CN104988153A - Plant root specific promoter and application thereof - Google Patents

Abstract

The invention discloses a plant root specific promoter and application thereof. The promoter is DNA molecules in the following (1) or (2) or (3) or (4), wherein the DNA molecules involved in (1) are formed by deoxyribonucleotide shown in the sequence 1 in a sequence table; the DNA molecules involved in (2) are formed by deoxyribonucleotide shown in the sequence 2 in the sequence table; the DNA molecules involved in (3) hybridize with a DNA sequence defined by (1) or (2); the DNA molecules involved in (4) in plant roots have the homology of 90% or more with the DNA sequence defined by (1) or (2) and can regulate and control specific expression of target genes in the plant roots. The plant root specific promoter can start specific expression of exogenous genes in the plant roots and has important significance in the traits inheritance improvement of crops with recombined root architectures and improved root functions. The invention further provides an expression cassette and a recombined expression vector comprising the promoter. The expression cassette and the recombined expression vector comprising the promoter can be used for culturing transgenic plant varieties which can specially express the target genes in the plant roots and are high in biological safety.

Description

A kind of roots of plants specificity promoter and application thereof

Technical field

The present invention relates to roots of plants specific gene promoter and application thereof, particularly relate to and derive from Barley Roots specific expression gene HvTIP2; The root-specific promoter of 1 upstream sequence and application thereof.

Background technology

Promotor is the section of DNA molecule being positioned at genetic transcription starting bit point upstream, and controlling gene is expressed under particular organization, etap and envrionment conditions.As engineered key controlling element, promotor determines phraseology and the transcriptional efficiency of foreign gene, plays an important role at genetically modified animals and plants rearing new variety and genetically modified organism reactor research and development field.

According to the mode of genetic expression, usually plant promoter is divided into constitutive promoter, tissue-specific promoter and inducible promoter three major types [Wang Guanlin, Fang Hongjun, 2002, plant genetic engineering philosophy and technique (second edition), Beijing, science tech publishing house].Promotor conventional in current plant genetic engineering is composition type expression promoter, foreign gene can a large amount of basic substance consumed in plant body at the omnibearing high expression of plant, grows bring disadvantageous effect to other Metabolic activity of transfer-gen plant and normal growth.The genetic expression that tissue-specific promoter drives is only limitted to some certain organs or tissue site, avoid the unnecessary waste of plant nutrition and energy, needs [the Song Yang at particular organization's organ mass expressing external gene can be met again, Zhou Junhui, Zhang Yongqiang. plant tissue specificity promoter is studied, biotechnology is circulated a notice of, and 2007 (6): 21-24].This kind of promotor is except having general promoter structure as except TATA box and CAAT box, also there is element [the Yamamoto YT of several control tissue specific expression simultaneously, Taylor CG, Acedo GN et al.Characterization of cis-acting sequences regulating root-specificgene expression in tobacco, The Plant Cell, 1991,3:371-382.].Existing research is reported in the promotor driving gene specific to express in the vascular bundle of plant, parenchyma, pollen, seed and endosperm tissue.

The root of plant has and absorbs moisture and inorganic salt, fixing plant, and under arid, saline and alkaline and heavy metal edaphic condition protective plant over-ground part, for growing of plant, there is important biological significance.In addition, root or the important nutritive substance of some species stores organ.Organizing specific expression gene is the valuable source of cloned tissue specificity promoter, gene differential expression bioinformatics method based on microarray data is that Rapid identification tissue-specific gene provides powerful tool (Yang XL, Xu HL, Li WH, et al.Screening and identification of seed-specific genes using digital differential displaytools combined with microarray data fromcommon wheat, BMC genomics, 12:513-514.).Utilize the promoters driven cytokinin degradative genes (CKX) of Arabidopis thaliana root specific expression gene AtWRKY6 specific expressed in tobacco and Arabidopis thaliana root, make root biomass increase by 60%, improve ability that the drought-resistant and preventing from heavy metal of plant coerces [ w, Erika N, Ireen K.Root-specific reduction of cytokinin causes enhanced root growth, droughttolerance, and leaf mineral enrichment in Arabidopsis and tobacco, Plant Cell, 2010,22 (12): 3905-3920.].

The root-specific reported is expressed promotor and is mainly derived from the blue mustard of plan, tobacco isotype plant, the root-specific promoter in farm crop source is utilized not only to contribute to illustrating the relevant basic theory problem of root development, more can regulate destination gene expression targetedly, improve the quality of plant, output and disease-resistant (inverse) ability, thus become the powerful tool of farm crop character improvement and rearing new variety, be with a wide range of applications.

