CN103243109A - Corn FK506 binding protein encoding gene ZmFKBP20-1 and application thereof - Google Patents
Corn FK506 binding protein encoding gene ZmFKBP20-1 and application thereof Download PDFInfo
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Abstract
The invention relates to a corn FK506 binding protein encoding gene ZmFKBP20-1 and an application thereof. The nucleotide sequence of the corn FK506 binding protein encoding gene ZmFKBP20-1 is expressed by SEQ ID NO.1. The invention further provides a corn FK506 binding protein, wherein the amino acid sequence of the corn FK506 binding protein is expressed by SEQ ID NO.2. The encoding gene ZmFKBP20-1 is cloned from the corn, and the gene is verified to have the function of adjusting the drought resistant function of plant at seed germination stage, early seedling development stage and seedling stage, and as shown in a test, the gene can be excessively expressed to obtain a transgenic crop with yield higher than that of a wild plant. The gene provided by the invention can be used for providing a theoretical foundation and a gene source for cultivating new plant species.
Description
Technical field
The present invention relates to a kind of corn FK506 in conjunction with protein coding gene ZmFKBP20-1 and application thereof, belong to molecular biology and technical field of biotechnology.
Background technology
Soil drought is one of essential environmental factors of restriction crop yield in the world wide, and the grain drop in production that annual global arid causes surpasses the summation of the underproduction that other all environmental factorss cause.On the earth about 1/3 land area belong to lack of water arid and half-dried morning the zone, half moistening even humid region also regular meeting has periodic, seasonal or provisional arid.Corn integrates grain, feed and industrial raw material, with regard to its gross output and cultivated area, has leapt to the first food crop of China, occupies critical role in development and national economy.The production of corn is subjected to the influence of arid especially obvious, and the research corn is to adaptation and the regulation mechanism of arid, and the drought-resistant corn kind is significant for cultivating.
It is complicated that the various Physiology and biochemistries of the plant that drought stress causes change, and the injury that arid causes plant also is many-sided.Plant can not freely escape as animal, thereby develops in the long-term evolution process and to have produced its distinctive mechanism and come this coercing in the response environment.From the genetics angle, the drought resistance of crop is normally by the quantity shape of controlled by multiple genes.From angle of physiology, the coefficient result of the normally a plurality of metabolic processes of the drought resistance of crop.
FK506 in conjunction with albumen (FKBP) be a kind ofly in organism, extensively exist, the composing type protein (Michnick et al., 1991) of high conservative.(Harding M.W. since 1989 find FKBP first, Galat A., Uehling D.E., Schreiber SL.A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.Nature (1989) 341,758-760), found many kinds of FKBP successively, its content can account for 0.2%～0.5%(Chen Peng of intracellular reactive Tot Prot in some kind, Chen Chonghong, Cheng Yuanrong.FKBPs progress [J].Strait Pharmaceutical Journal (2004) 16 (3): 6-9).Suitable/anti-isomerase (PPIase) activity that FKBP has peptide acyl proline(Pro) participates in Protein Folding, assembling transportation as molecular chaperones, participates in processes such as apoptosis, signal transduction in addition.Discovered FKBP (the Geisler M. that may in processes such as development of plants and degeneration-resistant reaction, play an important role in recent years, Bailly A.Tete-a-tete:the function of FKBPs in plant development.Trends Plant Sci (2007) 12,465-473.), therefore clear and definite their gene function has very important significance.
