CN101851284B - Plant resistance-associated membrane protein BjCRP1, genes and application thereof - Google Patents
Plant resistance-associated membrane protein BjCRP1, genes and application thereof Download PDFInfo
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Abstract
The invention relates to the field of plant gene engineering, in particular to a plant resistance-associated membrane protein BjCRP1, genes and the application thereof. The amino acid sequence of the plant resistance-associated membrane protein BjCRP1 according to the invention is as shown in SEQ 1D No.1. Due to the expression of BjCRP1 genes, a recombinant strain and a plant can obtain remarkable characteristics of heavy metal (CdCl2) resistance and high salt (NaCl) resistance at the same time; and a plant gene which has remarkable functions of heavy metal resistance and high salt resistance is rarely reported at present. The proof of the resistance provides base for the BjCRP1 genes in the application of plant resistance gene engineering, and plays an important role in culturing plants (wheat, corns, rice and vegetables) with enhanced comprehensive resistances (heavy metal and/or high salt and/or drought).
Description
Technical field
The present invention relates to plant genetic engineering field, particularly, the present invention relates to a kind of stress resistance of plant related membrane protein BjCRP1 and gene and application.
Background technology
Indian mustard (Brassica juncea) is that a kind of grow fast, that living weight is big Cd, Zn, the Pb that filter out at present restrain oneself-the concentration type plant.Though accumulation Cd ability does not have the Zn, the ultra accumulation of the Cd plant Thlaspi caerulescens that generally acknowledge in the world at present high in its body; But its growth velocity and the upperground part biomass are higher than Thlaspicaerulescens far away; Thereby Indian mustard is higher than Thlaspi caerulescens to the absorption total amount of Cd under the same terms, in phytoremediation, has bigger potentiality.
The ultra enriching heavy metal mechanism of plant relates generally to plant to the high absorption of metals ion, transportcapacity, and aspects such as compartmentation and sequestering action are wherein striden the proteic expression of membrane transport, regulated and control heavy metal super-enriched this characteristic has been played keying action.Acting membrane transport protein gene of output and gene family are identified out outside many compartmentations at metals ion (as in vacuole) at present or the born of the same parents, like CPx type ATP enzyme (Williams et al., Biochim BiophysActa; 2000,1465:104-126), NRAMP family member (Vidal et al., Cell; 1993,73:469-485), CE family member (Pence et al., Proc Natl Acad Sci USA; 2000,97:4956-4960; Persans et al., Proc Natl Acad Sci USA, 2001,98:9995-10000), ATP combines and transporter (ABC transporter) (Rea; Exp Bot, 1999,50:895-913), the CAX (Hirschi of two valency positively charged ion P proton antiport albumen such as Arabidopis thaliana; Plant Cell, 1999,11:2113-2122) and AtMHX1 (Shaul et al.; EMBO J, 1999,18:3973-3980).
Summary of the invention
The purpose of this invention is to provide a kind of stress resistance of plant related membrane protein and encoding sox thereof.
Another purpose of the present invention provides the recombinant vectors that comprises said gene.
A purpose more of the present invention provides the application of above-mentioned albumen and gene.
According to the aminoacid sequence of stress resistance of plant related membrane protein BjCRP1 of the present invention shown in SEQ ID No.1.
According to stress resistance of plant genes involved of the present invention, its above-mentioned membranin BjCRP1 that encodes, for example, its nucleotide sequence is shown in SEQ ID No.2.
Recombinant vectors according to the present invention comprises said gene, and for example, said carrier is pET32a-BjCRP1.
Can be used to improve the preventing from heavy metal of plant, anti-salt, drought resistance according to albumen of the present invention.
Can be used to improve the preventing from heavy metal of plant, anti-salt, drought resistance according to gene of the present invention.
Therefore, the method according to raising plant preventing from heavy metal of the present invention, anti-salt, drought resisting comprises the step that said gene is changed over to plant, also expresses.
