CN107460195A - Cabbage type rape als gene promoter and application - Google Patents

Cabbage type rape als gene promoter and application Download PDF

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CN107460195A
CN107460195A CN201710701957.2A CN201710701957A CN107460195A CN 107460195 A CN107460195 A CN 107460195A CN 201710701957 A CN201710701957 A CN 201710701957A CN 107460195 A CN107460195 A CN 107460195A
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promoter
plant
sequence
gene
cabbage type
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陈松
浦惠明
彭琦
张洁夫
陈锋
王晓东
张维
胡茂龙
龙卫华
高建芹
付三雄
周晓婴
熊冬琴
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Jiangsu Academy of Agricultural Sciences
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01006—Acetolactate synthase (2.2.1.6)

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Abstract

The present invention relates to cabbage type rape als gene promoter and application, belong to biological technical field.The present invention has cloned rape als gene promoter sequence, the promoter element included in sequence is predicted by bioinformatics software, and qualitative detection analysis is carried out to its promoter activity by building plant expression vector, as a result show that institute's cloned sequence can drive gus reporter gene to be expressed in each organ of tobacco seedling, show that the sequence has the expression activity and feature of very strong constitutive promoter, and strong composition type expression promoter can be used as to be applied in plant genetic engineering research.Cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium kermes alkali gene no promoters commonly used in the alternative research of plant genetic engineering at present of the promoter, build plant transgene expression vector, applied to the genetic engineering of dicotyledon, the bio-safety risk of genetically modified plants is reduced.

