CN110423753A - A kind of root knot specificity promoter T106-P and application by root-knot nematode induction - Google Patents
A kind of root knot specificity promoter T106-P and application by root-knot nematode induction Download PDFInfo
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- CN110423753A CN110423753A CN201910815082.8A CN201910815082A CN110423753A CN 110423753 A CN110423753 A CN 110423753A CN 201910815082 A CN201910815082 A CN 201910815082A CN 110423753 A CN110423753 A CN 110423753A
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Abstract
The invention discloses a kind of root knot specificity promoter T106-P by root-knot nematode induction and applications.Described is made of root knot specificity promoter T106-P nucleotide sequence shown in SEQ ID NO:1 of root-knot nematode induction.The primer pair includes the first primer and the second primer, and the sequence of the first primer is as shown in SEQ ID NO:2, and the sequence of second primer is as shown in SEQ ID NO:3.The present invention takes the tomato underground part of inoculation Meloidogyne enterolobii 21 days and the underground part progress transcript profile sequencing analysis of overground part and non-inoculating tomato, filters out when being infected by root-knot nematode in root specifically expressing and the significant candidate gene of expression differenceT106.The present invention have successfully been obtained it is a kind of when root-knot nematode is infected in the promoter T106-P that root knot is specific expressed, provide a kind of suitable promoter for the anti-nematode breeding of transgenosis, can be used for the genetically modified plants kind of the safe and efficient anti-root-knot nematode of breeding.
Description
Technical field
The invention belongs to plant genetic technical fields, and in particular to a kind of root knot specificity starting by root-knot nematode induction
Sub- T106-P and application.
Background technique
Root-knot nematode (MeloidogyneSpp.) be it is a kind of endanger root system of plant, it is serious with lateral root and fibrous root morbidity, and
Inducing plant root system distorts to form the obligate fixed endoparasitism nematode of one kind of root knot.Root-knot nematode is due to adaptable, propagation way
Diameter multiplicity, host range is extensive, and harmfulness is very big, referred to as " most destructive pathogen in the world ".Meloidogyne incognita
(M.incognita), M hapla (M.hapla), javanese root knot nematode (M.Javanica) and peanut root-knot nematode
(M.arenaria) be Meloidogyne the common root-knot nematode species of representative species and China, in recent years, enterolobium cyclocarpum root knot
Nematode (M.enterolobii) generation be in the trend being gradually increasing, with extensive host, can overcome and be currently known
All resistant genes, in the world using it as important quarantine object.
The root of root knot nematode disease main harm plant, in the usually asymptomatic table of initial stage overground part that root-knot nematode is infected
Existing, when aggrieved heavier, Symptoms are that dwarfing, yellow, wilting, growing way are weak.Root-knot nematode Population breeding ability is strong, Yi Chuan
It broadcasts, more hosts, stubbornness and hidden, prevention and treatment is difficult, causes and seriously threatens to agricultural production.Root-knot nematode damages caused by crop
On the one hand evil is by invading host plant tissue aspiration plant nutrient, the substances such as secretase and toxin, induction host plant hair
Raw pathological change；On the other hand it is that nematode invaded plants tissue causes wound, causes the pathogens such as fungi, bacterium, virus compound
It infects.
Root-knot nematode can colonize in 3000 various plants, wherein up to 100 kinds or more of industrial crops, including draft is planted
Object, xylophyta, dicotyledon and monocotyledon spread flowers, and vegetables are economical, medicinal material, fruit tree, the crops such as grain, point
Cloth in the torrid zone, subtropical zone and Temperate Region in China, caused by economic loss be up to 157,000,000,000 dollars.In recent years, the disease of root-knot nematode
Occur in rising trend, underproduction 30%-50% after generation reaches 70% or more, or even crop is caused to be had no harvest when serious.
Since the parasitic character of root-knot nematode makes the prevention and treatment of root knot nematode disease extremely difficult, traditional control argument be
Apply the chemistry, physics and biotic factor for being unfavorable for nematode in soil, acts on the root-knot nematode egg and two of exposure in the soil
Instar larvae reduces nematode and successfully invades the probability of host, to achieve the purpose that disease-control.Current main control method has chemistry
Prevention and treatment, biology and cultural control.
Chemical prevention is always the main body in disease control due to the advantages that quick, with strong points, when high due to existing
The negative interactions such as poison, high residue, pathogen resistance, many nematocides such as iron goes out gram, bromomethane etc. is gradually prevented or restricted from
It uses.
Biological control is mainly natural enemy and the microbial resources prevention and treatment nematode for utilizing nematode, is to have gradually developed in recent years
The control method relatively friendly to environment, but much researchs are only to be unfolded indoors, also lack practical in production practices
Using.
Cultural control includes plantation disease-resistant variety, efficent rotation, plantation predacious plant, grafting etc..Wherein shift of crops is anti-
Disease is widely used, it is the limitation using root-knot nematode host, but crop rotation itself also has certain limitation, it is uncomfortable
It should be in the prevention and treatment of the wider root-knot nematode of host range.
Since there are obvious drawbacks for chemistry, biology and cultural control, efficient, safety root knot nematode control technology has been explored
The direction made joint efforts as various countries phytopathologist.As the development of recombinant DNA technology, Exogenous DNA transfered system are built
It is vertical, plant tissue culture technique perfect, and to the continuous depth of nematode infection mode, plant itself Defense response architectural study
Enter, using disease-resistant variety due to having economic, safe and efficient, advantages of environment protection, has become the head of research and development
Choosing.Genetic engineering breeding is because its period is short, high-efficient, resistance is lasting, be easy to growing and cultivation, has many advantages, such as resistance of wide spectrum,
As breeding method of greatest concern in recent years.During genetic engineering breeding, promoter gene and anti-nematode major gene resistance
It is two indispensable elements, selects suitable promoter gene and anti-nematode major gene resistance, promoter is made to drive nematode resistance gene
It is expressed on demand in plant, is the key that genetic engineering breeding.
Constitutive promoter can drive target gene high efficient expression in the institute of organism is organized, make target gene
Expression quantity is constant in certain level, not by space-time limited or the induction of certain substance and expressed in plant.But due to composition
Type promoter drives foreign gene non-specific, continuous expression characteristic in recipient plant, has aggravated the burden of plant, and
It falls flat at the specific organization position or specific time for needing the gene great expression because expression quantity is too low, in reality
Some shortcomings are gradually exposed in the application of border.
Organ specific promoters are also by tissue-specific promoter, can regulate and control foreign gene and specifically organize in plant
High efficient expression is carried out in organ, typically exhibits the characteristic of growth adjustment, this species specificity is usually thin with specific plant tissue
Born of the same parents' structure and Chemical Physics signal are existing basis.
During genetic engineering breeding, it is often desirable to the foreign gene of insertion can in plant is specifically organized table
It reaches, improves Zonal expression amount, avoid the unnecessary waste of plant endotrophic, to reduce the burden of plant, mitigate to work
The influence of object economical character, to obtain useful character, this is just needed through effective tissue-specific promoter to target
The expression of gene is regulated and controled.Therefore tissue specific promoter becomes most rich promising foreign gene in transgenic research and opens
Dynamic element, has become molecular biology of plants research hotspot and Disciplinary Frontiers in recent years.
In terms of the transgenosis breeding for disease resistance of root-knot nematode, plant to root knot nematode resistance Mechanism Study obtained it is larger into
Exhibition, such as using plant to the disease-resistant or defense response genes of the structural resistance of root knot nematode disease, plant itself, have to root-knot nematode
The toxic protein gene of inhibiting effect, dsRNA the or siRNA structure that RNA interference can be generated to the pathogenic related gene of nematode,
Although obtaining some transgenic plants that can be significantly reduced root-knot nematode and infect, still do not formed so far it is a kind of for
The disease-resistant variety commercially produced, one of major reason are a lack of suitable gene promoter.
