CN104561026B - Application of the peanut AhFRDL1 genes in Genes For Plant Tolerance Al toxicity stress is improved - Google Patents

Abstract

The invention provides applications of the peanut citrate transporter GFP AhFRDL1 in Genes For Plant Tolerance Al toxicity stress is improved.The application is by the peanut citrate transporter GFP AhFRDL1 or the recombinant expression carrier containing the peanut citrate transporter GFP AhFRDL1 is transferred in plant cell, screening transgenic plant, render transgenic plant expresses peanut citrate transporter albumen, so as to improve the anti-Al toxicity stress ability of plant.First demonstration that peanut citrate transporter GFP AhFRDL1, which is one, regulates and controls citric acid to the exocrine gene of plant, and it take part in the biological process of plant resistant Al toxicity stress, it is that the further chief crop for cultivating resistance to aluminium poison on acid soil has long-range practice significance in the anti-Al toxicity stress that peanut citrate transporter GFP AhFRDL1 is applied to raising plant.

Description

Application of the peanut AhFRDL1 genes in Genes For Plant Tolerance Al toxicity stress is improved

Technical field

The invention belongs to genetic engineering field, the more particularly to one citrate transporter GFP AhFRDL1 that cultivates peanut is being carried Application in high Genes For Plant Tolerance Al toxicity stress.

Background technology

Aluminium is content most abundant metallic element in the earth's crust, is generally existed with slightly solubility silicate or alumina form, right Plant is not poisoned, but in acid condition（pH<5）, soluble aluminium（Mainly Al³⁺）Most plants can all be produced Poison, China's acid soil accounts for the 21% of the national soil gross area, aluminium be not only soil aciditiy on acid soil it is main come Source, simultaneously because the exchange capacity of aluminium accounts for the 20-80% of Cation Exchange Capacity in Soils, causes soil cationic to be easy to leaching loss, causes The nutrient deficiencies such as P, K, Ca, Mg, B, Mo, therefore, aluminium toxicity turn into the important limitation of restriction crop normal growth and development Factor（Wright RJ.1989.Soil aluminum toxicity and plant growth.Communications in Soil Science and Plant Analysis.20:1479-1497）.There are some researches show soybean root system pair under Acid-Al stress NO3- absorption is seriously obstructed（Lazol DB,Rincon M,Rufty JW,Mackown CT,Carter TE.Aluminum accumulation and associated effect on15NO₃ ^-influx in roots of two soybean genotypes differing in Al tolerance.Plant and Soil.164:291-295）；Acid-Al stress makes in root The concentration increase of phosphorus, is that aluminium makes phosphorus be deposited on the surface of root the reason for possible, so as to lower the operating of phosphorus（Ryan PR, Skerrett M,Findlay GP,Delhaize E,Teyman SD.1997.Aluminum activates an anion channel in the apical cells of wheat roots.Proceedings of the National Academy of Sciences.94:6547-6552）, thus the influence metabolic activity relevant with phosphorus；Wheat on acid soil Magnesium deficiency, symptom can only be eliminated using fertiliser containing magnesium, and can not improve overall nutrition；, not only can be with even if liming does not increase fertiliser containing magnesium Eliminate aluminium poison, magnesium deficiency also Lock-out, therefore think that acid soil wheat magnesium deficiency is induced by aluminium, its essence is aluminium is malicious, or can be with It is considered a kind of morphologic appearance of aluminium poison（Shi Jiebin, Qin Suichu, single hero .1997. acid soil wheat magnesium deficiencies and aluminium and calcium, The relation research Zhejiang Agriculture science .6 of potassium element：282-284）.Plant has gradually formed and permitted in long-term evolutionary process The physiological mechanism of many resistance Al toxicity stress, is turned wherein most direct physiological characteristic is plant by the citric acid of root system epidermal cell Albumen secreting outside citric acid is transported, the aluminium in soil is directly chelated, so as to reduce toxic effect of the aluminium to root system（Furukawa J,Yamaji N,Wang H,Mitani N,Murata Y,Sato K,Katsuhara M,Takeda K,Ma JF.2007.An aluminum-activated citrate transporter in barley.Plant Cell Physiology.48: 1081-1091）.

The clear citric acid transport protein of functional study belongs to MATE protein families in plant at present.MATE （Multidrug and Toxic Compound Extrusion）Family is a very big family, in bacterium, fungi, plant It is widely present with mammal, the family protein generally comprises 400-700 amino acid, and forms typical case 12 across thin The transmembrane region of after birth（Omote H,Hiasa M,Matsumoto T,Otsuka M,Moriyama Y.2007.The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations.Trends in Pharmacological Sciences.11:587-593）, these albumen in current plant are same Source property is relatively low, and the homologous uniformity of its albumen is only 40%.There are some researches show, when barley and sorghum are induced by Acid-Al stress, root system Substantial amounts of citric acid can be secreted.In many plants, it is this by transport vehicle of MATE families secrete citric acid mechanism be to The main mechanism of aluminum-resistant stress（Ma JF,Ryan PR,Delhaize E.2001.Aluminum tolerance in plants and the complexing roleof organic acids.Trends in Plant Science.6:273-278; Kochian LV,Pineros MA,Hoekenga OA.2005.The physiology,genetics and molecular biology of plant aluminum resistance and toxicity.Plant and Soil.274:175-195; Delhaize E,Gruber BD,Ryan PR.2007.The roles of organic anio permeases in aluminum resistance and mineral nutrition.Federation of European Biochemical Societies Letters.581:2255-2262;Furukawa J,Yamaji N,Wang H,Mitani N,Murata Y, Sato K,Katsuhara M,Takeda K,Ma JF.2007.An aluminum-activated citrate transporter in barley.Plant Cell Physiology.48:1081-1091;Magalhaes JV,Liu J, Guimaraes CT, wait .2007.A gene in the multidrug and toxic compound extrusion (MATE)family confers aluminum tolerance in sorghum.Nature Genetics.39:1156- 1161）.

