wherein, in sequence table, sequence 2 is made up of 1062 Nucleotide, is the 1-1062 position of sequence 1.
Recombinant vectors containing described DNA molecular (promotor), expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
In the present invention, described recombinant vectors be the multiple clone site (as Sac I and Xba I) of pC13P1 carrier insert containing as described in DNA molecular DNA fragmentation after the recombinant plasmid that obtains.
Further, described recombinant vectors obtains as follows: be connected with pEASY-T1 carrier by described DNA molecular, obtain intermediate carrier; With intermediate carrier described in restriction enzyme Sac I and Xba I double digestion, by also having the endonuclease bamhi of described DNA molecular to be connected with the skeleton fragment of the described pC13P1 carrier of Xba I double digestion through restriction enzyme Sac I with same, obtain described recombinant vectors.
Described pC13P1 carrier is based on pCAMBIA1301 carrier, transform the annular carrier obtained, the sequence not containing any promotor on described pC13P1 carrier, is used for meeting the needs studying promoter function.
Specifically, the sequence of described pC13P1 carrier is as shown in sequence in sequence table 3.
Described expression cassette by the described DNA molecular with promoter function, can be started the goal gene of expressing by described DNA molecular, and transcription termination sequence composition; Described DNA molecular is connected with described goal gene with functional way, and described goal gene is connected with described transcription termination sequence.
In one embodiment of the invention, described goal gene is specially gus gene (deriving from described pC13P1 carrier); Described transcription termination sequence is specially NOS transcription terminator (deriving from described pC13P1 carrier).
The application that described DNA molecular starts in destination gene expression in plant also belongs to protection scope of the present invention.
In the application, be expressed as root-specific described in express.
Further, described root-specific is expressed and be can be high expression level in root, and the expression amount be specially in root is significantly higher than its hetero-organization (as petiole, blade master pulse, flower, fruit pod shell).The expression of trace is only there is in its hetero-organization.
In the application, described goal gene can be gus gene.Described plant can be dicotyledons or monocotyledons.Described dicotyledons specifically can be Arabidopis thaliana or soybean.
In one embodiment of the invention, described plant is specially Arabidopis thaliana (Arabidopsis thaliana (L.) Heynh.) Columbia.
In addition, the primer pair of described DNA molecular (promotor) of increasing also belongs to protection scope of the present invention.
In the present invention, the primer pair of described DNA molecular (promotor) of increasing is as follows:
5'-ATGGAAATTGCAAATG-3'；
5'-GGTTTCTCACGTTGTAGTTG-3'。
Experiment proves, GmPRP2p-852 promotor provided by the present invention, can specific expressed in root of effectively start goal gene.The present invention is that plant provides a kind of means at the molecular improvement of root, just has the certain significance, have wide application space and market outlook at agriculture field to the degeneration-resistant research of plant.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of first round PCR primer.Arrow is depicted as object band.
Fig. 2 is the second agarose gel electrophoresis figure taking turns PCR primer GmPRP2p-1062.Arrow is depicted as object band.
Fig. 3 is the PCR detected result of GmPRP2p-1062 and GmPRP2p-852 promotor.Wherein, swimming lane M is the DNA molecular amount standard of DL5000; Swimming lane 1,2 is respectively the PCR primer of GmPRP2p-1062, GmPRP2p-852.
Fig. 4 is Sac I and the Xba I double digestion electrophorogram of recombinant plasmid pEASY-GmPRP2p-1062, pEASY-GmPRP2p-852 and pC13P1 carrier.Wherein, swimming lane M is the DNA molecular amount standard of DL2000; Swimming lane 1,2,3 is respectively pEASY-GmPRP2p-1062, pEASY-GmPRP2p-852 and pPC13P1.
Fig. 5 is the plasmid map of pC13P1 carrier.
Fig. 6 is pC13(Delta) plasmid map of GUS carrier.
