CN1164751C - Phosphoethanolamine N-methyltransferase gene and its application - Google Patents

Abstract

The present invention constructs a plant expression carrier lambda 455 and an x delta NA expression library of arabidopsis constructed by the carrier. The present invention utilizes agrobacterium tumefaciens to convert arabidopsis (A rho alpha beta iota delta omicron pi delta iota sigma) to obtain a series of mutants, wherein one mutant expresses presenility, light green of the color of leaves and male sterile. The gene is a gene of encoding phosphoethanolamine N-methyltransferase (pi EAMT) by gene clone, sequence determination and transgenic analysis, and the gene inhibits the sensitivity to high-concentration salts and high-temperature environment conditions of the mutant on the expression level, thereby, the temperature sensitive male sterility is generated. The gene has very important application value on the aspects of salt resistance of plants, resistance characteristics and hybridization seed preparation by utilizing the male sterility.

Description

Phosphoethanolamine N-methyltransferase gene and application thereof

Technical field

The invention belongs to plant genetic engineering field.Furthermore, the present invention relates to the structure in plant expression vector λ 455 and just/antisense expression of plants library; Relate to and utilize this technical system clone Phosphoethanolamine N-methyltransferase (phosphoethano lamine N-methyltransferase AtPEAMT) gene; Relate to and utilize this gene to improve plant anti-salt ability and the artificial male sterile farm crop of creation.

Technical background

The mRNA content of PEAMT increases under the condition of salt stress, and enzymic activity can increase (1) more than 10 times.Show that under condition of salt stress plant needs the synthetic to satisfy plant needs to glycinebetaine (abbreviation trimethyl-glycine) under condition of salt stress of a large amount of choline.As a kind of osmoregulation material, relevant glycinebetaine has goed deep into (2-5) in the research of plant salt tolerance alkali very much.But the functional study as key enzyme PEAMT in the synthesizing betaine approach and gene PEAMT thereof is less.The present invention adopt special plant expression vector utilization just/after the antisense expression system at first obtains the mutant of this gene, utilize mutant to study the function of this gene again, with respect to the method for utilizing fission yeast to have complementary functions, the function of research plant gene that can be directly deep more, according to mutation type surface, can find the new function of this gene simultaneously.

Summary of the invention

At above-mentioned research background, the objective of the invention is to utilize a kind of new plant expression vector and just/antisense expression system in the model plant Arabidopis thaliana, to obtain mutant and clone new gene.

Another object of the present invention is by the mutant phenotype of this model plant and homogenic correlation function research PEAMT gene and the effect on plant salt tolerance alkali and plants male sterility.

Another purpose of the present invention is with PEAMT gene-transformed plant of the present invention (particularly having the farm crop that important production application is worth), cultivates saline alkali tolerant plant and creates new male sterile parent, expands heterotic utilization.

For achieving the above object, realize that concrete technological step of the present invention is as follows:

One, makes up model plant Arabidopis thaliana cDNA expression library

Shown in figure one: utilize the middle stuffer of plasmid pJL453 displacement GEM-12, obtain λ 455.Contain the ColE1 ori in λ 455 plasmids, coding beta-lactamase (BLA) gene is taken this and can be duplicated and screen in intestinal bacteria and agrobacterium tumefaciens.By T-DNA, this carrier can be integrated exogenous dna fragment and be inserted in the Plant Genome; Two placed in-line 35S promoters and NOS terminator can make foreign gene efficiently express in transgenic plant; Selection markers gene neomycin phosphotransferase (NPT II) can be distinguished the true and false of transfer-gen plant.Contain two lox tumor-necrosis factor glycoproteinss among the λ 455, can under the proteic catalysis of cre, carry out the locus specificity reorganization.After invading E.coli bacterial strain trpC9830 (λ KC) (can produce cre albumen), λ 455 can the cyclisation generation can directly carry out agriculture bacillus mediated Plant Transformation plasmid.By this recombination system, obtain the cDNA expression library arabidopsis thaliana transformation of λ 455.

