CN104232664B - Aegilops varibilis tryptophan decarboxylase gene and its application - Google Patents

Abstract

The invention discloses a kind of Aegilops varibilis tryptophan decarboxylase nucleotide sequence and its application, described Aegilops varibilis tryptophan decarboxylase gene has the nucleotide sequence shown in SEQ.ID.NO.1.One tryptophan decarboxylase of Aegilops varibilis tryptophan decarboxylase gene code of the present invention, its anti-root-knot nematode performance also have been found, and this has important references value to tobacco breeding and the raising anti-root-knot nematode ability of tobacco.

Description

Aegilops varibilis tryptophan decarboxylase gene and its application

Technical field

The present invention relates to molecular biology and agriculture field, is specifically related to a kind of Aegilops varibilis tryptophan decarboxylase core Acid sequence and its application.

Background technology

Tryptophan decarboxylase (Tryptophan decarboxylase, TDC；EC4.1.1.28 aromatic series L- amino) is belonged to Acid decarboxylase (aromatic L-amino acid decarboxylase, AADC) family, it is to rely on cofactors phosphoric acid pyrrole to tremble One kind in the decarboxylase of aldehyde (Pyridoxal Phosphate, PLP) catalytic reaction.Rely on the other of phosphopyridoxal pyridoxal phosphate family Member also includes glutamic acid decarboxylase enzyme (Glutamate decarboxylase, GAD), histidine decarboxylase class (Histidine decarboxylase, HDC), aromatic amino acid decarboxylase (AADC) etc..Tryptophan decarboxylase substrate is specially changed Property it is higher, be only capable of being catalyzed L-Trp and its derivative such as 5HTP, 4- fluorotryptophans, 5- fluorotryptophans, 4- methyl color ammonia The decarboxylic reaction of acid.Oneself all TDC of report can be used as substrate using L-Trp.And to such as L- of the amino acid containing phenolic group side chain Tyr or Dopa is then without activity.

In plant, important as precursors of the tryptophan as secondary metabolite, these secondary metabolites include protective plant protecting agent, Alkaloid (such as vinblastine, camptothecine etc.), terpene.Tryptophan participates in Secondary Metabolism of Plant approach mainly by 2 kinds of centres Body, tryptamines and indoles -3- acetaldoximes.Wherein tryptamines is catalyzed L-Trp decarboxylation generation by tryptophan decarboxylase, and this reaction is wide General is present in plant, and is all proved to pest-resistant with plant by its multiple metabolite as substrate generation, disease-resistant, resists Inversely related, such as five hydroxytryptamine, tryptamines, isoquinolin etc..

1993, TDC was cloned in catharanthus roseus first, found to coerce when by biological factor and abiotic factor When, the enzymatic activity and TIAs accumulation have correlation.RISHI etc. has found the TDC bases of camplotheca acuminata (Camptotheca acuminata) Because being overexpressed in willow and tobacco, willow and tobacco normal growth are found, tryptamines accumulating level improves, polychaete worm and hawkmoth Food-intake and number are affected, with the expressions of corresponding TDC genes into negative correlation.And the research of the TDC genes to rice Show the TDC genes of rice and the expression of serotonin, there is direct relation to paddy disease-resistant.Overexpress rice TDC bases Cause, serotonin accumulation is found, improves the resistance to pathogen；With the diseased region of serotonin processing rice mutant, hair The growth of existing Bipolaris oryzae hypha,hyphaes is suppressed, and lesion locations are repaired；With (S)-α- (fluoromethyl) after tryptophan (S- α-FMT) (TDC Suicide substrate) handles Bipolaris oryzae dip-dyes Rice Seedling Leaves, find serotonin accumulation be suppressed, and to through S- α-FMT processing after blade, supplement tryptamines The metabolite brown material of position (contaminate), the secondary metabolite in blade recovers accumulation.Pepper (pepper Fruits) TDC genes are also proved to participate in plant disease-resistant process.But it up to the present, yet there are no TDC genes and participate in Genes For Plant Tolerance The report of root-knot nematode.