Barley HvAACT1 genes belong to MATE families, mainly the barley strain root expression after by Acid-Al stress, mainly The epidermal cell of the barley tip of a root is positioned at, while the heterogenous expression in frog's egg cell illustrates HvAACT1 genes and citrate exudation It is relevant.Overexpression and wild-type tobacco of the HvAACT1 genes in tobacco relatively add the bright gene and can improve plant root It is the secretory volume of citric acid, so as to produce obvious aluminum-resistant stress ability, hence it is demonstrated that barley HvAACT1 genes are one by aluminium The citrate transporter carrier of induction, can resist the Acid-Al stress in the presence of barley growth environment（Furukawa J,Yamaji N,Wang H,Mitani N,Murata Y,Sato K,Katsuhara M,Takeda K,Ma JF.2007.An aluminum-activated citrate transporter in barley.Plant Cell Physiology.48: 1081-1091）.

SbMATE genes in sorghum, are also proved to be a citrate transporter carrier induced by aluminium, can resist environment In Al toxicity stress particularly in acid soil, be mankind's life so as to mitigate infringement of the aluminium poison to sorghum, it is ensured that crop yield Deposit and ensure there is provided more preferable grain and safety（Magalhaes JV, Liu J, Guimaraes CT, wait .2007.A gene in the multidrug and toxic compound extrusion(MATE)family confers aluminum tolerance in sorghum.Nature Genetics.39:1156-1161;Magalhaes JV.2010.How a microbial drug transporter became essential for crop cultivation on acid soils:aluminium tolerance conferred by the multidrug and toxic compound extrusion(MATE)family.Annals of Botany.106:199-203）.

It is arabidopsis on the most clear plant of MATE gene studies.In arabidopsis, after being induced by aluminium, root system secretion Citric acid and malic acid also function to highly important effect to plant resistant Acid-Al stress.AtMATE genes are resisted with sorghum and barley The gene of Acid-Al stress has a very high similitude, one citric acid transport agent of the gene code, secretes citric acid aluminium chelate To resist Acid-Al stress.AtMATE is main to express in root, is knocking out the Arabidopsis Mutants AtMATE-Ko of AtMATE genes In, the citric acid of the root system secretion of aluminium induction is drastically reduced so that ability of the plant in terms of Acid-Al stress is resisted substantially weakens.Together Sample, AtALMT1 genes can also secrete malic acid and carry out aluminium chelate, knock out the mutation of the gene as the transport agent of malic acid Body AtALMT1-Ko equally can cause arabidopsis by aluminium toxicity, root growth due to being unable to the enough malic acid of secreting outside Substantially suppressed.The two genes of AtMATE and AtALMT1 are that the resistance to aluminium for participating in arabidopsis by separate process is coerced Compel reaction, the AtALMT1 genes of wherein encoding malate transport agent are played a part of will be larger.When in arabidopsis Two genes of AtMATE and AtALMT1 are knocked simultaneously when, double-mutant Atdouble-Ko is outwards divided by Acid-Al stress induction root system The citric acid and malic acid secreted all are significantly reduced, and cause plant to compare single mutant（AtMATE-Ko and AtALMT1-Ko）Produce More sensitive Al toxicity stress symptom（Liu JP,Magalhaes JV,Shaff J,Kochian LV.2009.Aluminum- activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.The Plant Journal.57:389-399;Liu JP,Luo XY,Shaff J,Liang CY,Jia XM,Li ZY,Magalhaes J, Kochian LV.2012.A promoter-swap strategy between the AtALMT and AtMATE genes increased Arabidopsis aluminum resistance and improved carbon-use efficiency for aluminum resistance.The Plant Journal.71:327-337）.

At present, identified function and the MATE gene family member relevant with the poison of resistance to aluminium mainly have barley HvAACT1, Sorghum SbMATE, arabidopsis AtMATE, ScFRDL2（Secale cereale）, corn ZmMATE1, rice Os FRDL4 etc. （Furukawa J,Yamaji N,Wang H,Mitani N,Murata Y,Sato K,Katsuhara M,Takeda K,Ma JF.2007.An aluminum-activated citrate transporter in barley.Plant Cell Physiology.48:1081-1091;Magalhaes JV, Liu J, Guimaraes CT, wait .2007.A gene in the multidrug and toxic compound extrusion(MATE)family confers aluminum tolerance in sorghum.Nature Genetics.39:1156-1161;Liu JP,Magalhaes JV,Shaff J,Kochian LV.2009.Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.The Plant Journal.57:389-399;Yokosho K,Yamaji N,Ma JF.2010.Isolation and characterisation of two MATE genes in rye.Functional Plant Biology.37:296-303;Yokosho K,Yamaji N,Ma JF.2011.An Al-inducible MATE gene is involved in external detoxification of Al in rice.The Plant Journal.68:1061-1069;Maron LG,Pineros MA,Guimaraes CT,Magalhaes JV,Pleiman JK,Mao CZ,Shaff J,Belicuas SNJ,Kochian LV.2010.Two functionally distinct members of the MATE(multi-drug and toxic compound extrusion)family of transporters potentially underlie two major aluminum tolerance QTLs in maize.The Plant Journal.61:728-740）.And in peanut, the MATE gene relevant with resistance to Acid-Al stress is gone back at present Not yet there is any report.Peanut is one of main oil crops that China plants extensively, and research Peanut Root System citrate transporter is carried Body gene will be the peanut varieties of resistance to aluminium seed selection and the cultivation of other resistance to aluminium crops from molecular breeding and technique for gene engineering angle The important theoretical foundation of offer and technical support.

The content of the invention

It is an object of the invention to provide peanut citrate transporter GFP AhFRDL1 in Genes For Plant Tolerance Al toxicity stress is improved Application.

The peanut citrate transporter GFP AhFRDL1 has（1）Or（2）Shown nucleotide sequence：

（1）Nucleotide sequence shown in SEQ ID NO.1；

（2）Nucleotide sequence shown in SEQ ID No.1 is substituted, lacks or added one or several nucleotides and formed The nucleotide sequence with equal function.

The peanut citrate transporter albumen of the peanut citrate transporter GFP AhFRDL1 codings has（1）Or（2） Shown amino acid sequence：

（1）Amino acid sequence shown in SEQ ID NO.2；

（2）Amino acid sequence shown in SEQ ID No.2 is substituted, lacks or added one or several amino acid and has Equal function by（1）Derivative protein.

The invention provides the peanut citrate transporter GFP AhFRDL1 in Genes For Plant Tolerance Al toxicity stress is improved Using.

The application is by the peanut citrate transporter GFP AhFRDL1 or containing the peanut citrate transporter egg White Gene A hFRDL1 recombinant expression carrier is transferred in plant cell, screening transgenic plant, render transgenic plant expression peanut Citrate transporter albumen, so as to improve the ability of the anti-Al toxicity stress of plant.

