CN105524928A - Peach PpeAMT1; 1 gene, transporter protein, expression vector and construction method of transporter protein - Google Patents

Abstract

The invention discloses a peach PpeAMT1; 1 gene, wherein the base sequence is as shown in SEQ ID NO: 1. The invention also discloses transporter protein encoded by the peach PpeAMT1; 1 gene, an expression vector pYES2-PpeAMT1; 1 and a construction method of the expression vector pYES2-PpeAMT1; 1. Through homologous cloning, a 1515bp fragment is cloned from a peach fruit; the fragment enjoys a relatively high homology with the MAT1 family members of other crops, and the amino acid sequence has one typical ammonium transporter functional domain which includes 12 transmembrane regions, so that the fragment is inferred to be a novel peach ammonium transporter PpeAMT1; 1 gene. The gene achieves the maximum expression level in the stages of young fruit formation and initial fruit development, and the expression level is significantly higher than that of annual leaf, phloem, flower and root while the expression level in the stage of fruit ripening is relatively low. The gene, which has the capacity of absorbing external NH4+, is inferred to take an important role in NH4+ dynamic equilibrium in the initial peach fruit development stage.

Description

Peach PpeAMT1; 1 gene, translocator, expression vector and construction process thereof

Technical field

The present invention relates to peach PpeAMT1; 1 gene, translocator, expression vector and construction process thereof, belong to molecular biology and biological technical field.

Background technology

Ammonium ion (NH ₄ ⁺) be topmost inorganic nitrogen existence form, be the nitrogen nutrition of plant preferential absorption, closely related with the growth of plant, growth and fruit quality.Ammonium is fertile to play an important role in physiology such as promotion fruit quality and output etc., uses the interior quality that appropriate ammonium fertilizer not only can strengthen fruit significantly, also can improve the exterior quality of fruit.But relevant report mainly concentrates on Physiology and biochemistry aspect, about fruit quality, output and NH ₄ ⁺absorption, transhipment and metabolism thereof the report of molecule mechanism fresh few.

Plant is to NH ₄ ⁺absorption be rely on the AMT transporter be positioned on cytoplasmic membrane realized, be divided into AMT1 and AMT2 two gene subtribes.Ninnemann etc. clone and identify first plant AMT1 member gene from Arabidopis thaliana, up to now, the report about such gene structure and function mainly concentrates on annual herb plant Arabidopsis thaliana, tomato, Root or stem of Littleleaf Indianmulberry, rape and paddy rice etc. for research material.Report display AMT1 gene mainly shows high expression level at the root of yearly plant body, leading root NH ₄ ⁺absorption (Preferentialexpressionofanammoniumtransporterandoftwoput ativenitratetransportersinroothairsoftomato, LauterFR, NinnemannO, BucherM, RiesmeierJW, FrommerWB, Proc.Natl.Acad.Sci.USA; Threefunctionaltransportersforconstitutive, diurnallyregulated, andstarvation-induceduptakeofammoniumintoArabidopsisroot s, GazzarriniS, LejayL, GojonA, NinnemannO, FrommerWB, PlantCell; Differentialregulationofthreefunctionalammoniumtransport ergenesbynitrogeninroothairsandbylightinleavesoftomato, vonWir é nN, LauterFR, NinnemannO, GillissenB, Walch-LiuP, EngelsC, JostW, FormmerWB, PlantJournal; Functionalcharacterizationofanammoniumtransportergenefro mLotusjaponicas, SalveminiF, MariniAM, RiccioA, PatriarcaEJ, ChiurazziM, Gene; Distinctexpressionandfunctionofthreeammoniumtransporterg enes (OsAMT1; 1 – 1; 3) inrice, SonodaY, IkedaA, SaikiS, vonWir é nN, YamayaT, YamaguchiJ, PlantCellPhysiol), or main at leaf expression, transhipment (Functionalcharacterizationofanammoniumtransportergenefro mLotusjaponicas, SalveminiF, the MariniAM of leading overground part, RiccioA, PatriarcaEJ, ChiurazziM, Gene; Characterizationofthreefunctionalhigh-affinityammoniumtr ansportersinLotusjaponicuswithdifferentialtranscriptiona lregulationandspatialexpression, D'ApuzzoE, RogatoA, Simon-RosinU, ElAlaouiH, BarbulovaA, BettiM, DimouM, KatinakisP, MarquezA, MariniAM, UdvardiMK, ChiurazziM, PlantPhysiology).But the function of plant AMT1 family gene member is not yet completely clear, AMT family gene is unknown especially at the concrete function of fruit development and quality responses and regulatory mechanism thereof.Because xylophyta has the characteristic of perennation, there is the distinct Nitrogen Absorption transport with herbaceous plant, their AMT homologous gene control methods and physiological function also difference to some extent, therefore, is separated and identifies that new AMT homologous gene is significant from xylophyta fruit.

