CN105695470A - Root specific expression AhMtan promoter and application thereof - Google Patents

Abstract

The invention relates to a root specific expression AhMtan promoter and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the root specific expression AhMtan promoter is shown as SEQ ID NO2 or SEQ ID NO3 or SEQ ID NO4.Peanut root specific promoters are cloned, core areas of the promoters are determined through a truncation experiment, the functions of all the promoters are verified, and a foundation is laid for application and development of the peanut biotechnology of China. An important molecular basis is provided for providing the efficient, stable and special expression promoter for obtaining a bivalent grub resisting peanut variety.

Description

Root-specific expresses AhMtan promoter and application thereof

Technical field

The present invention relates to biological technical field, particularly relate to root-specific and express promoter, its clone, its application of functional verification。

Background technology

Cultivation Semen arachidis hypogaeae (ArachishypogaeaL.) belongs to legume, is the important oil crop in the world and industrial crops, and 2015, China's peanut yield reached 16,700,000 tons, occupies first place in the world。The yield of Semen arachidis hypogaeae is subject to some biological and abiotic stress impacts, especially subterranean pest-insect, fungus, antibacterial etc.。There is wildness in Peanut Fields subterranean pest-insect in recent years, has had a strong impact on the yield and quality of Semen arachidis hypogaeae, has caused the Peanut over Large Areas underproduction。

Subterranean pest-insect refers to the polyphagous pest-insect of harm of a certain stage plant closely soil table stem, root, seed in life cycle, causes root system of plant impaired, affects plant absorption moisture and inorganic salt, causes that plant growing is slow, even dead。The kind of subterranean pest-insect is a lot, mainly has Holotrichia diomphalia Bates, Gryllotalpa, wireworm etc.。Wherein Holotrichia diomphalia Bates is the subterranean pest-insect that harm China is main, is also important worldwide insect, all once causes the underproduction of crop, harm Semen Maydis, Semen arachidis hypogaeae, Rhizoma Dioscoreae esculentae, Semen sojae atricolor, Fructus Pruni salicinae etc. in states such as India, Japan, the U.S.。

Holotrichia diomphalia Bates is the general name of coleoptera Scarabaeidae (Scarabaeoidae) larva, the most extensive with the harm of phytophagous Holotrichia diomphalia Bates。The Semen arachidis hypogaeae underproduction typically resulted in is 1～2 one-tenth, and more than serious plot reaches 5 one-tenth, even No kernels or seeds are gathered, as in a year of scarcity。Shandong Xintai City bureau of agriculture data show, during 2008～2011 years, peanut pod is on average injured rate respectively 15%, 25%, 28%, 35%, and in increasing trend year by year, indivedual plot almost have no harvest because of Holotrichia diomphalia Bates harm, cause great economic loss。2008, Guizhou golf course Holotrichia diomphalia Bates population outbreak, turfgrass chlorosis, wilting, root system necrosis, cause that turfgrass sheet is withered。

At present, mainly with physical control, chemical prevention and three kinds of means preventing and treating subterranean pest-insect Holotrichia diomphalia Batess of Biological control。Physical control is mainly the phototaxis according to adult, uses black light lamps adult, but what effect larva does not have。When carrying out chemical prevention, use the preventing and treating that Furadan, Le Siben, pungent sulfur woods, clothianidin chemical agent carry out, although the effect of poisoning Holotrichia diomphalia Bates is fine, but there is also certain drawback simultaneously。First, the contaminated environment that chemical agent is serious；Secondly, some insecticide is high residue, directly jeopardizes the health of the mankind, and the peasant especially used more has the even lethal situation that disables。

Nematicide, pathogenic fungi, pathogenetic bacteria etc. are mainly had for Biological control。Although the preventing and treating of these biologies is had longer research history, but it is not widely applied。Tracing it to its cause, some biology itself has pathogenic on the one hand, causes that Semen arachidis hypogaeae is caught an illness；On the other hand, as prevention effect all can be produced impact by the batch production of nematicide, storage temperature, storage time etc., pathogenic fungi and the major part in antibacterial are easily subject to the impact etc. of environment。For realizing the green prevention and control of Holotrichia diomphalia Bates, it is badly in need of opening up new controlling way。Along with the development of biotechnology, application Molecular tools cultivates insect pest new peanut variety, it has also become controlling way new at present。

By Biological control, resistant crop varieties and control the Integrated Pest preventing and treating based on insect growing environment by crop rotation, the application in extensive range in the world, this is mainly attributed to the development of resistant crop varieties, and biotechnology plays an important role in cultivating resistant variety。Anti insect gene mainly has three major types, vegetable source pesticide gene (Cowpea Trypsin Inhibitor (CpTI) gene, Plant Secondary Metabolites etc.)；Animal sources killing gene (chitinase gene, spider toxin gene etc.)；Microbial source killing gene (thuringiensis (Bt) insecticidal crystalline gene, cholesterol oxidase gene from rhodococcus equi etc.)。Both at home and abroad the research of insect pest transgenic peanuts being started late, the anti insect gene of application is also concentrated mainly on microbial source killing gene at present。

At present, tissue-specific promoter is widely used in genetic engineering。By genetic engineering means, the promoter of organizing specific expression is connected with disease-resistant gene, makes disease-resistant gene at specific part timing, high efficient expression, reach the purpose of prevention and elimination of disease and pests, the chance of more ecological safety and Security Strategies is provided for peasant。

