CN103320441B - Plant promoter and application thereof - Google Patents
Plant promoter and application thereof Download PDFInfo
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- CN103320441B CN103320441B CN201310238965.XA CN201310238965A CN103320441B CN 103320441 B CN103320441 B CN 103320441B CN 201310238965 A CN201310238965 A CN 201310238965A CN 103320441 B CN103320441 B CN 103320441B
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Abstract
The invention discloses a plant promoter and application thereof. The invention provides a DNA (deoxyribonucleic acid) molecule which is as shown in (1) or (2) or (3), (1): a DNA molecule shown in a sequence 1 in a sequence table; (2): a DNA molecule which is hybridized with the DNA sequence limited in (1) under the strict condition and has a promoter function; (3): a DNA molecule which has homology of above 90 percent with the DNA sequence limited in (1) or (2) and has a promoter function. The invention provides a promoter, which is capable of driving a gene to be expressed in a plant. The invention provides an economic, rapid and effective approach for genetic modification of plants, especially vegetables and flowers of cruciferae. The plant promoter is wide in application space and market prospect in the agricultural field.
Description
Technical field
The present invention relates to a kind of plant promoter and application thereof.
Background technology
Arabidopis thaliana (Arabidopsis thaliana) is a kind of cress, be widely used in plant genetics, developmental biology and molecular biological research, make it become a kind of typical model plant because it has the features such as plant is little, generation time is short, genome is little.
Promotor is one section of DNA sequence dna being positioned at that structure gene 5' holds upstream, can activate RNA polymerase, make it combine exactly with template DNA and have the specificity of transcription initiation.Transcribe initial be the critical stage of genetic expression, and the major issue of this one-phase is the interaction of RNA polymerase and promotor: the avidity of the structure influence of promotor it and RNA polymerase, thus have impact on the level of genetic expression.
Summary of the invention
The object of this invention is to provide a kind of plant promoter and application thereof.
The invention provides a kind of DNA molecular, is following 1) or 2) or 3):
1) DNA molecular shown in sequence 1 in sequence table;
2) under strict conditions with 1) DNA sequence dna that limits hybridizes and has the DNA molecular of promoter function;
3) with 1) or 2) DNA sequence dna that limits has more than 90% homology and have the DNA molecular of promoter function.
Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, hybridizes, then use 2 × SSC under 65 ° of C, and 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Recombinant vectors containing described DNA molecular, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.Described recombinant vectors specifically can be the recombinant plasmid multiple clone site that described DNA molecular inserts pCAMBIA1391z carrier obtained.
Described DNA molecular has the function of promotor, can be applied to biotechnology as promotor.
The present invention also protects described promotor starting the application in destination gene expression.Described expression can be to be expressed in plant, specifically can be the expression in the flower of plant and/or leaf and/or root.Described plant can be monocotyledons or dicotyledons.Described dicotyledons specifically can be cress.Described cress specifically can be Arabidopis thaliana, as Columbia ecotype Arabidopis thaliana.Described goal gene specifically can be gus gene, as the gus gene in pCAMBIA1391z carrier.
The present invention also protects a kind of method of expression alien gene in plant, is DNA fragmentation first is imported the plant that sets out, thus expresses described foreign gene in the described plant that sets out; Described DNA fragmentation first contains described DNA molecular and described foreign gene from upstream successively to downstream.First described DNA molecular can be connected with described foreign gene, form warm DNA, then build recombinant expression vector with fusion dna, import plant.Also described DNA molecular and described foreign gene can be inserted respectively the multiple clone site that the carrier that sets out is applicable to, obtain the recombinant expression vector driving described exogenous gene expression with described DNA molecular, then recombinant expression vector is imported plant.For the ease of identifying transgenic plant and screening, can process described recombinant expression vector, as having the antibiotic marker thing or chemical resistance reagent marker gene etc. of resistance.Recombinant expression vector is by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, the conventional biology methods conversion of plant such as agriculture bacillus mediated.Set out described in described DNA fragmentation first specifically imports by following recombinant vectors plant: the recombinant plasmid multiple clone site that described DNA molecular and described foreign gene insert pCAMBIA1391z carrier obtained, and described DNA molecular is positioned at the upstream of described foreign gene.Described plant can be monocotyledons or dicotyledons.Described dicotyledons specifically can be cress.Described cress specifically can be Arabidopis thaliana, as Columbia ecotype Arabidopis thaliana.Described " in plant expression alien gene " can be and express described foreign gene in the flower of plant and/or leaf and/or root.