Summary of the invention

The present invention makes public for the first time isolated ammonium transporter PpeAMT1 from Peach fruits; The full-length cDNA of 1 gene, and the Yeast expression carrier construction process that a kind of high expression peach AMT gene is provided.

The invention discloses a kind of peach PpeAMT1; 1 gene, its base sequence is as shown in SEQIDNO:1.

The invention also discloses above-mentioned peach PpeAMT1; The translocator of 1 genes encoding, its sequence is as shown in SEQIDNO:2.

The invention also discloses above-mentioned peach PpeAMT1; 1 expression vector pYES2-PpeAMT1; 1, it is characterized in that carrier is pYES2 plasmid, gene is the gene shown in SEQIDNO:1.

The invention also discloses above-mentioned peach PpeAMT1; 1 expression vector PpeAMT1; The construction process of 1, its step comprises:

A, by PCR method amplifying target genes fragment, and reclaim PCR primer, wherein PpeAMT1; The base sequence of 1 gene 5 ' end primer as shown in SEQIDNO:3, PpeAMT1; 1 gene 3 ' holds the base sequence of primer as shown in SEQIDNO:4;

B, respectively use KpnI and NotI double digestion PCR reclaim product and pYES2 plasmid, then connect with T4DNAligase, obtain expression vector pYES2-PpeAMT1; 1.

The present invention clones the fragment obtaining a 1515bp from peach by homologous clone method, BLAST display has higher homology with the AMT1 family member of other crops, aminoacid sequence has 1 typical ammoniumtransporter functional domain, comprise 12 cross-film districts, deducibility its be a new peach ammonium transporter PpeAMT1; 1 gene.Real-time quantitative PCR result shows, this gene is the highest at the expression level of young fruit formation and early stage of fruit development, be significantly higher than the expression level of annual leaf, phloem, flower and root, and lower at the expression level in fruit maturation period, infer that it mainly works in Fruit Development Process.In order to understand AMT1 further; 1 gene function, is connected on Yeast expression carrier pYES2, is built into recombinant plasmid pYES2-PpeAMT1; 1, and transformed yeast bacterium 31019b mutant (MATamepl △ mep2 △:: LEU2mep3 △:: KanMX2ura3, absorbs and transhipment NH owing to having lacked ₄ ⁺site, at extraneous NH ₄ ⁺as only nitrogen source and concentration lower than 5mmolL ^-1shi Buneng grows).Found that, this recombinant plasmid can recover the growth of yeast mutants, shows PpeAMT1; 1 gene has the extraneous NH of absorption ₄ ⁺ability, for inquire into further AMT1 family gene Peach fruits grow in NH ₄ ⁺absorption, transhipment and regulatory mechanism provide convenient and may.

Accompanying drawing explanation

Fig. 1 is the AMT1 of peach in embodiment 1, paddy rice, Arabidopis thaliana, tomato and Root or stem of Littleleaf Indianmulberry; 1 Argine Monohydrochloride sequence analysis figure.Caption: PpeAMT1; 1: peach AMT1; 1; OsAMT1; 1: paddy rice AMT1; 1; AtAMT1; 1: Arabidopis thaliana AMT1; 1; LeAMT1; 1: tomato AMT1; 1; LjAMT1; 1: Root or stem of Littleleaf Indianmulberry AMT1; 1; Consensus: Amino acid sequence identity.

Fig. 2 is peach PpeAMT1 in embodiment 2; 1 gene expresses figure at the real-time fluorescence quantitative RT-PCR of different tissues or organ.Caption: S1, developmental stage 1 (fruit Rapid development phase first time); S2, developmental stage 2 (stone phase); S3, developmental stage 3 (fruit expanding period); S4, developmental stage 4 (fructescence).

Fig. 3 is pYES2-PpeAMT1; The construction process schematic diagram of 1 expression vector.

Fig. 4 is pYES2-PpeAMT1 in embodiment 3; The digestion verification figure of 1 recombinant expression vector.

Fig. 5 is the growth conditions figure of yeast under the condition of different N source in embodiment 4.

Embodiment

The invention will be further described below.