Fig. 7 is transgenic arabidopsis T
1the Molecular Detection result in generation.Wherein, A, B represent respectively and turn pCAM-PRP2p-1062, pCAM-PRP2p-852 Arabidopsis plant detected result.In A and B, swimming lane M is the DNA molecular amount standard of DL2000plus; Swimming lane CK+ is positive control; Swimming lane CK-is negative control; Other swimming lanes are respectively different transgenic line, and what have object band (shown in arrow position) to occur is the positive.
Fig. 8 is transgenic arabidopsis T
3the vegetative growth phase different development stage GUS coloration result in generation.
Fig. 9 is transgenic arabidopsis T
3the generative growth phase different development stage GUS coloration result in generation.
Figure 10 is transgenic arabidopsis T
3gUS active level measurement result in the plant root in generation and leaf.Wherein, WT represents not genetically modified wildtype Arabidopsis thaliana Columbia; P1062-9, P1062-14, P1062-16 be three strains qualifications positive turn pCAM-PRP2p-1062 Arabidopsis plant; P852-5, P852-9, P852-30 be three strains qualifications positive turn pCAM-PRP2p-852 Arabidopsis plant.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Quantitative test in following embodiment, all arranges and repeats experiment for three times, results averaged.Primer synthesis in following embodiment and examining order complete by Hua Da company.
The cultivated soybean (Glycine max (L.) Merr.) Williams82: be recorded in " Xu Shuo. wild soybean salt stress is correlated with the functional analysis .2011 Chinese Academy of Agricultural Sciences Master's thesis of microRNA " literary composition, supplied by Institute of Crop Science, Chinese Academy of Agricultural Science.
PC13P1 carrier and pC13(Delta) GUS carrier: be annular carrier, provided by the Institute of Subtropical Agriculture, The Chinese Academy of Sciences Pedro S.C.F.Rocha researcher.Not containing any promotor on pC13P1 carrier, but containing gus reporter gene (as Fig. 5); PC13(Delta) GUS carrier is not containing gus gene, but containing kalamycin resistance and hygromycin resistance (as Fig. 6).The sequence of described pC13P1 carrier is as shown in sequence in sequence table 3; Described pC13(Delta) sequence of GUS carrier is as shown in sequence in sequence table 4.
Arabidopis thaliana (Arabidopsis thaliana (L.) Heynh.) Columbia: be recorded in " Guo Xiao beautiful .Columbia type Arabidopsis callus culture studies. Henan Agricultural Sciences; 01 phase in 2009 " literary composition, supplied by Institute of Crop Science, Chinese Academy of Agricultural Science.
Agrobacterium tumefaciens GV3101: be recorded in " Zhao Zhan, Li Wensheng. the impact of different Strains on Agrobacterium tumefaciens-mediated Transformation Efficiencyof Trichoderma harzianum. northern gardening, 03 phase in 2006 " literary composition, supplied by Institute of Crop Science, Chinese Academy of Agricultural Science.
The clone and sequence of embodiment 1, GmPRP2p total length promotor
With the cultivated soybean (Glycine max (L.) Merr.) Williams82 for material, CTAB method is adopted to extract soybean root system genomic dna.According to soybean gene group database lookup PRP2 gene and upstream sequence thereof, design special primer upstream primer F1 and downstream primer R1, with the STb gene extracted for template, carries out pcr amplification.
The 1-20 position of F1:5'-ATTTTCCGGACAAACTCTGG-3'(sequence 1);
The reverse complementary sequence of the 1219-1236 position of R1:5'-TGGGGGGTTTTCAACTGG-3'(sequence 1),
Response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 35 circulations; 72 DEG C extend 10min.
After reaction terminates, detect pcr amplification product with 1% agarose gel electrophoresis.
