CN102766638A - Application of tomato TFT6 and TFT7 transgenes in enhancing phosphorus-deficient soil environment endurance of plants - Google Patents
Application of tomato TFT6 and TFT7 transgenes in enhancing phosphorus-deficient soil environment endurance of plants Download PDFInfo
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- CN102766638A CN102766638A CN2012102358500A CN201210235850A CN102766638A CN 102766638 A CN102766638 A CN 102766638A CN 2012102358500 A CN2012102358500 A CN 2012102358500A CN 201210235850 A CN201210235850 A CN 201210235850A CN 102766638 A CN102766638 A CN 102766638A
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Abstract
The invention relates to an application of tomato TFT6 and TFT7 transgenes in enhancing the phosphorus-deficient soil environment endurance of plants. The DNA (deoxyribonucleic acid) sequences of TFT6 and TFT7 are obtained, the PCR (Polymerase Chain Reaction) measure is adopted to clone the coding regions of TFT6 and TFT7 genes from a tomato, the coding regions are sequenced and then connected to a modified vector pBI121, and the flower-dipping method is then adopted to transform plants. Under the condition of deficient phosphorus stress, compared with wild arabidopsis thaliana, TFT6 transgenetic arabidopsis thaliana and TFT7 transgenetic arabidopsis thaliana, all the transgenetic grafted plants, the overground parts of which over-express TFT6 and the roots of which over-express TFT7, have high phosphorus content and dry matter weight, thereby indicating that the deficient phosphorus stress-enduring capability of the tomato TFT6 and TFT7 transgentic grafted plants is greatly enhanced.
Description
Technical field
The invention belongs to plant genetic engineering field; Specifically tomato TFT6 and TFT7 gene are expressed in respectively in the overground part and root of plant through transgenic means and precise and tiny graft technology, thus the ability and the application thereof that further improve the low-phosphorous environment of the anti-soil of transgenic grafting plant.
Background technology
Soil is low-phosphorous to be one of major reason of China's grain drop in production.Low-phosphorous approach and the method for improving the soil is a lot, and some researchs show that improving the low-phosphorous genetic potential of the anti-soil of plant also is one of effective means.In recent years, research demonstration plant 14-3-3 albumen is resisted in the low-phosphorus stress process and possibly played a leading role the regulation and control plant.People utilize transgenic plant to do a few thing for material aspect the plant tolerant to low-phosphorus, but about 14-3-3 albumen aspect effect research less.Although 14-3-3 albumen is resisted in the low-phosphorus stress process and possibly played a leading role the regulation and control plant, the direct evidence of this respect is less.Tomato is the very strong crop of flexibility, and cultivation is all arranged from the north polar circle to the equator.Although tomato is very strong to various weather condition flexibility, coercing the influence of output of the poor environment factor is still very big, and low-phosphorus stress is one of these poor environments and the yield and quality that has a strong impact on tomato.Therefore, the anti-low-phosphorous ability of raising tomato is significant.14-3-3 protein gene family has 12 members in the tomato body, and TFT6 is No. six member wherein, and TFT7 is No. seven member wherein.
Summary of the invention
The technical problem that solves: the present invention is directed to the low-phosphorous present situation of soil; Through transgenic means and precise and tiny graft technology tomato TFT6 and TFT7 gene are expressed in respectively in the overground part and root of plant, thereby further improve the ability in the low-phosphorous environment of the anti-soil of transgenic grafting plant.
Technical scheme: change tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant, said plant is an Arabidopis thaliana.
Change tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant; Step is: a. makes up transgenic arabidopsis: extracting tomato RNA and rt is cDNA; Dna sequence dna through the TFT7 shown in TFT6 shown in the SEQ ID No.1 and the SEQ ID No.2; With the PCR means from the tomato vivo clone coding region of TFT6 and TFT7, wherein the primer of TFT6 is TTACGAGGAGATGGTAGAGTTC; AGAGCCAATGAGCTTAGAATC, the primer of TFT7 are ACCTCGTTCCTTCGTCCACTAC; AATTCAATGCGAGTCCAAGTC; , order-checking is connected to plasmid vector pBI121 after identifying; To dip in colored method arabidopsis thaliana transformation, contained the transgenic arabidopsis of TFT6 and TFT7 gene respectively again; B. after intending the Arabidopis thaliana seed sterilization vernalization of commentaries on classics TFT6 or TFT7 of grafting, be grown in vertical sterile agar plate; Agar plate is placed on 21 ℃ of temperature, and the culturing room of continuous light also keeps vertical cultivation 3 days, and then be placed on 23 ℃ of temperature, illumination in 8 hours and dark vertical cultivation down in 16 hours 3 days; Under the aseptic technique environment, overground part and the root of plant separated, culture plate cuts under the angle of level fully; In complete vertical angle grafting, overground part is connected with root system then, entangle with silicone tube the back; The plant of grafting was cultivated 6 days with agar plate, and until the binding site recovery from illness of grafting, plant moves into the water planting system of 1/5Hoagland then; Regrowth 3 days obtains the transgenic arabidopsis plant.