Root system of plant is important subterranean organ, can absorb moisture, inorganic salt etc. for needed for plant normal growth；The resistance protein etc. of some resistance gene expressions of plant root, can slow down some biological and abiotic stress impacts on plant, make aerial parts from the harm of heavy metal, arid, pathogenic bacterium etc.。Prior art has been cloned into some cry8 genoids to Holotrichia diomphalia Bates high virulence, and Holotrichia diomphalia Bates mainly gnaws the under ground portion of Semen arachidis hypogaeae, so finding the root-specific promoter at whole peanut growth Ji Dougao efficient expression, drive cry8 genoid efficient, specifically expressing in Roots of Peanut, reach the effect of control of grubs, it is possible not only to achieve the green prevention and control of Holotrichia diomphalia Bates, and provides important molecule basis for improveing peanut quality, cultivating anti-subterranean pest-insect new peanut variety。

Cui Shanshan (Cui Shanshan, Qiao Yake, Li Guilan, Deng. the checking [J] in Fructus Lycopersici esculenti of the myrosin gene pyk10 root-specific promoter. biotechnology is circulated a notice of, 2012 (11): 73-77.) finding after carrying out GUS dyeing after etc. converting Fructus Lycopersici esculenti with the pky10-GUS plant expression vector merged, pyk10 promoters driven gus gene is at root specifically expressing。Vanghan (VaughanSP, JamesDJ, LindseyK, etal.CharacterizationofFaRB7, anearroot-specificgenefromstrawberry (Fragaria × ananassaDuch.) andpromoteractivityanalysisinhomologousandheterologousho sts.JExpBot [J] .JournalofExperimentalBotany, 2006,57 (14): 3901-3910.) etc. find that Fructus Fragariae Ananssae FaRB7 promoter is root-specific promoter, provides a valuable root-specific promoter for crop gene engineering。Cu (CuR, ZhaoL, ZhangY, etal.Isolationofamaizebeta-gulcosidasegenePormoter [J] .PlantCellReports, 2006,25:1157-1165.) etc. cloned the Zmglu1 promoter of Semen Maydis, find afterwards, this promoters driven gus gene expression in Zea mays root is the highest。Having cloned a lot of root-specific promoters of acquisition at present, its source includes Nicotiana tabacum L., Oryza sativa L., Semen Maydis, arabidopsis, Fructus Lycopersici esculenti, Semen sojae atricolor etc., but Roots of Peanut specific promoter report is rarer。

Summary of the invention

The present invention constructs the promoter of analysis result clone's Semen arachidis hypogaeae root specifically expressing of gene numeral expression spectrum based on transcript profile order-checking, genes of interest can be made in root particular expression, be without fear of an attack from the rear in use。

The present invention clones Roots of Peanut specific promoter, and determines promoter nucleus by truncate experiment, verifies the function of each promoter, lays the foundation for China's Semen arachidis hypogaeae biotechnology applications and development；Promoter efficient, stable, specifically expressing is provided to provide important molecule basis for obtaining the anti-Holotrichia diomphalia Bates peanut varieties of bivalent。

Root-specific expresses promoter, and its nucleotide sequence is such as shown in SEQIDNO2, SEQIDNO3 or SEQIDNO4。

AhMtan gene, its nucleotide sequence is such as shown in SEQIDNO1。

A kind of expression vector, containing above-mentioned specific expression promoter。

The application in transgenic plant of the above-mentioned expression vector。

Described application, is inserted with anti insect gene in this expression vector, proceeds in plant by this expression vector so that it is express this anti insect gene in root, to prevent and treat subterranean pest-insect。

Above-mentioned root-specific expresses promoter application in transgenic plant。

Described application, for proceeding in plant by the expression vector expressing promoter and anti insect gene containing above-mentioned root-specific so that it is express this anti insect gene at seed or the tip of a root, to prevent and treat subterranean pest-insect。

The present invention has cloned the promoter sequence of a Semen arachidis hypogaeae 5'-methylthioadenosine/S-adenosine cysteine nucleosidase (5'-methylthioadenosine/S-adenosylhomocysteinenucleosidas e) upstream region of gene 1486bp, is AhMtan promoter by its preliminary designation。This promoter sequence being carried out bioinformatic analysis show, this section contains 12 ROOTMOTIFTAPOX1 elements, 2 OSE2ROOTNODULE elements, and both of which is the cis acting element relevant to root specifically expressing。Distribution situation according to root specifically expressing cis acting element, constructs the plant expression vector of 3 5' end series of deletions, after transformation of tobacco, that resistance Seedling of the card of acquisition is carried out GUS dyeing, and result shows, turns pAhMtan₁₂₄₇、pAhMtan₅₂₃、pAhMtan₂₃₅The Nicotiana tabacum L. root of carrier is all dyed to blueness, and blade is without blue spot, it was demonstrated that AhMtan promoter is root-specific promoter, and the promoter fragment of its lease core district 235bp remains to the specifically expressing instructing reporter gene at root。The nucleotide sequence of three promoteres is shown in shown in SEQIDNO2, SEQIDNO3, SEQIDNO4。

