CN102260677B - Rice seed glutelin GluC gene terminator and application thereof - Google Patents
Rice seed glutelin GluC gene terminator and application thereof Download PDFInfo
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Abstract
The invention discloses a rice seed glutelin GluC gene terminator and application thereof. The terminator is a deoxyribonucleic acid (DNA) molecule, and is 1) a DNA molecule consisting of a nucleotide sequence shown as a sequence 1 in a sequence table, 2) a molecule which is at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent homologous with the DNA sequence limited in 1), or 3) a molecule which hybridizes with a DNA sequence limited in 1) or 2) under strict conditions. The matching of the terminator and different promoters can improve the expression and accumulation level of exogenous genes in target plants; and the terminator lays a foundation for researches such as seed quality improvement by a biological technology, molecular medicine farming and the like, and has great application prospect.
Description
Technical field
The present invention relates to a kind of rice paddy seed gluten GluC gene terminator and application thereof.
Background technology
The recent development of Plant Biotechnology has not only realized the improvement (as improving crop yield, strengthening disease-resistant, pest-resistant, antiweed characteristic, improvement quality etc.) of traditional economical character, and makes plant become the bio-reactor of biological medicine and Industrial products.Characteristics such as most gramineous crops have that output height, production cost are low, storage endurance, industrial scale are controlled easily, direct-edible, and it possesses the ability of posttranslational modification in the body, thereby become the ideal carrier of s-generation transgene product.Utilize rice paddy seed production foreign protein with health role and the development of edibility vaccine research very fast in recent years, and obtained immense success.All successfully in paddy rice, express and the high level accumulation like vitamin A, glycinin (Glycinin), soybean ferritin (Ferritin), GLP, Hepatitis B virus vaccine and the pollen hypersensitivity vaccine etc. that have prevention and treatment iron-deficiency anaemia and improve autoimmune function with the prevent diabetes generation function that stimulates insulin secretion with lowering blood glucose effect.Yet, realize that further efficiently expressing of foreign gene must improve genetic expression simultaneously transcribing with post-transcriptional level.Promotor is mainly in transcriptional level control genetic expression, and terminator then plays regulating and controlling effect transcribing with post-transcriptional level simultaneously.
Although the terminator of widespread use at present like Nos, Ocs, and can start the high expression level of reporter gene in plant after the allogeneic promoter combination, can satisfy the research demand of biological chemistry, physiology and cellular localization aspect.Yet, less for other terminator researchs that can improve genetic expression.Because some Main Agronomic Characters and Secondary Metabolism of Plant product all are by controlled by multiple genes, the expression that improves individual gene is not obvious for the correlated character improving effect.Through traditional method for transformation, as repeat to transform or hybridization realizes that polygene transforms and but wastes time and energy.The polygene conversion system that development in recent years is got up can insert a plurality of genes simultaneously in an expression vector; For fear of the too high transgene silencing that causes of transgenic homology; These genes need be expressed by different promoters driven, and different terminating stops transcribing.
Summary of the invention
An object of the present invention is to provide a terminator, name is called tGluC, and this terminator is compared with the no terminator, can improve the expression of exogenous gene level.
Terminator provided by the present invention is following 1) or 2) or 3) dna molecular:
1) dna molecular of forming by the nucleotide sequence shown in the sequence in the sequence table 1;
2) with 1) sequence of the DNA that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or the molecule that has 99% homology at least at least;
3) under stringent condition with 1) or 2) molecule of the dna sequence dna hybridization that limits.
Said stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 2 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.5 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3PO
4With hybridize in the mixing solutions of 1mM EDTA, at 65 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, use 2 * SSC then, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 1 is made up of 516 Nucleotide in the sequence table.
