CN102888409A - Eucalyptus PGEF 10 gene and plant expression vector, host cell and application thereof - Google Patents
Eucalyptus PGEF 10 gene and plant expression vector, host cell and application thereof Download PDFInfo
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- CN102888409A CN102888409A CN2012102690322A CN201210269032A CN102888409A CN 102888409 A CN102888409 A CN 102888409A CN 2012102690322 A CN2012102690322 A CN 2012102690322A CN 201210269032 A CN201210269032 A CN 201210269032A CN 102888409 A CN102888409 A CN 102888409A
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Abstract
The invention provides a eucalyptus PGEF 10 gene. The cDNA sequence of the eucalyptus PGEF 10 gene is characterized by comprising the DNA (deoxyribose nucleic acid) sequence as shown in SEQIDNO:1 (sequence), or comprising polypeptide with the amino acid sequence as shown in SEQIDNO:2 at the code. The invention also provides a plant expression vector which includes the eucalyptus PGEF 10 gene or a fragment of the eucalyptus PGEF 12 gene. The invention also provides a host cell which includes the eucalyptus PGEF 12 gene or the fragment or the plant expression vector. The invention also provides the application of the gene, the plant expression vector or the host cell on prompting vegetative growth and improving plant biomass; and the application is mainly to transfer the gene or the fragment of the gene and/or the plant expression vector into the plant or infect the plant through the host cell. The expression index of PGEF10 in the plant is increased, therefore, the quantity of the leaves of the plant is increased, the diameter of the plant is improved, the root is prompted to grow, and the biomass is improved. The eucalyptus PGEF 10 gene, the plant expression vector and the host cell provided by the invention can be widely used for increasing the output of forests, vegetables and industrial crops.
Description
Technical field
The present invention relates to plant gene and application thereof, particularly relate to eucalyptus PGEF10 gene and plant expression vector thereof, host cell and its application on promotion vegetation growth of plant and raising plant-growth amount.
Background technology
Forest at Cleaning Air, check winds and fix drifting sand, conserve water and soil, keep the eubiosis and species diversity, provide the aspects such as high-quality timber and high yield and high quality Lin Guo to play an important role, have huge ecological benefits, social benefit and economic worth.The xylophyta genetic resourceses such as forest are abundant, and genetic diversity is complicated, have potential using value but not yet fully excavated gene with effective separation at present with many.In recent years along with the development of the subjects such as molecular biology, molecular genetics, genomics and information biology, foundation and the improvement of the technology such as library construction and triage techniques, gene clone technology and high throughput sequencing technologies, increasing forest gene obtains separating and identifying, for the molecular breeding of forest with and excavation and the application of Forest-tree gene lay a good foundation people such as (, Botany Gazette 46 (1): 79-107 (2011)) Li Shaofeng.
At present, following methods is mainly adopted in the Function Identification of the forest gene that separated: 1) carry out similarity relatively through the gene of identifying with homologous gene or the function of model plant, set up evolutionary tree; 2) carry out forecast analysis to protein structure and with function; 3) analytical procedure by transcriptional level or protein expression level, such as sxemiquantitative or quantitative RT-PCR, Western Blotting etc., the spatial and temporal expression of research purpose gene and to the response of environmental factor; 4) gene being connected with suitable element is made into expression cassette, and be assembled into corresponding plant expression vector, by genetic transfoumation expression cassette is transformed in model plant or the source species, thereby change the expression amount of target gene in the host, as because of the overexpression up-regulated, or because of Antisense Suppression or RNA interference down-regulated expression, and and by observing the proterties of transfer-gen plant, the function of the research gene (people such as what light source, plant genetic engineering, press of Tsing-Hua University (2007); The people such as Wang Guanlin, plant genetic engineering, Science Press (2009)).
