CN104988175B - Application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved - Google Patents
Application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved Download PDFInfo
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Abstract
The invention discloses a kind of application of tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved.Comprise the following steps：(1) the Agrobacterium tumefaciems engineering bacteria B of Agrobacterium tumefaciems engineering bacteria A and HsfA1a containing the tomato gene silencing vector of the gene overexpression carriers of HsfA1a containing tomato is built；(2) by the Agrobacterium tumefaciems engineering bacteria A mediated transformation target plant explants, HsfA1a gene transgenic plant are prepared；The Agrobacterium tumefaciems engineering bacteria B is contaminated into target plant cotyledon, HsfA1a gene silencing plant are prepared；(3) the HsfA1a gene transgenics plant and HsfA1a gene silencings plant are subjected to Osmotic treatment, observe the number and drought resisting phenotype of plant autophagosome.Present invention research is found, arid can induce the formation of wild-type tomatoes autophagosome, the induction of autophagosome is hindered after HsfA1a gene silencings, and HsfA1a gene overexpressions can dramatically increase autophagosome activity, therefore under drought stress, tomato HsfA1a is by inducing autophagosome to be formed so as to strengthen its drought resistance.
Description
Technical field
The invention belongs to biological technical field, specifically relate to a kind of tomato HsfA1a genes improve plant autophagosome activity and
Application in drought resistance.
Background technology
Since 21 century, modern agricultural development has become the main flow of world agriculture development.Industrialized agriculture is as existing
For agriculture principal mode, have become various countries' agricultural development emphasis.According to statistics, the China's Vegetable output value in 2011 reaches 1.26 trillion
Member, first more than total grain output value, turn into the maximum agricultural product of the Chinese output value.The overall development level of China's industrialized agriculture is also not
Height, facility structure is simple, and fund input deficiency, institution falls behind, the ability withstood natural calamities, in recent years, global gas
Wait and deteriorate, carbon dioxide raises, global warming, and the frequent natural calamity such as arid occurs, and the yield and quality of agricultural are made
Into great threat.
Tomato (Solanum lycopersicum L.) is annual or perennial in Solanaceae (Solanaceae) Solanum
Herbaceous plant, alias tomato, tomato, it is the vegetable crop that cultivation is most wide in world wide, consumption figure is maximum.Tomato variety
Type is enriched, and can not only eat raw and make vegetables but also can make fruit, and the lycopene composition in tomato can prevent various cancers, be in the world
One of most popular healthy food.Tomato occupies critical role in facilities vegetable, therefore improves tomato reply poor environment
Ability, cultivating resistant variety has important actual production meaning.
Thermal excited transcryption factor (Heat stress transcription factor, Hsf) is widely present in animal and plant body
It is interior, it is the signal of interest transduction element of the environment stresses such as plant response high temperature, specific heat shock element (Heat can be identified
Stress elements, HSEs), the expression of heat shock protein (heat shock proteins, Hsps) is mainly adjusted, in albumen
Heat shock protein plays an important role as important molecular chaperones in folding, distribution and the degraded of matter.When plant cell by
After such as high temperature, low temperature, arid, machinery wound adverse circumstance, Hsf can be phosphorylated activation, go to nucleus and downstream stress response base
Because of the HSE specific bindings in promoter, accelerate the transcriptional expression of these genes, environment stress is resisted so as to strengthen plant
Property.Plant Hsf is a complicated gene family, there is 21 in arabidopsis, has 25 in rice, tomato has 24.According to
Their structure is classified as the class of A, B and C tri-.
The research to Hsf is concentrated mainly in its response to environment stress and the regulation and control to downstream heat shock protein at present.
AtHsfA1a, AtHsfA1b of arabidopsis can make the heat resistance of plant notable by high temperature induction, and after AtHsfA2 gene delections
Decline, the research on the Hsf of tomato is concentrated mainly on A races and its effect in high temperature stress.Tomato HsfA1a is in high temperature
It is main regulatory factor in stress, under heat stress, HsfA1a, HsfA2 and HsfB1 can combine to form tripolymer induction
The Hsp such as Hsp70 and Hsp90 expression, the HsfA2 and HsfB1 expression after heat shock of HsfA1a RNAi plant significantly reduce, because
This heat resistance weakens.Effects of the tomato HsfA1a in other stress beyond high temperature is not yet reported, therefore is furtherd investigate
HsfA1a function, there is certain theory directive significance to research and development resistant variety.
The content of the invention
The present invention provides a kind of application of tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved, research
Applications of the tomato HsfA1a in other stress beyond high temperature.
