CN102604955A - Application of tandem repeat sequence capable of improving expression activity of plant gene - Google Patents

Abstract

The invention relates to application of a tandem repeat sequence capable of improving expression activity of a plant gene, namely application of a fragment containing five tandem repeat TTTACAC sequences on a zinc finger transcription factor At5g15840 gene promoter of Arabidopsis thaliana in improving expression of a target gene in a transgenic plant. When the tandem repeat sequence is deleted, the activity of the promoter is sharply lowered, and therefore, the tandem repeat sequence has the characteristic of enhancing gene expression. By use of the tandem repeat sequence provided by the invention, the transgene development of a plant is performed, so that the expression of the target gene is improved, and the important economic and social benefits are achieved.

Description

A kind of application that can improve the active tandem repetitive sequence of gene expression in plants

Technical field

The invention belongs to plant tandem repetitive sequence field, particularly about the one-stage serial Tumor-necrosis factor glycoproteins on a kind of zinc finger transcription factor At5g15840 gene that improves activity of gene expression, and the purposes of this tandem repetitive sequence in the plant transgene exploitation.

Background technology

The section of DNA fragment that tandem repetitive sequence is made up of several Nucleotide, its ubiquity and be randomly distributed in protokaryon and the eukaryotic gene group in, its multiplicity have the height variability.Tandem repetitive sequence has following characteristic usually: 1. nonrandom distribution in genome; 2. extensively exist in the genome, and the content of tandem repetitive sequence and genome size do not have dependency; 3. tandem repetitive sequence may reside in non-translational region (UTR), exon, intron or intergenic region; 4. the tandem repetitive sequence height makes a variation and presents polymorphum, therefore is widely used in genetic marker; 5. tandem repetitive sequence is different in the different regions performance of different taxonomical groups and same taxonomical group.

Tandem repetitive sequence has been participated in many biological procedureses, in molecular evolution, has important biological significance.At first; Tandem repetitive sequence can influence chromosome organization: there is the tandem repetitive sequence of (CT) 17 in wheat (Triticum aestium) 4A Chromosome G WM601 locus position; This sequence is relevant with the chromosomal tissue of 4A, and (Heredity 2002,89:127-132).Fruit bat Idefix and Gypsy repeat element are participated in chromosomal high-level organization and generegulation, and the SINE element can be through expression (Mol Cell 2008, the 29:154-156 of the special promotor activated gene of Looping; Cell2008,134:14-16); Second; Tandem repetitive sequence can also change dna structure, influence the function of kinetochore and telomere: in many species; Zone, chromosomal kinetochore comprises a large amount of tandem repetitive sequences usually; Repeat (AATGG) n like human kinetochore and can form double-stranded folding hair clip dna structure, and then (Genetica 1999,106:15-36) to influence centric tissue.And telomere repeats the length of keeping telomere, participates in nuclear tissue-equivalent appearance and play a significant role also that (Yeast 2009,26:125-138; PloS One 2008,3:e3249); The 3rd, tandem repetitive sequence influences genetic recombination: research shows that many tandem repetitive sequences are focuses of genetic recombination, and different repeating units and number of iterations all can influence the efficient of genetic recombination; The 4th, tandem repetitive sequence has also been participated in dna replication dna and cell cycle: in mouse cell, include (GA) ₂₇Specific fragment can stop the generation of dna replication dna.Equally, tandem repetitive sequence also can influence the enzyme of control cell cycle, for example; Some regulating cell round-robin gene (CHK1, hMSH3, hMSH6; BAX, IGFIIR, TGFbetaIIR; E2F4 h and BRCA2) in all comprise short tandem repetitive sequence, they the cell fidelity of reproduction keep and growth control in play a significant role.In case this tandem repetitive sequence is undergone mutation (insertion of repeating unit or disappearance), it is active all to influence these expression of gene, thereby cell cycle is got muddled, finally cause canceration etc. (In J Oncol 2000,16:133-139); The 5th, tandem repetitive sequence also influences translation: research shows some gene (infB of intestinal bacteria (E.coli) and Bacillus subtilus (B.subtilis); AceF/pdhC; Eno, rplI, OMPa; OMF and tolA) in the AGCT tandem repetitive sequence can cause translation frameshift (Electrophoresis 1998,19:515-527); At last, many tandem repetitive sequences can combine various adjusting albumen: for example, CGG on FMR1 gene series connection repetition can by some specific proteinses such as CGGBP albumen (J Biol Chem 1997,272:16761-16768); (Nucleic Acids Res2000,28:3197-3205) (Proc Natl AcadSci USA 1989 86:8737-8741) waits combination, thereby influences gene activity Pur albumen with the Egr transcription factor.

