CN105695470B

CN105695470B - Root-specific expresses AhMtan promoters and its application

Info

Publication number: CN105695470B
Application number: CN201610264251.XA
Authority: CN
Inventors: 耿丽丽; 张�杰; 冯艳芳; 束长龙; 彭琦; 宋福平; 梁影屏
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2016-04-26
Filing date: 2016-04-26
Publication date: 2018-11-13
Anticipated expiration: 2036-04-26
Also published as: CN105695470A

Abstract

The present invention relates to root-specific expression AhMtan promoters and its applications, belong to biotechnology.Root-specific expresses promoter, and nucleotide sequence is as shown in SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4.The present invention clones peanut root-specific promoter, and determines promoter nucleus by truncating to test, and verifies the function of each promoter, lays the foundation with development for China's peanut biotechnology applications；Efficient, stable, specifically expressed promoter is provided for the acquisition anti-grub peanut varieties of bivalent, and important molecule basis is provided.

Description

Root-specific expresses AhMtan promoters and its application

Technical field

The present invention relates to biotechnology, promoter is expressed more particularly to root-specific, clone, functional verification its Using.

Background technology

Cultivation peanut (Arachis hypogaea L.) belongs to legume, is the important oil crops in the world and economy Crop, 2015, China's peanut yield occupied first place in the world up to 16,700,000 tons.The yield of peanut is biological and abiotic by some The influence of stress, especially subterranean pest-insect, fungi, bacterium etc..Peanut Fields subterranean pest-insect occurs rampant in recent years, seriously affects The yield and quality of peanut causes the Peanut over Large Areas underproduction.

Subterranean pest-insect refers to the polyphagous pest-insect that a certain stage endangers the closely native table stem of plant, root, seed in the history of life, is made It is impaired at root system of plant, plant absorption moisture and inorganic salts are influenced, cause plant growth slow, or even is dead.Subterranean pest-insect There are many type, mainly there is grub, mole cricket, wireworm etc..Wherein grub is to endanger the main subterranean pest-insect in China, is also important Worldwide pest all once caused the underproduction of crop in states such as India, Japan, the U.S., endangered corn, peanut, sweet potato, soybean, Lee Son etc..

Grub is the general name of coleoptera Scarabaeidae (Scarabaeoidae) larva, is endangered with phytophagous grub the widest It is general.The peanut underproduction typically resulted in is 1~2 one-tenth, and serious plot is up to 5 one-tenth or more, or even No kernels or seeds are gathered, as in a year of scarcity.Shandong Xintai City bureau of agriculture Data show, the peanut pod aggrieved rate that is averaged is respectively 15%, 25%, 28%, 35% during 2008~2011 years, in increasing year by year Trend, individual plot is added to cause great economic loss because grub harm is almost had no harvest.2008, Guizhou golf course Qi Scarabaeiform population outbreak, turfgrass chlorosis, wilting, root system necrosis, causes turfgrass sheet withered.

Currently, three kinds of physical control, chemical prevention and biological control means is mainly used to prevent subterranean pest-insect grub.Physics is anti- The phototaxis mainly according to adult is controlled, with black light lamps adult, but to larva without what effect.Carry out chemical prevention When, the prevention carried out using Furadan, Le Siben, pungent sulphur woods, clothianidin chemical agent, although the effect of poisoning grub is fine, But there is also certain drawbacks simultaneously.First, the serious pollution environment of chemical agent；Secondly, some insecticides are high residues, The health of the mankind is directly jeopardized, the peasant especially applied more has the even lethal situation that disables.

Mainly there are nematode, disease fungus, pathogenetic bacteria etc. for biological control.Although to these biology preventions have compared with Long research history, but be not widely used.To find out its cause, on the one hand some biology itself have it is pathogenic, cause to spend Ingrain disease；On the other hand, such as the batch production of nematode, storage temperature, storage time can all have an impact control effect, disease Major part in fungal pathogens and bacterium is easy to be influenced by environment.To realize that the green prevention and control of grub, urgent need are opened up new Controlling way.With the development of biotechnology, insect pest new peanut variety is cultivated using Molecular tools, it has also become new at present is anti- Control approach.

Prevent by biological control, resistant crop varieties and by the Integrated Pest based on crop rotation control pest growing environment It controls, in world's application in extensive range, this is mainly attributed to the development of resistant crop varieties, and biotechnology is cultivating resistance It plays an important role in terms of kind.Anti insect gene mainly has three categories, and (cowpea trypsase inhibits vegetable source pesticide gene Agent (CpTI) gene, Plant Secondary Metabolites etc.)；Animal sources killing gene (chitinase gene, spider toxin gene etc.)；It is micro- Biological source killing gene (thuringiensis (Bt) insecticidal crystalline gene, cholesterol oxidase gene from rhodococcus equi etc.).Both at home and abroad It starts late to the research of insect pest transgenic peanuts, the anti insect gene applied at present is also concentrated mainly on microbial source desinsection base Cause.