Two, just producing/transgenic plant of antisense expression and the phenotype of t365

Make up λ 455 expression libraries of Arabidopis thaliana Columbia wild-type cDNA, approximately obtain～2 * 10 ⁶Individual recombinant chou after infecting E.coli trpC9830 (λ KC), is collected the cDNA plasmid library and is transformed agrobacterium tumefaciens GV3101 (pMP90RK).Agrobacterium tumefaciens grew on solid medium 3 days, then on the liquid medium within 28 ℃ cultivated 2 hours, by the vacuum filtration method ⁽⁶⁾Arabidopsis thaliana transformation Columbia wild-type plant.By continuous two generation 50mg/L kantlex screening, sowing T2 observes phenotype for seed.T3 is for seed analysis and the strain system that keeps the phenotype genetic stability.Wherein contain the mutant that the present invention relates to, called after t365.Its phenotype is seen accompanying drawing 2.

The genetic analysis of t365 mutant: by 117 T2 are detected for the PCR of plant, carry out Southern blot simultaneously and identify, segregation ratio is that the unit point that meets expection at 88: 29 inserts ratio (x ²=0.0028; P＞0.90), sowing is observed the wildtype phenotype t365 that contains the T-DNA insertion and is not contained the wildtype phenotype t365 that T-DNA inserts respectively, contain mutant that T-DNA inserts and take place to separate and separate than similar T1 for transfer-gen plant, the wildtype phenotype plant offspring who does not have T-DNA to insert is wild-type.The above results shows that the mutant phenotype of t365 is because the insertion sudden change of cDNA causes.

Three, gene clone: (accompanying drawing 3):

According to the sequences Design primer of 35S promoter and NOS terminator, the PCR reaction can amplify the cDNA fragment (accompanying drawing 3A) that is inserted in the Plant Genome, perhaps utilizes the strategy clone T-DNA of plasmid rescue to insert the genome sequence that relates to.The cDNA fragment of utilizing pcr amplification to obtain is made probe, and the cDNA library of sieve Arabidopis thaliana obtains 21 clones, and wherein the longest clone contains the PolyA fragment of 22A base for 1.457kb.5 '-RACE and sequential analysis proof are sieved to full-length cDNA fragment (Fig. 4).Relatively the sequence of cDNA and genomic dna finds that the t365 gene contains 12 exons, 492 amino acid of encoding, and molecular weight is 56.1KD.BLAST analyzes in the arabidopsis gene group and contains 3 copies.Coding S-adenosyl-L-methionine-phosphoethanolamine N-methyltransferase (PEAMT) in homologous sequence analysis revealed t365 and the spinach

Albumen has 86% amino acid identity, proves the t365 gene Phosphoethanolamine N-methyltransferase of encoding in Arabidopis thaliana.The generation that Northern Blot analyzes the t365 mutation type surface is owing to change the result that cDNA fragment in the plant and endogenous homologous gene suppress altogether over to.

Functional study shows: blade bleaching even dead phenomenon appear in the t365 mutant under condition of salt stress.(Fig. 5) differing temps continuous illumination condition is handled, and the t365 mutant shows temperature sensitive sterile phenotype.Under 20 ℃ of conditions,, under 26 ℃ of conditions, show as male sterile (Fig. 6) and prove that this gene is relevant with the study on temperature sensitive male sterility phenomenon for educating.Structure contains the segmental plant expression vector conversion of mutant insertion cDNA wildtype phenotype Arabidopis thaliana and obtains male sterile phenotype plant, proves that the t365 gene can obtain the male sterile transgenic plant by transgenic technology.

China exists per capita area of cultivated farmland constantly to reduce at present, the ever-increasing problem of soil salinization and alkalization and area, and large-area coastal beach and salinized soil are idle useless simultaneously, and the creation of salt/alkali-tolerance germplasm and the seed selection of kind will have huge potentiality to be exploited.

Along with heterotic continuous utilization, press for cost height, the inefficient problem that artificial emasculation causes in the first generation of hybrid production of hybrid seeds process that solve.The successful key of hybrid rice is the screening success of light, study on temperature sensitive male sterility system.The development of genetic engineering technique makes that using the t365 gene can cause the function of plant study on temperature sensitive male sterility to become possibility artificial creation male sterile transgenic plant.

