CN107488664B - Cabbage type rape root-specific promoter pBn21194 and its application - Google Patents

Abstract

The invention discloses a kind of cabbage type rape root-specific promoter pBn21194 and its applications.The promoter of gene Bn21194 has been cloned from cabbage type rape, construct plant expression vector pBI121-pBn21194, using the inflorescence dip method arabidopsis thaliana transformation of mediated by agriculture bacillus, the dyeing of GUS histochemical method is carried out to the positive transgenic strain of screening, the result shows that: specifically expressing of the pBn21194 with driving downstream gene in root, and the function of not expressed in other histoorgans.The promoter improves crop resistance in rape transgenosis, in terms of nutritional quality and in terms of manual creation, has good application potential.

Description

Cabbage type rape root-specific promoter pBn21194 and its application

Technical field

The invention belongs to plant genetic engineerings and field of biotechnology, and in particular to a kind of cabbage type rape Bn21194 base The promoter of cause is named as pBn21194 (same as below), which can be used for the expression of arabidopsis root-specific.

Background technique

Plant gene promoter is to be located at structural gene 5 ' to hold upstream region, the section of DNA sequence containing cis-acting elements Column play key effect in the expression regulation of gene: determining specificity, direction and the efficiency of downstream gene transcription.In addition, Promoter can play a key effect in building during high level expression heterologous expression vector, it determines foreign gene table The expression of the sequence of time and space, expression intensity, transcriptional efficiency and the gene that reach.So studying the function of promoter for gene Expression and regulation mechanism and increasingly mature plant genetic engineering have very important scientific meaning.

It is found by the analysis to various plants gene promoter area, the promoter of most functional protein genes all has There is common tactic pattern, is generally made of core promoter element and upstream element.Positioned at transcription initiation site upstream -20 The TATA box at the place~-30bp is the core element of promoter, is rich in the conserved sequence area for having AT, with DNA (deoxyribose core Acid) double-strand unwinding it is related, and determine the selection of transcripting start point, be most plant promoters correctly express it is required 's.The conserved sequence of the upstream TATA box is known as promoter upstream element, and the CAAT box and -80 including the place -75bp~- The general upstream promoter element such as GC box near 110bp and other special upstream elements, such as jasmonate response (Zhang Chunxiao, Wang Wenqi, Jiang Xiangning wait plant gene promoter to study to the elements such as element (JRE), ethylene response element (ERE) Be in progress [J] Acta Genetica Sinica, 2004,31 (12): 1455-1464；Lu Jing, Zhao Huayan, He Yikun, wait Plant Promoter and Its application study progress [J] natural science progress, 2004,8:002.).Wherein, CAAT box be than more conservative sequence, with The identification and combination of RNA (ribonucleic acid) polymerase are related, have stronger activation to genetic transcription.As long as being provided with this Conservative structural frames a bit, then can have the function of the starting downstream gene expression of response.

3 classes: constitutive promoter, tissue or organ specific promoters can be classified as according to the transcriptional profile of promoter And inducible promoter.

Stablize table in all developmental stages and tissue of the foreign gene of constitutive promoter driving in transgenic plants It reaches.Conversion to many dicotyledons is generally used the 35S promoter from Caulimovirus (CaMV) or comes from bacterium Nopaline synthase no promoter plasmid vector, and in monocot transformation the most commonly used is contain rice flesh Plasmid vector (Guan Liying, Liu Xianglin, the Yin Liping of filamentous actin Act promoter, maize ubiquitin Ubi promoter and 35S promoter The effective expression of foreign gene and its safety evaluatio [J] Capital Normal University journal in genetically modified plants: natural science edition, 2002,23(2):52-56)。

But many times high efficient expression of the foreign gene in recipient plant not only causes the waste of the energy in organism, And it is organized in expression may have toxic action to plant itself, or even cause Transgene-safty problem (Shirong J.Environment and Food Biosafety Assessment of Transgenic Plants[J].PROGRESS IN BIOTECHNOLOGY,1997,6).Therefore, inducible promoter and tissue-specific promoter research and application increasingly Attention by breeder.The inducible promoter identified in transgenic plants mainly includes abiotic stress induction type (Nie Lina, Chillon qin, Xu Zhaoshi wait plant for promoter, biotic inducible promoter and hormone inducible promoter etc. The clone of gene promoter and its Research progress on Function [J] plant genetic resources journal, 2008,9 (3): 385-391).But The application of inducible promoter also has certain limitation, handles the external condition that recipient plant carries out, such as heat shock, at hormone Reason etc. may cause the normal growth that a series of Physiology and biochemistry in organism reflects and be unfavorable for plant.In addition, chemical regulation It is used as the methyl prednisolone, estradiol and tetracycline of inducer in system all to ecological environment nocuousness, should not be used in production Practice.

