CN108424919A - The research and application of transporter gene PbrALMT9 regulation and control pear flesh malic acid contents - Google Patents

The research and application of transporter gene PbrALMT9 regulation and control pear flesh malic acid contents Download PDF

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CN108424919A
CN108424919A CN201810333150.2A CN201810333150A CN108424919A CN 108424919 A CN108424919 A CN 108424919A CN 201810333150 A CN201810333150 A CN 201810333150A CN 108424919 A CN108424919 A CN 108424919A
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pbralmt9
pears
transporter gene
tomato
pulp
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张绍铃
许林林
黄小三
齐开杰
谢智华
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Nanjing Agricultural University
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Nanjing Agricultural University
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Abstract

Researchs and application of the transporter gene PbrALMT9 provided by the invention in regulation and control pear flesh malic acid content, belong to field of plant genetic, the sequence of the transporter gene PbrALMT9 is as shown in SEQ ID No.1, have the function of improving fruit malic acid content, it can be applied to improve pulp malic acid content, according to the record of embodiment part, it is that malic acid content is apparently higher than wild type in tomato pulp that the pears transporter gene PbrALMT9, which is transferred to two overexpressions obtained in tomato plant, and two overexpressions are that the expression pattern of PbrALMT9 genes and the variation tendency of malic acid content are consistent in tomato.

Description

The research and application of transporter gene PbrALMT9 regulation and control pear flesh malic acid contents
Technical field
The invention belongs to field of plant genetic, and in particular to a transporter gene PbrALMT9 regulates and controls the operatic circle The research and application of meat malic acid content.
Background technology
Pears are a kind of important economic fruits, and commercial value is very big, and the commercial value of Pear Fruit is by the quality of fruit It determines.The quality of fruit is divided into exterior quality and interior quality, and the content of the components such as acid, sugar and aroma substance is to determine in fruit The principal element of fruit interior quality.Wherein, the organic acid content in pear fruit be influence pear fruit taste quality it is important because Element has important influence to consumption market, fruit juice production quality etc..
The main component of organic acid is malic acid in most kind pears ripening fruits in white pear system.For many years, to the greatest extent Pipe domestic and international researcher has carried out many researchs around pear fruit acidity, but most of research all concentrate on the composition of organic acid at Point and content and metabolic pathway in terms of, the research to determining the gene of pear fruit acidity is very few.
Aluminium activated form malic acid transport protein (ALMT) family is the big albuminoid being prevalent in plant cell, ALMT albumen plays extensive effect in plant growth.Recent studies indicate that multiple ALMT family members take part in apple The transhipment process of tartaric acid.In arabidopsis, although AtALMT6 and AtALMT9 are proved to be located in different eucaryotic cell structures, It is that they are involved in guard cell malic acid across the transhipment of tonoplast；AtALMT6 is one to Ca2+Sensitive malic acid channel And participate in the exchange of Stomatal Gas；And AtALMT9 is a Cl-Malic acid channel is not only involved in the opening and closing for adjusting stomata, moreover it is possible to Cope with drought stress.Multiple ALMT family members on grape and tomato, such as VvALMT9, SlALMT4, SlALMT5 and SlALMT9, the accumulation being all proved to malic acid are related.In addition to this, some ALMT families can be by Al3+Induction, is conducive to The transmembrane transport of malic acid, for example AtALMT1 is in Al3+The lower outflow that can promote malic acid in vacuole of stress, utilizes malate2-In conjunction with Al3+Compound is formed, so as to contact Al3+Poisoning.Same result is also in the crops such as rye and rape In be found.But there is not been reported for the gene for influencing pear fruit acidity.
Invention content
In view of this, the purpose of the present invention is to provide a transporter gene PbrALMT9 and its in regulation and control pear flesh apple Application in tartaric acid content, the transporter gene can improve malic acid content in pulp.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：One pears transporter gene PbrALMT9, The sequence of the transporter gene PbrALMT9 is as shown in SEQ ID No.1.
The present invention provides the protein of the transporter gene PbrALMT9 codings, the amino acid sequences of the protein Row are as shown in Seq ID No.2.
The present invention also provides the transporter gene PbrALMT9 or described transporter genes PbrALMT9 codings Application of the protein in improving pulp malic acid content.
Preferably, the pulp is pear flesh.
Preferably, the pulp is tomato pulp.
Preferably, include the following steps：1) the pears transporter gene PbrALMT9 is provided；2) by the pears transporter base Recombinant vector is obtained because PbrALMT9 is connect with carrier；3) recombinant vector is transferred in Agrobacterium tumefaciems and obtains recombination crown gall Agrobacterium；4) the recombination Agrobacterium tumefaciems is infected into the tomato that tomato obtains overexpression pears transporter gene PbrALMT9.
Preferably, step 1) is：Using pear flesh cDNA as template, carries out PCR amplification and obtain pears transporter gene PbrALMT9；The amplification primers are to including forward primer F1 and reverse primer R1；The sequence such as SEQ of the forward primer F1 Shown in ID No.3；The sequence of the reverse primer R1 is as shown in SEQ ID No.4.
The present invention also provides applications of the pears transporter gene PbrALMT9 in pears crossbreeding.
Preferably, the application is the acidity character of identification crossbreeding offspring's pulp, including：With crossbreeding offspring's CDNA is pears transporter gene PbrALMT9 described in template amplification；It is good to expand successfully judgement filial generation pulp acidity character.