The recombinant vectors, expression cassette, transgenic cell line, reorganization bacterium or the recombinant virus that contain said dna molecular also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of said dna molecular.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment.Like pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.When using said dna molecular to make up the recombinant plant expression vector; Before transcription of foreign genes nuclei originis thuja acid, can add any enhancement type promotor (like the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (like the promotor of seed specific expression), they can use separately or be used in combination with other plant promoter; In addition; When using dna molecular of the present invention to make up plant expression vector; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole exogenous gene sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector; Can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that can in plant, express, antibiotic marker gene (as is given the nptII gene to kantlex and associated antibiotic resistance; Give bar gene to weedicide phosphinothricin resistance; Give hph gene to the microbiotic hygromycin resistance; With the dhfr gene of giving the methatrexate resistance, give EPSPS gene to the Glyphosate IPA salt resistance) or anti-chemical reagent marker gene etc. (like anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.
Said recombinant vectors specifically can be arbitrary following carrier: pGluB-3-tGluC, pGluC-tGluC, p35S-tGluC, pUbi-tGluC.
Said pGluB-3-tGluC replaces with the recombinant vectors that the tGluC terminator shown in the sequence 1 obtains in the sequence table with the no terminator among the pGluB-3-nos; Said pGluC-tGluC replaces with the recombinant vectors that the tGluC terminator shown in the sequence 1 obtains in the sequence table with the no terminator among the pGluC-nos; Said p35S-tGluC replaces with the recombinant vectors that the tGluC terminator shown in the sequence 1 obtains in the sequence table with the no terminator among the pBI221; Said pUbi-tGluC replaces with the recombinant vectors that the tGluC terminator shown in the sequence 1 obtains in the sequence table with the no terminator among the pUbi-221, and said pUbi-221 replaces with the recombinant vectors that the Ubiquitin promotor obtains with the 35S promoter among the pBI221.
Said expression cassette is meant and starts by promotor, by said promotor that the goal gene of transcribing and the said dna molecular that is positioned at said goal gene downstream form.Wherein, said promotor can be constitutive promoter or organizing specific expression promotor (like endosperm specificity promoter).Said goal gene can be protein coding gene and/or non-protein coding gene; Said protein coding gene is preferably the quality-improving gene; Said non-protein coding gene is just rna gene and/or sense-rna gene.
Another object of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention; Be that the expression cassette that will contain said dna molecular imports in the purpose plant, obtain said destination gene expression level and be higher than the transgenic plant that following expression cassette imported said purpose plant: the said dna molecular in the described expression cassette is replaced with the expression cassette that the no terminator of rouge alkali synthetase obtains.
Said purpose plant specifically can be monocotyledons or dicotyledons.
Said monocotyledons specifically can be paddy rice, wheat, and corn, Chinese sorghum or barley, said dicotyledons specifically can be soybean, rape, cotton, tobacco, yam, sweet potato or tung oil tree.
Said transgenic plant are interpreted as not only to comprise the said expression cassette that contains said dna molecular are transformed the first-generation transgenic plant that the purpose plant obtains, also comprise its filial generation.For transgenic plant, can in these species, breed this and contain the expression cassette of said dna molecular, also available traditional breeding method shifts other kind that gets into same species with the expression cassette that this contains said dna molecular, in commercial variety.
Said dna molecular in cultivating transgenic plant application or as the application of terminator also in protection scope of the present invention.
Utilize terminator of the present invention to cooperate with different promotors; Can improve expression and the accumulation level of foreign gene in the purpose plant; But improved seed quality, the albumen that will have physiologically active or small peptide import initiative health function new variety in the seed, utilize seed to produce useful foreign protein or edibility vaccine as bio-reactor, increase agricultural-food science and technology added value etc.Terminator of the present invention and being applied as utilizes researchs such as bio-technology improvement seed quality, molecule medicine farm to lay a good foundation, and has great application prospect.
Description of drawings
Fig. 1 is the electrophoretogram of pcr amplification gluten GluC gene terminator (tGluC) from oryza sativa genomic dna.Wherein 1 is dna molecular amount standard, and 2 is the fragment of tGluC.Wherein, the standard molecular weight size is followed successively by from the bottom to top: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb.