Can be to avoid this problem using the tissue-specific promoter of itself in plant, the tissue of foreign gene is special By the biological safety for effectively improving genetically modified crops, (Song Yang, Zhou Junhui, Zhang Yongqiang plant tissue specificity are opened for opposite sex expression Mover studies [J] biotechnology notification, 2007,2007 (6): 21-24).In recent years, about tissue-specific promoter Research has been made significant headway, including the Organ specific expressions such as blade, bast, vascular bundle, root promoter and pollen, hero (Song Yang, Zhou Junhui, Zhang Yongqiang plant tissue specificity open the reproductive organs such as stamen, gynoecium, fruit, seed specific expression promoter Mover studies [J] biotechnology notification, 2007,2007 (6): 21-24).Pyk10 exists in such as Inke Nitz discovery arabidopsis Specifically expressing in root finds that its promoter has root-specific (Inke Nitz, Heike by GUS staining analysis Berkefeld,Piotr S.Puzio,et al.Pyk10,a seedling and root specific gene and promoter from Arabidopsis thaliana.Plant Science 161(2001)337-346.Takanori etc. The same expression that gus gene is detected by transgene tobacco, the promoter that screening obtains rRSP1, rRSP3, rRSP5 in rice are Root specific expression promoter.(HUANG Li-yu,ZHANG Fan,QIN Qiao,et al.Identification and validation of root-specific promoters in rice.Journal of Integrative Agriculture 2015,14(1):1–10.Li Chen etc. identifies and has studied soybean GmTIP gene and its starting sub-feature It was found that it is specific expressed in root, and under the treatment conditions such as ABA, expression significantly improves, and improves the resistance of plant, has very Good application prospect (Li Chen, Bingjun Jiang, Cunxiang Wu et al.The characterization of GmTIP,a root-specific gene from soybean,and the expression analysis of its promoter.Plant Cell Tiss Organ Cult(2015)121:259–274).The specificity promoter of root is enhancing Plant resistance to environment stress, reinforcement nutrient absorption etc. also have many other reports: Kynet Kon etc. utilizes LjNRT2 and AtNRT2.1 base The promoter of cause drives artificial chitinase gene, and the resistance to Fusarium oxysporum is obtained in transgene tomato and tobacco (Kynet Kong,So Makabe,Valentine Otang Ntui,et al.Synthetic chitinase gene driven by root-specific LjNRT2 and AtNRT2.1 promoters confers resistance to Fusarium oxysporum in transgenic tobacco and tomato.Plant Biotechnol Rep (2014)8:151–159).In addition, wheat expansin gene is transferred in tobacco by Ai Xiu etc. using root-specific promoter, It was found that enhancing the growth of root system and resistance (Ai Xiu Li, Yang Yang Han, Xin Wang, et to Water deficit al.Root-specific expression of wheat expansin gene TaEXPB23 enhances root growth and water stress tolerance in tobacco.Environmental and Experimental Botany 110(2015)73–84).Meanwhile Jin Seo Jeong etc. is also specific expressed in root by rice Os NAC10, hair Now which increase to arid resistance and improve yield (Jin Seo Jeong, Youn Shic Kim, Kwang Hun Baek,et al.Root-Specific Expression of OsNAC10 Improves Drought Tolerance and Grain Yield in Rice under Field Drought Conditions.Plant Physiology Vol.153, pp.185–197).The promoter IDE1 and IDE2 that the iron of the discovery barley such as Takanori lacks special responsive genes IDS2 have Root-specific, the function of inducing expression under iron deletion condition to adjust the metabolism of ferro element, and create plant to iron deficiency Tolerance improves nutritional quality (Takanori, Yuko, Reiko, et al.Identification of novel cis- acting elements,IDE1 and IDE2,of the barley IDS2 gene promoter conferring iron-deficiency-inducible,root-specific expression in heterogeneous tobacco plants.The Plant Journal(2003)36,780-793).It can be seen that root-specific promoter is in various plants Resistance is being improved, enhancing absorbs, and increases yield etc. and is all applied, there should be good development prospect.