Currently, tissue-specific promoter is widely used in genetic engineering.By genetic engineering means by organizing specific The promoter of expression is connected with disease-resistant gene, makes disease-resistant gene in privileged site timing, high efficient expression, reaches prevention pest and disease damage Purpose provides the chance of more ecological safeties and Security Strategies for peasant.

Root system of plant is important subterranean organ, can absorb moisture, inorganic salts etc. for needed for plant normal growth；Plant The resistance protein etc. of some resistance gene expressions of root can slow down some influences of biology and abiotic stress to plant, Make aerial part from the harm of heavy metal, arid, pathogenic bacteria etc..It has been cloned into some in the prior art to the high virulence of grub Cry8 genoids, and grub mainly gnaws the under ground portion of peanut, so finding in all high efficient expressions of entire peanut growth season Root-specific promoter, driving cry8 genoids efficient, specifically expressing in Roots of Peanut, has the function that control of grubs, not only may be used To realize the green prevention and control of grub, and important point is provided for improvement peanut quality, the anti-subterranean pest-insect new peanut variety of cultivation Subbase plinth.

(Cui Shanshan, Qiao Yake, Li Guilan wait myrosin gene pyk10 root-specific promoters in tomato to Cui Shanshan Verification [J] biotechnologys notification, 2012 (11):73-77.) etc. with the plant expression vector conversion kind of pky10-GUS fusions It is found after carrying out GUS dyeing after eggplant, pyk10 promoters drive gus gene in root specifically expressing.Vanghan(Vaughan SP,James DJ,Lindsey K,et al.Characterization of FaRB7,a near root-specific gene from strawberry(Fragaria×ananassa Duch.)and promoter activity analysis in homologous and heterologous hosts.J Exp Bot[J].Journal of Experimental Botany,2006,57(14):It 3901-3910.) etc. finds that strawberry FaRB7 promoters are root-specific promoter, is crop gene Engineering provides a valuable root-specific promoter.Cu(Cu R,ZhaoL,Zhang Y,et al.Isolation of a maize beta-gulcosidase gene Pormoter[J].Plant Cel lReports,2006,25:1157- Etc. 1165.) the Zmglu1 promoters for having cloned corn, find later, which drives expression of the gus gene in corn root Measure highest.It has cloned at present and has obtained many root-specific promoters, source includes tobacco, rice, corn, arabidopsis, tomato, big Beans etc., but peanut root-specific promoter report is rarer.

Invention content

The analysis result that gene numeral expression spectrum is constructed the present invention is based on transcript profile sequencing clones the special table in peanut root The promoter reached can make target gene in root particular expression, be without fear of an attack from the rear in use.

The present invention clones peanut root-specific promoter, and determines promoter nucleus by truncating to test, and verification is respectively opened The function of mover lays the foundation for China's peanut biotechnology applications with development；It is provided to obtain the anti-grub peanut varieties of bivalent Efficiently, stable, specifically expressed promoter provides important molecule basis.

Root-specific expresses promoter, nucleotide sequence such as SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4 institutes Show.

AhMtan genes, nucleotide sequence is as shown in SEQ ID NO1.

A kind of expression vector contains above-mentioned specific expression promoter.

The application of above-mentioned expression vector in transgenic plants.

The expression vector is transferred in plant, makes in its root inserted with anti insect gene in the expression vector by the application The anti insect gene is expressed, to prevent subterranean pest-insect.

The application of above-mentioned root-specific expression promoter in transgenic plants.

The application, for the expression vector for containing above-mentioned root-specific expression promoter and anti insect gene is transferred to plant In, so that it is expressed the anti insect gene in seed or the tip of a root, to prevent subterranean pest-insect.

The present invention has cloned a peanut 5'-methylthioadenosine/S- adenosine cysteine nucleosidases (5'- Methylthioadenosine/S-adenosylhomocysteine nucleosidase) upstream region of gene 1486bp startup Its preliminary designation is AhMtan promoters by subsequence.Bioinformatic analysis is carried out to the promoter sequence and shows the section Containing 12 ROOTMOTIFTAPOX1 elements, 2 OSE2ROOTNODULE elements, the two is all relevant with root specifically expressing Cis-acting elements.According to the distribution situation of root specifically expressing cis-acting elements, the plant of 3 ends 5' series of deletions is constructed Expression vector, after transformation of tobacco, by the card of acquisition, that resistance seedling carries out GUS dyeing, the results show that turning pAhMtan₁₂₄₇、 pAhMtan₅₂₃、pAhMtan₂₃₅The tobacco root of carrier is dyed to blue, and blade is without blue spot, it was demonstrated that AhMtan starts Son is root-specific promoter, and the promoter fragment of lease core area 235bp remains to the special table for instructing reporter gene in root It reaches.The nucleotide sequence of three promoters is shown in SEQ ID NO2, SEQ ID NO3, shown in SEQ ID NO4.

