CN102417904B - Method for cloning arabidopsis root specificity expressed gene ARSK1 promoter - Google Patents
Method for cloning arabidopsis root specificity expressed gene ARSK1 promoter Download PDFInfo
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Abstract
The invention relates to the field of plant genetic engineering, in particular to a method for cloning an arabidopsis root specificity expressed gene ARSK1 promoter. The method comprises the following steps of: obtaining an arabidopsis root specificity expressed gene ARSK1 and obtaining the arabidopsis root specificity expressed gene ARSK1 promoter. The method provided by the invention can be used for cloning the arabidopsis root specificity expressed gene ARSK1 promoter, the promoter, which is expressed by plant root specificity, can be used for driving the expression of related function gene, so that the promoter has the capacity of specifically changing root form of the plant and obtaining corresponding drought-resisting plant; and the livability of the promoter with the plant being transferred in is 60 percent to 70 percent higher than that of a wild type plant of a control group at same drought conditions.
Description
Technical field
The present invention relates to plant genetic engineering field, particularly a kind of cloning process of Arabidopis thaliana root-specific expressing gene ARSK1 promotor.
Background technology
Promotor is the most important factor in the Factor of gene expression, determines substantially whether a gene is expressed, when expressed and where express.By the mode of action and function, promotor can be divided into constitutive promoter, specificity promoter and inducible promoter three major types substantially.At present; oneself enters the further investigation stage regulatory mechanism of tissue-specific promoter, and such promotor also has the sequence of some regulation and control specifically expressings usually except having basic structure; these sequences provide binding site for transcription factor, come regulatory gene to express by the interaction with protein.Under the driving of tissue-specific promoter, the expression of gene only occurs in specific tissue or organ usually, and often shows the characteristic of growing adjusting.
Root is the vitals of plant absorbing moisture and nutritive substance, and the height that the different expression system of Gent can be used for studying plant oozes stress-tolerance, phytoremediation and rhizosphere secretion etc.The phosphoric acid transferase gene of Arabidopis thaliana
PHT1Promotor with
GUSMerge, at agriculture bacillus mediated lower arabidopsis thaliana transformation and Arabidopis thaliana, tissue chemical analysis shows that albumen only in root hair and the epiblem expression of Arabidopis thaliana and Arabidopis thaliana, illustrates that this promotor is that Gent is different.The root-specific promoter mas2 such as Borisjuk, GFP and tobacco calprotectin gene (calreticulin) vector construction hexose transport protein, transgene tobacco water planting result of study shows, root cells can not only efficiently produce some target protein matter, and target protein matter can be secreted in the liquid nutrient medium.
The root system of plant plays an important role in plant drought, and building up of the lifting of root water uptake power and Root morphology to improving the suction force of plant, strengthens its drought resistance and have great importance undoubtedly.Most plant function genes such as hormone are synthetic, metabolism related gene, plant-growth, developmental regulation gene all have expression at over-ground part and the underground part of plant, mode by simple transgenosis or gene silencing on the whole regulatory gene in the expression of plant materials, can not specific realization to the regulation and control of root system of plant function and growth.
In order to address the above problem, need to utilize the expression of the specific expressed promoters driven correlation function gene of root system of plant, the plant water-absorbent be can improve to reach, root system of plant length and quantity increased, do not affect again the purpose that plant shoot is grown, do not damaged vegetation growth of plant, can obtain corresponding drought resisting plant by genetically modified means thus.Need the clone to obtain root-specific expressing gene promotor in the present research.
Summary of the invention
The invention provides a kind of cloning process of Arabidopis thaliana root-specific expressing gene ARSK1 promotor.
The objective of the invention is to be achieved through the following technical solutions:
A kind of cloning process of Arabidopis thaliana root-specific expressing gene ARSK1 promotor, the method may further comprise the steps:
I, obtain Arabidopis thaliana root-specific Gene A RSK1:
A, extract the total RNA of Arabidopis thaliana root and the total RNA of Arabidopis thaliana Ye respectively from the Arabidopis thaliana plant, carry out respectively the reverse transcription PCR reaction again, amplification obtains Arabidopis thaliana root cNDA sequence and Arabidopis thaliana leaf cNDA sequence respectively;
B, respectively take described Arabidopis thaliana root cNDA sequence and Arabidopis thaliana leaf cNDA sequence as template, respectively according to Arabidopis thaliana root-specific gene design primer, carry out respectively again sxemiquantitative PCR reaction, screening obtains Arabidopis thaliana root-specific Gene A RSK1;
II, obtain Arabidopis thaliana root-specific expressing gene ARSK1 promotor:
A, on-line analysis obtain the promotor of Arabidopis thaliana root-specific expressing gene ARSK1, clone to obtain described Arabidopis thaliana root-specific expressing gene ARSK1 promotor in Arabidopis thaliana root DNA again.