By genetic engineering means, the promoter of the root specifically expressing of the present invention is connected with disease-resistant/anti insect gene, make disease-resistant/anti insect gene at root timing, high efficient expression, reach the purpose of prevention and elimination of disease and pests, the chance of more ecological safety and Security Strategies is provided for peasant。Proceeded to by above-mentioned reporter gene and in the specifically expressing result explanation of root, the expression vector of root specific expression promoter containing the present invention can be built, it is inserted into the killing gene to subterranean pest-insect high virulence, proceeds to plant, make this killing gene at root specifically expressing, to prevent and treat subterranean pest-insect, particularly Holotrichia diomphalia Bates is had high virulence cry8 genoid, adopts transgenic technology, proceed to peanut plant, it is made to express at root, control of grubs。

Accompanying drawing explanation

The pcr amplification result of Fig. 1 contig219746 upstream sequence

The amplification of Fig. 2 AhMtan genetic fragment

M:DL2000DNAMarker；1-5:PCR amplified production；CK is respective negative comparison

Fig. 3 AhMtan gene structure display

Gray shaded area: exon (E1-E5)；White space: intron (I1-I4)

3 '-racePCR the results of Fig. 4 AhMtangene

The bioinformatic analysis of Fig. 5 AhMtan promoter

The each truncate schematic diagram of Fig. 6 AhMtan root-specific promoter

Fig. 7 root-specific promoter plant expression vector construction flow chart

Fig. 8 plant expression vector pAhMtan1247 (left figure), pAhMtan523 (in), vector construction Fig. 1 of pAhMtan235 (right figure): each truncate PCR fragment；2: negative control；3: the double digestion (HindIII+BamHI) of corresponding plant vector；4:p2300GUSplus (HindIII+BamHI)；5: the double digestion (HindIII+BamHI) of corresponding replicating vector；M1:DM2000；M2:DM15000

Fig. 9 pAhMtan1247 (1-2), pAhMtan523 (3-4), pAhMtan235 (5-6) convert Agrobacterium LBA4404 pcr amplification result M:DL2000Marker；CK: respective negative compares；1,2:pAhMtan1247 converts the pcr amplification result of Agrobacterium；3,4:pAhMtan523 converts the pcr amplification result of Agrobacterium；5,6:pAhMtan235 converts the pcr amplification result of Agrobacterium,

The genetic conversion system of Figure 10 Nicotiana tabacum L.

A: leaf dish；B: induced bud；C: resistant buds extends；D, E: resistant buds is taken root。

Figure 11 turns the GUS coloration result of pAhMtan1247 Nicotiana tabacum L.

Figure 12 turns the GUS coloration result of pAhMtan523 Nicotiana tabacum L.

Figure 13 turns the GUS coloration result of pAhMtan235 Nicotiana tabacum L.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described in further detail。

1, experiment material

1.1 vegetable materials

Cultivation Semen arachidis hypogaeae (ArachishypogaeaL.) kind white sand 1016, is provided by Shandong Peanut Inst.。

Nicotiana tabacum L. (Nicotianabenthamiana), is preserved by this laboratory。

1.2 bacterial strains and plasmid

This research bacterial strain uses therefor and plasmid are in Table 1。The public all can be provided by following bacterial strain and plasmid。

Table 1 bacterial strain and plasmid

1.3 data bases and biosoftware

1.3.1 data base

NCBI:http: //www.ncbi.nlm.nih.gov/

1.3.2 biosoftware

DNAMAN6.0: sequence alignment analysis software

Primer5.0: primer-design software

1.4 reagent

1.4.1RNA extraction, reverse transcription and quantitative fluorescent PCR reagent

Extracting the TotalRNAExtractor of RNA purchased from Shanghai bio-engineering corporation, the first-class RNasefree product of other RNase-freeddH2O, rifle is purchased from AXYGEN company, and DNAse I is purchased from TIANGEN company。Reverse transcription and PCR kit for fluorescence quantitative are purchased from Beijing Tian Gen company FastQuantcDNA the first chain synthetic agent box and SYBRGreen fluorescence quantitative kit。

1.4.2 enzyme and other biochemical reagents

2 × TaqMix, PrimerStar exo+ polymerase are purchased from TaKaRa company；PMD-19TVector is purchased from TaKaRa company；DNAMarker molecular weight standard is purchased from Promega company；GUS dye liquor is purchased from Beijing Bing Da company；Other reagent are domestic analytical pure product。The recovery of DNA glue, PCR primer purification, plasmid Mini Kit are all purchased from Axygen company。

1.4.3DNA molecular weight

DL15000:15000,10000,7500,5000,2500,1000,250bp

DL5000:5000,3000,2000,1500,1000,750,500,250,100bp

DL2000:2000,1000,750,500,250,100bp

DL500:500,400,300,200,150,100,50bp

1.4.4 other reagent

TE buffer: 100mmol/LTrisCl (pH8.0), 10mmol/LEDTA (pH8.0)；

Acetosyringone (Acetosyringone, As) solution: be configured to 500 μm of ol/mL mother solutions with DMSO。

1.5 culture medium, antibiotic and hormone

1.5.1 culture medium

LB liquid medium: tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, 121 DEG C of sterilizing 21min。

Solid LB media: tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, agar powder 1.3%, 121 DEG C of sterilizing 21min。

Minimal medium: MS inorganic salt+3% sucrose+0.7% agar powder, 121 DEG C of sterilizing 21min。