Experimental result shows, is containing the CdCl of different concns
2In the substratum, the BL21 growing state that contains empty carrier pET32a is obviously not as the growing state of the BL21 that contains recombinant vectors pET32a-BjCRP1.0.5mmol/L CdCl with 1.0mmol/L
2During processing, BL21 (pET32a-BjCRP1) when reaching summit of growth the value of OD600 be respectively 0.9143 and 0.4514, and BL21 (pET32a) OD600 value when same condition is issued to summit of growth is merely 0.3014 and 0.1494.Work as CdCl
2When concentration was 1.5mmol/L, BL21 (pET32a-BjCRP1) and BL21's (pET32a) all can not normal growth, and this has shown BL21 (pET32a-BjCRP1) tolerance CdCl
2Mxm. be 1.5mmol/L.
In containing the LB liquid nutrient medium of different concns NaCl; When NaCl is 0.4mol/L; Before the time of coercing reached 5 hours, the growth of BL21 (pET32a) obviously was suppressed, and growing state is clearly better afterwards; BL21 (pET32a-BjCRP1) is in logarithmic phase always, does not receive the influence of NaCl basically.BL21 (pET32a) is when NaCl concentration is 0.6mol/L and 0.8mol/L; Almost can not grow; BL21 (pET32a-BjJ10-2) is 0.6mol/L at NaCl, and the OD600 value is 0.4743 during summit of growth, can not grow the same with BL21 (pET32a) when NaCl is 0.8mol/L.Therefore, think that the threshold value of BL21 (pET32a-BjJ10-2) tolerance NaCl is 0.8mol/L.
Different concns PEG6000 solution is coerced process result and is shown, the concentration of PEG6000 is when 10%-20%, and reorganization bacterium and contrast bacterium be the ability normal growth all, and the reorganization bacteria growing slightly is superior to contrasting bacterium, but is not so good as NaCl and CdCl
2Resistance obvious, explain that the BL21 bacterial strain itself has certain anti-PEG and coerces ability.When PEG concentration was 30%, BL21 (pET32a-BjJ10-2) and BL21 (pET32a) growth obviously was suppressed, but concentration difference is little, show that ability that the anti-PEG of reorganization bacterium BL21 (pET32a-BjJ10-2) coerces a little less than.
The heavy metal Cd of BjCRP1 transgene tobacco and high salt NaCl coerce experiment and show, compare the ability of coercing that expression BjCRP1 has significantly improved transfer-gen plant preventing from heavy metal Cd (200 μ M/L) and NaCl (250mmol/L) with the empty carrier contrast.And under the normal cultured condition the two no significant difference.
Explain that the BjCRP1 expression of gene can make recombinant bacterial strain and plant obtain significant preventing from heavy metal (CdCl simultaneously
2) and high salt tolerance (NaCl) characteristic, the function not only also rare report of plant gene of preventing from heavy metal but also high salt tolerance significantly at present.The application of BjCRP1 gene in the plant stress-resistance genetically engineered that prove of this resistance provides the foundation, and will in cultivating comprehensive resistance (heavy metal and/or high salt/or/arid) enhanced plant (wheat, corn, paddy rice and vegetables), play a significant role.
Description of drawings
The membrane spaning domain (TMHMM software) that Fig. 1: BjCRP1 infers.
Fig. 2: the RT-PCR amplification of BjCRP1 gene complete ORFs.
Fig. 3: clone's product P GM-BjCRP1 restriction enzyme digestion and electrophoresis detected result.
Fig. 4: recon PET-BjCRP1 restriction enzyme digestion and electrophoresis detected result.
Fig. 5: the PAGE collection of illustrative plates of BjCRP1 prokaryotic expression protein, 1-5 swimming lane are respectively the albumen supernatant expression amount that IPTG induces 0h, 2h, 4h, 6h and 8h; The 6-9 swimming lane is respectively the albumen precipitation expression amount that IPTG induces 0h, 2h, 4h and 6h, is fusion rotein for finding out this albumen, and the supernatant expression amount is obviously greater than deposition, and this albumen is 27.5 dalton, arrow indication this albumen for expressing.
Fig. 6: albuminised PAGE collection of illustrative plates on the BjCRP1 prokaryotic expression; The 1-5 swimming lane is respectively the albumen supernatant expression amount that IPTG induces 0h, 2h, 4h, 6h and 8h; The 6th swimming lane is the empty bacterial strain of BL21 is induced 6h through IPTG a expression amount; The 7th swimming lane is that BL21 (pET32a) induces the expression amount of 6h can find out that this albumen expression amount after inducing 6 hours through IPTG reaches maximum, arrow indication this albumen for expressing through IPTG.