Fig. 2 is that carrier pMD-tGluC identifies collection of illustrative plates through the double digestion of Sac I and EcoR I.Wherein 1 is dna molecular amount standard, and 2 is the endonuclease bamhi of pMD-tGluC.Wherein, the standard molecular weight size is followed successively by from the bottom to top: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb; 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb; 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb; 6.0kb, 8.0kb, 10.0kb, endonuclease bamhi is followed successively by from the bottom to top: tGluC, 516bp; PMD-tGluC carrier frame fragment, 2700bp.
Fig. 3 is the carrier structure synoptic diagram that contains tGluC.Wherein, A figure is the pGluB-3-tGluC structural representation, and B figure is the pGluC-tGluC structural representation, and C figure is the p35S-tGluC structural representation, and D figure is the pUbi-tGluC structural representation, and GluCT represents tGluC.
Fig. 4 is that the double digestion that contains the recombinant vectors of tGluC is identified collection of illustrative plates.Wherein, A-D figure is respectively carrier pGluB-3-tGluC, pGluC-tGluC, p35S-tGluC, pUbi-tGluC; 1 is dna molecular amount standard, and 2 is the endonuclease bamhi of each carrier; Fig. 4 A standard molecular weight size is followed successively by from the bottom to top: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb; 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb; 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb; 6.0kb, 8.0kb, 10.0kb, endonuclease bamhi is followed successively by from the bottom to top: tGluC, 516bp; PGluB-3-tGluC carrier frame fragment, 16.6kb; Fig. 4 B-4D standard molecular weight size is followed successively by from the bottom to top: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp; Fig. 4 B endonuclease bamhi is followed successively by from the bottom to top: tGluC and GUS merge back fragment, 2.4kb; PGluC-tGluC carrier frame fragment, 14.7kb; Fig. 4 C endonuclease bamhi is followed successively by from the bottom to top: tGluC, 516bp, p35S-tGluA-2 carrier frame fragment, 5.2kb; Fig. 4 D endonuclease bamhi is followed successively by from the bottom to top: tGluC, and 516bp, GUS and part Ubiquitin promotor merge fragment 2.5kb (wherein part Ubiquitin promotor size is 0.6kb), and pUbi-tGluC removes fragment 3.9kb behind above two portions.
Fig. 5 is for changeing pGluB-3-tGluC and the T that changes pGluC-tGluC
0For rice plant chimeric primers pcr amplification electrophoretogram.Wherein, 1 is dna molecular amount standard; 2 are the positive control that is template with corresponding conversion plasmid; 3 for being to be the negative control of template with unconverted strain, and 4～16 for changeing the regeneration plants of pGluB-3-GluC plant expression vector among the A, and 4～17 for changeing the regeneration plant of pGluC-tGluC plant expression vector among the B.
Fig. 6 is not genetically modified wild-type paddy rice Kitaake plant GUS coloration result.Wherein, 1,2,3,4 be respectively not genetically modified wild-type paddy rice Kitaake plant root, stem, leaf and seed coloration result.
Fig. 7 is T
0For transgenic paddy rice Kitaake/pGluB-3-nos plant GUS coloration result.Wherein, 1,2,3,4 be respectively the coloration result that changes pGluB-3-nos plant root, stem, leaf and seed.
Fig. 8 is T
0For transgenic paddy rice Kitaake/pGluB-3-tGluC plant GUS coloration result.Wherein, 1,2,3,4 be respectively the coloration result that changes pGluB-3-tGluC plant root, stem, leaf and seed.
Fig. 9 is T
0Measure the result for the seed bearing GUS fluorescence activity of transgenic paddy rice Kitaake/pGluB-3-tGluC institute.