The above report is all the example for carrying out promoter function analysis between different plants using transgenic technology, These examples show using model plant arabidopsis, tobacco etc. as carrier, by the expression analysis of genetically modified plants confirm come Function from the promoter of other plant is to be widely recognized as and received.

Rape is important edible oil source, and the resistance of rape, nutritional quality and resistant to lodging are improved by transgenosis Etc. characters be suitable for mechanization production, be the project of researcher the supreme arrogance of a person with great power, then screening root-specific express starting Son is just necessary.For the present invention by experimental analysis, it is special that discovery promoter pBn21194 shows very strong root-specific expression Property, in pBn21194 transgenic arabidopsis, the Reporter gene GUS driven is only all showed in the root of transgenic arabidopsis Very strong expression out, these data sufficiently show that pBn21194 is a very strong root-specific expression promoter, are planting It has a good application prospect in object transgenic breeding.

Summary of the invention

An object of the present invention is to be to provide a kind of cabbage type rape promoter pBn21194, the advantage is that two Aspect: firstly, can be accurately positioned regulated and controled gene from the endogenous tissue-specific promoter of rape, purpose base is driven Because expressing in the specific organization of genetically modified plants or period, the waste of plant self energy and substance is avoided；Secondly, improving The expression efficiency of foreign gene in transgenic plants, limits its expressive site.Therefore, which can be applied to the base of plant Because of engineering research and germ plasm resource Upgrading.

The second object of the present invention is to be the provision of the amplimer of cabbage type rape pBn21194 promoter.With wild cabbage Type rapeseed gene group DNA is template, carries out PCR amplification using the specific primer, obtains cabbage type rape promoter PBn21194 sequence, easy to operate, product is special, as a result reliable and stable.

The third object of the present invention is to be the provision of a kind of recombination containing plant efficient expression promoter pBn21194 Carrier pBI121-pBn21194.The carrier is sized for, and is easy to convert in plant, and the marker gene GUS expression being had is strong Degree is high, is easy detection, can get gus gene arabidopsis of high efficient expression in plant by this carrier arabidopsis thaliana transformation and turns base Because of plant.

The fourth object of the present invention is to be the provision of cabbage type rape promoter pBn21194 in plant (arabidopsis) root Application in system's expression.Under the driving of the promoter, the main specifically expressing in the root of plant of target gene, and in other portions It does not express position.This promoter with tissue specific expression is in the genetic engineerings such as creation fine germplasm resources and transgenosis peace There is good application value in complete.

In order to complete above-mentioned purpose, the present invention adopts the following technical scheme:

The clone of promoter pBn21194: PCR amplification, forward primer are carried out by template of cabbage type rape genomic DNA Bn21194P-Hind3-UP is 5'-CCCAAGCTTGAGAACCGGCAAGTAATCCA-3', reverse primer Bn21194P- BamH1-DN is preceding 3 alkali in 5'-CGCGGATCCTGTTATGAGATCTTCTTCGCT-3'(primer Bn21194P-Hind3-UP Base CCC is that base is protected in digestion, and AAGCTT thereafter is HindIII restriction enzyme site, remaining sequence is pBn21194 upstream amplification Sequence；In primer Bn21194P-BamH1-DN, preceding 3 base CGC is that base is protected in digestion, and GGATCC thereafter is BamHI digestion Site, remaining sequence are pBn21194 downstream amplification sequence), cabbage type rape pBn21194 sequence is obtained, such as SEQ ID NO.1 It is shown.

The construction method of recombinant vector pBI21-pBn21194, the steps include:

It is carried out with PCR product and plant expression vector pBI121 of Hind III and BamH the I enzyme to above-mentioned pBn21194 Double digestion, the CaMV35S promoter on pBI121 will be removed.Two kinds of 1% agarose gel electrophoresis of digestion products are detected, It cuts glue and is recycled with gel reclaims kit, T4 ligase is added and is attached.Heat shock method converts E. coli competent DH5 α Afterwards, then on the solid LB plate of (50ug/L) containing kanamycin it is screened, the monoclonal of picking is with carrier upstream primer PBI21-F:5'-TGTGGAATTGTGAGCGGATA-3' and aim sequence special primer pBn21194-R:5'- TGTTATGAGATCTTCTTCGCT-3' carries out positive detection, extracts the expression vector plasmid of positive colony, which attaches most importance to Group carrier pBI121-pBn21194.