Fig. 7: sxemiquantitative RT-PCR detects the BjCRP1 gene and under different stress conditions, transcribes abundance, and the Indian mustard of 5-6 sheet leaf age is used 200 μ M CdCl respectively
2, 250mMNaCl, 1000 μ M ZnCl
2Handle 12h with 20%PEG6000, do not have the processing of coercing with contrast and compare.Find that the BjCERP1 gene significantly responds heavy metal (Cd, Zn) and high salt (NaCl) and coercing of arid PEG and strengthens expression in leaf portion, at root, except that Cd coerced, other was coerced and all makes the BjCRP1 gene transcription level significantly strengthen expression.
Fig. 8: non-coercing and different concns CdCl
2Coerce down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjJ10-2), (a) growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1) under the non-stress conditions; (b) 0.5mmol/L CdCl
2Coerce down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (c) 1.0mmol/L CdCl
2Coerce down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (d) 1.5mmol/L CdCl
2Coerce down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1).
The growth curve of Fig. 9: BL21 under the NaCl stress (pET32a) and BL21 (pET32a-BjJ10-2), (a) 0.4mol/L NaCl coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (b) 0.6mol/L NaCl coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (c) 0.8mol/L NaCl coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1).
Figure 10: different concns PEG6000 coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1), and (a) 10%PEG6000 coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (b) 20%PEG6000 coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1); (c) 30%PEG6000 coerces down the growth curve of BL21 (pET32a) and BL21 (pET32a-BjCRP1).
Figure 11: the structural representation of pBI121-BjCRP1-GFP expression plasmid, nos-pro, rouge alkali synthetase promoter; NptII, the plain phosphoric acid transferase gene of new enzyme; Nos-ter, the rouge alkali synthetase terminator; CaMV 35S Pro, the cauliflower mosaic virus 35S promoter; GFP, green fluorescence protein gene; RB, right arm; LB, left arm.
Figure 12: plant expression vector recon pBI121-BjCRP1-GFP restriction enzyme digestion and electrophoresis detected result, M, 100bpDNA molecular weight Marker; 1-4, the enzyme that is respectively 4 recombinant clone plasmids is cut qualification result, wherein clones 3 and contains correct BjCRP1 insertion fragment.
Figure 13: sxemiquantitative RT-PCR detects on the BjCRP1 gene transcription level that the transformation of tobacco plant integrates and obtains to express, 1,2, two pBI121-BjCRP1-GFP transgenic lines independently; 3, wild-type tobacco; 4, the pBI121-GFP transgene tobacco.With the about 800bp segment of special primer amplification reporter gene GFP, cycle number is 25, and Actin is a house-keeping gene, is used for controlling original mold plate amount.
Figure 14: at 200 μ M CdCl
2Under the stress conditions, BjCRP1 and empty carrier transgene tobacco explants differentiation seedling ability are relatively.The no CdCl of Figure 14 (a)
2With 200 μ MCdCl
2Different genotype differentiation regrowth example under the culture condition, the no CdCl of Figure 14 (b)
2With 200 μ MCdCl
2Different genotype differentiation regrowth data analysis under the culture condition.
Figure 15: at 200 μ M CdCl
2With the comparison of 15 days cauline leaf of different genotype transgene tobacco growth under the 250mM NaCl stress conditions with the situation of taking root.
Embodiment
Experimental technique among the following embodiment like no specified otherwise, is ordinary method.
Embodiment 1: the acquisition of plant adversity resistance related protein BjCRP1 and encoding sox thereof
Following according to Indian mustard and nearly edge species RAMP4 family conserved regions nucleotide sequence design primer: 5 '-CGC
GGATCCGTTATCTGCAATTATGACAAC-3 ' is (underscore is the restriction enzyme site of BamH I) (F), 5 ' CCG
CTCGAGTTATGA CATGCCTCCGCTAG-3 ' is (underscore is the restriction enzyme site of Xho I) (R).
Through containing 200 μ mol CdCl
21/2Hoagland ' the s nutritive medium Indian mustard seedling of coercing, extract total RNA.