Arabidopis thaliana root-specific gene described in step I-B is respectively in the numbering of Arabidopis thaliana Information Resources Web Site: AT1G79580.1, AT1G62980.1, AT1G66470.1, AT1G27740.1, AT1G54970.1, AT3G10710.1, AT1G12560.1, AT2G26420.1, AT1G12040.1, AT1G62440.1, AT3G62680.1, AT2G26290.1.
The sequence of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor is SEQ ID No:1.
After the step II, carry out described Arabidopis thaliana root-specific expressing gene ARSK1 promoter function checking, may further comprise the steps:
A, on-line analysis obtain the transcription initiation site of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor, the functional element of taking advantage of a situation that contains;
B. described Arabidopis thaliana root-specific expressing gene ARSK1 promotor is carried out 5 ' end disappearance and process, reaction obtains ARSK1 promoter deletion body fragment through PCR;
C. described ARSK1 promoter deletion body fragment is made up respectively ARSK1 promoter deletion body GUS expression vector;
D, with described ARSK1 promoter deletion body GUS expression vector arabidopsis thaliana transformation plant, obtain the transformant plant, detect again.
The size of described ARSK1 promoter deletion body fragment is respectively 1981bp, 1460bp, 879bp, 762bp, 409bp, 358 bp;
Beneficial effect of the present invention is:
Because Arabidopis thaliana root-specific expressing gene ARSK1 promotor is cloned in the present invention, this promotor is the specific expressed promotor of root system of plant, can drive the expression of correlation function gene, so this promotor has Root Morphology, the corresponding drought resisting plant potentiality of acquisition that specifically change plant: the plant that changes this promotor over to, under equal drought condition, survival rate is than the high 60-70% of wild-type plant that compares control group.
Description of drawings
Among Fig. 1: the embodiment 1, the amplified production electrophoresis result of 12 Arabidopis thaliana root-specific genes.
Among Fig. 2: the embodiment 1, the gene mapping of arabidopsis gene ARSK1.
Among Fig. 3: the embodiment 1, the gene mapping of arabidopsis gene promotor ARSK1.
Among Fig. 4: the embodiment 1, the amplified production electrophoresis result of Arabidopis thaliana root-specific expressing gene ARSK1 promotor.
Among Fig. 5: the embodiment 1, the analytical results of the cis-acting elements that contains in the Arabidopis thaliana root-specific expressing gene ARSK1 promoter sequence.
Among Fig. 6: the embodiment 1, the structure diagram of 6 kinds of ARSK1 promoter deletion bodies.
Among Fig. 7: the embodiment 1, the structure diagram of 6 kinds of ARSK1 promoter deletion body GUS expression vectors.
Among Fig. 8: the embodiment 1,6 kinds of ARSK1 promoter deletion body GUS expression vectors are at the Gus colored graph of the expression amount of transformation of Arabidopsis thaliana body plant.
Embodiment
Embodiment 1:
A kind of cloning process of Arabidopis thaliana root-specific expressing gene ARSK1 promotor, the method may further comprise the steps:
I, obtain Arabidopis thaliana root-specific Gene A RSK1:
A, extract the total RNA of Arabidopis thaliana root and the total RNA of Arabidopis thaliana Ye respectively from the Arabidopis thaliana plant, carry out respectively the reverse transcription PCR reaction again, amplification obtains Arabidopis thaliana root cNDA sequence and Arabidopis thaliana leaf cNDA sequence respectively;
Concrete is operating as:
Root and the leaf of Arabidopis thaliana plant of getting respectively growth 20d is material, extracts respectively total RNA of Arabidopis thaliana root and leaf with the Trizol method, carries out respectively reverse transcription PCR and reacts, and obtains respectively Arabidopis thaliana root cDNA and Arabidopis thaliana leaf cDNA;
The system of described reverse transcription PCR reaction is: RNA: 1.2 μ g, and Oligo (dT) 15:2 μ l,
M-MLV buffer:5 μ l, dNTP Mix:1.25 μ l, M-MLV:1 μ l, RNase inhibitor:0.6 μ l, the ddH after DEPC processes
2O: supply 25 μ l;
The program of described reverse transcription PCR reaction is:
Add above-mentioned oligo (dT) 15 rear 70 ℃ of reactions 5 minutes, then add other reagent in the above-mentioned system of reverse transcription PCR, 37 ℃ were reacted 1 hour, and obtained described Arabidopis thaliana root cNDA sequence and Arabidopis thaliana leaf cNDA sequence.
