CN102807990A - Application of trkA genes in deinococcus radiodurans R1 to culture of drought-resistant plant - Google Patents
Application of trkA genes in deinococcus radiodurans R1 to culture of drought-resistant plant Download PDFInfo
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Abstract
本发明发现耐辐射异球菌Deinococcus radiodurans R1中的trkA基因（DR1666）具有增强原核生物及植物的抗逆能力。本发明构建了包含上述基因的重组载体，把它们分别导入原核及真核宿主细胞。实验证明，trkA基因（DR1666）在原核宿主细胞和油菜中表达后，均能增强干旱抗性。The present invention finds that the trkA gene (DR1666) in Deinococcus radiodurans R1 can enhance the stress resistance of prokaryotes and plants. The present invention constructs recombinant vectors containing the above genes, and introduces them into prokaryotic and eukaryotic host cells respectively. Experiments have shown that the expression of trkA gene (DR1666) in prokaryotic host cells and rapeseed can enhance drought resistance.
Description
Technical field
(DR1666, new function GeneID:1798231) are specifically related to the application of this gene aspect enhancement of plant drought resisting resistance to the present invention relates to radiation hardness abnormal cocci R1 (Deinococcus radiodurans R1) trkA gene.
Background technology
Potassium element is one of three essential big mineral elements (N, P, K) of plant, and plant materials is had the important physical effect.When plant receives salt stress, a large amount of Na
+Get in the plant materials from root, make K
+Absorption be suppressed, form low K
+/ Na
+Ratio, thus plant is damaged.In this case, if can regulate K in the plant materials
+/ Na
+Balance just can be resisted excessive N a
+The injury that plant is caused.
HKT (high-affinity K+transporter) gene family translocator has can regulate K in the plant materials
+/ Na
+The equilibrated ability extensively is present in plant, fungi, eubacterium and the archeobacteria.Radiation hardness abnormal cocci R1 (D.radiodurans R1) contains 2 HKT proteinoids, transports in close relations closely relatedly with plant Na+, and secular drying and strong oxidative stress are had extremely strong resistance.
Potassium is one of essential nutritive element of plant materials, and sodium is the functional element of plant-growth, and is useful on a small quantity for most plants, excessively then can produce murder by poisoning.Plant mainly leans on its intravital channel protein and transport vehicle to their absorption.Getting in touch closely of the K translocator of plant HKT translocator and fungi Trk translocator, procaryotic KtrB and TrkH, the Trk translocator is a main K absorption system under the low K concentration in the fungi.All possibly have the HKT translocator in the most plants, this gene family is not very big, and this family has only a member in Arabidopis thaliana.Sequential analysis infers that the translocator of this family possibly striden the K passage of membrane structure by 2 times of bacterium and evolved, and forms a present pore structure albumen of striding film and 4 circles for 8 times.The HKT family gene mainly is divided into 2 subfamilies; Subfamily 1 member is the low affinity translocator of specific Na; Major part is distributed on the cytoplasmic membrane of root system center pillar, especially on the xylem parenchyma cell film, is responsible for from xylem sap, obtaining Na again and stops its transportation to overground part; And the subfamily 2 members high affinity translocator that is K/Na, thereby especially under K shortage condition, be responsible for the absorption of Na is promoted the growth of crop, mainly be distributed on the cortex and endodermis cytolemma of root system.
Deinococcus radiodurans (D.radiodurans) is one of biology of the highest radiation resistance in the known organism so far.This bacterium has the extreme resistance of ionizing rays, UV radiation and dna damage reagent to lethal dose, the complete recovery of genome that can ionizing rays be caused up to a hundred dna double splitting of chain as before, and the ability of not undergoing mutation.Radiation hardness ability that it is extreme and DNA loss thereof are repaired molecular mechanism and are caused the very big interest of scientific circles; Not only the basic subject of exploring DNA reparation molecule mechanism there is very significance; And to the development that promotes new dna technique and in environment protection and biological prosthetic, human health, biotechnology, and even aspect such as extraterrestrial spatial development and utilization has a very big potential application foreground.1999 gene studies institute (TIGR) accomplish and announced D.radiodurans genom sequence (White1999).
Although the trkA encoded protein belongs to HKT proteinoid member in the known D.radiodurans R1 radiation hardness bacterial strain, possibly on evolving, have synergetic property with plant Na+ transhipment, do not see the research report of trkA at present in enhancement of plant drought resisting resistance function aspects.