Discover that some human genetic diseases are also relevant with tandem repetitive sequence.1991, people found (CGG) n and the repetition of (CAG) n trinucleotide relevant (Cell 1991,67:1047-1058 with human inheritance's disease first respectively in FMR1 (amentia 1) gene and androgen receptor gene; Nature 1991,352:77-79).These repeated amplifications can form different two mutants, and mutation rate is relevant with repeat length.These a large amount of repetitions are not neutral biologically; But participated in the generation of numerous disease: like CGG series connection multiplicity is that the FMR1 allelotrope of 55-200 causes degenerative sacred disease (Ment Retard Dev Disabil Res Rev 2004 usually; 10:25-30) with ovary sterile (Semin Reprod Med2000,18:59-66; Am J Med Genet 2000,97:189-194).And CGG series connection multiplicity greater than 200 times FMR1 allelotrope relevant with dysnoesia and autism usually (J Dev Behav Pediatr 2006,27:63-74).The series connection repetition can make protein produce polyglutamyl amine (polyQ) phenomenon to CAG in the coding region; Research shows that polyQ can make protein conformation change; And then cause toxicity; In, excitotoxicity disorderly and the cell like some mitochondrial functions transportation interrupt etc. all relevant with the proteic expression of polyQ (Annu Rev Neurosci2007,30:575-621).Certainly, tandem repetitive sequence causes that the phenomenon of disease also can occur in other zone of gene, repeats to cause that by 20 series connection on the cystatin B gene promoter (Nature 1997,386:847-851) like gradual muscle epilepsy (EPM1); And myotonia atrophica (DM1) is since the CTG that DMPK gene 3 '-UTR district increases connects repeat to cause (Curr Opin Neurol 2007,20:572-576).In addition, human DXZ4 tandem repetitive sequence is being controlled the deactivation of X chromosome, so cause some muscular dystrophies (Neuromuscul Disord 2009,19:17-20).

Moreover; Some important biological economy indexs are often also relevant with tandem repetitive sequence in the agriculture prodn: the milk yield like Dutch dairy determines that by 18 base multiple quantity on the tri-glyceride synthase gene promoter (Genetics 2004,167:1873-1881).It is thus clear that the change of repeat length can influence the variation of function in the tandem repetitive sequence, this possibly be a kind of adaptation of environment to external world during evolution.

Yet aspect plant, the research of tandem repetitive sequence aspect only is in the starting stage.Current, although people have also found some tandem repetitive sequences in plant, the research of its function aspects also not in animal deeply.But; Recent several pieces of plant aspects have caused that about the report that tandem repetitive sequence influences gene function people pay close attention to widely: the article report of on Science, delivering like Sureshkumar in 2009 etc.: Arabidopis thaliana IIL1 (At4g13430) but this expression of gene of TTC tandem repetitive sequence amplification remarkably influenced in the gene intron; And then (Science 2009,323:1060-1063) to influence the growth of plant.

Based on theoretical investigation and need of production practice, the inventor has identified a kind of tandem repetitive sequence that improves activity of gene expression, and it can be used in the industrialization development and application of plant genetic engineering.

Summary of the invention

The object of the invention is to provide the one-stage serial Tumor-necrosis factor glycoproteins on the Arabidopis thaliana zinc finger transcription factor At5g15840 gene; And this sequence is used to strengthen the purposes of destination gene expression in plant genetic engineering, lays the foundation for subsequent development efficiently expresses transgenic product.

The present invention has cloned the one-stage serial Tumor-necrosis factor glycoproteins (TTTACAC) of zinc finger transcription factor At5g15840 gene promoter area (SEQ ID NO.7) from Arabidopis thaliana ₅, and studied this tandem repetitive sequence enhancing β-D-glucuronidase (GUS) expression of gene.This tandem repetitive sequence is positioned at the At5g15840 gene translation initiation site ATG upper reaches-230 to-195 places, when this tandem repetitive sequence (TTTACAC) ₅During deletion, the active of this promotor sharply reduces.It is thus clear that this section tandem repetitive sequence (TTTACAC) ₅Has the effect that strengthens reporter gene GUS expression activity.

The present invention provides the one-stage serial Tumor-necrosis factor glycoproteins (TTTACAC) of described At5g15840 gene ₅In transgenic plant, improve the purposes of destination gene expression.Described plant optimization is Arabidopis thaliana or tobacco.Described goal gene is preferably gus gene.