Figure 10 is T
1Measure the result for the seed bearing GUS fluorescence activity of transgenic paddy rice Kitaake/pGluB-3-tGluC institute.
Figure 11 is T
0Measure the result for the seed bearing GUS fluorescence activity of transgenic paddy rice Kitaake/pGluC-tGluC institute.
Figure 12 carries out determination of activity for the 14 hours protoplastiss of paddy rice incubation that change p35S-tGluC and pBI221 are extracted GUS.Wherein, 1 for changeing three repetition MVs of pBI221, and 2,3,4 are respectively three measured values of changeing p35S-tGluC.
Figure 13 carries out determination of activity for the 14 hours protoplastiss of paddy rice incubation that change pUbi-tGluC and pUbi-221 are extracted GUS.Wherein, 1 for changeing three repetition MVs of pUbi-221, and 2,3,4 are respectively three measured values of commentaries on classics pUbi-tGluC.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The acquisition of embodiment 1, rice paddy seed gluten GluC gene terminator (tGluC)
CDNA sequence (GenBank number is AK64478) according to paddy rice gluten GluC gene; From GenBank, search the genomic dna sequence of gluten GluC gene; The sequence of 516bp is rice paddy seed gluten GluC gene terminator of the present invention (tGluC) after the terminator codon, design primer amplification tGluC.For ease of vector construction, on primer, add restriction enzyme site (shown in the underscore) respectively.The forward primer of tGluC is tGluC SacF:5 '-AA
GAGCTCACTAAATGTGTAACGATCTTAC-3 ' (Sac I), reverse primer is tGluC EcoR:5 '-A
GAATTCATGGCGCATGGCGCATGTGGG-3 ' (EcoR I).
(document Qu et al., J.Exp.Bot.2008 59:2417-2424) extract genomic dna in the blade to the CTAB method in a small amount, are template with it, are primer with tGluC SacF and tGluC EcoR, pcr amplification tGluC sequence from wild-type paddy rice Kitaake.The PCR response procedures is: 94 ℃ of preparatory sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 30 circulations, last 72 ℃ of 10min.Pcr amplification obtains the purpose band of 516bp, and is as shown in Figure 1.Reclaim amplified production, be directly connected on the pMD18-T carrier (available from TaKaRa company), check order, the result shows that the tGluC sequence size that amplification obtains is 516bp, has the nucleotide sequence of sequence 1 in the sequence table.To check order to detect and show and contain the segmental recombinant vectors called after of tGluC pMD-tGluC.PMD-tGluC identifies that through the double digestion of Sac I and EcoR I collection of illustrative plates is as shown in Figure 2.
The vector construction and the conversion of embodiment 2, rice paddy seed gluten GluC gene terminator (tGluC)
1, tGluC merges the plant expression vector construction of gus gene
With Sac I and EcoR I double digestion pMD-tGluC plasmid; Reclaim the 516bp endonuclease bamhi of tGluC, this fragment is inserted between Sac I and the EcoR I double digestion recognition site of the pGluB-3-nos that contains the GluB-3 promotor and between the Sac I of the pGluC-nos expression vector of GluC promotor and the EcoR I double digestion recognition site and makes up stable conversion carrier pGluB-3-tGluC (its structural representation is shown in Fig. 3 A) and pGluC-tGluC (its structural representation is shown in Fig. 3 B).