Summary of the invention
The objective of the invention is from D.radiodurans R1 genome to find can enhancement of plant to the resistant gene of high salt.And, make the plant that changes this gene over to obtain the ability of salt tolerant with this gene transferred plant.
The present invention is through following research; Find first; The high affine kalium ion transport body Deinococcus radiodurans R1 trkA gene of sequence shown in the SEQ ID NO:1 (DR1666 GeneID:1798231) has drought-resistant function of coercing, and can be used for cultivating the plant of drought-resistant proterties:
1, obtains to contain the recombinant strain of D.radiodurans R1 trkA gene (DR1666)
1) through PCR method, amplify D.radiodurans R1 trkA gene (DR1666) from D.radiodurans R1 (DSM20539) strain gene group, gene order number is: GeneID:1798231.Its size is 681bp, and 226 amino acid of this genes encoding are cloned it on carrier pGEMT-easy, have made up the recombinant plasmid pGEMT-trkA that contains complete trkA gene;
2) trkA gene (DR1666) is connected on the pRADZ3 shuttle plasmid; This plasmid contain can be in intestinal bacteria and the different coccus of radiation hardness all acting groEL promotor, make up complete trkA gene (DR1666) the recombinant plasmid pRADZ3-trkA G that contains the groEL promotor;
The recombinant plasmid pRADZ3-trkA G that 3) will import trkA gene (DR1666) changes in the acceptor e. coli jm109, obtains engineering strain JM-trkA (seeing embodiment 1 for details);
Anyone can both obtain said engineering strain through open channel.
2, contain the drought resisting experiment of D.radiodurans R1trkA engineering strain
Experiment confirm; After high density (3M) sorbyl alcohol (Sorbitol) impacts; The JM-trkA recombinant bacterial strain upgrowth situation that contains D.radiodurans R1 trkA gene (DR1666) is good, and colony count is higher than the JM-Z3 bacterial strain (seeing embodiment 2 and Fig. 4) that only contains empty plasmid.Engineering bacillus strain has the ballistic ability of tolerance 3M sorbyl alcohol (Sorbitol).(because of Sorbitol has water absorbability, the environment that the Sorbitol solution of high density can simulating drought sees embodiment 2 for details), this experiment shows: D.radioduransR1 trkA gene (DR1666) has the ability that strengthens the prokaryotic organism drought resisting.
3, trkA gene (DR1666) is expressed in rape and the arid resistance of transfer-gen plant is identified
1) D.radiodurans R1 trkA gene (DR1666) is connected among the plant expression vector pBI121, makes up recombinant plasmid pBI121-lea with trkA gene (DR1666);
2), obtained the transgene rape plant that drought-resistantly to coerce with in the pBI121-lea recombinant plasmid transformed rape;
This experiment shows: D.radiodurans R1 trkA gene (DR1666) has the purposes (seeing embodiment 3 and Fig. 5～8) of cultivating drought-resistant plant.
Description of drawings:
Fig. 1 is the PCR product checking electrophoretogram that contains D.radiodurans R1 trkA gene (DR1666) sequence;
Fig. 2 is the construction of prokaryotic expression vector checking electrophoretogram that contains D.radiodurans R1 trkA gene (DR1666) and groEL promotor;
Fig. 3 is the bacterium colony photo that contains the empty carrier and the colibacillary upgrowth situation of the prokaryotic expression carrier that contains D.radiodurans R1trkA gene (DR1666) sequence before high density 3M sorbyl alcohol (Sorbitol) impacts, wherein:
A is the e. coli jm109 bacterial strain that contains empty expression vector;
B is the intestinal bacteria recombinant bacterial strain that contains D.radiodurans R1 trkA gene (DR1666) expression vector;
Fig. 4 is the bacterium colony photo of the growing state of intestinal bacteria (E.coli) in containing 4M NaCl substratum that contains prokaryotic expression carrier and the empty carrier of D.radiodurans R1 trkA gene (DR1666), and the bacterial strain among the figure is following:
A is the e. coli jm109 bacterial strain that contains empty expression vector;
B is the intestinal bacteria recombinant bacterial strain that contains D.radiodurans R1 trkA gene (DR1666) expression vector.
Fig. 5 is drought stress non-transgenic rape and the growth conditions photo that changes trkA gene (DR1666) rape in the time of 5 days.
Fig. 6 is drought stress non-transgenic rape and the growth conditions photo that changes trkA gene (DR1666) rape in the time of 30 days.
Among Fig. 5 and Fig. 6, the left side is non-transgene rape, and the right is to change trkA gene (DR1666) rape.
