CN100388878C - Method for promoting plant growth and/or increasing plant resistance - Google Patents
Method for promoting plant growth and/or increasing plant resistance Download PDFInfo
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- CN100388878C CN100388878C CNB2005100693805A CN200510069380A CN100388878C CN 100388878 C CN100388878 C CN 100388878C CN B2005100693805 A CNB2005100693805 A CN B2005100693805A CN 200510069380 A CN200510069380 A CN 200510069380A CN 100388878 C CN100388878 C CN 100388878C
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Abstract
The present invention discloses a method promoting plant growth and / or increasing plant resistance, which comprises the step that an encoding gene of plant glutathione transferase is transduced into a plant in order to obtain the transgenic plant of which the growth is accelerated and / or the resistance is increased. The plant glutathione transferase can be from corn, arabidopsis thaliana, saline land alkali moss or paddy rice, and the best plant glutathione transferase is from the corn. The best plant glutathione transferase is ZmGST 7 which can promote the growth of transgenic arabidopsis thaliana under a normal condition, and can increase the plant resistance under the osmotic stress of low concentration. The method of the present invention has important practicability and high economic benefit for cultivating novel anti-reverse garden plants and farm crops.
Description
Technical field
The present invention relates to a kind of method that promotes plant growing and/or improve plant resistance to environment stress in the biological technical field.
Background technology
The abiotic environment factor (as arid, saline and alkaline etc.) has a strong impact on the normal growth of crops and grows and output, and environmental pollution and population explosion then make the supply of grain more be becoming tight.Thereby needs are cultivated the cereal crops of high resistance and high yield and high quality.The Molecular Study of under adverse circumstance, reacting for plant, present difficult point is known to coerce response gene very little, therefore study the mechanism of plant to the adverse circumstance response, most important is exactly to find more to coerce response gene, and studies their effects in adverse circumstance.
(glutathione S-transferase GST) is the very abundant enzyme of a class to the plant glutathione transferase in plant, and the gene family coding of height difference ancient by.The major function of GST is the electrophilic group of the harmful substance of some endogenous or external source in the catalysis biological body and thin basic combination of glutathione, makes its decomposition, or forms material soluble in water, excretes.
Liu Xinfang etc. find that a GST of arabidopsis is relevant to the tolerance of ultraviolet radiation damage with plant; Liping Wangs etc. are from saline land cloning a GST relevant with salt tolerant the alkali paulin.The homologous gene GST8 of Bianchi etc. research tobacco Nt107 gene in arabidopsis, in dehydration fast and progressive when arid, the expression of GST8 increases gradually.Infer that the possible effect of GST8 is the murder by poisoning of eliminating the active oxygen that is caused by drought stress.
To corn GST research, start from Frear the research to corn antiweed atrazine GST in 1970, find that corn GST has the conjugation that promotes interior GSH of corn body and triazine herbicide atrazine, thereby eliminate its toxicity rapidly corn.BZ2 discovers to corn glutathione transferase gene, and the BZ2 encoded protein works in the biosynthetic final step of anthocyan, finally makes anthocyanidin aggegation in the vacuole of corn.At present, have been found that 42 glutathione transferases in corn, be divided three classes according to the similitude of sequence: type i, II, III, the functional study of wherein most corn glutathione transferase genes is report not.Corn glutathione transferase ZmGST7 is made up of 227 amino acid residues, its encoding gene ZmGST7, and by 840 base compositions, its coded sequence is 25-708 bit base (GenBank AJ010440).At present, do not find that it has the report that promotes plant growing and/or have degeneration-resistant function.
Summary of the invention
The purpose of this invention is to provide a kind of method that promotes plant growing and/or improve plant resistance to environment stress.
Promotion plant growing provided by the present invention and/or improve the method for plant resistance to environment stress is that the encoding gene with the plant glutathione transferase imports the purpose plant, obtains growing accelerating and/or transfer-gen plant that resistance improves.
Described plant glutathione transferase can derive from corn, arabidopsis, saline land alkali paulin or paddy rice, is preferably corn, and described plant glutathione transferase is preferably ZmGST7.
The encoding gene of described plant glutathione transferase imports the purpose plant by plant expression vector; Described plant expression vector is Ti class plasmid vector or viral vectors.
The encoding gene of described plant glutathione transferase can add any general promotor, strengthen promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation nucleotide.