Summary of the invention
The first object of the present invention is to provide a kind of root knot specificity promoter T106-P induced by root-knot nematode；The
Two are designed to provide one group for expanding the primer of the root knot specificity promoter T106-P by root-knot nematode induction
It is right；Third is designed to provide a kind of recombination containing the root knot specificity promoter T106-P by root-knot nematode induction
Expression vector；4th is designed to provide a kind of expression cassette；5th is designed to provide the root induced by root-knot nematode
Tie the application of specificity promoter T106-P.
The first object of the present invention is achieved in that the root knot specificity promoter induced by root-knot nematode
T106-P nucleotide sequence shown in SEQ ID NO:1 is constituted.
The second object of the present invention is achieved in that the primer pair includes the first primer and the second primer, described
The sequence of the first primer is as shown in SEQ ID NO:2, and the sequence of second primer is as shown in SEQ ID NO:3.
The third object of the present invention is achieved in that the recombinant expression carrier is the recombinant plasmid that will be constructed
T106-P-TA is usedHindIII HeNcoI double digestion, glue recycle Insert Fragment T106-P promoter, use simultaneouslyHindIII HeNcoI pair
Digestion pCambia1304 carrier, the pCambia1304 carrier segments of recycling removal 35S promoter, connects Insert Fragment T106-P
The pCambia1304 carrier segments of promoter and removal 35S promoter, obtain recombinant plasmid P1304-T106-P；Described heavy
In group expression vector, the root knot specificity promoter T106-P by root-knot nematode induction is connected to the upstream of gus gene.
The fourth object of the present invention is achieved in that the expression cassette includes described in claim 1 by root knot
The root knot specificity promoter T106-P of nematode-inducible.
The fifth object of the present invention is achieved in that the root knot specificity promoter induced by root-knot nematode
Application of the T106-P in the genetically modified plants kind for obtaining safe and efficient anti-root-knot nematode.
In previous genetically modified plants using it is more be composition type expression promoter, they are in plant genetic engineering
Using earliest, widest a kind of promoter, feature is that expression has duration, expression quantity substantially constant, but this external source base
Cause expression efficiency in recipient plant body is low, expression product is unstable, or even phenomena such as gene inactivation or silencing occurs, influences to plant
The normal growth and development of object, in addition it is dead, so that genetically modified plants can not put into practical application.Select tissue-specific promoter
Plant expression vector is constructed, is solved the above problems to regulate and control foreign gene timing, positioning, quantitative expression in plant
One Critical policies.
In root-knot nematode and plant interaction, it is its parasitism harm that root-knot nematode induction plant, which forms megabacterium and keeps,
Key, finding out control megabacterium and being formed facilitates deep to understand that root-knot nematode is caused a disease machine with the plant major gene resistance kept
Reason, at the same clone by root-knot nematode induce in root knot specifically expressed gene promoter, help to obtain efficiently anti-
Tie lines worm transgenic line.
The present invention takes the tomato underground part of inoculation Meloidogyne enterolobii 21 days and the underground of overground part and non-inoculating tomato
Portion carries out transcript profile sequencing analysis, filters out when being infected by root-knot nematode in root specifically expressing and expression difference is significantly waited
Select geneT106。
InterceptionT106Upstream 1.5kb segment utilizes position of the bioinformatics software prediction transcription initiation site where possible
It sets, designs specific primer, carry out the amplification of promoter destination region, amplification obtainsT106Promoter gene fragment 2506bp.
By the 35S promoter of the predicted gene promoter fragment expanded replacement pCambia1304, new plant table is constructed
Up to carrier P1304-106-P.Tomato is converted, normotrophic transgenic positive tomato seedling is obtained.
Successful transgene tomato transplantation of seedlings is screened into soil, is inoculated with Meloidogyne enterolobii and Root Knot line respectively
It is carried out after worm 21 daysGUSGene dyeing verifying T106-P promoter activity, it is found that the promoter T106-P of prediction can startGUSBase
Because specific expressed at root knot.Fluorescence quantitative PCR detection proves, can start with 35S promoterGUSGene is at each position of plant
Stablize expression to compare, T106-P promoter is at root knotGUSGene relative expression quantity is significantly higher than the root system and ground of no root knot
Upper part.
The experimental results showed that the present invention has successfully been obtained and a kind of opens when root-knot nematode is infected root knot is specific expressed
Mover T106-P provides a kind of suitable promoter for the anti-nematode breeding of transgenosis, can be used for the safe and efficient anti-root knot of breeding
The genetically modified plants kind of nematode.
The present invention provides a kind of root knot specificity promoter induced by root-knot nematode, nucleotides sequence is classified as SEQ ID
T106-P promoter sequence shown in No.1.
Use primer amplified geneT106Promoter fragment, primer sequence such as SEQ ID No.2/SEQ ID
Shown in No.3, the T106-P promoter nucleotide sequence expanded is as shown in SEQ ID No.1.The T106-P expanded is started
The PCR product of subsequence carries out glue recycling, and the TA for next step is cloned.
TA cloning vector selects pEASY-T1 to obtain after T106-P promoter gene segment is connected to carrier pEASY-T1
To recombinant plasmid T106-P-TA, useHindIII HeNcoI pair of T106-P-TA double digestion, the standard of verifying connection promoter fragment
True property, for constructing needed for plant expression vector in next step.
The recombinant plasmid T106-P-TA of building is usedHindIII HeNcoI double digestion, glue recycling Insert Fragment T106-P are opened
Mover is used simultaneouslyHindIII HeNcoThe pCambia1304 of I double digestion pCambia1304 carrier, recycling removal 35S promoter is carried
Body segment, connection Insert Fragment T106-P promoter and the pCambia1304 carrier segments for removing 35S promoter, are recombinated
Plasmid P1304-T106-P.
PCR verifying is carried out to the plant expression vector P1304-T106-P built, it is ensured that T106-P promoter fragment is just
True is connected toGUSBefore gene, use simultaneouslyHindIII HeNcoI couple of plant expression vector P1304- built
T106-P carries out double digestion verifying.
Successful P1304-T106-P plant expression vector will be constructed by electric shocking method to be transferred in agrobacterium strains EHA105.
Coated plate carries out resistance screening by YM culture medium flat plate (25 mg/L of rifampin, 50 mg/L of kanamycins).
The YM culture medium, which refers to, weighs quantitative YM dry powder according to the specification of YM culture medium, is put into the deionized water of 1 L
Middle dissolution, 121 DEG C of 20 min of high-temperature sterilization.It takes out and cools after sterilizing, 50 mg/L kanamycins and 50 mg/L rifampins are added.
Single colonie is chosen, 28 DEG C are shaken 48 h of bacterium until bacterium solution OD value reaches between 0.6-0.8.
It is centrifuged resuspension, when the OD value of Agrobacterium bacterium solution reaches between 0.6-0.8, culture bottle is taken out, in super-clean bench
Bacterium solution is poured into 50 sterile mL centrifuge tubes, 5000 r/min, 4 DEG C of 12 min of centrifugation.Centrifuge tube is taken out after centrifugation, super
Supernatant is outwelled on net workbench, resuspension fluids culture medium is added, is resuspended to OD value 0.3, the AS of 100 μm of ol/L concentration is added
(acetosyringone) mixes spare.
The resuspension fluids culture medium refers to, 20 g/L D-Glucoses and 10 g/L sucrose are added in MS basal medium,
117 DEG C of 20 min of high-temperature sterilization.
It carries out recombination bacterium solution PCR using specific primer to screen, primer sequence such as SEQ ID No.4/SEQ ID No.5 institute
Show, 7 single colonies of picking on the plate of conversion P1304-T106-P carrier, PCR verifies to obtain 2 plants of positive strain, will be positive
Bacterial strain is sent to sequencing company sequencing, and -80 DEG C of preservation sequencings correctly recombinate bacterium solution.A part of streak plate saves, for next
The co-cultivation of step and Tomato Calli obtains transgene tomato seedling, to verify T106-P promoter activity.
It is carried out disinfection using sodium-hypochlorite process to tomato seeds.
Aseptic tomato seed is inoculated into hair seedling culture medium and is cultivated.
The hair seedling culture medium refers to that+8 g/L agar of 1/2 MS+30 g/L sucrose, medium pH=5.8,121 DEG C high temperature go out
20 min of bacterium.
