CN104560985B - Root-specific promoter GmTIPp 1546 and its application from soybean - Google Patents

Abstract

The invention discloses a kind of root-specific promoter GmTIPp 1546 from soybean and its application.Promoter provided by the present invention is at least one of following：a）The 509th 2054 nucleotide sequence at least containing sequence in ordered list 2, and extend since the 509th of sequence 2 according to the nucleotide sequence of sequence 2 to 5 ' ends of sequence 2, obtain any one DNA fragmentation that length is 1546 to 2054bp；b）With a）The nucleotide sequence of restriction has more than 90% homology, and the DNA fragmentation with promoter function；c）Under strict conditions with a）Or b）The nucleotide sequence hybridization of restriction, and the DNA fragmentation with promoter function.Promoter provided by the present invention can effectively start target gene it is specific expressed in root.Molecular improvement of the present invention for plant in root provides a kind of means, and the degeneration-resistant research to plant just has the certain significance, and has wide application space and market prospects in agriculture field.

Description

From the root-specific promoter GmTIPp-1546 of soybean and its application

Technical field

The present invention relates to a kind of from the root-specific promoter GmTIPp-1546 of soybean and its application.

Background technology

Promoter is the part of gene, controls starting and the expression degree of gene expression.In transgenic breeding, outside Accuracy controlling of the source gene in plant is mainly what is realized by suitable promoter.At present, in genetic engineering extensively Constitutive promoter is used, but is due to that the expression that constitutive promoter drives target gene in plant tissue is constant With it is lasting, do not limited by space-time and extraneous factor, can consume intracellular matter and energy excessively, produce a large amount of heterologous eggs White matter or metabolite, destroy the original physiological metabolism balance of plant, cause plant growth abnormal or even dead.With composing type Promoter is compared, and its foreign gene for being driven of root-specific promoter is expressed in recipient plant root, is overcome composing type and is opened Mover starts consuming excessively for the cellular material that foreign gene non-specificity is expressed and caused due to continuing, efficiently.With turn The raising of gene plant safety standard, including all commercialization transgenic crops, it is desirable to must be to promoter used Expression activity, potential restructuring ability, surrounding enviroment and safety effects are carried out with detailed research.

Soybean is that a kind of important grain and oil raise dual-purpose crop, in China soybean producing region mostly in arid, saline and alkaline and high and cold Deng region, the soybean varieties resistance of reverse of application is not strong, and serious drought and waterlogging pest and disease damage etc. is abiotic and biotic stress coerces notable shadow Ring soybean yields.What is used at present in genetically engineered soybean is all CaMV35S promoters, and root-specific promoter is applied to soybean and turned In gene, studied for soybean transgene and more abundant promoter element is provided, while being also the drought-enduring, salt tolerant of cultivation, cold-resistant etc. Genetically engineered soybean new varieties provide a kind of means.

The content of the invention

It is an object of the invention to provide a kind of DNA molecular with promoter function.

DNA molecular provided by the present invention with promoter function, from pulse family Glycine the cultivated soybean （Glycine max(L.)Merr.）Williams82, is following a）-c）In any DNA fragmentation：

a）At least 509-2054 nucleotide sequences containing sequence in ordered list 2, and from the 509th of sequence 2 Start to 5 ' ends of sequence 2 to extend according to the nucleotide sequence of sequence 2, obtain any one that length is 1546 to 2054bp DNA fragmentation；

b）With a）The nucleotide sequence of restriction has more than 90% homology, and the DNA fragmentation with promoter function；

c）Under strict conditions with a）Or b）The nucleotide sequence hybridization of restriction, and the DNA fragmentation with promoter function.

Above-mentioned stringent condition can be in 6 × SSC, 0.5%SDS solution, to hybridize at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Last nucleotides of the promoter is the 2054th of sequence 2.

In the present invention, the DNA molecular with promoter function is 509-2054 cores of sequence 2 in sequence table DNA molecular shown in thuja acid, is named as GmTIPp-1546.

Wherein, sequence 2 is made up of 2054 nucleotides, is 1-2054 of sequence 1.

Contain the DNA molecular（Promoter）Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium fall within this The protection domain of invention.

In the present invention, the recombinant vector is the multiple cloning sites in pC13P1 carriers（Such as Kpn I and Pst I）Insertion The recombinant plasmid that the DNA molecular is obtained.