The invention provides a promotor, this promotor can drive gene to express in plant.The present invention helps lend some impetus to the research of people to Arabidopis thaliana development growth, and provides an economy, fast and effectively approach for the genetic modification of the plant particularly vegetable or flower of Cruciferae.The present invention has wide application space and market outlook at agriculture field.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS.
Fig. 2 is the GUS coloration result of transfer-gen plant.
Fig. 3 is the GUS coloration result of Columbia ecotype Arabidopis thaliana.
Fig. 4 is the GUS coloration result turning empty carrier plant.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.Columbia ecotype Arabidopis thaliana: Arabidopsis Biological Resource Center (ABRC), seed number is CS6673.PCAMBIA1391z carrier (also known as pCAMBIA1391z plasmid), is purchased from Cambia company, catalog number VZC0329.
The acquisition of embodiment 1, bHLH14pro sequence
With the genomic dna of the blade of Columbia ecotype Arabidopis thaliana for template, be there is the DNA molecular of promoter function, as shown in the sequence 1 of sequence table by pcr amplification and order-checking discovery one section.By DNA molecular called after bHLH14 promotor (also known as bHLH14pro) shown in the sequence 1 of sequence table.
The acquisition of embodiment 2, transfer-gen plant and qualification
One, the structure of recombinant expression vector
1, with the genomic dna of the blade of Columbia ecotype Arabidopis thaliana for template, adopt F1 and R1 composition primer pair carry out pcr amplification, reclaim pcr amplification product.
F1：5’-aa
ctgcagcctatgtcgatatgcgccgagaaggac-3’；
R1：5’-cgc
ggatccctttccagtgatcccgaccaaagaga-3’。
2, use the pcr amplification product of restriction enzyme PstI and BamHI double digestion step 1, reclaim digestion products.
3, with restriction enzyme PstI and BamHI double digestion pCAMBIA1391z carrier, the carrier framework of about 14kb is reclaimed.
4, the digestion products of step 2 is connected with the carrier framework of step 3, obtains recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS.According to sequencing result, structrual description carries out to recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS as follows: the DNA molecular shown in sequence 1 inserting sequence table between PstI and the BamHII restriction enzyme site of pCAMBIA1391z carrier.The structural representation of recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS is shown in Fig. 1.
Two, the acquisition of transfer-gen plant
1, recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS is imported Agrobacterium GV3101, obtain recombinational agrobacterium.
2, (namely the flower of Arabidopis thaliana is at OD600 by flower infusion method, recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS to be imported Columbia ecotype Arabidopis thaliana
nmsoak 30 seconds in the bacterium liquid of the recombinational agrobacterium that the step 1 of=0.8 obtains), the seed of results is T
0for the seed of Arabidopis thaliana (also known as T
1for seed).
3, by T
1for planting seed in the enterprising row filter of MS culture medium flat plate containing 20mg/L Totomycin, obtain the T that 30 strains have hygromycin resistance
1for plant (numbering is followed successively by 1-30).
4, T is treated
1be transplanted to when growing to 4-6 sheet leaf for plant and vermiculite grown 45 days (24 DEG C; 16 h light/8 h dark), extract 30 strain T respectively
1for the genomic dna of the leaf of plant, with the corresponding bHLH14pro of F2() and R2(correspondence gus gene) form primer pair and carry out PCR qualification, if show the pcr amplification product of about 1300bp, then plant is transfer-gen plant.