Seed hair seedling culture medium in grow 10-15 days after, cut tender aseptic tomato seedling leaf at about 2-5 mm ×
The tissue block of 2-5 mm size, is put into induced medium, is spaced moderate, about 5 mm.
The induced medium refers to+8 g/L agar of MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose, 121 DEG C
20 min of high-temperature sterilization.
Squamous subculture is carried out every 10 days picking foresythia dense callus, it is good that growth conditions are obtained after two generation of subculture
Good, the callus of color cadmium yellow, can be used as receptor progress Agrobacterium tumefaciems infects conversion.
Take callus after resuspension and added AS(acetosyringone) Agrobacterium tumefaciems liquid in, 100 rpm, 20
DEG C, impregnate 30 min.Callus material is placed in superclean bench in the culture dish for be covered with aseptic filter paper and is air-dried, completed
The transfection of recombinational agrobacterium.
Callus material after infecting is transferred to co-culture medium, 18 DEG C of dark culture 2-3 d.
The co-culture medium refers to MS+20 g/L sucrose+10 g/L glucose+100 μm of ol/L AS of+8 g/L agar,
118 DEG C of 20 min of high-temperature sterilization.
Callus is transferred in screening and culturing medium, the callus of the successfully plant expression vector with resistant gene is converted
Tissue can be grown on the culture medium of the resistance containing screening, and without the callus that is transferred to resistant gene, then slowly browning is die.
The screening and culturing medium refers to+8 g/L agar+600 of MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose
mg/L cef+ 20 mg/L Hyg.Wherein IAA, Cef and Hyg will add again after 121 DEG C of 20 min of high-temperature sterilization of culture medium.
New bud is grown by breaking up on screening and culturing medium, is transferred to root induction on root media.
The root media refers to 1/2MS+0.1 mg/L IBA+150 mg/L Cef, 121 DEG C of 20 min of high-temperature sterilization.
PCR detection, primer sequence are carried out across transgenic plant of the aligning primer to acquisition with verifying plant expression vector
As shown in SEQ ID No.4/SEQ ID No.5, verifying purpose gene T106-P promoter is successfully transferred in tomato dna group.
The positive transgenic tomato plant to grow fine in root media is transplanted in cultivation soil, strong sprout is carried out.
The cultivation soil refers to that Nutrition Soil is mixed with sand in 1:1 ratio, 121 DEG C of 20 min of high-temperature sterilization.
When Transgenic Tomato Plants grow fine, every basin transgene tomato seedling is inoculated with 2000 respectively in the same circumstances
Meloidogyne enterolobii and Meloidogyne incognita second instar larvae.After inoculation 1 month, transgene tomato grows root knot, carries outGUSBase
Because active tissue staining detects.
GUSColoration result shows that T106-P promoter is only after Meloidogyne enterolobii and Meloidogyne incognita infect
Blue by dye at root knot, the blade without the Tomato Root System and overground part that are infected by root-knot nematode is not all by dye indigo plant.35S is opened
Mover transgene tomato seedling is that entire root system is all blue by dye after root-knot nematode is infected, at root knot and at non-root knot.
The RNA of Transgenic Tomato Plants is extracted, reverse transcription is at cDNA, fluorescence quantitative PCR detectionGUSThe relative expression of gene
Amount.
Testing result is shown, in T106-P promoter Transgenic Tomato Plants, at root knotGUSGene relative expression quantity
It is significantly higher than root system and overground part at no root knot,GUSGene in the overground part of T106-P Transgenic Tomato Plants and without root knot at
Root system does not detect expression substantially.And in the Transgenic Tomato Plants of 35S promoter, root system at root knot, without root knot and
Overground partGUSGene relative expression quantity no significant difference,GUSGene relative expression quantity is almost the same.
In summary the result shows that, T106-P promoter provided by the invention and 35S promoter are in plant tissue each section
Middle expression is different, compared to 35S constitutive promoter, is existed by the root knot specificity promoter T106-P that root-knot nematode induces
Special at root knot, stable expression.This shows that T106-P promoter provided by the invention can be not only used for Meloidogyne enterolobii
Breeding for disease resistance, can also be applied to current root-knot nematode dominant population Meloidogyne incognita breeding for disease resistance work.
Detailed description of the invention
Fig. 1 is T106-P promoter fragment electrophoretogram；
Fig. 2 is that T106-P promoter fragment glue recycles electrophoretogram；
Fig. 3 is T106-P-TA carrier double digestion electrophoretogram；
Fig. 4 is carrier pCambia1304 structure chart；
Fig. 5 is construction of expression vector P1304-T106-P schematic diagram；
Fig. 6 is that plant expression vector P1304-T106-P PCR identifies electrophoretogram；
Fig. 7 is plant expression vector P1304-T106-P and carrier pCambia1304 double digestion identifies electrophoretogram；
Fig. 8 is that bacterium solution PCR identifies electrophoretogram after plant expression vector P1304-T106-P turns Agrobacterium；
Fig. 9 is transgene tomato seedling；
Figure 10 is that the PCR of Transgenic Tomato Plants detects electrophoretogram；
Figure 11 is the strong sprout of transgene tomato seedling and the inoculation of Meloidogyne enterolobii；
Figure 12 isGUSDyeing；
Figure 13 is fluorescence quantitative PCR detectionGUSGene relative expression quantity.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
The root knot specificity promoter T106-P nucleotide as shown in SEQ ID NO:1 induced as root-knot nematode
Sequence composition.
One group of the present invention for expanding the primer of the root knot specificity promoter T106-P by root-knot nematode induction
Right, the primer pair includes the first primer and the second primer, and the sequence of the first primer is as shown in SEQ ID NO:2, institute
The sequence of the second primer is stated as shown in SEQ ID NO:3.
Recombinant expression carrier of the present invention containing the root knot specificity promoter T106-P induced by root-knot nematode
Recombinant plasmid T106-P-TA for that will construct is usedHindIII HeNcoI double digestion, glue recycle Insert Fragment T106-P promoter, together
Shi YongHindIII HeNcoI double digestion pCambia1304 carrier, the pCambia1304 carrier segments of recycling removal 35S promoter,
It connects Insert Fragment T106-P promoter and removes the pCambia1304 carrier segments of 35S promoter, obtain recombinant plasmid
P1304-T106-P；In the recombinant expression carrier, the root knot specificity promoter T106- induced by root-knot nematode
P is connected to the upstream of gus gene.
Expression cassette of the present invention includes the root knot specificity promoter T106-P induced by root-knot nematode.
The application of root knot specificity promoter T106-P of the present invention by root-knot nematode induction is described by root
The root knot specificity promoter T106-P of nematode-inducible is tied in the genetically modified plants kind for obtaining safe and efficient anti-root-knot nematode
Application.
With specific embodiment, the present invention will be further described below:
Biomaterial
Meloidogyne enterolobii and Meloidogyne incognita, this laboratory save.
Vegetable material tomato variety is Rutgers, this laboratory saves.
Key instrument and reagent
Instrument: PCR instrument, centrifuge, electrophoresis apparatus, electroporated instrument, constant incubator, water-bath, tissue culture bottle, culture dish, alcohol
Lamp, tweezers, scalpel, fluorescence quantitative PCR instrument (CFX96, BIO-RAD)
Reagent: Tris, dense HCl, EDTA-Na2, NaCl, CTAB, mercaptoethanol, LA-Taq DNA polymerase, agarose, TAE
Buffer, full formula gold size QIAquick Gel Extraction Kit, pEASY-T1 Simple Cloning Kit(Quan Shijin), DH5 α competence, LB training
Support base (liquid/plate), Omega plasmid extraction kit, DNA A-Tailing KIT(TakaRa), pCAMBIA1304, limit
Property restriction endonuclease processedEcoRⅠ、HindIII HeNcoI, T4 ligase, Agrobacterium EHA105, heteroauxin (indole-3-acetic
Acid, IAA), 6-benzyl aminopurine (6-Benzylaminopurine, 6-BA), 2,4 one dichlorophenoxyacetic acids (2,4-
Dichlorophenoxyacetic acid, 2,4-D), zeatin (zeatin, ZT), acetosyringone
(Acetosyringone, AS), rifampin (Rifampin, Rif), kanamycin sulfate (Kanamycin Monosulfate,
KM), ampicillin (Ampicillin), 10% liquor natrii hypochloritis of hygromycin (Hygromycin, Hyg), (domestic point of alcohol
Analyse pure reagent), sucrose, glucose, MS culture medium, YM culture medium,GUSStaining kit (coolaber), reverse transcription reagent box
Box, which is extracted, purchased from Dalian treasured biotech firm, SYBR dyestuff, TRIZOL is purchased from Quan Shi King Company.