The specifically expressed promoter of root of the present invention is connected with disease-resistant/anti insect gene by genetic engineering means, makes to resist Disease/anti insect gene achievees the purpose that prevent pest and disease damage, more ecological safeties is provided for peasant in root timing, high efficient expression With the chance of Security Strategies.It is transferred to by above-mentioned reporter gene and the specifically expressing result in root illustrates, can built The expression vector of root specific expression promoter containing the present invention, is inserted into the killing gene to the high virulence of subterranean pest-insect, is transferred to Plant makes the killing gene in root specifically expressing, to prevent subterranean pest-insect, especially has high virulence cry8 class bases to grub Cause is transferred to peanut plant using transgenic technology, so that it is expressed in root, control of grubs.

Description of the drawings

The PCR amplification result of Fig. 1 contig219746 upstream sequences

The amplification of Fig. 2 AhMtan genetic fragments

M：DL2 000 DNA Marker；1-5：Pcr amplification product；CK is corresponding negative control

Fig. 3 AhMtan gene structure displays

Gray shaded area：Exon (E1-E5)；White space：Introne (I1-I4)

3 '-racePCR the results of Fig. 4 AhMtan gene

The bioinformatic analysis of Fig. 5 AhMtan promoters

Fig. 6 AhMtan root-specific promoters respectively truncate schematic diagram

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

Fig. 8 plant expression vectors pAhMtan1247 (left figure), pAhMtan 523 (in), pAhMtan235 (right figure) Vector construction Fig. 1：It is each to truncate PCR fragment；2：Negative control；3：The double digestion (Hind III+BamH I) of corresponding plant vector； 4：p2300GUSplus(Hind III+BamH I)；5：The double digestion (Hind III+BamH I) of corresponding replicating vector； M1:DM2000；M2:DM15000

Fig. 9 pAhMtan1247 (1-2), pAhMtan 523 (3-4), pAhMtan235 (5-6) convert Agrobacterium LBA4404 PCR amplification results M：DL2000Marker；CK：Corresponding negative control；1,2：PAhMtan1247 converts Agrobacterium PCR amplification result；3,4：PAhMtan 523 converts the PCR amplification result of Agrobacterium；5,6：PAhMtan235 converts Agrobacterium PCR amplification as a result,

The genetic conversion system of Figure 10 tobaccos

A：Leaf dish；B：Induced bud；C：Resistant buds extend；D,E：Resistant buds are taken root.

Figure 11 turns the GUS coloration results of pAhMtan1247 tobaccos

Figure 12 turns the GUS coloration results of 523 tobaccos of pAhMtan

Figure 13 turns the GUS coloration results of pAhMtan235 tobaccos

Specific implementation mode

With reference to embodiment, the present invention is described in further detail.

1, experiment material

1.1 vegetable material

Peanut (Arachis hypogaea L.) kind white sand 1016 is cultivated, is provided by Shandong Peanut Inst..

Tobacco (Nicotiana benthamiana), is preserved by this laboratory.

1.2 bacterial strains and plasmid

This research bacterial strain uses therefor and plasmid are shown in Table 1.Following bacterial strains can provide the public with plasmid.

1 bacterial strain of table and plasmid

1.3 databases and biosoftware

1.3.1 database

NCBI：http://www.ncbi.nlm.nih.gov/

1.3.2 biosoftware

DNAMAN 6.0：Sequence alignment analysis software

Primer 5.0：Primer-design software

1.4 reagent

1.4.1 RNA extractions, reverse transcription and quantitative fluorescent PCR reagent

The Total RNA Extractor for extracting RNA are purchased from Shanghai bio-engineering corporation, other RNase-free The RNase free products such as ddH2O, pipette tips are purchased from AXYGEN companies, and DNAse I is purchased from TIANGEN companies.Reverse transcription and fluorescence are fixed It measures PCR kit and is purchased from Beijing Tiangeng company FastQuant cDNA the first chain synthetic agent box and SYBR Green fluorescent quantitations Kit.

1.4.2 enzyme and other biochemical reagents

2 × Taq Mix, Primer Star exo+ polymerases are purchased from TaKaRa companies；PMD-19T Vector are purchased from TaKaRa companies；DNA Marker molecular weight standards are purchased from Promega companies；GUS dye liquors reach company purchased from Beijing ice；Other examinations Agent is domestic analysis net product.The recycling of DNA glue, PCR product purifying, small amount plasmid extraction kit are purchased from Axygen public affairs Department.

1.4.3 DNA molecular amount

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 reagents

TE buffer solutions：100mmol/L Tris Cl (pH8.0), 10mmol/L EDTA (pH8.0)；

Acetosyringone (Acetosyringone, As) solution：It is configured to 500 μm of ol/mL mother liquors with DMSO.

1.5 culture mediums, 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 Sterilize 21min.

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

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

Bud inducement cultivation base：20mg/L 6-BA+50mg/L kanamycins+500mg/L carbenicillins；

Elongation medium：20mg/L 6-BA+50mg/L kanamycins+500mg/L carbenicillins；

Root media：0.5mg/L NAA+50mg/L kanamycins+500mg/L carbenicillins.