People are to Arabidopis thaliana (He Z. before, Li L., Luan S.Immunophilins and parvulins.Superfamily of peptidyl prolyl isomerases in Arabidopsis.Plant Physiol (2004) 134,1248-1267.) and paddy rice (Gollan P.J., Bhave M.Genome-wide analysis of genes encoding FK506-binding proteins in rice.Plant Molecular Biology (2010) 72,1-16.) the FKBP gene family carried out the bioinformatic analysis of system, find 23 FKBP genes in the Arabidopis thaliana, have 29 in the paddy rice.Some members of (Arabidopis thaliana, paddy rice, corn, wheat, tomato) FKBP family are cloned successively in plant, and their functional study also obtains certain progress.The signal transduction that plant FKBP participates in cell may be with many environmental factors such as light, to coerce the factor, virus infraction etc. relevant.Be subjected under the situation of environment stress, plant FKBP gene may play regulating and controlling effect.Wheat TaFKBP73 and TaFKBP77 and Arabidopis thaliana AtFKBP62 and AtFKBP65 gene at present research are more deep, and experiment shows that these four expression of gene all can be subjected to induce (mda can be induced the abiotic stress Expression of Related Genes) of extraneous injury, NaCl, mda.Wherein the expression of TaFKBP77 and AtFKBP65 also is subjected to thermal induction, and all the other the two do not possess this characteristic.AtFKBP62 and AtFKBP65 are organizing specific expression, and heat shock protein HSP90 can be combined by the TPR structural domain in AtFKBP62, but is not combined with AtFKBP65.Magiri(2006) etc. discover that rice Os FKBP64, the expression of OsFKBP65 and OsFKBP75 are induced by 42 ℃ of heat shocks, and express and have tissue specificity, thereby three kinds of FKBPs that can infer paddy rice may be three kinds of heat shock proteins.In addition, plant FKBP also participates in the growth and development of plant process.The grain that overexpression TaFKBP73 and TaFKBP77 can change transgenic wheat strain system in wheat heavily reaches diastatic activity in the seed.Arabidopis thaliana AtFKBP72(PAS1) propagation that can regulating cell, in the PAS1 mutant, blade and merismatic cell fission present unequal state.Arabidopis thaliana AtFKBP42 is proved to be the transportation of regulation and control growth hormone, and its sudden change can produce short and small and two kinds of mutation type surfaces of spiral growth.FKBP12 can do mutually with transcription factor HAP5, thereby participates in the direction of growth (Yu Y, the Li Y of regulation and control pollen tube, Huang G, Meng Z, Zhang D, Wei J, Yan K, Zheng C, Zhang L.PwHAP5, a CCAAT-binding transcription factor, interacts with PwFKBP12and plays a role in pollen tube growth orientation in Picea wilsonii.J Exp Bot. (2011) 62,4805-4817.).Spot thatch FKBP12 gene PEG handles the back expression amount significantly to be increased, and (Zhang Muqing haves mercy on into, Wu Yang, Chen Rukai to infer this gene participation drought-resisting regulating.Clone and the expression in the sugarcane drought resisting thereof of spot thatch FKBP12 gene.Whole nation plant molecular breeding symposial summary collection (2009) P118).In important and undergraduate course food crop corn, FKBP research is very few.Hueros etc. (1998) report ZmFKBP66 is present in kytoplasm and the nucleus, and can be combined with calmodulin, but concrete function fails to prove (Hueros G, Rahfeld J, Salamini F, Thompson R.A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein.Planta (1998) 205,121-131.).
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of corn FK506 in conjunction with protein coding gene ZmFKBP20-1 and application thereof.
A kind of corn FK506 is in conjunction with protein coding gene ZmFKBP20-1, and nucleotide sequence is shown in SEQ ID NO.1.
The present invention extracts total RNA from the corn seedling blade of the corn seedling of 400mM treatment with mannitol and normal growth, then the total RNA reverse transcription of 2 micrograms is become cDNA, makes up corn drought stress cDNA library and normal corn cDNA library.Use anti-phase Northern differential screening method, the cDNA that obtains a total length is 859bp, and wherein open reading frame partly is 561bp, pushes away for having 186 amino acid whose one section sequences thus.This aminoacid sequence is compared in international gene pool, show that with the protein-bonded amino acid identity of the FK506 of the Chinese sorghum of having delivered, paddy rice and Arabidopis thaliana be 94%, show through above-mentioned screening step to have obtained the protein-bonded gene of coding FK506
A kind of corn FK506 is in conjunction with albumen, and aminoacid sequence is shown in SEQ ID NO.2.
A kind of carrier that inserts nucleotide sequence shown in the above-mentioned SEQ ID No.1.
A kind of reconstitution cell has inserted nucleotide sequence shown in the above-mentioned SEQ ID No.1 or has contained the carrier of SEQ ID No.1 sequence.
The application in cash crop production improvement of above-mentioned encoding gene ZmFKBP20-1, carrier or reconstitution cell.
The present invention isolates a certain FK506 of coding in conjunction with the full-length cDNA of albumen (ZmFKBP20-1) from corn, be connected on the expression vector, utilize Agrobacterium infestation method arabidopsis thaliana transformation, this gene can improve transgenic arabidopsis reaches the phase in strong sprout in early days at seed germination phase and seedling development drought resistance.
Beneficial effect
The present invention has cloned encoding gene ZmFKBP20-1 from corn, and verified that this gene has in the seed germination phase, seedling development is early stage and become the effect of regulating plant drought resisting function seedling stage, through test, by this gene of overexpression, can obtain the genetically modified crops higher than wild-type plant output (shown in Fig. 1-5).Gene of the present invention can be the cultivation new crop varieties theoretical foundation and gene source is provided.