Application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved.Comprise the following steps：
(1) Agrobacterium tumefaciems engineering bacteria A and HsfA1a containing the tomato base of the gene overexpression carriers of HsfA1a containing tomato is built
Because of the Agrobacterium tumefaciems engineering bacteria B of silent carrier；
(2) by the Agrobacterium tumefaciems engineering bacteria A mediated transformation target plant explants, HsfA1a genes is prepared and turn
Gene plant；The Agrobacterium tumefaciems engineering bacteria B is contaminated into target plant cotyledon, HsfA1a gene silencing plant are prepared；
(3) the HsfA1a gene transgenics plant and HsfA1a gene silencings plant are subjected to drought stress processing, seen
Examine the number and drought resisting phenotype of plant autophagosome.
The application of the present invention refers to base sequence such as SEQ TD NO：The albumen of HsfA1a gene codes shown in 1 is regulating and controlling
The application of at least one of plant autophagosome activity and drought resistance.Autophagosome number is embodied in increase or decrease and do
Non-irrigated resistance enhancing reduces.
Expression quantity of the HsfA1a genes in plant is lower, under drought stress in the plant leaf blade autophagosome number
It is fewer, it is more sensitive to arid；Expression quantity of the HsfA1a genes in the plant is higher, the plant leaf blade under drought stress
The number of interior autophagosome is more, strengthens the resistance of arid.
The method provided by the present invention for cultivating drought resisting genetically modified plants, particularly may be divided into following (A) or (B)；
(A) genetically modified plants with the increase of autophagosome number and/or plant drought resistance enhancing purpose character are cultivated, including
Following steps：
A) base sequence such as SEQ TD NO are imported into target plant：HsfA1a genes shown in 1 is overexpressed, and is obtained
The transfer-gen plant of HsfA1a gene overexpressions；
B) by the transfer-gen plant of HsfA1a gene overexpressions compared with untreated target plant, obtain with autophagosome
Number increases and/or the genetically modified plants of plant drought resistance enhancing purpose character.
(B) genetically modified plants for weakening purpose character with the reduction of autophagosome number and/or plant drought resistance are cultivated, including
Following steps：
A) base sequence such as SEQ TD NO are imported into target plant：HsfA1a genes shown in 1 carries out suppression expression, obtains
To HsfA1a gene silencing transfer-gen plants；
B) by HsfA1a gene silencings transfer-gen plant compared with untreated target plant, obtain with autophagosome number
Reduction and/or plant drought resistance weaken the genetically modified plants of purpose character.
In the above method (A), HsfA1a genes can import purpose by the recombinant expression carrier containing the gene and plant
In thing, recombinant expression carrier can be carried with the derivative plant expression of existing pFGC5941, pCAMBIA1300 and pBI121 etc. or other
Body, during using plant expression vector construction recombinant vector, composing type, organizing specific type or inducible promoter can be used.
Right in purpose plant in the above method (B), HsfA1a genes carry out suppression expression, can be any reduce
The method of the expression of HsfA1a genes described in purpose plant.In the present invention, HsfA1a genes are carried out in purpose plant
It is what is realized by way of virus induced gene silencing to suppress expression.
The tomato HsfA1a genes in following two any one：
A, the base sequence of the tomato HsfA1a genes such as SEQ TD NO：Shown in 1；
B, its any base sequence and SEQ TD NO：Sequence shown in 1 has more than 90% homology and coding such as SEQ TD
NO：The DNA molecular of amino acid sequence shown in 2.
The preparation method of Agrobacterium tumefaciems engineering bacteria A and Agrobacterium tumefaciems the engineering bacteria B is as follows：
(a) total serum IgE is extracted from tomato young leaflet tablet；
(b) by the tomato total serum IgE reverse transcription obtained in step (a) into cDNA；
(c) cDNA obtained using step (b) is template, SlHsfA1aOEF (SEQ TD NO：And SlHsfA1aOER 3)
(SEQ TD NO：4) HsfA1a genes are expanded for primer (restriction enzyme site containing AscI and KpnI), be connected to
PFGC1008-HA carriers obtain OE carriers；SlHsfA1aF(SEQ TD NO：And SlHsfA1aR (SEQ TD NO 5)：6) it is primer
(restriction enzyme site containing XbaI and BamHI) expands to HsfA1a genes, is connected to pTRV2 carriers and obtains VIGS carriers；
(d) the OE carriers that step (c) obtains are transferred in Agrobacterium tumefaciems EHA105, obtain the mistake of gene plant containing HsfA1a
The Agrobacterium tumefaciems engineering bacteria A of expression vector；The VIGS carriers that step (c) obtains are transferred in Agrobacterium tumefaciems GV3101, obtained
The Agrobacterium tumefaciems engineering bacteria B of HsfA1a gene silencing vectors must be contained.