For model plant, generally select to be Arabidopis thaliana (Arabidopsis thaliana) or tobacco (Nicotiana tabacum).Mouradov etc. (Proceedings of the National Academy of Sciences 95:6537-6542 (1998)) will be at pine (Pinus radiata)
DEEDLY(
NLY), one with Arabidopis thaliana
LEAFY(
LFY)/
FLORICAULAThe gene of gene height homology changes Arabidopis thaliana over to, finds to change over to
LFY::NLYPlant can complementary development pathway in corresponding gene
LFYDisappearance, thus show
NLYWith
FLYThe similarity on function.Sonoda etc. (Plant Biotechnology 26:115-120 (2009)) change eucalyptus camaldulensis HD-Zip class II transcription factor EcHB1 gene over to tobacco, transfer-gen plant staple length and dry weight increase, and the increment of blade, root and stem all is higher than contrast.The genetic background of this two kind of plant is clear, genetic transformation system is also comparatively ripe, especially Arabidopis thaliana, the plant that is sequenced the earliest as genome, there is a large amount of research its genome sequence, pathways metabolism and the aspects such as molecular mechanism of growing, can provide comparatively perfect reference data for proterties and the Analysis on Mechanism of transgenic progeny.Therefore, the credible result of the method identified gene function by the genetic transformation model plant is high, and the result of gained is more approaching apart from the application of molecular breeding.
Eucalyptus is that Myrtaceae (Myrtaceae) eucalyptus belongs to the general name of (Eucalyptus).View and admire, use material, pulpwood, medicine to be used for the fine tree species of one as a kind of collection, eucalyptus is planted widely owing to its fast growth, transplanting survival rate advantages of higher.Excellent genes by molecular biology and the screening of genomics means and evaluation eucalyptus, and therefrom excavation has the gene that can promote vegetation growth of plant and improve the phytomass using value, except can laying the foundation by the good eucalypt species of genetic engineering modified acquisition high yield, can also provide for the molecular breeding of other forests, vegetables and crop good genetic resources.
Summary of the invention
Technical problem to be solved by this invention is, a kind of eucalyptus PGEF10 gene and plant expression vector thereof, host cell and the application on promotion vegetation growth of plant and raising plant-growth amount are provided, with effective promotion vegetation growth of plant, improve phytomass.
For solving the problems of the technologies described above, first aspect the invention provides a kind of eucalyptus PGEF10 gene, and its cDNA sequence has following feature:
Has the dna sequence dna shown in the SEQ ID NO:1; Perhaps
Its coding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2.
Second aspect, the present invention also provides a kind of plant expression vector, and it comprises aforesaid eucalyptus PGEF10 gene or its fragment.
Further, described plant expression vector is pCAMBIA1301.Preferably, described plant expression vector is recombinant vectors pCAMBIA1301-PGEF10.
The third aspect, the present invention also provides a kind of host cell, and it comprises aforesaid eucalyptus PGEF10 gene or its fragment or as above each described plant expression vector.
Further, described host cell is selected from intestinal bacteria, Agrobacterium or vegetable cell.In a preferred embodiment, described host cell is selected from the cell of intestinal bacteria Trans1-T1 bacterial strain, Agrobacterium GV3101 bacterial strain or dicotyledons.
Fourth aspect, the present invention also provides the application aspect promotion vegetation growth of plant and raising phytomass of a kind of aforesaid gene, plant expression vector or host cell, specifically described gene or its fragment and/or plant expression vector are transformed into plant, perhaps use described host cell infected plant.
In one embodiment, by means commonly known in the art, Agrobacterium tumefaciens mediated conversion method for example, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 are transformed into plant, the transformation generation of this plant is compared with the control plant of quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 not, and the growth of the vegetative organ such as blade is promoted, and shows as the number of blade and increases, the plant diameter increases, and simultaneously aerial growth measures raising.
In second embodiment, by means commonly known in the art, Agrobacterium tumefaciens mediated conversion method for example, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 are transformed into plant, the transformation generation of this plant is compared with the control plant of quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 not, the growth of root is promoted, and the length that shows as root increases.
In the 3rd embodiment, by means commonly known in the art, Agrobacterium tumefaciens mediated conversion method for example, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 are transformed into plant, compare with the control plant of quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 not after the conversion of this plant, nourishing and growing under condition of tissue culture is promoted, and increment is improved.