Embodiment 1
Separate promoter sequence
1, DNA of plants extracts
It is extracted using CTAB method.Specific operation process is as follows:
Solution is prepared: preparing CTAB extracting solution: 100 mmol/L Tris-HCl(pH8.0 according to following ratio), 20 mmol/L
EDTA-Na2,1.4 mol/L NaCl, 2 % CTAB use preceding addition 0.1%(V/V) beta -mercaptoethanol.Wherein Tris-HCl
Need to be formulated as the solution of 1 M and 0.5 M concentration in advance respectively with EDTA-Na2, adjust pH to desirable value, after sterilizing again with its
His ingredient prepares the CTAB solution for completing respective concentration together.
1) the fresh tomato root tissue for taking out 1.0 g or so, is placed in ready clean mortar in advance, quickly falls
Enter liquid nitrogen, rapid grind into powder, centre is continuously added liquid nitrogen, is transferred to the CTAB extracting solution for filling 1.2 mL, 65 DEG C of warm bath
In centrifuge tube, and the beta -mercaptoethanol of 30 μ L is added, mixes well, 65 DEG C of 50 min of warm bath.
2) it is cooled to room temperature, 4 DEG C of 12000 rpm is centrifuged 15 min.
3) it takes supernatant in new centrifuge tube, adds isometric chloroform: isoamyl alcohol (24:1), be vortexed concussion 30s, room temperature
12000 rpm are centrifuged 15 min.
4) supernatant is taken, adds the dehydrated alcohol of two volumes, slowly turns upside down, it is seen that milky flocculent deposit, centrifugation
After remove supernatant, respectively wash one time with 70 %, 100 % ethyl alcohol, natural air drying.
5) plus 600 μ L ddH2O dissolving DNAs slightly precipitate, and add the RNA enzyme of 2 μ L, 10 mg/mL, 37 DEG C of 1 h of warm bath.
6) it takes 500 μ L supernatants into new centrifuge tube, the isopropanol of 350 μ L is added, overturn for several times repeatedly, room temperature is put
10 min are set, 4 DEG C, 13000 rpm are centrifuged 15 min.
7) supernatant is taken, the dehydrated alcohol of 3M NaAc (pH5.2) and 2 times of volumes of 1/3 volume, -20 DEG C of 2 h of placement are added
Or overnight.
8) 4 DEG C, 13000 rpm are centrifuged 10 min, in tube bottom it can be seen that milky flocculent deposit, carefully outwells ethyl alcohol,
It is dry with the extra liquid of pipette tips sucking gently.
9) the sterile ddH2O dissolving DNA precipitating of 30-60 μ L is added, is placed in -20 DEG C and saves backup.
2, promoter sequence amplification, recycling
The determination of 2.1 design of primers and restriction enzyme site
The promoter activity of plant expression vector pCambia1304 verifying prediction is selected, the promoter fragment replacement of clone need to be used
Fall the 35S promoter of carrier pCambia1304.Promoter purpose sequence is combined according to plasmid vector pCambia1304 map (Fig. 1)
Column, select promoter T106-P restriction enzyme site for I site Hind III and Nco.Utilize primer-design software Primer Premier
And Oligo7 design primer, in addition expanding promoter sequence T106-P after restriction enzyme site sequence and protection base.Final design
Amplimer it is as follows:
T106-P-F:5 '-CCCAAGCTTGAGCATGGAGCATGACGTATAACG-3 ' (SEQ ID No.2)
T106-P-R:5 '-CATGCCATGGAACTTTGGAATCCTATGCCTTTG-3 ' (SEQ ID No.3)
PCR reaction system:
PCR response procedures: 95 DEG C, 3 min；94 DEG C, 1 min of 35 circulations, 60 DEG C, 1 min, 72 DEG C, 3 min；72℃,10
min。
The PCR product obtained after amplification is detected with 1.2 % agarose gel electrophoresis, and T106-P as shown in Figure 1 starts sub-pieces
Section electrophoretogram, T106-P promoter sequence length are 2506 bp, the promoter aim sequence as downstream experiment.
The recycling of 2.2 target fragments
Target fragment is recycled using plastic recovery kit, specific operation process is as follows:
1) it uses 1xTAE to configure the Ago-Gel of 0.8 % concentration as buffer, PCR stoste is subjected to agarose gel electrophoresis,
To isolate target gene product.
2) in ultraviolet bale cutting instrument, blob of viscose where cutting target fragment band, about 0.1 g is put into clean and sterile 1.5 mL
In centrifuge tube.
3) 3 times of bulk solution GSB solution are added, i.e. 300 mL melt glue 5-10 min in 55 DEG C of water-baths, until blob of viscose is complete
Melt, monoploid product isopropanol is added, is i.e. 100 mL can increase the yield of DNA in the gel solution melted.
4) when the gel solution wait melt is down to room temperature, solution is added in centrifugal column and stands 1 min, 13000 rpm from
Heart 1min abandons efflux.
5) 650 μ L solution W B, 13000 rpm are added and are centrifuged 1 min, abandon efflux.
6) step 5) is repeated, blank pipe is centrifuged 2 min, completely removes remaining WB.
7) centrifugal column is placed in a clean 1.5 mL centrifuge tube, uncaps and stand 3 min, ethyl alcohol is thoroughly dried, in column
The deionized water after 30 μ L preheating is added in centre, is stored at room temperature 3 min.
8) 13000 rpm are centrifuged 2 min, elute DNA, are stored in -20 DEG C.
T106-P promoter fragment glue recycles result T106-P promoter fragment glue as shown in Figure 2 and recycles electrophoretogram.Glue returns
Receipts target fragment size is 2506 bp, and the TA as next step is cloned.
3, promoter sequence TA is cloned
The connection of 3.1 target fragments and carrier
Specific operation process is as follows:
4.5 μ L of PCR recovery product
PEASY-T1 vector(10 ng/ μ L) 0.5 μ L
Solution I 5.0 μL
It is soft to mix, 25 DEG C of 30 min of connection.
The conversion and screening of 3.2 connection products
Specific operation process is as follows:
1) DH5 α competence is taken out from ultra low temperature freezer to be placed on ice to melt, 10 μ L connection products are gently added to 100
In μ L bacillus coli DH 5 alpha competence, after mixing gently, 30 min are placed on ice.
2) 42 DEG C of 90 s of heat shock, ice bath stands 3 min immediately.
3) LB liquid medium of 500 μ L preheating, 37 DEG C, 220 r/min shaken cultivation, 1 h is added.
4) 3000 rpm are centrifuged 1 min, sop up 200 μ L supernatants, and bacterium solution is coated on containing Amp(100 after taking 100 μ L to recover
μ g/mL) LB culture medium flat plate, plate is inverted, cultivates 12-16h in 37 DEG C of constant incubators.
5) bacterial plaque is put into the training of the liquid LB containing antibiotic Amp by the single white bacterial plaque of picking on superclean bench
Support base in, 37 DEG C, 190 rpm shake bacterium 8-12 h.
3.3 plasmids extract
The plasmid in Escherichia coli bacteria liquid is extracted with Quan Shijin Plasmid DNA Mini Kit, specific operation process is as follows:
1) bacterium solution being incubated overnight is taken, 13000 rpm are centrifuged 1 min, take out supernatant, can be collected by centrifugation several times.