Beneficial effects of the present invention：Pears transporter gene PbrALMT9 provided by the invention has and improves fruit malic acid The function of content can be applied to improve pulp malic acid content, and the albumen of the pears transporter gene PbrALMT9 codings is fixed On tonoplast, the malic acid dianions that can dissociate in combination cell form polymer, and pass through special cross-film Malic acid dianions are transported in vacuole by ion channel, to increase the accumulation of malic acid in vacuole.According to implementation The record of example part, it is tomato fruit that the pears transporter gene PbrALMT9, which is transferred to two overexpressions obtained in tomato plant, Malic acid content is apparently higher than wild type in meat, and two overexpressions are the expression pattern and apple of PbrALMT9 genes in tomato The variation tendency of tartaric acid content is consistent.In addition, pears transporter gene PbrALMT9 provided by the invention is also used as molecule mark Note is applied to pears crossbreeding, improves the content of pear flesh malic acid.
Description of the drawings
Fig. 1 is the techniqueflow chart of pears transporter gene PbrALMT9 Function Identifications of the present invention；
Fig. 2 is that gene PbrALMT9 gene expression patterns in pear fruit growth course change with malic acid content in fruit The relational graph of trend；
Fig. 3 is gene PbrALMT9 subcellular localization schematic diagrames；
Fig. 4 is gene PbrALMT9 vector construction schematic diagrames；
Fig. 5 is that gene PbrALMT9 converts tomato process schematic；
Fig. 6 is that gene PbrALMT9 converts the regeneration plant PCR qualification figures obtained after tomato；
Fig. 7 is T2For expression analysis-Semiquatitative RT-PCR assay knot of foreign gene PbrALMT9 in tomato transgenic plant Fruit is schemed；
Fig. 8 is gene PbrALMT9 in wild type and T2For Transgenic tomato fruit (OE7-2-6 and OE14-1-4 two System) expression analysis figure during dynamic development；
Fig. 9 is wild type and T2For apple in Transgenic tomato fruit (OE7-2-6 and OE14-1-4 two are) growth course The variation diagram of tartaric acid content；
Figure 10 is in wild type (WT) and T2It is sent out for Transgenic tomato fruit (OE7-2-6 and OE14-1-4 two are) dynamic During educating, the activity analysis figure of key enzyme in Malic Metabolism approach；
Figure 11 is in wild type (WT) and T2It is sent out for Transgenic tomato fruit (OE7-2-6 and OE14-1-4 two are) dynamic During educating, the expression analysis figure of the gene of key enzyme in encoding malate metabolic pathway.
Specific implementation mode
The present invention provides a pears transporter gene PbrALMT9, and the sequence of the transporter gene PbrALMT9 is such as Shown in SEQ ID No.1.
The pears transporter gene PbrALMT9 preferably derives from Dangshan pear (Pyrus in the present invention bretschneideri Rehd cv.‘Dangshansuli’).Pears transporter gene PbrALMT9 provided by the invention is in SEQ It is the code area of gene at 73~1882bp of sequence shown in ID No.1.
The pears transporter gene PbrALMT9 is prepared by the following in the present invention：It is carried from Dangshan pear pulp Take the RNA for obtaining Dangshan pear；The RNA reverse transcriptions are obtained into cDNA；Using the cDNA as template, with forward primer F1 and instead The pears transporter gene PbrALMT9 is obtained to primer R1 amplifications.
The extraction of the RNA of the Dangshan pear uses the plant tissue RNA extraction method of this field routine in the present invention , without other particular determinations, Plant Total RNA Isolation Kit are used in specific implementation process of the present invention Plus (being purchased from Fu Ji Bioisystech Co., Ltd, Chengdu) kit carries out the RAN extractions of Dangshan pear pulp, according to kit Specification operates.
For the present invention after obtaining Dangshan pear RNA, reverse transcription obtains cDNA；The reverse transcription in the present invention uses ability The method of domain routine, the specific TransScript One-Step gDNA Removal and with reference to Quan Shi King Companies CDNA Synthesis SuperMix reverse transcription reagent box specifications carry out reverse transcription operation.
The present invention is after obtaining the cNDA, using the cDNA of acquisition as template, is expanded with forward primer F1 and reverse primer R1 Increase and obtains the pears transporter gene PbrALMT9.The sequence such as SEQ ID of the forward primer F1 in the present invention Shown in No.3；The sequence of the reverse primer R1 is as shown in SEQ ID No.4.In specific implementation process of the present invention, the expansion Increasing system is preferably 50 μ l systems, and the amplification system includes 100ng template DNAs, 5 × Q5 reaction buffers (Q5 Reaction Buffer), 10mM dNTPs, 1 U Q5 exo+ polymerases (Q5 High-Fidelity DNA Polymerase), 1.0 μM of forward primers and 1.0 μM of reverse primer.Heretofore described 5 × Q5 buffer solutions and Q5 high are protected True polymerization enzyme is preferably purchased from New England Biolabs companies.The program of heretofore described amplified reaction is preferably： 98 DEG C, 30s；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 60s, 35 cycles；After the completion of cycle, 72 DEG C of extensions 5min.Heretofore described amplified reaction is preferably completed in Roche480 (Applied Biosystem) amplification instrument.
For the present invention after amplification obtains the pears transporter gene PbrALMT9, the product that preferred amplification obtains carries out fine jade Sequencing obtains the nucleotide sequence of the pears transporter gene PbrALMT9 after sepharose electrophoresis, recycling.
The present invention also provides the protein of pears transporter gene PbrALMT9 codings；The amino acid of the protein For sequence as shown in SEQ ID No.2, the protein of the pears transporter gene PbrALMT9 codings includes 1809 open reading The molecular weight of frame, 602 amino acid, isoelectric point 7.66, prediction is 67.59kDa.Heretofore described albumen is that film positions egg In vain, the content of pulp malic acid can be improved.
The present invention also provides applications of the pears transporter gene PbrALMT9 in improving pulp malic acid content.
The pulp is preferably pear flesh or tomato pulp in the present invention.