The present invention is described in further detail below in conjunction with accompanying drawing.

Description of drawings

Accompanying drawing 1. plant expression vector λ 455 and expression of plants library construction

Accompanying drawing 2.T365 gene mutation body phenotype, wherein (A) and (B) contrast of the wild-type of expression growth 35 and t365 mutant plant respectively, the t365 mutant shows leaf look pale green (B); (C) and (D) the lotus Zuo leaf of expression growth wild-type contrast in 40 days and t365 mutant respectively, wherein the t365 plant shows the phenomenon (D) of early ageing; (E) and (F) show wild-type and t365 mutant plant blossom blocky period, wherein t365 mutant fruit pod (F) does not have seed; (G) and (H) be respectively the enlarged view of the floral organ of wild-type and mutant, (H) show the male sterile of t365 plant.

Accompanying drawing 3. gene clone strategies (A) can amplify the cDNA fragment of insertion by PCR by 35S and NOS design primer.

(B) genomic dna that is close to T-DNA insertion site can separate by the plasmid rescue method.B，BamH?I；C，Cla?I；H，Hind?III；R，EcoRI；S，SacI；X，Xhol?I

Accompanying drawing 4.T365 gene order and encoding amino acid sequence

Col-0 wild-type (A) and T365 gene mutation body (B) phenotype under the accompanying drawing 5.NaCl stress conditions

Col-0 wild-type (A) and T365 gene mutation body (B) phenotype under accompanying drawing 6. condition of different temperatures

Accompanying drawing 7. homologies are Arabidopis thaliana T365 and spinach PEAMT protein relatively

Specific embodiments

1, the structure of plant expression vector λ 455

Be used to make up the plasmid of pJL453: pSE936 ⁽⁷⁾, pBluescript II KS (+/-) (Stratagene), pBI101 (Clontech, Palo Alto, CA), pUC118 (BoehringerMannheim), and pSS ⁽⁸⁾Bgl II (blunt ended)-Sal I (blunt ended) double digestion pSE936 obtains the EcoR V restriction enzyme site that the lox site changes pBluescript II KS over to, obtains pJL420; EcoR I and Sac I enzyme are cut pBI101 and are obtained the NOS terminator, and the restriction enzyme site that is connected to the EcoR I-Sac I of pJL420 generates pJL422; The EcoR I restriction enzyme site that destroys pJL422 with the Klenow fragment (Promega) of dna polymerase i generates pJL42; Cut through Sac I-Sal I (blunt ended) enzyme, the gained fragment is connected on the restriction enzyme site of the Sac I of pAUC118 and Sma I and generates pJL452; PJL452 replaces BamH I-EcoR I double digestion pJL429 through BamH I-EcoR I double digestion and (generates pJL453 from pSS through the fragment of KlenowFragment (Promega cuts Not I restriction enzyme site); The λ GEM-12 (Promega) that cuts with Not I enzyme after Not I linearizing is connected generation cDNA expression vector λ 455.

With the over-ground part of QuickPrep Micro mRNA Purification Kit (Pharmacia) extraction Arabidopis thaliana Columbia wild-type, through the synthetic cDNA of TimeSaver cDNA Synthesis Kit (Pharmacia).With Wizard Lamda Preps DNA PurificationSystem (Promega) preparation λ 455 DNA.50 μ L enzymes are cut system, the EcoR I of 30 units, complete degestion 4 μ g λ 455 DNA, through 1 hour dephosphorization treatment of 30 ℃ of water-baths of HK phosphatase (EpicentreTechnologies), add 5 μ L 3M NaAC and 110 μ L-20 ℃ pre-cooled ethanols, 20 ℃ of insulation 30min, 16, the centrifugal 15min of 900g adds 1mL 70% (v/v) ethanol washing and precipitating, is suspended in after drying up in the 8 μ L TE damping fluids.2 μ g cut through EcoR I enzyme, and λ 455 dna vectors that phosphorylation is handled spend the night in 25 μ L linked systems with 15 μ L cDNA and are connected, and pack through one Ready-To-Go Lamda PackagingReaction System (Pharmacia).