Embodiment
The plasmid of being lifted in following examples, bacterial strain only are used for the present invention is done further explain, flesh and blood of the present invention are not limited.All unreceipted concrete experiment conditions; Be according to normal condition well known to those skilled in the art; For example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
Plasmid, the bacterium source lifted among the embodiment are following:
Cloning vector pGEMT-easy: be Promrga company commercially available prod;
Shuttle plasmid pRADZ3: for preserving in this laboratory;
Plant expression vector pBI121: be Clontech company commercially available prod;
E. coli jm109: be the full formula in Beijing King Company commercially available prod.Agrobacterium tumefaciens EHA105 is preserved by this laboratory.
The expression of embodiment 1D.radiodurans R1 trkA gene (DR1666) sequence in intestinal bacteria
According to the 1 pair of PCR Auele Specific Primer of trkA gene (DR1666) sequences Design in the D.radiodurans R1 genome of having announced:
Up?5＇ATTAACTAGT?GTGTGCTCTACACTCAGC3＇
Down?5＇ACGCCATATG?TTATTCCCCCAGATACCG3＇
From D.radiodurans R1 genomic dna, amplify target gene sequences through PCR method.Reaction conditions: 94 ℃ of 10min, [94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 1min] 35 circulations, 72 ℃ of 10min.The PCR product is cloned on carrier pGEMT-easy called after pGEMT-trkA, and sequence verification after glue reclaims; Obtain to contain the trkA gene (DR1666) of sticky end through the SpeI/NdeI double digestion then and contain the pRADZ3 carrier of promotor groEL; TrkA gene (DR1666) is connected on the pRAD Z3 carrier, makes up coli expression carrier pRADZ3-trkA G, this expression vector transformed into escherichia coli JM109; Cut through PCR, enzyme; Sequence verification insertion sequence correct (seeing Fig. 1,2) is with this bacterial strain called after JM-trkA.
The E.coli JM109 called after JM-Z3 that will contain the pRADZ3 empty plasmid.
Embodiment 2 contains the drought resisting experiment of D.radiodurans R1 trkA gene (DR1666) recombinant bacterial strain
One, experimental technique
1, the intestinal bacteria with 2 reorganization that obtain among the embodiment 1 are inoculated in respectively in the 20mL LB liquid nutrient medium (containing the Amp microbiotic); Shake (37 ℃) after bottle incubated overnight; Transfer again in the LB of 100mL liquid nutrient medium, keep the unanimity of inoculum size as far as possible, be cultured to OD
600About about 0.5 (keep OD as far as possible
600Value is consistent).
2, get the bacterium liquid of 10mL centrifugal after, in the sorbitol solution of isopyknic 3M, impact 2h, each sample is used aseptic deionized water doubling dilution to 10 immediately
-4, get 10 μ L points at LB solid culture primary surface, through 37 ℃ of cultivation 16h, observe bacterium colony formation situation and also take a picture.
Two, experimental result
Fig. 3 shows, before the sorbitol solution of 3M is impacted, contains the JM-trkA bacterial strain and the JM-Z3 strain growth state basically identical that contains empty plasmid of D.radiodurans R1 trkA gene (DR1666); Behind the 4M NaCl fluid challenge, the JM-trkA recombinant bacterial strain upgrowth situation that contains D.radiodurans R1 trkA gene (DR1666) is good, and colony count is more than the JM-Z3 bacterial strain (see figure 4) that only contains empty plasmid.The Sorbitol Powder of 3M is hypertonic solution, but simulating drought is coerced.
Three, experiment conclusion
D.radiodurans R1 trkA gene (DR1666) has strengthened the ability of prokaryotic organism drought resistings.
Embodiment 3 trkA genes (DR1666) are expressed in rape and the arid resistance of transfer-gen plant is identified
(1) agrobacterium mediation converted rape experiment
1. the competent preparation of agrobacterium tumefaciens EHA105
1) picking list bacterium colony is inoculated in 5mL YEB liquid nutrient medium (containing Rifampin Rif50mg/L), and 28 ℃, the 250rpm shaking culture is spent the night;
2) get 2mL bacterium liquid, add in the 50mL YEB liquid nutrient medium (containing Rif50mg/L), 28 ℃, the 250rpm shaking culture is to OD
600About about 0.6;
3) bacterium liquid is gone in the aseptic centrifuge tube of 50mL ice bath 30min, the centrifugal 5min of 5000 * g;
4) abandon supernatant, deposition is with 2mL 20mM CaCl
2Resuspended, every part 100 μ L branch installs in the 1.5mL centrifuge tube, preserves subsequent use in the liquid nitrogen.