PGluB-3-nos and pGluC-nos be according to document Qu et al., J.Exp.Bot.2008, and the described method of 59:2417-2424 makes up: with 65 genomic dna in the paddy rice platform is template, with GluB-3 forward primer 5 '-CCC
AAGCTTATTTTACTTGTACTGTTTAACC-3 ' (HindIII) with GluB-3 reverse primer 5 ' AAA
CCCGGGAGCTTTCTGTATATGCTAATG-3 ' (Sma I) is a primer; Pcr amplification obtains the GluB-3 promotor; The GluB-3 promotor with HindIII and Sma I double digestion, is inserted into the GUS upper reaches of pGPTV-35S-HPT, and the recombinant vectors that obtains is pGluB-3-nos; With 65 genomic dna in the paddy rice platform is template, with GluC forward primer 5 '-GGG
AAGCTTGTTCAAGATTTATTTTTGG-3 ' (HindIII) with GluC reverse primer 5 '-ACGC
GTCGACAGTTATTCACTTAGTTTCCC-3 ' (Sal I) is a primer, and pcr amplification obtains the GluC promotor, and the GluC promotor with HindIII and Sal I double digestion, is inserted into the GUS upper reaches of pGPTV-35S-HPT, and the recombinant vectors that obtains is pGluC-nos.PGPTV-35S-HPT is foreign gene with GUS, and no is a terminator.
TGluC is building up to pBI221 (Chen et al. respectively; Mol.Breed.2003 makes up transient expression carrier p35S-tGluC (its structural representation is shown in Fig. 3 C) and pUbi-tGluC (its structural representation is shown in Fig. 3 D) 11:287-293) and between the Sac I of pUbi-221 and the EcoR I double digestion recognition site.Wherein pUbi-221 is that 35S promoter with pBI221 replaces with the recombinant vectors that the Ubiquitin promotor obtains.
Above 4 kinds of recombinant vectorss that obtain are carried out double digestion identify on the basis that PCR identifies, as shown in Figure 4.
2, change the acquisition of pGluB-3-tGluC, pGluC-tGluC, pGluB-3-nos and pGluC-nos rice regeneration plant
The pGluB-3-tGluC that make up to accomplish and pGluC-tGluC expression vector pass through enzyme cut with its exactness of sequence verification after; Utilize freeze-thaw method to import respectively among the Agrobacterium EHA105; Concrete grammar is following: draw 7 μ l expression vector plasmids and add in the 100 μ l EHA105 Agrobacterium competent cells, flick mixing and be placed on freezing 7min in the liquid nitrogen, change over to afterwards in 37 ℃ of water-baths and leave standstill 3min; Add the 800ulYEB liquid culture then and leave standstill recovery cultivation 3～5 hours based on 28 ℃; Get 400 μ l bacterium liquid and coat (kantlex 50mg/L, Rifampin Rif50mg/L) on the YEB resistant panel, be inverted in 28 ℃ and cultivated 2～3 days.After the bacterium colony PCR that the single bacterium colony of a little Agrobacterium of picking carries out target gene detects positive bacteria is dropped on the YEB resistant panel line and expand numerous cultivation; Utilize the callus of Agrobacterium infestation method rice transformation kind kitaake; Use the hygromycin selection kanamycin-resistant callus tissue, and further Culture and Differentiation becomes intermediate house behind the seedling.
Utilize the hygromycin selection method (according to document Hiei et al., Plant J.1994, the said method of 6:271-282 is carried out), obtain 13 strain T
0In generation, changeed pGluB-3-tGluC rice plant and 14 strain T
0In generation, changeed the pGluC-tGluC rice plant.Utilize PCR method that commentaries on classics pGluB-3-tGluC paddy rice and the commentaries on classics pGluC-tGluC paddy rice that above-mentioned screening obtains carried out the PCR Molecular Detection, the PCR primer is the chimeric primers of tGluC and gus gene sequence, that is: tGluC sequence reverse primer tGluC EcoR:5 '-A
GAATTCForward primer GUSF185:5 '-TCGTCGGTGAACAGGTATGG-3 ' that ATGGCGCATGGCGCATGTGGG-3 ' is inner with the GUS sequence.The PCR response procedures is: 94 ℃ of preparatory sex change 5min, 94 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 1min, 30 circulations, last 72 ℃ of 10min.The purpose band that pcr amplification obtains 0.7kb is detection positive (as shown in Figure 5).The result shows that 13 strains commentaries on classics pGluB-3-tGluC plant (being expressed as Kitaake/pGluB-3-tGluC) PCR detection all is positive, and 14 strains are changeed pGluC-tGluC plant (being expressed as Kitaake/pGluC-tGluC) PCR detection and all are positive.