2, Agrobacterium tumefaciens mediated arabidopsis thaliana transformation

λ phage competent cell (reference method preparation) is with the freshly prepd E.coli bacterial strain of 1 μ L trpC9830 (λ KC) ⁽¹⁰⁾(contain 10mM MgCl at 1mL LB substratum ₂) 37 ℃ cultivate 30 minutes altogether after, get 150mL coated plate (50 μ gmL ^-1Ampicillin and 50 μ gmL ^-1Kanamycin), 37 ℃ of overnight incubation are suspended in 250mL LB (containing ampicillin and the kanamycin) substratum, and 37 ℃ of shaking table shaking culture 1 hour are extracted plasmid DNA purification with polyethyleneglycol ⁽⁹⁾Electricity swashs conversion agrobacterium tumefaciens bacterial strain GV3101 (pMP90RK), with vacuum filtration method arabidopsis thaliana transformation Col-0 wild-type.Before transforming, plant cultivates under the B5 medium 1/3,23 ℃, continuous illumination condition.Transformed plant (T ₀) receive T after the self-pollination ₁Seed adopts 50 μ gmL ^-1Kanamycin screens on the 1/2MS substratum.The plant of anti-kanamycin is transferred in the soil individual plant and receives and kind obtain T2 for seed.

3, genetic analysis and molecular biology identification

Utilize 35S-F ₂(5 ' ACCACGTCTTCAAAGCAAGTG3 ') and NOS-R ₂(5 ' TATGATAATC

ATCGCAAGACCG3 ') fragment is made primer, extracts mutant DNA and carries out the PCR detection.Reaction conditions is as follows: 94 ℃ of sex change 3 minutes, 45 circulations (94 ℃ 1 minute, 58 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute) were extended 10 minutes for last 72 ℃.Reclaim dna fragmentation and make probe, southern blot identifies.5 ' the end of T365 cDNA uses 5 '-RACE Kit (Boehringer Mannheim) to identify.3 primer: SP1 (5 ' GTTTGTGAGCACTGGTGGAC3 ') SP25 ' TGGCCAAAGACACGCT that adopts

CATAG3’)、P3(5’GACATAAGCTCCAATGCACTTG3’)。Clone PCR products is in (Stratagene) carrier of pBluescriptII SK (+), with 37 3A dna sequencing instrument and DYEnamic Direct dGTP Sequencing Kit (Amersham) order-checking.For checking t365 whether with mutant phenotype be divided into from, utilize T-DNA carrier primer 35S-F ₂With the special primer T365-R of T365cDNA fragment (5 ' GATGAGAA-CTTTACCTCCCG3 ') pcr amplification mutant gene, make up plant expression vector arabidopsis thaliana transformation Col-0 wildtype phenotype plant again, relevant with the t365 gene at T1, T2 for the phenotype proof mutant phenotype that obtains t365.Extract plant genome DNA and RNA, conventional Southern and Nothern Blot identify the situation of the transcript and expression of mutator gene.

4, sowing Col-0 and t365 mutant are at continuous illumination ((80 to, 120 μ E m ^-2Sec ^-1), cultivate respectively under 20 ℃, 23 ℃, the 26 ℃ conditions, observe the male sterile phenotype.The result shows that mutant can be educated under 20 ℃ of conditions, and mutant is sterile fully under 26 ℃ of conditions, and this gene is subjected to temperature-induced.

5, sowing Col-0 and t365 mutant spray the NaCl of 50 μ M, 100 μ M, 200 μ M, 400 μ M, 500 μ M concentration in same nutrition pot, observe phenotype and photograph.Test-results show lower concentration (50 μ M) NaCl can induce obvious phenotypes difference, and the bleaching of mutant blade is dead.This gene is when expression is suppressed, and mutant is salt tolerant alkali not obviously.This gene is relevant with the salt adverse circumstance.

Reference:

1、Nuccio，M.L.，Ziemak，M.J.，Henry，S.A.，Weretilnyk，E.A.，andHanson，A.D.(2000).cDNA?cloning?of?phosphoethanolamine?N-methyltransferase?from?spinach?by?complementation?in?Schizosaccharomycespombe?and?characterization?of?the?recombinant?enzyme.J?Biol?Chem?275，14095-14101

2、Bart?Clase，Rudy?Dekeyser?et?al.，(1990)Characterization?of?a?ricegene?showing?organ-specific?expression?in?response?to?salt?stress?anddrought.The?Plant?Cell?Vol?2：19-27.