The pC13P1 carriers are that obtained annular carrier is transformed based on pCAMBIA1301 carriers, the pC13P1 The sequence of any promoter is not contained on carrier, the need for meeting research promoter function.

Specifically, the sequence of the pC13P1 carriers is as shown in sequence 3 in sequence table.

The expression cassette can be started the mesh of expression by the DNA molecular by the DNA molecular with promoter function Gene, and transcription terminator composition；The DNA molecular is connected with functional way with the target gene, and described Target gene is connected with the transcription terminator.

In one embodiment of the invention, the target gene is specially gus gene（Carried from the pC13P1 Body）；The transcription terminator is specially NOS transcription terminators（From the pC13P1 carriers）.

The application that the DNA molecular starts in plant in destination gene expression falls within protection scope of the present invention.

In the application, it is described to be expressed as root-specific expression.

Further, the root-specific is expressed as in root high expression, and specially the expression quantity in root is significantly higher than other groups Knit（Such as petiole, blade master pulse, flower, Fruit pod shell）, expression quantity is extremely low in its hetero-organization.

In the application, the target gene can be gus gene.The plant can be that dicotyledon or unifacial leaf are planted Thing.The dicotyledon concretely arabidopsis or soybean.

In one embodiment of the invention, the plant is specially arabidopsis（Arabidopsis thaliana(L.) Heynh.）Columbia.

In addition, expanding the DNA molecular（Promoter）Primer pair fall within protection scope of the present invention.

In the present invention, the DNA molecular is expanded（Promoter）Primer pair be as follows：

5'-GGTACCGAGTATAAAACCCAAAATC-3'；

5'-CTGCAGTTTGGCACCTCACCTCAC-3'。

It is demonstrated experimentally that GmTIPp-1546 promoters provided by the present invention can effectively start target gene in root It is specific expressed.Molecular improvement of the present invention for plant in root provides a kind of means, just has one to the degeneration-resistant research of plant Determine meaning, there is wide application space and market prospects in agriculture field.

Brief description of the drawings

Fig. 1 is the agarose gel electrophoresis figure of first round PCR primer.Arrow show purpose band.

Fig. 2 is the second wheel PCR primer GmTIPp-2054 agarose gel electrophoresis figure.Arrow show purpose band.

Fig. 3 is the PCR testing results of GmTIPp-2054 and GmTIPp-1546 promoters.Wherein, swimming lane M is DL2000plus DNA molecular amount standard；Swimming lane 1,2 is respectively to enter performing PCR with primer pair F-2054/R and F-1546/R to expand The electrophoretogram of the PCR primer of gained.

Fig. 4 be recombinant plasmid pMD18-GmTIPp-2054 and pMD18-GmTIPp-1546, and pC13P1 carriers Kpn The double digestion electrophoretograms of I and Pst I.Wherein, swimming lane M is DL2000plus DNA molecular amount standard；Swimming lane 1,2,3 is respectively PMD18-GmTIPp-2054, pMD18-GmTIPp-1546 and pPC13P1.

Fig. 5 is the plasmid map of pC13P1 carriers.

Fig. 6 is pC13（Delta）The plasmid map of GUS carriers.

Fig. 7 is transgenic arabidopsis T₁The Molecular Detection result in generation.Wherein, A, B represent respectively turn pCAM-TIPp-2054, PCAM-TIPp-1546 Arabidopsis plant testing results.In A and B, swimming lane M is DL2000plus DNA molecular amount standard；Swimming lane CK+ is positive control；Swimming lane CK- is negative control；Other swimming lanes are respectively different transgenic lines, purposeful band（Arrow Shown position）Occur for the positive.

Fig. 8 is transgenic arabidopsis T₃The vegetative growth phase different development stage GUS coloration results in generation.

Fig. 9 is transgenic arabidopsis T₃The generative growth phase different development stage GUS coloration results in generation.

Figure 10 is transgenic arabidopsis T₃GUS active level measurement results in the plant root and leaf in generation.Wherein, WT is represented The wildtype Arabidopsis thaliana Columbia of non-transgenosis；T2054-7, T2054-10, T2054-18, which are that three plants of identifications are positive, to be turned PCAM-TIPp-2054 Arabidopsis plants；T1546-19, T1546-43 and T1546-45 be three plants of identifications it is positive turn pCAM- TIPp-1546 Arabidopsis plants.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

Quantitative test in following embodiments, is respectively provided with three repetition experiments, results averaged.In following embodiments Primer is synthesized and examining order is completed by Hua Da company.