The functional analysis of promoter pBn21194 and its application in the expression of arabidopsis root system:

The promoter cis-acting elements is carried out by promoter function element forecasting software PlantCARE online pre- Survey analysis, the results showed that the promoter sequence cloned contains the upstreams such as TATA box core promoter sequence, CAAT box and opens Mover element and root-specific Expression element；The plant table by the Reporter gene GUS of the promoter regulation is constructed on this basis Up to carrier pBI121-pBn21194；Using the inflorescence dip method arabidopsis thaliana transformation of mediated by agriculture bacillus, Jing Kana mycin and PCR are bis- Screening obtains transgenic positive plant again；The dyeing of GUS histochemical method is carried out to positive transgenic strain, the results showed that, this is opened The gus gene of mover driving is only expressed in the root of arabidopsis.It can be seen that the gus gene of promoter driving has centainly Expression specificity, there is expression of the driving downstream gene in root, the function that other organizer official ranks are not expressed；This has The promoter of tissue specific expression manual creation fine germplasm resources, in terms of have well application Value；As using promoter driving resistance or nutrient absorption related gene, building pBn21194 first drives target gene Over-express vector, transformation receptor plant, then target gene can be improved target gene and exist under the driving of pBn21194 promoter Expression quantity in above-mentioned tissue is to achieve the purpose that improve resistance or enhance the correlation merits such as nutrient absorption.

Compared with prior art, the present invention has the following advantages and beneficial effects:

1. the promoter can expression of the specificity driving downstream target gene in plant roots, without in its hetero-organization, device It is expressed in official, there is tissue expression specificity.

2. piece being the important nutrition organs of plant, breeding expert can be changed using the specific expressed target gene of pBn21194 Become the character of plant resistance to environment stress and nutrient absorption, such as moisture or other nutritional ingredients absorb in the soil for enhancing drought resistance, reinforcement, There is good application potential for modern agriculture.PBn21194 promoter utilizes transgene improvement crop quality, people in rape Work, which creates fine germplasm resources etc., has good application potential.

Detailed description of the invention

Fig. 1 is cabbage type rape Bn21194 promoter gene fragment electrophoretogram

Swimming lane 1 is the nucleic acid Marker of DL2000；Swimming lane 2 is using genomic DNA as the starting sub-pieces of the PCR amplification of template Section result.

Fig. 2 is that pBn21194 promoter sequence analyzes result figure

TATA-box: core promoter element；CAAT-box: promoter, enhancer core element；CGTCA-motif/ TGACG-motif: methyl jasmonate responds cis-acting elements；TGA-element: auxin response element；GARE-motif: Gibberellin response element；O2-site: it participates in Zn-ef ficiency and is metabolized cis-acting elements；ARE: the required cis acting member of anaerobic induction Part；TC-rich repeats: defence and stress response cis-acting elements are participated in；MBS: drought-induced MYB bound site is participated in Point；MRE: the MYB binding site of photoresponse is participated in；G-Box: photoresponse controlling element.

Fig. 3 is the mass spectrogram of plant expression vector pBI121.

Fig. 4 is the area the T-DNA schematic diagram of carrier construction pBI121-pBn21194.

Fig. 5 is the transgenic plant PCR qualification result figure of pBI121-pBn21194

Swimming lane 6 is the nucleic acid Marker of DL2000；Swimming lane 1-5,7,8 be transgenic arabidopsis；Swimming lane 9 is pBI121- The control of pBn21194 positive plasmid；Swimming lane 10 is wildtype Arabidopsis thaliana.

Fig. 6 is pBn21194 promoter transgenic arabidopsis GUS histochemical stain result figure

A:1 days young plants, B:3 days young plants, C:5 days young plants, D:7 days young plants, E:10 days young plants, F:14 days young plants, G:21 days Young plant, H: flower, I: silique and seed.