According to the method described above; Change pGluB-3-nos, pGluC-nos over to wild-type paddy rice Kitaake respectively; Obtaining 9 strains changes the positive plant (being expressed as Kitaake/pGluB-3-nos) of pGluB-3-nos, and the positive plant (being expressed as Kitaake/pGluC-nos) of pGluC-nos is changeed in 10 strains.Change the PCR detection method of the rice plant of pGluB-3-nos and pGluC-nos: is primer with tnos sequence reverse primer tnosR:5 '-GATCTAGTAACATAGATGAC-3 ' with the inner forward primer GUSF185:5 '-TCGTCGGTGAACAGGTATGG-3 ' of GUS sequence; The chimeric sequences of amplification tnos and gus gene, the purpose band that obtains 500bp is positive transformant.
The seed that T0 ties for transfer-gen plant and be T by the plant that this seed grows up to
1In generation, the rest may be inferred, T
2, T
3Represent the 2nd generation of transfer-gen plant and the 3rd generation respectively.
3, change the acquisition of p35S-tGluB-5, pUbi-tGluB-5, pBI221 and pUbi-221 protoplastis
Method through PEG mediation changes p35S-tGluC, pUbi-tGluC, pBI221 and pUbi-221 carrier over to process with the etiolated seedling of wild-type paddy rice Kitaake protoplastis respectively (according to document Chen et al; Mol Plant Pathol.2006; 7:417-427); Three in each carrier repeats, 28 ℃ incubation 14-16 hour.
The functional verification of embodiment 3, rice paddy seed gluten GluC gene terminator (tGluC)
1, T
0GUS histological chemistry for transgenic paddy rice is detected
To the transgenic paddy rice Kitaake/pGluB-3-nos of PCR test positive and the T of Kitaake/pGluB-3-tGluC
0Carrying out histochemical stain for plant, is contrast with not genetically modified wild-type paddy rice Kitaake plant.Concrete steps are: with the T of transgenic paddy rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluC
0Partial blade, root, cane tissue for plant and not genetically modified wild-type paddy rice Kitaake plant are cut into small pieces; The filling stage seed that to bloom back 17 days is with scalper longitudinal incision from the middle part.The sample of handling well is soaked in GUS staining reaction liquid (0.1M sodium phosphate buffer (pH 7.0), 10mMNa
2-EDTA (pH7.0), the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash, 1.0mM X-Gluc, 0.1%Triton X-100), 37 ℃ were reacted 0.5-4 hour.Preserve in 70% ethanol, observe.Observe under the stereoscope and take pictures.Result such as Fig. 6, Fig. 7, shown in Figure 8: root, blade, the cane of not genetically modified wild-type paddy rice Kitaake plant and the aleurone layer of the back 17 days seed of blooming and inferior aleurone layer are not all observed GUS and are expressed; T
0All do not observe GUS for root, blade, the cane of transgenic paddy rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluC plant and express T
0For transgenic paddy rice Kitaake/pGluB-3-nos bloom back 17 days seed only have at aleurone layer and inferior aleurone layer expressed, and T
0For the aleurone layer of transgenic paddy rice Kitaake/pGluB-3-tGluC, inferior aleurone layer and all high-visible expression of whole endosperm blueness.The result shows: tGluC compares with the no terminator and has strengthened the expression intensity of GUSB (GUS) reporter gene in the rice paddy seed endosperm, but does not change gus reporter gene specific expressed in endosperm.