By adopting technique scheme, the present invention has following beneficial effect: confirm by experiment, provided by the inventionly come from the PGEF10 gene of eucalyptus and the polypeptide of coding has the function that promotes vegetation growth of plant and improve phytomass.Specifically, the expression amount that improves PGEF10 in plant can increase plant leaf, plant diameter, promotes the growth of root, and improves biomass.Technical scheme of the present invention can be widely used in improving the output of forest, vegetables and cash crop.
Above-mentioned nourishing and growing refers to the growth and development process of plant in the vegetative phase (Vegetative Phase of Development) of growing.This process is from the embryo occurs, extend to the whole growth cycle of plant always, comprise formation and the extension of the prolongation of seed germination and seedling formation, root and stem and branch, blade, and the secondary thickening of root and the stem (people such as Taiz, Plant Physiology, Sinauer Associates, the third edition (2002); The people such as Leyser, Mechanisms in Plant Development, Blackwell Science Ltd. (2003)).
Description of drawings
Fig. 1 is the result that the aminoacid sequence of the polypeptide of PGEF10 coding utilizes conservative regional data base (CDD v3.05-42589 PSSMs) output of CD-Search retrieval albumen of NCBI website.
Fig. 2 is unconverted Col-0 Arabidopis thaliana and crosses and express PGEF10 Arabidopis thaliana plant at the rear 28 days aerial growth situation contrast photo of transplanting.
Fig. 3 is unconverted Col-0 Arabidopis thaliana and crosses and express PGEF10 Arabidopis thaliana plant at transplanting rear 28 days lotus throne diameter and number of blade statistics histogram.
Fig. 4 is the photo of unconverted Col-0 Arabidopis thaliana root growth situation.
Fig. 5 was the photo of expressing PGEF10 Arabidopis thaliana plant root growth situation.
Fig. 6 is unconverted Col-0 Arabidopis thaliana and crosses the long statistics of the root histogram of expressing the PGEF10 Arabidopsis thaliana Seedlings.
Embodiment
The present invention will be described in more detail below by specific embodiment and with reference to accompanying drawing.The embodiment that it should be understood that the following stated only is for explanation rather than restriction the present invention.
One, PGEF10 gene cloning and sequential analysis
1. the preparation of eucalyptus cDNA
Choose fresh eucalyptus (
Eucalyptus) blade, for example eucalyptus belongs to tail alpine ash kind DH32-29(
Eucalyptus urophylla * grandisCv DH32-29) blade, it is freezing to put into rapidly liquid nitrogen, then puts into-80 ° of C Ultralow Temperature Freezers (SANYO, MDF-382E) and saves backup.
According to existing hexadecyl trimethyl ammonium bromide (Cetyltrimethyl Ammonium Bromide, CTAB) extraction method (people such as Chang, Plant Molecular Biology Reporter 11 (2): 113-116 (1993)) extract the total RNA of eucalyptus blade.The RNA that uses the DNA enzyme (Takara, catalog number (Cat.No.) D2270A) do not contain the RNA enzyme to process to extract with remove that the inside mixes genomic dna.
Get the eucalyptus blade total tissue RNA of about 5 μ g, adopt the reverse transcription test kit, SuperScript III First-Strand Synthesis System for RT-PCR(catalog number (Cat.No.) 18080-051 such as Invitrogen company), synthetic eucalyptus cDNA(DNA sequence is shown in SEQ ID NO:1).Synthetic cDNA saves backup in-20 ° of C.
2. polymerase chain reaction method amplification PGEF10 gene
According to polymerase chain reaction (Polymerase Chain Reaction, PCR) method (people such as Mullis, Cold Spring Harbor symposia on quantitative biology 51:263-273 (1986)), use high-fidelity hot polymerization synthase, Platinum Pfx DNA Polymerase(catalog number (Cat.No.) such as invitrogen: 11708-039) or the KOD FX(catalog number (Cat.No.) of TOYOBO: KFX-101X5), design is with the primer that contains the restriction enzyme site joint, the sequence that described primer is corresponding is SEQ ID NO:3 and SEQ ID NO:4, take above-mentioned eucalyptus cDNA as template, clone PGEF10 gene.