Aegilops varibilis is the nearly edge species of wheat, can produce fertile offspring when carrying out distant hybridization with wheat, be wheat The valuable source of breeding improvement.Variable goat No. 1 is had proved to be to cereal cyst nematode (H.avenae) and root-knot nematode (Meloidogyne naasi) resistant dual anti-material.Nearly edge species (barley, rice, wheat, Ophiorrhiza japonica, short handle Grass) nucleotide sequence design degenerate primer, using cDNA as template amplification nucleic acid fragment, and the nucleotide sequence to obtain design RACE Primer carries out the sequence amplification at 5 ' and 3 ' ends, obtains full length sequence.Total length primer is designed, expands complete genome sequence, and build original Nuclear expression carrier, enzyme activity is determined, it is found that it has tryptophan decarboxylase active；Binary expression vector is built, this sequence is imported In tobacco, it is found that the positive plant of conversion significantly improves to the resistance of root-knot nematode.

The content of the invention

It is an object of the invention to provide a kind of Aegilops varibilis tryptophan decarboxylase gene and its application, it is characterised in that Described Aegilops varibilis tryptophan decarboxylase gene has the nucleotide sequence shown in SEQ.ID.NO.1.

Another object of the present invention is to provide Aegilops varibilis tryptophan decarboxylase gene in tobacco seed selection or anti-root knot line Application in worm.

Another aspect of the present invention further relates to a kind of Aegilops varibilis tryptophan decarboxylase, it is characterised in that the variable goat The amino acid sequence of careless tryptophan decarboxylase is SEQ ID NO.2.

One tryptophan decarboxylase of Aegilops varibilis tryptophan decarboxylase gene code of the present invention, its anti-root-knot nematode performance It was proved already, this has important references value to tobacco breeding and the raising anti-root-knot nematode ability of tobacco.

Brief description of the drawings

The anti-root-knot nematode detection of Figure 1A eTDC transformed plants.

The Suicide substrates of Fig. 2 (S- α-FMT) suppress Aegilops varibilis TDC activity

Embodiment

If not specified, technological means used is well known to those skilled in the art in embodiment conventional meanses.

Embodiment 1：

1. clone obtains total length 1533bpTDC nucleotide sequences, 510 amino acid of the sequential coding, albumen 56.1kDa

First, material and reagent

Vegetable material：The root of No. 1 seedling of Aegilops varibilis

Primer：

dfTDC：5'-AGCCCVAAYTTCTTCGCSTT-3

drTDC：5'-TCCTCCTGCAGYGAYGANCC-3'

AeTDC5-1：5'-CCACTCCACGTTCTTTAAGGCTTGTCG

AeTDC5-2：5'-GAGTACCTCAAGAACGACGCTACCGATT

AeTDC3-1：5'-GAGTCCACACGCTCTACGCCATCAA

AeTDC3-2：5'-ACACAACCAAGCGTGGGATGTCAGA

fAeTDC：5'-ATGGGCAGCTTGGRCACC

rAeTDC：5'-TTAATCCATGATACTGCTCGTG

rBar:5'-TCAAATCTCGGTGACGGGCA

fBar:5'-GTCTGCACCATCGTCAACCACTA

Reagent：RNA extracts kits are purchased from Quan Shi King Companies；Reverse transcriptase (M-MLV), DNA Marker, pEASY- T1cloningKit, pEASY-Blunt E1Expression Kit are Quan Shi King Companies product：SMARTe^TM RACE cDNA Amplification Kit, Taq enzyme are BD-Clontech Products；

2nd, method

1st, TDC core sequences expand

1.1 plant Total RNAs extractions and reverse transcription

(1) seed of material is subjected to surface sterilizing processing (in the sodium hypochlorite of 3% concentration and 0.01% first Soaked 3 times with aqua sterilisa again after 5min is soaked in Tween20 mixed liquors, then seed is uniformly elaborated on the filter paper of constant moisture It is placed in the culture dish of 5cm diameters, seedling is issued in 20 DEG C or so of room temperatures and 16h/8h periodicity of illumination environment.

(2) clip sends out the seedling root of 20 days after kind, is milled in liquid nitrogen, and RNA is extracted by kit protocol.