B, respectively take described Arabidopis thaliana root cNDA sequence and Arabidopis thaliana leaf cNDA sequence as template, respectively according to 12 Arabidopis thaliana root-specific gene design primers, carry out again sxemiquantitative PCR reaction, screening obtains Arabidopis thaliana root-specific Gene A RSK1;
Described 12 Arabidopis thaliana root-specific genes are at Arabidopis thaliana Information Resources Web Site (TAIR), the numbering of (network address is http://www.arabidopsis.org/) and to put down in writing the literature reference of this gene as follows:
SMB（AT1G79580.1）：Tom?Bennett,?Albert?van?den?Toorn,?Gabino?F,(2010).?SOMBRERO,?BEARSKIN1,?and?BEARSKIN2?Regulate?Root?Cap?Maturation?in?Arabidopsis?.?Plant?Cell.22:?640–654；
EXP18（AT1G62980.1）：Hyung-Taeg?Cho,?Daniel?J.?Cosgrove,(2002).?Regulation?of?Root?Hair?Initiation?and?Expansin?Gene?Expression?in?Arabidopsis.Plant?cell.?14:3237–3253；
RHD6?（AT1G66470.1）：Jill?S.?Parker,Alison?C.?Cavell,Liam?Dolan,(2000).?Genetic?Interactions?during?Root?Hair?Morphogenesis?in?Arabidopsis.?Plant?Cell.?12:1961–1974；
RSL4 （AT1G27740.1）：Keke?Yi,?Beno t?Menand,?Elizabeth?Bell,Liam?Dolan,(2009).?A?basic?helix-loop-helix?transcription?factor?controls?cell?growth?and?size?in?root?hairs.nature?genetics.10:1038；
PRP1 （AT1G54970.1）：Su-Kyung?Won,?Yong-Ju?Lee,?Ha-Yeon?Lee,?Yoon-Kyung?Heo,?Misuk?Cho,?and?Hyung-Taeg?Cho,(2009).?cis-Element-?and?Transcriptome-Based?Screening?of?Root?Hair-Speci c?Genes?and?Their?Functional?Characterization?in?Arabidopsis.Plant?Physiology.150:1459-1473；
RHS12（AT3G10710.1）：Su-Kyung?Won,?Yong-Ju?Lee,?Ha-Yeon?Lee,?Yoon-Kyung?Heo,?Misuk?Cho,?and?Hyung-Taeg?Cho,(2009).?cis-Element-?and?Transcriptome-Based?Screening?of?Root?Hair-Speci c?Genes?and?Their?Functional?Characterization?in?Arabidopsis.Plant?Physiology.150:1459-1473；
EXP7 （AT1G12560.1）：Hyung-Taeg?Cho1,Daniel?J.?Cosgrove,(?2002).Regulation?of?Root?Hair?Initiation?and?Expansin?Gene.?Plant?Cell.?14:3237–3253；
PIP5K3（AT2G26420.1）：Irene?Stenzel,Till?Ischebeck,Sabine?Konig,(2008).?The?Type?B?Phosphatidylinositol-4-Phosphate?5-Kinase?3?Is?Essential?for?Root?Hair?Formation?in?Arabidopsis?thaliana.?Plant?Cell.?20:?124–141；
LRX1 （AT1G12040.1）：Nicolas?Baumberger,?Christoph?Ringli?,?Beat?Keller,(2001). The?chimeric?leucine-rich?repeat/extensin?cell?wall?protein?LRX1?is?required?for?root?hair?morphogenesis?in?Arabidopsis?thaliana.?Genes?Dev.?15:?1128-1139；
LRX2 （AT1G62440.1）：N.Baumberger,M.Steiner,U.Ryser,(2003).Synergistic?interaction?of?the?two?paralogous?Arabidopsis?genes?LRX1and?LRX2?in?cell?wall information?duiing?root?hair?development.Plant?Journal,35:71-81；
PRP3 （AT3G62680.1）： Christine?Bernhardt?,Mary?L.?Tierney,(2000).?Expression?of?AtPRP3,?a?Proline-Rich?Structural?Cell?Wall?Protein?from?Arabidopsis,?Is?Regulated?by?Cell-Type-Specific?Developmental?Pathways?Involved?in?Root?Hair?Formation1.?Plant?Physiology.?122:?705–714；
ARSK1（AT2G26290.1）：Inhwan?Hwang,Howard?M.Goodman,(1995).An?Arabidopsis?thaliana?root-specific?kinase?homolog?is?induced?by?dehydration,ABA,and?NaCl.Plant?Journal.8(1):37-43；
Utilize the sxemiquantitative PCR reaction primer of described 12 the Arabidopis thaliana root-specific genes of Oligo6.0 software design; Because it is more to detect gene, for improving conventional efficient, when design of primers, should make annealing temperature keep concentrated, consistent as far as possible, namely annealing temperature is 51 ℃; Concrete primer sequence is as follows:
SMB：
Upper：GGAGGAGGAGCTTCTTTAC?，
Lower：CCAGAAACGACCAGTCTC?CA；
EXP18：
Upper：CG?CAAGTGCCTGGTTATTTTA，
Lower：CGG?AGCAGCATTATAAGCGT；
RHD6：
Upper：GGCACTCGTTAATGACCATC，
Lower：G?GTGATCAGATTCGAATTCC；
RSL4：
Upper：GGACGTTTTTGTTGATGGTG，
Lower：CG?TACATCCATA?GATCATCC；
PRP1：
Upper：GCTGATTATTACGCTCCCTC?，
Lower：GTT?GGATCACTGTAGATGAC；
RHS12：
Upper：CGC?TGAGCTTCTTGACCTAAC，
Lower：CGGT?AGAATTGGCGTTGTGC?；
EXP7：
Upper：GTT?CGTCGCTGGATACTATC，
Lower：GCAA?CGTTCCAAGCATAGAT；
PIP5K3：
Upper：GCTGCCG?AGATTAGAATAGT，
Lower：GCACCACCA?CTCTCCAAAA?G；
LRX1：
Upper：GGTTCAGATGTTTGCTCCT，
Lower：CTTCTAGGCTCTTCATGTTA?C；
LRX2：
Upper：GGC?TCTGATGTTTGTTCCTAC，
Lower：GG?TTAGAGAGCTGACAAATGC；
PRP3：
Upper：CCACCCGTTT?ACAAGCCTAC，
Lower：GAGCTCCAAGTTCTTGTCCG；
ARSK1：
Upper：CG?CAAGCAAATACGAAGGAG，
Lower：CGTATGTTCGCCTTCAG?GTC；
The system of described sxemiquantitative PCR reaction is:
Ex?Taq?buffer?：2?μl，dNTP?Mix?(2.5?mM?each)?：1.6?μl，Primer?Mix?(5?μM?each)?：2.4?μl，cDNA?：2.4?μl，Ex?Taq?：0.2?μl，ddH2O?：11.4?μl；
The program of described sxemiquantitative PCR reaction is: 94 ℃ of denaturation 5 min; 94 ℃ of sex change 45 s, 55 ℃ of annealing 45 s, 72 ℃ are extended 1 min, totally 30 circulations; 72 ℃ are extended 7 min; 4 ℃ of preservations.
Obtain respectively the amplified production of 12 Arabidopis thaliana root-specific genes by described sxemiquantitative PCR reaction; Amplified production with above-mentioned 12 Arabidopis thaliana root-specific genes detects again, and screening obtains Arabidopis thaliana root-specific Gene A RSK1;
Concrete is operating as:
The amplified production of above-mentioned 12 Arabidopis thaliana root-specific genes is carried out electrophoresis on 1% sepharose; Electrophoresis result as shown in Figure 1, swimming lane M is DNA marker II, swimming lane 1 is confidential reference items actin, swimming lane 2-13 is respectively the amplified production of above-mentioned 12 Arabidopis thaliana root-specific genes; Swimming lane 4 shows that the expression amount of described Arabidopis thaliana root-specific Gene A RSK1 in the root of Arabidopis thaliana is large, expresses hardly in the leaf of Arabidopis thaliana.
II, obtain Arabidopis thaliana root-specific expressing gene ARSK1 promotor:
A, on-line analysis obtain described Arabidopis thaliana root-specific expressing gene ARSK1 promotor, clone to obtain described Arabidopis thaliana root-specific expressing gene ARSK1 promotor in Arabidopis thaliana root DNA again.
Concrete is operating as:
A. analyze the promotor that obtains Arabidopis thaliana root-specific expressing gene ARSK1 in the NCBI website:
Find Arabidopis thaliana ARSK1 gene order in the NCBI website, network address is:
http://www.ncbi.nlm.nih.gov/nuccore/NM_128186.2；
Analyze the map view of ARSK1 gene in the NCBI website, network address is:
http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=3702&chr=2&MAPS=rna-r&cmd=focus&fill=40&query=uid(7448160)&QSTR=NM%5F128186%2E2；
Fig. 2 is the gene mapping of arabidopsis gene ARSK1, can find out that thus this gene contains 5 exons, 4 introns;
Analyze the map view of ARSK1 gene promoter in the NCBI website, network address is:
http://www.ncbi.nlm.nih.gov/projects/mapview/maps.cgi?TAXID=3702&CHR=2&MAPS=rna-r&QSTR=NM_128186.2&QUERY=uid%287448160%29&BEG=11%2C194%2C200&END=11%2C196700&oview=default；
Fig. 3 is the collection of illustrative plates of ARSK1 gene promoter, and the 1st front about 2500 the fragment of exon is promoter region;
Find the sequence of the Arabidopis thaliana ARSK1 upstream region of gene of having announced in the NCBI website, network address is:
http://www.ncbi.nlm.nih.gov/nuccore/NC_003071.7?from=11194200&to=11196700&report=fasta；
The page of above website links has shown the 1st front sequence of about 2500 of exon of Arabidopis thaliana ARSK1 gene, present embodiment intercepting 1981bp wherein, see sequence table SEQ ID No:1 for details, as the final described Arabidopis thaliana root-specific expressing gene ARSK1 promotor out that needs the clone;
B. obtain described Arabidopis thaliana root-specific expressing gene ARSK1 promotor at Arabidopis thaliana root dna clone;
Get growth 20-28d, be preferably the Arabidopis thaliana plant of 25d, adopt the CTAB method to extract Arabidopis thaliana root DNA; Carry out the PCR reaction take above-mentioned Arabidopis thaliana root DNA as template, obtain the promotor of Arabidopis thaliana ARSK1 gene; This Arabidopis thaliana is that mouse ear mustard belongs to the Latin literary fame
Arabidopsis thaliana
Described PCR reaction system is:
5×?buffer：5μl，dNTP?Mix?(2.5?mM?each)：?2?μl，Primer?Mix?(5?μM?each)?：2?μl，cDNA：?1?μl，PrimeSTAR?：0.25?μl，ddH
2O?：14.75?μl；
The primer of described PCR reaction is sequence:
Upper (band restriction enzyme site Hind III):
5-AAGCTT CCATGATTAACCTACGCTTC---3，
Lower(band restriction enzyme site Nco I): 5-CCATGG GC CAT TTT CAA CTT CTT CTT TTG----3;
Described PCR response procedures is:
94 ℃ of denaturation 5 min; 98 ℃ of sex change 10s, 50 ℃ of annealing 15 s, 72 ℃ are extended 2 min, totally 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations;