Specific embodiment

Method therefor is conventional method unless otherwise instructed in following embodiments, and the primer holds up section's biology by Wuhan Science and Technology Ltd.'s synthesis, sequencing are completed by Wuhan Qing Ke Biotechnology Co., Ltd, and quick restriction endonuclease is purchased from Thermo Fisher Scientific, DNA gel QIAquick Gel Extraction Kit, DNA Marker etc. are purchased from Dalian treasured biotech firm；It is used in experiment Cabbage type rape variety in double No. 11, Colombia wildtype Arabidopsis thaliana Arabidopsis Thaliana, Escherichia coli sense It is this by state bacterial strain DH5 α, Agrobacterium competence bacterial strain GV3101 and improved plant genetic expression vector pBI121 etc. Laboratory saves.

Embodiment 1: the primer sequence design of rape promoter pBn21194:

The upstream Bn21194 gene (Sequence ID:XM_009109941.1) obtained according to rape genome sequencing Genetic interval sequence 1466bp design pair of primers carry out PCR amplification, amplification obtain rape Bn21194 5 ' upstream promoters Sequence, forward primer Bn21194P-Hind3-UP:5'-CCCAAGCTTGAGAACCGGCAAGTAATCCA-3' used, reversely draws Object Bn21194P-BamH1-DN:5'-CGCGGATCCTGTTATGAGATCTTCTT CGCT-3'.Primer Bn21194P-Hind3- In UP, preceding 3 base CCC is that base is protected in digestion, and AAGCTT thereafter is HindIII restriction enzyme site, remaining sequence is PBn21194 upstream amplification primer sequence；In primer Bn21194P-BamH1-DN, preceding 3 base CGC is that base is protected in digestion, GGATCC afterwards is BamH I restriction enzyme site, remaining sequence is pBn21194 downstream amplification primer sequence.

Embodiment 2: the preparation of rape promoter pBn21194:

Rape used in the present invention is double No. 11 in cabbage type rape (Brassica napus L.), rape seed in crop field, Normal field management.Extract rape leave chip base because of a group DNA using CTAB method, using the rape complete genome DNA of extraction as template into Row polymerase chain reaction PCR (Polymerase chain reaction).PCR system are as follows: 2 × Mix buffer, 25 μ L, Bn21194P-Hind3-UP 1 μ L, Bn21194P-BamH1-DN 1 μ L, DNA 1 μ L, ddH₂O 22μL.PCR program are as follows: 94 DEG C 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1.5min, 35 circulations；72℃ 10min；4℃ ∞.PCR product size is 1466bp (see Fig. 1) is identified through 1.0% agarose gel electrophoresis and is carried out purification and recovery, detectable concentration by kit specification.

PBI121 vector plasmid and the PCR product of recycling are complete through Hind III and BamH I double digestion, and through 1% fine jade The recycling of sepharose electrophoresis detection.The digested plasmid of recycling is mixed with the PCR product of digestion by the molar concentration rate of 1:3, is added 1 μ L of 1 μ L of T4 ligase and 10 × reaction buffer supplies distilled water to 10 μ L, and 16 DEG C are attached overnight.Heat is utilized later The method of swashing, connection product is added in E. coli competent DH5 α, gently plays even, is statically placed in 42 DEG C of heat shocks after 30min on ice 1min is added 600 μ L LB liquid mediums after standing 2min on ice, is placed in 37 DEG C of shaking tables, and 200rpm is incubated for 1h, later uniformly It is coated on the solid LB plate for adding 50 μ g/L cards to receive mycin, in 37 DEG C of inversion overnight incubations after drying.The monoclonal grown is to carry Body upstream primer PBI21-F:5'-TGTGGAATTGTGAGCGGATA-3' and aim sequence special primer pBn21194-R:5'- TGTTATGAGATCTTCTTCGCT-3' carries out positive colony detection, send the Wuhan section of holding up raw through PCR test positive monoclonal Object Science and Technology Ltd. is sequenced, and is analyzed the results show that obtaining rape Bn21194 full length gene promoter, sequence is Nucleotide sequence shown in SEQ ID NO:1, is named as pBn21194.