2, T
0And T
1GUS fluorescence activity for transgenic paddy rice is measured
To the transgenic paddy rice Kitaake/pGluB-3-nos of PCR test positive and the T of Kitaake/pGluB-3-tGluC
0For 17 days the T of filling stage seed, Kitaake/pGluC-nos and Kitaake/pGluC-tGluC behind the plant blossom
0For 17 days filling stage seed and the T of Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluC behind the plant blossom
1Carry out the GUS quantitative analysis for 17 days filling stage seed behind the plant blossom.Method according to the fluorescence detection method of Jefferson etc. (Jefferson, Plant Mol.Biol.Report, 1987,5:387-405) carry out.Be specially: 3 seeds are got in every strain, place 3 1.5ml eppendorf pipes respectively, with pestle seed are pulverized, and add 200 μ l extract (50mM buffer solution of sodium phosphate (pH7.0), 10mM beta-mercaptoethanol, 10mM Na
2EDTA (pH8.0), 0.1%SDS, 0.1%Triton X-100), the vibration mixing.4 ℃ 13, the centrifugal 5min of 000rpm gets supernatant in new pipe.10 μ l supernatants add 90 μ l reaction solutions (1mM 4-MUG is dissolved in the GUS extract), and 37 ℃ were reacted 60 minutes, and added 900 μ l stop buffer (0.2M Na
2CO
3), the room temperature termination reaction.Do typical curve with 4-MU, under 360nm excitation wavelength and 460nm absorbing wavelength, detect relative 4-MU content with F-4500 (Hitachi) type spectrophotofluorometer.With the bovine serum albumin is contrast, utilizes BIO-Rad ProteinAssay KitII (available from Bio Rad Laboratories, numbering GPR6611) to measure protein contnt.
To transgenic line T
0The Dai Suojie seed carries out GUS determination of activity result such as Fig. 9 and shown in Figure 11.T at 13 Kitaake/pGluB-3-tGluC
0In the strain system (Fig. 9), the active mxm. of GUS is 31.55pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 13.76pmol 4-MU min
-1μ g
-1Albumen, MV are 24.0 ± 5.7pmol 4-MUmin
-1μ g
-1Albumen.And the T of 9 contrast Kitaake/pGluB-3-nos
0In the strain system, the active mxm. of GUS is 17.38pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 2.39pmol 4-MU min
-1μ g
-1Albumen, MV are 11.2 ± 5.0pmol 4-MU min
-1μ g
-1Albumen.13 strains changeing the pGluB-3-tGluC carrier are that two of GUS expression amounts are less than changeing pGluB-3-nos plant peak; All the other are all greater than changeing pGluB-3-nos plant peak; The former peak is peaked 1.8 times of the latter; Be 13.18 times of its minimum value, MV is 2.1 times of contrast MV.
T at 14 Kitaake/pGluC-tGluC
0In the strain system (Figure 11), the active mxm. of GUS is 64.63pmol4-MU min
-1μ g
-1Albumen, Schwellenwert are 19.85pmol 4-MU min
-1μ g
-1Albumen, MV are 39.7 ± 12.2pmol 4-MU min
-1μ g
-1Albumen.And the T of 10 contrast Kitaake/pGluC-nos
0In the strain system, the active mxm. of GUS is 43.55pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 6.46pmol 4-MU min
-1μ g
-1Albumen, MV are 20.38 ± 10.64pmol 4-MU min
-1μ g
-1Albumen.The former peak is peaked 1.48 times of the latter, is 10.0 times of its minimum value, and MV is 2.0 times of contrast MV.
To transgenic line part T
1It is shown in figure 10 that the Dai Suojie seed carries out GUS determination of activity result.The T of 9 Kitaake/pGluB-3-tGluC
1Strain is that the active mxm. of GUS is 45.25pmol 4-MU min
-1μ g
-1Albumen, Schwellenwert are 11.81pmol 4-MU min
-1μ g
-1Albumen, MV are 30.5 ± 9.9pmol 4-MU min
-1μ g
-1Albumen.And the T of 7 contrast Kitaake/pGluB-3-nos
1In the strain system, then the active mxm. of GUS is 21.52pmol4-MU min
-1μ g
-1Albumen, Schwellenwert are 2.82pmol 4-MU min
-1μ g
-1Albumen, MV are 9.9 ± 5.8pmol4-MU min
-1μ g
-1Albumen.The former MV is 3.07 times of contrast.