Description of drawings
Fig. 1 is that transgenic arabidopsis seed and wild-type Arabidopis thaliana seed are in the sprouting situation that contains on the MS substratum of 100mM N.F,USP MANNITOL;
Wherein: WT represents wild-type Arabidopis thaliana seed, and T represents the transgenic arabidopsis seed.
Fig. 2 is transgenic arabidopsis seed and wild-type Arabidopis thaliana seed seed germination rate statistical conditions under the different concns treatment with mannitol;
Wherein: X-coordinate is represented the mannitol concentration gradient, and ordinate zou is represented seed germination rate;
WT represents wild-type Arabidopis thaliana seed, and T represents the transgenic arabidopsis seed.
Fig. 3 is that transgenic arabidopsis seedling and wild-type Arabidopis thaliana seedling are at the growing state that contains on the MS substratum of different concns N.F,USP MANNITOL;
Wherein: WT represents the wild-type Arabidopis thaliana, and T represents transgenic arabidopsis.
Fig. 4 is transgenic arabidopsis and wild-type Arabidopis thaliana average root increment under arid is handled;
Wherein: X-coordinate is represented the mannitol concentration gradient, and ordinate zou is represented the average root increment; WT represents the wild-type Arabidopis thaliana, and T represents transgenic arabidopsis.
Fig. 5 be transgenic arabidopsis strong sprout with to impinging upon the growing state of arid under handling;
Wherein: left column is represented the normal growth condition, and 12 days growth conditions of dehydration is shown in right tabulation;
WT represents the wild-type Arabidopis thaliana, and T represents transgenic arabidopsis.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is further elaborated, but institute of the present invention protection domain is not limited thereto.
Biological material source:
Corn B73 is available from China Agricultural University international corn improvement center;
Intestinal bacteria (E.coli) DH5 α and BM25.8, plasmid pTriplEx2, Pmd18-T are all available from Promega company;
Carrier PBI121 is available from Clontech company;
Reagent source:
Dna gel reclaims test kit, DNA restriction enzyme, T4DNA ligase enzyme, ExTaq enzyme, DNA Blunting Kit, DNAMarkerDL2000+15000 available from the biological Dalian of treasured company limited (Takara product);
TRIzol reagent (No.15596-026) is available from Life Technologies company.
Description of equipment
2720 Thermal Cycler gene-amplificative instraments are used in the PCR reaction, available from Applied Biosystems company;
Agarose gel electrophoresis uses UVP Gel Documentation gel analysis system, available from Gene company;
Agarose gel electrophoresis uses DYY-8C type electrophoresis apparatus, available from Beijing 61 instrument companies.
Embodiment 1: corn FK506 is in conjunction with the acquisition of protein coding gene ZmFKBP20-1
1. the processing of corn seedling: 10 days seedling of corn B73 growth, 400mM treatment with mannitol 18 hours is collected blade;
2. the extraction of total RNA: adopt RNAeasy plant mini kit(promoga company product) extract total RNA;
3. use SMART cDNA Library Construction Kit(available from Clontech company) make up corn drought stress cDNA library and contrast the cDNA library;
4. use anti-phase Northern differential screening method screening that the cDNA segment of differential expression is arranged; Concrete steps are with reference to (Wang Guanlin, the grand sincere work in side, Science Press, 1998) in " plant genetic engineering philosophy and technique ".
5. the pTriplEx2 carrier that will carry the cDNA segment of differential expression changes intestinal bacteria BM25.8 over to; Concrete steps are with reference to (Wang Guanlin, the grand sincere work in side, Science Press, 1998) in " plant genetic engineering philosophy and technique ".
6. sequencing: checked order by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, obtain the cDNA segment of a 859bp, be corn FK506 in conjunction with protein coding gene ZmFKBP20-1, nucleotide sequence is shown in SEQ ID NO.1, wherein open reading frame partly is 561bp, pushes away for having 186 amino acid whose one section sequences thus.
7. homology retrieval: utilize BLAST software that the sequence among isolated sequence and the Genbank is compared, determine that it belongs to corn FK506 binding-protein gene.