Operate with PrimeScript TM 1st Strand cDNA Synthesis Kit and by the test kit specification sheets: get the total RNA of 1-5 μ g Indian mustard, obtain article one chain cDNA.
The reverse transcription product of getting the acquisition of 1 μ l step 3) is a template, under the guiding of 5 ' end primer and 3 ' end primer, synthesizes the cDNA of BjCRP1 with the method for PCR.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, obtain the band that molecular weight is about 250bp, conform to, reclaim test kit (worker is given birth in Shanghai) with sepharose and reclaim this fragment with expected results.
Should reclaim fragment and be connected, will connect product transformed into escherichia coli TOP10 competent cell, and, obtain containing and reclaim segmental recombinant plasmid, name PGM-BJJ10-2 according to the Pyocianil resistance marker screening positive clone on the PGM-T carrier with carrier PGM-T.With T7 on this recombinant plasmid vector and SP6 promoter sequence be primer to its carry out the nucleotide sequencing sequencing result show increase BjCRP1 opening code-reading frame (ORF) totally 207 bases; Coding has 68 amino acid whose protein; TMHMM software infers that the membrane spaning domain of BjCRP1 is as shown in Figure 1; The 38th to the 60th amino acids for striding the film district, N end 1-37 amino acids is in film, C end 61-68 amino acids is outside film.
1, the structure of prokaryotic expression carrier PET-BjCRP1:
The correct PGM-T that contains the BjCRP1 gene fragment directly carries out double digestion with BamH I, two kinds of enzymes of Xho I with order-checking; Reclaim endonuclease bamhi and carry out the cloning vector pET32-a (+) that double digestion crosses with BamH I, two kinds of enzymes of Xho I and be connected; Transformed into escherichia coli BL21 (DE3) pLysS; And identify that through plasmid enzyme restriction filter out positive colony and check order, therefrom filtering out sequence not have the pET-BjCRP1 recon that suddenlys change.
2, the BjCRP1 expressing protein detects:
With recombinant plasmid; Empty plasmid all is transferred among e. coli bl21 (DE3) pLysS; The picking empty bacterial strain that contains the pET-BjCRP1 plasmid, contain empty plasmid and do not contain plasmid adds 10mL LB liquid nutrient medium (containing corresponding microbiotic) 37 ℃ of 200rpm incubated overnight in the triangular flask of sterilization respectively; By dilution in 1: 50, the adding final concentration was 0.2% glucose with bacterium liquid, continued 37 ℃ of 200rpm and cultivated 2h, made it OD600 and reached 0.5-0.6, and it is subsequent use to collect 1mL bacterium liquid.Adding final concentration is the IPTG of 1.0mM, and at 37 ℃ of abduction delivering 0h, 2h, 4h, 6h and 8h, each collects bacterium liquid 1mL, abandon after centrifugal supernatant stay thalline place-20 ℃ subsequent use.
Take out the thalline of-20 ℃ of preservations, add 100 μ l 10mM Tris-HCl (PH8.0), place on ice; Add 2 μ l2%-beta-mercaptoethanols again, mixing; Ultrasonication 50 times; 12000rpm, 4 ℃ of centrifugal 15min go cleer and peaceful deposition; Deposition adding 100 μ l 10mMTris-HCl (PH8.0) and 2 μ l, 2% beta-mercaptoethanol mixing are abundant, add 20 μ l, 4 * sample-loading buffer again, and supernatant directly adds 20 μ l, 4 * sample-loading buffer.
Protein sample (bacterium liquid) is dissolved in the sample-loading buffer, place boiling water 10min, centrifugal 10sec; Dispose 12% separation gel respectively, 5% concentrated glue is treated to extract after its polymerized at room temperature finishes comb and is gone up appearance; Concentrate the glue part with the 80V constant voltage, the separation gel part is with 120V constant voltage electrophoresis.After treating tetrabromophenol sulfonphthalein electrophoresis to gel bottom, stop electrophoresis, shell glue then, fix and dye, decolour.The PAGE collection of illustrative plates of BjCRP1 prokaryotic expression protein is as shown in Figure 5; Albuminised PAGE collection of illustrative plates people is shown in Figure 6 on the BjCRP1 prokaryotic expression; Induce the expression amount of 6h can find out that this albumen expression amount after inducing 6 hours through IPTG reaches maximum, arrow indication this albumen through IPTG for expressing.