1.5.2 antibiotic

Ampicillin (Ampicillin, Amp) stores mother liquor 100mg/mL, uses concentration 100mg/L；Kanamycins (Kanamycin, Km) stores mother liquor 100mg/mL, uses concentration 100mg/L；Streptomycin sulphate (Streptomycin, Sm) stores up Mother liquor 100mg/mL is deposited, concentration 100mg/L is used；Carbenicillin (Carbenicillin, Cb) stores mother liquor 500mg/mL, Use concentration 500mg/L.3 sterilizings of all antibiotic multigelations, -20 DEG C of preservations.

1.5.3 hormone

6-BA(6-Benzylaminopurine)：0.5g 6-BA are weighed, adds 300 μ L 2mol/L NaOH, is completely dissolved Constant volume is to 10mL afterwards, and mother liquor 50mg/L, 3 sterilizings of multigelation, -20 DEG C preserve；

TDZ(Thidiazuron)：0.05g TDZ are weighed, adds 100 μ L 2mol/L NaOH, is completely dissolved rear constant volume and arrives 10mL, mother liquor 5mg/L, 3 sterilizings of multigelation, -20 DEG C of preservations；

NAA(Naphthylacetic)：0.1g 6-BA are weighed, adds 100 μ L 2mol/L NaOH, is completely dissolved rear constant volume To 10mL, mother liquor 10mg/L, 3 sterilizings of multigelation, -20 DEG C of preservations.

2 research methods

The preparation of 2.1 vegetable materials

Roots of Peanut, stem, leaf are derived from the tissue-cultured seedling of 2 week old, and Immature Cotyledons are to collect 25-55d different sizes after gynophore is buried Peanut pod, mixed after peeling.After the sampling of all vegetable materials in liquid nitrogen quick freeze, preserved in -80 DEG C of refrigerators.

Osmotic treatment：When peanut growth to 15d or so, the seedling that selection is healthy and strong and growing way is consistent uses 15%PEG6000 The processing of solution simulating drought takes the seedling root of 0,2,4,6 and 8h of processing, -80 DEG C of preservations to be used for Total RNAs extraction and fluorescence respectively Quantitative PCR analysis.Experiment sets 3 repetitions.

Aspergillus flavus is handled：Plantation is inoculated with aspergillus spore in greenhouse after 3 weeks after peanut peels off kind of skin.It is received with sterile water Collect the Aspergillus flavus spore of 2~3d in PDA culture medium, adjustment spore concentration is 6 × 108/ml, is sprayed on the root of peanut, respectively Take it is untreated and processing after 1,3,5 and 7d seedling root, in -80 DEG C preservation, for quantitative fluorescent PCR analyze.Experiment sets 3 weights It is multiple.

The extraction and purifying of 2.2 Roots of Peanut, stem, leaf RNA

2.2.1 TRIzol methods extract RNA

With TRIzol reagents extraction Roots of Peanut, stem, leaf, rataria total serum IgE, it is as follows：

1. taking in the appropriate mortar for being organized in and liquid nitrogen being added, it is ground to rapidly powdered.

2. being transferred in 1.5ml centrifuge tubes, 1ml TRIzol reagents are added, acutely shakes 30s, is stored at room temperature 5min.

3. adding 200 μ l chloroforms, 30s is acutely vibrated, places 8min on ice.

4.4 DEG C, 12 000rpm centrifuge 10min, take honest and upright and thrifty 400 μ l.

5. adding 400 μ l isopropanols, mixes well, be placed at room temperature for 10min.

6.4 DEG C, 12 000rpm centrifuge 10min, abandon supernatant.

7. 75% ethyl alcohol of 700 μ l is added, washing precipitation, 4 DEG C, 12 000rpm centrifuge 1min.

8. the absolute ethyl alcohol of 700 μ l is added, washing precipitation, 4 DEG C, 12 000rpm centrifuge 3min, abandon supernatant.

9. room temperature is dried, appropriate RNase-free ddH are added₂O dissolves.

2.2.2 the purifying of RNA

DNA is removed with DNase I and the RNase Inhibitor of TaKaRa companies, purification system is：

With the RNA purification kits of TIANGEN companies, operating procedure is as follows：The absolute ethyl alcohol of 0.5 times of volume is added to be mixed, Transfer adsorbs CR3, and subsequent process is carried out referring to TIANGEN companies RNAprep pure series specifications, until eluted rna point Son.

2.2.3 the detection of RNA purity

The integrality of electrophoresis detection total serum IgE, it is that Peanut genome is used in combination using actF/R as primer in template to take 1 μ l total serum IgEs DNA is positive control, 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′.

2.2.4 real-time PCR analyze the expression characterization of AhMtan genes

2.3.1 the synthesis of cDNA

1. preparing the mixed liquor of DNA removals：

Total RNA 2μL

5×gDNA Buffer 2μL

Add DEPC water to 10 μ L

2. brief centrifugation, 42 DEG C are incubated 3min, on ice chilling；

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

4. reaction system mixed liquor is added in the mixed liquor of DNA removals, mix well.

Cooled on ice after 5.42 DEG C of incubations 15min, 95 DEG C of heat preservation 3min, -20 DEG C of preservations.