The cultivated soybean（Glycine max(L.)Merr.）Williams82：It is recorded in " Xu Shuo wild soybean salt stress phases Close microRNA functional analysis .2011 Chinese Academy of Agricultural Sciences Master's thesis " text, by crop section of the Chinese Academy of Agricultural Sciences Learn research institute's supply.

PC13P1 carriers and pC13（Delta）GUS carriers：It is annular carrier, by the Chinese Academy of Sciences, subtropical zone agricultural is raw State research institute Pedro S.C.F.Rocha researchers provide.Any promoter is not contained on pC13P1 carriers, but contains GUS reports Accuse gene（Such as Fig. 5）；pC13（Delta）GUS carriers do not contain gus gene, but contain kalamycin resistance and hygromycin resistance （Such as Fig. 6）.The sequence of the pC13P1 carriers is as shown in sequence 3 in sequence table；The pC13（Delta）The sequence of GUS carriers As shown in sequence 4 in sequence table.

Arabidopsis（Arabidopsis thaliana(L.)Heynh.）Columbia：It is recorded in " the beautiful .Columbia of Guo Xiao The text of type Arabidopsis callus culture studies Henan Agricultural Sciences, 01 phase in 2009 " one, by crop section of the Chinese Academy of Agricultural Sciences Learn research institute's supply.

Agrobacterium tumefaciems GV3101：It is recorded in that " Zhao Zhan, Li Wen life are different, and Agrobacterium tumefaciens strain type is lost to Trichoderma Pass the northern gardening of influence of transformation efficiency, 03 phase in 2006 " text, supplied by Institute of Crop Science, Chinese Academy of Agricultural Science.

The clone and sequence of embodiment 1, GmTIPp total length promoters

With the cultivated soybean（Glycine max(L.)Merr.）Williams82 is material, and Soybean Root is extracted using CTAB methods It is genomic DNA.According to soybean gene group database lookup TIP genes and its upstream sequence, special primer sense primer is designed F1 and anti-sense primer R1, using the STb gene of extraction as template, enters performing PCR amplification.

F1：5'-TCAGGAGGACGAATAGCCCA-3'（1-20 of sequence 1）；

R1：5'-CAACACCAGCGAACACGAAA-3'（The reverse complementary sequence of 2145-2163 of sequence 1）,

Response procedures：94℃5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min30s, 35 circulations；72 DEG C of extension 10min.

After reaction terminates, pcr amplification product is detected with 1% agarose gel electrophoresis.

As a result Fig. 1 is seen, wherein, swimming lane M is DL2000plus DNA molecular amount standard, and swimming lane 1 is PCR primer.With recovery Kits reclaim PCR primer, with pMD18-T-Simple carriers after PCR primer recovery（TaKaRa, catalog number (Cat.No.)：D103A） Connection, connection product Transformed E .coli competent cell DH5 α（TIANGEN, catalog number (Cat.No.)：CB101）, plasmid is extracted, is expanded through PCR The recombinant plasmid for increasing to the positive sends to sequencing.Sequencing result shows that PCR primer is 2163bp, as shown in sequence 1, including 109bp TIP gene C DS sequences（2055-2163 of sequence 1）.

In order to obtain the promoter fragment without TIP gene C DS sequences, since translation initiation codon ATG, new design One anti-sense primer R2 carries out second and takes turns PCR amplifications, with first round PCR primer（Sequence 1）For template, using F1 and R2 as primer Carry out second and take turns PCR amplifications.

F1：5'-TCAGGAGGACGAATAGCCCA-3'（1-20 of sequence 2）；

R2：5'-TTTGGCACCTCACCTCAC-3'（The reverse complementary sequence of 2037-2054 of sequence 2）.

Response procedures：94℃5min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2min15s, 35 circulations；72 DEG C of extension 10min.

After reaction terminates, pcr amplification product is detected with 1% agarose gel electrophoresis.

As a result Fig. 2 is seen, wherein, swimming lane M is DL2000 DNA molecular amount standard, and swimming lane 1 is PCR primer.Use reclaim reagent PCR primer is reclaimed in box purifying, and PCR primer is connected after reclaiming with pMD18-T-Simple carriers, connection product Transformed E .coli senses By state cell DH5 α, plasmid is extracted, is expanded through PCR and sends to sequencing for positive recombinant plasmid.Sequencing result has obtained 2054bp GmTIPp total length promoter sequences, as shown in sequence 2, be named as GmTIPp-2054.Correct recombinant plasmid name will be sequenced For pMD18-GmTIPp.