Choose the Indian mustard seedling of 5-6 sheet leaf age and use 200 μ M CdCl respectively
2, 250mMNaCl, 1000 μ MZnCl
2After handling 12h with 20%PEG6000, after not having the young plant of coercing processing and carry out rinsing 2-3 time with sterilized water with contrast, the root leaf separates rapidly, and liquid nitrogen flash freezer carries out total RNA extraction.
Use the BjCRP1 special primer respectively: upstream primer: 5 '-TTTTGTTCCTGA GACCACCAC-3 '; Downstream primer: 5 '-TATGACATGCCTCCGCTAG-3 ' and Actin muscle β-Actin primer: upstream primer: 5 '-CTTAACCCTAAGGC TAACAG-3 '; Downstream primer: 5 '-TCCTCCGATCC AGACACT-3 ' increases.β-Actin is as confidential reference items, with the unanimity of control masterplate amount.CDNA template after at first reversing with 1 μ l contrast is in charge of pcr amplification with Actin primer and BjCRP1 primer respectively.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect (Fig. 7).Find that the BjCERP1 gene significantly responds heavy metal (Cd, Zn) and high salt (NaCl) and coercing of arid PEG and strengthens expression in leaf portion, at root, except that Cd coerced, other was coerced and all makes the BjCRP1 gene transcription level significantly strengthen expression.Explain the BjCERP1 gene not only significantly respond heavy metal (Cd, Zn) coerce up-regulated expression, and significantly respond the up-regulated expression of coercing of high salt (NaCl) and arid factor PEG, possibly all play an important role aspect plant preventing from heavy metal and salt and the drought stress.
The resistance analysis of embodiment 4 recombination bacillus coli BL21 (pET32a-BjCRP1)
Recombinant plasmid pET32a-BjCRP1 and contrast empty plasmid pET32a are transformed into respectively among expression strain BL21 (DE3) plysS, and the screening positive bacteria falls on the agar plate that contains paraxin and ammonia benzyl mycin.New activatory BL21 of picking (pET32a-BjCRP1) and the single bacterium colony of contrast BL21 (pET32a) are inoculated in the LB substratum respectively, and 37 ℃ of following shaking culture are spent the night.Getting overnight culture respectively is inoculated in 1: 50 ratio and is cultured to the OD600 value in the LB liquid nutrient medium that contains paraxin and ammonia benzyl mycin and is 0.6 and is used to coerce experiment.
1. the preventing from heavy metal of recombination bacillus coli BL21 (pET32a-BjCRP1) is coerced.
Get BL21 (pET32a-BjCRP1) and contrast BL21 (pET32a) 600 μ l respectively, be added to and contain CdCl
2In antibiotic 30ml LB liquid nutrient medium, CdCl
2Final concentration is respectively 0.5mmol/L, 1.0mmol/L, 1.5mmol/L, and the contrast that does not contain the factor of coercing is set simultaneously, and each processing is provided with 3 repetitions, and 37 ℃ of shaking culture are every at a distance from 1h mensuration OD600 value, survey altogether 9 hours.
The result shows that BL21 (pET32a-BjCRP1) and BL21 (pET32a) cultivate and gets into logarithmic phase immediately in not containing the LB substratum of the factor of coercing; The growth impetus is suitable; All (Fig. 8 a) is transferred to the CdCl that contains different concns and work as to OD600 value when reaching summit of growth about 1.7
2In the time of in the substratum, the growing state of BL21 (pET32a) obviously is not so good as BL21 (pET32a-BjCRP1), the CdCl of 0.5mmol/L and 1.0mmol/L
2During processing; BL21 (pET32a-BjCRP1) when reaching summit of growth the value of OD600 be respectively 0.9143 and 0.4514; (Fig. 8 b c), also can find out CdCl simultaneously and BL21 (pET32a) OD600 value when same environment is issued to summit of growth is merely 0.3014 and 0.1494
2The concentration of handling increases, and the time that BL21 (pET32a-BjCRP1) reaches the growth mxm. obviously shortens.Work as CdCl
2When concentration was 1.5mmol/L, BL21 (pET32a-BjCRP1) and BL21's (pET32a) all can not normal growth (Fig. 8 d), this explanation BL21 (pET32a-BjCRP1) tolerance CdCl
2Mxm. between 1-1.5mmol/L.