2) colourless solution RB(A containing RNase is added) 250 μ L, concussion suspended bacterial precipitating, until fungus block all suspends.
3) 250 μ L of blue solution LB is added, mild overturning mixes 4-6 times, cracks thallus sufficiently, color is bright by half
Become bright blue, then indicates bacterium solution cracking completely, be usually no more than 5 min.
4) 350 μ L of yellow solution NB is added, mixes gently 5-6 times, until color has blue to become yellow completely, is formed
Yellow is aggregated block, is stored at room temperature 2 min.
5) 13000 rpm are centrifuged 10 min, and for careful Aspirate supernatant into centrifugal column, 13000 rpm are centrifuged 1 min, abandon
Efflux.
6) 650 μ L solution W B, 13000 rpm are added and are centrifuged 1 min, abandon efflux.
7) step 6 is repeated, empty 13000 rpm of centrifugal column is centrifuged 2 min, removes remaining WB.
8) centrifugal column is put in clean centrifuge tube, is dried, 30 μ L aseptic deionized waters are added in the center of column, it is quiet
3 min are set, 13000 rpm are centrifuged 2 min, elute Plasmid DNA.
The detection of 3.4 double digestions
Double digestion detection is carried out to the plasmid T106-P-TA extracted using restriction enzyme Hind III and Nco I, it is specific to grasp
It is as follows to make process:
Digestion system:
4 μ L of TA plasmid
10xK 2 μL
0.1% BSA 2 μL
HindⅢ/NcoⅠ 1 μL
It is settled to 20 μ L
It is stayed overnight for 37 DEG C after soft mixing, glue recycling is carried out to the segment after double digestion with 0.8 % Ago-Gel.T106-P-TA
Carrier double digestion result T106-P-TA carrier double digestion electrophoretogram as shown in Figure 3, verifies the accuracy of junction fragment, under being used as
The building of one step plant expression vector.
Embodiment 2
Plant expression vector construction
The building of plant expression vector is using carrier pCambia1304 as skeleton, and carrier pCambia1304 structure is as shown in figure 4, structure
Plant expression vector P1304-T106-P is built, expression vector P1304-T106-P is constructed shown in schematic diagram 5.
1, the digestion of pCambia1304 carrier
Double digestion is carried out to carrier pCambia1304 using restriction enzyme Hind III and Nco I, obtains P1304 H/N.It presses
3.4 digestion systems and reaction condition carry out digestion operation in embodiment/1.
2, the connection of T4 enzyme and conversion
2.1 connection
The linear carrier after target fragment and double digestion is attached using T4 ligase, linked system is as follows:
16.5 μ L of promoter fragment
P1304 H/N 4 μL
10×T4 ligase buffer 2.5 μL
2 μ L of T4 ligase
2.2 conversion
Overall reaction system is 25 μ L, soft to mix, and 16 DEG C of connections are overnight.The good plasmid of structure is imported into bacillus coli DH 5 alpha impression
State.By in the embodiment one 3.2 carry out conversion operations.
3, the identification of recombinant plasmid
3.1 PCR identification
Design primer carries out PCR identification to the plant expression vector P1304-106-P built, and just whether detection promoter sequence
It is true instead of the 35S promoter in carrier pCambia1304, and be properly inserted before Reporter gene GUS region.
Therefore designed preceding primer location should be located on promoter aim sequence, and rear primer is needed positioned at pCambia1304 carrier
The region GUS.Using software Primer Premier and Oligo7 design primer, the primer sequence of final design is as follows:
P1304-106-P-F:5 '-TTTCTCAAGTAAAATCTACCCAA-3 ' (SEQ ID No.4)
P1304-106-P-R:5 '-TTCTACAGGACGTAAACTAGCT-3 ' (SEQ ID No.5)
PCR reaction system:
PCR response procedures: 95 DEG C, 3 min；94 DEG C, 1 min of 35 circulations, 54 DEG C, 1 min, 72 DEG C, 1 min；72℃,10
min。
The PCR product obtained after amplification is detected with 1.2 % agarose gel electrophoresis.Plant expression vector P1304-T106-
P PCR qualification result plant expression vector P1304-T106-P PCR as shown in Figure 6 identifies electrophoretogram, to P1304-T106-P into
Row verifying, it is ensured that promoter fragment is correctly connected to the upstream of gus gene.
The identification of 3.2 double digestions
Double digestion identification is carried out to the binary vector P1304-106-P built using restriction enzyme Hind III and Nco I.
Digestion operation is carried out by 3.4 digestion systems in embodiment one and reaction condition.Plant expression vector P1304-T106-P and
Carrier pCambia1304 double digestion qualification result plant expression vector P1304-T106-P as shown in Figure 7 and carrier
PCambia1304 double digestion identifies electrophoretogram.
Embodiment 3
Recombinant plasmid transformed EHA105 Agrobacterium
1, electricity turns the preparation of EHA105 Agrobacterium
Specific operation process is as follows:
1) bacterial strain EHA105, YM plate (25 mg/L containing Rif) scribing line culture Agrobacterium EHA105 are taken out from -80 DEG C of refrigerators.
2) picking single bacterium is fallen in 2 mL YM fluid nutrient mediums (25 mg/L containing Rif), and 28 DEG C are activated overnight.
3) bacterium solution after taking 1 mL to activate is inoculated in 50 mL YM fluid nutrient mediums, and 28 DEG C, 200 rpm cultivate to OD value
For 0.4-0.5.
4) bacterium solution is transferred in sterile 50 mL centrifuge tube, 5000 rpm, 4 DEG C of 10 min of centrifugation, abandons liquid, sterilized in 20 mL
And the agrobatcerium cell that suspends in 0.1 M NaCl of pre-cooling.
5) after ice sets 30 min, 5000 rpm, 4 DEG C of 10 min of centrifugation abandon liquid, collect thallus.
6) 10 % glycerol after 2 mL sterilizing is added and is pre-chilled, are dispensed by 100 μ L to clean and sterile 1.5 mL centrifuge tube
In, it is put into liquid nitrogen freezes rapidly, stored in -80 DEG C.
2, Agrobacterium EHA105 is converted
With electric shocking method by the recombinant plasmid transformed correctly constructed into Agrobacterium EHA105, specific operation process is as follows:
1) electric shock cup is immersed in the alcohol of 200 mL of 70 %, is then used sterile water rinse 3-5 times, is placed on ice after drying
It is spare.
2) the EP pipe for filling 100 μ L competence Agrobacterium EHA105 cells is taken out from liquid nitrogen container, ice bath thaws.
3) 2 μ L of recombinant plasmid is added, mixes gently, 30 min of ice bath.
4) mixed liquor is taken out, is transferred in electric shock cup precooled in advance, electric shock cup is put into electric shock tank, 2100V high pressure
Electric shock, pins the shock button in equipment with hand, is that electric shock is completed when hearing a sound.
5) cup that shocks by electricity is taken out, the YM fluid nutrient medium of 700 μ L pre-cooling is added, gently piping and druming mixes, and suction bacterium solution is transferred to dry
In net sterile 1.5 mL centrifuge tube.
6) 28 DEG C, 200 rpm shaking table recovery 2-4 h.
7) 10000 rpm are centrifuged 1 min, collect thallus, sop up 500 μ L supernatants, are blown and beaten with pipette tips and thallus is resuspended, piping and druming
100 μ L bacterium solutions are taken to be coated on the YM plate containing 50 mg/L of Rif 25 mg/L and kana after uniformly, 28 DEG C of inversion cultures.
3, plant expression vector imports the PCR identification of Agrobacterium
Whether bacterium colony PCR detection recombinant plasmid is successfully transferred in Agrobacterium, extracts plasmid.Primer P1304-106-P-F(SEQ ID
No.4)/P1304-106-P-R(SEQ ID No.5).PCR reaction is by PCR reaction system and reaction described in embodiment 2 3.1
Program carries out PCR operation.Bacterium solution PCR qualification result is planted as shown in Figure 8 after plant expression vector P1304-T106-P turns Agrobacterium
Object expression vector P1304-T106-P turns bacterium solution PCR identification electrophoretogram after Agrobacterium.