Change the grafting plant of tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant; Obtain the dna sequence dna (seeing table 2) of TFT6 and TFT7; With the PCR means from the tomato vivo clone coding region of TFT6 and TFT7 gene;, order-checking is connected to transformed carrier pBI121 after identifying; Transform plant and further identify (see figure 1) to dip in colored method again, the overground part that utilizes precise and tiny graft technology will change the TFT6 arabidopsis thaliana then carries out grafting and identifies (seeing table 3) with the root system that changes the TFT7 plant, and TFT6 and TFT7 are expressed in respectively in the overground part and root of plant specifically.
Beneficial effect: under the condition of low-phosphorus stress; Compare with wild-type Arabidopis thaliana, commentaries on classics TFT6 gene Arabidopis thaliana and commentaries on classics TFT7 gene Arabidopis thaliana; Transgenic grafting plant (overexpression TFT7 in overground part overexpression TFT6 and the root) all has higher phosphorus content and dry matter weight, shows that the grafting plant that changes tomato TFT6 and TFT7 gene has the ability (seeing Fig. 2 and Fig. 3) that improves plant tolerant to low-phosphorus stress largely.
Description of drawings
Fig. 1 is for changeing the evaluation of TFT6 and TFT7 plant.L6-1 to L6-6 is for changeing 6 of TFT6 gene Arabidopis thaliana not homophyletic systems; L7-1 to L7-6 is for changeing 7 of TFT7 gene Arabidopis thaliana not homophyletic systems;
Fig. 2 is under the low-phosphorus stress, compares with wild-type Arabidopis thaliana, commentaries on classics TFT6 gene Arabidopis thaliana and commentaries on classics TFT7 gene Arabidopis thaliana, changes TFT6 and intravital dry weight of TFT7 grafting plant and phosphorus content;
Fig. 3 is under the low-phosphorus stress, compares with wild-type Arabidopis thaliana, commentaries on classics TFT6 gene Arabidopis thaliana and commentaries on classics TFT7 gene Arabidopis thaliana, changes the phloem sucrose transhipment of TFT6 and TFT7 grafting plant and the speed of root system secretion proton.
Embodiment
The applicant has at first made up the transgenic arabidopsis plant of difference overexpression TFT6 and TFT7; Research shows compares (Columbia) with the wild-type arabidopsis thaliana; Changeing TFT6 and TFT7 arabidopsis thaliana has improved low-phosphorous patience; Difference is, TFT6 mainly on the ground portion work (regulation and control overground part sucrose is transported) and TFT7 mainly in root, work (regulation and control root system secretion proton) in root.Again it; The overground part that the applicant utilizes precise and tiny graft technology will change the TFT6 arabidopsis thaliana carries out grafting with the root system that changes the TFT7 plant; TFT6 and TFT7 are expressed in respectively in the overground part and root of plant specifically, have not only increased the transhipment of transgenic grafting plant shoot sucrose, also improved the rhizosphere souring ability of transgenic grafting plant; Angle from " coordinating up and down " between overground part and the root system has improved the anti-low-phosphorous ability of plant widely.Therefore; The applicant has studied TFT6 and the role of TFT7 and orthofunction effect each other in the plant tolerant to low-phosphorus process through transgenic method and precise and tiny graft technology; Can applicable gene and means be provided for the ability that improves plant tolerant to low-phosphorus through molecular biology method and the low-phosphorous environment of improving the soil, also lay further research basis for the ability that improves the tomato tolerant to low-phosphorus stress through the molecular breeding means.