Preferably, the recombinant expression carrier of the GFP of citrate transporter containing the peanut AhFRDL1 is pBAR1- AhFRDL1, its construction method is as follows：

（1）Extract peanut sample RNA and reverse transcription obtains cDNA, using cDNA as template, with AhFRDL1-F and AhFRDL1- R is primer, enters performing PCR；

AhFRDL1-F：5’-TGGTAGAGAAAATGGCTGAGAAG-3’（SEQ ID NO.3）；

AhFRDL1-R：5’-AACATGATTGCATTCATCTCCAT-3’（SEQ ID NO.4）；

（2）By step（1）Obtained PCR primer clone is connected on pMD20-T carriers, obtains carrier pMD20-T- AhFRDL1；

（3）Using carrier pMD20-T-AhFRDL1 as template, using 35s-AhFRD1-F and 35s-AhFRD1-R as primer, enter Performing PCR；

35s-AhFRD1-F：5’-ACGCCCCGGGATGGCTGAGAAGCAG-3’（SEQ ID NO.5）；

35s-AhFRD1-R：5’-CGGGATCCTTTCTCCATAGGAATTCCCA AG-3’（SEQ ID NO.6）；

（4）By step（3）Obtained PCR primer is cloned into pGEM-T carriers, obtains carrier pGEM-T-AhFRDL1；

（5）Carrier pGEM-T-AhFRDL1 and pBAR1 is subjected to double digestion with XmaI and BamHI simultaneously, AhFRDL1 is reclaimed Purpose fragment and pBAR1 carrier segments, connection build recombinant expression carrier pBAR1-AhFRDL1.

Preferably, the plant is peanut, arabidopsis.

The present invention has the advantages that：

First demonstration that peanut citrate transporter GFP AhFRDL1 has the biology work(of resistance Acid-Al stress Energy.AhFRDL1 genes are transferred to two mutant AtALMT1-Ko, AtMATE- of arabidopsis aluminum-resistant afunction by the present invention In Ko and double-mutant Atdouble-Ko, transfer-gen plant is obtained, and using wild type and each Arabidopsis Mutants as control, obtain Arrive：

（1）Different transgenic arabidopsis have different aluminum-resistant growth optimum concentrations under culture medium condition.Double-mutant Atdouble-Ko has knocked out the Gene A tALMT1 and AtMATE of two resistance Acid-Al stress in arabidopsis simultaneously, to the response of aluminium poison most Sensitivity, in 50 μM of AlCl₃Transgenic arabidopsis aluminum-resistant effect is notable during processing；Single mutant AtMATE-Ko is in 400 μM of AlCl₃ During processing, transgenic arabidopsis aluminum-resistant effect is notable；Single mutant AtALMT1-Ko is in 200 μM of AlCl₃During processing, transgenosis Arabidopsis aluminum-resistant effect is notable.

（2）Transgenic arabidopsis can be obviously promoted root growth compared with mutant.Double-mutant Atdouble-Ko turns Gene arabidopsis root system relative increase 42%-50%, single mutant AtMATE-Ko transgenic arabidopsis root system relative increases 45%- 62%, single mutant AtALMT1-Ko transgenic arabidopsis root system relative increases 21%-37%.

（3）Transgenic arabidopsis root system citrate exudation amount compared with mutant is dramatically increased.Double-mutant Atdouble- Ko transgenic arabidopsis root system secreting outside citric acid amounts are 2.6-5.4 times of mutant, single mutant AtMATE-Ko transgenosis Arabidopsis root system secreting outside citric acid amount is 1.4-1.8 times of mutant, single mutant AtALMT1-Ko transgenic arabidopsis Root system secreting outside citric acid amount is 1.3 times of mutant；And the malic acid amount and phase of three kinds of transgenic arabidopsis root system secretions The malic acid amount that mutant is secreted is answered without significant difference.

（4）AhFRDL1 genes are that first be proved at present in the peanut plant without sequencing being capable of aluminum-resistant poison Gene, mainly resists Acid-Al stress by root system secreting outside citric acid.

First demonstration that Peanut Root System citrate transporter GFP AhFRDL1, which is one, regulates and controls citric acid to plant The gene of secreted in vitro, and the biological process of plant resistant Acid-Al stress is take part in, with molecular biology and genetic engineering Technology indicates the gene from multi-angle checking and root system secretion citric acid is closely related, and this is not only further deeply to promote to plant The research of the resistance to aluminium molecular mechanism of thing provides important foundation, and to be cultivated from plant molecular breeding or genetic engineering aspect The yield and quality of the malicious crop of resistance to aluminium and then raising crop has established important basis, therefore can turn Peanut Root System citric acid Transport GFP AhFRDL1 to be applied in the anti-Al toxicity stress of raising plant, aluminum-tolerant plant is further cultivated, with long-range reason By and practice significance, its application prospect is very wide.

Brief description of the drawings

Fig. 1 is the lower citric acid deletion mutant of various concentrations aluminium processing（AtMATE-Ko）Transgenic arabidopsis is with respect to root system Increment, the corresponding Al of relative root volume of each arabidopsis strain³⁺Concentration is from left to right followed successively by 50,100,150,200, 250,300,350,400,450μM。

Fig. 2 is the lower malic acid deletion mutant of various concentrations aluminium processing（AtALMT1-Ko）Transgenic arabidopsis is with respect to root system Increment, the corresponding Al of relative root volume of each arabidopsis strain³⁺Concentration is from left to right followed successively by 25,50,100,150, 200,250μM。

Fig. 3 is the lower double-mutant of various concentrations aluminium processing（Atdouble-Ko）Transgenic arabidopsis with respect to root volume, The corresponding Al of relative root volume of each arabidopsis strain³⁺Concentration is from left to right followed successively by 50,100,150,200 μM..

Fig. 4 is optimum concentration Acid-Al stress（400μM AlCl₃）Handle citric acid deletion mutant（AtMATE-Ko）Transgenosis Arabidopsis root growth situation.

Fig. 5 is optimum concentration Acid-Al stress（200μM AlCl₃）Handle malic acid deletion mutant（AtALMT1-Ko）Turn base Because of arabidopsis root growth situation.

Fig. 6 is optimum concentration Acid-Al stress（50μM AlCl₃）Handle double-mutant（Atdouble-Ko）Transgenic arabidopsis Root growth situation.