Embodiment 4
Plant Tissue Breeding
1, seed disinfection
Firstly, taking 50 mL centrifuge tubes after a sterilizing, take tomato seeds into centrifuge tube, the ethyl alcohol of 75 % is added, gently shakes
30 s are shaken, ethyl alcohol is outwelled, is added sterile water rinse 1-2 times, adds appropriate 5 % sodium hypochlorite (NaClO) after outwelling sterile water
Solution jiggles 20 min.Then, with sterile water rinse 5-6 times, until it can't smell the taste of sodium hypochlorite.Finally,
Seed after disinfection is placed on the culture dish for being covered with aseptic filter paper, uncaps and dries up in superclean bench.
2, the hair seedling of tomato
The culture medium of culture aseptic tomato seedling is named as FM.Culture medium prescription are as follows:+8 g/L agar of 1/2 MS+30 g/L sucrose,
Medium pH=5.8.It is dispensed into tissue culture bottle, every bottle of 30 mL, 121 DEG C of 20 min of sterilizing in autoclave is put into after packing.Wait train
Support 20 aseptic tomato seeds of every bottle of inoculation after base solidifies, 25 DEG C of illumination cultivations in growth cabinet.The tomato as shown in Fig. 9 A
The hair seedling of seed.
3, the culture of callus
After seed grows about 10-15 days in hair seedling culture medium, tomato seedling leaf is taken to carry out cutting seedling.In the glass for being covered with aseptic filter paper
A small amount of sterile water is poured into glass culture dish, moistens the filter paper in culture dish, is taken out the tomato seedling leaf in tissue culture bottle with tweezers, is put
On filter paper, blade is cut into the tissue block of about 2-5 mm × 2-5 mm size, there is the edge of a knife in 4 face of tomato leaf.The leaf cut
Piece is connected on induced medium (YD), and tissue block gap is about 5 mm or so.The formula of the culture medium of callus induction is MS+2
+ 8 g/L agar of mg/L ZT+0.2 mg/L IAA+30 g/L sucrose.After sealed membrane sealing, labeled species title, culture medium name
Title and date etc., are cultivated in tissue culture room, and after 1 week one subculture of replacement, culture 3-4 weeks, callus be can be used to
Carry out subsequent genetic transformation operation.The induction of the Tomato Calli as shown in Fig. 9 B.
Embodiment 5
The transfection and co-cultivation of recombinational agrobacterium
1, the preparation of recombinational agrobacterium
The preparation of 1.1 culture mediums
Quantitative YM dry powder is weighed according to the specification of YM culture medium, is put into the deionized water of 1 L and dissolves, and pours into the bottle of 1 L
121 DEG C of 20 min of high-temperature sterilization in son.It takes out and cools after the completion of sterilizing, 50 mg/L kanamycins and 50 mg/L benefit good fortune are added
It is flat.Re-suspension liquid is fluid nutrient medium, and 20 g/L D-Glucoses and 10 g/L sucrose, 117 DEG C of height are added in MS basal medium
20 min of temperature sterilizing.
1.2 shake bacterium
When callus tissue culture is good, sterile tissue culture bottle is taken, the YM fluid nutrient medium of about 50 mL is added, to fluid nutrient medium
In add 500-1000 μ L Agrobacterium mother liquor, sealed membrane sealing after, 200 r/min, 28 DEG C are incubated overnight.
1.3 centrifugations are resuspended
When the OD value of Agrobacterium bacterium solution reaches between 0.6-0.8, culture bottle is taken out, is poured into bacterium solution in super-clean bench sterile
In 50 mL centrifuge tubes, 5000 r/min, 4 DEG C of 12 min of centrifugation.Centrifuge tube is taken out after centrifugation, is outwelled on superclean bench
Clear liquid, be added re-suspension liquid, be resuspended to OD value 0.3 or so, the AS(acetosyringone of 100 μm of ol/L concentration is added) mix it is spare.
2, it transfects
It takes 100 sterile mL wide-mouth triangular flasks that callus is put into triangular flask in superclean bench, pours into appropriate weight
The bacterium solution of AS is hanged and has added, Agrobacterium bacterium solution is more than 3 mm of callus or so, after sealed membrane sealing, 100 rpm, 20 DEG C
Rock 30 min.Organization material is placed in superclean bench in the culture dish for be covered with aseptic filter paper and is air-dried.
3, it co-cultures
Culture medium for co-culturing is named as GP.The formula of GP culture medium is+10 g/L glucose+8 of MS+20 g/L sucrose
+ 100 μm of ol/L AS of g/L agar.118 DEG C of 20 min of sterilizing in high-pressure sterilizing pot, wherein AS needs to be added after sterilization.Screening
Before, sterile filter paper first is opened on each co-culture medium upper berth one, then the material after infecting is connected on culture medium, 18 DEG C
Dark culture 2-3 days.
4, it regenerates and takes root
4.1 screening
Screening and culturing medium is named as SX.Screening and culturing based formulas is MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose+8
20 mg/L Hyg of+600 mg/L cef+ of g/L agar.Wherein IAA, Cef and Hyg will add again after medium sterilization.It will be total to
Callus after culture is inoculated into screening and culturing medium, is placed in illumination box and is cultivated, the primary sieve of replacement in 15 days or so
Culture medium is selected, is replaced 2 times altogether.The resistance screening as shown in Fig. 9 C.
4.2 differentiation
Differential medium SR1 are as follows:+400 mg/L of+8 g/L agar of MS+2 mg/L ZT+0.2 mg/L IAA+30 g/L sucrose
cef.Every 10 days subcultures are primary, newly sprout until growing.
4.3 taking root
When bud grows to 3-5 cm, bud is transferred in root media (ST).Prescription of rooting medium are as follows: 1/2MS+0.1 mg/L
IBA+150 mg/L Cef.IBA and cef is added after sterilizing, is dispensed into tissue culture bottle, daily 16 h of illumination, 26 DEG C of cultures, induction
It takes root.The root induction as shown in Fig. 9 D.
5, the Molecular Detection of tomato is converted
5.1 conversion tomato DNA are extracted
Transgene tomato seedling leaf DNA is extracted using CTAB method.Specific operation process is mentioned by the embodiment one 1 progress DNA
Extract operation.
5.2 transgene tomato seedling PCR verifying
Verify transgene tomato seedling and use primer P1304-106-P-F(SEQ ID No.4)/P1304-106-P-R(SEQ ID
No.5).PCR reaction carries out PCR operation by PCR reaction system described in embodiment 2 3.1 and response procedures.Transgene tomato is planted
The PCR of the PCR testing result Transgenic Tomato Plants as shown in Figure 10 of strain detects electrophoretogram, shows promoter T1061-P purpose
Gene is successfully transferred in tomato dna group, obtains 18 plants of P1304-106-P transgene tomato seedling altogether, wherein positive 12 plants of seedling,
Positive rate is 66.7%；The transgenic seedling that P1304-35S-P is obtained shares 30 plants, and positive seedling is 22 plants, positive rate 73.3%.
The positive rate for the Transgenic Tomato Plants that T106-P plant binary expression vector and 35S control group obtain has been above 60% or more.
6, the transplanting of transgene tomato seedling
The successful positive tomato seedling of well-developed in root media and conversion is moved into soil and is cultivated.It will be glued with clear water
Culture medium on root is rinsed well, prepares the cultivation soil that Nutrition Soil is mixed with sand 1:1, transgene tomato seedling plantlet of transplant is arrived
In cultivation soil.The strong sprout of transgene tomato seedling as shown in Figure 11 A.
7, transgene tomato seedling is inoculated with root-knot nematode
When Transgenic Tomato Plants wait move into cultivation soil grow fine, in the same circumstances, it is inoculated with 2000 respectively as ear
Beans root-knot nematode and Meloidogyne incognita second instar larvae.After the transgene tomato seedling as shown in Figure 11 B is inoculated with root-knot nematode 1 month.