Embodiment 2: the acquisition of transgenic arabidopsis and detection thereof
The structure of recombinant expression vector
1. the corn FK506 that gets 1 acquisition of 2 μ l above-described embodiments is connected with the pMD18-T carrier in conjunction with protein coding gene ZmFKBP20-1, and operation steps is undertaken by Promega company product pMD18-T Vector system specification sheets.Transformed into escherichia coli DH5 α bacterial strain then, transferring is coated with overnight incubation on the LB flat board that contains penbritin (100 μ g/ml) of 5-bromo-4-chloro-3-indoles-β-D-galactoside and X-gal in the surface.The picking white colony, overnight incubation and carry out bacterium colony PCR and identify in the LB liquid nutrient medium; Alkaline process extraction plasmid DNA is carried out sequencing simultaneously, makes reorganization pMD18-T carrier;
2. cut reorganization pMD18-T carrier with restriction enzyme BamHI and restriction enzyme SalI enzyme, be connected with the carrier PBI121 that restriction enzyme SalI enzyme is cut then with through restriction enzyme BamHI.Connect product and transform DH5 α cell, cultivate at the LB solid plate that contains kantlex (50 μ g/ml) then, bacterium colony is carried out the restriction analysis of PCR evaluation and plasmid DNA; Make recombinant expression vector PBI121-ZmFKBP20-1.
The acquisition of transgenic arabidopsis
Recombinant expression vector PBI121-ZmFKBP20-1 is transformed the competent cell of Agrobacterium GV3101, the mono-clonal that picking changes the Agrobacterium of recombinant expression vector PBI121-ZmFKBP20-1 over to is inoculated in the LB liquid nutrient medium that contains the 50mg/L kantlex, 28 ℃ of shaking culture two days.With centrifugal 5 minutes of fermented liquid 3000rpm/min, gained Agrobacterium precipitation suspended with the liquid that infects that contains 5wt% sucrose and 0.03wt%SilwetL-77.(detailed process also can be referring to Cheng Zhendong, Wei Zhiming, Xu Zhihong.Agrobacterium tumefaciens is to the conversion of swede type rape and the regeneration of transfer-gen plant.Botany Gazette, 1994,36 (9): 657-663)
The environmental wild-type Arabidopis thaliana (Col-0) of colored dip method conversion Colombia is stained with in employing (can be available from U.S. Arabidopis thaliana Biological resources center, ABRC, http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome. htm), gather in the crops the seed (T that this present age, the transgenic arabidopsis plant was tied
1Generation), the seed of sprouting in the MS substratum screening that contains 50mg/LKan (kantlex).With the T that sprouts
1Move on in the compost results seed (T for seedling
2Generation), the T that obtains isozygotying through identical screening process then
3In generation, changeed the transgenic arabidopsis seed of PBI121-ZmFKBP20-1.At last with T
3Directly be seeded in the compost T that grows for the transgenic arabidopsis seed
3Blooming about two weeks of growth under the long day condition for the transgenic arabidopsis plant.
To T
3Carry out transfer-gen plant for transgenic arabidopsis and identify that comprise: resistance substratum preliminary screening, pcr amplification further screen positive plant, West-blotting identifies positive plant; Finally obtain selecting the positive plant band strain clearly system from identifying by West-blotting, gather in the crops its seed, make the transgenic arabidopsis seed, carry out degeneration-resistant phenotypic evaluation.
It is as follows that pcr amplification further screens the PCR primer of positive plant:
Primer 1:5 '-ATGGAAGAGATGATAGATC-3 ';
Primer 2: 5 ' CTATTTTCCCTTTCCCTTC-3 '.
Transgenic arabidopsis drought resisting phenotypic evaluation
This experiment arranges following two contrasts:
Wild-type contrast: the wild-type Arabidopis thaliana (Col-0) that does not change any plasmid over to;
Empty carrier contrast: according to obtaining T
3In generation, change the method for the transgenic arabidopsis of recombinant expression vector PBI121-ZmFKBP20-1 over to, and empty carrier PBI121 is changed in the wild-type Arabidopis thaliana, carries out succeeding transfer culture then and obtain T
3In generation, changeed the transgenic arabidopsis of PBI121, makes empty carrier contrast seed.
1, the experiment of the seed germination under N.F,USP MANNITOL (Mannitol) the simulating drought stress conditions
Get wild-type Arabidopis thaliana seed, empty carrier contrast seed and transgenic arabidopsis seed, carry out the seed germination experiment at the MS substratum, wherein contain different concns N.F,USP MANNITOL (being respectively 100mM, 200mM, 300mM and 400mM) in the substratum, experiment condition is to be illumination in 16 hours the photoperiod, 8 hours dark; Light intensity is 300-400umol/M
2/ S; Temperature under the illumination condition is 22-24 ℃, and relative humidity is 70-90%; Temperature under the dark condition is 18-20 ℃, and relative humidity is greater than 90%.
At the 7th day statistics seed germination rate, experiment repeated 3 times, and (WT represents wild-type Arabidopis thaliana seed to measurement result, and T represents the transgenic arabidopsis seed as depicted in figs. 1 and 2; Empty carrier contrast seed is consistent with the phenotype of wild-type Arabidopis thaliana seed, so do not show empty carrier contrast among the figure), the germination rate when transgenic arabidopsis seed mannitol concentration in substratum is 100mM, 200mM, 300mM and 400mM is significantly higher than the germination rate of wild-type Arabidopis thaliana seed and empty carrier contrast seed.