Tandem repetitive sequence of the present invention derives from the gene of Arabidopis thaliana, can strengthen the expression of external source gus gene through checking.The report of existing some tandem repetitive sequence regulate gene expressions, as (CA) n tandem repetitive sequence in first intron of EGFR gene can influence transcriptional activity (Int J Biol Markers 2000,15:105-110); The tandem repetitive sequence of Arabidopis thaliana AtHKT1 gene promoter area plays enhancement to this genetic expression, this to the salt tolerance of keeping plant most important (Plant Cell Physiol 2011,52:149-161).Therefore, tandem repetitive sequence of the present invention equally also can be regulated and control the expression of goal gene.

Description of drawings

Fig. 1 is zfP promoter fragment clone electrophorogram, M (molecular weight standard thing): DL2000;

Fig. 2 is zfP1 promoter fragment clone electrophorogram, M (molecular weight standard thing): DL2000;

Fig. 3 is zfP2 promoter fragment clone electrophorogram, M (molecular weight standard thing): DL2000;

Fig. 4 for the promoter expression vector that makes up (zfP::GUS and zfP Δ:: GUS) structural representation.

Fig. 5 contains histochemical stain behind the During Agrobacterium tobacco leaf transient expression of expression vector.

Fig. 6 is the relative reactivity of fluorometry quantitative expression carrier.

Fig. 7 is zfP::GUS transgenic arabidopsis plant GUS histochemical stain figure.

Fig. 8 is the zfP Δ:: GUS transgenic arabidopsis plant GUS histochemical stain figure.

Embodiment

Embodiment 1: the structure of total length promotor and disappearance promotor

With Arabidopis thaliana (Arabidopsis thaliana, Columbia ecotype) blade is material, and (EMBO J 1987 6:3901-3907) extracts the total DNA of plant with the CTAB method.With the total DNA of plant is template, through the promoter fragment of special primer amplification zinc finger transcription factor At5g15840 gene.Synthetic primer Z1 (5 '-AAGCTTGATCTTAGAAACATGTCCT-3 ', SEQ ID NO.1 has introduced the HindIII site), Z2 (5 '-GGATCCTCTTGCAGCTAGTTGA-3 '; SEQ ID NO.2 has introduced the BamHI site), Z3 (5 '-GTCGACATCTCCTCCTATGCA-3 ', SEQ ID NO.3; Introduced the SalI site); Z4 (5 '-GTCGACATAGGCCTTCCCAAAAGCT-3 ', SEQ ID NO.4 has introduced the SalI site).Wherein primer Z1/Z2 amplifies 1091bp total length promoter fragment (called after zfP), like Fig. 1; The 866bp fragment (called after zfP1) of primer Z1/Z3 amplification is like Fig. 2; The 190bp fragment (called after zfP2) of primer Z2/Z4 amplification is like Fig. 3.PCR product zfP directly is connected with the pGEM-T cloning vector, transforms DH5 α intestinal bacteria competence bacterial strain, screening positive clone (containing total length promoter fragment zfP).PCR product zfP1 is connected with the pGEM-T cloning vector; Behind the screening positive clone with SalI and BamHI double digestion; Get big fragment; Be connected with the zfP2 of BamHI double digestion again with through SalI, transform DH5 α intestinal bacteria competence bacterial strain, PCR method is identified positive colony (containing disappearance promoter fragment zfP Δ).

The structure of embodiment 2:GUS fusion expression vector

With Restriction Enzyme HindIII and BanHI two kinds of positive colonies among the double digestion embodiment 1 and binary expression vector pBI121 (can bounties Gene Tech. Company Limited buys from sky, Beijing) respectively; Total length promotor after enzyme cut and disappearance promoter fragment are connected with the pBI121 endonuclease bamhi respectively; The structure promoter expression vector (zfP::GUS and zfP Δ:: GUS) (Fig. 4); Be transformed into them in the DH5 α intestinal bacteria competence bacterial strain respectively, expand numerous.