The PCR product can downcut the purpose band behind 1% agarose gel electrophoresis, and uses the MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0(catalog number (Cat.No.) of TaKaRa: D823A) reclaim purifying.The fragment that reclaims uses flat terminal clone's test kit to be connected to cloning vector, pEASY-Blunt Cloning Kit(Beijing Quanshijin Biotechnology Co., Ltd for example, catalog number (Cat.No.): CB101), behind the conversion intestinal bacteria, send Sangon Biotech's order-checking with positive bacterium colony.Choosing the correct single bacterium colony of order-checking preserves.
3. PGEF10 gene conservative region is analyzed
Use such as CD-Search(conserved structure domain search (Conserved Domain Search); The people such as Marchler-Bauer, Nucleic Acids Research 39 (D): 225-229 (2011); Also referring to NIH (National Institutes of Health, NIH) state-run medical library (National Library of Medicine, NLM) (the National Center for Biotechnology Information of NCBI, NCBI) on the World Wide Web Site to the explanation of CD-Search and conserved structure regional data base (Conserved Domain Database)) and so on analysis tool, the peptide sequence (aminoacid sequence is SEQ ID NO:2) of PGEF10 coding is carried out the conserved structure domain analysis.Fig. 1 has showed and utilizes CD-Search with the result of a kind of algorithm at CDD lane database region retrieval.
Two, PGEF10 crosses the nourish and grow observation of state of the acquisition of table Arabidopis thaliana plant and ground segment thereof
1. the structure of recombinant vectors pCAMBIA1301-PGEF10
Carry out plasmid double digestion, purpose fragment by the tool using enzyme and reclaim and be connected, PGEF10 is cloned into plant expression vector, such as pCAMBIA1301, so that this gene is positioned under the control of promotor such as CaMV 35S promoter.
Implement according to following working method, detailed step can be made amendment according to method known to those skilled in the art: use in a small amount extraction agent box (Sangon Biotech (Shanghai) Co., Ltd. of SanPrep pillar plasmid, catalog number (Cat.No.): SK8192) from the intestinal bacteria of above-mentioned conversion, extract the recombinant plasmid that contains PGEF10, use restriction enzyme
BglII(NEB, catalog number (Cat.No.): R0144) and
PmlI(NEB, catalog number (Cat.No.): R0532) carry out double digestion; Other gets the pCAMBIA1301 plasmid, uses
BglII and
PmlI carries out double digestion.Enzyme is cut product and is carried out 1% agarose gel electrophoresis, downcut respectively PGEF10 and the big or small pCAMBIA1301 skeleton band that is about 10000 bp that size is about 800 bp, and use MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0(Takara, catalog number (Cat.No.): D823A) reclaim.Be that 3:1 mixes with above-mentioned two fragments according to the mol ratio of PGEF10:pCAMBIA1301, use T4 dna ligase (Fermentas, catalog number (Cat.No.): #EL0011) connect rear conversion intestinal bacteria, send Sangon Biotech's order-checking with positive bacterium colony.Choose the correct single bacterium colony of order-checking and use in a small amount extraction agent box extraction plasmid of SanPrep pillar plasmid, the plasmid of gained is recombinant vectors pCAMBIA1301-PGEF10.
2. the screening of the genetic transformation of Arabidopis thaliana and transformation generation
2.1 contain the preparation of the agrobacterium tumefaciens GV3101 cell of recombinant vectors pCAMBIA1301-PGEF10
According to the method well known to those skilled in the art (people such as Wang, Agrobacterium Protocols, Human Press, the 2nd edition (2006)), the competent cell of preparation agrobacterium tumefaciens GV3101, and use the heat shock conversion method that recombinant vectors pCAMBIA1301-PGEF10 is transformed agrobacterium tumefaciens, preparation contains the agrobacterium tumefaciens GV3101 cell of pCAMBIA1301-PGEF10.
2.2 the genetic transformation of Arabidopis thaliana and the screening of transformation generation
Get the environmental Columbia-0(Col-0 of Arabidopis thaliana (Arabidopsis thaliana)) seed, with the method well known to those skilled in the art (people such as Weigel D, Arabidopsis:A Laboratory Manual, Cold Spring Harbor Laboratory Press (2002)) planting seed is planted at compost.Planting environment: temperature 20-23oC, humidity 70-85%, illumination 16 h/ days, intensity of illumination 4500 Lux.