Summary of the invention

The object of the present invention is to provide a kind of root-specific promoter and application thereof of farm crop source.

Roots of plants specificity promoter provided by the present invention, derive from barley (Hordeum vulgare), be extending to 3 ' end according to the nucleotide arrangement mode of sequence 1 of 5 ' end the 745-1258 nucleotide sequence at least containing sequence 1 in sequence table, length is the deoxynucleoside acid fragment of 514 to 1258bp.

Described promotor, its nucleotide sequence is preferably one of following sequence:

1) DNA molecular be made up of the deoxyribonucleotide shown in sequence in sequence table 1;

2) DNA molecular be made up of the deoxyribonucleotide shown in sequence in sequence table 2;

3) with have 1)-2) and in the DNA molecular that can hybridize under stringent hybridization condition of the Nucleotide of arbitrarily described sequence.

Described high high stringency conditions can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS, hybridizes and wash film at 65 DEG C.

4) with 1) or 2) DNA sequence dna that limits has the homology of more than 90% and can regulate and control the DNA molecular of goal gene specifically expressing in the root of plant.

The step of described promotor cloning process is as follows:

1) according to HvTIP2; The mRNA transcription initiation site upstream sequence of 1 gene, designs two upstream primer 1310F1 and 1310F2 and shared downstream primer 1310R, and holds at upstream and downstream primer 5' and introduce HindIII and Nco I restriction endonuclease sites respectively;

With barley gene group DNA for masterplate, the promoter fragment of two different lengthss that increased by PCR method.

Described PCR reaction system is: template DNA 500ng, and each 1 μ l of upstream and downstream primer (10 μMs), 10 × PCR Buffer is (containing Mg ²⁺) 2 μ l, dNTP (2.5mM) 1.6 μ l, Ex-Taq archaeal dna polymerase (5U/ μ l) 0.2 μ l, adds distilled water to 20 μ l.PCR program is: 98 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations; 72 DEG C extend 10min.

2) cloning process comprises PCR primer recovery and plasmid restructuring further, be specially: pcr amplification product is separated through the agarose gel electrophoresis of 2%, the recovery product of object band is connected with pMD19-T carrier, utilize and connect product conversion competent escherichia coli cell, obtain positive colony through blue hickie screening.

Expression cassette containing above-mentioned promotor, recombinant expression vector all belong to protection scope of the present invention.

Promotor of the present invention is that root-specific expresses promotor.Utilize plant expression vector, promoter sequence of the present invention is building up to any goal gene upstream, import vegetable cell, transgenic cell line and the transfer-gen plant of root-specific expression can be obtained.The plant tissue of conversion by using Ti-plasmids, directly delivered DNA, microinjection, conventional biology methods transformed plant cells or the tissue such as agriculture bacillus mediated, and is cultivated into plant by the plant expression vector carrying sequence of the present invention.The plant host be converted both can be the monocotyledonss such as corn, paddy rice, wheat, also can be the dicotyledonss such as soybean, rape, cotton.

The root-specific that the invention provides farm crop source expresses promotor, can be applicable to cultivate specifically expressing goal gene, biological safety are high in roots of plants transgenic plant kind or bio-reactor.

Accompanying drawing explanation

Fig. 1 HvTIP2; The differential expression schematic diagram of 1 gene in different tissues;

G1-G3: be root, stem, the leaf of barley seedlings; G4-G8: the root of Adult plant barley, stem, leaf, flower and internode; G8: young tender seed

The enzyme of Fig. 2 plant expression vector pCAM-Hv1310p1, pCAM-Hv1310p2 cuts qualification schematic diagram;

Swimming lane 1,3: be respectively enzyme cut before expression vector pCAM-Hv1310p1, pCAM-Hv1310p2

Swimming lane 2,4: be respectively enzyme cut after expression vector

The GUS dyeing schematic diagram of Fig. 3 pCAM-Hv1310p1 and pCAM-Hv1310p2 transgenic line;

Every picture group sheet is root, stem, flower and foliage organ from top to bottom successively

The GUS quantitative assay schematic diagram of Fig. 4 pCAM-Hv1310p1 and pCAM-Hv1310p2 transgenic line.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.In embodiment, bacterial strain uses therefor and reagent are the conventional material in laboratory.

The acquisition of embodiment 1, promotor of the present invention and functional verification thereof

1, HvTIP2; The acquisition of 1 promotor full length sequence

Does the present invention utilize PLEXdb database (http://www.plexdb.org/plex.php? database=Barley) BB3 gene expression chip data and gene digitizing differential display technique, screen the aquaporin gene HvTIP2 of a Barley Roots specifically expressing; 1, through real-time fluorescence quantitative RT-PCR analysis, prove that the transcriptional expression of this gene has root-specific and regulation and control of being grown, the expression amount in Adult plant is apparently higher than seedling stage.