2.3.2 real-time PCR

1. the foundation of standard curve

The dilution of the plasmid gradient of AhMtan, reference gene actin, dilution gradient are respectively 10^-3、10^-4、10^-5、10^-6、 10^-7、10^-8, it is template to take 1 μ L, carries out real-time PCR reactions.Reaction condition is 95 DEG C of 15min；95 DEG C of 10s, 60 DEG C 30s collects fluorescence signal, 40 cycles.

2.real-time PCR analyses

Using Roots of Peanut, stem, leaf, rataria cDNA as template, 2 each pairs of primers of table are analyzed.Reaction condition is 95 DEG C 15min；95 DEG C of 10s, 60 DEG C of 30s collect fluorescence signal, 40 cycles.Data analysis is carried out using relative quantification method, with Actin is reference gene, using 2^-△△CtIt is calculated.

2 Primer of table and sequence

2.4 CTAB methods extract Peanut genome

The extraction of peanut genome is using CTAB methods (the Doyle J J.A rapid DNA isolation improved procedure for small quantities of fresh leaf tissue[J].Phytochem Bull,1987, 19:11-15.), extraction genome is put in -20 DEG C to store for future use, the method with reference to Doyle etc. is slightly modified.

1. taking appropriate peanut leaf, it is fully ground, is transferred in 1.5mL centrifuge tubes in liquid nitrogen, is rapidly added 600 μ L 65 DEG C CTAB Extraction buffers (contain 0.5% beta -mercaptoethanol), overturn mixing, 65 DEG C of water-bath 10min, during which gentle inversion is mixed It is even twice.

2. the KAc of 250 μ L 5M is added, it is placed in 30min on ice, 12000rpm centrifuges 10min.

3. taking supernatant, the isopropanol of 0.6 times of volume, soft mixing is added to be placed at room temperature for 10min.

4.12000rpm centrifuges 10min, abandons supernatant, and the washing of 75% ethyl alcohol is placed at room temperature for 10min, the 30 sterile water dissolutions of μ L.

5. electrophoresis detection, -20 DEG C save backup.With Hind III, BamH I, several enzymes such as Sac I, EcoR I are respectively to gene Group is handled, and mixing recycling, -20 DEG C save backup.

The clone of 2.5 root-specific promoters

2.5.1 design of primers

3 Primer of table and sequence

2.5.2 the amplification of promoter

Using 2.5 digestion mixing Post genomes as template, expanded.

After electrophoresis detection purpose band size is correct, gel extraction connects pMD-19T Vector, send sequencing.

1.PCR product glue recycles

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

2) the ED-A solution of 3 times of gel volumes is added, melts in 75 DEG C of water-bath.

3) the ED-B solution of 0.5 times of ED-A liquor capacity is added after gel piece melts completely.

4) recovery column is transferred to after mixing, 12000rpm centrifuges 1min.

5) residul liquid-removing is removed, 500 μ L W are added₁, 12000rpm centrifugations 1min.

6) residul liquid-removing is removed, 700 μ L W are added₂, 12000rpm centrifugations 1min.

7) for sky from 1 time, 12000rpm centrifuges 1min.

8) 30 μ L ultra-pure waters are added into recovery column, stand 1min.

9) 12000rpm centrifuges 1min, spare.

10) agarose gel electrophoresis detects.

11) recycling segment is connect with the pMD-19T of TaKaRa companies.Linked system is：52 × Solution of μ L I, 0.5 μ L pMD-19T carriers, 4.5 μ L recovery products, 10 μ L of total volume, 4 DEG C of 6h after mixing.

Preparation (the CaCl of TG1 competent cells₂Method)

1) picking E.coli TG1 single bacteriums are fallen in 5mL LB liquid mediums, and 37 DEG C are incubated overnight；

2) it is inoculated in 100mL LB liquid mediums by 1% inoculum concentration, 37 DEG C, 220rpm cultivates about 2hr (OD₆₀₀= 0.5-0.6)；

3) 4 DEG C, 5000rpm centrifuges 10min；

4) supernatant is abandoned, the 0.1mol/L CaCl of 40mL precoolings are added₂Suspension cell places 30min on ice；

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

6) supernatant is abandoned, the 0.1mol/L CaCl being pre-chilled with 2mL₂, cell is resuspended in 2mL glycerine, often pipe plus 300 μ after mixing L, in -70 DEG C of preservations.

The conversion of TG1

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

2) 10 μ L connection products are added thereto, place 45min on ice；

3) 42 DEG C of thermal shock 90s, it is rapid to take out, 1min is placed on ice；

4) add the LB liquid medium of 600 μ L non-resistants, the recovery 45min in 37 DEG C of incubators；

5) it is applied on the LB solid mediums containing corresponding resistant, 10~12h is cultivated in 37 DEG C of incubators, until there is single bacterium colony It grows.

2. identifying positive transformant

1) single bacterium colony is chosen, is placed in 600 LB liquid mediums of the μ L containing corresponding antibiotic, 37 DEG C, 220rpm trains 4h, bacterium solution Template for PCR identifications.

2) PCR identification systems and condition are as follows：

3) bacterium solution of PCR positive colonies is sent to sequencing.