Embodiment 1: the cloning process of Peach fruits gene

1, the extraction of fruit total serum IgE:

(1) getting 1mLCTAB extracting solution is dispensed in 2mL centrifuge tube, 65 DEG C of preheatings;

(2) take 0.5g Peach fruits material, proceed in the centrifuge tube containing CTAB extracting solution in liquid nitrogen after grinding immediately, vortex mixed, 65 DEG C of temperature bath 5min;

(3) isopyknic chloroform/primary isoamyl alcohol (24:1) is added immediately, vortex mixed, room temperature, the centrifugal 10min of 12000rpm;

(4) supernatant is drawn in a new centrifuge tube, repeating step 3;

(5) get supernatant in a new centrifuge tube, add the 8MLiCl of 1/3 volume, 4 DEG C of precipitates overnight;

(6) 4 DEG C, the centrifugal 20min of 12000rpm;

(7) abandon supernatant, precipitation uses 70% washing with alcohol, then uses absolute ethanol washing;

(8) by resolution of precipitate in 100 μ LSTE (65 DEG C of preheatings), use chloroform/isoamyl alcohol extraction immediately once;

(9) get supernatant, add the dehydrated alcohol of 2 times of volumes ,-20 DEG C of precipitation 2h;

(10) 4 DEG C, the centrifugal 15min of 12000rpm;

(11) precipitation uses 70% ethanol and absolute ethanol washing once respectively, by 20 μ LRNase-free water dissolution after dry, preserves for a long time in-70 DEG C of refrigerators

2, peach PpeAMT1; The Cloning and sequence analysis of 1 gene

(1) PrimeScript of TaKaRa company is utilized ^tMthe reverse transcription of fruit total serum IgE is the first chain cDNA by RTreagentKit Reverse Transcription box, as pcr template.

(2) peach PpeAMT1 is designed; 1 full length gene CDS amplimer:

PpeAMT1；1-FP1:5’-ATGGCGACGTGGGCTACCTTAGA-3’(SEQIDNO：5)

PpeAMT1；1-RP1:5’-CTAAACGGAAGTGGGTGTGGAAG-3’(SEQIDNO：6)

(3) PCR reaction system is:

Response procedures is: 95 DEG C of denaturations, 3min; 95 DEG C of sex change, 30S, 59 DEG C of annealing, 30S, 72 DEG C of extensions, 2min, 35 circulations; Last 72 DEG C, extend 10min, 4 DEG C of insulations.

The CDS sequence obtaining total length is 1515bp, comprises initiator codon ATG to terminator codon TAG, 505 amino acid of encoding.This aminoacid sequence is compared in the Genebank of NCBI, shows the AMT1 with the Arabidopis thaliana delivered, tomato, Root or stem of Littleleaf Indianmulberry and paddy rice; The amino acid identity of 1 albumen is respectively 80%, 79%, 83% and 69%, as shown in Figure 1.

Embodiment 2: peach PpeAMT1; 1 gene is in the real-time fluorescence quantitative RT-PCR detection of expression of different tissues or organ:

Extract the annual blade of peach, phloem, root, peach blossom and Peach fruits and grow different development stage RNA, utilize real-time fluorescence quantitative PCR to measure PpeAMT1; 1 gene, at the expression level of different tissues or organ and fruit different development stage, utilizes SYBRPremixExTaq (TaKaRa) fluorescence dye to enter real-time fluorescence quantitative PCR analysis in the cDNA template of ABI7500 real-time fluorescence quantitative PCR instrument to different tissues or organ and fruit different development stage.

PpeAMT1；1-FP2：5’-GTCGTCTCCCACTGGTTCTG-3’(SEQIDNO：7)，

PpeAMT1；1-RP2：5’-GAACGTGCCCAAGACTACCA-3’(SEQIDNO：8)。

PCR reaction system is:

Response procedures is: 95 DEG C of denaturations, 30sec; 95 DEG C of sex change, 5sec, 59 DEG C of annealing, 34sec, 40 circulations; Last 72 DEG C, extend 30sec.

Fig. 3 shows, PpeAMT1; 1 gene young fruit formed and the expression level of early stage of fruit development the highest, be significantly higher than the expression level of annual leaf, phloem, flower and root, and lower at the expression level in fruit maturation period.This illustrates, PpeAMT1; 1 gene may participate in the process that early stage of fruit development ammonium ion absorbs and transports.

Embodiment 3: peach PpeAMT1; 1 gene yeast expression vector pYES2-AMT1; The structure of 1

1, according to isolated peach PpeAMT1; The nucleotide sequence of 1 gene, design primer:

PpeAMT1; 1-FP3:5 '-GAGAGGTACCATGGCGACGTGGGCTACCTTAGA-3 ' (SEQIDNO:3) (introducing KpnI restriction enzyme site),

PpeAMT1; 1-RP3:5 '-GAGAGCGGCCGCCTAAACGGAAGTGGGTGTGG-3 ' (SEQIDNO:4) (introducing NotI restriction enzyme site).

With the first chain cDNA of total serum IgE reverse transcription for template, carry out polymerase chain reaction.