The results are shown in Figure 1, wherein, swimming lane M is the DNA molecular amount standard of DL2000, and swimming lane 1 is PCR primer.PCR primer is reclaimed with recovery kits, PCR primer reclaims rear and pEASY-T1 carrier (Quan Shi King Company, catalog number (Cat.No.): CT101) connect, connect product conversion E.coli competent cell DH5 α (TIANGEN, catalog number (Cat.No.): CB101), extracting plasmid, is that positive recombinant plasmid sends to order-checking through pcr amplification.Sequencing result shows, PCR primer is 1236bp, as shown in sequence 1, comprising 174bp PRP2 gene C DS sequence (the 1063-1236 position of sequence 1).
In order to obtain not containing the promoter fragment of PRP2 gene C DS sequence, from translation initiation codon ATG, newly devise a downstream primer R2 to carry out second and take turns pcr amplification, with first round PCR primer (sequence 1) for template, take turns pcr amplification with F1 and R2 for primer carries out second.
The 1-20 position of F1:5'-ATTTTCCGGACAAACTCTGG-3'(sequence 1);
The reverse complementary sequence of the 1043-1062 position of R2:5'-GGTTTCTCACGTTGTAGTTG-3'(sequence 1).
Response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2min, 35 circulations; 72 DEG C extend 10min.
After reaction terminates, detect pcr amplification product with 1% agarose gel electrophoresis.
The results are shown in Figure 2, wherein, swimming lane M is the DNA molecular amount standard of DL2000, and swimming lane 1 is PCR primer.Reclaim PCR primer with recovery kits, PCR primer is connected with pEASY-T1 carrier after reclaiming, and connects product conversion E.coli competent cell DH5 α, extracts plasmid, is that positive recombinant plasmid sends to order-checking through pcr amplification.Sequencing result obtains the GmPRP2p total length promoter sequence of 1062bp, as shown in sequence 2, and called after GmPRP2p-1062.By recombinant plasmid called after pEASY-GmPRP2p-1062 correct for order-checking.
The structure of embodiment 2, serial recombinant expression vector containing GmPRP2p-1062 promotor and 5 ' deletion fragment
One, the acquisition of soybean GmPRP2p-1062 promotor and sequence 5 ' deletion sequence
The present inventor lacks 5 ' of GmPRP2p-1062 sequence.According to GmPRP2p-1062 sequence (sequence 2), devising 1 upstream primer F-852 respectively, take R2 as downstream primer, and the recombinant plasmid pEASY-GmPRP2p-1062 obtained with embodiment 1, for template, carries out pcr amplification respectively.
Primer sequence is as follows:
The 211-226 position of F-852:5'-ATGGAAATTGCAAATG-3'(sequence 2);
The reverse complementary sequence of the 1043-1062 position of R2:5'-GGTTTCTCACGTTGTAGTTG-3'(sequence 2).
Response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 1min, 35 circulations; 72 DEG C extend 10min.
After reaction terminates, detect pcr amplification product with 1% agarose gel electrophoresis.
Result as shown in Figure 3.Gained PCR primer checked order, result shows: the sequence of primer pair F-852/R2 amplification gained PCR primer is the 211-1062 position (called after GmPRP2p-852) of sequence 2.
Above PCR primer is connected with pEASY-T1 carrier, by gained recombinant plasmid called after pEASY-GmPRP2p-852.
The structure of the serial recombinant expression vector two, containing GmPRP2p-1062 promotor and 5 ' deletion fragment
By the pEASY-GmPRP2p-1062 that embodiment 1 obtains, and the pEASY-GmPRP2p-852 that step one obtains carries out Sac I and Xba I (pEASY-T1 carrier carries restriction enzyme site) double digestion respectively, reclaim digestion products, gained 2 kinds is reclaimed fragment to be connected (restriction enzyme mapping as shown in Figure 4) with the skeleton large fragment of the pC13P1 carrier (plasmid map as shown in Figure 5) through same double digestion respectively, obtain 2 kinds of recombinant plasmids.The correct plasmid sample presentation order-checking of qualification will be cut through enzyme.The recombinant plasmid called after pCAM-PRP2p-1062 inserting the DNA fragmentation containing sequence 2 in ordered list between the multiple clone site Sac I and Xba I of pC13P1 carrier is shown by through order-checking; The recombinant plasmid called after pCAM-PRP2p-852 of the DNA fragmentation inserting the 211-1062 position Nucleotide containing sequence 2 in ordered list between the multiple clone site Sac I and Xba I of pC13P1 carrier will be shown through order-checking.