The clone of 2.6 AhMtan full length genes

2.6.1 the clone of AhMtan full length genes

According to sequence on peanutDB, design primer is shown in sequence table 4.Full-length gene is longer, is unfavorable for obtaining purpose product, So it is that 1000-2000bp is expanded to be divided into 5 sections of sizes, it is easier to obtain corresponding segment.It is with digestion Peanut genome Template carries out PCR amplification with 4 primer of table.PCR amplification program is：94℃10min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 30 cycles；72℃10min.The correct purpose band of size is chosen, recycles, be cloned into pMD-19T Vector, be sequenced and spell It connects.

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

2.6.2 the determination of AhMtan genes tail structure

Use SMARTer^TMRACE kits, method detailed is referring to illustrating.It is carried out by primer of AhMtan 3'-race PCR amplification.PCR amplification program is as follows.By the correct purpose band of size, sequencing is sent after connecting pMD-19T, with AhMtan genes It compares and determines 3' tail structures.

5 Primer of table and sequence

The bioinformatic analysis of 2.7 AhMtan upstream region of gene promoters

Using bioinformatics method, the upstream promoter sequence of these three genes is predicted with following two software And analysis：PLACE Signal Scan program(http://www.dna.affrc.go.jp/PLACE/ ) and Plantcare databases (http signalscan.html://bioinformatics.psb.ugent.be/ webtools/plantcare/html/search_CARE.html)。

The structure of 2.8 root-specific promoter plant expression vectors

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

It is purposive according to bioinformatic analysis as a result, for each Gent nonequivalent element position in promoter sequence Design truncates primer, and Hind III and BamH I restriction enzyme sites are added respectively at the ends 5' of upstream and downstream primer.Primer sequence is as follows, Lower-case portion is added restriction enzyme site：

6 Primer of table and sequence

PCR amplification system and condition are as follows：

PCR product purifying plus A tails, connect carrier T after purification again, convert TG1, the same 2.1.4 of positive transformant identification method, Positive colony is delivered into sequencing.

2. plasmid extraction

1) sequencing is identified that correct bacterial strain is forwarded in LB culture solutions of the 5mL containing corresponding antibiotic, 37 DEG C, 220rpm, Shaken cultivation about 5h；

2) 12000rpm centrifuges 1min, collects thalline, abandons supernatant；It is repeated once.

3) the Buffter S1 that 250 μ L have added RNase A are added into thalline, bacterial precipitation is resuspended.

4) add 250 μ L Buffter S2, turn upside down 5 times, thalline is made fully to crack, stand 3min.

5) add 350 μ L Buffter S3, mildly and fully spin upside down 6-8 times, stand 3min, 12000rpm centrifugations 10min。

6) to supernatant after centrifugation is added in pipe is prepared, 12000rpm centrifuges 1min, abandons filtrate.

7) add 500 μ L Buffter W1,12000rpm centrifugation 1min, abandon filtrate.

8) add 700 μ L Buffter W2,12000rpm centrifugation 1min, abandon filtrate, be repeated once.

9) pipe will be prepared to put back into 2mL centrifuge tubes, 12000rpm centrifuges 1min.

10) pipe will be prepared and be put into 1.5mL centrifuge tubes, 60 μ L ultra-pure waters are added, be stored at room temperature 1min, 12000rpm centrifugations 1min。

3. the digestion of carrier and target fragment

Digestion system：

37 DEG C of digestion 2h, electrophoresis detection.

4. target fragment recycles same 2.6.2

5. the target gene of recycling is connect with carrier large fragment, reaction system is as follows：

p2300GUSplus 0.5μL

4.5 μ L of target fragment

SolutionⅠ 5μL

4 DEG C of connections overnight after mixing.

6. a pair e. coli tg1 is converted, PCR is identified, plasmid extraction method is same as above.

7. the plant expression vector digestion built is identified, system is as follows：

2.9 plant expression vectors convert Agrobacterium

1. the preparation of Agrobacterium tumefaciems competent cell

1) Agrobacterium scribing line is incubated on the solid LB tablets of the resistance containing Sm, about 40h is cultivated in 30 DEG C of incubators extremely There is single bacterium colony to grow.

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

3) 1mL bacterium solutions are seeded to 50mL and contain Sm^rLB liquid medium in, 28 DEG C, 220rpm cultivate 5-6h, until OD₆₀₀ =0.8；

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

5) plus the sterile water washing thalline of 20mL precoolings, 4500rpm centrifuge 10min, collect thalline, are repeated 2 times.

6) plus thalline, soft mixing, 200 μ L/ pipes, -70 DEG C of storages are resuspended in the sterile water of 2mL precoolings, 50% glycerine of 2mL It is spare.

2. electroporated recombinant plasmid is to LBA4404 competent cells

1) it takes 3 μ L recombinant plasmids to be added separately in 100 μ L competent cells, is softly mixed, be placed in about 15min on ice；

2) bacterium solution is transferred in the electric shock cup of 0.2cm precoolings, conversion condition is：2000V,25μF,200Ω；

3) 600 μ L liquid LB are added in electric shock cup, soft mixing, in the 1.5mL Ep pipes moved to, 28 DEG C, 8h；

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

5) 28 DEG C of incubator culture 40h, until there is single bacterium colony to grow.