2. the NaCl of recombination bacillus coli BL21 (pET32a-BjJ10-2) coerces.
Get BL21 (pET32a-BjCRP1) and contrast BL21 (pET32a) 600 μ l respectively; Be added to and contain in NaCl and the antibiotic 30mlLB liquid nutrient medium; The NaCl final concentration is respectively 0.4mol/L, 0.6mol/L, 0.8mol/L, and each processing is provided with 3 repetitions, 37 ℃ of shaking culture; Every at a distance from 1h mensuration OD600 value, 9 totally hours.
The result shows, when NaCl is 0.4mol/L, before the time of coercing reached 5 hours; The growth of BL21 (pET32a) obviously is suppressed; Growing state is clearly better afterwards, and BL21 (pET32a-BjCRP1) is in logarithmic phase always, and (Fig. 9 a) not receive the influence of NaCl basically.BL21 (pET32a) is when NaCl is 0.6mol/L and 0.8mol/L; Almost can not grow; BL21 (pET32a-BjCRP1) is 0.6mol/L at NaCl; The OD600 value is 0.4743 during summit of growth, is 3 times (Fig. 9 b) of BL21 (pET32a) basically, can not grow the same with BL21 (pET32a) (Fig. 9 c) when NaCl is 0.8mol/L.Therefore, think that the threshold value of BL21 (pET32a-BjCRP1) tolerance NaCl is 0.8mol/L.
3. the drought stress of recombination bacillus coli BL21 (pET32a-BjCRP1).
Get BL21 (pET32a-BjCRP1) and contrast BL21 (pET32a) 600ml respectively, be added to and contain in PEG6000 and the antibiotic 30mlLB liquid nutrient medium, the PEG6000 final concentration is respectively 10%, 20%, 30%.Behind 37 ℃ of shaking culture 24h, the bacterium liquid 600 μ l that get respectively again after coercing are transferred to 37 ℃ of following shaking culture in the 30mlLB substratum, and each processing is provided with 3 repetitions, and are every at a distance from its OD600 value of 1h mensuration.
The result shows that the concentration of PEG6000 is when 10%-20%, and reorganization bacterium and contrast bacterium be the ability normal growth all; The OD value all reaches 1.0 (Figure 10 a during summit of growth; B), explain that the BL21 bacterial strain itself has certain anti-PEG and coerces ability, but the reorganization bacteria growing slightly is superior to contrasting bacterium.When PEG concentration is 30%; BL21 (pET32a-BjCRP1) and BL21 (pET32a) growth obviously are suppressed; But concentration difference little (Figure 10 c), show that ability that reorganization bacterium BL21 (pET32a-BjCRP1) shows that the anti-PEG of reorganization bacterium BL21 (pET32a-BjJ10-2) coerces a little less than.
The resistance analysis of embodiment 5BjCRP1 transgene tobacco
Step 1: the structure of plant expression vector and Agrobacterium-mediated Transformation
According to the polyclone restriction enzyme site XbaI/BamHI sequence on two valency carrier pBI121-GFP (seeing Figure 11), designed the ORF district PCR primer of BjCRP1: upstream primer 5 '-gggTCTAGAATGGTTATCTGCAATTATGACAAC-3 '; Downstream primer 5 '-ggcGGATCCTTATGACATGCCTCCGCTAG-3 '; With the BjCRP1 full length cDNA sequence on the prokaryotic expression carrier PET-BjCRP1 carrier is template its ORF zone of increasing respectively; The dna fragmentation of amplification is after the XbaI/BamHI enzyme is cut; Carry out cyclisation with the pBI121-GFP carrier of XbaI/BamHI double digestion and be connected, the transformed competence colibacillus bacillus coli DH 5 alpha is at the enterprising row filter of LB substratum that contains 50 μ g/mL kantlex.37 ℃ of incubated overnight.5-10 mono-clonal of difference picking; Cultivate amplification, extract plasmid and carry out XbaI/BamHI double digestion (Figure 12), get respectively to contain and insert segmental clone (worker is given birth in the Shanghai) checking of checking order; Check order row consistent with known array; Obtain the pBI121-BjCRP1-GFP plant expression vector, wherein contain CaMV 35S constructive expression's strong promoter and GFP green fluorescent protein reporter gene, expressed BjCRP1 and GFP constitute fusion rotein.Use heat shock method (TzVi Tzfira et al; Plant Molecular BiologyReporter; 1997; 15:219-235) pBI121-BjCRP1-GFP is imported among Agrobacterium (Agrobacteriatumefaciencs) EHA105,, and through the bacterial plaque round pcr mono-clonal is transformed Agrobacterium and prove conclusively at the enterprising row filter of LB substratum that contains 30 μ g/mL Rifampins (Rif) and 50 μ g/mL kantlex (Kan).