In the present invention, when the pears transporter gene PbrALMT9 is applied to improve malic acid content in tomato pulp When, preferably include the following steps：1) it using pear flesh cDNA as template, carries out amplification and obtains pears transporter gene PbrALMT9； 2) the pears transporter gene PbrALMT9 is connect with expression vector and obtains recombinant expression carrier；3) recombinant expression is carried Body, which is transferred in Agrobacterium tumefaciems, obtains recombination Agrobacterium tumefaciems；4) the recombination Agrobacterium tumefaciems tomato is infected to be overexpressed The tomato of pears transporter gene PbrALMT9.
In the present invention, the amplification described in step 1) obtains the specific method and step ginseng of pears transporter gene PbrALMT9 See the method for above-mentioned acquisition pears transporter gene PbrALMT9, details are not described herein.
The present invention is after obtaining the pears transporter gene PbrALMT9, by the pears transporter gene PbrALMT9 and enzyme It cuts expression vector connection and obtains recombinant expression carrier.The expression vector is preferably pBI121 carriers in the present invention.
In specific implementation process of the present invention, the construction method of the recombinant vector preferably includes following steps：
Secondary PCR amplification is carried out by template of the pears transporter gene PbrALMT9；
The product that the secondary PCR expands is connect with cloning vector, obtains recombinant cloning vector；
The recombinant cloning vector is subjected to double digestion, obtained endonuclease bamhi is connect with digestion expression vector, is obtained Recombinant expression carrier.
The primer of the secondary PCR amplification includes preferably forward primer F4 and reverse primer R4 in the present invention；It is described The sequence of forward primer F4 is as shown in SEQ ID No.9；The sequence of the reverse primer F4 is as shown in SEQ ID No.10；This hair The system and program of the amplification of secondary PCR described in bright obtain the system and program of pears transporter gene PbrALMT9 with aforementioned amplification Unanimously.
The product that secondary PCR amplification obtains connect with cloning vector and obtains after secondary PCR expands by the present invention Obtain recombinant cloning vector.The cloning vector is preferably pEASY-Blunt Zero carriers in the present invention；In the present invention The method and parameter that the secondary PCR amplified production is connect with cloning vector adopt with the conventional methods in the field with parameter, Without other particular/special requirements；In specific implementation process of the present invention, Quan Shi King Companies pEASY-Blunt Zero are can refer to Cloning Kit kit specifications are operated.
The present invention carries out the digestion piece that obtains after double digestion after obtaining recombinant cloning vector, by the recombinant cloning vector Section is connect with digestion expression vector obtains recombinant expression carrier.In the present invention, the restriction endonuclease used in the double digestion is preferred For XbaI and KpnI；The temperature of the double digestion is preferably 37 DEG C, and the time of the double digestion is preferably 4~5h.At this In invention, the system total volume of the double digestion is preferably 40 μ l, including 15 μ l of clone's recombinant vector, 10 × buffer solution, 5 μ l, Each 2 μ l of XbaI and KpnI, 16 μ l of distilled water.In the present invention, the digestion expression vector obtains after carrying out double digestion by expression vector , the double digestion system and parameter of the expression vector are consistent with recombinant cloning vector.In the present invention, the expression vector is preferred It is pBI121 carriers.
Heretofore described endonuclease bamhi and the total volume of digestion expression vector coupled reaction system are preferably 10 μ l, wrap Include 6 μ l of endonuclease bamhi, 2 μ l of digestion expression vector, 10 × T4 connections buffer solution, 1 μ l, 1 μ l of T4 ligases；Heretofore described company The temperature connect is preferably 16 DEG C；The time of the connection is preferably 14~16h.
The recombinant expression carrier is preferably converted coli strain by the present invention after obtaining recombinant expression carrier DH5 α, carry out whether recombinant expression carrier described in bacterium colony PCR sequence verifications builds success；The bacterium colony PCR is surveyed in the present invention The method of sequence verification uses the bacterium colony PCR sequence verification methods of this field routine.
The recombinant expression carrier is transferred in Agrobacterium tumefaciems and is recombinated after obtaining recombinant expression carrier by the present invention Agrobacterium tumefaciems；In the present invention, the method that the recombinant expression carrier is transferred to Agrobacterium tumefaciems is preferably freeze-thaw method.This hair In bright, the Agrobacterium tumefaciems is preferably Agrobacterium tumefaciems GV3101.
The recombination Agrobacterium tumefaciems is infected tomato and obtained table by the present invention after obtaining the recombination Agrobacterium tumefaciems Up to the tomato of pears transporter gene PbrALMT9.In the present invention, the recombination Agrobacterium tumefaciems infects the method for tomato and is：With The bacterium solution immersion of the recombination Agrobacterium tumefaciems is co-cultured after infecting tomato cotyledon, and screening, which obtains, is overexpressed pears transporter base Because of the tomato of PbrALMT9.The recombination Agrobacterium tumefaciems is obtained bacterium solution, the bacterium solution by the present invention after fluid enlargement culture Concentration be preferably OD600=0.3-0.8.The time that the immersion is infected in the present invention is preferably 8~10min, described It impregnates in infection processs with concussion.The amount of bacterium solution preferably can be completely soaked tomato when heretofore described immersion is infected Cotyledon.
In the present invention, the culture medium of the co-cultivation is preferably the MS culture mediums for adding zeatin and AS；The present invention Described in co-culture the additive amount of AS in culture medium and be preferably 80~120mg/L, the amount of the addition of the zeatin is preferred For 1.5~2.5mg/L.The time of heretofore described co-cultivation is preferably 1~3d, the temperature 24~26 of the co-cultivation DEG C, described co-culture is preferably light culture.