Fig. 7 is citric acid deletion mutant（AtMATE-Ko）400 μM of AlCl of transgenic arabidopsis₃Handle root system citric acid （A）And malic acid（B）Secretory volume.

Fig. 8 is malic acid deletion mutant（AtALMT1-Ko）200 μM of AlCl of transgenic arabidopsis₃Handle root system citric acid （A）And malic acid（B）Secretory volume.

Fig. 9 is double-mutant（Atdouble-Ko）50 μM of AlCl of transgenic arabidopsis₃Handle root system citric acid（A）And apple Tartaric acid（B）Secretory volume.

Figure 10 is（A）Citric acid deletion mutant（AtMATE-Ko）、（B）Malic acid deletion mutant（AtALMT1-Ko） With（C）Double-mutant（Atdouble-Ko）The relative expression quantity of AhFRDL1 genes in transgenic arabidopsis.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.

Unless otherwise specified, experiment material, reagent and instrument used in the embodiment of the present invention etc. are commercially available, if Do not particularly point out, the conventional meanses that technological means used is well known to the skilled person in embodiment.

Arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double-mutant Atdouble-Ko referring to Liu JP, Magalhaes JV,Shaff J,Kochian LV.2009.Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance.The Plant Journal.57:389-399。

The clone of the peanut AhFRDL1 genes of embodiment 1

（1）Peanut water planting culture

14 (being purchased from the Chinese Academy of Agricultural Sciences) are spent into 10% dioxygen water sterilization 30 minutes in peanut seed Shandong, after cleaning up It is placed in saturation calcium sulphate soln about 8 hours, and lucifuge of ventilating.It is placed in afterwards in the pallet for being covered with blotting paper, pours appropriate water Lucifuge is placed in artificial culturing room afterwards.It is 30 DEG C of illumination 14 hour that culturing room, which sets condition, dark 10 hours 22 DEG C.1 day or so Peanut sends to move into quartz sand after budlet and cultivated, and is transferred in mill water culture nutrient solution and cultivates after 2 leaves are grown.Mill water culture nutrient solution Formula is as follows：KH₂PO₄（0.5mM）、K₂SO₄（0.75mM）、KCl（0.1mM）、MgSO_4·7H₂O（0.65mM)、CaSO_4·2H₂O (2mM)、H₃BO₃（1μM）、MnSO_4·4H₂O（1μM）、ZnSO_4·7H₂O（1μM）、CuSO_4·5H₂O（0.1μM）、(NH₄)₆Mo₇O_24· 4H₂O(0.005μM)、EDTA-Fe（80μM）, pH value is 5.8-6.

（2）Peanut RNA is extracted and reverse transcription is cDNA

The Peanut Root System and leaf sample that are stored in -80 DEG C are put into the mortar with Liquid nitrogen precooler, liquid nitrogen grinding is added Plant sample is until into powdered, taking out about 0.1g powder samples in 1.5ml without in RNase centrifuge tubes, adding 1ml Trizol. It is vortexed and shakes 15s, be stored at room temperature 5min, add 200 μ l chloroforms, fully mix, stand 5min, 4 DEG C centrifuges, 12000rpm, 15min, takes supernatant to enter in 1.5ml centrifuge tube, adds 500 μ l isopropanols, mixes, 10min is stored at room temperature, in 4 DEG C 12000rpm centrifuges 10min, outwells supernatant after taking-up, adds 1ml75% ethanol, is vortexed the concussion several seconds, 4 DEG C of 7500rpm from Heart 5min, repeated washing twice, is outwelled ethanol and sucked after remaining supernatant, centrifuge tube is air-dried at room temperature.Add in right amount DEPC water and RNase inhibitor, obtain RNA, and reverse transcription is cDNA.

（3）Full length gene is cloned

Design expands the primer of the full length gene, is respectively：

AhFRDL1-F：5’-TGGTAGAGAAAATGGCTGAGAAG-3’（SEQ ID NO.3）；

AhFRDL1-R：5’-AACATGATTGCATTCATCTCCAT-3’（SEQ ID NO.4）.

PCR reaction systems are：2 μ l, 10 × reaction buffer 2 μ l, dNTP (10mM) 0.5 μ l are taken after cDNA is diluted into 5 times, MgCl2（50mM）0.5 μ l, forward primer AhFRDL1-F（0.01mM）0.5 μ l, reverse primer AhFRDL1-R（0.01mM）0.5μ L, ddH2O13.7 μ l, Taq DNA polymerase（Invitrogen companies）0.3μl.

Above-mentioned PCR reaction systems are mixed into laggard performing PCR reaction, program is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, 30 circulations；72 DEG C of extension 10min.

Agarose gel electrophoresis obtains expected about 1.6kb DNA bands after PCR reactions, carries out glue reclaim purifying, obtains DNA total lengths.Then DNA recovery obtained is connected to pMD20-T carriers（TaKaRa companies）, 16 DEG C overnight, are transformed into large intestine Bacillus DH5 α competent cells（TIANGEN companies）, be applied to the LB plates of the antibiotic containing Amp, 37 DEG C of incubated overnights, choose monoclonal in 37 DEG C of LB liquid shakes bacterium overnight, send bacterium solution to be sequenced（Shanghai bio-engineering corporation）The correctness of sequence is determined, and extracts correct matter Grain, obtains carrier pMD20-T-AhFRDL1.AhFRDL1 gene orders are as shown in SEQ ID No.1（2034bp）, orresponding amino acid Sequence is SEQ ID No.2.

The structure of the recombinant expression carrier of embodiment 2

1st, carrier construction

（1）Restriction enzyme site XmaI and BamHI are added by primer, carried out by template of carrier pMD20-T-AhFRDL1 PCR；

Primer sequence is：

35s-AhFRD1-F：5’-ACGCCCCGGGATGGCTGAGAAGCAG-3’（SEQ ID NO.5）；

35s-AhFRD1-R：5’-CGGGATCCTTTCTCCATAGGAATTCCCAAG-3’（SEQ ID NO.6）；

（2）PCR primer is cloned into pGEM-T carriers, after conversion DH5 α competent cells, positive colony is selected, cultivates Bacterium simultaneously extracts plasmid, obtains carrier pGEM-T-AhFRDL1；

（3）Carrier pGEM-T-AhFRDL1 and pBAR1 carrier are subjected to double digestion with XmaI and BamHI simultaneously, returned respectively Receive AhFRDL1 genes purpose fragment and pBAR1 carrier segments, then with T4 ligases connection AhFRDL1 genes purpose fragment and PBAR1 carrier segments after digestion, build the carrier that 35S strong promoters drive gene expression；

（4）Connection product is transformed into DH5 α competent cells, positive colony is selected, plasmid is extracted, digestion is detected and surveyed Sequence；

（5）After determining that sequence is correct, correct plasmid pBAR1-AhFRDL1 electricity is transformed into GV3101 Agrobacteriums, in case Follow-up test.