Step 2: tobacco transforms
Select for use tobacco bred W38 (Nicotiana tabacum c.v.W38) as transgene receptor, adopt Ye Panfa (Horsch RB et al, Science, 1985,227:1229-1231) transformation of tobacco kind W38.With MS (Murashige&Shoog) is minimum medium, and wherein breaking up regeneration culture medium is T
0: MS+1mg/L6-BA+0.1mg/L NAA+100mg/L kantlex+250mg/L Cef (cephamycin)+8g/L agar, PH5.8; Root media is T
1: 1/2MS+0.1mg/L NAA+100mg/L kantlex+250mg/L Cef+8g/L agar, PH5.8.
Picking carries single bacterium colony of the Agrobacterium EHA105 of pB121-BjCRP1-GFP improved carrier, is inoculated in 5ml and contains in the YEB liquid nutrient medium of Rif20mg/l and Kan 50mg/L, and 28 ℃ of shaking culture are spent the night.Get the Agrobacterium that activation is spent the night, in the LB liquid nutrient medium that contains Rif30mg/L and Kan 50mg/L, continue to be cultured to the OD600 value and be approximately 0.6-0.8 by 1: 50 dilution proportion.The centrifugal 5min of 5000rpm collects thalline, washs thalline once with the 1/2MS liquid nutrient medium, and it is diluted to OD600 value 0.3-0.35.
Choose the tobacco aseptic seedling of about 30 days seedling ages, downcut mature leaf, produce leaf dish explant with the punch tool of diameter 6mm.Freshly prepd explant is dropped in the off-the-shelf Agrobacterium bacterium liquid, after vibration is infected 15-20 minute, take out and to blot the raffinate that is attached to the leaf panel surface with filter paper, what be placed on then that the surface is covered with one deck filter paper does not contain antibiotic T
0The dark place is cultivated two days altogether on the substratum, and then forwards T to
0The enterprising row filter of substratum is cultivated, and is every at a distance from 2-3 week using T
0The substratum subculture once.When treating resistant buds length, T is changed in its cutting-out to 1-1.5cm
1Root induction and obtain resistant plant on the substratum.
Transgenic cigarette seedling and the wild-type seedling of choosing 3-4 sheet leaf age carries out that total RNA extracts and cDNA is synthetic.Be PCR with GFP upstream primer p1:5 '-cccGGATCCAAGGAGATATAAC-3 ' and downstream primer p2:5 '-CCCGAGCTCTTATTTGTATAGTTCATCC-3 '; Reaction system 25 μ L reaction systems; Get cDNA template 1 μ L, the condition of PCR reaction is: 94 ℃ of preparatory sex change 10min; 94 ℃ of 1min, 55 ℃ of 2min, 72 ℃ of 2min, 25 circulations; 72 ℃ are extended 10min.Like Figure 13; BjCRP1 transgenic strain (swimming lane 1,2) and empty carrier transgenic seedling (swimming lane 4) have the about 800bp of GFP amplified fragments, and contrast WT does not have amplified band; Explain that fusion gene BjCRP1-GFP is incorporated into respectively in the tobacco gene group, and on transcriptional level, obtain to express.
Step 3:T
0Analyze for tobacco heavy metal and salt resistance
Heavy metal Cd influences transfer-gen plant tissue culture differentiation regenerated: the about 1cm of difference clip
2Empty carrier (pBI121-GFP) and BjCRP1 (pBI121-BjCRP1-GFP) rotaring gene plant blade, be inoculated in T
0(add has 200 μ M/L CdCl to division culture medium
2) on, 10 of every bottle graft kinds, each inoculates 3 bottles, and 25 ℃, the 16h/d illumination cultivation.About 35d (my god) after, calculate differentiation seedling number (Figure 14).