The present invention preferably carries out the tomato sieve for being overexpressed pears transporter gene PbrALMT9 after the co-cultivation Choosing.The screening in the present invention is preferred to be carried out using the solid medium of addition kanamycins and cephalosporin, Neng Gou The tomato grown in the solid medium of addition kanamycins and cephalosporin is to be overexpressed pears transporter gene PbrALMT9 Tomato.
The present invention preferably carries out the table excessively after the tomato for obtaining the overexpression pears transporter gene PbrALMT9 Culture of rootage up to pears transporter gene PbrALMT9 tomatoes and transplanting.Heretofore described culture of rootage and transplanting use ability The method of domain routine.
The present invention also provides applications of the pears transporter gene PbrALMT9 in pears crossbreeding, the applications Specially identify the acidity character of crossbreeding offspring's pulp.In the present invention, it is with the pears transporter gene PbrALMT9 Molecular labeling, by using the cDNA of crossbreeding offspring be the result of pears transporter gene PbrALMT9 described in template amplification come really Determine the pulp acidity character of crossbreeding offspring；If expanding successfully, judge that filial generation pulp acidity character is good.
With reference to embodiment to pears transporter gene PbrALMT9 provided by the invention and its in regulation and control pulp malic acid Application in content is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
1 pears transporter gene PbrALMT9 clones of embodiment and expression analysis
Extracting RNA and reverse transcription from Dangshan pear pulp, the first chain of gained cDNA is for expanding PbrALMT9 genes Overall length.Amplification gene PbrALMT9 primer pairs are：Forward primer：PbrALMT9F1：5’- ATGGCAGCAAAATTGGGTTCCTTC-3’(SEQ ID NO.3)；Reverse primer：PbrALMT9R1：5’- GGATTTCAGACAGTTGAGCAATCG-3’(SEQ ID NO.4).The amplification reaction system of 50 μ l includes 100ngTemplateDNA, 5 × Q5 reaction buffer (Q5 Reaction Buffer), 10mM dNTPs, 1 U Q5 high-fidelities Polymerase (Q5 High-Fidelity DNA Polymerase), above-mentioned buffer solution and Q5 exo+ polymerases are purchased from New England Biolabs companies, 1.0 μM of forward primers F1,1.0 μM of reverse primer R1.
PCR reactions are completed on Roche480 (Applied Biosystem) amplification instrument by following procedure：98 DEG C, 30s； 98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 60s, 35 cycles；After the completion of cycle, 72 DEG C of extension 5min.PCR amplification A single PCR band product is generated, after 1% agarose gel electrophoresis, with AxyPrepDNA gel reclaims kits (being purchased from Axygen companies, the U.S.) recycling specific band, extraction step are referred to explanation.The DNA solution of recovery purifying with PEASY-Blunt Zero carriers (being purchased from TRANS companies) are attached reaction (be referred to and illustrate to operate), connect reactant PbrALMT9 genes recovery product and the molar ratio of pEASY-Blunt Zero carriers are 4 in system：1.Connection reaction total volume is 5 μ l include the pEASY-Blunt Zero carriers of the PCR recovery products and 1 μ l of 4 μ l purifying.(20 DEG C~37 DEG C) reactions 5 of room temperature Minute, after reaction, connection product is placed in 3 minutes on ice, then by 5 μ l connection products, with heat shock method (reference《Molecule Cloning: A Laboratory Manual》The third edition, Science Press, 2002) conversion bacillus coli DH 5 alpha, in the LB containing 50mg/L kanamycins Screening positive clone in solid plate, 5 monoclonal sequencings of picking.Obtain the pears transporter gene as shown in SEQ ID No.1 The nucleotide sequence of PbrALMT9.
BLASTX analyzes the gene order and known ALMT (all documents delivered) sequence, finds to clone The amino acid sequence of PbrALMT9 genes and multiple species ALMT9 DNA homologs (including：Arabidopsis AtALMT9, tomato SlALMT9 and grape VvALMT9).It is whether related to Malic Metabolism in pear flesh for analysis PbrALMT9 genes.Determine pears The malic acid content in 6 periods during fruit dynamic development, and use real-time fluorescence quantitative PCR (forward primer：F2：5’- TCCTGCTGAGCGAAGACAAG-3’(SEQ ID NO.5)；Reverse primer：R2：5’-CCAGCTCCTTTGGTCGACTT-3’ (SEQ ID NO.6)) analyze PbrALMT9 genes corresponding period expression.Real-time fluorescence quantitative PCR is in RocheIt is carried out in 480 II PCR instruments.20 μ l real-time fluorescence quantitative PCR systems are as follows：10 μ l of SYRB fluorescent dyes (being purchased from Roche companies), 1 μ l of 300ng cDNA, each 7 μ l of 1 μ l and RNase-Free water of above-mentioned up/down trip primer；Response procedures It is as follows：95 DEG C of 10min, 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 30s, 45 cycles, after the completion of cycle, 72 DEG C of 10min.As a result as schemed Shown in 2, in S1 to the S3 periods in pear fruit growth course, the malic acid content of pear fruit pulp first drops to be risen again, and in S3 Phase reaches maximum value, then rapid to decline, and reaches minimum in S4 periods, and in S4 to S6 stages of development, pear fruit pulp In malic acid content gradually rise.In S1 periods in pear fruit growth course, the expression quantity of PbrALMT9 genes is very high, with Afterwards in S1 to S2 stages of development, the expression quantity range of decrease of PbrALMT9 genes is larger, is slightly increased again in S2 to S3 stages of development, so And drastically reduced in S3 to S4 periods, and reach minimum in S4 periods, in S4 to S6 stages of development, PbrALMT9 genes Expression quantity slightly increases again.The variation tendency one of malic acid content in the expression pattern and pulp of pears transporter gene PbrALMT9 It causes, therefore illustrates that PbrALMT9 genes and Malic Metabolism have certain correlation.