3、Bjarne?Landfald，Arne?R.Strom(1986)Cholinr-gycine?betainepathway?confers?a?high?evel?of?osmotic?tolerance?in?E.coli.J.Bacteriol.165：849-855.

4、Csonka?Laszlo?N(1989)Physiological?and?genetic?responses?ofbacteria?to?osmotic?stress.Microbiol.Rev.53：121-147.

5、Jordi?Gomez，Demetrio?S.M?et?al(1988)A?gene?induced?by?the?planthormone?abscisci?acid?in?response?to?water?stress?encodes?a?glycine-richprotein.Nature?334：262-264.

6、Bechtold，N.，Ellis，J.，and?Pelletier，G.(1993).In?plantaAgrobacterium-mediated?gene?transfer?by?infiltration?of?Arabidopsis?thalianaplants.CR?Acad.Sci.(Paris)316，1194-1199.

7、Elledge，S.J.，Mulligan，J.T.，Ramer，S.W.，Spottswood，M.，andDavis，R.W.(1991).Lambda?YES：a?multifunctional?cDNA?expression?vectorfor?the?isolation?of?genes?by?complementation?of?yeast?and?Escherichia?colimutations.Proc.Natl.Acad.Sci.88，1731-1735.

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Claims (5)

1, a kind of gene τ 365 of the Phosphoethanolamine N-methyltransferase of encoding has nucleotide sequence as follows:

ACTCTTTTCGCTCTCAGATCTGAAACCTTATCTGAATTACATTTCCCCGATTCGACGTTG 60

TGCGAATTGATGCAGCAGAGAGGACGATCCGTCAACCGCAGATCACGAAGCTTCTCTAGA 120

TCCAGACTCGCCGTCGAAGGCCACTGAATTACTCTCTCCCCTCTCTTGGCAGATCTTCTT 180

CTTCGTTTTTTCCCGACAAACGACATTTCCGAAATGGCTGCATCGTACGAAGAAGAGCGT 240

GATATTCAGAAGAATTACTGGATAGAGCATTCCGCTGATCTGACTGTTGAAGCTATGATG 300

CTTGACTCGAGAGCTTCTGATCTCGACAAGGAAGAACGTCCTGAGGTACTCTCTTTGCTC 360

CCTCCATATGAAGGCAAATCAGTGTTGGAACTTGGAGCTGGTATTGGTCGTTTCACTGGT 420

GAATTAGCTCAAAAGGCTGGTGAACTCATTGCTCTTGACTTCATTGATAACGTTATCAAG 480

AAGAATGAAAGTATCAATGGGCATTACAAGAATGTCAAGTTTATGTGTGCTGATGTTACA 540

TCCCCTGACCTCAAGATCACTGATGGATCTCTTGACTTGATTTTCTCCAACTGGCTGCTC 600

ATGTATCTTTCTGACAAAGAGGTGGAGCTTTTGGCAGAAAGGATGGTCGGTTGGATCAAG 660

GTTGGAGGATACATTTTCTTCCGTGAATCTTGCTTCCACCAATCAGGGGACAGTAAGCGG 720

AAATCCAACCCCACTCACTACCGTGAACCCCGTTTCTATTCCAAGGTCTTTCAAGAGTGT 780

CAGACTCGGGATGCTGCTGGAAATTCATTTGAGCTCTCTATGATCGGATGCAAGTGCATT 840

GGAGCTTATGTCAAGAACAAGAAGAATCAGAATCAGATTTGTTGGATATGGCAGAAGGTC 900

AGCTCAGAAAATGACAGAGGCTTCCAACGTTTCTTGGACAATGTCCAATACAAATCCAGT 960

GGAATCCTACGCTATGAGCGTGTCTTTGGCCAAGGGTTTGTGAGCACTGGTGGACTTGAG 1020

ACAACCAAAGAATTTGTGGAGAAAATGAATCTGAAACCAGGACAGAAAGTCTTAGATGTT 1080

GGGTGTGGCATTGGTGGAGGTGACTTCTACATGGCTGAGAAGTTTGATGTTCACGTTGTT 1140

GGTATCGATCTTTCTGTCAACATGATCTCTTTCGCATTGGAACGTGCTATTGGACTCAGC 1200

TGCTCGGTTGAGTTTGAGGTTGCTGATTGCACCACAAAACACTACCCAGATAATTCGTTT 1260

GATGTCATTTACAGCCGTGACACTATTCTGCACATCCAAGACAAACCAGCCTTGTTTAGG 1320

ACTTTCTTCAAATGGCTTAAACCGGGAGGTAAAGTTCTCATCAGCGACTACTGTAGAAGC 1380

CCCAAAACTCCATCTGCTGAGTTTTCAGAGTACATCAAACAGAGAGGATATGATCTCCAT 1440

GACGTTCAAGCTTATGGACAGATGCTAAAAGACGCTGGCTTCACTGATGTGATCGCAGAG 1500

GACCGTACTGATCAGTTTATGCAAGTCCTGAAACGTGAATTAGACAGGGTGGAGAAAGAA 1560

AAGGAAAAATTCATCTCCGACTTCTCCAAAGAGGATTACGATGACATTGTTGGAGGATGG 1620

AAGTCAAAGCTGGAGAGGTGTGCATCGGATGAGCAGAAATGGGGACTTTTCATCGCCAAC 1680

AAGAATAAGCAAATCGAAATCTACTACTTCTATGTTTTCTTCTTTCTTTTGTTTGTTCAA 1740

TAAAAATGTCATTTCCGCTAGGTGATGATATATGCCTGTAGTGTGTTATGTGGACTTGTT 1800

GAGTGGTGTTAAATCTTGTTTCCCTTATGTGATATGTAAACCGAATTATGTTTGGTCTTA 1860

GTTTAACTCTTTGATTTGAAGAAAAAAAAAAAAAAAAAAAA.

2, as follows by gene τ 365 its Nucleotide deduced amino acid of the described a kind of Phosphoethanolamine N-methyltransferase of encoding of claim 1:

M A A S Y E E E R D I Q K N Y W I E H S A D L T V E A M M L D S

R A S D L D K E E R P E V L S L L P P Y E G K S V L E L G A G I G R

F T G E L A Q K A G E L I A L D F I D N V I K K N E S I N G H Y K

N V K F M C A D V T S P D L K I T D G S L D L I F S N w L L M Y L

S D K E V E L L A E R M V G W I K V G G Y I F F R E S C F H Q S G

D S K R K S N P T H Y R E P R F Y S K V F Q E C Q T R D A A G N S

F E L S M I G C K C I G A Y V K N K K N Q N Q I C W I W Q K V S s

E N D R G F Q R F L D N V Q Y K S S G I L R Y E R V F G Q G F V S

T G G L E T T K E F V E K M N L K P G Q K V L D V G C G I G G G D

F Y M A E K F D V H V V G I D L S V N M I S F A L E R A I G L S C

S V E F E V A D C T T K H Y P D N S F D V I Y S R D T I L H I Q D

K P A L F R T F F K W L K P G G K V L I S D Y C R S P K T P S A E

F S E Y I K Q R G Y D L H D V Q A Y G Q M L K D A G F T D V I

A E D R T D Q F M Q V L K R E L D R V E K E K E K F I S D F S K

E D Y D D I V G G W K S K L E R C A S D E Q K W G L F I A N K N

3, a kind of plant expression vector λ 455, it is a kind of middle stuffer that utilizes plasmid pJL453 displacement GEM-12, obtain containing the ColE1 ori, coding beta-lactamase (BLA) gene, the plant expression vector of two placed in-line 35S promoters and NOS terminator.

4, the application of the gene τ 365 of the described coding Phosphoethanolamine N-methyltransferase of claim 1 in cultivate plants salt tolerant alkali and plants male sterility.

5, the application of the described plant expression vector λ 455 of claim 3 in cultivate plants salt tolerant alkali and plants male sterility.

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