Total serum IgE is extracted from tomato young leaflet tablet and uses Tiangen Plant total RNA extraction kit,
Its step is：
(1) take 0.1g blades to be ground in liquid nitrogen, add 1mL lysates, be vortexed and mix；
(2) equal slurry samples are placed into 5min at 15-30 DEG C so that nucleic acid-protein compound is kept completely separate；
(3) 4 DEG C, 12,000rpm centrifugation 5min, supernatant is abandoned, is transferred in a new centrifuge tube without RNase.
(4) 200 μ L chloroforms are added, cover lid, acutely shake 15s, room temperature places 3min；
(5) 4 DEG C, 12,000rpm centrifugation 10min, sample can be divided into three layers：The organic phase of yellow, intermediate layer and colourless
Aqueous phase, for RNA mainly in aqueous phase, the volume of aqueous phase is about the 50% of lysate RZ reagents used.Aqueous phase is transferred in new pipe,
Carry out next step operation；
(6) absolute ethyl alcohol of 0.5 times of volume is slowly added to, well mixed (now agreeing precipitation can occur).By what is obtained
Solution and precipitation are transferred to adsorption column CR3 together, 4 DEG C, 12,000rpm centrifugation 30s, discard the waste liquid in collecting pipe；
(7) 500 μ L protein liquid removals RD are added into adsorption column CR3,4 DEG C, 12,000rpm centrifugation 30s, abandon waste liquid；
(8) 600 μ L rinsing liquid RW are added into adsorption column CR3, the static 2min of room temperature, 4 DEG C, 12,000rpm centrifuge 30s,
Abandon waste liquid；
(9) step (8) is repeated；
(10) adsorption column is put into 2mL collecting pipes, 4 DEG C, 12,000rpm centrifugation 2min, removes remaining waste liquid；
(11) adsorption column CR3 is transferred in a new centrifuge tube, adds 50 μ L RNase-Free ddH2O, room temperature are put
2min is put, 4 DEG C, 12,000rpm centrifuge 2min；
(12) OD is determined with ultraviolet specrophotometer260/OD280Checking R NA sample sizes and purity, and use Ago-Gel
Electrophoresis is tested to RNA quality.
The reverse transcription of tomato total serum IgE is used into Toyobo ReverTra Ace qPCR RT Kit into cDNA.
The primer sequence is as follows：
SlHsfA1aOEF：5′-TTGGCGCGCCATGGAGCCGAATTCTTAT-3′(SEQ TD NO：3),
SlHsfA1aOER sequence is：5′-GGGGTACCGATCATATGTTTTTGTTG-3′(SEQ TD NO：4),
SlHsfA1aF：5′-GCTCTAGACATCTCAGTCATCATCTC-3′(SEQ TD NO：5),
SlHsfA1aR：5′-CGCGGATCCGCGCCGTCTGCAACATTG-3′(SEQ TD NO：6).
The observational technique of autophagosome number is as follows in the present invention：
Using single red sulphonyl cadaverine (monodansylcadaverine, MDC) dyeing and transmission electron microscope (transmission
Electron microscopy, TEM).
MDC is dyed, and specific method is：
(1) tomato leaf is cut into 2mm × 4mm small pieces, inserts in 100 μM of MDC solution, vacuumizes, dark lower placement
30min；
(2) washed twice with PBS, be finally placed in PBS, autophagosome with the Laser Scanning Confocal Microscopes of LSM 780 (Zeiss,
Germany) observation.
TEM method is：
(1) tomato leaf is cut into 1mm × 3mm small pieces, stayed overnight with 2.5% 4 DEG C of fixations of glutaraldehyde；
(2) 0.1M is used, pH7.0 phosphate buffer washs three times, each 15min；
(3) 1-2h is fixed with 1% osmic acid；With 0.1M, pH7.0 phosphate buffer washs three times, each 15min；
(4) dewater treatment, every kind of concentration processing are carried out with 30%, 50%, 70%, 80%, 90% and 95% ethanol
15min, then with 100% Ethanol Treatment 20min, it is last excessively to pure acetone processing 20min；
(5) with embedding medium and acetone mixture (V/V=1/1) processing 1h；
(6) with embedding medium and acetone mixture (V/V=3/1) processing 3h；
(7) pure embedding medium processing is stayed overnight；
(8) will get up by the sample embedding for permeating processing, 70 DEG C are heated overnight, that is, obtain embedded sample, sample
Cut into slices in Leica EM UC7 type ultramicrotome, obtain 70-90nm section, cut into slices through lead citrate solution and acetic acid
The alcohol saturated solution of uranyl 50% respectively dyes 5-10min, is observed in Hitachi H-7650 (Japan) type transmission electron microscope.