Embodiment 3: the sequence analysis of rape promoter pBn21194 and function prediction:

It will be in the pBn21194 sequence core promoter element forecasting software PLACE that cloned and be sequenced PlantCARE(Lescot M,Déhais P,Thijs G,et al.PlantCARE,a database of plant cis- acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic acids research,2002,30(1):325-327.http:// Bioinformatics.psb.ugent.be/webtools/plantcare/html/ the prediction point of function element) is carried out online Analysis.The result shows that as shown in Fig. 2, pBn21194 contains core element TATA box and CAAT necessary to promoter in eukaryote box.Promoter sequence discovery is further analyzed, other than necessary core element, there is also more in pBn21194 sequence Kind promoter function element: TGA-element (AACGAC): auxin-responsive element auxin response element； GARE-motif (CGTCA): gibberellin-responsive element gibberellin response element；CGTCA-motif/ TGACG-motif:cis-acting regulatory element involved in the MeJA- Responsiveness, methyl jasmonate respond cis-acting elements；O2-site (GATGATGTGG): cis-acting Regulatory element involved in zein metabolism regulation participates in Zn-ef ficiency and is metabolized cis- work Use element；ARE (TGGTTT): cis-acting regulatory element essential for the anaerobic Induction anaerobic induction necessity cis-acting elements；TC-rich repeats(ATTTTCTTCA)cis-acting Element involved in defense and stress responsiveness participates in defence and the cis- work of stress response Use element；MBS (CAACTG): MYB binding site involved in drought-Inducibility participates in arid The MYB binding site of induction；G-Box (CACGTT): regulatory element involved in light Responsiveness photoresponse controlling element.Inventor analyzes due to multiple anaerobic induction types, hormone induction and degeneration-resistant induction The presence of element, pBn21194 may be expressed in root, and have certain effect to root system of plant nutrition, resistance.

Embodiment 4: the building of plant expression vector pBI121-pBn21194 and Agrobacterium tumefaciens strain GV3101 conversion

The construction method of carrier pBI121-pBn21194 is connected with carrier using T4 using the good target fragment of digestion Enzyme connection.It the steps include:

1) Hind III and BamH I double digestion plant expression vector pBI121 are used, and is examined through 1% agarose gel electrophoresis Survey time receives；

2) the PCR product pBn21194 recycled presses molar concentration rate with the digested plasmid of recycling equally after above-mentioned double digestion 3:1 mixing, is added 1 μ L of T4 ligase and 10 × reaction buffer, 1 μ L, supplies distilled water to 10 μ L, 16 DEG C are connected overnight E. coli competent DH5 α is converted after connecing；

3) monoclonal grown is with carrier upstream primer PBI121-F:5'-TACGTCGCCGTCCAGCTCGA-3' and purpose Sequence specific primers Bn21194P-BamH1-DN:5'-CGCGGATCCTGTTATGAGATCTTCTTCGCT-3' carries out positive gram Grand detection send Wuhan Qing Ke Biotechnology Co., Ltd to be sequenced through PCR test positive monoclonal, and analysis result is aobvious Show, obtain rape Bn21194 full length gene promoter, sequence is nucleotide sequence shown in SEQ ID NO:1, the weight of extraction Group plasmid is named as pBI121-pBn21194.

Agrobacterium competent cell GV3101 is converted using freeze-thaw method, steps are as follows:

1) the Agrobacterium competent cell GV3101 for being stored in -80 DEG C is placed on ice to melt；

2) 3 μ L (100ng) expression vector plasmid pBI121-pBn21194 are taken with liquid-transfering gun, submergence pipette tips are added to impression In state cell, it is placed in static 30min on ice, suddenly freezes 1min in liquid nitrogen, then the water-bath 5min in 37 DEG C of thermostat water baths；

3) 600 μ L LB liquid mediums (tryptone 10g, yeast extract 5g, NaCl 10g) is added, 28 DEG C, 200rpm, shaken cultivation 4h；

4) it is coated on the solid LB training of additional 50 μ g/mL kanamycins, 50 μ g/mL gentamicins and 50 μ g/mL rifampins It supports on base (tryptone 10g, yeast extract 5g, NaCl 10g, Agar 1.5%), 36-48h is cultivated in 28 DEG C of inversions；

5) monoclonal grown is with plant expression vector upstream primer PBI121-F:5'-TACGTCGCCGTCCAGCTCGA- 3' and aim sequence special primer Bn21194P-BamH1-DN:5'-CGCGGATCCTGTTATGAGATCTTCTTCGCT-3' into The detection of row positive colony, the monoclonal through PCR test positive shake bacterium to OD600=1.8-2.0 (ultraviolet specrophotometer inspection Survey), bacterium is protected in -80 DEG C of ultra low temperature freezers, in case follow-up study uses with 50% isometric glycerol.