Experimental example 6
The active tissue staining detection of GUS
1, tissue staining
Transgene tomato seedling is inoculated with Meloidogyne enterolobii 1 month, after Tomato Root System grows root knot, carries out the active tissue of GUS
Dyeing detection.The T106-P promoter and 35S promoter transgene tomato root tissue and overground part tissue for taking root knot are put into 1.5
It in mL centrifuge tube, is dyed using GUS dyeing liquor, 37 DEG C overnight.
GUS coloration result shows that T106-P promoter is at the root knot after Meloidogyne enterolobii infects by dye Lan Rutu
Shown in 12 A, the blade without the Tomato Root System and overground part that are infected by Meloidogyne enterolobii is not all by dye Lan Rutu
Shown in 12 A B.At the root knot after Meloidogyne incognita infects by dye indigo plant as shown in Figure 12 C, without by Root Knot
The Tomato Root System of nematode infection and the blade of overground part are all not by dye indigo plant as shown in Figure 12 C D.35S promoter transgenosis kind
Eggplant seedling is entire root system after Meloidogyne enterolobii infects, at root knot and at non-root knot all by dye indigo plant as shown in Figure 12 C.
2, the fluorescence quantitative PCR detection of gus gene
2.1 transgenic plant RNA are extracted
The extraction of plant tissue RNA uses Quan Shijin (Beijing) TransZol Up Plus RNA Kit.Specific operation process is such as
Under:
1) it after weighing the vegetable material of superfreeze, is transferred quickly in the mortar of Liquid nitrogen precooler, with pestle by plant material
Material is fully ground into powdered, and liquid nitrogen is continuously replenished in centre.
2) powder of grinding is transferred in centrifuge tube, 1 mL Transzol Up is added in every 100 mg sample, mixes.
3) 5 min are stored at room temperature.
4) chloroform of 0.2 mL is added in every pipe, acutely shakes 30 s, is incubated for 3 min at room temperature.
5) 12000 r, 4 DEG C of 15 min of centrifugation, collect top layer's colorless supernatant liquid (about 500 μ L) to 1.5 new mL from
In heart pipe.
6) isometric dehydrated alcohol is added, is gently mixed by inversion.
7) liquid after mixing is all added in centrifugal column, 14000 r room temperatures are centrifuged 30 s, discard efflux and (divide 2-3
Secondary progress).
8) plus 500 μ L CB9,14000 r of room temperature are centrifuged 30 s, discard efflux.
9) step 8 is repeated.
10) plus 500 μ L WB9,14000 r of room temperature are centrifuged 30 s, discard efflux.
11) step 10 is repeated.
12) 14000 r of room temperature is centrifuged 2 min, is stored at room temperature 10 min.
13) centrifugal column taking-up is put in RNase-free Tube, adds 30 μ L RNase-free Water in centrifugal column
Center, is stored at room temperature 1 min, and 14000 r of room temperature is centrifuged 1 min.
14) in BioMateTM3S spectrophotometer (Thermo Fisher Scientific, the U.S.), Agilent
2100(Agilent, the U.S.) and Qubit2.0(Thermo Fisher Scientific, the U.S.) on detect quality, guarantee RNA
Quality and integrality, OD standard reach 1.8≤OD260/280≤2.2.
15) RNA solution is saved backup as -80 DEG C.
The part that root knot is formed after the inoculation of promoter T106-P transgenic positive plant is No. 1 sample, does not form root knot
Root system be No. 2 samples, aerial part be No. 3 samples.In the positive plant of 35S transgenosis, the root system for having root knot to be formed is No. 4
Sample, the root system for not forming root knot is No. 5 samples, and aerial part is No. 6 samples.
2.2 cDNA synthesis
Using the PrimeScript RT reagent Kit with gDNA Eraser of precious biological (Dalian) Engineering Co., Ltd
(Perfect Real Time) kit carries out reverse transcription reaction, and synthesis detection cDNA used, the amount of the total serum IgE of reversion is 1
μg.Specific operation process is as follows:
1) it is formulated as follows reaction system:
2) 42 DEG C, 2 min(or react 5 min at room temperature)
3) 4 DEG C of preservations
4) it is formulated as follows reaction system:
It after reagent is mixed, is placed in PCR instrument and is reacted, reaction condition is 37 DEG C, 15 min；85 DEG C, 5 s；4 DEG C, 1
min.The cDNA of reverse transcription -20 DEG C are placed in after reaction to save backup.
2.3 fluorescence quantitative PCR detection
Fluorescence quantitative PCR detectionGUSThe primer sequence of gene is as follows:
GUS- F:ACACCGACATGTGGAGTGAA(SEQ ID No.6)
GUS- R:TCATTGTTTGCCTCCCTGCT(SEQ ID No.7)
Actin- F:TGTCCCTATTTACGAGGGTTATGC(SEQ ID No.8)
Actin- R:AGTTAAATCACGACCAGCAAGAT(SEQ ID No.9)
Quantitative fluorescent PCR reaction system
The response parameter of quantitative fluorescent PCR is arranged
Amplification cycles parameter: 95 DEG C of 3 min of initial denaturation；95 DEG C of 20 s of denaturation；59 DEG C of annealing extend 20 s；65 DEG C of acquisition fluorescence letters
Number；Recurring number is 44 times.
Solubility curve parameter: heating up from 59 DEG C, and temperature is every to promote 0.5 DEG C for a circle collection fluorescence signal, altogether
Acquire the fluorescence signal of 80 circulations.3 repetitions are arranged in each sample, record Ct value, calculate relative expression levels.
Quantitative fluorescent PCR data processing, with 2—△CtMethod is rightGUSThe expression quantity of gene is calculated.GUSGene it is opposite
Expression quantity=GUSGene C T value-Actin CT value.
Fluorescent quantitative PCR result fluorescence quantitative PCR detection as shown in figure 13GUSGene relative expression quantity.
T106-P promoter transgenic positive tomato plant is inoculated with after Meloidogyne enterolobii at root knot i.e. No. 1 sampleGUSGene relative expression quantity is 0.0057, root system i.e. No. 2 sample at non-root knotGUSGene relative expression quantity is 0.0015,
Overground part i.e. No. 3 sample after T106-P transgenic positive tomato plant inoculation Meloidogyne enterolobiiGUSGene relative expression
Amount is 0.0007；
35S promoter transgenic positive tomato plant is inoculated with after Meloidogyne enterolobii at root knot i.e. No. 4 samplesGUSGene phase
It is 0.0052 to expression quantity, root system i.e. No. 5 sample at non-root knotGUSGene relative expression quantity is 0.0048,35S promoter
The relative expression quantity of overground part i.e. No. 6 sample is 0.0051 after transgenic positive tomato plant inoculation Meloidogyne enterolobii.
From fluorescence quantitative PCR detectionGUSThe result of gene relative expression quantity can be seen that in T106-P promoter transgenosis
In tomato plant, at root knotGUSGene relative expression quantity is significantly higher than root system and overground part at no root knot,GUSGene exists
The overground part of T106-P Transgenic Tomato Plants and expression is not detected substantially without root system at root knot.And in 35S promoter
In Transgenic Tomato Plants, root system and overground part at root knot, without root knotGUSGene relative expression quantity no significant difference,GUS
Gene relative expression quantity is almost the same.
The above results show T106-P promoter, and the expression in plant tissue each section is different from 35S promoter, phase
Compared with 35S constitutive promoter, by root-knot nematode induction root knot specificity promoter T106-P root knot it is special, stablize table
It reaches.