The target plant is dicotyledon or monocotyledon, preferred dicotyledon tomato in of the invention.
Drought stress processing the step of be：
When HsfA1a gene transgenics plant and its blank control or HsfA1a gene silencings plant and its blank control grow
To five leaves wholeheartedly when, water to saturation, afterwards every the once respective relative soil moisture content and according to mistake of measurement in 1~4 day
Water, which keeps the skin wet, to be made between HsfA1a gene transgenics plant and its blank control or HsfA1a gene silencings plant is empty with it
Soil relative water content is consistent between white control, and soil is until processing terminates.
Soil relative water content is controlled to be gradually reduced from 100% to 30% in stress during drought stress.
10~15 days drought stress processing times.
It is further preferred that, drought stress processing time is 13 days in stress during drought stress every measurement in 1 day once.
HsfA1a gene transgenics plant is using wild-type tomatoes plant as blank control；HsfA1a gene silencings plant with
PTRV empty carriers transfection tomato plant is blank control.
Drought stress processing terminates the rear control group with not carrying out drought stress processing under identical planting conditions and is compared,
Observe HsfA1a gene transgenics plant, HsfA1a gene silencings plant and its respective blank control and without Stress treatment
The properties difference of plant.
Found through present invention research, the arid formation that can induce wild-type tomatoes autophagosome, after HsfA1a gene silencings
The induction of autophagosome is hindered, and HsfA1a gene overexpressions can dramatically increase autophagosome activity, therefore under drought stress, tomato
HsfA1a is by inducing autophagosome to be formed so as to strengthen its drought resistance.
Brief description of the drawings
Fig. 1 is HsfA1 races gene expression in HsfA1a gene silencing plant.
Fig. 2 is that Western blot detect HsfA1a overexpression plant.Wherein 1#, 2#, 3#, 4# represent four independent strains
System.
Fig. 3 is the phenotype under tomato HsfA1a silence plant drought stresses.PTRV is adjoining tree, and HsfA1a plants for silence
Strain, scale=10cm.
Fig. 4 is the phenotype that tomato HsfA1a is overexpressed under plant drought stress.WT is the wild-type tomatoes of non-transgenosis
Ailsa Craig, OE are that HsfA1a is overexpressed plant, and 1# and 3# are two strains.Scale=10cm.
Fig. 5 is tomato HsfA1a silences plant and the electrical conductivity being overexpressed under plant drought stress.PTRV is adjoining tree,
HsfA1a is silence plant；WT is that wild-type tomatoes the Ailsa Craig, OE of non-transgenosis are that HsfA1a is overexpressed plant, 1#
It is two strains with 3#.
Fig. 6 A and Fig. 6 B are that MDC is dyed under tomato HsfA1a silence plant drought stresses.Fig. 6 A are MDC coloration results, figure
6B is the statistical result to A figures.PTRV is adjoining tree, and HsfA1a is silence plant.Scale=25 μm.
Fig. 7 A and Fig. 7 B are that tomato HsfA1a is overexpressed MDC dyeing under plant drought stress.Fig. 7 A are MDC coloration results,
Fig. 7 B are the statistical result to A figures.WT is that wild-type tomatoes the Ailsa Craig, OE of non-transgenosis are that HsfA1a is overexpressed plant
Strain, 1# and 3# are two strains.Scale=25 μm.
Fig. 8 A and Fig. 8 B are TEM results under tomato HsfA1a silence plant drought stresses.A figures are representational TEM results,
B figures are the statistical result to A figures.Autophagosome is indicated with arrows, and pTRV is adjoining tree, and HsfA1a is silence plant.Scale=1
μm.Cp, chloroplaset；S, starch；V, vacuole.
Fig. 9 A and Fig. 9 B are that tomato HsfA1a is overexpressed TEM results under plant drought stress.Fig. 9 A tie for representational TEM
Fruit, Fig. 9 B are the statistical result to A figures.Autophagosome is indicated with arrows, and WT is the wild-type tomatoes Ailsa of non-transgenosis
Craig, OE are that HsfA1a is overexpressed plant, and 1# and 3# are two strains.Scale=1 μm.Cp, chloroplaset；S, starch；V, liquid
Bubble.