The amplified production of above-mentioned PCR reaction is carried out electrophoresis on 1% sepharose; Electrophoresis result as shown in Figure 4, swimming lane M is DNA marker III, the stronger stripe size of brightness is 1981bp among the swimming lane 1-2;
III, the checking of Arabidopis thaliana root-specific expressing gene ARSK1 promoter function:
A, on-line analysis obtain the transcription initiation site of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor, the functional element of taking advantage of a situation that contains;
Predict the transcription initiation site of described rice root-specific expressing gene ARSK1 promotor in the on-line analysis of Softberry website, above-mentioned Softberry on-line analysis network address is:
http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind；
Analytical results shows: the transcription initiation site of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor may be arranged in the base T from the 1866th at 5 of sequence table SEQ ID No:1 ' end;
The on-line analysis of Place website predict that described rice root-specific expressing gene ARSK1 promotor contains cis-acting elements, above-mentioned Place on-line analysis network address is http://www.dna.affrc.go.jp/PLACE/signalscan.html;
Fig. 5 is the analytical results of the cis-acting elements that contains of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor: described Arabidopis thaliana root-specific expressing gene ARSK1 promotor (SEQ ID No:1 in the sequence table) be TATABOX from 5 ' end 1834-1840 position Nucleotide, from 5 ' end 1567-1570 position, the 1621-1624 position, 1696-1699 position Nucleotide is CAATBOX; Nucleotide is the different Expression element NODCON2GM of Gent from 5 ' end 1102-1106 position; The 285-289 position, the 521-525 position, the 1219-1223 position, the 1572-1576 position, the 1581-1585 position, 1623-1627 position Nucleotide is the different expression related elements of Gent ROOTMOTIFTAPOX1.
B. described Arabidopis thaliana root-specific expressing gene ARSK1 promotor is carried out 5 ' end disappearance and process, reaction obtains 6 kinds of ARSK1 promoter deletion body P1, P2, P3, P4, P5, P6 fragment through PCR;
Concrete is operating as:
Take Arabidopis thaliana root cNDA sequence as template, design primer according to above-mentioned 6 kinds of ARSK1 promoter deletion body P1, P2, P3, P4, P5, P6 respectively, carry out again the PCR reaction, obtain respectively the amplified production of above 6 kinds of ARSK1 promoter deletion bodies, recycle again, obtain 6 kinds of ARSK1 promoter deletion body fragments; Be the details of above 6 kinds of ARSK1 promoter deletion bodies in the following table; Fig. 6 is the structure diagram of above 6 kinds of EXP18D promoter deletion bodies.
The system of described PCR reaction is:
5 * buffer:5 μ l, dNTP Mix (2.5 mM each): 2 μ l, Primer Mix (5 μ M each): 2 μ l, Arabidopis thaliana root cDNA: 1 μ l, PrimeSTAR:0.25 μ l, ddH
2O:14.75 μ l;
The program of described PCR reaction is:
94 ℃ of denaturation 5 min; 98 ℃ of sex change 10 s, 50 ℃ of annealing 15 s, 72 ℃ are extended 2 min, totally 30 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations;
C. above-mentioned 6 kinds of EXP18D promoter deletion body P1, P2, P3, P4, P5, P6 fragments are made up respectively ARSK1 promoter deletion body GUS expression vector:
A. respectively above-mentioned 6 kinds of ARSK1 promoter deletion body fragments are connected on the PMD18-T plasmid vector, carry out again conversion processing, shaking culture processing, plasmid extraction processing, obtain 6 kinds of ARSK1 promoter deletion body fragment-PMD18-T plasmid vectors: P1-PMD18-T plasmid vector, P2-PMD18-T plasmid vector, P3-PMD18-T plasmid vector, P4-PMD18-T plasmid vector, P5-PMD18-T plasmid vector, P6-PMD18-T plasmid vector;
Linked system in the described connection is: the solution I: 1 μ l, PMD18-T plasmid vector: 0.5 μ l, 6 kinds of ARSK1 promoter deletion body fragments: 4.5 μ l; 16 ℃ of connections of this linked system are spent the night, and obtain respectively 6 kinds and connect product;