2 PbrALMT9 gene subcellular localizations of embodiment
The present embodiment studies the subcellular localization of PbrALMT9 genes using protoplasts of Arabidopsis thaliana broken by ultrasonic.Expanded using RT-PCR Increase and the entire ORF of PbrALMT9 genes (reading frame), and two digestions of XbaI and BamHI are added respectively at its amplimer both ends Site, amplimer are (forward primer：F3：5’-GCTCTAGAATGGCAGCAAAATTGGGTTCCTTC-3’(SEQ ID NO.7)；Reverse primer：R3：5’-GGGGATCCGGATTTCAGACAGTTGAGCAATCG-3’(SEQ ID NO.8)).50 μ l's Amplification system includes 100ng Template DNA, 5 × Q5 reaction buffer (Q5 Reaction Buffer), 10mM DNTPs, (above-mentioned buffer solution and Q5 high are protected 1 U Q5 exo+ polymerases (Q5 High-Fidelity DNA Polymerase) True polymerization enzyme is purchased from New England Biolabs companies), 1.0 μM of above-mentioned primers.PCR is reacted in Roche480 It is completed by following procedure on (Applied Biosystem) amplification instrument：98 DEG C, 30s；98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C extend 60s, 35 cycle；After the completion of cycle, 72 DEG C of extension 5min.Amplified production is mounted in pEASY-Blunt Zero first On carrier, to obtain a pEASY-Blunt Zero-PbrALMT9 recombinant vector.XbaI and BamHI double digestions are used simultaneously PEASY-Blunt Zero-PbrALMT9 and pUC19-35S-GFP carriers, recovery product simultaneously connects, to obtain pUC19- 35S-PbrALMT9-GFP recombinant vectors, and it is transferred to Agrobacterium GV3101.Subcellular is carried out using protoplasts of Arabidopsis thaliana broken by ultrasonic The step of positioning, is as follows：
(1) tablet is drawn, chooses monoclonal (Agrobacterium containing recombinant plasmid) in 5mlYEB culture mediums (40 μ containing kanamycins G/ml, rifampin 25mg/L), 28 DEG C of shake cultures 24-36 hours, until OD600 is about 0.6.
(2) it is 1 by volume：1000 ratios, the inoculation above-mentioned Agrobacterium bacterium solutions of 100 μ l are in 100ml YEB culture mediums (containing card That mycin 40 μ g/ml, rifampin 25mg/L) in, 28 DEG C of shake cultures 12-24 hours.
(3) a large amount of extraction purification kits of plasmid (being purchased from the Lars Wei Ge Bioisystech Co., Ltd, Beijing) is utilized to carry greatly Plasmid simultaneously purifies, and extraction step is referred to explanation.The high concentration plasmid that extraction is completed adjusts about 1 μ g/ μ l of concentration, be stored in- 80 DEG C of ultra low temperature freezers.
(4) Arabidopsis leaf that 3-4 weeks is grown under the conditions of short-day is disposably cut along main lobe arteries and veins vertical direction with blade It is cut into the strip of 1-1.5mm or so, and is immediately placed on 10ml enzymolysis liquids (containing 1.5% cellulase, 0.4% pectase, 0.1% BSA, 20mM MES, 10mM CaCl2,20mM KCl, 0.4M mannitol) in, it is digested 4-5 hours under the conditions of being then protected from light.With dry Blade residue in the nylon filter net filtration enzymolysis liquid of 75 μm of net holes collects protoplast in the round bottom sterile tube of 10ml In.
(5) room temperature, centrifuges 10 minutes (the plus/minus speed of centrifuge is 1) by 80-100 revs/min.
(6) supernatant is abandoned, W5 solution (MES containing 2mM, 154mM NaCl, 125mM CaCl2,5mM that 5ml now matches is added KCl), gently mixing.
(7) room temperature, centrifuges 3 minutes (the plus/minus speed of centrifuge is 1) by 80-100 revs/min.
(8) supernatant is abandoned, 2ml MaMg solution (mannitol containing 0.4M, 0.1%100mM MgCl2,4mM MES) is added.
(9) step (7) is repeated.
(10) supernatant is abandoned, 1ml MaMg solution suspensions precipitation is added, lightly shakes up, is protected from light and is placed in 30 minutes on ice.
(11) step (7) is repeated.
(12) supernatant is abandoned, 1ml MaMg solution suspensions precipitation is added, lightly shakes up, and protoplast is dispensed to 10ml In centrifuge tube (often 100 μ l of pipe).
(13) it is added in 20 μ l high concentrations plasmids and the protoplast of packing, and is added the 40% of isometric (120 μ l) PEG4000 is mixed well, and is placed at room temperature for 20 minutes.
(14) 3ml W5 solution is added, mixed liquor in (13) is suspended, mixing, room temperature, 80-100 revs/min, centrifugation 10 Minute (the plus/minus speed of centrifuge is 1).
(15) supernatant is abandoned, addition 1ml W5 solution suspensions precipitate, 25 DEG C, after light culture 24, use laser confocal microscope (ZEISS, LSM-780) visual report assignment of genes gene mapping situation.
The results are shown in Figure 3, wherein Fig. 3 a, GFP genes (zero load control) light field (a figure left side 1), chloroplaset (a figure left side 2), Imaging under details in a play not acted out on stage, but told through dialogues (a figure left side 3), right figure are the imaging after three's superposition；Fig. 3 b, gene PbrALMT9 are in light field (a figure left side 1), leaf Imaging under green body (a figure left side 2), details in a play not acted out on stage, but told through dialogues (a figure left side 3), right figure is the imaging after three's superposition.
From the figure 3, it may be seen that have fluorescence (Fig. 3 a) in the entire cell of unloaded control vector, and in the cell of recombinant vector conversion Fluorescence (Fig. 3 b) can only be detected on tonoplast, illustrate that the albumen of pears transporter gene PbrALMT9 codings is one and is positioned at Albumen on tonoplast.