Embodiment 1:
Materials and methods
Vegetable material, growth conditions, low-phosphorus stress are handled
The means that we cultivate tomato are water planting, and nutritive medium is that extension rate is 1/5 Hoagland solution.We utilize MS plate (1%wt agar and 1%wt sucrose) to cultivate Arabidopis thaliana.The processing means are normal growth (CK: phosphorus concentration is 1mM) and low-phosphorus stress (LP: phosphorus concentration is 1 μ M).For growth test; We get the consistent Arabidopis thaliana of the growth of normal sprouting after 5 days is material; Its MS plate growth that moves on to normal MS plate and processing after 10 days, is got Arabidopis thaliana plant (seedling age 15 days) after liquid nitrogen is fixing, and 80 ℃ of refrigerators of Chu Cun Yu – are with usefulness to be analyzed.Each test triplicate is got 10 Arabidopis thaliana plant at every turn.In addition, each test repeats (the biological repetition) at least twice.
Make up transgenic arabidopsis
Extracting tomato RNA and rt is cDNA.We obtain the dna sequence dna of TFT6 and TFT7 from the website of GENEBANK, with the PCR means from the tomato vivo clone coding region of TFT6 and TFT7, after order-checking is identified, be connected to carrier pBI121.The method of all molecule manipulations is seen " molecular cloning experiment guide " second edition.We are to dip in colored method (Floral dip:asimplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The plant journal 1998,16,735-743.) arabidopsis thaliana transformation.Through after the series of selection, cultivation, evaluation, we are to be the material analysis that makes an experiment with isozygotying of transgenic T3 generation at last.Primer used in the experiment is seen table 1.
The PCR primer that uses in table 1 experiment
The cDNA sequence of table 2 tomato TFT6 and TFT7 gene
Precise and tiny graft technology between the transgenic arabidopsis plant
After intending the Arabidopis thaliana seed sterilization vernalization of commentaries on classics TFT6 or TFT7 of grafting, be grown in vertical sterile agar plate (fully vertically not at all can tiltedly).These planks are placed on 21 ℃ of temperature, and the culturing room of continuous light (120 μ E) also keeps vertical cultivation 3 days, and then be placed on 23 ℃ of temperature, illumination in 8 hours (60 μ E) and dark vertical cultivation down in 16 hours 3 days.Under the aseptic technique environment, under microscopical help, with ultra-thin scalpel the overground part of plant is separated with root and (to cut like the then near undercut of needs overground part; As need root system then to cut on near-earth top); Culture plate cuts under the angle of complete level, then rapidly and carefully in complete vertical angle grafting, with pincet overground part is connected with root system; Entangle with little silicone tube the back; The plant of grafting was cultivated 6 days with duplicate agar plate and under same growth conditions, and until the binding site recovery from illness of grafting, plant moves into the water planting system of 1/5Hoagland then; Regrowth 3 days, the transgenic arabidopsis plant of 15 days seedling ages of process grafting is used for experimental analysis so.
Table 3 changes the evaluation of TFT6 and TFT7 grafting plant.
Remarks: WT (wild-type Arabidopis thaliana); OE-TFT6 (changeing the TFT6 Arabidopis thaliana); OE-TFT7 (changeing the TFT7 Arabidopis thaliana); OE-TFT6/TFT7 (changeing TFT6 and TFT7 grafting Arabidopis thaliana); REU (relative expression unit); N.D. (do not express).
Sucrose phloem transport velocity
According to this method (The Arabidopsis nitrate transporter NRT1.7; Expressed in phloem; Is responsible for source-to-sink remobilization of nitrate.The Plant Cell 2,009 21 2750-2761.) collects the Arabidopsis leaf phloem exudate that exsomatizes.After downcutting Arabidopsis leaf, the end of petiole cuts in damping fluid with new blade that (damping fluid contains 5mMNa again
2EDTA, pH7.5, osmotic pressure is adjusted to 270mosmol with sorbyl alcohol).Blade rinses out dirt with a large amount of aseptic edta buffer liquid, is placed on then in the new edta buffer liquid of 200 μ L.Discharge secretory product therebetween at phloem, blade places culturing room to accept illumination.After one hour ooze out, analyze the sucrose content in the buffered soln that contains the bast exudate.