Description

Cabbage type rape als gene promoter and application
Technical field
The present invention relates to cabbage type rape als gene promoter and application, belong to biological technical field.
Background technology
Rape is one of most important oil crops, and the oil crops that China is important in the world.China's rapeseed cultivation Area and total output occupy first place in the world.In the 60 to 70's, China's rape annual planting area is ten thousand mu about more than 3000, per mu yield 30-40 Kilogram；Have developed to 100,000,000-1.1 hundreds of millions mu, per mu yield 90-100kg the nineties, rapeseed total output is more ten thousand tons up to 1100-1200. Rape is that the fifth-largest crop of the China after paddy and wheat, corn and soybean (will exceed soybean, the Yangtze river basin such as Hubei etc. quickly Save, rape is the second largest crop after rice), and unique winter oil crops.China's rapeseed area, total yield account for / 3rd of the world, it is maximum Rape-seed production state.The Yangtze river basin is rape producing region the biggest in the world, and area, total yield account for World's a quarter.Canola oil accounts for China's edible oil 50% or so, is the most important edible vegetable oil in China.
In view of rape is in consequence in oil crops, rape scientific research is also by Genetic breeders Pay much attention to.The past genetic improvement of rape in decades obtains notable achievement, and high yield, high-quality double-low rapeseed kind are in production Leading position is already taken up.In recent years, the rapid development of modern biotechnology and molecular biology research, some biological skills Art is basic with being also used widely in application study in rape, the molecular mark of rape, dense genetic map Structure, the Genes location of important character gene and clone, and the research field such as genetic engineering improvement all make remarkable progress. Wherein the genetic engineering research of rape and commercial applications are worldwide than earlier.Such as last century Mo, high bay The transgene rape of acid just comes into Commercialization application.And antiweed glyphosate transgene rape is national in Canada etc. To being widely applied, huge economic benefit is generated, is the most successful case of transgene rape.And domestic transgene rape Research also obtains some progress in pest-resistant, transgenosis high oleic acid and Transgenic Resistant Herbicide etc..But at present in China The transgene rape kind for not having commercialization still is applied in production.
The research of genetic engineering be unable to do without promoter with application, and the promoter that excavating has application value is always plant molecular The important subject of breeding.Research about rape promoter in recent years constantly some document reports.The scape third of the twelve Earthly Branches (2012) Expanded with inverse PCR method and obtain galactinol synthase gene (B n G O L S 1) promoter, there is promoter work( Can, gus gene can be driven to be expressed in Oil Rape Tissue；The amplification of Xiao Danwang (2016) PCR method obtains rape hemolytic phosphatidyl Transferase gene (LPAT) promoter；Zhu Bin (2013) PCR amplification method obtains the gene families of cabbage type rape MAP K 7 and opened Mover；Wang Xuanpeng (2013) obtains the gene promoters of cabbage type rape R ab G D I 3, the startup according to genome sequence Son can drive reporter gene g u s specifically expressings only in flower pesticide, be tissue-specific promoter；Sun is learned (2016) etc. and passed through forever PCR clones obtain rape cysteine proteinase family gene BnCP51 promoter sequence, should by transgenosis functional verification Promoter is only expressed in bud and flower pesticide organ, and a kind of Organ specific expression promoter；Shao Tiemei etc. (2016) Rape oil body protein promoter is cloned into using PCR method based on rapeseed gene group information, the promoter has seed specific Expression characterization, this is also the specific expressed promoter of another rape seed after rape Napin promoters.Believe with The passage of time, the promoter for having increasing rape are found and applied.
It is mainly at present the promoter of cauliflower blossom disease poison using more promoters in rapeseed gene engineering research (CaMV35S), this is a promoter from the constitutive expression of plant DNA virus.It is past generally in transgenic protocol Toward secondary 2 or more than 2 expression cassettes of conversion, still, in view of driving 2 or more using same promoter in cell The expression of gene, the phenomenon for being easily caused transgene silencing occur.Therefore, excavate the promoter of new high expression activity also just into One importance of plant genetic engineering research, especially excavate the promoter of the relatively low plant origin of bio-safety risk Seem and be even more important.
Acetolactate synthestase (ALS) is catalysis branched chain amino acid such as valine, leucine and isoleucine life in plant The enzyme of thing synthesis first step reaction.Research finds to suppress the ALS activity in plant cell, hinders branched-chain amino acid (valine, bright Propylhomoserin and isoleucine) biosynthesis, so as to suppress the division of plant cell and growth, serious meeting causes Plant death.Base A series of herbicide extensive uses in production for ALS enzymes are have developed successively in some chemical companies of this principle.And ALS Gene is also by the common concern of biologists.The als gene of some plants or crop is cloned.However, relevant als gene There is not been reported for the research and application of promoter.
The content of the invention
Technical problem
It is contemplated that clone's cabbage type rape (Brassica napus) als gene promoter region sequence, and pass through Transgenosis verifies its promoter sequence size, activity and function, to screen the composition type expression promoter of plant origin, can replace Cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium kermes alkali gene commonly used in being studied for current plant genetic engineering Nos promoters, plant transgene expression vector is built, be plant genetic engineering applied to the genetic engineering of dicotyledon Using the reliable new selection of offer, and the bio-safety risk of genetically modified plants may be reduced.
Technical scheme
A kind of cabbage type rape als gene promoter, sequence signature include the sequence and mutual with it shown in SEQ NO.1 Mend, it is homologous, or the sequence is by insertion, the nucleotide sequence with identical function for being mutated several nucleotides and being formed.
Present invention additionally comprises the recombinant vector containing the cabbage type rape als gene promoter nucleotide sequence, and Plant host cell containing the recombinant vector or plant host tissue.
The method that the promoter is expressed in plant, it is characterized in that：