Dip in colored dip method (people such as Zhang, Nature Protocols 1 (2): 641-646 (2006)) arabidopsis thaliana transformation and screen positive plant from the offspring with existing.According to the method, the seed that gather in the crops the present age after transforming is that T1 is for transgenic seed.Get Arabidopis thaliana T1 and containing 50 mg/L hygromycin B (Hygromycin B), Murashige and Skoog substratum (the MS substratum of 1% sucrose and 0.8 g/L for the transgenic seed sterilization is rear; The people such as Murashige, Physiologia plantarum 15 (3): 473-497 (1962)) upper screening and culturing is 12 days, and the seedling that the energy normal growth is grown is transgenic positive T1 for plant.Transgenic positive T1 is carefully transferred on the compost for the plant seedling, cultivate in above-mentioned culture condition, the seed that the plant maturation is gathered in the crops afterwards is T2 for transgenic seed.
In order further to confirm positive transfer-gen plant, the positive plant that can use PCR method (people such as Mullis, Cold Spring Harbor symposia on quantitative biology 51:263-273 (1986)) that screening is obtained carries out Molecular Identification.After seedling grows 8-10 sheet blade, get the long blade of an agreement that contracts a film or TV play to an actor or actress 1 cm and extract genomic dna as template, super neomycin phosphotransferase gene (the Hygromycin phosphotransferase of design amplification, hpt) special primer, be that the hpt-F3 of SEQ ID NO:5 and hpt-R3 that sequence number is SEQ ID NO:6 increase to the hpt gene such as sequence number, the plant corresponding to sample that can successfully amplify about 750 bp bands can be defined as positive transfer-gen plant.
3. PGEF10 crosses the observation of table Arabidopis thaliana plant ground segment growth conditions
Get above-mentioned T2 for transgenic seed, containing 25 mg/L hygromycin B after the sterilization, screening and culturing is 12 days on the MS substratum of 1% sucrose and 0.8 g/L agar, and the seedling that the energy normal growth is grown is transgenic positive T2 for plant.Other gets after the seed disinfection of non-transgenic Col-0 Arabidopis thaliana MS substratum identical in nutritive ingredient but that do not contain hygromycin B and cultivated 12 days, in contrast.Choose the normal positive transgenosis seedling of form and contrast seedling and carefully transfer to respectively on the compost, in aforesaid culture condition, cultivate.Transplant and add up plant lotus throne diameter and the number of blade (sample size〉15) after 28 days, and take the photo of upgrowth situation, as shown in Figure 2.According to statistics shown in Figure 3, cross express PGEF10 transformation of Arabidopsis thaliana offspring (PGEF10-T2) plant compared with unconverted Col-0 plant, lotus throne diameter and blade number average have remarkable increase, and wherein lotus throne diameter PGEF10-T2 is 7.7 ± 1.6 cm, and Col-0 is 5.2 ± 1.1 cm; Number of blade PGEF10-T2 is 22.1 ± 6.2, and Col-0 is 14.5 ± 5.8, as shown in Figure 3.In t test, P value＜0.01 illustrates that the more unconverted plant of PGEF10-T2 plant is significantly improved in these two indexs.
Three, PGEF10 crosses expression Arabidopis thaliana plant root growth phenotype
Crossing the Arabidopis thaliana transgenic seed of expressing PGEF10 can obtain according to above-described method.And can use vertical flat plate to detect and analyze the Arabidopis thaliana plant root growth phenotype of expressing PGEF10.