(3) reverse transcription synthesis cDNA

Total serum IgE sample is determined to RNA quality by spectrophotometric determination sample in 260nm and 280nm absorption value And purity, and sample is diluted to 1.0 μ g/ μ L, reverse transcription reaction system is as follows：

37 DEG C of 1h, 70 DEG C of 15min, 4 DEG C of 5min reactions terminate the rear of short duration centrifugation several seconds, are placed in standby in -20 DEG C of refrigerators.

The amplification of 1.2 target gene target fragments

Using reverse transcription product cDNA as template, the HVA1 gene pieces for treating silence are expanded respectively using two pairs of different primers Section, reaction system are as follows：

Response parameter is 95 DEG C of 5min；94 DEG C of 40s, 68-62 DEG C of 40s, 72 DEG C of 40s, each 2 circulation；94 DEG C of 40s, 60 DEG C 40s, 72 DEG C of 40s, 29 circulations, last 72 DEG C of extensions 10min.

The PCR primer recovery of 1.3 purpose fragments

(1) 1.5% agarose gel electrophoresis of the PCR primer of target gene, comparison DNA Marker cuts purpose band, It is transferred in the clean centrifuge tubes of 2mL.

(2) press and add 600 μ L glue reclaim Binding Buffer per 100mg gels, with being placed 15 minutes in 60 DEG C of water-baths, Mixing is shaken per 2-3 minutes once.

(3) gel of thawing is transferred in recovery column, and recovery column is put into collecting pipe, at room temperature 10,000rpm from The heart 1 minute.

(4) recovery column is removed, the solution in collecting pipe is outwelled, recovery column is put into collecting pipe again, adds Wash Solution700 μ L, at room temperature 10,000rpm centrifugations are repeated after 1 minute and washed once.

(5) recovery column is put into another clean 1.5mL centrifuge tube, adds 30~50 μ L Elution Buffer In on recovery column film, being stored at room temperature 2 minutes, 10,000rpm centrifuge 1 minute.

The connection and conversion of 1.4 purpose fragments

(1) target fragment of recovery is connected with cloning vector pEASY-T1, and reaction system is as follows：

Target fragment 0.5-4 μ L

pEASY-T1Cloning Vector 1μL

Add water to 5 μ L

After standing 5min in 22 DEG C of metal bath, connection product is used as conversion.

(2) the μ L of coupled reaction liquid 5 are transferred in the μ L of competent escherichia coli cell 200, mix, avoid vibrating, on ice Place 30 minutes.

(3) make competent cell heat shock in 42 DEG C of water-baths 90 seconds, ice bath 5 minutes, be then quickly added into 37 DEG C in advance The LB fluid nutrient medium 1mL of heat, centrifuge tube, which is placed horizontally on 37 DEG C of shaking tables, slowly to be shaken 45 minutes.

(4) 4,000rpm is centrifuged 5 minutes at room temperature, is left about 100 μ L of supernatant, cell is suspended again with sterile pipette tips, Bacterial suspension is applied on the LB solid plates containing Ampr (100 μ g/mL).

(5) in order that bacterium solution is fully absorbed, cover, be inverted after plate is placed about 1 minute, 37 DEG C are cultivated, and 12-16 is small When after it is observed that bacterium colony is grown.

The bacterium colony PCR identifications of 1.5 recons

(1) the new bacterium colony grown on picking plate, lines on new LB solid plates and (contains 100 μ g/mL Amp), 37 DEG C Culture, it is observed that linear bacterium colony is grown after 12-16 hours.And the bacterium colony newly grown is done into picking and does PCR amplification identifications on a small quantity. Reaction system is as follows：

Response parameter is 95 DEG C of 5min；94 DEG C of 40s, 58 DEG C of 40s, 72 DEG C of 40s, 30 circulations；Last 72 DEG C of extensions 10min。

(2) 1% agarose gel electrophoresis detect PCR primer, and by the transformant of positive colony, monoclonal is chosen to LB liquid (containing 100 μ g/mL Amp), 37 DEG C, 200rpm/min concussion and cultivates to OD600=0.5~1.0 send invitrogen companies Shanghai Branch company completes sequencing.