The structure of embodiment 2, the recombinant expression carrier containing GmTIPp-2054 promoters and its 5 ' deletion fragments

First, the acquisition of the deletion sequence of soybean GmTIPp-2054 promoter sequences 5 '

The present inventor is lacked to the 5 ' of GmTIPp-2054 sequences.Due to pMD18-T-Simple carriers Without restriction enzyme site, according to GmTIPp-2054 sequences（Sequence 2）, the upstream for having separately designed the restriction enzyme sites of 2 band Kpn I is drawn Thing（F-2054、F-1546）With the anti-sense primer R of the restriction enzyme sites of 1 band Pst I, the recombinant plasmid pMD18- obtained with embodiment 1 GmTIPp is template, and performing PCR amplification is entered respectively.

Primer sequence is as follows：

F-2054：5'-GGTACCTCAGGAGGACGAATAGCCCA-3'（Sequence after underscore is the 1-20 of sequence 2 Position）；

F-1546：5'-GGTACCGAGTATAAAACCCAAAATC-3'（Sequence after underscore is the 509- of sequence 2 527）；

R：5'-CTGCAGTTTGGCACCTCACCTCAC-3'（Sequence after underscore is 2037-2054 of sequence 2 Reverse complementary sequence）.

The response procedures expanded into performing PCR are expanded using primer pair F-2054/R：94℃5min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 2min15s, 35 circulations；72 DEG C of extension 10min.

Enter the response procedures of performing PCR amplification using primer pair F-1546/R：94℃5min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C 2min, 35 circulations；72 DEG C of extension 10min.

After reaction terminates, pcr amplification product is detected with 1% agarose gel electrophoresis.

As a result it is as shown in Figure 3.Two kinds of PCR primers of gained are sequenced respectively, as a result shown：Primer pair F-2054/R expands Increase gained PCR primer sequence for "GGTACC1-2054 of+sequence 2+CTGCAG”；PCR obtained by primer pair F-1546/R The sequence of product for "GGTACC509-2054 of+sequence 2+CTGCAG”。

Further by the GmTIPp-2054 sequences of gained（1-2054 of sequence 2）5 ' deletion sequences --- sequence 2 509-2054, be named as GmTIPp-1546.Both the above PCR primer is connected with pMD18-T-Simple carriers respectively Connect, according to the difference of exogenous sequences in gained recombinant plasmid, pMD18-GmTIPp-2054 and pMD18- are named as successively GmTIPp-1546。

2nd, the structure of recombinant expression carrier

Step one is obtained into pMD18-GmTIPp-2054 and pMD18-GmTIPp-1546 and carries out Kpn I and Pst I respectively Double digestion, reclaim digestion products, by two kinds of gained reclaim fragments respectively with the pC13P1 carriers by same double digestion（Plasmid Collection of illustrative plates is as shown in Figure 5）Skeleton large fragment be connected（Restriction enzyme mapping is as shown in Figure 4）, obtain two kinds of recombinant plasmids.It will be reflected through digestion Fixed correct plasmid sample presentation sequencing.It will show to insert between the multiple cloning sites Kpn I and Pst I of pC13P1 carriers through sequencing The recombinant plasmid of 1-2054 nucleotides of sequence 2 is named as pCAM-TIPp-2054 in sequence table；It will show through sequencing 509-2054 nucleotides of sequence 2 in insetion sequence table between the multiple cloning sites Kpn I and Pst I of pC13P1 carriers Recombinant plasmid is named as pCAM-TIPp-1546.

In recombinant expression carrier pCAM-TIPp-2054 and pCAM-TIPp-1546, corresponding promoter（GmTIPp-2054 And GmTIPp-1546）Gus gene upstream is respectively positioned on, the transcription of gus gene is driven, while being respectively connected with end in gus gene downstream Only its transcription NOS terminator.

Embodiment 3, the acquisition for turning GmTIPp-2054, GmTIPp-1546 arabidopsis and expression characterization research

First, flower-dipping method arabidopsis thaliana transformation and identification

YEP fluid nutrient mediums：Solvent is water, and solute and its concentration are as follows：5g/L NaCl, 5g/L yeast extracts, 10g/ L tryptones；pH7.0.Each concentration is the final concentration of respective components in the medium.