In recombinant expression vector pCAM-PRP2p-1062 and pCAM-PRP2p-852, corresponding promotor (GmPRP2p-1062 and GmPRP2p-852) is all positioned at gus gene upstream, drive transcribing of gus gene, be all connected with in gus gene downstream simultaneously and stop its NOS terminator of transcribing.
Embodiment 3, the acquisition turning GmPRP2p-1062 and GmPRP2p-852 Arabidopis thaliana and expression characterization research
One, flower-dipping method arabidopsis thaliana transformation and qualification
YEP liquid nutrient medium: solvent is water, solute and concentration as follows: 5g/L NaCl, 5g/L yeast extract, 10g/L Tryptones; PH7.0.Each concentration is respective components final concentration in the medium.
1/2MS solid medium: solvent is water, solute and concentration as follows: 2.17g/L MS salt (Phyto Tech, catalog number (Cat.No.): M524), 7g/L agar, 30g/L sucrose, 1ml/L MS organic (Phyto Tech, catalog number (Cat.No.): M533), pH5.8.Each concentration is respective components final concentration in the medium.
1, with two kinds of recombinant plasmid pCAM-PRP2p-1062 and pCAM-PRP2p-852 that embodiment 2 obtains, and pC13P1 empty carrier transform Agrobacterium tumefaciens GV3101 competent cell respectively, obtain 3 kinds of recombinational agrobacterium.3 kinds of recombinational agrobacterium are inoculated in respectively YEP liquid nutrient medium (containing kantlex 50mg/L), 28 DEG C, 220rmp shaking culture, obtain 3 kinds of recombinational agrobacterium bacterium liquid of OD600=0.4-0.6.
2, by pC13(Delta) GUS carrier (plasmid map is as shown in Figure 6) transform Agrobacterium tumefaciens GV3101 competent cell, obtain recombinational agrobacterium.Recombinational agrobacterium is inoculated in YEP liquid nutrient medium (containing kantlex 50mg/L), 28 DEG C, 220rmp shaking culture, obtains the recombinational agrobacterium bacterium liquid of OD600=0.4-0.6.
3,3 kinds of recombinational agrobacterium bacterium liquid step 1 obtained respectively with the recombinational agrobacterium bacterium liquid in step 2 by volume 1:1 mix, add 0.5%(v/v) silwet L-77, obtain 3 kinds of Agrobacterium mixed solutions altogether, be used for contaminate plant.
4, the preparation of plant is contaminated: by Arabidopis thaliana (Arabidopsis thaliana(L.) Heynh.) seed of Columbia is at 10%(v/v) sterilize in the aqueous solution of clorox 15min, after aseptic washing 3-4 time, by seed on 1/2MS solid medium, after 4 DEG C of cultivation 3d, be placed in 22 DEG C of incubators to cultivate, the dark 8h of illumination 16h/.The plant growing 2 weeks is transplanted to continued growth in soil matrix from substratum, and the Arabidopis thaliana growing into flowering period is used for contaminating.
5, contaminate: in the 3 kinds of Agrobacterium mixed solutions prepared in step 3 by the Arabidopsis plant growing into the florescence in above-mentioned steps 4, soak 1min, plant shading after dip-dye is spent the night, contaminate after second day, recover normally to cultivate, adopt same method to carry out second time to plant after one week and contaminate.Until results T
1for seed.