Embodiment
Institute is conventional method experimentally unless otherwise specified in following embodiments.
Experiment material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The present invention is further described below with reference to specific embodiment.
Embodiment 1
The clone of tomato HsfA1a genes and vector construction
1. tomato Total RNAs extraction
The total serum IgE of tomato young leaflet tablet is extracted using Tiangen Plant total RNA extraction kit, its
Step is：
(1) take 0.1g blades to be ground in liquid nitrogen, add 1mL lysates, handled afterwards with homogenate instrument；
(2) equal slurry samples are placed into 5min at 15-30 DEG C so that nucleic acid-protein compound is kept completely separate；
(3) 4 DEG C, 12,000rpm centrifugation 5min, supernatant is removed, is transferred in a new centrifuge tube without RNase.
(4) 200 μ L chloroforms are added, cover lid, acutely shake 15s, room temperature places 3min；
(5) 4 DEG C, 12,000rpm centrifugation 10min, sample can be divided into three layers：The organic phase of yellow, intermediate layer and colourless
Aqueous phase, for RNA mainly in aqueous phase, the volume of aqueous phase is about the 50% of lysate RZ reagents used.Aqueous phase is transferred in new pipe,
Carry out next step operation；
(6) absolute ethyl alcohol of 0.5 times of volume is slowly added to, well mixed (now agreeing precipitation can occur).By what is obtained
Solution and precipitation are transferred to adsorption column CR3 together, 4 DEG C of 12,000rpm centrifugation 30s, discard the waste liquid in collecting pipe；
(7) 500 μ L protein liquid removals RD are added into adsorption column CR3,4 DEG C, 12,000rpm centrifugation 30s, abandon waste liquid；
(8) 600 μ L rinsing liquid RW are added into adsorption column CR3, the static 2min of room temperature, 4 DEG C, 12,000rpm centrifuge 30s,
Abandon waste liquid；
(9) step (8) is repeated；
(10) adsorption column is put into 2mL collecting pipes, 4 DEG C, 12,000rpm centrifugation 2min, removes remaining waste liquid；
(11) adsorption column CR3 is transferred in a new centrifuge tube, adds 50 μ L RNase-Free ddH2O, room temperature are put
2min is put, 4 DEG C, 12,000rpm centrifuge 2min；
(12) OD is determined with ultraviolet specrophotometer260/OD280Checking R NA sample sizes and purity, concentration are 835ng/ μ
L, OD260/OD280=2.12.
2. gene cloning
Using Toyobo ReverTra Ace qPCR RT Kit, by 1 μ g tomato total serum IgE reverse transcriptions into cDNA.According to
The coded sequence of HsfA1a genes, design amplify the primer (SlHsfA1aOEF and SlHsfA1aOER in table 1) of complete frame,
And add restriction enzyme site (AscI and KpnI) respectively on primer；And VIGS primers (SlHsfA1aF in table 1 and
SlHsfA1aR restriction enzyme site (XbaI and BamHI)), and on primer is added, then HsfA1a genes are expanded
Increase, then digestion, be connected respectively to pFGC1008-HA and obtain over-express vector OE and be connected on pTRV2 carriers to obtain silence
Carrier VIGS.It is sequenced by Shanghai Sani bio tech ltd, sequencing result such as SEQ TD NO：Shown in 1, coded egg
Bai Xulie such as SEQ TD NO：Shown in 2, the results showed that the sequence announced in the sequence and solgenomics cloned
(Sl08g005170) it is consistent.
3. Agrobacterium tumefaciems engineering bacteria is built
OE carriers are transferred in Agrobacterium tumefaciems EHA105, obtain the crown gall agriculture of the gene overexpression carriers of HsfA1a containing tomato
Bacillus engineering bacteria A；VIGS carriers are transferred in Agrobacterium tumefaciems GV3101, obtain the root of the gene silencing vectors of HsfA1a containing tomato
Cancer Agrobacterium engineering bacteria B.
The SlHsfA1a of table 1 is overexpressed and VIGS primer sequence tables
Embodiment 2
Build tomato HsfA1a gene overexpression plant
1 culture aseptic seedling
Tomato seeds soak (or with 28 DEG C of 200r/min of shaking table) 6-8h with running water, then with 75% alcohol disinfecting
30sec, sterilizes 15min (using 28 DEG C of 200r/min of shaking table) in 10%NaClO afterwards, and sterile purified water is rinsed 3 times and shifted
To sterilizing vessel, 1/2MS culture mediums are inoculated in.Culture is transferred to illumination cultivation room, growth of seedling to germinateing under 25 DEG C of dark conditions
Condition is 25 DEG C, 16h illumination/8h is dark, intensity of illumination 1800lx.