Described conversion processing is: connect product 5 μ l with above-mentioned every kind and add in the 100 μ l competent escherichia coli cell DH5 α competent cells, mixing was placed 30 minutes on ice gently; 42 ℃ of heat shocks are 45 seconds again, place on ice 2 minutes; Add again 500 μ lLB nonreactive substratum, in 37 ℃ of rotating speed 150rpm shaking culture 1 hour; With rotating speed 4000rpm centrifugal 5 minutes again, sop up 400 μ l left and right sides supernatant liquors with rifle, remaining is coated on the foster base that contains penbritin with spreading rod; In 37 ℃ of incubated overnight, obtain single bacterium colony again;
Described shaking culture is treated to: picking is the above-mentioned single bacterium colony that grows respectively, adds the LB liquid nutrient medium, and 37 ℃ of overnight shakings are cultivated; Get respectively again bacterium liquid and carry out bacterium liquid PCR, select the correct bacterium liquid of checking;
Described plasmid extraction is treated to: the bacterium liquid that above-mentioned checking is correct carries out the plasmid extraction processing with the DV801A type plasmid DNA a small amount of purification kit of precious biotechnology (Dalian) company limited, obtains 6 kinds of ARSK1 promoter deletion body fragment-PMD18-T plasmid vectors: P1-PMD18-T plasmid vector, P2-PMD18-T plasmid vector, P3-PMD18-T plasmid vector, P4-PMD18-T plasmid vector, P5-PMD18-T plasmid vector, P6-PMD18-T plasmid vector;
B. respectively above-mentioned 6 kinds of ARSK1 promoter deletion body fragment-PMD18-T plasmid vectors are carried out enzyme with restriction endonuclease Hind III Nco I respectively and cut processing, obtain 6 kinds of ARSK1 promoter deletion body enzymes and cut small segment, molecular weight (not comprising Hind III Nco I restriction enzyme site) is about respectively: P1 is 1981bp, P2 is 1460bp, P3 is 879bp, P4 is 762bp, and P5 is 409bp, and P6 is 358bp;
C. expression vector PCAMBIA1391 is carried out enzyme with restriction endonuclease Hind III Nco I and cut processing, obtain molecular weight and be the carrier large fragment about 10780bp;
Above-mentioned enzyme is cut in the processing, and the enzyme system of cutting is: 10 * buffer:2 μ l; Hind III: 1 μ l, Nco I: 1 μ l, plasmid: 10 μ l, ddH
2O; 6 μ l; In 37 ℃ of reactions 3 hours;
D. respectively above-mentioned 6 kinds of ARSK1 promoter deletion body enzymes being cut small segment, molecular weight is that carrier large fragment about 10780bp recycles, and carries out respectively ligation again, obtains respectively connecting product;
In the above-mentioned ligation, linked system is: 10 * buffer:1 μ l, and the carrier large fragment: 1 μ l,
Every kind of ARSK1 promoter deletion body enzyme is cut small segment: 7 μ l, T4DNA ligase enzyme: 1 μ l; 16 ℃ of connections are spent the night;
Connect product with reference to the method among III-C-a with 6 kinds and carry out respectively conversion processing, shaking culture processing, plasmid extraction processing, obtain 6 kinds of ARSK1 promoter deletion body GUS expression vectors: plasmid vector PCAMBIA-GUS-ARSK1-P1, plasmid vector PCAMBIA-GUS-ARSK1-P2, plasmid vector PCAMBIA-GUS-ARSK1-P5, plasmid vector PCAMBIA-GUS-ARSK1-P4, plasmid vector PCAMBIA-GUS-ARSK1-P5, plasmid vector PCAMBIA-GUS-ARSK1-P6;
It is the structure diagram of above 6 kinds of ARSK18D promoter deletion body GUS expression vectors among Fig. 7.
D, with described 6 kinds of ARSK1 promoter deletion body GUS expression vector arabidopsis thaliana transformation plant, obtain T3 for the transformant plant, detect again.
A. change described 6 kinds of ARSK1 promoter deletion body GUS expression vectors over to Arabidopis thaliana, obtain T3 for Arabidopis thaliana plant transformant;
Test materials:
Arabidopis thaliana (Arabidopsis thaliana) is the Columbia-0 wild-type, bacterial strain and plasmid: intestinal bacteria (Esche-richia coli) DH5a bacterial strain, Agrobacterium (Agrobacterium turnefaciens) EHA105(GV3101) bacterial strain;
Main agents:
70% ethanol for disinfection, bleaching and disinfecting agents (50% household bleach, 50% sterilized water, 0.05% Tween-20), Silwet L-77, sucrose (SILVER REAGENT), glucose (SILVER REAGENT), edible sucrose, N.F,USP MANNITOL etc.;
Substratum:
LB substratum 1L:10 g Tryptones (Tryptone); 5 g yeast extracts (Yeast Extract); 5 g NaC1;
The infiltration substratum: 5% sucrose, 0.3%-0.5% Silwet L-77, PH 5.7;
Screening culture medium: 1/2MS; 8 g/L agar; 50 μ g Kan; PH 5.8.