Applications of the 3 pears transporter gene PbrALMT9 of embodiment in regulating and controlling tomato pulp malic acid content
Plant Transformation overexpression vector is built
It is analyzed according to the restriction enzyme site on the coding region sequence of the multiple cloning sites of pBI121 carriers and PbrALMT9 genes, Select XbaI and KpnI as restriction endonuclease.By PCR amplification is carried out using the clone of PbrALMT9 genes as template, (forward direction is drawn first Object：F4：5’-GCTCTAGAATGGCAGCAAAATTGGGTTCCTTC-3’(SEQ ID NO.9)；Reverse primer：R4：5’- GGGGTACCGGATTTCAGACAGTTGAGCAATCG-3 ' (SEQ ID NO.10)), then PCR product is connected to pEASY- On BluntZero carriers, to one recombinant vector pEASY-Blunt Zero-PbrALMT9 of structure.By above-mentioned recombinant vector Double digestion is carried out at 37 DEG C, double digestion system total volume is 40 μ l, wherein containing pEASY-Blunt Zero-PbrALMT9 matter 15 μ l of grain, 10 × buffer solution, 5 μ l, XbaI and each 2 μ l of KpnI, 16 μ l of distilled water, digestion purify recycling after 4~5 hours.pBI121 Carrier double digestion system is same as above.Coupled reaction system total volume is 10 μ l, wherein PbrALMT9 genes and carrier pBI121 volumes 1 μ l of respectively 6 μ l and 2 μ l, 10 × T4 connection buffer solution, 1 μ l of T4 ligases, 16 DEG C connect 14-16 hours.Connection product converts Coli strain DH5 α are screened on the LB solid plates containing 100mg/L kanamycins, are chosen monoclonal and are carried out PCR detections, PCR detection architectures are 20 μ l, including PCRMasterMix (2x) (being purchased from Thermo companies) 10 μ l, primer (forward primer：F4： 5’-GCTCTAGAATGGCAGCAAAATTGGGTTCCTTC-3’(SEQ ID NO.9)；Reverse primer：R4：5’- GGGGTACCGGATTTCAGACAGTTGAGCAATCG-3 ' (SEQ ID NO.10)) each 1 μ l, bacterium solution template 1 μ l, 7 μ of sterile water l；Response procedures are 95 DEG C, 3min, 95 DEG C and are denaturalized 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 90s, and 35 recycle, after the completion of cycle 72 DEG C of extension 10min.The monoclonal extracting plasmid being positive carries out double digestion and is sequenced, with AxyPrep plasmid extraction kits (being purchased from Axygen companies, the U.S.) extracting plasmid, operating procedure is according to kit operation instructions.Sequencing determines reading frame base After mutation, that is, indicate that pBI121-PbrALMT9 overexpression vectors are built successfully.Carrier construction process schematic is shown in Fig. 4.Using PBI121-PbrALMT9 recombinant vectors are imported into Agrobacterium tumefaciems GV3101 and are preserved by freeze-thaw method.
The positive identification of tomato genetic transformation and Transgenic Tomato Plants
The genetic transformation process of Agrobacterium tumefaciens mediated tomato is as shown in figure 5, be as follows：
A tomato seeds) are sowed：First with 70% alcohol treatment tomato seeds 30 seconds, then with sterile water washing 3 times, then use 2.5% sodium hypochlorite is handled 5 minutes, and finally with sterile water washing 4 times, seed is placed on germination medium.
B) preculture：The tomato seedling after being sowed 7-10 days on germination medium is taken, cotyledon is cut and surrounding scratches, is cut Good cotyledon is placed on precultivation medium (cotyledon is face-up), 25 DEG C of light cultures 1 day.C) Agrobacterium tumefaciems is cultivated：It takes super The Agrobacterium tumefaciems bacterium solution preserved in low temperature refrigerator, it is flat in the LB solids containing 50mg/L kanamycins and 100mg/L rifampins Lining out.
D) the preparation of bacterium solution：The scribing line bacterium colony in step 3 is scraped, is added in MS fluid nutrient mediums, 28 DEG C, 200 revs/min Shaken cultivation waits for bacterial concentration OD600It can then be infected when between 0.5-0.8.
E it) infects：Cotyledon after preculture is placed in the Agrobacterium tumefaciems bacterium solution in step 4, immersion infects 8-10 points During which clock constantly shakes.
F it) co-cultures：Tomato cotyledon after infecting, blots the bacterium solution on surface on aseptic filter paper, is then inoculated in total training It supports on base (cotyledon is face-up), 25 DEG C of light cultures 2 days.
G) screening and culturing：Tomato cotyledon after co-culturing 2 days, is washed one time with the cephalosporin solution containing 500mg/L, Then sterile water wash 3-5 times is then transferred to the solid culture for being added to 100mg/L kanamycins and 500mg/L cephalosporins Callus growth is carried out on base.
H) culture of rootage：When adventitious bud on culture medium to be screened grows to 1cm or so, cuts and be transferred to and be added to 100mg/ On the root media of L kanamycins and 500mg/L cephalosporins.
I) tomato seedling transplanting is buried：After transformation seedlings after under growth root cover with culture bottle, hardening of uncapping 3 days, then from taking root Culture medium takes out, and is transplanted to after sterilizing in Nutrition Soil.