6, by the T of results
1after seed disinfection, plant on the 1/2MS substratum containing 50mg/L Totomycin, after 4 DEG C of cultivation 3d, be placed in 22 DEG C of incubators to cultivate, the dark 8h of illumination 16h/, filtering out can the plant of normal growth, the growth plant of 2 weeks is transplanted to continued growth in soil matrix from substratum, results T
2for seed.Extract T simultaneously
1for the genomic dna of plant, carry out PCR detection.Specific as follows: to take genomic dna as template, for the resistant transgenic Arabidopis thaliana proceeding to pCAM-PRP2p-1062, adopt primer pair F-1062/R2 to carry out PCR detection, obtain size and be about the Arabidopis thaliana of 1062bp object band (sequence 2) for positive.For the resistant transgenic Arabidopis thaliana proceeding to pCAM-PRP2p-852, adopt primer pair F-852/R2 to carry out PCR detection, obtain size and be about the Arabidopis thaliana of 852bp object band (the 211-1062 position of sequence 2) for positive.Each process arranges the negative control replacing template with not genetically modified arabidopsis thaliana genomic dna all simultaneously, and replaces the positive control of template with corresponding plasmid.PCR detected result as shown in Figure 7.
7, the strain of object band is detected, its T
2method plantation same in step 6 is adopted to cultivate for seed, results T
3seed, selects T
3the seed of homozygous lines that generation is no longer separated is used for follow-up test.
Two, the research of GmPRP2p-1062 and GmPRP2p-852 Arabidopis thaliana expression characterization is turned
1, the tissue expression of vegetative growth phase different development stage
Get the T of growth 1d, 3d, 5d, 7d, 10d, 20d
3gUS tissue staining is carried out for transgenic Arabidopsis plants.Simultaneously with not genetically modified wildtype Arabidopsis thaliana Columbia with proceed to the Arabidopsis plant of pC13P1 empty carrier in contrast.Experiment in triplicate.
GUS histochemical stain is with reference to the method for Jefferson.Specific as follows: tissue sample to be dyed to be put into and detects liquid, 37 DEG C of incubation 12-16h.After 70%, 90% ethanol soaks 2h respectively, 70% ethanol spends the night, to remove the chlorophyll in chlorenchyma.Under microscope or the coloration result of the test sample that detects by an unaided eye, and take a picture.Blueness under white background is GUS expression sites.Wherein, liquid formula is detected as follows: 50mmolL
-1phosphoric acid buffer (formula: 4.095g Na
2hPO
4with 2.537g NaH
2pO
4constant volume is in the 1L aqueous solution), pH7.0,10mmolL
-1eDTA, 0.1%(w/v) Triton X-100,2mmolL
-1the Tripotassium iron hexacyanide, 2mmolL
-1yellow prussiate of potash, 5%(w/v) X-Gluc.Each concentration is that respective components is detecting the final concentration in liquid.
Result as shown in Figure 8, as can be seen from the figure, is turning in GmPRP2p-1062 and GmPRP2p-852 Arabidopsis plant, from seed germination, at its different growing stage, in root and hypocotyl, the expression activity of gus gene detected, almost do not detect in blade.And not genetically modified wildtype Arabidopsis thaliana Columbia in contrast all expresses without GUS with the Arabidopsis plant proceeding to pC13P1 empty carrier.Repeat experimental result three times consistent.
2, the tissue expression of generative growth phase
By the growth T of 2 weeks
3transgenic Arabidopsis plants is transplanted to continued growth in soil matrix from substratum, and blooming, fruiting period carries out GUS tissue staining to its each histoorgan (leaf, flower, really), and method is with step 1.Simultaneously with not genetically modified wildtype Arabidopsis thaliana Columbia with proceed to the Arabidopsis plant of pC13P1 empty carrier in contrast.Experiment in triplicate.