2. recombinant plasmid dna changes Agrobacterium over to
1) the pBI121-trkA DNA with about 1 μ g joins in the 100 μ L EHA105 competent cells mixing, ice bath 5min respectively;
2) centrifuge tube is put freezing 8min in the liquid nitrogen, go to temperature bath 5min in 37 ℃ of water-baths rapidly;
3) add 1mL YEB liquid nutrient medium, 250rpm recovery 4～5h on 28 ℃ of shaking tables;
4) get an amount of bacterium liquid and be applied on the YEB solid medium that contains Rifampin (Rif) 50mg/L and kantlex (Kan) 100mg/L, put 28 ℃ and cultivate 24～48h.
3. the extraction of Agrobacterium DNA
1) picking colony (YEB: Tryptones 5g/L, yeast extract 1g/L, sucrose 5g/L, sal epsom 0.5g/L) in the YEB liquid nutrient medium that contains Rifampin (Rif) 50mg/L and kantlex (Kan) 100mg/L, 28 ℃, 250rpm shaking culture 20h;
2) get 1.5mL bacterium liquid centrifugal after, abandon supernatant, with STE solution (Tris-HCI10mM pH8.0; NaCl10mM, EDTA10mM pH8.0) wash 2 times, abandon supernatant; The solution I (50mM glucose, 25mM Tris-HCl pH8.0, the 10mM EDTA pH8.0 that add precooling; RNase A100 μ g/mL) 300 μ L aspirate mixing repeatedly with pipettor, and room temperature leaves standstill 10min;
3) (solution II of the fresh configuration of adding turns upside down mixing several times for 0.2M NaOH, 1%SDS) 600 μ L;
4) solution III (5M potassium acetate pH5.2, Glacial acetic acid min. 99.5 11.5mL adds water to 100mL) the 450 μ L of adding precooling, abundant mixing, ice bath 5min, 4 ℃ of centrifugal 5min of 12000 * g, supernatant adds the isopropanol precipitating of 0.6 times of volume;
5) deposition adds 50 μ L TE (Tris-HCl10mmol/L, 1mmol/L EDTA is pH8.0) after the dissolving, through PCR, Bam HI, the checking of Sac I double digestion.
4. the preparation of rape aseptic seedling and agriculture bacillus mediated trkA gene (DR1666) genetic transformation
1) cultivation of rape aseptic seedling
Swede type rape (Brassica napus L.) (84100-18) seed soaks 10min with aqua sterilisa on Bechtop; 10% NaClO sterilization 12min; Again with 0.1% mercuric chloride solution sterilization 7-8min; With sterilization washing 3-5 time, and be placed in the Semen Brassicae campestris of sterilization equably on the pre-culture medium of no hormone (7.5% agar).Illumination cultivation, 4-5d rape in age seedling is suitable for genetic transformation most.
2) cultivate in advance
The scissors of alcohol with 75% and high-temperature sterilization sterilization is cut cotyledon and vegetative point, and clip rape aseptic seedling next-door neighbour vegetative point is the long hypocotyl of about 0.5cm down, places pre-culture medium (MS+6-BA2mg/L+2,4-D1mg/L+AgNO
32.5mg/L+AS19.62mg/L) on, 2d is cultivated in illumination in advance.
3) cultivation of Agrobacterium
In 50mL LB liquid nutrient medium (containing kantlex 100mg/L, Rifampin 50mg/L), add 0.1mLlea-EHA105 bacterium liquid; About 28 ℃ of following 220rpm shaking culture 16h, the centrifugal 10min of 4000 * g abandons supernatant under the room temperature; Thalline suspends with the MS liquid nutrient medium (containing AS100 μ mol/L) of sterilization; The 5-20 that is diluted to original volume doubly at 28 ℃ of following 220rpm shaking culture 1h, makes the OD of bacterium liquid
600Reach about 0.5.
4) cultivate altogether
Put into bacterium liquid to the hypocotyl of cultivating 2d in advance and contaminate 1min; The medication spoon is pulled hypocotyl out, is placed on the thieving paper of sterilization, blots the unnecessary bacterium liquid on the hypocotyl; Be put into again on the pre-culture medium that is coated with sterilization filter paper; Seal petridish with sealing film, about 25 ℃, cultivate about 2d (the dark cultivation) altogether in the dark place.