Get above-mentioned T2 for transgenic seed, point has been sowed at 25 mg/L hygromycin B after the sterilization, on the vertical MS culture medium flat plate of 0.5% sucrose and 0.8 ~ 1% plant gel (Phytagel), and 10 seeds of each dull and stereotyped point.Other gets after the seed disinfection of non-transgenic Col-0 Arabidopis thaliana point and is sowed at identical nutritive ingredient but does not contain on the vertical MS culture medium flat plate of hygromycin B, 10 seeds of each dull and stereotyped point, in contrast.Postvaccinal flat board is placed in temperature 20-23oC, and humidity 70-85% illumination 16 h/ days, cultivates in the environment of intensity of illumination 2000 Lux.Cultivate the root added up afterwards seedling in 12 days long (sample size〉15), and take respectively the photo of upgrowth situation, such as Fig. 4 and shown in Figure 5, in conjunction with statistics shown in Figure 6, PGEF10-T2 seedling root length is 5.1 ± 0.4 cm, and Col-0 seedling root length is 3.2 ± 0.6 cm.In t test, P value＜0.01 illustrates that the root length of the more unconverted plant seedling of PGEF10-T2 plant has remarkable increase.
Four, PGEF10 crosses the growth conditions of expressing under the Arabidopis thaliana plant sterile culture condition
Crossing the Arabidopis thaliana transgenic seed of expressing PGEF10 can obtain according to above-described method.
Get above-mentioned T2 for transgenic seed, containing 25 mg/L hygromycin B after the sterilization, screening and culturing is 12 days on the MS substratum of 1% sucrose and 0.8 g/L agar, and the seedling that the energy normal growth is grown is transgenic positive T2 for plant.Other gets after the seed disinfection of non-transgenic Col-0 Arabidopis thaliana MS substratum identical in nutritive ingredient but that do not contain hygromycin B and cultivated 12 days, in contrast.Choosing the normal positive transgenosis seedling of form and contrasting seedling carefully transfers in the culturing bottle respectively, described culturing bottle is the glass jar of diameter 8 cm, high 12 cm, in have 60 ml to contain the MS substratum of 1% sucrose and 0.8 g/L agar, use the bottle cap of ventilation.Culturing bottle is placed in temperature 20-23oC, and humidity 70-85% illumination 16 h/ days, cultivates in the environment of intensity of illumination 3500 Lux.Cultivate the fresh weight added up afterwards plant in 35 days (sample size〉15).According to statistics, PGEF10-T2 plant fresh weight 3.52 ± 0.6 g, Col-0 plant fresh weight 2.88 ± 0.3 g.In t test, P value＜0.01 illustrates the more unconverted plant of PGEF10-T2 plant under condition of tissue culture, and biomass (fresh weight) has remarkable increase.
Claims (9)
1. an eucalyptus PGEF10 gene is characterized in that, its cDNA sequence has following feature:
Has the dna sequence dna shown in the SEQ ID NO:1; Perhaps
Its coding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO:2.
2. plant expression vector, it is characterized in that: it comprises eucalyptus PGEF10 gene as claimed in claim 1 or its fragment.
3. plant expression vector according to claim 2, it is characterized in that: described plant expression vector is pCAMBIA1301.
4. plant expression vector according to claim 3 is characterized in that, described plant expression vector is recombinant vectors pCAMBIA1301-PGEF10.
5. host cell is characterized in that: comprise as claimed in claim 1 eucalyptus PGEF10 gene or its fragment or such as each described plant expression vector in the claim 2 ~ 4.
6. host cell according to claim 5, it is characterized in that: described host cell is selected from intestinal bacteria, Agrobacterium or vegetable cell.
7. the application of gene as claimed in claim 1 aspect promotion vegetation growth of plant and raising phytomass is characterized in that, described gene or its fragment are transformed into plant.
One kind such as claim 2 or 3 or 4 described plant expression vectors in the application that promotes aspect vegetation growth of plant and the raising phytomass, it is characterized in that, described plant expression vector is transformed into plant.
One kind such as claim 5 or 6 described host cells in the application that promotes aspect vegetation growth of plant and the raising phytomass, it is characterized in that, with described host cell infected plant.
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| CN101410515A (en) * | 2004-09-22 | 2009-04-15 | 阿博根有限公司 | Compositions and methods for modulating lignin of a plant |
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| CN101410515A (en) * | 2004-09-22 | 2009-04-15 | 阿博根有限公司 | Compositions and methods for modulating lignin of a plant |
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| TUSKAN,G.A.,等: "predicted protein [Populus trichocarpa]", 《GENBANK登录号XM_002315823》, 26 February 2009 (2009-02-26) * |
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