(3) unigene6752 and sequencing result and nearly edge species TDC genes are compared into analysis.

The 2 TDC full-length clones of Aegilops varibilis 1

2.1 5 ' terminal sequence expands

(1) RNA extractions are same as above.Using Total RNA as template, it is anti-to carry out reverse transcription using 5 ' RACE Adaptor primers Should, synthesis 1st Strand cDNA.

(2) it is anti-using upstream outer specific primer (AeTDC5-1) and 5 ' RACE Outer Primer progress 1st PCR Should.Reaction system is as follows：

Response parameter is 94 DEG C of 30s, 72 DEG C of 3min, and 5 circulate；94 DEG C of 30s, 70 DEG C of 30s, 72 DEG C of 3min, 5 circulations； 94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 3min, 27 circulations.

(3) 5 μ L1st PCR reaction solutions are taken, 10 times are diluted, from the nested primer RACE of AeTDC5-2 and 5 ' inner Bis- amplifications of Primer, same amplification system, response parameter is ibid.

(4) PCR primer detects through 1% agarose gel electrophoresis.Purpose band is tapped rubber and reclaimed, connection pEASY-T1 clones Carrier, converts competent escherichia coli cell, and picking positive colony send Shanghai branch company of invitrogen companies to complete sequencing.

2.2 3 ' terminal sequence expands,

(1) RNA extractions are same as above.Using Total RNA as template, it is anti-to carry out reverse transcription using 3 ' RACE Adaptor primers Should, synthesis 1st Strand cDNA.

(2) it is anti-using upstream outer specific primer (AeTDC3-1) and 3 ' RACE Outer Primer progress 1st PCR Should, reaction system and program are same as above.

(3) 5ul1st PCR reaction solutions are taken, 10 times are diluted, from the nested primer RACE of AeTDC3-2 and 5 ' inner Bis- amplifications of Primer, same amplification system, response parameter is ibid.

(4) PCR primer electrophoresis detection, recovery, pEASY-T1 cloning vectors are connected, converted, picking, sample presentation sequencing is same as above.

(5) DNAMAN splices total length, searches ORF reading frames on NCBI, designs special total length primer.

2.3 TDC total lengths expand

(1) with No. 1 young root extraction total serum IgE of Aegilops varibilis, the double-strand for inverting cDNA is as follows for template reaction system：

Response parameter is 95 DEG C of 5min；94 DEG C of 40s, 62 DEG C of 40s, 72 DEG C of 40s, 35 circulations；Last 72 DEG C of extensions 10min。

(2) 1% agarose gel electrophoresis detect PCR primer, and the transformant of positive colony is sent in invitrogen companies Extra large branch company completes sequencing.

2nd, No. 1 TDC binary vector con- struction of Aegilops varibilis and the genetic transformation to tobacco

1 material

Reagent：DNA Marker, pEASY-T1cloning Kit, Taq enzyme are Quan Shi King Companies product, and glue reclaim is tried Agent box (Tiangeng)

E. coli jm109, Agrobacterium EHA105

2 methods

2.1 AeTDC binary plants expression vector establishments and genetic transformation tobacco

(1) total length PCR primer amplification AeTDC full length sequences, cloning vector is connected to by the target fragment after amplification Peasy-T1。

(2) 5 μ L plasmids are added in Agrobacterium competent cell EHA105, ice bath 30min, 1min in liquid nitrogen, 42 DEG C of heat Swash 90s, ice bath 5min, aseptic operating platform adds 1ml, 200rpm, 28 DEG C of YEP fluid nutrient mediums and shakes bacterium 3h, 5000rpm centrifugation 10min, coated plate (contain Kan), choose monoclonal, PCR detections.

(3) bacterium is shaken：Choose some bacterium solutions with the toothpick after sterilizing or matchstick etc., be put into above-mentioned YEP liquid training together Support in base, be subsequently placed on oscillator and shake 16-17h of bacterium (180r/min), until OD600=0.5-0.8.

(4) leaf dish is cut out from blade with the 0.5mm card punch after sterilization, then leaf dish is put into Agrobacterium suspension and trained Support 5min.