2 prepare explant, culture Agrobacterium
6d after germination, the cotyledon of aseptic seedling is cut with knife, cotyledon carries a bit of petiole, inserts accompanied culture base
Preculture 1d in KCMS (lucifuge, overnight, accompanied culture overlong time easily cause to infect excessively).Containing antibiotic
Picking Agrobacterium single bacterium colony on LB flat boards, it is inoculated in the LB that 20mL contains antibiotic, 28 DEG C, 200r/min is incubated overnight to right
Number mid-term (OD600 ≈ 1.0, about 16-24h).Bacterium is first shaken, then cuts cotyledon (being inoculated between 12-20h).
3 transformation tissue cultures
3.1 infect
Cultured Agrobacterium tumefaciems engineering bacteria A bacterium solutions go to 10mL centrifuge tubes (sealed membrane sealing), 4 DEG C of 4000r/min
Centrifuge 10min；Culture medium is poured out, suspension medium MS0.2 is added, shakes up.The cotyledon explant of 3~4 wares has been transferred to
In MS0.2 sterilizing culture dish, pouring into the bacterium solution that has suspended, (7-8mLMS0.2, wherein 2-3mL fall to OD600 ≈ 0.2~0.3
Enter culture dish) secretly lower inoculation 4-5min, and gently rock culture dish.Explant is transferred in sterilizing filter paper, blotted residual
The bacterium solution stayed, on tieback to KCMS, 22 DEG C co-culture 2d (lucifuge).Reverse side is upward
3.2 selection cultures, regeneration
After co-cultivation, by explant being transferred on MRS1 (2Z+) carefully, it is transferred in MRS2 (0.2Z+) and cultivates after 2~3 weeks,
The material of pollution is cleared up in time during selection culture, until growing regeneration bud.
4. regeneration bud is taken root, transplanted
When bud length to be regenerated is to 1cm or so, bud is cut (it can not cut, in order to avoid injury position of taking root), it is put into training of taking root
Support and taken root in base.Hardening is carried out to good, the long transformation seedlings to 5cm or so of taking root after 2 weeks, transplanted into disposal plastic cup,
Transplanting obtains tomato HsfA1a gene overexpression plant into flowerpot after surviving.
Embodiment 3
Build tomato HsfA1a gene silencing plant
Culture medium and antibiotic compound method in the present embodiment is as follows：
The preparation of YEB culture mediums：5g beef extracts, 5g peptones, 1g yeast extracts, 5g sucrose, 0.5g MgSO4·
7H2O, 1L is settled to distilled water, regulation pH value is that 7.0,121 DEG C of autoclaving 20min are standby.Every liter of YEB solid mediums
Add agar powder 15g, it is other to be divided into the same fluid nutrient medium of part.
Kanamycins：50mg/mL, filtration sterilization, -20 DEG C of packing preserve.
Rifampin：50mg/mL methanol dissolves, filtration sterilization, and -20 DEG C of packing preserve.
Gentamicin：25mg/mL is purchased from Shanghai bioengineering Co., Ltd, -20 DEG C of preservations.
Infect liquid preparation：10mM magnesium chlorides, 10mM MES, pH=5.7, used time add 150 μM/L acetosyringones.
It is as follows to infect method：
(1) the Agrobacterium GV3101 (Agrobacterium tumefaciems engineering bacteria B) containing target gene is drawn into flat board, 36-48h occurs single
Bacterium colony；
(2) picking single bacterium falls within the 10mL pipes containing 4mL YEB culture mediums and shakes bacterium, and 200rpm shakes bacterium under the conditions of 28 DEG C
24h；
(3) 1 is pressed:100 ratio adds the bacterium solution of step (2) anti-containing kanamycins, rifampin, 3 kinds of gentamicin
Expand training in the 50mL YEB culture mediums of raw element to OD600=0.8-1.0 (about 12h)；
(4) 4 DEG C, 4000g, 10min is centrifuged, abandons supernatant；
(5) precooling aqua sterilisa 20mL is washed, 4000g, 4 DEG C, 10min centrifugations, abandons supernatant；
(6) repeat step (5) is once；
(7) precooling infect liquid dissolving be precipitated to OD600=1-1.5；
(8) room temperature places 3h, pTRV1：PTRV2-HsfA1a=1:1 mixed infection, pTRV1：PTRV2=1:1 mixing is invaded
Dye is as control；
(9) when tomato seedling two panels cotyledon planishes, infected with injection.