Embodiment 5: genetic transformation and genetically modified plants sieve of the plant expression vector pBI121-pBn21194 in arabidopsis Choosing

1, flower-dipping method arabidopsis thaliana transformation:

1) main titbit top is cut off after arabidopsis bolting, it is consistent when collateral generation and when being in bud stage, prepare material into Row conversion；

2) contain the LB liquid of 50 μ g/mL kanamycins, 50 μ g/mL gentamicins and 100 μ g/mL rifampins with 200mL Culture medium, inoculation carry the Agrobacterium pBI121-pBn21194 of target gene, 28 DEG C, 200rpm, cultivate 12-18h；

3) loaded in centrifugal bottle, 5000rpm is centrifuged 15min, siphons away supernatant with liquid-transfering gun and abandon it bacterium solution；

4) 100mL re-suspension liquid (5% sucrose, 0.02% surfactant Silwet L-77) is used, Agrobacterium is resuspended； Arabidopsis titbit is immersed into 60s in bacterium solution, and gentle agitation；

5) it after having disseminated, is covered overnight with plastic film, improves transformation efficiency, can converted again after 5-7d primary；

6) after about one month, seed is mature, and harvest is placed on 37 DEG C of baking oven 7d, in 4 DEG C of vernalization 3d after threshing, is labeled as T0 is for seed.

2, positive transgenic plant is screened:

1) take the pBn21194 transgenic arabidopsis seed of appropriate (no more than the 1/5 of EP pipe) through threshing in 1.5mL EP Guan Zhong；

2) plus supernatant is siphoned away and is abandoned with liquid-transfering gun by 75% alcohol of 1mL, concussion washing 1min, 8000rpm centrifugation 30s It；

3) plus the sodium hypochlorite of 1mL 10%, concussion washing 5min, 8 000rpm centrifugation 30s are inhaled supernatant with liquid-transfering gun It walks and abandons it；

4) add 1mL dd H₂O, concussion washing 1min, 8 000rpm centrifugation 30s, supernatant is siphoned away and abandon it with liquid-transfering gun；

5) it repeats step 4 three times, adds 1mL ddH₂O, under dark condition, 4 DEG C, vernalization 3d；

6) bed board plantation is in 1/2MS culture medium (the MS powder 2.15g of the kanamycins containing 25mg/L；Sucrose 10g；Agar 0.8%；PH 5.8) on；

Etc. 7) when growing two panels true leaf, growth is normally transplanted seedlings in hot-house culture alms bowl (Nutrition Soil: vermiculite: perlite =3:1:1)；

8) plant for surviving normal growth waits for that blade is taken before bolting to extract genomic DNA, with plant expression vector upstream Primer PBI121-F:5'-TACGTCGCCGTCCAGCTCGA-3' and aim sequence special primer Bn21194P-BamH1-DN: 5'-CGCGGATCCTGTTATGAGATCTTCTTCGCT-3' screens positive transgenic plant, turns base through PCR test positive Because material is T1 generation (see Fig. 5), single plant harvest, and number consecutively preservation.Equally by T1 for transgenic line through anti-kanamycins Screening is accredited as T2 generation with PCR, single plant harvest, and number consecutively saves.In T2 generation, is screened again later, obtain homozygosis turns base Because of strain.

Embodiment 6: the functional analysis of cabbage type rape pBn21194 promoter

Using expression of the histochemical staining method detection GUS in plant tissue's cell.In T2 generation prepared by Example 5, turns The different times of gene arabidopsis young plant, specific Proper Sampling Period are as follows: 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, and meanwhile it is right The flower and silique of arabidopsis have also carried out sampling dyeing.After sampling into small centrifuge tube or culture dish, appropriate GUS dyeing liquor is added (50mmol/L PBS,pH 7.0；0.5mol/L X-Gluc；50mmol/L potassium ferricyanide；50mmol/L potassium ferrocyanide；10mmol/L EDTA, 0.001%Triton X-100 and 20% methanol).In 37 DEG C Insulation reaction 1h or more is stayed overnight；Then it is transferred to after decolourizing in 70% ethyl alcohol, is seen using stereoscope (Olympus SZX7) It examines and takes pictures.Plant is dyed to the position that blue position is exactly gus gene expression, passes through and detects gus gene under different space-times Expressive site and expression intensity explore the expression pattern of pBn21194.