The genetic transformation of 2.10 tobaccos

1. the culture of tobacco

1) the tobacco seed ethyl alcohol of 1mL 80% is impregnated into 2min.

2) ethyl alcohol is outwelled, the NaClO solution of 1mL 5% is added, jog impregnates 5min.

3) NaClO solution is outwelled, remaining liq is removed as possible with pipettor, with aseptic water washing 2 times.

4) it is seeded on MS basal mediums, 28 DEG C, illumination cultivation.

2. the activation of Agrobacterium

It is taken out from -70 DEG C of refrigerators and contains pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅The Agrobacterium of carrier, bacterial strain The same 2.4.1 of activation process.

3. Agrobacterium infects tobacco

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

2) it uses aseptic water washing 3 times, leaf dish is placed in light culture 2 on the co-cultivation culture medium of 50 μm of ol/L acetosyringones It；

3) leaf dish after co-cultivation is gone into selective differentiation culture medium (MS+2mg/L 6-BA+0.2mg/L NAA+500mg/ L Cb+50mg/L Km) on, it is placed in illumination cultivation in 28 DEG C of greenhouses, replaces a subculture within every 30 days；

4) wait for that budlet that leaf dish differentiates grows to 1cm, cuts budlet and is transferred to (MS+ in selection root media 0.2mg/L NAA+300mg/L Cb+50mg/L Km) continue to cultivate, replace a subculture within every 15~20 days.

2.11 GUS histochemical stains and microexamination

1. conversion plant tissue is put into 24 orifice plates, GUS dyeing liquors are added；

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

3. stained tissue is fixed 2h in FAA fixers, decolourize in absolute ethyl alcohol 1 day (removal chlorophyll)；

GUS dyeing knots are observed under SZX16 type fluorescence stereomicroscopes and the super depth of field three-dimensional microscopic system of vhx-2000 types Fruit.

3 results and analysis

The clone of 3.1 root-specific promoters

The upstream sequence of quasi- clone contig219746, according to this laboratory transcript profile sequencing result and Blast as a result, setting Meter primer (being shown in Table 3) obtains the segment such as Fig. 1 using digestion Peanut genome as the PCR amplification of template.It is connect with PMD-19T, Send sequencing.See SEQ ID NO：Shown in 3.

The sequencing result of contig219746 sequences is carried out in NCBI to similitude comparison respectively, is as a result shown： Contig219746 and potato 5'-methylthioadenosine/S- adenosine cysteine nucleosidases (Solanum tuberosum5'- Methylthioadenosine/S-adenosylhomocysteine nucleosidase, Genbank No：XM_ 003625013.1) similarity is 72%, and functional annotation result is Solanum tuberosum 5'- Methylthioadenosine/S-adenosylhomocysteine nucleosidase-like (LOC102601008), MRNA, its preliminary designation is AhMtan genes by we.Table 7 has carried out the preliminary designation of above-mentioned contig and gene always Knot.

The functional annotation of 7 gene of table and name

The bioinformatic analysis of 3.2 AhMtan roots specific expression genes and promoter

3.2.1 AhMtan genomic organizations

This laboratory is sequenced through transcript profile, obtains the est sequence (Contig219746) of a 1237bp, enterprising in NCBI Row compares, and annotates as 5'-methylthioadenosine/S- adenosine cysteine nucleosidases (Solanum tuberosum5'- methylthioadenosine/S-adenosylhomocysteine nucleosidase).By the sequence and http:// The Peanut genome sequence announced on the websites peanutbase.org is compared, according to comparison result design primer (table 4), with Peanut genome is template, carries out segmentation amplification, and amplification obtains 5 segments (Fig. 2) of different sizes, send sequencing, splices.? Similarity system design is carried out in ncbi database, the albumen and potato 5'-methylthioadenosine/S- adenosine cysteine nucleosidases (Solanum tuberosum5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase, Genbank No：XM_003625013.1 similitude) is up to 72%.It is AhMtan genes, code area by the unnamed gene For 1074bp.AhMtan gene DNA sequences compare analysis with cDNA sequence and find, AhMtan full length genes are 3520bp (see SEQ ID NO1), including 5 exons, 4 intrones, code area 1074bp, encode 358 amino acid, terminator codon is TAA (Fig. 3).Use SMARTer^TMRACE kits determine the tail structure of AhMtan genes, are set according to the downstream sequence of gene Primer (table 5) is counted, using the cDNA of kit synthesis as template, is expanded, after size is correct, send sequencing, with the gene order It is compared, determines that 3'UTR length is 156bp (Fig. 4).

The boundary characteristic of 4 intrones, size, AT contents, site are counted (table 8).The AT of all intrones contains Amount both greater than 60%, highest is 70.5%.The shearing site of introne I1, I2, I14 meet the shearing rule of AGGT-AG.