2, get 5 μ LPCR products to be connected with pMD18-T carrier, operation steps is undertaken by TakaRa Products pMD18-TVectorsystem specification sheets.Then transformation of E. coli DH5 α bacterial strain, the LB grow on plates containing penbritin (100 μ g/ μ L) being coated with isopropylthio-β-D-galactoside (IPTG) and the bromo-4 chloro-3-indoles-β-D-galactoside (X-gal) of 5-on surface spends the night.Picking white colony, overnight incubation in LB liquid nutrient medium.The little extraction reagent kit of TIANGEN rapid plasmid is utilized to carry out plasmid extraction and carry out sequencing.

3, cut from pMD18-TVector carrier by this gene with KpnI and NotI two restriction enzymes, the pYES-2 carrier cut with identical KpnI and NotI enzyme connects.Connect product conversion DH5 α cell, then spend the night at the LB grow on plates containing penbritin (100 μ g/ μ L).Picking white colony, through PCR, overnight incubation in LB liquid nutrient medium, identifies that correct with plasmid DNA digestion verification is positive single bacterium colony, as shown in Figure 4.By the recombinant expression vector called after pYES2-PpeAMT1 built; 1.

Embodiment 4: peach PpeAMT1; The Function Identification of 1 gene

1, yeast conversion and cultivation

Adopt electric conversion instrument MicroPulser (Bio-Rad company) by pYES2-PpeAMT1; 1 and empty carrier pYES2 proceed to yeast ammonium transporter deletion mutant strain 31019b competent cell respectively, the 1M sorbyl alcohol of 1mL precooling is added after electric shock, 30 DEG C of degree incubation 2h, 30 DEG C of dark culturing 3d on yeast selection substratum (0.17%YNB+2mM arginine+2%D-semi-lactosi+2% agar) solid plate.Picking list bacterium colony, in liquid yeast selective medium, 36h are cultivated in 30 DEG C of concussions, identify that correct with plasmid DNA digestion verification is positive single bacterium colony through PCR.

2, yeast has complementary functions

Recombinant expression vector pYES2-PpeAMT1 will be transformed respectively; 1 and the 31019b thalline of empty carrier pYES2 be seeded in YNB fitting of fluids substratum, 30 DEG C of concussions are cultured to OD ₆₀₀to 1.0, become 10 through 10 times of gradient dilutions ^-1, 10 ^-2with 10 ^-3three concentration, the bacterium liquid 5 μ L point sample getting 4 concentration gradients is respectively in 2mM arginine or 0.02,0.2 and 2mM different concns NH ₄on the solid medium (0.17%YNB+2%D-semi-lactosi+2% agar) that Cl (pH5.8) is only nitrogen source, in 30 DEG C of dark culturing, checking 31019b mutant recovers transhipment NH ₄ ⁺growing state, checking peach PpeAMT1; 1 gene absorbs extraneous NH ₄ ⁺ability.

As shown in Figure 5, yeast ammonium transporter deletion mutant strain 31019b absorbs and transhipment NH owing to having lacked ₄ ⁺site, at extraneous NH ₄ ⁺as only nitrogen source and concentration lower than 5mmolL ^-1shi Buneng grows.In the solid medium being only nitrogen source with 2mM arginine (0.17%YNB+2%D-semi-lactosi+2mM arginine+2% agar), transform pYES2-PpeAMT1; 1 and the bacterial strain of empty carrier pYES2 can normal growth, with 0.02,0.2 or 2mMNH ₄cl is on the substratum (ammonium+2% agar of 0.17%YNB+2%D-semi-lactosi+different concns) of only nitrogen source, and the yeast transforming empty carrier pYES2 can not normal growth, and has transformed recombinant expression vector pYES2-PpeAMT1; The yeast of 1 can normal growth, and along with the continuous rising of ammonium concentration, yeast growth must be better, and peach ammonium transporter PpeAMT1 is described; 1 has absorption NH ₄ ⁺function, can NH be mediated ₄ ⁺absorption.

Claims (4)

1. a peach PpeAMT1; 1 gene, its base sequence is as shown in SEQIDNO:1.

2. a peach PpeAMT1; 1 translocator, its sequence is as shown in SEQIDNO:2.

3. a peach PpeAMT1; 1 expression vector pYES2-PpeAMT1; 1, it is characterized in that carrier is pYES2 plasmid, gene is the gene shown in SEQIDNO:1.

4. a peach PpeAMT1; 1 expression vector pYES2-PpeAMT1; The construction process of 1, its step comprises:

A, by PCR method amplifying target genes fragment, and reclaim PCR primer, wherein PpeAMT1; The base sequence of 1 gene 5 ' end primer as shown in SEQIDNO:3, PpeAMT1; 1 gene 3 ' holds the base sequence of primer as shown in SEQIDNO:4;

B, respectively use KpnI and NotI double digestion PCR reclaim product and pYES2 plasmid, then connect with T4DNAligase, obtain expression vector pYES2-PpeAMT1; 1.