2nd, electricity conversion（Agrobacterium）

（1）Use the electroporated instrument of BIO-RAD, 1.8kV voltages, the Ω of resistance 200；

（2）1 μ L plasmids pBAR1-AhFRDL1 is added in 100 μ L Agrobacterium competent cells, mixes, places on ice 60sec；

（3）Mixed liquor is transferred in the electric shock of precooling cup, electric shock cup is put into electric reburner rapidly；Press " Pulse " Key, hears rapid taking-up electric shock cup after sound；

（4）1mL YEB fluid nutrient mediums are added immediately, and liquid is transferred in new 1.5mL centrifuge tubes；

（5）28 DEG C of multiple culture 2h that relax；

（6）In super-clean bench, prepare the flat board containing corresponding antibiotic；

（7）Bacterium solution is fetched, gently mixed in super-clean bench, appropriate bacterium solution is taken（Such as 100 μ L）Drop on flat board, with cooling Spreader on flat board by bacterium solution even spread, culture dish mouthful is sealed with sealed membrane；

（8）Flat board is placed on 28 DEG C of incubator culture 48h；

（9）Choosing colony is detected and preserved from flat board, and flat board then is placed on into 4 DEG C of refrigerator preservations.

Peanut AhFRDL1 gene pairs arabidopsis single mutant AtALMT1-Ko, the AtMATE-Ko of embodiment 3 and double-mutant The Atdouble-Ko experiment that has complementary functions

1.1 are transferred to the acquisition of the Arabidopsis plant of AhFRDL1 genes

The recombinant expression carrier pBAR1-AhFRDL1 that embodiment 2 is obtained is transformed into aluminum-resistant respectively by transgenic technology In three Arabidopsis Mutants of afunction：（1）The Gene A tALMT1 of the resistance to aluminium of arabidopsis（Malic acid transport agent）Knock out Single mutant AtALMT1-Ko；（2）The Gene A tMATE of the resistance to aluminium of arabidopsis（Citric acid transport agent）The single mutant of knockout AtMATE-Ko；（3）The arabidopsis double-mutant Atdouble-Ko that AtALMT1 and AtMATE is knocked out simultaneously.

Concrete operation step is as follows：Arabidopsis seed is soaked with 1% agarose, by seed liquid-transfering gun point to being mixed with leech In the Nutrition Soil of stone（The ratio of vermiculite and Nutrition Soil is 1:1, soil is filled in plastic tub, and soil is poured with water until reaching saturation）, use Covered rearing with plastic film lives soil.22 DEG C of greenhouse, 16h illumination 8h dark processings are placed on, and pours and uses nutrient solution.Treat that first inflorescence is long To 5-15cm, second inflorescence is transplanted seedlings into another basin on the first appearance.While transplanting seedlings, the recombination expression built is carried Body pBAR1-AhFRDL1 is transferred in Agrobacterium GV3101, and conversion is completed when arabidopsis grows to first inflorescence of appearance.

Recombinant expression carrier pBAR1-AhFRDL1 GV3101 Agrobacterium inoculations will be transferred to the LB culture mediums in 5mL （Contain kanamycins）In, 30 DEG C of 200rpm concussion incubated overnights in constant-temperature shaking culture device；Second day, by incubated overnight In Agrobacterium inoculation 5mL to 500mL LB nutrient solutions, 50mg/L Rif are added（Rifampin antibiotic）,50g Sucrose（Sugarcane Sugar）, 500ul Silwet L-77, culture；3rd day, above-mentioned bacterium solution centrifugation 20min is taken, abandoning supernatant uses conversion medium （0.433g MS culture mediums, 5g sucrose, 25ul Silwet L-77 are added in per 100mL sterilized waters）Resuspension is precipitated to OD values and reached To 0.8 or so.Filter paper is put well on greenhouse platform, plant is kept flat on estrade, and the inflorescence immersion of arabidopsis is contained into agriculture bar The arabidopsis being immersed in Agrobacterium is taken out after 1 minute in the nutrient solution of bacterium, kept flat, and is lived with covered rearing with plastic film, in greenhouse In（22℃）Overnight.Second day, Arabidopsis plant vertical growth poured use nutrient solution daily, and arabidopsis restore normal growth.Work as kind When sub ripe, by seed collection to 15ml or 50ml centrifuge tube.

The seed of harvest is put into 1.5mL sterile tube, 75% ethanol infiltration 30s, vibration mixing, aseptic water washing one It is secondary, 1.5% hypochlorite disinfectant 60s（Period does not stop concussion, suctions out in time）, aseptic water washing 6 times adds 1mL after the completion of sterilization Aqua sterilisa, is put into 4 DEG C of refrigerators, vernalization 2 days.MS culture mediums（Formula is shown in Table 1-1）The good seed of vernalization is cultivated, one is grown very Ye Shi, transplants seedlings into Nutrition Soil（Vermiculite is with Nutrition Soil with 1:1 ratio is well mixed, 120 DEG C, 20min sterilizings）Middle culture（Start Preservative film is covered, moisture is kept）, water daily, plant to be planted sprays 100mg/L herbicide when growing to 4-6 piece leaves, every three days Spray is once, continuous to spray 5 times, and part arabidopsis is dead, continues to plant the seedling of survival, treats maturation, individual plant collects seed and obtains T1 For transgenic seed.

Table 1-1MS culture medium prescriptions

Note：Plus pH value is adjusted before agar powder to 7.0

Acquisitions of the 1.2 transfer-gen plant T3 for seed

Same method obtains T2 for transgenic seed, and the T2 for the transgenic line chosen is put into 1.5mL's for seed In sterile tube, 75% ethanol infiltration 30S, vibration mixing, aseptic water washing once, 1.5% hypochlorite disinfectant 60s（Period does not stop Concussion, is suctioned out in time）, aseptic water washing 6 times, addition 1mL aqua sterilisas, are put into 4 DEG C of refrigerators, vernalization 2 days after the completion of sterilization.Will Vermiculite and Nutrition Soil sterilizing（120 DEG C, 20min）.By vermiculite and Nutrition Soil with 1:1 ratio is well mixed, and is filled basin, is poured before dibbling Water is to saturation.