(1) described recombinant vector is imported into plant cell,
(2) described plant cell growth is made into the dicotyledon of plant.
In described method, the plant is tobacco.
The cabbage type rape als gene promoter can be with driving function gene in each organ of plant especially tobacco The application of expression.
The cabbage type rape als gene promoter can also substitute cauliflower flower in the genetic engineering of dicotyledon In terms of mosaic virus (CaMV) 35S promoter or Agrobacterium kermes alkali gene no promoters structure plant transgene expression vector Using.
Beneficial effect
The present invention has cloned cabbage type rape (Brassica napus) als gene promoter sequence, passes through biological information The promoter element that software prediction sequence is included is learned, and its promoter activity is carried out by building plant expression vector Qualitative detection, the results showed that institute's cloned sequence can drive gus reporter gene table in tobacco seedling root, stem, each organ of blade Reach, show that the sequence has the expression activity and feature of very strong constitutive promoter, and can be opened as strong constitutive expression Mover is applied in plant genetic engineering research.
Cauliflower mosaic virus (CaMV) 35S commonly used in the alternative research of plant genetic engineering at present of the promoter starts Son and Agrobacterium kermes alkali gene no promoters, build plant transgene expression vector, the gene work applied to dicotyledon Journey, reduce the bio-safety risk of genetically modified plants.
Brief description of the drawings
Fig. 1, als gene promoter pcr amplification product electrophoretogram
The first column is DNA molecular amount standard from left to right：5000bp,3000bp,2000bp,1000bp,750bp,500bp, 200bp, 100bp, the second column are PCR primer.
Fig. 2, recombinant vector schematic diagram.
Fig. 3, recombinant vector is through restriction enzymes double zyme cutting collection of illustrative plates
The first column is DNA molecular amount standard from left to right：5000bp,3000bp,2000bp,1000bp,750bp,500bp, 200bp, 100bp, the second column are recombinant plasmid pCAMBIA1304-P, and third column is restriction enzyme HindIII, NcoI double Digestion result.
Fig. 4, transgene tobacco resistant budses induction (left side) and culture of rootage (right side).
Fig. 5, transgene tobacco GUS activity qualitative detection results, a left side 1,2 is transgene tobacco seedling；The right side 1 is non-transgenic cigarette Careless seedling control.
Embodiment
1st, promoter sequence clone analyzes with promoter element
ALS of the present invention according to rape M3423Gene order, utilize NCBI websites (https:// Www.ncbi.nlm.nih.gov/ its corresponding rapeseed gene group sequence) is retrieved, extracts ALS3The end of gene coding region 5 ' upstream The sequence of about -2000 bases, utilize professional website (http://molbiol-tools.ca/Promoters.htm) carry out Promoter prediction is analyzed, and according to analysis result, is chosen the sequence of the base of upstream -1000 rich in promoter element or so, is designed Specificity amplification primer.Sense primer is mainly with ALS3Gene upstream sequence is template, and anti-sense primer is with M342 ALS3Gene 5 ' end non-coding region sequences are template, expand ALS3The upstream size about 1048bp of gene DNA sequence dna.
1.1 promoter sequences expand and clone：With Atrazine resistant Brassica napus M342 (it is public, see patent of invention " 201310054645.9, a kind of Atrazine resistant Brassica napus directive breeding method based on ALS target enzymes ") genomic DNA is template, if Count primer pair LPF (5'-AAG CTT GTG GAG CTG ATC TTA CCG ACC GAA C-3')/LPR (5'-CCA TGG GGT TAG AGG AGA GAG AGA TGA TGA A-3'), ' end with the addition of restriction enzyme to the primer pair 5 respectively Site：In 5 ' ends of sense primer addition restriction enzyme HindIII restriction enzyme site, add in 5 ' ends of anti-sense primer Add NcoI restriction enzyme site；Rape is expanded with the high-fidelity DNA polymerase produced with Beijing Quanshijin Biotechnology Co., Ltd ALS3The DNA sequence dna of the promoter region of gene：Amplification system：5 × Trans Start FastPfu Buffer10 μ l, base Because of group DNA1 μ a l, 10 μM of primer LPF 1 μ l, 10 μM of 1 μ l, 2.5mM dNTPs of primer LPR 5 μ l, Trans Start The μ l of FastPfu archaeal dna polymerases 1, supply ddH2O to 50 μ l；Temperature program(me)：95 DEG C, 3min, 95 DEG C, 30sec, 55 DEG C, 30sec, 72 DEG C of 1min, after 35 circulations, 72 DEG C, 5min, agarose gel electrophoresis checks amplified production (see Fig. 1), uses Beijing The PCR primer for the glue reclaim kit, recovery purifying 1Kb or so that Quan Shijin Bioisystech Co., Ltd produces simultaneously is cloned into Beijing On the T/A carriers pEASY-T1 that Quan Shijin Bioisystech Co., Ltd produces, bacillus coli DH 5 alpha is converted, it is purposeful to obtain restructuring The intermediate carrier pEASY-P of sequence, chooses positive colony sequencing.
1.2 sequence analysis：The clone of acquisition is actual to obtain the ALS that length is 1048bp after sequencing removes carrier sequence3 Gene upstream sequence (see sequence 1).
Sequence 1, the main related cis-acting elements of the promoter sequence of acquisition and part
Pass through Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/ Plantcare/html/) the ALS to being cloned into3Gene upstream sequence carries out bioinformatic analysis, as a result shows that the sequence contains There are a variety of cis-acting elements related to promoter, such as CAAT-box, TATA-box etc., show the ALS3Gene upstream sequence Architectural feature with eukaryotic promoter, thus it is speculated that this section of sequence may have promoter activity.Part cis element is concluded In list 1.
Table 1, the main related cis-acting elements that ALS promoter sequences contain
Element title Position Sequence Function
ABRE-motif 328 CACGTG Abscisic acid response element
Box4 202 ATTAAT Light response element
CAAT-box 696,840 CAAAT Promoter region or the cis-acting elements in enhancer region
810 CAAT Promoter region or the cis-acting elements in enhancer region
CGTCA-motif 296 CGTCA Methyl jasmonic acid response element
G-box 328 CACGTG Cis light response element
1008 CACGTT Cis light response element
HSE 631 AGAAAATTCG Cis heat shock response element
TATA-box 116,183,186,465 TAATA Core promoter element
562 ATATAA Core promoter element
647,650 TAATA Core promoter element
687 TTTTA Core promoter element
753 ATATAA Core promoter element
858,912 TAATA Core promoter element
996 TATAAA Core promoter element
TGA-element 586 AACGAC Auxin response element
2nd, promoter function checking expression vector establishment
For the cloned ALS of further checking3The biological function of gene upstream sequence, sequence restructuring is arrived 5 '-end of gus reporter gene, is thus constituted by ALS on pCAMBIA1304 carriers (Australian CAMBIA companies)3 One complete gus gene expression cassette of gene promoter driving.