(5) unnecessary bacterium solution is blotted with filter paper, blade is placed on the IAA0.1mg/L culture mediums of MS+6-BA1.0mg/L ten altogether 2d is cultivated, then switches into additional bar5mg/L, screening and culturing on Ticarcillin/Clavulanate Acid 250mg/L culture medium, (25 ± 1) DEG C, 16h light week Phase.

Kalamycin resistance bud (should be green) is differentiated after (6) 2 weeks, cuts, is gone to containing bar5mg/L from base portion by bud, Culture of rootage on Ticarcillin/Clavulanate Acid 250mg/L MS+0.1mg/LIAA, the plant after taking root move into warm indoor growing.

2.2 transformed plants are identified and Resistance Identification

(1) using CTAB methods extraction conversion and the STb gene of untransformed tobacco plant leaf.

With plasmid (containing target gene) for positive control, the tobacco leaf not converted is negative control, enters performing PCR Expand (primer r/fAeTDC, r/fBar), then enter row agarose gel electrophoresis detection (system is the same).

(2) root of clip transformation of tobacco, double-edged razor blade section and slide, covered, tabletting, in fluorescence microscope Lower observation.

(3) root-knot nematode will be contained to individual with each (5 plants) transplantings of adjoining tree through fluorescence and PCR test positive plant Basin alms bowl in, seedling is dug out after 2 months, clean, photograph, count root-knot nematode number, as a result as shown in Figure 1.

3rd, TDC Suicide substrate (S- α-FMT) suppresses No. 1 TDC activity of Aegilops varibilis

(1) seed of material is subjected to surface sterilizing processing (in the sodium hypochlorite of 3% concentration and 0.01% first Soaked 3 times with aqua sterilisa again after 5min is soaked in Tween20 mixed liquors, then seed is uniformly elaborated on the filter paper of constant moisture It is placed in the culture dish of 5cm diameters, seedling is issued in 20 DEG C or so of room temperatures and 16h/8h periodicity of illumination environment.

(2) after 20d, 6 plants consistent of seedling of growth conditions is chosen, is divided into two groups, one group is used as control, and another group is processing Sample, after the additional 100 μm of ol/LS- α-FMT of the horgland aqueous solution, water planting 48h, 72h, it will compare and processing seedling replanting turns Into the flowerpot with a soil containing root-knot nematode.

(3) after 60d, the seedling of control group and treatment group is dug out, running water rinses, and records root-knot nematode growing state. As a result it is as shown in Figure 2.

Described above is the preferred embodiments of the present invention, it is noted that is come for those skilled in the art Say, on the premise of principle of the present invention is not departed from, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Sequence table

Application Project

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<120> Title :Aegilops varibilis tryptophan decarboxylase gene and its application

<130> AppFileReference :

<140> CurrentAppNumber :

<141> CurrentFilingDate :