1/2MS solid mediums：Solvent is water, and solute and its concentration are as follows：2.17g/L MS salt（Phyto Tech, mesh Record number：M524）, 7g/L agar, 30g/L sucrose, 1ml/L MS are organic（Phyto Tech, catalog number (Cat.No.)：M533）, pH5.8.It is each dense Spend the final concentration in the medium for respective components.

1st, two kinds of recombinant plasmids pCAM-TIPp-2054, pCAM-TIPp-1546 obtained with embodiment 2, and pC13P1 Empty carrier converts Agrobacterium tumefaciems GV3101 competent cells respectively, obtains 3 kinds of recombinational agrobacteriums.By 3 kinds of recombinational agrobacteriums point YEP fluid nutrient mediums are not inoculated in（50mg/L containing kanamycins）, 28 DEG C, 220rmp shaken cultivations obtain OD600=0.4-0.6 3 kinds of recombinational agrobacterium bacterium solutions.

2nd, by pC13（Delta）GUS carriers（Plasmid map is as shown in Figure 6）Convert Agrobacterium tumefaciems GV3101 competence thin Born of the same parents, obtain recombinational agrobacterium.Recombinational agrobacterium is inoculated in YEP fluid nutrient mediums（50mg/L containing kanamycins）, 28 DEG C, 220rmp shaken cultivations, obtain OD600=0.4-0.6 recombinational agrobacterium bacterium solution.

3rd, 3 kinds of recombinational agrobacterium bacterium solutions for obtaining step 1 respectively with the recombinational agrobacterium bacterium solution in step 2 by volume 1:1 mixing, adds 0.5%（v/v）Silwet L-77, are obtained 3 kinds of Agrobacterium mixed liquors, for contaminating plant.

4th, the preparation of plant is contaminated：By arabidopsis（Arabidopsis thaliana(L.)Heynh.）Columbia kind Son is 10%（v/v）15min is sterilized in the aqueous solution of sodium hypochlorite, after sterile wash 3-4 times, by seed kind in 1/2MS solids On culture medium, after 4 DEG C of culture 3d, it is placed in 22 DEG C of incubators and cultivates, illumination 16h/ dark 8h.The plant of growth 2 weeks is from culture Continued growth in soil matrix is transplanted in base, the arabidopsis for growing into florescence is used for contaminating.

5th, contaminate：3 kinds of Agrobacteriums that the Arabidopsis plant that the florescence is grown into above-mentioned steps 4 is prepared in step 3 1min is soaked in mixed liquor, the plant shading after dip-dye is stayed overnight, after contaminating second day, recover normal culture, using same after one week Quadrat method carries out second to plant and contaminated.Until harvest T₁For seed.

6th, by the T of harvest₁After seed disinfection, plant on the 1/2MS culture mediums of the hygromycin containing 50mg/L, 4 DEG C are cultivated After 3d, it is placed in 22 DEG C of incubators and cultivates, illumination 16h/ dark 8h filter out the plant for being capable of normal growth, will grows 2 weeks Plant is transplanted to continued growth in soil matrix from culture medium, harvests T₂For seed.T is extracted simultaneously₁For the genome of plant DNA, enters performing PCR detection.It is specific as follows：Using genomic DNA as template, the resistant transgenic for being transferred to pCAM-TIPp-2054 Arabidopsis, enters performing PCR detection, it is about 2054bp purpose bands to obtain size using primer pair F-2054/R（Sequence 2）Plan south Mustard is the positive.Resistant transgenic arabidopsis for being transferred to pCAM-TIPp-1546, performing PCR inspection is entered using primer pair F-1546/R Survey, it is about 1546bp purpose bands to obtain size（509-2054 of sequence 2）Arabidopsis for the positive.Each processing is simultaneously The negative control that template is replaced with the WT lines genomic DNA of non-transgenosis is set, and template is replaced with correspondence plasmid Positive control.PCR testing results are as shown in Figure 7.

7th, the strain of purpose band is detected, its T₂Use method same in step 6 to plant culture for seed, harvest T₃ Seed, selects T₃The seed for the homozygous lines that generation no longer separates is used for follow-up experiment.