Co-culture culture medium :+50 μm of ol/L acetosyringones of minimal medium；

Bud inducement culture medium: 20mg/L6-BA+50mg/L kanamycin+500mg/L Carbenicillin；

Elongation medium: 20mg/L6-BA+50mg/L kanamycin+500mg/L Carbenicillin；

Root media: 0.5mg/LNAA+50mg/L kanamycin+500mg/L Carbenicillin。

1.5.2 antibiotic

Ampicillin (Ampicillin, Amp) stores mother solution 100mg/mL, working concentration 100mg/L；Kanamycin (Kanamycin, Km) stores mother solution 100mg/mL, working concentration 100mg/L；Streptomycin sulfate (Streptomycin, Sm) stores mother solution 100mg/mL, working concentration 100mg/L；Carbenicillin (Carbenicillin, Cb) stores mother solution 500mg/mL, working concentration 500mg/L。3 sterilizings of all antibiotic multigelations ,-20 DEG C of preservations。

1.5.3 hormone

6-BA (6-Benzylaminopurine): weigh 0.5g6-BA, adds 300 μ L2mol/LNaOH, is completely dissolved rear constant volume to 10mL, and mother solution is 50mg/L, 3 sterilizings of multigelation ,-20 DEG C of preservations；

TDZ (Thidiazuron): weigh 0.05gTDZ, adds 100 μ L2mol/LNaOH, is completely dissolved rear constant volume to 10mL, and mother solution is 5mg/L, 3 sterilizings of multigelation ,-20 DEG C of preservations；

NAA (Naphthylacetic): weigh 0.1g6-BA, adds 100 μ L2mol/LNaOH, is completely dissolved rear constant volume to 10mL, and mother solution is 10mg/L, 3 sterilizings of multigelation ,-20 DEG C of preservations。

2 research methoies

The preparation of 2.1 vegetable materials

Roots of Peanut, stem, leaf take from the tissue cultured seedling of 2 week old, and Immature Cotyledons, for collecting the different size of peanut pod of 25-55d after fruit pin buries, mixes after peeling。All vegetable materials sampling after in liquid nitrogen quick freeze, at-80 DEG C of Refrigerator stores。

Osmotic treatment: when peanut growth to about 15d, select healthy and strong and that growing way is consistent seedling, process with 15%PEG6000 solution simulating drought, take the seedling root of process 0,2,4,6 and 8h respectively,-80 DEG C of preservations, for Total RNAs extraction and quantitative fluorescent PCR analysis。Test sets 3 repetitions。

Aspergillus flavus processes: Semen arachidis hypogaeae is planted in greenhouse after peelling off seed coat, inoculates aspergillus spore after 3 weeks。Collecting the Aspergillus flavus spore of 2～3d in PDA culture medium with sterilized water, adjustments spore concentration is 6 × 108/ml, is sprayed on the root of Semen arachidis hypogaeae, takes after untreated and process the seedling root of 1,3,5 and 7d respectively, in-80 DEG C of preservations, for quantitative fluorescent PCR analysis。Test sets 3 repetitions。

2.2 Roots of Peanuts, stem, the extraction of leaf RNA and purification

2.2.1TRIzol method extracts RNA

Extract Roots of Peanut, stem, leaf, rataria total serum IgE with TRIzol reagent, specifically comprise the following steps that

1. take and be organized in right amount in the mortar adding liquid nitrogen, be ground to rapidly Powdered。

2. being transferred in 1.5ml centrifuge tube, add 1mlTRIzol reagent, acutely shake 30s, room temperature stands 5min。

3. add 200 μ l chloroforms, thermal agitation 30s, place 8min on ice。

4.4 DEG C, 12000rpm is centrifuged 10min, takes honest and upright and thrifty 400 μ l。

5. adding 400 μ l isopropanols, fully mix, room temperature places 10min。

6.4 DEG C, 12000rpm is centrifuged 10min, abandons supernatant。

7. adding 75% ethanol of 700 μ l, washing precipitation, 4 DEG C, 12000rpm is centrifuged 1min。

8. adding the dehydrated alcohol of 700 μ l, washing precipitation, 4 DEG C, 12000rpm is centrifuged 3min, abandons supernatant。

9. room temperature is dried, and adds appropriate RNase-freeddH₂O dissolves。

2.2.2RNA purification

Removing DNA with the DNase I of TaKaRa company and RNaseInhibitor, purification system is:

With the RNA purification kit of TIANGEN company, operating procedure is as follows: the dehydrated alcohol adding 0.5 times of volume is mixed, and transfers the possession of and adsorbs CR3, and subsequent process carries out referring to TIANGEN company RNApreppure series description, until eluted rna molecule。

2.2.3RNA the detection of purity

The integrity of electrophoresis detection total serum IgE, taking 1 μ l total serum IgE is template, with actF/R for primer, and is positive control by peanut genome, detects the presence of DNA pollution。ActF/R is that primer is as follows, amplified production 384bp:

ActF:5 '-ATGAAGGAGAAGCTAGCTTA-3 '；

ActR:5 '-AACACTGTACTTCCTCTCTG-3 '。

2.2.4real-timePCR the expression characterization of AhMtan gene is analyzed

2.3.1cDNA synthesis

1. the mixed liquor that preparation DNA removes:

TotalRNA2μL

5×gDNABuffer2μL

Add DEPC water to 10 μ L

2. brief centrifugation, hatches 3min for 42 DEG C, chilling on ice；

3. prepare reaction system mixed liquor according to following system:

4. reaction system mixed liquor is joined in the DNA mixed liquor removed, fully mix。

Hatch 15min for 5.42 DEG C, cooled on ice after 95 DEG C of insulation 3min ,-20 DEG C of preservations。

2.3.2real-timePCR

1. the foundation of standard curve

AhMtan, reference gene actin the dilution of plasmid gradient, dilution gradient respectively 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8, taking 1 μ L is template, carries out real-timePCR reaction。Reaction condition is 95 DEG C of 15min；95 DEG C of 10s, 60 DEG C of 30s, collect fluorescence signal, 40 circulations。

2.real-timePCR analyzes

With Roots of Peanut, stem, leaf, rataria cDNA for template, 2 each pairs of primers of table are analyzed。Reaction condition is 95 DEG C of 15min；95 DEG C of 10s, 60 DEG C of 30s, collect fluorescence signal, 40 circulations。Adopt relative quantification method to carry out data analysis, with actin for reference gene, adopt 2^-△△CtIt is calculated。

Table 2 Primer and sequence

2.4CTAB method extracts Peanut genome

Extracting of peanut genome adopts CTAB method (DoyleJJ.ArapidDNAisolationprocedureforsmallquantitiesoff reshleaftissue [J] .PhytochemBull improved, 1987,19:11-15.), extraction genome being put in-20 DEG C store for future use, the method with reference to Doyle etc. is slightly modified。

1. take appropriate Folium Arachidis hypogaeae, be fully ground in liquid nitrogen, transfer in 1.5mL centrifuge tube, it is rapidly added the extract with CTAB buffer (beta-mercaptoethanol containing 0.5%) of 600 μ L65 DEG C, reverse mixing, 65 DEG C of water-bath 10min, period gentle inversion mixes twice。

2. add the KAc of 250 μ L5M, be placed in the centrifugal 10min of 30min, 12000rpm on ice。

3. taking supernatant, add the isopropanol of 0.6 times of volume, softly mix, room temperature places 10min。

4.12000rpm is centrifuged 10min, abandons supernatant, 75% washing with alcohol, and room temperature places 10min, and 30 μ L sterilized water dissolve。

5. electrophoresis detection ,-20 DEG C save backup。Respectively genome being processed with several enzymes such as Hind III, BamH I, Sac I, EcoR I, mixing is reclaimed, and-20 DEG C save backup。

The clone of 2.5 root-specific promoters

2.5.1 design of primers

Table 3 Primer and sequence

2.5.2 the amplification of promoter

With 2.5 enzyme action mixing Post genome for template, expand。

After electrophoresis detection purpose band size is correct, cuts glue and reclaim, connect pMD-19TVector, send order-checking。

1.PCR product glue reclaims

1) under long-wave ultra violet lamp, cut target DNA fragment, be placed in 1.5mLEp pipe, calculated for gel weight, it is calculated with 1mg=1 μ L；

2) add the ED-A solution of 3 times of gel volumes, melt in the water-bath of 75 DEG C。

3) after gel piece melts completely, add the ED-B solution of 0.5 times of ED-A liquor capacity。

4) proceeding to recovery post after mixing, 12000rpm is centrifuged 1min。

5) remove residual liquid, add 500 μ LW₁, 12000rpm is centrifuged 1min。

6) remove residual liquid, add 700 μ LW₂, 12000rpm is centrifuged 1min。

7) empty from 1 time, 12000rpm is centrifuged 1min。

8) in recovery post, add 30 μ L ultra-pure waters, stand 1min。

9) the centrifugal 1min of 12000rpm, standby。

10) agarose gel electrophoresis detection。

11) it is connected reclaiming fragment with the pMD-19T of TaKaRa company。Linked system is: 5 μ L2 × Solution I, 0.5 μ LpMD-19T carrier, and 4.5 μ L reclaim product, cumulative volume 10 μ L, mix rear 4 DEG C of 6h。

Preparation (the CaCl of TG1 competent cell₂Method)

1) the mono-bacterium colony of picking E.coliTG1 is in 5mL LB liquid medium, 37 DEG C of incubated overnight；

2) being inoculated in 100mLLB fluid medium by 1% inoculum concentration, 37 DEG C, 220rpm cultivates about 2hr (OD₆₀₀=0.5-0.6)；

3) 4 DEG C, 5000rpm is centrifuged 10min；

4) abandon supernatant, add the 0.1mol/LCaCl of 40mL pre-cooling₂Suspension cell, places 30min on ice；

5) 4 DEG C, 5000rpm is centrifuged 10min, collects cell；

6) supernatant is abandoned, with the 0.1mol/LCaCl of 2mL pre-cooling₂, 2mL glycerol, re-suspended cell, after mixing, often pipe adds 300 μ L, in-70 DEG C of preservations。

The conversion of TG1

1)-70 DEG C of refrigerators take out TG1 competent cell, are immediately placed on ice；

2) 10 μ L are connected product to add wherein, place 45min on ice；

3) 42 DEG C of thermal shock 90s, take out rapidly, place 1min on ice；

4) the 600 nonresistant LB fluid mediums of μ L, recovery 45min in 37 DEG C of incubators are added；