Embodiment 3: Agrobacterium-mediated Transformation

With the expression vector that builds (zfP::GUS and zfP Δ:: GUS) change among the Agrobacterium host cell EHA105 (can obtain from national Microbial resources storehouse www.matrs.com, product IDs is 20110114049).Concrete grammar is following: the expression vector of structure (zfP::GUS and zfP Δ:: GUS) coli strain is inoculated into 10ml and contains in Km (50 μ g/ml) the LB liquid nutrient medium, after 37 ℃ of incubated overnight, the upgrading grain, for follow-up conversion Agrobacterium subsequent use.EHA105 agrobacterium strains inoculation 5ml contains in Sm (50 μ g/ml) the YEP liquid nutrient medium, and 28 ℃ of following 200rpm cultivated 48 hours.Centrifugal collection thalline is with the freezing CaCl of 0.1M ₂Resuspended, place 20 minutes on ice after, centrifugal 2 minutes of 4 ℃ of following 5000rpm, the thalline of collection is with the freezing CaCl of 200 μ l 0.1M ₂Suspend.Add then the expression vector plasmid prepare (zfP::GUS and zfP Δ:: GUS); Placed on ice 20 minutes, and placed 10 minutes for-70 ℃, 37 ℃ of placements add 800 μ l and do not contain antibiotic YEP liquid nutrient medium after 5 minutes; Cultivate after 4 hours for 28 ℃; Centrifugal collection thalline is coated with the rod coating and contains on the YEP solid medium flat board of Km (50 μ g/ml) and Sm (50 μ g/ml) with glass, cultivates 2 days for 28 ℃.With Auele Specific Primer bacterium colony is carried out PCR and identify screening positive clone.

Embodiment 4: transient expression

To contain expression vector (zfP::GUS and zfP Δ:: agrobacterium strains GUS) (making up among the embodiment 3) is inoculated in the YEP substratum that contains Km (50 μ g/ml) and Sm (50 μ g/ml); Be cultured to logarithmic phase; Centrifugal collection thalline is resuspended in MMA solution (10mM MES, 100 μ M Syringylethanones, 10mMMgCl ₂), and the adjustment bacterial concentration is to OD ₆₀₀=1.2,28 ℃ left standstill 2 hours.Get the tobacco middle part at 2 monthly ages and do not launch blade fully, the back side is positioned on the MS solid medium up.200 μ l Agrobacterium infiltrates are dropped on the tobacco leaf, cultivate in 25 ℃ of growth cabinets.GUS histochemical stain and fluorescence activity mensuration are carried out in sampling after 72 hours.

Embodiment 5:GUS is active to be detected

Histochemical stain: the blade among the embodiment 4 is taken out, blot to be placed on thieving paper and contain in the 1mMX-Gluc staining fluid, 37 ℃ of incubations 5 hours, sample is observed photograph after 75% ethanol dehydration.Like Fig. 5.

Fluorometry: the blade among the embodiment 4 is taken out, blot, get blade with scalpel and contaminate the position,, add the GUS extracting solution (50mMNa of 3 times of volumes through liquid nitrogen grinding with thieving paper ₃PO ₄The 10mM beta-mercaptoethanol; 0.1%Triton X-100; 1%Sarcosyl), get supernatant after centrifugal, add 1mM MUG (4-methyl umbelliferone-β-D-glucuronic acid) reaction, it is active that the fluorophotometric appearance is measured GUS, like Fig. 6.The total length promotor zfP that contains tandem repetitive sequence has high expression activity, and the promotor zfP Δ activity of tandem repetitive sequence sudden change sharply reduces.

Embodiment 6: Arabidopis thaliana transforms and identifies

Get contain expression vector (zfP::GUS and zfP Δ:: Agrobacterium GUS) (making up among the embodiment 3) bacterium liquid 20 μ l, add 5ml YEP, 28 ℃ of constant temperature joltings are spent the night; This 5ml bacterium liquid is poured in the YEP liquid nutrient medium that 250ml contains Km (50 μ g/ml) and Sm (50 μ g/ml), and 28 ℃ of constant temperature joltings are spent the night, when the concentration of Agrobacterium should reach OD600=1.8; Centrifugal 15 minutes of 4000rpm; Supernatant discarded add to be soaked into substratum (1/2MS+5% sucrose), resuspended after; Add tensio-active agent Silwet-L77, make its final concentration reach 0.02%.The inflorescence of Arabidopis thaliana plant is immersed in the bacterium liquid, soaks into lasting 2min, take off film after spending the night with the preservative film covering.Normal growth is collected seed.Seed dries the back earlier with 70% ethanol disinfection 10 minutes, again with 10 Youxiaolin sterilization 5 minutes, washes 5 volumes with aqua sterilisa at last.The Arabidopis thaliana seed points that sterilization is good is cultivated in 25 ℃ of growth cabinets on MS solid medium (containing 30 μ g/ml Km microbiotic), illumination 16 hours/dark 8 hours, 2-3 week rear blade greening and root elongation be doubtful transfer-gen plant.These plant are carried genomic dna, further verify with GUS special primer (upstream primer 5 '-GATGTCACGCCGTATGT-3 ', SEQ ID NO.5 and downstream primer 5 '-CACACTCTGTCTGGCT-3 ', SEQ ID NO.6) pcr amplification.The histochemical stain of transfer-gen plant is carried out with reference to embodiment 5, and the result sees Fig. 7, Fig. 8.This shows that the transfer-gen plant that contains the total length promotor zfP of tandem repetitive sequence is expressed higher GUS activity, and the promotor zfP Δ of tandem repetitive sequence sudden change detects almost less than the GUS that expresses active.