By the seed dibbling after vernalization, dibbling is uniform, and basin is lived with covered rearing with plastic film after dibbling, is placed on arabidopsis training Cultivate, 22 DEG C of indoor temperature, illumination 16h dark 8h photo-irradiation treatments, after plant emergence, plastic sheeting is opened, suitably in foster room Water.Arabidopsis sprays 100mg/L herbicide when growing to 4-6 piece leaves, spray every three days once, continuous to spray 5 times, and south is intended in part Mustard is dead, continues to plant the seedling of survival, and when arabidopsis is ripe, individual plant collects seed, obtains T3 for transgenic homozygous body kind Son, in case follow-up test is used.

According to the method described above, respectively obtain be transferred to AhFRDL1 genes arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double-mutant Atdouble-Ko transgenic arabidopsis T3 is for seed.

Influence of the 1.3 different Acid-Al stress to arabidopsis transfer-gen plant root growth

Test material：Wildtype Arabidopsis thaliana WT seeds, arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double prominent Modification A tdouble-Ko seeds, are transferred to the transgenic arabidopsis T3 of AhFRDL1 genes for seed.Wherein, AhFRDL1 bases are transferred to The arabidopsis single mutant AtMATE-Ko of cause different transgenic arabidopsis strains are AtAm2, AtAm8, AtBm10；It is transferred to The arabidopsis single mutant AtALMT1-Ko of AhFRDL1 genes different transgenic arabidopsis strains be AtAa10, AtAa18, AtBa14；The different transgenic arabidopsis strains for being transferred to the arabidopsis double-mutant Atdouble-Ko of AhFRDL1 genes are AtBd1、AtBd2、AtBd4。

Seed is put into 1.5mL sterile tube, 75% ethanol infiltration 30s, vibration mixing, aseptic water washing once, 1.5% Hypochlorite disinfectant 60s（Period does not stop concussion, suctions out in time）, aseptic water washing 7 times, addition 1mL aqua sterilisas after the completion of sterilization, It is put into 4 DEG C of refrigerators, vernalization 2 days.Configure nutrient medium（Formula is shown in Table 1-2）, different aluminum concentration processing are added after sterilizing and are adjusted It is 4.2 to save pH.AtALMT1-Ko sets 0,25,50,100,150,200,250 μM of Al³⁺Seven aluminum concentration gradients, AtMATE-Ko 0,50,100,150,200,250,300,350,400,450 μM of Al is set³⁺Ten aluminum concentration gradients, Atdouble-Ko is set 0,50,100,150,200μM Al³⁺Five aluminum concentration gradients, it is after a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices solidification that the good seed point of vernalization is dense to different aluminum processing Media surface is spent, each gradient sets the repetition of three plates, and is horizontally placed to culture in the condition of suitable arabidopsis growth.Temperature：22 DEG C, humidity：70% or so, intensity of illumination：120μmol/m²/ s, long-day conditions：16h illumination, 8h is dark.

Table 1-2 arabidopsis culture medium nutrient formulations（pH4.2）

Note：Arabidopsis nutrient solution pH value is transferred to 4.2

Quantitative main root is long after cultivating 7 days, compares different tests material root growth situation when different aluminum is handled, it is determined that most Quantitatively root is repeated after suitable aluminium concentration for the treatment of long, analyze transfer-gen plant root growth situation.Parameter：With respect to root growth Amount：RNRG%

RNRG%=plus aluminium ambient growth root length/control are not added with aluminium root length × 100

As shown in Figure 1-Figure 3, different transgenic arabidopsis have different aluminum-resistant growth optimum concentrations under culture medium condition.It is single Mutant AtMATE-Ko is in 400 μM of AlCl₃During processing, transgenic arabidopsis aluminum-resistant effect is notable（Fig. 1）.Single mutant AtALMT1-Ko is in 200 μM of AlCl₃During processing, transgenic arabidopsis aluminum-resistant effect is notable（Fig. 2）.Double-mutant Atdouble- Ko has knocked out the Gene A tALMT1 and AtMATE of two resistance Acid-Al stress in arabidopsis simultaneously, most sensitive to aluminium poison response, 50 μM AlCl₃Transgenic arabidopsis aluminum-resistant effect is notable during processing（Fig. 3）.

It is determined that after most suitable aluminum concentration processing, same method com-parison and analysis different tests material is in control（It is not added with aluminium）Processing and The difference of the most suitable lower root growth situation of aluminum concentration processing, analysis AhFRDL1 gene pairs arabidopsis root length under Acid-Al stress grows Influence.

As Figure 4-Figure 6, transgenic arabidopsis can be obviously promoted root growth compared with mutant.Single mutant AtMATE-Ko transgenic arabidopsis root system increases 45%-62% with respect to mutant（Fig. 4）, single mutant AtALMT1-Ko transgenosis Arabidopsis root system increases 21%-37% with respect to mutant（Fig. 5）, double-mutant Atdouble-Ko transgenic arabidopsis root systems are relative Mutant increases 42%-50%（Fig. 6）.