Tomato after infecting is placed in 22/19 DEG C, the 16h/8h photoperiods, 200 μm of ol m–2s–1The phjytotron training of light intensity
Support, obtain tomato HsfA1a gene silencing plant, while prepare pTRV empty carriers transfection tomato plant in case control, five leaves are wholeheartedly
When start Osmotic treatment.
Embodiment 4
The tomato HsfA1a gene silencings plant that embodiment 2 and embodiment 3 are prepared and the overexpression plant arid side of body
Compel processing
The specific method of tomato HsfA1a gene silencing plant drought stress processing is as follows：
PTRV empty carriers are transfected into tomato plant and tomato HsfA1a gene silencing plant are divided into two groups, one group is control group
(including pTRV blank controls and silence plant), one group is experimental group (comprising pTRV blank controls and silence plant).
When tomato grow to five leaves wholeheartedly when experimental group and control group while watering to saturation, control group plant is normally poured afterwards
Water, until experiment terminates；After experimental group relative soil moisture content (VWC) and root are measured once every 1 day ZigWSN recorder
Being kept the skin wet according to fluid loss is consistent VWC between silence plant and pTRV blank controls, until processing terminates, experimental group is whole
Relative soil moisture content is controlled to be slowly lowered to 30% or so from 100% in individual stress during drought stress, the drought stress time is 13
My god.
Tomato HsfA1a gene overexpression plant drought stress processing when experimental group and control group in non-transgenosis
Wild-type tomatoes Ailsa Craig are as blank control, and other processing are the same as tomato HsfA1a gene silencing plant.
Experiment terminates that electrical conductivity is taken pictures and determined after there is phenotype.As a result as shown in Fig. 1~5.
Fig. 1 is HsfA1 races gene expression in HsfA1a gene silencing plant.
Fig. 2 is that Western blot detect HsfA1a overexpression plant.Wherein 1#, 2#, 3#, 4# represent four independent strains
System.
Fig. 3 is the phenotype under tomato HsfA1a silence plant drought stresses.PTRV is adjoining tree, and HsfA1a plants for silence
Strain, scale=10cm.
Fig. 4 is the phenotype that tomato HsfA1a is overexpressed under plant drought stress.WT is the wild-type tomatoes of non-transgenosis
Ailsa Craig, OE are that HsfA1a is overexpressed plant, and 1# and 3# are two strains.Scale=10cm.
Fig. 5 is tomato HsfA1a silences plant and the electrical conductivity being overexpressed under plant drought stress.PTRV is adjoining tree,
HsfA1a is silence plant；WT is that wild-type tomatoes the Ailsa Craig, OE of non-transgenosis are that HsfA1a is overexpressed plant, 1#
It is two strains with 3#.
As seen from the figure, its expression quantity of HsfA1a silences plant is only the 40% of pTRV, and the expression of its homologous gene with
PTRV compares indifference, and it is HsfA1a single-gene silences to show silence plant.Western blotting results show,
Four strains of HsfA1aOE plant energy normal expression, and 1# and 3# expression quantity is higher.After Osmotic treatment, pTRV plant only bottom
Old leaf committee is here, and pTRV-HsfA1a plant seriously committee here, illustrate that significantly reduce tomato after silence HsfA1a resists to arid
Property.After Osmotic treatment, here WT plant there is serious committee, and HsfA1aOE-1# and HsfA1aOE-3# only lower blade yellow are withered
Listless, drought resistance is apparently higher than WT plant.Under normal moisture supply, the electrolytic leakage of all plant is similar, pTRV-
The electrolytic leakage of HsfA1a plant adds 58.7%, HsfA1aOE plant than pTRV and significantly improves drought resistance, together
WT is compared, and HsfA1aOE-1# and HsfA1aOE-3# electrolytic leakage reduce by 47.1% and 53.3% respectively.
Embodiment 5
Tomato HsfA1a gene silencings plant and the lower autophagosome Activity determination of overexpression plant arid
Stress during drought stress tomato HsfA1a gene silencings plant and is overexpressed plant drought stress 6 days with embodiment 4,
Sampling detects the activity of autophagosome with MDC dyeing and TEM, with the autophagosome of adjoining tree activity for 1, experimental result see Fig. 6~
Fig. 9.