5) inducing culture
Be put into inducing culture (MS+6-BA2mg/L+AgNO to the hypocotyl of cultivating 2d altogether
32.5mg/L) on, seal petridish with sealing film, 25 ℃ of left and right sides illumination cultivation, per 2 all subcultures 1 time are until growing the callus that expands.
6) select to cultivate
Transfer to screening culture medium (MS+6-BA2mg/L+AgNO to the callus that expands
32.5mg/L+Kan100mg/L) on, seal petridish with sealing film, 25 ℃ of left and right sides illumination cultivation, per 2 all subcultures 1 time are until growing seedling.
7) root culture
When high, separate them from callus to 1-2cm when young shoot is long, transfer to that (1/2MS+NAA0.5mg/L+Kan25mg/L) carries out root culture on the root media.Under the antibiotic-screening condition, about 87% bud is at 2 weeks back formation root.
8) refining seedling and transplanting
Seedling after taking root is long to 5-6cm when high, and half opens wide the culturing bottle lid, refines seedling; Treat that seedling adapts to after the external environment, transfer in the vermiculite of indoor sterilization plantation and cultivate, and water with the 1/2MS nutrient solution.When growth of seedling during, sprigging is grown in earth, and then carry out next step experiment to 7-9cm.
(2) drought resistance that contains trkA gene (DR1666) sequence transgene rape is identified
1, experiment purpose
In view of this nucleotide sequence has been proved to be in intestinal bacteria drought stress is had resistance, further transfer-gen plant is carried out drought resistance and identify.
2, experimental technique
Carried out the experiment of 30 strain transgene rape plant and 30 strain non-transgenic rape plant, method is to the transgene rape plant in seedling stage and non-transgenic rape plant is disposable does not respectively rewater after watering sufficient nutritive medium, and carries out following experiment contrast:
1) growing state of observation rape plant
Began to start at from the same day of watering after the sufficient nutritive medium, do not rewater and nutritive medium in the 2nd day～the 27th day;
Rose in the 28th day and recover to water, and watered sufficient nutritive medium, only take the circumstances into consideration afterwards to water the 28th day same day.
2) measure leaf water content
From stopping to water second day, every other day measure blade relative water content (Relativewatercontent);
The measuring method of water cut: after taking off about 0.1g blade from plant, take by weighing fresh weight (FW) immediately, then blade is immersed 4h in the deionized water; Taking-up is inhaled with filter paper and is removed surface-moisture; Take by weighing saturated fresh weight (TW), then blade is put into baking oven and dry to constant weight in 70 ℃, take by weighing dry weight (DW).
Relative water content (RWC) is calculated as follows:
RWC(%)＝(FW-DW)/(TW-DW)×l00%。
3, experimental result
1) the growing state comparing result of rape plant
When coercing the 6th day dried morning, the non-transgenic rape is own through beginning wilting at blade; The transgene rape blade is full, and growth is normal; (see figure 5)
Drought stress is in the time of the 12nd day, and the dehydration of non-transgenic rape is serious, and blade is wilted; Transgene rape is normal growth still;
Drought stress is in the time of the 18th day, and the non-transgenic rape leaf is wilted, flavescence; The blade of transgene rape just begins to wilt; Drought stress is in the time of the 30th day, and the non-transgenic rape is withered dead, transgene rape blade wilting (see figure 6);
Rose in the 28th day and recover to water, dead at the 30th day non-transgenic rape; And recovering growth after the rape rehydration of commentaries on classics trkA gene (DR1666) rapidly, it is green that blade turns.
Table 1 transgene rape and non-transgenic rape are to the comparison of drought stress
2) leaf water content is measured the result
Can find out from following table 2: doing 30 days that early coerce processing, non-transgenic rape leaf relative water content sharply descends, and it is obviously slow than non-transgenic rape to change trkA gene (DR1666) rape leaf dehydrating speed, and the blade relative water content descends gradually;
Along with the prolongation in arid treatment time, the difference of transgene rape and non-transgenic rape relative water content is obvious gradually; When coercing the 30th day dried morning, the blade relative water content of non-transgenic rape reduces to 32.67%, and the blade relative water content of transgene rape is 72.98%, is about more than 2 times of non-transgenic rape water cut.
Table 2 is transgene rape and the comparison of non-transgenic rape leaf relative water content under drought stress
Annotate: data are 30 plant of transgene rape and each 30 MV that plant is calculated respectively of non-transgenic rape in the table 2.
4, experiment conclusion
Above-mentioned all results show that all the trkA gene (DR1666) of being cloned has the resistance to drought stress.
Claims (10)
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