(a). the cultivation of Arabidopis thaliana and processing:
With the Arabidopis thaliana planting seed of vernalization 3～4 d (vegetable mould: vermiculite=1:1) in the Artificial Soil that soaks with PNS (Plant Nutrition Solution) nutrient solution, and with on the preservative film cover, place artificial culture chamber illumination cultivation, take off film (culturing room's condition: relative humidity 70% after 1 month, constant temperature (23 ± 2) ℃, intensity of illumination 4 000 lx, periodicity of illumination are dark 8 h, illumination 16 h); Treat plant culturing to nascent about 5～15 cm of inflorescence, when secondary inflorescence has just formed the bud shape, the nascent inflorescence of removal is of value to growing of secondary inflorescence, is convenient to infiltration and transforms; The infiltration conversion processing should be finished in removing nascent inflorescence 3～5 d; And need fully water the front 1 d plant that infiltration transforms, so that plant stomata fully opens when transforming;
(b). the cultivation of bacterium liquid: the Agrobacterium bacterium liquid 10mL that has transformed 6 kinds of ARSK1 promoter deletion body GUS expression vectors that will prepare in 1 d evening before conversion, changes large bottle incubated overnight over to; Agrobacterium liquid OD600 is about 2.0 when taking out use on the 2nd day; In room temperature centrifugal treating (5000rpm, 15 min), abandon supernatant liquor, the Agrobacterium precipitation is suspended in the infiltration nutrient solution of about 3 times of volumes, make OD600 about 0.8;
(c). Agrobacterium is infected the Arabidopis thaliana inflorescence: infected 15 seconds in the over-ground part immersion agrobacterium suspension with plant; Or draw agrobacterium suspension with glue head dropper and drop by drop every flower is infected; The plant that to infect with preservative film again (T0 is for plant) cover gets up to keep humidity, puts dark 10 h of cultivation in the people culturing room, then places normal culture condition, can remove preservative film behind 2～3 d; 1 all right and lefts can water after transforming, and regularly continue to infect several times, continue to be cultured to the plant maturation, and sowing (T1 generation) was placed in the dry environment about 1 week, can screen transformant;
(d). Arabidopis thaliana T1 is for the screening of seed: T1 is used first 70% alcohol-pickled sterilization for seed, use the bleaching and disinfecting agents solution-treated again; Seed after the sterilization is coated on the screening and culturing primary surface equably, behind 4 ℃ of placement 2～3 d, moves in the artificial culture chamber and cultivates; Then the transformant of determining is changed over to and be cultured to maturation in the soil, results seed (T2 is for seed);
(e). above-mentioned T2 is screened, cultivates according to the method among the d for seed, and T3 is for seed for results;
(f). above-mentioned T3 is obtained T3 for homozyous seed for seed through twice screening, in the MS culture medium culturing, obtain T3 for transformation of Arabidopsis thaliana body plant after the sterilization;
B. described T3 is carried out the GUS histochemical stain for transformation of Arabidopsis thaliana body plant and process, observe coloration result;
(a). preparation GUS staining fluid and destainer:
Prepare described GUS staining fluid: get 5 mg X-gluc and be dissolved in 410 μ l diformazan methane amides, be distributed into 20 uL/ pipe, place-20 ℃ of refrigerators to store, the time spent gets a pipe and adds following solution and be made into staining fluid (now with the current):
50 mM sodium phosphate buffers (pH7.0): 760 ul (autoclaving), 50 mM K
3[Fe (CN)
6]: 10 μ l(filtration sterilizations), 50 mM K
4[Fe (CN)
6]: 10 μ l(filtration sterilizations), 0.5 M EDTA: 10 μ l(autoclavings),
Triton X-100: 1 μ l, methyl alcohol: 200 ul;
Described destainer is the ethanolic soln of concentration 70%;
(b). get a-(f) the step Arabidopis thaliana plant transformant of cultivating ten days puts into the processing of dyeing of described GUS staining fluid, temperature is 37 ℃, the time is more than 3 hours;
(c). decolour with destainer and to process 12-24 hour, observe at microscopically, take pictures; The result is as follows:
Fig. 8 is that above 6 kinds of ARSK1 promoter deletion body GUS expression vectors are at the Gus colored graph of the expression amount of transformation of Arabidopsis thaliana body plant; Fig. 8 comprises P1-P6 totally 6 parts, and the plant in the left side of every part is the Arabidopis thaliana plant transformant that changes corresponding ARSK1 promoter deletion body GUS expression vector over to, the right side be the wild-type plant in contrast; By the P1-P3 part as seen, ARSK1 promoter deletion body GUS expression vector is expressed at the root of Arabidopis thaliana plant specifically; By the P1-P6 part as seen, along with the shortening of promoter sequence length,
GusThe expression amount of gene in root reduces gradually; When promotor shortens to 762bp, no longer have activity, illustrate-764bp ~-may there be the controlling element relevant with the different expression of Gent in the 647bp zone.