2nd, the research of GmTIPp-2054, GmTIPp-1546 arabidopsis expression characterization is turned

1st, the tissue expression of vegetative growth phase different development stage

Take growth 1d, 3d, 7d, 20d T₃GUS tissue stainings are carried out for transgenic Arabidopsis plants.While not turn base The wildtype Arabidopsis thaliana Columbia of cause and the Arabidopsis plant of pC13P1 empty carriers is transferred to as control.Experiment is in triplicate.

Method of the GUS histochemical stains with reference to Jefferson.It is specific as follows：Tissue sample to be dyed is put into inspection Survey in liquid, 37 DEG C of incubation 12-16h.70%, 90% ethanol is soaked after 2h respectively, and 70% ethanol is stayed overnight, to remove in chlorenchyma Chlorophyll.Under microscope or the test sample that detects by an unaided eye coloration result, and take a picture.Blueness under white background is GUS Expression sites.Wherein, detection formula of liquid is as follows：50mmol·L^-1Phosphate buffer（Formula：4.095g Na₂HPO₄And 2.537g NaH₂PO₄Constant volume is into the 1L aqueous solution）, pH7.0,10mmolL^-1EDTA, 0.1%（w/v）Triton X-100,2mmolL^-1The potassium ferricyanide, 2mmolL^-1Potassium ferrocyanide, 5%（w/v）X-Gluc.Each concentration is end of the respective components in detection liquid Concentration.

As a result as shown in figure 8, it can be seen that being planted in all GmTIPp-2054 and GmTIPp-1546 arabidopsis that turn In strain, since being sprouted seed, in its different growing stage, the expression activity of gus gene is detected in root and hypocotyl（It is blue Color）, almost do not detected in its rotaring gene plant blade.And the wildtype Arabidopsis thaliana of the non-transgenosis as control Columbia is expressed with the Arabidopsis plant for being transferred to pC13P1 empty carriers without GUS.Three times repetition experimental result is consistent.

2nd, the tissue expression of generative growth phase

The T of 2 weeks will be grown₃Transgenic Arabidopsis plants are transplanted to continued growth in soil matrix from culture medium, are opening Fruiting period is spent to its each histoorgan（Leaf, flower, fruit）GUS tissue stainings are carried out, method is with step 1.Simultaneously with non-transgenosis Wildtype Arabidopsis thaliana Columbia and the Arabidopsis plant of pC13P1 empty carriers is transferred to as control.Experiment is in triplicate.

As a result as shown in figure 9, in reproductive growth period, turn in GmTIPp-2054 arabidopsis in petiole, blade master pulse, not Ripe Fruit pod and ripe Fruit pod shell, which have, detects a small amount of GUS expression activities, and GUS is turning GmTIPp-1546 arabidopsis In plant, GUS expression activities are not almost detected in its leaf, flower, fruit.And intend as the wild type of the non-transgenosis of control Southern mustard Columbia is expressed with the Arabidopsis plant for being transferred to pC13P1 empty carriers without GUS.Three times repetition experimental result is consistent.

3rd, the measure of GUS activity

The root and leaf of growth 20d transgenic Arabidopsis plants are taken respectively, and each transfer-gen plant takes 3 strains, determines GUS activity in root and leaf.Simultaneously with the wildtype Arabidopsis thaliana Columbia of non-transgenosis（It is designated as WT）It is empty with pC13P1 is transferred to The Arabidopsis plant of carrier is used as control.It is specific as follows：

（1）4-methyl umbelliferone（4-MU）The making of standard curve

4-methyl umbelliferone（4-MU）The preparation of standard solution：Use 0.2M Na₂CO₃The aqueous solution prepares various concentrations respectively 4-MU standard solutions, 1mM4-MU, 10 μM of 4-MU, 1 μM of 4-MU, 100nM4-MU, 50nM4-MU, 10nM4-MU, 5nM4- MU.Wherein, 4-MU standard items are sigma Products, and catalog number (Cat.No.) is M1381.Determine in exciting light 365nm, transmitting light 455nm Under fluorophotometric value.Standard curve is drawn according to the concentration of 4-MU standard items and correspondence fluorophotometric value.

（2）The preparation of testing sample

The root taken and leaf are respectively put into mortar（Precooling）, homogenate is ground to form with 1ml GUS extract solutions, is transferred to In 1.5ml centrifuge tube, 15000rpm 4 DEG C, centrifuges 10min, collects supernatant, testing sample is obtained, for detecting.