The SIET method is measured the proton secreting rate
The proton secreting rate adopts SIET method (ion specific electrode technology) to carry out non-damage and measures.The principle of this method and plant and instrument are specifically asked for an interview this method (Root growth inhibition by NH4
+In Arabidopsis is mediated by the root tip and is linked to NH4
+Efflux and GMPase activity.Plant, Cell and Environment 2010,33,1529-1542).All mensuration is responsible for measuring by rising sun moon Science and Technology Ltd. (BeiJing, China).
The mensuration and the statistical study of conventional physical signs
Dry weight and phosphorus content are measured and are seen this method [A " nonsterile " method for selecting and growing Arabidopsis thaliana transformants (T2 Transgenic Lines) resistant to kanamycin.Plant Molecular Biology Reporter 2008; 26,350-357].Data use SPSS13.0 Duncan ' sMultipleRangeTest to carry out statistical study (P < 0.05).
Result and conclusion
The result of Fig. 1 shows that tomato TFT6 and TFT7 gene successfully change in the wild-type Arabidopis thaliana body; The result of table 3 shows that TFT6 and TFT7 gene are expressed in the overground part and root of Arabidopis thaliana respectively; The result of Fig. 2 and Fig. 3 shows; Compare with wild-type Arabidopis thaliana, commentaries on classics TFT6 gene Arabidopis thaliana and commentaries on classics TFT7 gene Arabidopis thaliana; TFT6 and TFT7 are expressed in respectively in the overground part and root of plant specifically, have not only increased the transhipment of transgenic grafting plant shoot sucrose, also improved the rhizosphere souring ability of transgenic grafting plant; Angle from " coordinating up and down " between overground part and the root has improved the anti-low-phosphorous ability of plant widely.Therefore; The applicant has studied TFT6 and the role of TFT7 and orthofunction effect each other in the plant tolerant to low-phosphorus process through transgenic method and precise and tiny graft technology; Can applicable gene and means be provided for the ability that improves plant tolerant to low-phosphorus through molecular biology method and the low-phosphorous environment of improving the soil, also lay further research basis for the ability that improves the tomato tolerant to low-phosphorus stress through the molecular breeding means.
Sequence table
< 110>Nanjing Soil Inst., Chinese Academy of Sciences
< 120>change tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant
<130>
<160> 6
<170> PatentIn?version?3.3
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aatccgattg?ggacttgctc?tcaatttctc?tgtgttttac?tacgagattt?tgaattctcc 660
tgatcgtgcc?tgtaatctcg?ccaaacaggc?ctttgatgag?gcaattgccg?agttggacac 720
attgggcgaa?gagtcctaca?aggatagcac?tctgatcatg?cagcttcttc?gtgataacct 780
cactttatgg?acctctgata?tgcaggatga?tggaactgat?gagatcaaag?aagctacacc 840
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tctatgttga?tttgctgttg?tatttggatt?atttcctttt?tctcaaagga?atgacttact 1080
gggaagatgg?atgtaccttt?taatccacaa?atttgttagc?ttctccattt?catattacat 1140
ggtcatagat?tc 1152
<210> 2
<211> 1001
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ggccgctgag?aggagagaaa?tggagaagga?aagagaaaaa?caggtttact?tggctaggct 60
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gggtcatgag?cagaatgtta?agagaataaa?gacttacaga?cagagagttg?aagacgagct 300
tacaaaaata?tgcagcgaca?ttttgtcagt?gatcgatgag?cacctcgttc?cttcgtccac 360
tactggagaa?tctactgtct?tctactacaa?gatgaaggga?gattactatc?gctatttagc 420
agagttcaaa?gcaggggatg?atcgtaaaga?ggcgtctgaa?cagtctctta?agctttatga 480
ggctgctact?gccactgcta?gttcagatct?tgctccgact?catccaatca?gacttggact 540
cgcattgaat?ttctcagttt?tctactatga?gattctgaat?tcacccgaga?gggcatgcca 600
cttggctaag?caggcatttg?atgaagctat?tgccgaactt?gatagcctca?gtgaagaatc 660
ctacaaggac?agcaccctta?tcatgcagct?tctaagggat?aatctcactt?tgtggacatc 720
agatcttgaa?gagggaggtg?agcattctaa?gggtgatgag?cgccagggag?agaactagtt 780
gaagttcaag?aggtgtcggt?gaatcattgt?gatgctggct?cctcaagaga?catgttagct 840
tgtggaaagt?ctttgttttc?atataatgtt?cagggattca?tgcctattgg?ctttgtactt 900
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< 213>artificial sequence
<400> 3
ttacgaggag?atggtagagt?tc 22
<210> 4
<211> 21
<212> DNA
< 213>artificial sequence
<400> 4
agagccaatg?agcttagaat?c 21
<210> 5
<211> 22
<212> DNA
< 213>artificial sequence
<400> 5
acctcgttcc?ttcgtccact?ac 22
<210> 6
<211> 21
<212> DNA
< 213>artificial sequence
<400> 6
aattcaatgc?gagtccaagt?c 21
Claims (3)
1. change tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant.