The clone that promoter sequence is cloned in pEASY-T1 (Beijing Quanshijin Biotechnology Co., Ltd) acquisitions opens The intermediate carrier pEASY-P of promoter sequences.The plasmid extraction kit produced with Beijing Quanshijin Biotechnology Co., Ltd, point The clone for indescribably taking the present invention to obtain has the intermediate carrier pEASY-P plasmids and plant expression vector of als gene upstream sequence PCAMBIA1304 plasmids.
And the restriction enzyme HindIII and NcoI to be produced with NEB companies distinguish thorough double digestion pEASY-P and PCAMBIA1304 DNAs.The size about 1000bp promoters cut on agarose gel electrophoresis recovery pEASY-P plasmids Fragment and the pCAMBIA1304 of linearisation, the latter's size is 11600bp.In linearisation pCAMBIA1304 vector plasmid processes In, the original CaMV35S promoter sequences of gus gene, size 760bp are cut away by double digestion.Then connected again by T4 Enzyme reaction is by ALS3On the pCAMBIA1304 carriers that gene upstream sequence restructuring linearizes to double digestion, connection product conversion is big Enterobacteria DH5 α, obtain recombinant clone pCAMBIA1304-P (see Fig. 2).Selected clone culture, recombinant plasmid is extracted, is limited Property endonuclease digestion checking processed a, it can be seen that size is about the double enzyme digestion products of 1Kb (see Fig. 3).Prove promoter sequence Recombinate in destination carrier.Further through Sequence analysis, it is determined that the ALS cloned3Gene upstream sequence is accurately inserted Enter the corresponding restriction enzyme site of pCAMBIA1304 carriers, no missing, mismatching phenomenon, the recombinant vector can be used for the transgenosis of tobacco Experiment, carry out the functional verification work of promoter.
3rd, Transgenic Tobacco is tested
For the biological function of further checking institute cloning promoter, the expression vector pCAMBIA1304-P of structure is turned Enter Agrobacterium EHA105.Tobacco is transfected using leaf disc transformation method.
Specific experiment process is as follows：
3.1. Agrobacterium prepares：Picking carrying carrier pCAMBIA1304-P Agrobacterium EHA105 single bacterium colony, is inoculated in In 5ml 50mg/L containing Rif20mg/l and Kan LB fluid nutrient mediums, 28 DEG C of shaken cultivations are stayed overnight.Take and activate overnight agriculture bar Bacterium, by 1：50 dilution proportion continues culture to OD600 into the LB fluid nutrient mediums of the 50mg/L containing Rif20mg/L and Kan Value is about 0.6~0.8.6000rpm centrifuges 5min, thalline is collected, with the fluid nutrient mediums of 3%MS containing sucrose washing thalline one It is secondary, and it is diluted to OD600 values 0.3~0.35.
3.2 tobacco leaf disc genetic transformations：Choose tobacco (Nicotiana benthamiana) nothing of seedling age about 30 days or so Vaccine, it is explant to cut tobacco leaf into the blade of size about 0.8cm square with scalpel under aseptic condition, and input is ready for In good Agrobacterium bacterium solution, after 3-5 minutes are infected in vibration, the raffinate for being attached to blade surface is blotted in taking-up with filter paper, Ran Houfang Dark place co-cultures on base (1 × MS, sucrose 3%, agar powder 1%, BA 2.0mg/L, IAA 0.5mg/L, pH 5.8) is co-cultured 48 hours, 25 DEG C of cultivation temperature.
The regeneration of 3.3 transgene tobacco seedlings：After co-cultivation, turn outer plant to bud inducement cultivation base (1 × MS, sucrose 3%, BA2.0mg/L, IAA0.5mg/L, carbenicillin 500mg/L, hygromycin 20mg/L, agar powder 1%, pH 5.8) it is enterprising Row bud inducement cultivation, periodicity of illumination illumination in 16 hours, 8 hours dark, 25 DEG C of cultivation temperature.Every 2-3 weeks subculture once.Subculture When culture medium with resistant budses inducing culture.After adventitious bud is grown, and when bud grows to 1-1.5cm, bud is cut to change to and taken root Culture medium (1 × MS, sucrose 3%, IAA0.5mg/L, carbenicillin 500mg/L, hygromycin 20mg/L, agar powder 1%, pH 5.8) root induction on, illumination are the same with temperature conditionss.This process along with bud growth and take root, see Fig. 4.
4th, gus gene expression activity detects：Take by the above-mentioned 3.3 transgene tobacco seedling obtained, developed the color and tried with gus gene Agent, carry out the qualitative detection of gus gene expression activity.Tobacco seedling is completely taken out, is immersed in the nitrite ion of gus gene activity In, nitrite ion composition is：50mMNa2HPO4And NaH2PO4The buffer solution of composition, pH value 7.2, contains：0.1%Triton X- 100,2mMK3Fe (CN) 6,2mMK4Fe (CN) 6,10mMEDTA, 2mM X-Gluc.After placing 3-6 hours at room temperature, with anhydrous second Alcohol decolorization, the background color of place to go blade.If blue reaction just occurs in the tissue of gus gene expression activity, warp The experiment that develops the color finds that the tobacco seedling root of transgenic positive, stem, blade, complete stool obvious blueness occur and reacted, and transgenosis Negative seedling is in then light yellow.Taken pictures through stereomicroscope, as a result see Fig. 5.This result shows the ALS that the present invention clones The upstream sequence of gene has very strong constitutive promoter expression activity and feature, has certain application value.
The present invention has cloned rape als gene promoter sequence, and promoter element is predicted by bioinformatics software, And qualitative detection is carried out to its promoter activity by building plant expression vector, the results showed that institute's cloned sequence can drive Gus reporter gene high can express expression in tobacco seedling root, stem, foliage organ, show that the sequence has very strong composing type The activity of promoter, and strong composition type expression promoter can be used as to be applied in plant genetic engineering research.The promoter can Substitute in current plant genetic engineering research and commonly use cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium kermes alkali gene Nos promoters, plant transgene expression vector is built, applied to the genetic engineering of dicotyledon, reduces genetically modified plants Bio-safety risk.
SEQUENCE LISTING
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agattgatga tctacaaagg aaatcatcac gtgaattgaa aatctcgaag ttccatagct 360
ggttacagag atcgaaggag aagccttttc gattcagtgt ggaagaagaa caggaactct 420
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cccatcaaca tttgcgttta atgaaaccat gagtttcgtt cctgaatgac atgtgtcagc 540
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gtagcctaaa cgttcttagt gcacattttt aaatgcaaat atattacgga tttggtccta 720
atttctgtaa aataaaacta cttcattgtt ttatataaat aactctattg ctgcagtttc 780
ccaactttgt tgcttagatt caggtctcac aatcaagaaa tcaagaacag ttagatccac 840
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Claims (9)