F2：5’-cctatgtcgatatgcgccgag-3’；
R2：5’-cacgggttggggtttctacag-3’。
Result shows, 30 strain T
1transfer-gen plant is for plant.
Three, the cultivation of wildtype Arabidopsis thaliana
The Colombia's Arabidopsis thaliana ecotype growing to 4-6 sheet leaf is transplanted to and vermiculite grows 45 days (24 DEG C; 16 h light/8 h dark).
Four, the cultivation of empty carrier Arabidopis thaliana is turned
Replace recombinant plasmid pCAMBIA1391z-bHLH14pro-GUS to carry out step 2 with pCAMBIA1391z carrier, obtain turning empty carrier plant.
Five, GUS staining analysis
The T of 4-6 sheet leaf will be grown in step 2
1for transfer-gen plant complete stool and root and grow the T of 45 days on vermiculite
1for the flower (30 strain) of transfer-gen plant, step 3 is grown to the Colombia Arabidopsis thaliana ecotype complete stool of 4-6 sheet leaf and on vermiculite, is grown the flower (20 strain) of Colombia's Arabidopsis thaliana ecotype of 45 days, grows to the T of 4-6 sheet leaf in step 4
1in generation, turns empty carrier plant complete stool and on vermiculite, grows the T of 45 days
1the flower (30 strain) that generation turns empty carrier plant, carries out GUS staining analysis respectively.
The flower of 30 strain transfer-gen plants, complete stool to be all shown as blueness with root tissue, dyeing the results are shown in Figure in 2(Fig. 2, A is flower, B is complete stool, C is root).Columbia ecotype Arabidopis thaliana and turn empty carrier plant each organize and be not all colored, the flower of Columbia ecotype Arabidopis thaliana and the coloration result of complete stool are shown in that Fig. 3 is (in Fig. 3, A is for flower, B is complete stool), the coloration result of the flower and complete stool that turn empty carrier plant see Fig. 4 (in Fig. 4, A for flower, B be complete stool).Result shows, the promotor shown in sequence 1 can drive gus gene spending really, express in leaf and root tissue.
Claims (7)
1. a DNA molecular is the DNA molecular shown in sequence in sequence table 1.
2. the recombinant vectors containing DNA molecular described in claim 1, expression cassette, transgenic cell line or recombinant bacterium.
3. recombinant vectors as claimed in claim 2, is characterized in that: described recombinant vectors is the recombinant plasmid multiple clone site that DNA molecular described in claim 1 inserts pCAMBIA1391z carrier obtained.
4. DNA molecular described in claim 1 is as the application of promotor.
5. promotor described in claim 1 is starting the application in destination gene expression.
6. the method for expression alien gene in plant, is that DNA fragmentation first is imported the plant that sets out, thus expresses described foreign gene in the described plant that sets out; Described DNA fragmentation first is followed successively by DNA molecular described in claim 1 and described foreign gene from upstream to downstream; The described plant that sets out is Arabidopis thaliana.
7. method as claimed in claim 6, is characterized in that: set out described in described DNA fragmentation first is imported by recombinant vectors plant; Described recombinant expression vector is the recombinant plasmid multiple clone site that DNA molecular according to claim 1 and described foreign gene insert pCAMBIA1391z carrier obtained; Described DNA molecular is positioned at the upstream of described foreign gene.
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| CN106947764B (en) * | 2017-03-23 | 2020-04-21 | 中国农业科学院作物科学研究所 | Plant root specific promoter and application thereof |
| CN107354160B (en) * | 2017-08-04 | 2020-02-07 | 首都师范大学 | Plant promoter and application thereof |
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| PT1440151E (en) * | 2001-09-05 | 2008-03-17 | Monsanto Technology Llc | Seed specific 7s alpha promoter for expressing genes in plants |
| CN101280313B (en) * | 2008-05-21 | 2010-06-02 | 中国农业大学 | Root specific promoter and recombinant expression vector thereof |
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