As shown in Figure 9, in reproductive growth period, the base portion of the petiole and fruit pod shell that turn the Arabidopis thaliana of GmPRP2p-1062 has and a small amount of GUS expression activity detected result; The base portion turning the petiole of GmPRP2p-852 Arabidopsis plant, blade master pulse and fruit pod shell also detects the expression of a small amount of gus gene.And not genetically modified wildtype Arabidopsis thaliana Columbia in contrast all expresses without GUS with the Arabidopsis plant proceeding to pC13P1 empty carrier.Repeat experimental result three times consistent.
3, the mensuration of GUS activity
Get root and the leaf of the transgenic Arabidopsis plants of growth 20d respectively, each transfer-gen plant gets 3 strains, and the GUS measured in root and leaf is active.Simultaneously be designated as WT with not genetically modified wildtype Arabidopsis thaliana Columbia() and proceed to pC13P1 empty carrier Arabidopsis plant in contrast.Specific as follows:
(1) making of 4-methyl umbelliferone (4-MU) typical curve
The preparation of 4-methyl umbelliferone (4-MU) standard solution: use 0.2M Na
2cO
3the aqueous solution prepares the 4-MU standard solution of different concns respectively, 1mM4-MU, 10 μMs of 4-MU, 1 μM of 4-MU, 100nM4-MU, 50nM4-MU, 10nM4-MU, 5nM4-MU.Wherein, 4-MU standard substance are sigma Products, and catalog number (Cat.No.) is M1381.Be determined at exciting light 365nm, the fluorophotometric value under utilizing emitted light 455nm.According to concentration and the corresponding fluorophotometric value drawing standard curve of 4-MU standard substance.
(2) preparation of testing sample
The root got and leaf are put into mortar (precooling) respectively, grinds to form homogenate with the GUS extracting solution of 1ml, proceed in the centrifuge tube of 1.5ml, 15000rpm, 4 DEG C, centrifugal 10min, collect supernatant liquor, obtain testing sample, for detecting.
Wherein, GUS extract recipe is as follows: 50mM phosphoric acid buffer (formula: 4.095g Na
2hPO
4and 2.537gNaH
2pO
4constant volume is in the 1L aqueous solution), pH7.0,10mM Na
2-EDTA, pH8.0,10mM beta-mercaptoethanol, 0.1%(w/v) Triton X-100.Each concentration is the final concentration of respective components in GUS extracting solution.
(3) GUS determination of activity
A.GUS reacts
GUS reaction solution: 4.08g4-methyl umbelliferone-β-D-Glucose aldehydic acid glycosides (4-Methylumbellifery-β-D-Glucuronide is called for short 4-MUG) constant volume, in 10ml GUS extracting solution, is used for carrying out enzymatic reaction.Wherein, 4-MUG is INALCO Products, and catalog number (Cat.No.) is 1758-1630.
GUS termination reaction liquid: 0.2M Na
2cO
3the aqueous solution, is used for termination reaction.
Reaction process: add in the centrifuge tube of 1.5ml by the GUS reaction solution of 90 μ l, 37 DEG C of preheatings, add the supernatant liquor extracted in 10 μ l above-mentioned steps (1), after 37 DEG C of reaction 60min, add the GUS termination reaction liquid of 900 μ l.Meanwhile, the contrast that each sample has 0min to react, measures 365nm/455nm fluorophotometric value.According to 4-methyl umbelliferone (4-MU) typical curve that surveyed fluorophotometric value and step (1) obtain, " the 4-MU change in concentration of unit time " that calculate.
B. determining the protein quantity
Get above-mentioned testing sample (supernatant liquor extracted) 10 μ l, add 200 μ l Xylene Brilliant Cyanine Gs, room temperature reaction 15min, microplate reader measures the light absorption value of 595nm.