The result shows that tGluC compares with the no terminator and strengthened the expression level of foreign gene in paddy endosperm.
3, rice protoplast GUS fluorescence activity is measured
Carry out determination of activity (shown in Figure 12) to changeing p35S-tGluC and 14 hours wild-type paddy rice Kitaake protoplastis of pBI221 incubation extraction GUS.1 for changeing three repetition MV 0.51pmol 4-MU min of pBI221
-1μ g
-1Albumen, 2,3,4 then are respectively three measured values 1.77,0.58 and the 0.93pmol 4-MUmin that changes p35S-tGluC
-1μ g
-1Albumen is respectively 3.47,1.13 and 1.82 times of contrast.
Carry out determination of activity (shown in Figure 13) to changeing pUbi-tGluC and 14 hours wild-type paddy rice Kitaake protoplastis of pUbi-221 incubation extraction GUS.1 for changeing three repetition MV 9.22pmol 4-MU min of pUbi-221
-1μ g
-1Albumen, 2,3,4 then are respectively three measured values 29.9,13.1, the 10.9pmol 4-MUmin that changes pUbi-tGluC
-1μ g
-1Albumen is respectively 3.24,1.42 and 1.18 times of contrast.
The result shows that tGluC compares with the no terminator and strengthened the expression level of foreign gene in paddy rice.
Claims (8)
1.DNA molecule is characterized in that: the dna molecular of said dna molecular for forming by the nucleotide sequence shown in the sequence in the sequence table 1.
2. the recombinant vectors, expression cassette, transgenic cell line, reorganization bacterium or the recombinant virus that contain the said dna molecular of claim 1.
3. expression cassette according to claim 2 is characterized in that: said expression cassette is by promotor, start the goal gene of transcribing and the described dna molecular of claim 1 that is positioned at said goal gene downstream is formed by said promotor.
4. expression cassette according to claim 3 is characterized in that: said promotor is composition type expression promoter or organizing specific expression promotor, and said organizing specific expression promotor is an endosperm specificity expression promoter.
5. according to claim 3 or 4 described expression cassettes, it is characterized in that: said goal gene is protein coding gene and/or non-protein coding gene; Said protein coding gene is the quality-improving gene; Said non-protein coding gene is just rna gene and/or sense-rna gene.
6. method of cultivating transgenic plant; Be that arbitrary described expression cassette among the claim 3-5 is imported in the purpose plant, obtain said destination gene expression level and be higher than the transgenic plant that following expression cassette imported said purpose plant: the described dna molecular of claim 1 in the described expression cassette of claim 3 is replaced with the expression cassette that the no terminator obtains;
Said purpose plant is a paddy rice.
7. the application of the described dna molecular of claim 1 in cultivating transgenic plant, said plant is a paddy rice.
8. the described dna molecular of claim 1 is as the application of terminator.
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| CN (1) | CN102260677B (en) |
-
2011
- 2011-07-15 CN CN201110197906A patent/CN102260677B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| Le Qing Qu and Fumio Takaiwa.Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice.《Plant Biotechnology Journal》.2004,113-125. * |
| Qu LQ,et al..Expression pattern and activity of six glutelin gene promoters in transgenic rice.《Journal of Experimental Botany》.2008,第59卷(第9期),2417-2424. * |
| Wu CY,et al..Promoters of rice seed storage protein genes direct endosperm-specific gene expression in transgenic rice.《Plant Cell Physiol.》.1998,第39卷(第8期),885-889. * |
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| CN102260677A (en) | 2011-11-30 |
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