1.4 Acid-Al stress are to arabidopsis transfer-gen plant root system citric acid, the influence of malic acid secretory volume

Test material：Wildtype Arabidopsis thaliana WT seeds, arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double prominent Modification A tdouble-Ko seeds, are transferred to the transgenic arabidopsis T3 of AhFRDL1 genes for seed.Wherein, AhFRDL1 bases are transferred to The arabidopsis single mutant AtMATE-Ko of cause different transgenic arabidopsis strains are AtAm2, AtAm8, AtBm10；It is transferred to The arabidopsis single mutant AtALMT1-Ko of AhFRDL1 genes different transgenic arabidopsis strains be AtAa10, AtAa18, AtBa14；The different transgenic arabidopsis strains for being transferred to the arabidopsis double-mutant Atdouble-Ko of AhFRDL1 genes are AtBd1、AtBd2、AtBd4。

Normal incubation medium culture arabidopsis：Seed is put into 1.5mL sterile tube, 75% ethanol infiltration 30S, vibration is mixed Close, aseptic water washing once, 1.5% hypochlorite disinfectant 60s（Period does not stop concussion, suctions out in time）, aseptic water washing 7 times disappears 1mL aqua sterilisas are added after the completion of poison, are put into 4 DEG C of refrigerators, vernalization 2 days.Configure nutrient medium（Formula is shown in Table 1-2）, sterilizing After be not added with aluminium processing regulation pH be 4.2.After the solidification of falling culture medium flat plate, the good seed point of vernalization is cultivated on normal incubation medium 10d。

Collect root exudates：By the Arabidopsis plant in above-mentioned culture medium, every 20 plants are put in one and are secreted equipped with root system In the 2mL centrifuge tubes of thing collection liquid（Collection liquid composition：The 0.66mM CaCl of filtration sterilization₂Middle add after suitable aluminum concentration is adjusted PH is 4.2）, under the conditions of normal culture environment（Temperature：22 DEG C, humidity：70% or so, intensity of illumination：120μmol/m²/ s, Long-day conditions（16h illumination, 8h is dark））Collect 24h.Collection added after finishing in collection liquid micro microbial inhibitor with Organic acid is prevented to be degraded, -80 DEG C of preservations.

Lyophilized concentration：The least concentration for needed for the root exudates for making to be collected into meets or exceeds organic acidity test, by three Pipe（I.e. 60 plants）The root exudates gathered is merged into a pipe, and is placed in lyophilized instrument lyophilized concentration, and the place of drying is preserved, concentration It is yellow powder material afterwards, it is to be measured.

1mL high purity waters are taken fully to dissolve after lyophilized concentrate, high-efficient liquid phase chromatogram HPLC determines lemon in root exudates Acid, malic acid content, and do data analysis, the secretory volume of relatively more different arabidopsis material citric acids under Acid-Al stress.

As shown in figs. 7 to 9, transgenic arabidopsis root system citrate exudation amount compared with mutant is dramatically increased.Single mutation Body AtMATE-Ko transgenic arabidopsis root system secreting outside citric acid amounts are 1.4-1.8 times of mutant（Fig. 7）, single mutant AtALMT1-Ko transgenic arabidopsis root system secreting outside citric acid amounts are 1.3 times of mutant（Fig. 8）, double-mutant Atdouble-Ko transgenic arabidopsis root system secreting outside citric acid amounts are 2.6-5.4 times of mutant（Fig. 9）.And three kinds turn The malic acid amount that the malic acid amount of gene arabidopsis root system secretion is secreted with corresponding mutant is without significant difference.It is alphabetical in Fig. 7-9 Abc represents the variance analysis between each pillar numerical value, and same letter represents then to represent without significant difference, difference letter between numerical value There is significant difference.

Influence of 1.5 Acid-Al stress to transfer-gen plant AhFRDL1 gene expressions

1.5.1 test material

Wildtype Arabidopsis thaliana WT seeds, arabidopsis single mutant AtALMT1-Ko, AtMATE-Ko and double-mutant Atdouble-Ko seeds, are transferred to the transgenic arabidopsis T3 of AhFRDL1 genes for seed.Wherein, AhFRDL1 genes are transferred to Arabidopsis single mutant AtMATE-Ko different transgenic arabidopsis strains are AtAm2, AtAm8, AtBm10；It is transferred to AhFRDL1 The arabidopsis single mutant AtALMT1-Ko of gene different transgenic arabidopsis strains are AtAa10, AtAa18, AtBa14；Turn Enter AhFRDL1 genes arabidopsis double-mutant Atdouble-Ko different transgenic arabidopsis strains for AtBd1, AtBd2, AtBd4。

1.5.2 RNA is extracted

Arabidopsis plant is quickly removed after root exudates has been collected, gently clean dip in it is dry after with separately collecting arabidopsis Top（Such as：Leaf）And underground part（Such as：Root）About 100mg, is quickly charged with 2mL without RNase centrifuge tube, is put into liquid nitrogen quick freeze simultaneously In -80 DEG C of preservations.2 small steel balls of sterilizing are added in the centrifuge tube for preserve plant sample, and is placed in whirlpool instrument and repeatedly shakes Swing grinding.After the sample of all pipes is ground, RNA extracts kits are used（Tiangeng biochemical technology（Beijing）Co., Ltd, goods Number DP432）Extract RNA.

RNA extracts kits extract RNA：

450 μ L RL are added in the centrifuge tube of the ground samples of 2mL (to add using preceding add in beta -mercaptoethanol, 1mL RL 10 μ L beta -mercaptoethanols）Acutely concussion is mixed.All solution are transferred on filtering note CS（Filter column CS is placed in collecting pipe）, 12000rmp centrifuges 5min, and the careful supernatant drawn in collecting pipe is into RNase-Free centrifuge tube, and hair washing avoids connecing as far as possible Touch the pellet cell debris in collecting pipe.It is slowly added to the absolute ethyl alcohol of 0.5 times of supernatant volume（Usually 225 μ L）, mixing will Obtained solution and precipitation is transferred in adsorption column CR3 together, 12000rmp centrifugation 60s, is outwelled the waste liquid in collecting pipe, will be adsorbed Post CR3 is put back in collecting pipe.350 μ L protein liquid removals RW1,12000rmp centrifugation 60s are added into adsorption column, are outwelled in collecting pipe Waste liquid, adsorption column CR3 is put back in collecting pipe.80 μ L DNase I working solutions are added to adsorption column CR3 centers（10μL DNase I storing liquids are put into new RNase-Free centrifuge tube, are added 70 μ L RDD solution and are softly mixed）, room temperature placement 15min.350 μ L protein liquid removals RW1,12000rmp centrifugation 60s are added into adsorption column CR3, the waste liquid in collecting pipe is outwelled, will Adsorption column CR3 is put back in collecting pipe.500 μ L rinsing liquids RW (addition ethanol) are added into adsorption column CR3,2min is stored at room temperature, 12000rmp centrifuges 60s, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.500 μ L are equally added again Rinsing liquid RW, is stored at room temperature 2min, 12000rmp centrifugation 2min, outwells waste liquid.Adsorption column CR3 is placed in room temperature and places several points Clock, thoroughly to dry rinsing liquid remaining in sorbing material.Adsorption column CR3 is put into new RNase-Free centrifuge tube In, 50 μ LRNase-Free dd H are vacantly added dropwise to the middle part of adsorbed film₂O, room temperature places 2min, 12000rmp centrifugations 2min, obtains arabidopsis RNA.