In the present embodiment, use various restriction enzymes, T4DNA ligase enzyme, PrimeSTAR enzyme all available from Takara company; Various Marker are available from Takara company; Various PCR reaction kits are available from Takara company; DNA purification kit, PMD18-T plasmid vector test kit, expression vector PCAMBIA1391 test kit are all available from Promega company; It is DV805A that dna gel reclaims the test kit model, available from precious biotechnology (Dalian) company limited; Other routine biochemistry reagent such as CTAB, Trizol and test kit are glad through biotech company of section through biotech company of HTC of section and Beijing available from Beijing.
In the present embodiment, various PCR primers are synthetic to be finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd with dna sequencing work.
In the present embodiment, PTC-100 Peltier Thermal Cycler gene-amplificative instrament is used in various PCR reactions, available from MJ Research Inc company; Described agarose gel electrophoresis uses UVP Gel Documentation gel analysis system, available from genome company; Described agarose gel electrophoresis uses the DYCP-31DN electrophoresis apparatus, available from Beijing 61 instrument companies.
The genetically engineered elementary operation methods such as in the present embodiment, the DNA extraction of employing, PCR, enzyme are cut, connected, conversion are recorded in the specification sheets of each test kit; Above-mentioned elementary operation method also can be with reference to (work such as (U.S.) J. Pehanorm Brooker, Science Press publish in 2002) in " the molecular cloning experiment guide third edition ", (Wang Guanlin in " plant genetic engineering philosophy and technique ", the Fang Hongjun work, Science Press, 1998).
Claims (2)
1. the cloning process of an Arabidopis thaliana root-specific expressing gene ARSK1 promotor, it is characterized in that: the method may further comprise the steps:
I, obtain Arabidopis thaliana root-specific Gene A RSK1:
A, extract the total RNA of Arabidopis thaliana root and the total RNA of Arabidopis thaliana Ye respectively from the Arabidopis thaliana plant, carry out respectively the reverse transcription PCR reaction again, amplification obtains Arabidopis thaliana root cDNA sequence and Arabidopis thaliana leaf cDNA sequence respectively;
B, respectively take described Arabidopis thaliana root cDNA sequence and Arabidopis thaliana leaf cDNA sequence as template, respectively according to Arabidopis thaliana root-specific gene design primer, carry out respectively again sxemiquantitative PCR reaction, screening obtains Arabidopis thaliana root-specific Gene A RSK1;
II, obtain Arabidopis thaliana root-specific expressing gene ARSK1 promotor:
A, on-line analysis obtain the promotor of Arabidopis thaliana root-specific expressing gene ARSK1, clone to obtain described Arabidopis thaliana root-specific expressing gene ARSK1 promotor in Arabidopis thaliana root DNA again;
Arabidopis thaliana root-specific gene described in step I-B is respectively in the numbering of Arabidopis thaliana Information Resources Web Site: AT1G79580.1, AT1G62980.1, AT1G66470.1, AT1G27740.1, AT1G54970.1, AT3G10710.1, AT1G12560.1, AT2G26420.1, AT1G12040.1, AT1G62440.1, AT3G62680.1, AT2G26290.1;
The sequence of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor is SEQ ID No:1;
After the step II, carry out described Arabidopis thaliana root-specific expressing gene ARSK1 promoter function checking, may further comprise the steps:
A, on-line analysis obtain the transcription initiation site of described Arabidopis thaliana root-specific expressing gene ARSK1 promotor, the functional element of taking advantage of a situation that contains;
B. described Arabidopis thaliana root-specific expressing gene ARSK1 promotor is carried out 5 ' end disappearance and process, reaction obtains ARSK1 promoter deletion body fragment through PCR;
C. described ARSK1 promoter deletion body fragment is made up respectively ARSK1 promoter deletion body GUS expression vector;
D, with described ARSK1 promoter deletion body GUS expression vector arabidopsis thaliana transformation plant, obtain the transformant plant, detect again.
2. the cloning process of Arabidopis thaliana root-specific expressing gene ARSK1 promotor according to claim 1, it is characterized in that: the size of described ARSK1 promoter deletion body fragment is respectively
1981bp，?1460bp，?879bp，?762bp，?409bp，358?bp。
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| CP002685.1;lin et al;《Genbank》;20110613 * |
| lin et al.CP002685.1.《Genbank》.2011, |
| lin，X et al.NM.128186.2.《Genbank》.2011, * |
| 姚秋林.水稻根特异性表达启动子的克隆及功能分析.《中国优秀硕士学位论文全文数据库 农业科技辑》.2011,(第6期),2.2.2-2.2.11部分 |
| 水稻根特异性表达启动子的克隆及功能分析;姚秋林;《中国优秀硕士学位论文全文数据库 农业科技辑》;20110615(第6期);2.2.2-2.2.11部分；3.1-3.8部分 * |
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