Bovine serum albumin (BSA) standardized solution (1mg/ml) is provided (TIANGEN, catalog number (Cat.No.) PA102) by Bradford quantification of protein test kit.Get 0,0.5,1.5,2.5,3.5,4.5,5.5,6.5 μ l BSA standardized solution respectively, each standard 0.15M NaCl aqueous solution constant volume is to 10 μ l, add 200 μ l Xylene Brilliant Cyanine Gs, room temperature reaction 15min, microplate reader measures the light absorption value of 595nm.Protein content in the above-mentioned testing sample of concentration conversion (supernatant liquor extracted) of according to standard sample.
C.GUS activity calculates
According to the detected result of A and B, the GUS calculating testing sample is active, represents with " nmol/min/mg albumen ".Specifically, with " the 4-MU change in concentration of unit time " divided by " protein content in testing sample ", obtain the 4-MU change in concentration of the protein units time of unit mass, the GUS be in the unit time is active.
Test in triplicate, results averaged.
Result is as Figure 10, and turn in GmPRP2p-1062, GmPRP2p-852 Arabidopsis plant all, the GUS expression activity in root is significantly higher than in leaf, and the GUS expression activity of its transfer GmPRP2p-852 Arabidopsis plant in root is the highest.And not genetically modified wildtype Arabidopsis thaliana Columbia(WT in contrast) and proceed to the root of Arabidopsis plant of pC13P1 empty carrier and Ye Zhongjun expresses without GUS.
Claims (10)
1.DNA molecule is following a)-c) in arbitrary DNA fragmentation:
A) at least containing the 211-1062 position nucleotide sequence of sequence 2 in ordered list, and extend according to the 5 ' end of the nucleotide sequence of sequence 2 to sequence 2 from the 211st of sequence 2, obtain any one DNA fragmentation that length is 852 to 1062bp;
B) nucleotide sequence and a) limited has more than 90% homology, and has the DNA fragmentation of promoter function;
C) under strict conditions with nucleotide sequence hybridization a) or b) limited, and there is the DNA fragmentation of promoter function.
2. DNA molecular according to claim 1, is characterized in that: described DNA molecular is the DNA molecular shown in the 211-1062 position Nucleotide of sequence in sequence table 2.
3. the recombinant vectors containing DNA molecular described in claim 1 or 2, expression cassette, transgenic cell line or recombinant bacterium.
4. recombinant vectors according to claim 3, is characterized in that: the recombinant plasmid of described recombinant vectors for obtaining after the insertion of the multiple clone site place of pC13P1 carrier contains the DNA fragmentation of DNA molecular described in claim 1 or 2.
5. expression cassette according to claim 3, is characterized in that: described expression cassette, by the described DNA molecular with promoter function, is started the goal gene of expressing by described DNA molecular, and transcription termination sequence composition; Described DNA molecular is connected with described goal gene with functional way, and described goal gene is connected with described transcription termination sequence.
6. DNA molecular described in claim 1 or 2 starts the application in destination gene expression in plant.
7. application according to claim 6, is characterized in that: described in be expressed as root-specific express.
8. the application according to claim 6 or 7, is characterized in that: described plant is dicotyledons or monocotyledons.
9. application according to claim 8, described dicotyledons is Arabidopis thaliana or soybean.
10. the primer pair of DNA molecular described in amplification claim 1 or 2.
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| CN201310466928.4A CN104560986A (en) | 2013-10-09 | 2013-10-09 | Soybean-derived root-specific promoter GmPRP2p-852 and application thereof |
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Non-Patent Citations (5)
| Title |
|---|
| HONG J. C. ET AL.: "GenBank J05208", 《NCBI》 * |
| JONG CHAN HONG ET AL.: "Characterization of a Proline-rich Cell Wall Protein Gene Family of Soybean", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| ROBERT E. WYATT ET AL.: "Patterns of soybean praline-rich protein gene expression", 《THE PLANT CELL》 * |
| 张庆林等: "大豆球蛋白G1启动子的克隆及表达活性分析", 《分子植物育种》 * |
| 许文亮等: "一类新的编码PRPs 基因的分离及其在棉花纤维等组织细胞中的表达", 《生物化学与生物物理进展》 * |
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