According to the HvTIP2 of barley whole genome sequence prediction; 1 gene (morex_contig_51093 is the locus item of this gene in barley gene group) sequence, infers and HvTIP2; The promoter sequence of 1, at HvTIP2; 1 gene transcription start site upstream 1258bp Position Design primer:

1310F1:5 '-AAGCTTCGAAGGAAAGGCATTAGAAA-3 ' (HindIII); Reverse primer design is at HvTIP2; 1 gene transcription start site: 1310R:5 '-CCATGGTGCGGATGATTACCACAA-3 ' (Nco I).Prime end adds the restriction enzyme site of Hind III and Nco I respectively.

CTAB method is adopted to extract the leaves genomic DNA of barley variety Golden Promise.The genomic dna 500ng getting extraction is template, and with 1310F1 and 1310R for primer, carry out PCR reaction, PCR program is: 98 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 90s, 35 circulations; Last 72 DEG C extend 10min.

PCR primer is after the agarose gel electrophoresis of 1%, result shows that PCR obtains the band of about 1258bp, reclaimed purifying, be connected to pMD19-T cloning vector, and transform Escherichia coli strain DH5 α, extract recombinant plasmid and carry out sequence verification, result shows that fragment that above-mentioned PCR obtains has the nucleotide sequence of sequence 1, will have the promotor called after Hv1310p of the nucleotide sequence of sequence 1.

The online promoter Analysis instrument of PLACE and PLANTCARE is utilized to carry out forecast analysis to the cis-acting elements of Hv1310p1, find that this sequence is except containing basal promoter elements such as TATA frame and CAAT boxs, also there is 1 root hair specific element RHERPATEXPA7,4 different Expression element ROOTMOTIFTAPOX1 of Gent, DRE/CRT (transcription factor) the cis-acting elements DRECRTCOREAT of 1 environment-stress response, 2 MYC and MYB recognition sites, the two is all by ABA and arid induction.

2, HvTIP2; 1 promotor 5 ' holds the acquisition of deletion mutantion sequence

In order to carry out functional analysis to Hv1310p1, the leaves genomic DNA utilizing barley variety Golden Promise is template, at HvTIP2; 1 upstream region of gene 514bp place design forward primer 1310F2:5 '-AAGCTTCTGCATTATCAGACAGAACT-3 ' (adding HindIII site), utilize 1310R to carry out pcr amplification for reverse primer, result shows that 1310F2 and 1310R amplification obtains the fragment of about 514bp.By the fragment that above-mentioned PCR obtains, be connected to pMD19-T cloning vector, transform Escherichia coli strain DH5 α, obtain recombinant plasmid and carry out sequence verification, result shows, the fragment that above-mentioned PCR obtains has the nucleotide sequence of sequence 2, will have the promotor called after Hv1310p2 of the nucleotide sequence of sequence 2.

3, Hv1310P1 promotor and 5 ' deletion mutant thereof drive the structure of the plant expression vector of gus gene expression

Utilize hyg hygromycin gene as the selectable marker gene of expression vector, adopt HindIII/NcoI double digestion pCAMBIA1301 basal expression carrier and the pMD19-T cloning vector containing promoter fragment, reclaim the promoter fragment that the large fragment of pCAMBIA1301 plasmid enzyme restriction and cloning vector enzyme are cut respectively, then corresponding endonuclease bamhi is connected with T4DNA ligase enzyme, build the plant expression vector merging gus gene, and transformation of E. coli.By plant expression vector called after pCAM-Hv1310p1 and pCAM-Hv1310p2 respectively utilizing 1258bp and 514bp long fragment promotor to build.Positive colony carrier further through enzyme cut with sequence verification after, import agrobacterium strains EHA105.

4, the conversion of tobacco and the acquisition of transgenic positive plant

Adopting agriculture bacillus mediated leaf disc transformation method that the expression vector newly built is proceeded to Nicotiana tabacum, by the PCR Molecular Detection to hyg selectable marker gene, is that positive T0 is transplanted to continued growth in nutrient soil pot for transfer-gen plant by qualification result.

5, the quantitative and qualitative analysis of transgene tobacco GUS activity detects

With unconverted tobacco for contrast, the GUS transfer-gen plant in the Adult plant stage to 2 kinds of different fragments length promoters driven carries out histochemical stain comparative analysis.Get the root of transgene tobacco, stem, leaf, floral organ, in 37 DEG C of GUS dye liquors, temperature bath is spent the night, and to fade 5min, then forward in 100% ethanol until background is colourless through 70% ethanol.GUS formula for dye liquor is 100mM NaH2PO4,10mM Na2EDTA, 0.5mM K4 [Fe (CN) 6] 3H2O, 0.5mM K3 [Fe (CN) 6], 0.1%TritonX-100.