2, transgenic arabidopsis seedling phase arid phenotypic evaluation
To be displaced downwardly on the MS flat board that contains 100mM, 200mM and 300mM N.F,USP MANNITOL at aseptic condition the seedling of the firm growth unanimity that flattens of the normal cotyledon that germinates on the MS flat board, put between cultivation, vertically place flat board, experiment condition is to be illumination in 16 hours the photoperiod, 8 hours dark; Light intensity is 300-400umol/M
2/ S; Temperature under the illumination condition is 22-24 ℃, and relative humidity is 70-90%; Temperature under the dark condition is 18-20 ℃, and relative humidity is greater than 90%.Cultivate and take pictures after 1 week and to measure root long, experiment repeats 3 times.(WT represents the wild-type Arabidopis thaliana to measurement result, and T represents transgenic arabidopsis as shown in Figure 3 and Figure 4; The empty carrier contrast is consistent with the phenotype of wild-type contrast, so do not show empty carrier contrast among the figure), the growing state of the seedling of transgenic arabidopsis when mannitol concentration is 100mM, 200mM, 300mM in substratum significantly is better than wild-type Arabidopis thaliana and empty carrier contrast.
3, transgenic arabidopsis becomes arid phenotypic evaluation in seedling stage
Will be in matrix normal growth and the consistent transgenic arabidopsis strain system of growth become seedling to carry out the processing of dehydration arid jointly with the wild-type Arabidopis thaliana.(WT represents the wild-type Arabidopis thaliana, and T represents transgenic arabidopsis as shown in Figure 5; The empty carrier contrast is consistent with the phenotype of wild-type Arabidopis thaliana, and Gu Tuzhong does not show the empty carrier contrast), under normal growth conditions, wild-type and the growth of overexpression strain system do not have difference.Natural dehydration through 12 days is handled, and the Cheng Miao of wild-type Arabidopis thaliana and transgenic arabidopsis has shown the phenotype that arid resistance weakens: wild-type Arabidopis thaliana strain system generally wilts very fast, and surviving rate is low.By contrast, transgenic arabidopsis is wilted slower, the surviving rate height.Compare with wild-type, the drought-resistant ability of transgenic arabidopsis strain system obviously improves.
Genetically engineered elementary operation methods such as PCR, the enzyme that adopts among the embodiment cut, connected, conversion if no special instructions all can be by being recorded in (work such as (U.S.) J. Sa nurse Brooker in " the molecular cloning experiment guide third edition ", Science Press, published in 2002), (Wang Guanlin in " plant genetic engineering philosophy and technique ", the grand sincere work in side, Science Press, 1998) method of record operation.
Claims (5)
1. a corn FK506 is in conjunction with protein coding gene ZmFKBP20-1, and nucleotide sequence is shown in SEQ ID NO.1.
2. a corn FK506 is in conjunction with albumen, and aminoacid sequence is shown in SEQ ID NO.2.
3. carrier that has inserted nucleotide sequence shown in the SEQ ID No.1.
4. reconstitution cell has inserted nucleotide sequence shown in the SEQ ID No.1 or has contained the carrier of SEQ ID No.1 sequence.
5. the described encoding gene ZmFKBP20-1 of claim 1, the described carrier of claim 3 or the described reconstitution cell of claim 4 application in cash crop production improvement.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105315355A (en) * | 2015-09-08 | 2016-02-10 | 山东省农业科学院玉米研究所 | Clone and expression of maize FK506 binding protein gene and application of maize FK506 binding protein gene |
| CN110628780A (en) * | 2019-09-30 | 2019-12-31 | 山东大学 | Corn cell cycle converter gene ZmCCS52B and application thereof |
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2013
- 2013-05-13 CN CN2013101754336A patent/CN103243109A/en active Pending
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| GENBANK: "LOC100280699ZZ NP_001147090.1", 《GENBANK》 * |
| GENBANK: "zea mays full-length cDNA clone ZM-BF0030A19 mRNA complete cds", 《GENBANK》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105315355A (en) * | 2015-09-08 | 2016-02-10 | 山东省农业科学院玉米研究所 | Clone and expression of maize FK506 binding protein gene and application of maize FK506 binding protein gene |
| CN110628780A (en) * | 2019-09-30 | 2019-12-31 | 山东大学 | Corn cell cycle converter gene ZmCCS52B and application thereof |
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