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

gtttttcagc aagataccta cttacaagga atgggcagct tgggcaccaa ccccatttcc 60

ttctccgcca tccccgacga caaggcggcg ttcgagccgc tcaaccccga agatgtccgt 120

gcatatctcc acaaggccgt cgacttcatc tccgactact acaccaatgt cgagtccatg 180

ccggttctcc ctaacgtgaa gcccgggtac ctgcaagacg agctcagcgc gtccccaccg 240

acatactctg cgccgttcga cgtcaccatg aaggagctca ggacctccgt cgtccccggc 300

atgacgcact gggctagccc caacttcttc gccttcttcc cctccaccaa cagcgccgca 360

gcgatcgccg gcgacctcat cgcctcggcc atgaacactg ttggattcac gtggcaggcc 420

tcacctgcag ccaccgagat ggaggtcctt gctcttgact ggcttgcgca gctcctgcgc 480

ctacccgcca ccttcatgaa ccgcactagc actggtcgtg gcaccggcgg tggcgtcatc 540

cttggcacaa cgagtgaggc aatgctcgtc acgctagtcg ccgcccgtga cgcggcgctg 600

cgtcggagcg gctctgtcgg agtgtctgac atcccacgct tggttgtgta tgctgccgac 660

caaacccact ccacgttctt taaggcttgt cgcctcgcag gcttcgaccc cgccaacatc 720

cggtccatcc ctaccgggtc ggaaaccaac tatgggctcg acccggcaaa gcttctcgag 780

gtcatgcaag ctgatgccga cgccggtctc gtgccaacat atgtctgcgc gaccgtggga 840

accacatctt ccaacgcagt cgacccggtc ggtgccgtcg cggacgtggc ctccatcttc 900

aatgcatggg tccacgtgga tgctgcctat gctggcagcg catgtatctg cccggagttc 960

cgccaccatc ttgatggcgt agagcgtgtg gactccatta gcatgagccc acacaaatgg 1020

ctactcacat gcctcgattg cacatgtctc tacgtccgtg atgctcaccg actaagcgac 1080

tcgttggaga ccaacccgga gtacctcaag aacgacgcta ccgattccgg cgaggtcacc 1140

gatcttaagg acatgcaggt cggcgtcggt cggcgcttcc gtgggctcaa gctttggatg 1200

gtcatgcgca cctatggtac cgcaaagctc caagagcaca tccgtagtga cgttgccatg 1260

gccaagatgt ttgaagattt cgtccgtgcc gacgataggt ttgaggtggt cgtaccgagg 1320

aactttgctc ttgtgtgctt taggatcaag gcaagtggag ccatgacgga ggaggatgcc 1380

gacgaggcga accgcttgct aatggagaat ctcaacaaga ctggcaaggc ttatcttgcc 1440

cacacggtgg tcggtgacaa atttgtgctc cgtttcgccg ttggatcgtc gctgcaggag 1500

gaaaggcacg tgagaagtgc atgggacctc atcaagaaga ccacgagcag tatcatggat 1560

taagtgcact gaccaactgt tcaggatctt tctag 1595

<212> Type : DNA

<211> Length : 1595

SequenceName : 1

SequenceDescription :

Sequence

--------

<213> OrganismName :

<400> PreSequenceString :

MGSLGTNPIS FSAIPDDKAA FEPLNPEDVR AYLHKAVDFI SDYYTNVESM PVLPNVKPGY 60

LQDELSASPP TYSAPFDVTM KELRTSVVPG MTHWASPNFF AFFPSTNSAA AIAGDLIASA 120

MNTVGFTWQA SPAATEMEVL ALDWLAQLLR LPATFMNRTS TGRGTGGGVI LGTTSEAMLV 180

TLVAARDAAL RRSGSVGVSD IPRLVVYAAD QTHSTFFKAC RLAGFDPANI RSIPTGSETN 240

YGLDPAKLLE VMQADADAGL VPTYVCATVG TTSSNAVDPV GAVADVASIF NAWVHVDAAY 300

AGSACICPEF RHHLDGVERV DSISMSPHKW LLTCLDCTCL YVRDAHRLSD SLETNPEYLK 360

NDATDSGEVT DLKDMQVGVG RRFRGLKLWM VMRTYGTAKL QEHIRSDVAM AKMFEDFVRA 420

DDRFEVVVPR NFALVCFRIK ASGAMTEEDA DEANRLLMEN LNKTGKAYLA HTVVGDKFVL 480

RFAVGSSLQE ERHVRSAWDL IKKTTSSIMD 510

<212> Type : PRT

<211> Length : 510

SequenceName : 2

SequenceDescription :

Claims (4)

1. A kind of 1. Aegilops varibilis tryptophan decarboxylase gene, it is characterised in that described Aegilops varibilis tryptophan decarboxylase base Because having the nucleotide sequence shown in SEQ.ID.NO.1.
2. 2. a kind of Aegilops varibilis tryptophan decarboxylase gene as claimed in claim 1 is in tobacco seed selection or anti-root-knot nematode Application.
3. A kind of 3. Aegilops varibilis tryptophan decarboxylase gene, it is characterised in that the Aegilops varibilis tryptophan decarboxylase gene The amino acid sequence of coding is SEQ ID NO.2.
4. 4. a kind of carrier, it is characterised in that contain the Aegilops varibilis tryptophan decarboxylase base described in the claims 1 or 3 Cause.