In described expression vector, start the promotor that the encoding gene of described plant glutathione transferase transcribes and can be the cauliflower mosaic virus 35S promoter.Described expression vector is preferably 35S-ZmGST7.
In described expression vector, start the promotor that the encoding gene of described plant glutathione transferase transcribes and also can be arabidopsis rd29A gene promoter.Described expression vector is preferably RD29AP-ZmGST7.
For the ease of genetically modified plants or transgenic plant cells being identified and being screened, can process employed carrier, as the antibiotic marker gene (gentamicin, kanamycin etc.) that adds alternative mark (gus gene, GFP and luciferase gene etc.) or have resistance.For the safety that genetically modified plants discharge, when making up plant expression vector, also can not carry any marker gene, carry out specific PCR molecular marker screening in seedling stage.
Contain the expression vector of the encoding gene of described plant glutathione transferase can be by using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated or particle gun, and the plant transformed tissue cultivating become plant.
Experimental result shows that corn gene ZmGST7 can promote the growth of transgenic arabidopsis under normal operation, and can improve plant resistance to environment stress under the osmotic stress of low concentration.Method of the present invention has important practical value and economic benefit for cultivating novel anti-contrary ornamental plant and crops.
Description of drawings
Figure 1A for BamH I, the HindIII of 35S-ZmGST7, Xhol respectively enzyme cut qualification result
Figure 1B for EcoR I, the Nco I of RD29AP-ZmGST7, Kpn I, Sal I respectively enzyme cut qualification result
Embodiment
Experimental technique among the following embodiment is conventional method if no special instructions.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
T
0Expression is through being stained with the plant after flower infects, T
1Expression T
0The seed that produces for selfing reaches by the plant that it grew up to T
2Expression T
1The seed that produces for selfing and by plant that it grew up to.
Embodiment 1, utilize ZmGST7 to promote the arabidopsis growth and improve the arabidopsis resistance
1, changes the acquisition of ZmGST7 plant
(1) plant expression vector of structure ZmGST7
The ZmGST7 clone that contains who is utilized comes from suppressing the subtracted library that difference subtracts the cross method structure.Concrete library constructing method is all according to the PCR-select of Clontech company
TMThe condition that cDNA Subtraction Kit kit is recommended is carried out, and the ZmGST7cDNA fragment that obtains is connected on the pGEM T-easy carrier and (purchases the company in Promega), and is transformed into DH5 α Bacillus coli cells.All clones find that through the order-checking back one of them clone comprises complete ZmGST7 gene coded sequence, called after pGEM T-easy-ZmGST7.
Cut pGEM T-easy-ZmGST7 plasmid with EcoR I enzyme, obtain the ZmGST7 fragment, be connected between the EcoR I recognition site of pGEM-7Zf (+) carrier (purchasing company) in Promega, evaluation through the gene direction, with Xba I and Sac I double digestion, must arrive two ends and contain the ZmGST7 fragment of Xba I and Sac I restriction enzyme site, this fragment is connected to restriction enzyme Xba I and Sac I removes between the recognition site of restriction enzyme Xba I on pBI221 (the purchasing company) carrier of gus gene and Sac I in BioLabs, cut evaluation through restriction enzyme EcoR I and HindIII enzyme, obtain containing the recombinant vector pBI221-ZmGST7-35S of ZmGST7 gene order.PBI221-ZmGST7-35S cuts with Pst I enzyme, EcoR I is partially digested, obtain containing the ZmGST7 fragment of 35S promoter and NOS terminator, at last this fragment is connected between the recognition site that plant expression vector p3301 goes up the restriction enzyme Pst I of (p3301 available from Australian CAMBIA company) and EcoR I, again with BamH I, HindIII, Xhol respectively enzyme cut evaluation, enzyme is cut qualification result shown in Figure 1A, the band that BamH I enzyme is cut and obtained a size is 13kb; Band that the HindIII enzyme is cut and obtained a size is 11.5kb and the band of a 2kb; The band that the Xhol enzyme is cut and obtained a size is 10.6kb, the band of the band of a 1.8kb and a 560bp; Show obtain structure correct start the recombinant expression carrier 35S-ZmGST7 that ZmGST7 transcribes by the cauliflower mosaic virus 35S promoter.