5) it is applied on the LB solid medium containing corresponding resistant, in 37 DEG C of incubators, cultivates 10～12h, grow to there being single bacterium colony。

2. identify positive transformant

1) choosing single bacterium colony, be placed in 600 μ L containing in corresponding antibiotic LB liquid medium, 37 DEG C, 220rpm trains 4h, and bacterium solution is for the PCR template identified。

2) PCR identification system and condition are as follows:

3) order-checking is sent by the bacterium solution of PCR positive colony。

The clone of 2.6AhMtan full length gene

2.6.1AhMtan the clone of full length gene

According to sequence on peanutDB, design primer is shown in sequence table 4。Full-length gene is longer, is unfavorable for obtaining purpose product, is sized to 1000-2000bp expands so being divided into 5 sections, it is easier to obtain corresponding fragment。With enzyme action Peanut genome for template, with table 4 primer, carry out pcr amplification。Pcr amplification program is: 94 DEG C of 10min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 30 circulations；72 DEG C of 10min。Choose the purpose band that size is correct, reclaim, be cloned into pMD-19TVector, check order and splice。

The primer sequence of the AHMtan gene clone in table 4 the application

2.6.2AhMtan the determination of gene tail structure

Use SMARTer^TMRACE test kit, method detailed is referring to saying。Pcr amplification is carried out for primer with AhMtan3'-race。Pcr amplification program is as follows。By purpose band correct for size, send order-checking after connecting pMD-19T, determine 3' tail structure with AhMtan gene comparison。

Table 5 Primer and sequence

The bioinformatic analysis of 2.7AhMtan upstream region of gene promoter

Adopt bioinformatics method, with following two software, the upstream promoter sequence of these three gene is predicted and analyzes: PLACESignalScanprogram (http://www.dna.affrc.go.jp/PLACE/signalscan.html) and Plantcare data base (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/sear ch_CARE.html)。

The structure of 2.8 root-specific promoter plant expression vectors

1. the acquisition of root-specific promoter different length deletion fragment

According to bioinformatic analysis result, for Gent nonequivalent element position each in promoter sequence, autotelic design truncate primer, the 5' end at upstream and downstream primer adds HindIII and BamHI restriction enzyme site respectively。Primer sequence is as follows, and lower-case portion is added restriction enzyme site:

Table 6 Primer and sequence

PCR amplification system and condition are as follows:

PCR primer purification adds A tail, again connects carrier T after purification, converts TG1, the same 2.1.4 of positive transformant authentication method, positive colony is delivered order-checking。

2. plasmid extraction

1) order-checking being identified, correct bacterial strain is forwarded to 5mL containing in corresponding antibiotic LB culture fluid, 37 DEG C, 220rpm, shaken cultivation is about 5h；

2) 12000rpm, centrifugal 1min, collect thalline, abandon supernatant；It is repeated once。

3) in thalline, add the 250 μ L BuffterS1 having added RNaseA, resuspended bacterial precipitation。

4) add 250 μ LBuffterS2, turn upside down 5 times, make thalline fully crack, stand 3min。

5) adding 350 μ LBuffterS3, gentleness also spins upside down 6-8 time fully, stands the centrifugal 10min of 3min, 12000rpm。

6) to preparing the centrifugal rear supernatant of pipe addition, 12000rpm is centrifuged 1min, abandons filtrate。

7) add the centrifugal 1min of 500 μ LBuffterW1,12000rpm, abandon filtrate。

8) add the centrifugal 1min of 700 μ LBuffterW2,12000rpm, abandon filtrate, be repeated once。

9) putting back in 2mL centrifuge tube by preparing pipe, 12000rpm is centrifuged 1min。

10) putting into 1.5mL centrifuge tube by preparing pipe, add 60 μ L ultra-pure waters, room temperature stands the centrifugal 1min of 1min, 12000rpm。

3. the enzyme action of carrier and purpose fragment

Enzyme action system:

37 DEG C of enzyme action 2h, electrophoresis detection。

4. the fragment of order reclaims same 2.6.2

5. being connected with carrier large fragment by the genes of interest reclaimed, reaction system is as follows:

p2300GUSplus0.5μL

Purpose fragment 4.5 μ L

SolutionⅠ5μL

Mix latter 4 DEG C overnight to connect。

6. pair e. coli tg1 convert, PCR identify, plasmid extraction method ibid。

7. being identified by the plant expression vector enzyme action built, system is as follows:

2.9 plant expression vectors convert Agrobacterium

1. the preparation of Agrobacterium tumefaciems competent cell

1) by streak culture for Agrobacterium on the solid LB flat board containing Sm resistance, 30 DEG C of incubators are cultivated about 40h to there being single bacterium colony to grow。

2) picking list bacterium colony, is inoculated in 5mL and contains 100mg/LSm^rLB liquid medium in, 30 DEG C, 220rpm cultivates 40h。

3) 1mL bacterium solution is seeded to 50mL containing Sm^rLB liquid medium in, 28 DEG C, 220rpm cultivate 5-6h, to OD₆₀₀=0.8；

4) 4 DEG C, 4500rpm is centrifuged 10min, collects thalline；

5) adding the sterilized water washing thalline of 20mL pre-cooling, 4500rpm is centrifuged 10min, collects thalline, repeats 2 times。

6) adding the sterilized water of 2mL pre-cooling, the resuspended thalline of 2mL50% glycerol, softly mix, 200 μ L/ pipes ,-70 DEG C of storages are standby。