The result shows that (Fig. 6), the gus gene of the promoter driving only high efficient expression in the root of arabidopsis, in other groups It knits and is not expressed in organ.Therefore, which drives downstream gene high efficient expression in plant roots, and in other portions It does not express position.This promoter with tissue specific expression has good application value in plant genetic engineering.Such as It can be driven by building containing the promoter and improve degeneration-resistant, resistant to lodging, nutrient absorption gene the carrier of plant, transformation receptor is planted Strain, artificial creation's resistance, resistant to lodging, good quality and high output material are applied to agricultural production.

Sequence table

<110>Inst. of Oil Crops, Chinese Academy of Agriculture

<120>cabbage type rape root-specific promoter pBn21194 and its application

<130>cabbage type rape root-specific promoter pBn21194 and its application

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<170> SIPOSequenceListing 1.0

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<213>artificial sequence (Artificial Sequence)

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gagaaccggc aagtaatcca ctccactcag acacgattaa tcgataggcg aattgttaaa 60

tttaattatt cattatgtat caaatgaaaa ttatgattat tttggtgaaa gatgtaaaca 120

aaattatttt gcaagtggta tgccattttc tttctttttt gggtaaaaag ttgtatgcca 180

ttaaacgggg ggaaagtagt tttactttta tcagattaga gaggtttatt acaactcaca 240

gatgatgttg acaaaataaa ctttattaca gttgaaaaaa tgaaacaaaa aaagttgatt 300

aaagttgctt tcttattgtc tttctacgca tgtgattggg aagattagca tggacagcaa 360

tatctggaat attgttcaaa tgattatata cttttataat atatatacaa tttttgtttt 420

aatatatact tagcatattt aaatgtgata ataaatatgt ggacatatcc aatctagtgt 480

atgttgttta tctttaaagt aaacatataa tatttacatg ggtatataaa agtgcttaaa 540

atgtactaga aagaattctt tttttataaa atgagaaaca gatctgttaa caaaagatct 600

caatacataa ataaaaagat ttgtatgtgt tgagaacatg ccaatgtaat gttcaataat 660

gatgtttgta tgaatagatg aatattaaca agtggattaa taaatgctgg tgttttttgt 720

tcccaatttg gtcaacacga aaaatgcata acataagtac aaaatttata caatatggta 780

taaactataa agtagttgtg aattggaata cattccacgc aagataattt aatgaaacca 840

gttatgtaat tcagaaattg taatcttttt attcaagcat tgttattaag aaaatgtagc 900

agcagcataa tttggggttt taatccttat aaaagttgaa aatatttaag gaagacatga 960

gagtttctaa gtagtaaaat taattagcat ctgaacgacc aaacaagatt tattaagagt 1020

tcaatccata taaaaatcga ccaaactaat aatcagaagt ctgaaatgtt gtttctcaca 1080

ttttttcaaa cataactttg taaaactata tacacacacg ttttgatggt aacatatgtc 1140

tttctggtta tagcaattaa attctgatgt tcttttggaa atctcattat tccctatagt 1200

cgtcagcctt gacgcaacat ttctaaaata attcgatgac aataccaatg aaacgataaa 1260

ttaaccttaa acaaatacaa agaaatagat cacgtttcct tgtcaaagcc gccacttctt 1320

gatggtcaag taaacaaata cattcataca taaggcaggc acattcacat atatatacaa 1380

agagcaagtc caagtgtatg agttatgaac aaaacagagt caagaagaga gaatttaaaa 1440

cagatagcga agaagatctc ataaca 1466

Claims (4)

1. cabbage type rape promoter pBn21194, which is characterized in that the nucleotide sequence of the promoter such as SEQ ID NO:1 It is shown.

2. containing the recombinant vector pBI121-pBn21194 of promoter pBn21194 described in claim 1.

3. the amplimer of promoter pBn21194 described in claim 1, it is characterised in that: forward primer Bn21194P- Hind3-UP is 5'-CCCAAGCTTGAGAACCGGCAAGTAATCCA-3', and reverse primer Bn21194P-BamH1-DN is 5'- CGCGGATCCTGTTATGAGATCTTCTTCGCT-3'。

4. promoter pBn21194 described in claim 1 is in driving target gene answering in specifically expressing in arabidopsis root With.