1. a kind of cabbage type rape als gene promoter, sequence signature is complementary including the sequence shown in SEQ NO.1 and with it, It is homologous, or the sequence is by insertion, the nucleotide sequence with identical function for being mutated several nucleotides and being formed.
2. the recombinant vector containing nucleotide sequence described in claim 1.
3. the plant host cell containing recombinant vector described in claim 2 or plant host tissue.
4. a kind of method for the promoter that claim 1 is expressed in plant, it is characterized in that：
(1) recombinant vector described in claim 2 imports plant cell,
(2) described plant cell growth is made into the dicotyledon of plant.
5. in accordance with the method for claim 4, it is characterised in that the dicotyledon is tobacco.
6. the application of cabbage type rape als gene promoter described in claim 1.
During 7. cabbage type rape als gene promoter driving function gene described in claim 1 is expressed in each organ of plant Application.
During 8. cabbage type rape als gene promoter driving function gene described in claim 1 is expressed in each organ of tobacco Application.
9. cabbage type rape als gene promoter substitutes cauliflower in the genetic engineering of dicotyledon described in claim 1 In terms of mosaic virus (CaMV) 35S promoter or Agrobacterium kermes alkali gene no promoters structure plant transgene expression vector Application.
CN201710701957.2A 2017-08-16 2017-08-16 Cabbage type rape als gene promoter and application Pending CN107460195A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679956A (en) * 2019-01-09 2019-04-26 西南大学 Promoter sequence, recombinant vector and application based on BnaCnng52950D gene
CN109735560A (en) * 2019-01-29 2019-05-10 江苏省农业科学院 A kind of carrier construction method of the selection reporter gene expression frame containing complete plant origin and application
CN111471684A (en) * 2020-05-07 2020-07-31 海南波莲水稻基因科技有限公司 Plant constitutive promoter A L Spro and application thereof

Citations (3)

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