2. electroporated recombiant plasmid is to LBA4404 competent cell

1) take 3 μ L recombiant plasmid and be added separately in 100 μ L competent cells, be softly mixed, be placed in about 15min on ice；

2) bacterium solution being transferred in the electric shock cup of 0.2cm pre-cooling, conversion condition is: 2000V, 25 μ F, 200 Ω；

3) 600 μ L liquid LB are added in electric shock cup, softly mix, in the 1.5mLEp pipe moved to, 28 DEG C, 8h；

4) thalline is applied on the solid LB media containing corresponding antibodies；

5) 40h cultivated by 28 DEG C of incubators, grows to there being single bacterium colony。

The genetic transformation of 2.10 Nicotiana tabacum L.s

1. the cultivation of Nicotiana tabacum L.

1) by the soak with ethanol 2min of tobacco seed 1mL80%。

2) outwelling ethanol, add the NaClO solution of 1mL5%, jog soaks 5min。

3) outwell NaClO solution, as far as possible remove remaining liq with pipettor, with aseptic water washing 2 times。

4) it is seeded on MS basal medium, 28 DEG C, illumination cultivation。

2. the activation of Agrobacterium

Take out containing pAhMtan from-70 DEG C of refrigerators₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅The Agrobacterium of carrier, the same 2.4.1 of bacterial strain activation process。

3. Agrobacterium infecting Nicotiana tabacum L.

1) tobacco leaf is cut into the leaf dish of diameter 1cm, and with Agrobacterium 28 DEG C, 220rpm infects 20min。

2) using aseptic water washing 3 times, what leaf dish was placed in 50 μm of ol/L acetosyringones co-cultures light culture 2 days in culture medium；

3) the leaf dish after co-culturing is forwarded in selective differentiation culture medium (MS+2mg/L6-BA+0.2mg/LNAA+500mg/LCb+50mg/LKm), be placed in illumination cultivation in 28 DEG C of greenhouses, within every 30 days, change a subculture；

4) treat that budlet length that leaf dish differentiates is to about 1cm, cut budlet and transfer to (MS+0.2mg/LNAA+300mg/LCb+50mg/LKm) in selection root media and continue to cultivate, within every 15～20 days, changing a subculture。

2.11GUS histochemical stain and microexamination

1. conversion plant tissue is put in 24 orifice plates, add GUS dyeing liquor；

Incubation in dark 12h in 2.37 DEG C of incubators；

3. in FAA fixative, stained tissue being fixed 2h, decolour 1 day (removal chlorophyll) in dehydrated alcohol；

GUS coloration result is observed under the super depth of field three-dimensional microscopic system of SZX16 type fluorophor stereomicroscope and vhx-2000 type。

3 results and analysis

The clone of 3.1 root-specific promoters

Intend the upstream sequence of clone contig219746, according to this laboratory transcript profile sequencing result and Blast result, design primer (see table 3), the pcr amplification being template with enzyme action Peanut genome, obtain the fragment such as Fig. 1。It is connected with PMD-19T, send order-checking。See shown in SEQIDNO:3。

The sequencing result of contig219746 sequence is carried out respectively in NCBI similarity comparison, result shows: contig219746 and Rhizoma Solani tuber osi 5'-methylthioadenosine/S-adenosine cysteine nucleosidase (Solanumtuberosum5'-methylthioadenosine/S-adenosylhomocys teinenucleosidase, GenbankNo:XM_003625013.1) similarity is 72%, functional annotation result is Solanumtuberosum5'-methylthioadenosine/S-adenosylhomocys teinenucleosidase-like (LOC102601008), mRNA, its preliminary designation is AhMtan gene by we。The preliminary designation of above-mentioned contig and gene has been summed up by table 7。

The functional annotation of table 7 gene and name

The bioinformatic analysis of 3.2AhMtan root specific expression gene and promoter

3.2.1AhMtan genomic organization

This laboratory checks order through transcript profile, obtain the est sequence (Contig219746) of a 1237bp, NCBI compares, annotates as 5'-methylthioadenosine/S-adenosine cysteine nucleosidase (Solanumtuberosum5'-methylthioadenosine/S-adenosylhomocys teinenucleosidase)。This sequence is compared with the Peanut genome sequence of announcement on http://peanutbase.org website, primer (table 4) is designed according to comparison result, with Peanut genome for template, carry out segmentation amplification, amplification obtains 5 fragments varied in size (Fig. 2), send order-checking, splicing。Ncbi database carries out similarity system design, the similarity of this albumen and Rhizoma Solani tuber osi 5'-methylthioadenosine/S-adenosine cysteine nucleosidase (Solanumtuberosum5'-methylthioadenosine/S-adenosylhomocys teinenucleosidase, GenbankNo:XM_003625013.1) is up to 72%。Being AhMtan gene by this unnamed gene, coding region is 1074bp。AhMtan gene DNA sequence is analyzed with cDNA sequence comparison and is found, AhMtan full length gene is 3520bp (see SEQIDNO1), comprises 5 exons, 4 introns, coding region is 1074bp, encodes 358 aminoacid, and termination codon is TAA (Fig. 3)。Use SMARTer^TMRACE test kit determines the tail structure of AhMtan gene, downstream sequence design primer (table 5) according to gene, with the cDNA of test kit synthesis for template, expand, after size is correct, send order-checking, compare with this gene order, it is determined that 3'UTR length is 156bp (Fig. 4)。