J) the Molecular Identification of transgene tomato positive seedling：The tomato resistant plant that will be obtained takes appropriate blade extraction DNA, With plant genome DNA extracts kit (being purchased from Tiangeng biochemical technology Co., Ltd, Beijing) extraction.Operating procedure is with reference to reagent Box specification.Then using the DNA of said extracted as template, the positive seedling (Fig. 6 a) of PCR amplification identification is carried out with specific primer.Mirror It is set to possible positive plant and independently harvests seed (T1Generation).The seed of harvest is sowed again and identifies positive seedling (T2Generation) (figure 6b).Harvest T2For sample of the fruit as follow-up test of positive transgenic tomato.The tomato fruit of wild type (WT) is collected simultaneously It is implemented as Sample storage.After full-bloom stage, a sample was adopted every 10 days, the maturation until fruit reddens.
Tomato genetic transformation used medium is shown in Table 1
Solid culture based formulas used in 1 tomato genetic transformation of table
4 Semiquatitative RT-PCR assay of embodiment detects the overexpression of PbrALMT9 genes
1. the RNA of conversion tomato and wild-type tomato fruit that embodiment 3 obtains is extracted, using Trizol kits, Steps are as follows：
(1) mortar is pre-chilled：Mortar is impregnated 10 minutes with 0.5M NaOH, then is washed 3-4 times with sterilizing DEPC, and liquid feeding nitrogen is pre- It is cold；
(2) 1g fruits are weighed in the mortar of precooling, after liquid nitrogen grinding, add 1mlTrizol mixings.
It (3) 4 DEG C, 12000 revs/min, centrifuges 15 minutes, takes supernatant, add 200 μ l chloroforms, firmly shake 15 seconds, be incubated 2 points Clock, 12000 revs/min, centrifuges 15 minutes by 4 DEG C.
(4) supernatant is taken, 500 μ l isopropanols are added, is incubated 10 minutes.It 4 DEG C, 12000 revs/min, centrifuges 10 minutes.
(5) supernatant is abandoned, the alcohol (being matched with sterilizing DEPC water) of 1ml75%, jog are added.4 DEG C, 7200 revs/min, centrifugation 5 Minute.
(6) supernatant is abandoned, is air-dried, precipitation 30-50 μ lDEPC water dissolutions take the concentration and quality of 1-2 μ l detections RNA.-80 DEG C preserve.
2. by RNA reverse transcriptions at cDNA, transgene tomato exogenous gene expression amount uses Semi quantitative PCR analysis, used to draw Object is PbrALMT9 gene specific primer (forward primers：F2：5’-TCCTGCTGAGCGAAGACAAG-3’(SEQ ID NO.5)； Reverse primer：R2：5 '-CCAGCTCCTTTGGTCGACTT-3 ' (SEQ ID NO.6)), response procedures are 94 DEG C, 3 minutes；94 DEG C denaturation 30 seconds, 60 DEG C anneal 45 seconds, 72 DEG C extend 45 seconds, 30 cycle；72 DEG C, extend 10 minutes.Internal reference base is done with Actin Cause, primer sequence are：Forward primer Actin-F：5'-CAATGTGCCTGCCATGTATG-3’(SEQ ID NO：11)'；Reversely Primer Actin-R：5'-CCAGCAGCTTCCATTCCAAT-3'(SEQ ID NO：12).
The result shows that in 8 T filtered out2For PbrALMT9 genes in transgenic line expression than wild Type is high, and especially No. OE7-2-6 is remarkably reinforced (Fig. 7) with No. OE14-1-4 two expressions for being, therefore, selects the two System is used for further physiological function evaluation.
The physiological function evaluation of 5 Transgenic tomato fruit of embodiment
Using real time fluorescent quantitative method, during OE7-2-6, OE14-1-4 and WT tamato fruit dynamic development The expression pattern of PbrALMT9 genes is analyzed (Fig. 8), while measuring the content of malic acid in each corresponding period tamato fruit (Fig. 9).The result shows that during tamato fruit dynamic development, PbrALMT9 genes that OE7-2-6 and OE14-1-4 two are Expression be all significantly higher than WT, while two overexpressions are that malic acid content is also apparently higher than wild type in tamato fruit, And the expression pattern of PbrALMT9 genes is consistent with the variation tendency of malic acid content.As it can be seen that overexpression PbrALMT9 genes It can promote the accumulation of malic acid in fruit.
In order to further prove the relationship of PbrALMT9 genes and the accumulation of malic acid in fruit.The present embodiment determines kind During solanberry reality dynamic development, with the relevant 3 kinds of enzymes of Malic Metabolism (including malic dehydrogenase (malic Dehydrogenase, MDH), phosphoric acid enol pyruvic acid carboxylase (Phosphoenolpyruvate carboxylase, PEPC) and malate dehydrogenase (malic enzyme, ME)) enzyme activity (Figure 10), and to encode above three enzyme key gene (NAD-MDH, PEPC and NAD-ME) has carried out expression pattern analysis (Figure 11).The results show that although in Transgenic tomato fruit In, the activity of MDH and PEPC are higher than wild type, and the activity of ME is lower than wild type；But correspond to the expression mould of encoding gene in conjunction with it Formula is it is found that during tamato fruit dynamic development, and the activity of tri- kinds of enzymes of MDH, PEPC and ME is not by respective encoding gene Regulated and controled.
In summary conclusion is it is found that during Transgenic tomato fruit dynamic development, NAD-MDH, PEPC and NAD-ME These three genes have no correlation with apple acid accumulation in tamato fruit, can promote to demonstrate overexpression PbrALMT9 genes Into the conclusion of the accumulation of malic acid in fruit.