2. application according to claim 1 is characterized in that said plant is an Arabidopis thaliana.
3. change tomato TFT6 and TFT7 gene and improving the low-phosphorous environmental applications of the anti-soil of plant, it is characterized in that step is:
A. make up transgenic arabidopsis: extracting tomato RNA and rt is cDNA; Dna sequence dna through the TFT7 shown in TFT6 shown in the SEQ ID No.1 and the SEQ ID No.2; With the PCR means from the tomato vivo clone coding region of TFT6 and TFT7, wherein the primer of TFT6 is TTACGAGGAGATGGTAGAGTTC; AGAGCCAATGAGCTTAGAATC, the primer of TFT7 are ACCTCGTTCCTTCGTCCACTAC; AATTCAATGCGAGTCCAAGTC; , order-checking is connected to plasmid vector pBI121 after identifying; To dip in colored method arabidopsis thaliana transformation, contained the transgenic arabidopsis of TFT6 and TFT7 gene respectively again;
B. after intending the Arabidopis thaliana seed sterilization vernalization of commentaries on classics TFT6 or TFT7 of grafting, be grown in vertical sterile agar plate; Agar plate is placed on 21 ℃ of temperature, and the culturing room of continuous light also keeps vertical cultivation 3 days, and then be placed on 23 ℃ of temperature, illumination in 8 hours and dark vertical cultivation down in 16 hours 3 days; Under the aseptic technique environment, overground part and the root of plant separated, culture plate cuts under the angle of level fully; In complete vertical angle grafting, overground part is connected with root system then, entangle with silicone tube the back; The plant of grafting was cultivated 6 days with agar plate, and until the binding site recovery from illness of grafting, plant moves into the water planting system of 1/5Hoagland then; Regrowth 3 days obtains the transgenic arabidopsis plant.
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Cited By (3)
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| CN104604539A (en) * | 2015-01-30 | 2015-05-13 | 山东省林业科学研究院 | Grafting method for arabidopsis |
| CN112575005A (en) * | 2021-01-04 | 2021-03-30 | 昆明理工大学 | Method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment |
| CN114085843A (en) * | 2020-07-29 | 2022-02-25 | 青岛农业大学 | Application of TFT5 gene in improving resistance of plants to botrytis cinerea infection |
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| CN102220371A (en) * | 2011-05-16 | 2011-10-19 | 中国科学院南京土壤研究所 | Application of tomato TFT1 gene to improving salt tolerance of crops |
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| CN102220371A (en) * | 2011-05-16 | 2011-10-19 | 中国科学院南京土壤研究所 | Application of tomato TFT1 gene to improving salt tolerance of crops |
| CN102391369A (en) * | 2011-12-07 | 2012-03-28 | 中国科学院南京土壤研究所 | Stress tolerance related 14-3-3 protein GRF9 and application thereof |
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| WEI FENG XU: "Expression Profiling of the 14-3-3 Gene Family in Response to Salt Stress and Potassium and Iron Deficiencies in Young Tomato (Solanum lycopersicum) Roots: Analysis by Real-time RT–PCR", 《ANNALS OF BOTANY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104604539A (en) * | 2015-01-30 | 2015-05-13 | 山东省林业科学研究院 | Grafting method for arabidopsis |
| CN114085843A (en) * | 2020-07-29 | 2022-02-25 | 青岛农业大学 | Application of TFT5 gene in improving resistance of plants to botrytis cinerea infection |
| CN114085843B (en) * | 2020-07-29 | 2023-05-02 | 青岛农业大学 | Application of TFT5 gene in improving resistance of plants to botrytis cinerea infection |
| CN112575005A (en) * | 2021-01-04 | 2021-03-30 | 昆明理工大学 | Method for improving heavy metal cadmium stress resistance of tobacco and reducing cadmium enrichment |
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