Figure 14 (a) example under no Cd stress conditions, empty carrier and BjCRP1 transgene tobacco explant regeneration seedling no significant difference, and at 200 μ M/L CdCl
2Under the stress conditions, BjCRP1 crosses expression tobacco regrowth obviously more than contrast.Figure 14 (b) shows empty carrier and the statistical study of BjCRP1 transgene tobacco regrowth under normal and Cd stress conditions.Under the normal cultured condition, contrast is counted indifference (t detects, p＞0.05) with BjCRP1 transgene tobacco regrowth, and under the Cd stress conditions, BjCRP1 transgene tobacco regrowth digital display work is higher than the empty carrier contrast and gives birth to seedling (t detects, p＜0.01).
The resistance analysis of transfer-gen plant under heavy metal and the condition of salt stress: the about 1cm of difference clip
2Empty carrier (pBI121-GFP) and BjCRP1 (pBI121-BjCRP1-GFP) rotaring gene plant blade, be inoculated in T
0On the division culture medium, 25 ℃, the 16h/d illumination cultivation.Treat to select when regeneration plant grows to 2-3cm the regrowth of neat and consistent, be inoculated in T respectively
1, T1+200 μ M CdCl
2With T1+250mM NaCl root media, 4-5 of every bottle graft kind, each inoculates 3 bottles.Observe growing way after 15 days and take root situation (Figure 15).
Figure 15 example is at heavy metal Cd Cl
2Under the NaCl stress conditions, BjCRP1 transgene tobacco growing way is taken root, and situation is obviously excellent to be contrasted with empty carrier.Cross expression BjCRP1 tobacco under stress conditions, root has many root regenerations, and the empty carrier contrast does not almost have root regeneration and goes out.Under the normal cultured condition, the two no significant difference, root all have a large amount of roots to bear.The ability of coercing that BjCRP1 has obviously improved the preventing from heavy metal Cd and the high salt NaCl of transgene tobacco of expressing was described.
Sequence table
< 110>Agricultural University Of Hebei
< 120>a kind of stress resistance of plant related membrane protein BjCRP1 and gene and application
<160>2
<210>1
<211>68
<212>PRT
< 213>Indian mustard (Brassica juncea)
<400>1
MTTSKRLADR?KIEKFDKNIT?KRGFVPETTT?KKGKDYPVGP?ILLGFFVFVV
TATSGGMS 68
<210>2
<211>207
<212>DNA
< 213>Indian mustard (Brassica juncea)
<400>2
atgacaactt?caaagaggct?agcagacagg?aagattgaga?agtttgacaa?gaacataact 60
aaaagaggtt?ttgttcctga?gaccaccacc?aagaagggca?aggattaccc?tgttggaccc 120
attctcctcg?gcttctttgt?cttcgtcgtc?attggctcat?ctctcttcca?gatcatcagg 180
accgcaacta?gcggaggcat?gtcataa 207
Claims (4)
1. aminoacid sequence albumen application aspect raising plant preventing from heavy metal, anti-salt, drought resisting shown in SEQ ID No.1.
2. aminoacid sequence proteic gene application aspect raising plant preventing from heavy metal, anti-salt, drought resisting shown in SEQ ID No.1.
3. a method that improves plant preventing from heavy metal, anti-salt, drought resisting is characterized in that, said method comprises aminoacid sequence proteic gene transferred plant, and step of expressing shown in SEQ ID No.1.
4. method according to claim 3 is characterized in that, aminoacid sequence is proteinic gene constructed to the pBI121-GFP carrier shown in SEQ ID No.1, gets recombinant plasmid.
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| CN108192914A (en) * | 2018-01-30 | 2018-06-22 | 西安泽睿环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rape of S.plumbizincicola SpNramp5 genes |
| CN109496815A (en) * | 2018-12-20 | 2019-03-22 | 中国烟草中南农业试验站 | A kind of abductive approach of tobacco drop Cd stress trace |
| CN114349834B (en) * | 2022-01-07 | 2023-08-15 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Cysteine-rich toxic protein, expression vector and application thereof in inhibiting plant virus infection |
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