By above-described embodiment it is found that pears transporter gene PbrALMT9 provided by the invention, has and improve fruit malic acid The function of content can be applied to improve pulp malic acid content.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Agricultural University Of Nanjing
<120>The research and application of transporter gene PbrALMT9 regulation and control pear flesh malic acid contents
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tgggaagtac gtgcagtcag ggctgcgtgt ttctgcattc gaggctaaaa gctatttttc 1680
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Gly Phe Pro Asn Ile Glu Glu Gln Glu Pro Ser Ser Gly Ser Thr Arg
35 40 45
Cys Cys Thr Phe Arg Ser Val Ser Asp Arg Ile Val Gly Trp Cys Arg
50 55 60
Thr Val Gln His Val Trp Lys Arg Ala Val Arg Met Gly Gln Ser Asp
65 70 75 80
Pro Arg Lys Ile Ile Phe Ala Ala Lys Met Gly Leu Ala Leu Met Ile
85 90 95
Ile Ser Leu Leu Ile Phe Leu Lys Glu Pro Phe Lys Gln Leu Ser Arg
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Tyr Ser Val Trp Ala Ile Leu Thr Val Val Val Val Phe Glu Phe Ser
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Ile Gly Ala Thr Leu Ser Lys Gly Phe Asn Arg Gly Leu Gly Thr Phe
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Ser Ala Gly Gly Leu Ala Leu Val Val Ala Asn Leu Cys Gly Leu Ala
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Gly Glu Trp Glu Glu Val Val Ile Phe Ala Ser Ile Phe Ile Val Gly
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Phe Leu Ala Thr Tyr Ala Lys Leu Tyr Pro Thr Met Lys Pro Tyr Glu
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Tyr Gly Phe Arg Val Phe Leu Leu Thr Phe Cys Phe Ile Met Val Ser
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Gly Tyr Arg Thr Arg Glu Phe Val His Thr Ala Val Ser Arg Phe Phe
210 215 220
Leu Ile Ala Leu Gly Ala Gly Val Gly Leu Gly Val Asn Ile Cys Ile
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Phe Pro Ile Trp Ala Gly Glu Asp Leu His Asn Leu Val Val Lys Asn
245 250 255
Phe Val Gly Val Ala Lys Ser Leu Glu Gly Val Val Asn Ser Tyr Leu
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Asn Cys Val Glu Tyr Glu Arg Val Pro Ser Lys Ile Leu Thr Tyr Gln
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Ala Ser Asp Asp Pro Leu Tyr Ser Gly Tyr Arg Ser Ala Val Glu Ser
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Thr Ser Gln Glu Asp Thr Leu Met Gly Phe Ala Ile Trp Glu Pro Pro
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His Gly Arg Tyr Arg Met Leu Arg Tyr Pro Trp Lys Asn Tyr Gly Lys
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Val Gly Gly Ala Leu Arg His Cys Ala Phe Met Val Met Ala Leu His
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Gly Cys Ile Leu Ser Glu Ile Gln Ala Pro Ala Glu Arg Arg Gln Val
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Phe Cys Arg Glu Leu Gln Arg Val Gly Tyr Glu Gly Ala Lys Val Leu
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Arg Glu Leu Gly Asn Lys Leu Lys Thr Met Glu Lys Ile Gly Pro Ile
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Asp Ile Leu Asn Glu Val His Glu Ala Ala Glu Asp Leu Gln Lys Lys
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Gly Ser Arg Pro Lys Glu Leu Gly Glu Pro Gln Asp Leu Leu Asn Val
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Asp Asp Glu Glu Asn Tyr Phe Arg Glu Tyr Lys Ser Leu Ser Glu Ala
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Val Leu Asp Leu Ser Ser Phe Pro Val Pro Gln Ser Trp Asp Ser Gln
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Met Pro Ala Lys Pro Asn Gly Val Gly Ser Val Pro Thr Glu Val Asn
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His Ser Asn Leu Pro Ala Gly Val Ser Ser Gly Ser Met Phe Met Lys
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Gln Ile Ser Trp Pro Ala Gly Leu Lys Phe Lys Ala Asp Glu Pro Pro
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Ala Thr Phe Thr Ser Leu Leu Ile Glu Phe Val Ala Arg Leu Gln Asn
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Claims (9)

1. a pears transporter gene PbrALMT9, which is characterized in that the sequence such as SEQ of the transporter gene PbrALMT9 Shown in ID No.1.
2. the protein of pears transporter gene PbrALMT9 codings described in claim 1, which is characterized in that the protein Amino acid sequence is as shown in Seq IDNo.2.
3. protein described in pears transporter gene PbrALMT9 described in claim 1 or claim 2 is improving pulp apple Application in acid content.
4. application according to claim 3, which is characterized in that the pulp is pear flesh or tomato pulp.
5. application according to claim 4, which is characterized in that when the pulp is tomato pulp, the application is structure The tomato for being overexpressed pears transporter gene PbrALMT9 is built, is included the following steps：
1) pears transporter gene PbrALMT9 described in claim 1 is provided；
2) the pears transporter gene PbrALMT9 is connect with carrier and obtains recombinant vector；
3) recombinant vector is transferred in Agrobacterium tumefaciems and obtains recombination Agrobacterium tumefaciems；
4) the recombination Agrobacterium tumefaciems is infected into tomato, obtains the tomato for being overexpressed pears transporter gene PbrALMT9.
6. application according to claim 5, which is characterized in that step 1) is：Using pear flesh cDNA as template, PCR is carried out Amplification obtains pears transporter gene PbrALMT9；The amplification primers are to including forward primer F1 and reverse primer R1；It is described The sequence of forward primer F1 is as shown in SEQ ID No.3；The sequence of the reverse primer R1 is as shown in SEQ ID No.4.
7. applications of the pears transporter gene PbrALMT9 described in claim 1 in pears crossbreeding.
8. applying according to claim 7, which is characterized in that the application is to identify the acidity of crossbreeding offspring's pulp Shape.
9. application according to claim 8, which is characterized in that the identification includes：CDNA with crossbreeding offspring is Pears transporter gene PbrALMT9 described in template amplification；It is good to expand successfully judgement filial generation pulp acidity character.
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Application publication date: 20180821