Table 8 AhMtan genes, 4 intrones big size, site, AT contents and boundary characteristic statistical result

3.2.2 the bioinformatic analysis of AhMtan promoters

(Fig. 5) is analyzed to the cis-acting elements of AhMtan promoters in Plantcare lines database, wherein containing 12 with root specifically expressing relevant ROOTMOTIFTAPOX1,2 with root specifically expressing related elements OSE2ROOTNODULE；3 (GATA- and ACGT- are to determine the specifically expressed transcription factor binding site of histoorgan by a GATA-motif, 3 ACGT-motif Point)；2 inducer response element W-box ((T) TGAC (C)).In order to verify AhMtan promoter functions and determine the promoter Minimum active region three truncations are devised according to the position of the different related elements of AhMtan promoter region Gents and estimated number Promoter fragment (Fig. 6).

Initiation codon ATG is indicated with band * italics；With gray background indicate 12 it is related to root specifically expressing ROOTMOTIFTAPOX1；With yellow background indicate 2 it is equal with root specifically expressing related elements OSE2ROOTNODULE；2 inductions Object response element W-box ((T) TGAC (C)), 3 GATA-motif, 3 ACGT-motif are indicated with underscore and into rowers Note.

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

It is basic carrier with p2300GUSplus, is replaced on p2300GUSplus with each truncation of root-specific promoter CaMV35S promoters, build corresponding plant expression vector, and flow chart is shown in that Fig. 7, building process are as follows：

Using the PCR product of AhMtan upstream sequences as template, primer pair AhMtan is used respectively_1247f/AhMtanr、 AhMtan_523f/AhMtanr、AhMtan_235f/ AhMtanr expands the startup sub-pieces of the upstreams AhMtan 1247bp, 523bp and 235bp Section (Fig. 8), corresponding swimming lane is all swimming lane 1), PCR product is connected into pMD-19T, send sequencing, as a result with the sequence that has verified that It is completely the same.

By the pMD-AhMtan of acquisition_1247f、pMD-AhMtan_523f、pMD-AhMtan_235f(swimming lane 5) and carrier is carrier P2300GUSplus (swimming lane 4) BamH I and Hind Ш double digestions, by with cohesive end target fragment and digestion after it is big Fragment vector connects, and obtains plant expression vector pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅, with BamH I and Hind Ш respectively carries out 3 carriers (swimming lane 3) double digested, the band of acquisition 1247bp, 523bp and 235bp, and band respectively Size is consistent with PCR product, and sequencing proves plant expression vector pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅Correctly.It will pAhMtan₁₂₄₇、pAhMtan₅₂₃And pAhMtan₂₃₅Agrobacterium LBA4404 is converted, PCR amplification obtains 1247bp, 523bp respectively With the promoter fragment (Fig. 9) of 238bp.Three sequences are shown in SEQ ID NO respectively：3,SEQ ID NO：2,SEQ ID NO：1.

3.4 AhMtan carry genetic transformation of the Agrobacterium to tobacco of root-specific promoter

Tobacco leaf disc (Figure 10 A) is infected with the Agrobacterium containing plant expression vector, induced bud generates (figure after about 10 days 10B), cultivate and be transferred to induced bud on new differential medium (Figure 10 C) for 4 weeks or so, about two weeks time take root (Figure 10 D, E), obtain and can be used for the card of Molecular Detection that resistance seedling.

Transformed plant is taken to carry out GUS tissue chemical analysis, discovery turns pAhMtan₁₂₄₇、pAhMtan₅₂₃、pAhMtan₂₃₅Table Blue (Figure 11,12,13) are dyed to up to the tobacco root of carrier, and do not observe blue in blade, only in partial blade (Figure 11 A and Figure 12 C) observes blue spot at edge, it may be possible to by mechanical damage according at blue precipitate.Prove that AhMtan is opened Mover is root-specific expression promoter, and the AhMtan promoters of shortest 235bp (contain 3 ROOTMOTIFTAPOX1 elements With 1 OSE2ROOTNODULE element) still have and instructs gene in the specifically expressed function in tobacco root.

Claims

1. root-specific expresses promoter, nucleotide sequence is as shown in SEQ ID NO2, SEQ ID NO3 or SEQ ID NO4.

2. a kind of expression vector expresses promoter containing root-specific described in claim 1.

3. the application of expression vector in transgenic plants described in claim 2, the genetically modified plants are tobacco or peanut, The application is that the root-specific in expression vector expresses the expression that promoter starts downstream gene in the tissue of root.

4. application according to claim 3, inserted with anti insect gene in the expression vector, which is transferred to plant In, make to express the anti insect gene in its root, to prevent subterranean pest-insect.

5. the application of root-specific expression promoter described in claim 1 in transgenic plants, the genetically modified plants are Tobacco or peanut, the application are that the root-specific expression promoter in expression vector starts downstream gene in the tissue of root Expression.

6. application according to claim 5, promoter and pest-resistant is expressed for root-specific described in claim 1 will be contained The expression vector of gene is transferred in plant, it is made to express the anti insect gene in the tip of a root, to prevent subterranean pest-insect.