Cut pGEM T-easy-ZmGST7 with Not I enzyme, obtain the ZmGST7 fragment, be connected between the recognition site of restriction enzyme Not I of pBlue scriptIIKS/SK (+) (purchasing company) carrier in MBI Fermentas, evaluation through the gene direction, with Xbal and Sac I double digestion, must arrive two ends and contain the ZmGST7 fragment of Xbal and SacI restriction enzyme site, this fragment is connected to restriction enzyme Xbal and Sac I removes between the recognition site of restriction enzyme Xbal on pBI221 (the purchasing company) carrier of gus gene and Sac I, obtain containing the recombinant vector pBI221-ZmGST7 of ZmGST7 gene order in BioLabs.Cut 35S promoter with HindIII and Xbal enzyme except that the pBI221-ZmGST7 carrier.According to (MOLECULAR AND GENERAL GENETICS such as Yamaguchi Shinozaki, 1993,5 ' terminal sequence of the rd29A gene of 236:331-340) delivering, designed the special primer that a pair of two ends have HindIII and Xbal restriction enzyme site respectively (P1:5 '-AAAAGCTTACGCATGATTTGATGGAG-3 ' (sequence 1), P2:5 '-AGTCTAGAAACCCTTTATTCCTGATGATTG-3 ' (sequence 2)), synthetic by Shanghai bio-engineering corporation.Carry out pcr amplification from arabidopsis thaliana genomic dna, reaction system comprises 1 μ l dna profiling for being 25 μ l, 2.5 μ l10 * buffer, 2.5 μ l dNTPs (2mM), 1 μ l primer P1 (10mM), 1 μ l primer P2 (10mM), 0.3 μ lTaq enzyme (5U/ μ l), 16.7 μ l ddH
2O.Reaction condition is 94 ℃ of pre-sex change 3 minutes; 94 ℃, 45 seconds, 65 ℃, 45 seconds, 72 ℃, 1 minute, extended 10 minutes in 72 ℃ of insulations after 35 circulations.The PCR product is cut with HindIII and Xbal enzyme, obtain containing the RD29A Promoter fragment of HindIII and Xbal restriction enzyme site, this fragment is connected on the pBI221-ZmGST7 that contains ZmGST7 that removes 35S promoter, cut through the HindIII enzyme, EcoR I is partially digested, obtain containing the ZmGST7 fragment of RD29A promotor and NOS terminator, be connected at last between the recognition site of restriction enzyme HindIII on the plant expression vector p3301 and EcoR I, use EcoR I again, Nco I, Kpn I, Sal I enzyme respectively cuts evaluation, enzyme is cut qualification result shown in Figure 1B, band that Nco I enzyme is cut and obtained a size is 11.5kb and the band of a 1.92kb; Band that Kpn I enzyme is cut and obtained a size is 11.2kb and the band of a 2.27kb; Band that EcoR I enzyme is cut and obtained a size is 12.26kb and the band of a 1.2kb; The band that Sal I enzyme is cut and obtained a size is 10.9kb, the band of the band of a 2.26kb and a 280bp; Show obtain structure correct start the recombinant expression carrier RD29AP-ZmGST7 that ZmGST7 transcribes by arabidopsis rd29A gene promoter.
(2) transform plant
Recombinant expression plasmid 35S-ZmGST7 or RD29AP-ZmGST7 are transformed Agrobacterium GV3101, and extract plasmid and do that enzyme is cut and pcr amplification detects, the screening positive strain is used to transform plant.