The above examples are only used to illustrate the technical scheme of the present invention rather than limits, although referring to preferred embodiment to this hair
It is bright to be described in detail, those skilled in the art should understand that, it can modify to technical solution of the present invention
Or equivalent replacement, without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110>Yunnan Prov Agriculture University
<120>a kind of root knot specificity promoter T106-P and application by root-knot nematode induction
<130> 2019
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 2626
<212> DNA
<213>T106-P promoter sequence
<400> 1
tgttcttttt catcatatat ttgttcgtat tatatggtgt ttttctattt ttataattag 60
ttaaaaataa tttttttaaa tcaagaatta tggtcctagt tttagtattt tattttatat 120
aaattttttt ttatatctag gcatgtacac atgtgatctt ttatattttt cccaagatgc 180
aacccatctt tagttttaag tatgcattta taaatttaat ttttgtttca aattttattt 240
gacttttggg tagattttac ttgagaaaaa aatgaaaacc aaaaaagtag tcgctttaat 300
atcaaagtag tagttttatt gttgtttttc ttttttctaa aaggaaccaa atttttttta 360
aaatcatacc cgataacatg tttttttttc tttttcgata acatgttttt aggtgattgt 420
agacattgaa aataactcat atttgattaa tgagatatct ttctttccta ttacttatat 480
aattctatta aatatataaa atttcactca tacgtttatg ttattcatat aaaattaaaa 540
agaaaaaaat tatcattttg aatttatttc ttttaaaaga cttatgctga ggatgaacaa 600
attatatatt tgaaacatgt gaaaagataa tattagcaac ttgttattgt tgttgactaa 660
caaatcttta taatattcgg ttaaaatctt ataatcacaa atattcattt ataaaagaaa 720
tataaaatta tgtgattcat aaatatgaca ttttaaacta tttttatgtc ttgtttatga 780
actataattg attatttagt agtattatat aaattttacc agttattaca atttatggag 840
ttttttttta taaaaaaata tacaacttta aattttaaat tacttaaata aaatttcaac 900
tttatattat aatttaattt aacatcataa tttcatactg tctgtccaat gacatatcgt 960
gtttgacttg gttggggaca ttttgttgtt catcttgtat ttcaaacata cttagaacaa 1020
aactttgaac atctttgtaa gaaaacaaag tttaagaaca ttttcacaac tagaaaatta 1080
aaactagtag cgaaactaga atcagaaaca gatttaaaaa aaatatacga aatatttttc 1140
ttaaagaaga attgttatta atatgtgtct aaagaatctt actgattcca acaaactttg 1200
cagaaacacg aagttaccaa gtttaatgaa ccagtgaaga acaaaagaat agaagaactg 1260
aaattaattc acaaatctaa agtttgtaaa acacgtacca gaatttggaa aattttaaaa 1320
ggaaaaggat caagtccact gaattcacag tgtcccctta aggaaattat tcccctctag 1380
tatccgaggt ttgatttgga atatgacctc ccagggtaaa atgatctcaa tcaccagagt 1440
atagatacca aaaactccgg tgtcagcgag ccactcaacg gcaataaagt acacttagca 1500
tactagattt agtagttgaa taagaaatcc atgaattcta tttaaaatga gaggaaatcc 1560
ctcaatttat agaaaacaaa gaaaagtgcg aaaaggttct tattgtgcct taccggaaag 1620
gtcacaaacc tttggaaaag tcacaatgtt tcagaaaggt cgtcaccttt cataaaagtc 1680
acaacttacc ataaaagtca caacttttca taaaagtcac aacatttcat aaaagtcacg 1740
actcttcata aaagtcacaa catttcataa aagtcacaac tcttcataaa agtcttcata 1800
aaagtcgcaa ctatttattt tccattcaca cctttttaaa atccaacaat cccttgcatg 1860
aatgtggaat gactcgaaga caaagaaacg gacaagtatg tgtactttac aagcaagaac 1920
taattgcatc tggataagta ggtttctcct tggactttcc gtagtgaaca tatgttggat 1980
atactcgaaa aatcggtaga tgcgatattt ttgaaccgtc gaactttggt gtatacctag 2040
acaaccatat gtcacacaat taactcttta ccatttgtgg ttcttacggt tgtgttcgtt 2100
ttatcaatga acacctcctg gtttcatgag tgtatagaga gatggacttt tgacaatcat 2160
cttctttgaa gcggcttaca cttcacactc acataggtga tttctaaccg tgttatcgcg 2220
tagatatact atttggtcaa ctttgccaaa cttagcaaat cattaaaacc attaatcttt 2280
attaactcat taacaaacct taatgttgta tccttgtccc tgagcattgt cttcatcatg 2340
agaatggatt gagtttattg acaatgctga accgtcattc acaactttat ttttctcctt 2400
gaatctagct cttgggatct ccagtctgct agatagagtt atcgccatga tgacttgtcc 2460
taagccgtaa attcattctc ttggatgatc ttttaacttt ctctctagtt aggtcttttt 2520
gtaagtggat ccgacacatt atcctttaac tttacatagt caattctgat aattccacta 2580
gagagtagtt ttctaacagt atcatgtcta tgtcgtatat gacgag 2626
<210> 2
<211> 33
<212> DNA
<213>primer T106-P-F
<400> 2
cccaagcttg agcatggagc atgacgtata acg 33
<210> 3
<211> 33
<212> DNA
<213>primer T106-P-R
<400> 3
catgccatgg aactttggaa tcctatgcct ttg 33
<210> 4
<211> 23
<212> DNA
<213>primer P1304-106-P-F
<400> 4
tttctcaagt aaaatctacc caa 23
<210> 5
<211> 22
<212> DNA
<213>primer P1304-106-P-R
<400> 5
ttctacagga cgtaaactag ct 22
<210> 6
<211> 20
<212> DNA
<213> GUS-F
<400> 6
acaccgacat gtggagtgaa 20
<210> 7
<211> 20
<212> DNA
<213> GUS-R
<400> 7
tcattgtttg cctccctgct 20
<210> 8
<211> 24
<212> DNA
<213> Actin-F
<400> 8
tgtccctatt tacgagggtt atgc 24
<210> 9
<211> 23
<212> DNA
<213> Actin-R
<400> 9
agttaaatca cgaccagcaa gat 23
Claims (5)
1. a kind of root knot specificity promoter T106-P induced by root-knot nematode, it is characterised in that described to be lured by root-knot nematode
The root knot specificity promoter T106-P led nucleotide sequence shown in SEQ ID NO:1 is constituted.
2. one group for expanding the primer of the root knot specificity promoter T106-P described in claim 1 by root-knot nematode induction
It is right, it is characterised in that the primer pair includes the first primer and the second primer, the sequence of the first primer such as SEQ ID
Shown in NO:2, the sequence of second primer is as shown in SEQ ID NO:3.
3. a kind of recombinant expression containing the root knot specificity promoter T106-P described in claim 1 by root-knot nematode induction
Carrier, it is characterised in that the recombinant expression carrier is that the recombinant plasmid T106-P-TA that will be constructed is usedHindIII HeNcoI pair
Digestion, glue recycle Insert Fragment T106-P promoter, use simultaneouslyHindIII HeNcoI double digestion pCambia1304 carrier, recycling
Remove the pCambia1304 carrier segments of 35S promoter, connection Insert Fragment T106-P promoter and removal 35S promoter
PCambia1304 carrier segments obtain recombinant plasmid P1304-T106-P；It is described by root in the recombinant expression carrier
The root knot specificity promoter T106-P of knot nematode-inducible is connected toGUSThe upstream of gene.
4. a kind of expression cassette, it is characterised in that the expression cassette includes described in claim 1 by root-knot nematode induction
Root knot specificity promoter T106-P.
5. a kind of application of the root knot specificity promoter T106-P described in claim 1 by root-knot nematode induction, feature
It is that the root knot specificity promoter T106-P by root-knot nematode induction is obtaining turning for safe and efficient anti-root-knot nematode
Application in gene plant kind.
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| CN1222196A (en) * | 1996-06-04 | 1999-07-07 | 莫根国际股份有限公司 | Nematode-inducible plant gene promotor |
| WO1998031822A1 (en) * | 1997-01-20 | 1998-07-23 | Plant Genetic Systems, N.V. | Pathogen-induced plant promoters |
| CA2674495A1 (en) * | 2007-02-06 | 2008-08-14 | Basf Plant Science Gmbh | Nematode inducible plant mtn3-like gene promoters and regulatory elements |
| CN102812025A (en) * | 2009-12-04 | 2012-12-05 | 先正达参股股份有限公司 | Spiro Fused 1 -amino - Piperidine Pyrrolidine Dione Derivatives With Pesticidal Activity |
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