(table 8) is added up in the boundary characteristic of 4 introns, size, AT content, site。The AT content of all introns both is greater than 60%, and the highest is 70.5%。The shearing site of intron I1, I2, I14 meets the shearing rule of AGGT-AG。

Size, site, AT content and the boundary characteristic statistical result that 4 introns of table 8AhMtan gene are big

3.2.2AhMtan the bioinformatic analysis of promoter

Plantcare line data base, the cis acting element of AhMtan promoter is analyzed (Fig. 5), wherein contains 12 ROOTMOTIFTAPOX1s relevant to root specifically expressing, 2 and root specifically expressing related elements OSE2ROOTNODULE；3 GATA-motif, 3 ACGT-motif (GATA-and ACGT-is the Binding site for transcription factor determining histoorgan specifically expressing)；2 inducer response element W-box ((T) TGAC (C))。In order to verify AhMtan promoter function and determine the minimum active region of this promoter, according to the position of the AhMtan promoter region different related elements of Gent and estimated number, devise the promoter fragment (Fig. 6) of three truncates。

Start codon ATG band * italics represents；12 ROOTMOTIFTAPOX1s relevant to root specifically expressing are represented with gray background；With yellow background represent 2 with root specifically expressing related elements OSE2ROOTNODULE equal；2 inducer response element W-box ((T) TGAC (C)), 3 GATA-motif, 3 ACGT-motif are used that underscore represents and is labeled。

The structure of 3.3AhMtan root-specific promoter plant expression vector and conversion Agrobacterium

Carrier based on p2300GUSplus, replaces the CaMV35S promoter on p2300GUSplus with each truncate of root-specific promoter, builds corresponding plant expression vector, and flow chart is shown in Fig. 7, and building process is as follows:

With the PCR primer of AhMtan upstream sequence for template, use primer pair AhMtan respectively_1247f/AhMtanr、AhMtan_523f/AhMtanr、AhMtan_235f/ AhMtanr expands the promoter fragment (Fig. 8) of AhMtan upstream 1247bp, 523bp and 235bp, and corresponding swimming lane is all swimming lane 1), PCR primer is connected pMD-19T, send order-checking, result is completely the same with the sequence having verified that。

The pMD-AhMtan that will obtain_1247f、pMD-AhMtan_523f、pMD-AhMtan_235f(swimming lane 5) and carrier is carrier p2300GUSplus (swimming lane 4) BamHI and Hind Ш double digestion, be connected the purpose fragment with sticky end with large fragment carrier after enzyme action, it is thus achieved that plant expression vector pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅, respectively 3 carriers (swimming lane 3) being carried out double digested with BamHI and Hind Ш, obtain the band of 1247bp, 523bp and 235bp respectively, and stripe size is consistent with PCR primer, order-checking proves plant expression vector pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅Correctly。By pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅Converting Agrobacterium LBA4404, pcr amplification obtains the promoter fragment (Fig. 9) of 1247bp, 523bp and 238bp respectively。Three sequences are shown in SEQIDNO:3, SEQIDNO:2, SEQIDNO:1 respectively。

3.4AhMtan carries the Agrobacterium genetic transformation to Nicotiana tabacum L. of root-specific promoter

Tobacco leaf disc (Figure 10 A) is infected with the Agrobacterium containing plant expression vector, after about 10 days, induced bud produces (Figure 10 B), cultivate and induced bud was transferred to (Figure 10 C) on new division culture medium in about 4 weeks, the time of about two weeks takes root (Figure 10 D, E), it is thus achieved that can be used for that resistance Seedling of the card of Molecular Detection。

Take transformed plant and carry out GUS tissue chemical analysis, it has been found that turn pAhMtan₁₂₄₇、pAhMtan₅₂₃、pAhMtan₂₃₅The Nicotiana tabacum L. root of expression vector is all dyed to blueness (Figure 11,12,13), and in blade, do not observe blueness, only observe blue spot at partial blade edge (Figure 11 A and Figure 12 C), it may be possible to the blue precipitate shone by mechanical damage。Proving that AhMtan promoter is root-specific expression promoter, the AhMtan promoter (containing 3 ROOTMOTIFTAPOX1 elements and 1 OSE2ROOTNODULE element) of the shortest 235bp still has the function instructing gene at Nicotiana tabacum L. root specifically expressing。

Claims (7)

1. root-specific expresses promoter, and its nucleotide sequence is such as shown in SEQIDNO2, SEQIDNO3 or SEQIDNO4。

2.AhMtan gene, its nucleotide sequence is such as shown in SEQIDNO1。

3. an expression vector, containing the specific expression promoter described in claim 1。

4. the application in transgenic plant of the expression vector described in claim 3。

5. application according to claim 4, is inserted with anti insect gene in this expression vector, proceeds in plant by this expression vector so that it is express this anti insect gene in root, to prevent and treat subterranean pest-insect。

6. the root-specific described in claim 1 expresses promoter application in transgenic plant。

7. application according to claim 6, for proceeding in plant by the expression vector expressing promoter and anti insect gene containing the root-specific described in claim 1 so that it is express this anti insect gene at seed or the tip of a root, to prevent and treat subterranean pest-insect。