Positive agrobacterium strains is inserted the YEB medium respectively, cultivated OD 5～6 hours for 28 ℃
600About 0.5 o'clock, 5000g, collected somatic cells in centrifugal 15 minutes,, add volumn concentration and be 0.02% surfactant silwet L-77 (available from GE Silicones company) and be used to transform plant with the solution re-suspended cell that contains 1 * MS macroelement and 5% sucrose.Arabidopsis thaliana transformation adopts is stained with the method that flower infects (floral dipping), transforms the dark low temperature in back (16 degrees centigrade) cultivation and changes growth under the normal condition of culture after 24 hours over to, is T
0For plant.Results T
0For the seed (T on the plant
1For seed), with 7000 T
1For the seed vernalization 3 days of tiling on the MS medium, directly be seeded in the nutrition soil then and grow, normal growth is after 20 days, with weed killer herbicide grass fourth phosphine (PPT) the spraying screening transformed plant of 0.5 ‰ (percents by volume).Transformed plant still can normal growth after spraying antiweed, extract the genomic DNA of transformed plant simultaneously, utilize primer AAGACCTGGGCAACAAGAGCGA and TGGCGAAGAAATAGAGACACACCTTA to carry out pcr amplification ZmGST7 gene, utilize primer CCAGAAACCCACGTCATGCC and CAGGAACCGCAGGAGTGGA to carry out pcr amplification herbicide resistance gene bar gene, further identify transformed plant (no corresponding Z mGST7 gene and herbicide resistance gene amplified band in the unconverted plant), the result obtains 100 strain T
1For transformed plant.T
1Gather in the crops T for transformed plant respectively according to strain system
2For seed.T
2After the seed plantation, the evaluation transformed plant that uses the same method, and results T
3For seed, the result obtains the T of 50 35S-ZmGST7 transformation plants
3T for seed (each 1000 in each strain system) and 50 RD29AP-ZmGST7 transformation plants
3For seed (each 1000 in each strain system).
2, the growth traits of changeing the ZmGST7 plant is observed
The T that will contain the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for seed, commentaries on classics RD29AP-ZmGST7
3At first be seeded on the MS medium that contains 7mg/L grass fourth phosphine 4 ℃ of vernalization 3 days, illumination cultivation growth 4 days, the seedling that screening can be survived for seed (respectively getting 10 transformation plants, each 30 in each strain system); The seed of unconverted plant (30) be seeded on the MS medium, 4 ℃ of vernalization 3 days, illumination cultivation growth 4 days, seedling in contrast.The transgenosis seedling and the contrast seedling of screening are transferred on the MS medium simultaneously, and culture dish is uprightly placed, and the arabidopsis seedling was inverted growth after 4 days, and each strain system gets 10 strains and observes relatively plant root, and the result shows the T of the commentaries on classics 35S-ZmGST7 that contains ZmGST7
3Root long (average out to 2.74cm) for the plant seedling is obviously compared the length of shining (average out to 2.15cm), and variance analysis reaches utmost point significance level (1% significance level), and contains the T of the commentaries on classics RD29AP-ZmGST7 of ZmGST7
3Grow (average out to 2.26cm) difference little (table 1) compared with the control for the root of plant seedling.
Table 1. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
3Be inverted the root of growth after 4 days long (cm) relatively for plant and unconverted plant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 2.43 | 2.40 | 2.5 | 3.33 | 2.73 | 3.23 | 2.80 | 2.67 | 2.67 | 2.63 | 2.74 |
| Change RD29AP-ZmGST7 | 2.13 | 2.33 | 2.23 | 2.53 | 2.35 | 2.46 | 2.35 | 2.17 | 1.96 | 2.10 | 2.26 |
| Unconverted | 2.20 | 1.98 | 2.15 | 2.10 | 2.03 | 2.13 | 2.20 | 2.17 | 1.95 | 2.12 | 2.15 |
With the above-mentioned T that contains the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for seed, commentaries on classics RD29AP-ZmGST7
3At first be seeded on the MS medium that contains 7mg/L grass fourth phosphine 4 ℃ of vernalization 3 days, illumination cultivation growth 7 days, the seedling that screening can be survived for seed (respectively getting 10 transformation plants, each 30 in each strain system); The seed of unconverted plant (30) be seeded on the MS medium, 4 ℃ of vernalization 3 days, illumination cultivation growth 4 days, seedling in contrast.The transgenosis seedling and the contrast seedling of screening are sowed in the sub-small flower simultaneously, after growing into 20 days, obviously observe the commentaries on classics 35S-ZmGST7 plant comparison that contains ZmGST7 and shine bolting, bloomed 3-4 days in advance, it is then consistent with the contrast bolting time to change the RD29AP-ZmGST7 plant.Table 2 shows the difference of the Ahau measurement plant bolting height after transplanting.The difference of changeing 35S-ZmGST7 plant and contrast reaches utmost point significance level (p＜0.01).