Wherein, GUS extract recipes are as follows：50mM phosphate buffers（Formula：4.095g Na₂HPO₄With 2.537gNaH₂PO₄Constant volume is into the 1L aqueous solution）, pH7.0,10mM Na₂- EDTA, pH8.0,10mM beta -mercaptoethanol, 0.1% （w/v）Triton X-100.Each concentration is final concentration of the respective components in GUS extract solutions.

（3）GUS determinations of activity

A.GUS reacts

GUS reaction solutions：4.08g4- methyl umbelliferones-β-D-Glucose aldehydic acid glycosides（4-Methylumbellifery-β-D- Glucuronide abbreviations 4-MUG）Constant volume is into 10ml GUS extract solutions, for carrying out enzymatic reaction.Wherein, 4-MUG is INALCO Products, catalog number (Cat.No.) is 1758-1630.

GUS terminating reaction liquid：0.2M Na₂CO₃The aqueous solution, for terminating reaction.

Course of reaction：In the centrifuge tube that 90 μ l GUS reaction solutions are added to 1.5ml, 37 DEG C of preheatings add the 10 above-mentioned steps of μ l Suddenly（1）After the middle supernatant extracted, 37 DEG C of reaction 60min, 900 μ l GUS terminating reaction liquid is added.Meanwhile, each sample Product have the control that 0min reacts, and determine 365nm/455nm fluorophotometric values.According to surveyed fluorophotometric value and step（1）Obtain 4-methyl umbelliferone（4-MU）Standard curve, calculates obtained " the 4-MU change in concentration of unit interval ".

B. determining the protein quantity

Take above-mentioned testing sample（The supernatant extracted）10 μ l, add 200 μ l Coomassie brilliant blues, react at room temperature 15min, 595nm light absorption value is determined on ELIASA.

Bovine serum albumin（BSA）Standard liquid（1mg/ml）There is provided by Bradford quantification of protein kit （TIANGEN, catalog number (Cat.No.) PA102）.0,0.5,1.5,2.5,3.5,4.5,5.5,6.5 μ l BSA standard liquids are taken respectively, each Standard sample 0.15M NaCl aqueous solution constant volumes to 10 μ l, add 200 μ l Coomassie brilliant blues, 15min are reacted at room temperature, in ELIASA Upper measure 595nm light absorption value.The above-mentioned testing sample of concentration conversion of according to standard sample（The supernatant extracted）In egg Bai Hanliang.

C.GUS activity is calculated

According to A and B testing result, the GUS activity of testing sample is calculated, is represented with " nmol/min/mg albumen ".Specifically For, with " the 4-MU change in concentration of unit interval " divided by " protein content in testing sample ", obtain the albumen of unit mass The 4-MU change in concentration of unit interval, the GUS activity as in the unit interval.

Test in triplicate, results averaged.

As a result such as Figure 10, GmTIPp-2054 and GmTIPp-1546 Arabidopsis plants are turned all, the GUS tables in root It is all remarkably higher than up to activity in leaf, and has stronger expression activity in each transfer-gen plant root.And as control not The wildtype Arabidopsis thaliana Columbia of transgenosis（WT）With the root and Ye Zhongjun of the Arabidopsis plant for being transferred to pC13P1 empty carriers without GUS is expressed.

Claims (7)

1.DNA molecules, it is characterised in that：The DNA molecular is shown in 509-2054 nucleotides of sequence 2 in sequence table DNA molecular.

2. the recombinant vector containing DNA molecular described in claim 1.

3. the expression cassette containing DNA molecular described in claim 1.

4. the recombinant bacterium containing DNA molecular described in claim 1.

5. recombinant vector according to claim 2, it is characterised in that：The recombinant vector is at many grams of pC13P1 carriers The recombinant plasmid obtained after DNA molecular described in Long Weidianchu insertion claims 1.

6. expression cassette according to claim 3, it is characterised in that：The expression cassette is as described in promoter function DNA molecular, is started the target gene of expression, and transcription terminator composition by the DNA molecular；The DNA molecular is with work( Energy property mode is connected with the target gene, and the target gene is connected with the transcription terminator.

7. DNA molecular described in claim 1 starts the application in destination gene expression in plant；

It is described to be expressed as root-specific expression；

The plant is arabidopsis or soybean.