Fig. 6 A and Fig. 6 B are that MDC is dyed under tomato HsfA1a silence plant drought stresses.Fig. 6 A are MDC coloration results, figure
6B is the statistical result to A figures.PTRV is adjoining tree, and HsfA1a is silence plant.Scale=25 μm；Fig. 7 A and Fig. 7 B are kind
Eggplant HsfA1a is overexpressed MDC under plant drought stress and dyed.Fig. 7 A are MDC coloration results, and Fig. 7 B are the statistical result to A figures.
WT is that wild-type tomatoes the Ailsa Craig, OE of non-transgenosis are that HsfA1a is overexpressed plant, and 1# and 3# are two strains.Mark
Chi=25 μm；Fig. 8 A and Fig. 8 B are TEM results under tomato HsfA1a silence plant drought stresses.A figures are representational TEM knots
Fruit, B figures are the statistical result to A figures.Autophagosome is indicated with arrows, and pTRV is adjoining tree, and HsfA1a is silence plant.Scale
=1 μm.Cp, chloroplaset；S, starch；V, vacuole；Fig. 9 A and Fig. 9 B are that tomato HsfA1a is overexpressed TEM knots under plant drought stress
Fruit.Fig. 9 A are representational TEM results, and Fig. 9 B are the statistical result to A figures.Autophagosome is indicated with arrows, and WT is non-transgenosis
Wild-type tomatoes Ailsa Craig, OE is that HsfA1a is overexpressed plant, and 1# and 3# are two strains.Scale=1 μm.Cp, leaf
Green body；S, starch；V, vacuole.
As a result show, arid can induce the formation of wild-type tomatoes autophagosome, be hindered after HsfA1a gene silencings certainly
The induction of body is bitten, and HsfA1a gene overexpressions can dramatically increase autophagosome activity, therefore under drought stress, tomato HsfA1a leads to
Induction autophagosome is crossed to be formed so as to strengthen its drought resistance.
Claims (6)
1. application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved, the plant is tomato.
2. application as claimed in claim 1, it is characterised in that comprise the following steps：
(1) Agrobacterium tumefaciems engineering bacteria A and HsfA1a containing the tomato gene for building the gene overexpression carriers of HsfA1a containing tomato sinks
The Agrobacterium tumefaciems engineering bacteria B of silent carrier；
(2) by the Agrobacterium tumefaciems engineering bacteria A mediated transformation target plant explants, HsfA1a gene transgenics are prepared
Plant；The Agrobacterium tumefaciems engineering bacteria B is contaminated into target plant cotyledon, HsfA1a gene silencing plant are prepared；
(3) the HsfA1a gene transgenics plant and HsfA1a gene silencings plant are subjected to drought stress processing, observation is planted
The number and drought resisting phenotype of strain autophagosome.
3. apply according to claim 2, it is characterised in that the base sequence of the tomato HsfA1a genes such as SEQ ID
NO：Shown in 1.
4. apply according to claim 2, it is characterised in that be the step of drought stress processing：
When HsfA1a gene transgenics plant and its blank control or HsfA1a gene silencings plant and its blank control length to five
Leaf wholeheartedly when, water to saturation, afterwards every the once respective relative soil moisture content and according to fluid loss of measurement in 1~4 day
Keeping the skin wet makes between HsfA1a gene transgenics plant and its blank control or HsfA1a gene silencings plant and its blank pair
Soil relative water content is consistent according between, until processing terminates.
5. apply according to claim 4, it is characterised in that in stress during drought stress control soil relative water content from
100% gradually reduces to 30%.
6. apply according to claim 4, it is characterised in that 10~15 days drought stress processing times.
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| WO2008110848A2 (en) * | 2007-03-15 | 2008-09-18 | Plant Bioscience Limited | Plant responses |
| CN103275990A (en) * | 2013-02-06 | 2013-09-04 | 中国农业科学院作物科学研究所 | Arabidopis thaliana heat shock transcription factor gene HSFA 2 and coding protein and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008110848A2 (en) * | 2007-03-15 | 2008-09-18 | Plant Bioscience Limited | Plant responses |
| CN103275990A (en) * | 2013-02-06 | 2013-09-04 | 中国农业科学院作物科学研究所 | Arabidopis thaliana heat shock transcription factor gene HSFA 2 and coding protein and application thereof |
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| Title |
|---|
| Arabidopsis heat shock factor HsfA1a directly senses heat stress, pH changes, and hydrogen peroxide via the engagement of redox state;Yanfang Liu，等;《Plant Physiology and Biochemistry》;20130105;第64卷;第92－98页 * |
| 番茄热激转录因子研究进展;王国栋，等;《植物生理学报》;20130320;第49卷(第3期);第217－224页 * |
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