Table 2. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
3Comparison for the Ahau plant bolting height (cm) behind plant and the unconverted plantlet of transplant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 6.00 | 7.16 | 7.34 | 8.20 | 7.70 | 6.50 | 7.98 | 6.66 | 6.58 | 6.30 | 7.04 |
| Change RD29AP-ZmGST7 | 5.22 | 4.98 | 5.56 | 3.80 | 3.32 | 3.80 | 4.76 | 3.26 | 3.30 | 3.46 | 4.15 |
| Unconverted | 2.63 | 3.83 | 3.50 | 2.50 | 2.46 | 3.00 | 2.67 | 3.10 | 4.60 | 4.50 | 3.28 |
3, the resistance of changeing the ZmGST7 plant is observed
The T that will contain the commentaries on classics 35S-ZmGST7 of ZmGST7
3For seed, contain the T of the commentaries on classics RD29AP-ZmGST7 of ZmGST7
3(respectively get 10 transformation plants for seed, each 30 in each strain system) at first is seeded on the MS medium that contains 7mg/L grass fourth phosphine, (30 in the seed of unconverted plant, contrast) is seeded in the MS medium, 4 ℃ of vernalization 3 days, illumination cultivation growth 4 days, the seedling that can survive is transferred on the MS medium of the mannitol (100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L) that contains variable concentrations again, culture dish is uprightly placed, the arabidopsis seedling is inverted growth, and the root of measuring crooked growth downwards after 4 days is long.The result shows the T of the commentaries on classics RD29AP-ZmGST7 that contains ZmGST7
3Seedling for plant is coerced the long comparison of down relative root according to longer at the mannitol of low concentration, especially reaches utmost point significance level when 200mmol/L mannitol.The T that contains the commentaries on classics 35S-ZmGST7 of ZmGST7
2Coerce the long comparison of down relative root according to long for plant at the mannitol of low concentration, but that the amplitude of difference is not changeed the seedling of RD29AP-ZmGST7 is obvious, only when 200mmol/L mannitol, reaches significance level (table 3-table 6).
Table 3. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
34 days root of growth long (cm) under the mannitol of 100mmol/L is coerced compares for plant and unconverted plant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 1.73 | 2.03 | 1.83 | 1.10 | 1.70 | 2.13 | 2.27 | 2.07 | 1.90 | 1.83 | 1.86 |
| Change RD29AP-ZmGST7 | 1.67 | 2.03 | 2.07 | 2.00 | 1.90 | 1.97 | 2.17 | 2.00 | 1.93 | 1.83 | 1.96 |
| Unconverted | 1.10 | 1.80 | 1.93 | 1.80 | 1.50 | 1.23 | 1.20 | 1.40 | 2.07 | 1.86 | 1.59 |
Table 4. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
34 days root of growth long (cm) under the mannitol of 200mmol/L is coerced compares for plant and unconverted plant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 1.53 | 1.90 | 1.37 | 1.50 | 1.13 | 1.83 | 1.47 | 1.80 | 1.57 | 1.40 | 1.55 |
| Change RD29AP-ZmGST7 | 1.63 | 1.53 | 1.63 | 1.77 | 2.33 | 2.03 | 1.53 | 1.70 | 1.60 | 1.43 | 1.72 |
| Unconverted | 0.83 | 1.03 | 1.17 | 1.63 | 0.90 | 1.53 | 1.17 | 1.23 | 1.30 | 0.67 | 1.15 |
Table 5. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
34 days root of growth long (cm) under the mannitol of 300mmol/L is coerced compares for plant and unconverted plant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 0.87 | 0.87 | 1.20 | 1.00 | 0.70 | 1.20 | 1.07 | 1.10 | 1.00 | 1.13 | 1.01 |
| Change RD29AP-ZmGST7 | 1.07 | 0.80 | 0.80 | 0.80 | 0.93 | 0.77 | 0.70 | 1.03 | 0.97 | 0.90 | 0.88 |
| Unconverted | 0.80 | 0.83 | 0.70 | 0.53 | 0.50 | 0.83 | 0.70 | 0.80 | 0.60 | 0.85 | 0.71 |
Table 6. contains the T of the commentaries on classics 35S-ZmGST7 of ZmGST7
3T for plant, commentaries on classics RD29AP-ZmGST7
34 days root of growth long (cm) under the mannitol of 400mmol/L is coerced compares for plant and unconverted plant
| T 3For strain be | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | On average |
| Change 35S-ZmGST7 | 0.40 | 0.53 | 0.50 | 0.57 | 0.33 | 0.47 | 0.53 | 0.50 | 0.43 | 0.40 | 0.47 |
| Change RD29AP-ZmGST7 | 0.43 | 0.50 | 0.50 | 0.57 | 0.50 | 0.60 | 0.43 | 0.50 | 0.30 | 0.53 | 0.49 |
| Unconverted | 0.30 | 0.53 | 0.37 | 0.37 | 0.37 | 0.50 | 0.40 | 0.37 | 0.37 | 0.40 | 0.40 |
The result of present embodiment shows that corn gene ZmGST7 can obviously promote plant growing, and can improve the resistance of plant under low-level osmotic stress.