SEQUENCE?LISTING

 

< 110>Jiangsu University

 

< 120>a kind of application that can improve the active tandem repetitive sequence of gene expression in plants

 

<130>

 

<160> 7

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 25

<212> DNA

< 213>artificial sequence

 

<400> 1

aagcttgatc?ttagaaacat?gtcct 25

 

 

<210> 2

<211> 22

<212> DNA

< 213>artificial sequence

 

<400> 2

ggatcctctt?gcagctagtt?ga 22

 

 

<210> 3

<211> 21

<212> DNA

< 213>artificial sequence

 

<400> 3

gtcgacatct?cctcctatgc?a 21

 

 

<210> 4

<211> 25

<212> DNA

< 213>artificial sequence

 

<400> 4

gtcgacatag?gccttcccaa?aagct 25

 

 

<210> 5

<211> 17

<212> DNA

< 213>artificial sequence

 

<400> 5

gatgtcacgc?cgtatgt 17

 

 

<210> 6

<211> 16

<212> DNA

< 213>artificial sequence

 

<400> 6

cacactctgt?ctggct 16

 

 

<210> 7

<211> 1095

<212> DNA

< 213>Arabidopis thaliana

 

<400> 7

ctctgtgatc?ttagaaacat?gtcctgaaaa?ttccatacaa?gtgtcgtttg?tattagatta 60

 

cttccacagg?ttgagatcta?ataaagctac?aataaatagt?atagagtatc?atcataaacc 120

 

caaattacag?agatgtgaca?acactcatga?gtcatgtttt?taactactta?ctataatggt 180

 

taccaagtgc?aaatttctac?atactatata?tgataaatct?aattattgct?catgtggact 240

 

ccaaaatgcc?ttttaagttt?taacttgtgc?gtcaggtaaa?ttctaatttg?tagtctcaag 300

 

actacttggc?ggattcgagt?ttgatcctag?aaaatccacc?gtctctatgt?ttttcatgtc 360

 

acttttccga?tatgattctc?attaccatga?ctttatgaac?cagattaaac?attataacac 420

 

ttttcatcag?aaaatccttc?gaaagtttca?attgcaaatc?tttctaaatg?atgcagatgc 480

 

attcacaaat?aatggaacaa?caactatacc?atattcacga?gtttgtctaa?cctttgtata 540

 

ggtagtcaac?ccataacagt?tggtgatggc?tctgacactc?gaagccttac?tcggagagat 600

 

acctgaacag?taatcacaag?gttcaggatg?aatattcaac?cacttaaact?ttgtataaag 660

 

ccaaagagat?aaaacgaatc?tagctttact?ttaaataaaa?tgcatatgaa?aatagtaaaa 720

 

ggtgatacga?aaaaatagta?acaatttgcc?tgcaacacca?tggcattatc?cggaccactt 780

 

cctcttgaga?atctcagtat?ggcaagtggc?aaaacctaag?caacttgtga?acgggtccca 840

 

acgaagaagt?gcataggagg?agatgtttac?actttacact?ttacacttta?cactttacac 900

 

ataggccttc?ccaaaagctc?aactagctgc?aagaggatcc?aataacatgt?aagagccact 960

 

aacgctgtgc?cacgtgtagg?cactcaggat?tcgatctccc?ctctacttat?tctctcacac 1020

 

cagatataag?ctttattagc?cccttctttc?agataccagc?tcccacacca?tcaaacttac 1080

 

tacatctgag?ttatt 1095

 

 

Claims (3)

1. Arabidopis thaliana ( Arabidopsis thaliana) zinc finger transcription factor At5g158405 series connection of gene promoter the preceding paragraph multiple TTTACAC sequence improves the purposes in the destination gene expression in transgenic plant.
2. 2. purposes according to claim 1, wherein said plant are Arabidopis thaliana or tobacco.
3. 3. purposes according to claim 2, wherein said goal gene are β-D-glucuronidase (GUS) gene.

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