Further employing coomassie brilliant blue makes gus protein detection by quantitative.Get the root of transgene tobacco, stem, leaf, spend each 100mg, after liquid nitrogen grinding, add 1mL gus protein extracting solution, 4 DEG C, the centrifugal 5min of 12000rpm, get 500 μ L supernatant liquors in new EP pipe, be placed in for subsequent use on ice.Get 100 μ L supernatant liquors in new EP pipe, add GUS zyme extract 400 μ L and 500 μ L MUG (4-methylumbelliferyl β-D-glucuronide) reaction substrate (concentration 2mmol/L) of preheating, 37 DEG C of water-baths and respectively at 0,15,30,45,60min time get 200 μ L reaction solutions in new EP pipe and add 800 μ L Na2CO3 (concentration 0.2mol/L) stop buffers in keeping in Dark Place on ice.By microplate reader at excitation wavelength 365nm, under wavelength of transmitted light 455nm, measure the content of fluorescence-causing substance MU (4-methylumbelliferone).GUS fluorescence activity measures and represents GUS enzymic activity with the nmole number of every milligram of proteolysis substrate MUG in the unit time.

Result shows, and the transgenic tobacco plant root of two kinds of vector all has obvious GUS chemical staining, and does not show GUS activity in stem, leaf and flower tissue, shows HvTIP2; The promotor of 1 gene can drive exogenous gene expression specifically in root tissue.The detection by quantitative result of GUS fluorescence activity also finds, the root organ of transgene tobacco shows higher GUS activity (>30nmol.mg-1min-1), and the pole low Poison activity level of ﹤ 1.2nmol.mg-1min-1 in stem, leaf and floral organ, be the interference of other background impurity in sample.In addition, the transgene tobacco root GUS activity level of two kinds of carriers does not have marked difference, illustrates that two kinds of promotors of different fragments length all have and starts activity preferably.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (7)

1. a Barley Roots specificity promoter, is characterized in that, this promotor is following 1) or 2) or 3) or 4) DNA molecular:

1) DNA molecular be made up of the deoxyribonucleotide shown in sequence in sequence table 1;

2) DNA molecular be made up of the deoxyribonucleotide shown in sequence in sequence table 2;

3) with 1) or 2) DNA sequence dna that the limits DNA molecular of hybridizing;

4) with 1) or 2) DNA sequence dna that limits has the homology of more than 90% and can regulate and control the DNA molecular of goal gene specifically expressing in the root of plant.

2. promotor according to claim 1, it is characterized in that effectively driving foreign gene specific expressed in roots of plants, the step of described promotor cloning process is as follows:

According to HvTIP2; The mRNA transcription initiation site upstream sequence of 1 gene, design two upstream primer 1310F1 (5 '-AAGCTTCGAAGGAAAGGCATTAGAAA-3 ') and 1310F2 (5 '-AAGCTTCTGCATTATCAGACAGAACT-3 ') and a shared downstream primer 1310R (5 '-CCATGGTGCGGATGATTACCACAA-3 '), and hold at upstream and downstream primer 5' and introduce Hind III and Nco I restriction endonuclease sites respectively;

With barley gene group DNA for masterplate, the primer pair utilizing upstream primer to form with downstream primer respectively, the promoter fragment of two different lengthss that increased by PCR method.

3. promotor according to claim 2, is characterized in that, described PCR reaction system is: template DNA 500ng, each 1 μ l, the 10 × PCR Buffer (Mg of upstream and downstream primer (10 μMs) ²⁺plus) 2 μ l, dNTP Mixture (2.5mM) 1.6 μ l, Ex-Taq archaeal dna polymerase (5U/ μ l) 0.2 μ l, adds distilled water to 20 μ l.PCR program is: 98 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 35 circulations; 72 DEG C extend 10min.

4. promotor according to claim 2, it is characterized in that, described promotor cloning process comprises the recovery of PCR primer further and plasmid is recombinated, be specially: pcr amplification product is separated through the agarose gel electrophoresis of 2%, the recovery product of object band is connected with pMD19-T carrier, product conversion competent escherichia coli cell will be connected, obtain positive colony through blue hickie screening.

5. one kind contains expression cassette and the expression vector of promotor described in claim 1-4 any one.

6. cultivating the application in goal gene root-specific expression plant containing promotor described in claim 1-4 any one for one kind.

7. one kind contains the application of promotor in transgenic plant rearing new variety described in claim 1-4 any one.