Sequence table
<160>2
<210>1
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
aaaagcttac gcatgatttg atggag 26
<210>2
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
agtctagaaa ccctttattc ctgatgattg 30
Claims (6)
1. method that promotes plant growing and/or improve plant resistance to environment stress, be to import the purpose plant by the encoding gene that the cauliflower mosaic virus 35S promoter starts the corn glutathione transferase ZmGST7 transcribe, obtain growing and accelerate and/or transfer-gen plant that resistance improves; Described growth is accelerated to shifting to an earlier date flowering stage; Described resistance rises to the elongation of root under the hyposmosis stress conditions.
2. method according to claim 1 is characterized in that: the encoding gene of described corn glutathione transferase ZmGST7 imports the purpose plant by plant expression vector; Described plant expression vector is Ti class plasmid vector or viral vectors.
3. method according to claim 2, it is characterized in that: described expression vector is 35S-ZmGST7, this carrier is that the ZmGST7 gene coded sequence is connected between the EcoR I recognition site of pGEM-7Zf (+) carrier, with Xba I and Sac I double digestion, must arrive two ends and contain the ZmGST7 fragment of Xba I and Sac I restriction enzyme site, this fragment is connected between the recognition site of restriction enzyme Xba I on the pBI221 carrier of removing gus gene and Sac I, the recombinant vector that obtains is cut with Pst I enzyme, EcoR I is partially digested, obtain containing the ZmGST7 fragment of 35S promoter and NOS terminator, this fragment is connected between the recognition site of the restriction enzyme PstI of carrier p3301 and EcoR I, obtains expression vector 35S-ZmGST7.
4. a method that improves plant resistance to environment stress is that the encoding gene that will be started the corn glutathione transferase ZmGST7 that transcribes by arabidopsis rd29A gene promoter imports the purpose plant, obtains the transfer-gen plant that resistance improves; Described resistance rises to the elongation of root under the hyposmosis stress conditions.
5. method according to claim 4 is characterized in that: the encoding gene of described corn glutathione transferase ZmGST7 imports the purpose plant by plant expression vector; Described plant expression vector is Ti class plasmid vector or viral vectors.
6. method according to claim 5, it is characterized in that: described expression vector is RD29AP-ZmGST7, this carrier is that the ZmGST7 gene coded sequence is connected between the recognition site of restriction enzyme Not I of pBluescript II KS/SK (+) carrier, with Xbal and Sac I double digestion, must arrive two ends and contain the ZmGST7 fragment of Xbal and SacI restriction enzyme site, this fragment is connected between the recognition site of restriction enzyme Xbal on the pBI221 carrier of removing gus gene and Sac I, the recombinant vector that obtains cuts except that 35S promoter with HindIII and Xbal enzyme; The RD29A Promoter fragment that will contain HindIII and Xbal restriction enzyme site is connected on the recombinant vector of above-mentioned removal 35S promoter, cut through the HindIII enzyme, EcoR I is partially digested, obtain containing the ZmGST7 fragment of RD29A promotor and NOS terminator, be connected on the plant expression vector p3301, obtain recombinant expression carrier RD29AP-ZmGST7.
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| CN101798340B (en) * | 2009-02-09 | 2013-06-19 | 中国科学院遗传与发育生物学研究所 | Plant growth coordinating and disease-resistant related gene, coded protein and application thereof |
| CN101748144B (en) * | 2010-01-22 | 2011-12-28 | 昆明理工大学 | Torch pear haloduric gene PpGST and application thereof |
| WO2012008042A1 (en) * | 2010-07-16 | 2012-01-19 | 花王株式会社 | Method for imparting stress tolerance to plant, composition for imparting stress tolerance to plant and utilization thereof |
| US9499830B2 (en) * | 2011-07-25 | 2016-11-22 | National University Corporation NARA Institute of Science and Technology | Gene inducing elongation of roots or increasing biomass, and use therefor |
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