1.5.3RNA the measure and reverse transcription of concentration and purity

OD260/OD280 should be 1.7-2.1, less than 1.7, illustrate there is albumen or phenol pollution.OD260/OD230 should be greater than 2.0, less than the value, illustrate there is the pollution of the small molecules such as sugar, salt, guanidine.

Reverse transcription（The synthesis of first chain of cDNA）：According to reverse transcription reagent box (M-MLV reverse transcriptases, the general Lip rivers of Promega Mai Ge Bioisystech Co., Ltd, article No. M1701) specification operation, by 2 μ g total serum IgE samples, 5 μ L Oligo (dt) 18primer（The primers of Oligo (dt) 18）It is added in centrifuge tube, plus DEPC water is gently mixed to the μ L of cumulative volume 15, can not be acute It is strong；70 DEG C of stringent heat 5min, are immediately placed on 2min on ice；Reactant is collected in bottom after of short duration centrifugation, is subject to lower reaction Thing enters in pipe：1.25 μ L dNTP mixed liquors（10mM）, 5 μ L M-MLV5 × buffer solution, 1 μ L M-MLV Reverse Transcriptase（M-MLV reverse transcriptase）, 0.5 μ L RNase inhibitor（RNase inhibitor）, DEPC water complements to 25μL；42 DEG C of warm bath 1h；Cooled on ice.CDNA can be directly used for PCR reactions and can be deposited 3 months at -20 DEG C.

Real-time PCR：The primer of gene PCR uses Primer premier5.0 and Oligo6 Software for Design, primer Synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

Primer sequence is：

AhFRD1RTF：5’-GAAGATGTTAAAGTCCTCCAT-3’（SEQ ID NO.7）；

AhFRD1RTR：5’-ACCAGCAGATGACATGATAAG-3’（SEQ ID NO.8）.

The cDNA formed using reverse transcription is separately added into SYBR Green Realtime as template in each reaction system PCR Master Mix12.5 μ L, upstream and downstream primer each 1 μ L, template cDNA are 3 μ L, and overall reaction system is 25 μ L, remaining with going out Bacterium water is supplied.Reactant is put into the 7500System fluorescent quantitation instruments that ABI companies produce and reacted, the time is about two Individual hour.Response procedures are as follows：Step（1）：50℃2min；Step（2）：95℃1min；Step（3）：95 DEG C of 15s, 60 DEG C 15s, 72 DEG C of 45s, 40 circulations；Step（4）：From 60 DEG C to 98 DEG C, rise 1 DEG C per 0.5min and circulated.PCR reactions are complete Afterwards, using SPSS11.0 analysis softwares, divided from instrument upper derivate according to EXCEL processing under 0.05 level of signifiance Analysis.

As shown in Figure 10, AhFRDL1 genes are able to detect that in the root and leaf of each transgenic arabidopsis mutant plants Great expression, and the expression of the gene is can't detect in wild type and non-transgenic Arabidopsis Mutants, explanation AhFRDL genes are successfully transferred to Arabidopsis Mutants and expressed.

First demonstration that Peanut Root System citrate transporter GFP AhFRDL1, which is one, regulates and controls citric acid to plant The gene of secreted in vitro, and the biological process of plant resistant Acid-Al stress is take part in, with molecular biology and genetic engineering Technology illustrates that the gene and root system secretion citric acid are closely related from multi-angle checking, and this is not only that further further investigation is planted The Study on Molecular Mechanism of the resistance to aluminium of thing provides important foundation, and to be trained in terms of plant molecular breeding and genetic engineering Educate aluminium crop and then raising crop yield and quality improve and established important basis, therefore Peanut Root System citric acid can be turned Transport GFP AhFRDL1 to be applied in the anti-Al toxicity stress of raising plant, aluminum-tolerant plant is further cultivated, with long-range reality Meaning is trampled, its application prospect is very wide.

Although above having made to retouch in detail to the present invention with general explanation, embodiment and experiment State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art 's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed Scope.

Claims (4)

1. applications of the peanut citrate transporter GFP AhFRDL1 in Genes For Plant Tolerance Al toxicity stress is improved,

The nucleotide sequence of the peanut citrate transporter GFP AhFRDL1 is as shown in SEQ ID NO.1；

The plant is peanut, arabidopsis.

2. application according to claim 1, it is characterised in that the application is by the peanut citrate transporter albumen base Because of AhFRDL1 or the recombinant expression carrier containing the peanut citrate transporter GFP AhFRDL1 is transferred in plant cell, sieve Select genetically modified plants, render transgenic plant expression peanut citrate transporter albumen.

3. application according to claim 2, it is characterised in that the GFP of citrate transporter containing the peanut AhFRDL1 Recombinant expression carrier be pBAR1-AhFRDL1.

4. application according to claim 3, it is characterised in that the recombinant expression carrier is pBAR1-AhFRDL1 structure Construction method is as follows：

(1) peanut sample RNA and reverse transcription are extracted and obtain cDNA, using cDNA as template, using AhFRDL1-F and AhFRDL1-R as Primer, enters performing PCR；

AhFRDL1-F：5’-TGGTAGAGAAAATGGCTGAGAAG-3’；

AhFRDL1-R：5’-AACATGATTGCATTCATCTCCAT-3’；

(2) the PCR primer clone obtained step (1) is connected on pMD20-T carriers, obtains carrier pMD20-T-AhFRDL1；

(3) using carrier pMD20-T-AhFRDL1 as template, using 35s-AhFRD1-F and 35s-AhFRD1-R as primer, carry out PCR；

35s-AhFRD1-F：5’-ACGCCCCGGGATGGCTGAGAAGCAG-3’；

35s-AhFRD1-R：5’-CGGGATCCTTTCTCCATAGGAATTCCCAAG-3’；

(4) PCR primer for obtaining step (3) is cloned into pGEM-T carriers, obtains carrier pGEM-T-AhFRDL1；

(5) carrier pGEM-T-AhFRDL1 and pBAR1 is subjected to double digestion with XmaI and BamHI simultaneously, reclaims AhFRDL1 purposes Fragment and pBAR1 carrier segments, connection build recombinant expression carrier pBAR1-AhFRDL1.