CN102888409B - Eucalyptus PGEF 10 gene and plant expression vector, host cell and application thereof - Google Patents

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 SEQ ID NO:1 (sequence), or comprising polypeptide with the amino acid sequence as shown in SEQ ID NO: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

Eucalyptus PGEF10 gene and plant expression vector, host cell and application

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 it is in the application promoting vegetation growth of plant and improve in plant-growth amount.

Background technology

Forest at Cleaning Air, check winds and fix drifting sand, conserve water and soil, maintain the eubiosis and species diversity, provide in high-quality timber and high yield and high quality Lin Guo etc. and play an important role, there is huge ecological benefits, social benefit and economic worth.The xylophyta genetic resourceses such as forest enrich, and genetic diversity is complicated, have potential using value with many but not yet obtain at present fully excavating and the gene be effectively separated.In recent years along with the development of the subjects such as molecular biology, molecular genetics, genomics and information biology, the foundation of the technology such as library construction and triage techniques, gene clone technology and high throughput sequencing technologies and improvement, increasing forest gene obtains separation andpreconcentration, for forest molecular breeding with and the excavation of Forest-tree gene and application lay a good foundation people such as (, Botany Gazette 46 (1): 79-107 (2011)) Li Shaofeng.

At present, mainly following methods is adopted to the Function Identification of the forest gene be separated: 1) gene through having identified carries out similarity system design with the homologous gene of model plant or function, sets up evolutionary tree; 2) to protein structure with and function carry out forecast analysis; 3) by the analytical procedure of 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 the response to environmental factor; 4) being connected with suitable element of gene is made into expression cassette, and be assembled into corresponding plant expression vector, by genetic transfoumation, expression cassette is transformed in model plant or source species, thus change the expression amount of target gene in host, as the up-regulated because of overexpression, or because of Antisense Suppression or RNA interference and 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 as Arabidopis thaliana (Arabidopsis thaliana) or tobacco (Nicotiana tabacum).Mouradov etc. (Proceedings of the National Academy of Sciences 95:6537-6542 (1998)) will at pine (Pinus radiata) dEEDLY( nLY), one with Arabidopis thaliana lEAFY( lFY)/ fLORICAULAthe gene of gene very high homology proceeds to Arabidopis thaliana, finds to proceed to lFY::NLYplant can corresponding gene in complementary development pathway lFYdisappearance, thus show nLYwith fLYsimilarity functionally.Eucalyptus camaldulensis HD-Zip class II transcription factor EcHB1 gene is proceeded to tobacco by Sonoda etc. (Plant Biotechnology 26:115-120 (2009)), transfer-gen plant staple length and dry weight increase, and the increment of blade, root and stem is all higher than contrast.The genetic background of this two kind of plant is clear, genetic transformation system is also comparatively ripe, especially Arabidopis thaliana, as the plant that genome is sequenced the earliest, there is large quantifier elimination the aspect such as its genome sequence, pathways metabolism and the molecular mechanism of growing, can provide comparatively perfect reference data for the proterties of transgenic progeny and Analysis on Mechanism.Therefore, high by the credible result of the method identified gene function of genetic transformation model plant, the Application comparison of the result distance molecular breeding of gained is close.

Eucalyptus is the general name that Myrtaceae (Myrtaceae) eucalyptus belongs to (Eucalyptus).View and admire as a kind of collection, use material, pulpwood, medicine for the fine tree species of one, eucalyptus, due to its fast growth, transplanting survival rate advantages of higher, is planted widely.Screened by molecular biology and genomics means and identify the excellent genes of eucalyptus, and therefrom excavate the gene having and can promote vegetation growth of plant and improve phytomass using value, except laying the foundation by the excellent eucalypt species of genetic engineering modified acquisition high yield, excellent genetic resources can also be provided for the molecular breeding of other forests, vegetables and crop.

Summary of the invention

Technical problem to be solved by this invention is, a kind of eucalyptus PGEF10 gene and plant expression vector, host cell are provided and are promoting the application in vegetation growth of plant and raising plant-growth amount, effectively to promote 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, its cDNA sequence has following feature:

There is the DNA sequence dna shown in SEQ ID NO:1; Or

Its coding has the polypeptide of the aminoacid sequence shown in SEQ ID NO:2.

Second aspect, the present invention also provides a kind of plant expression vector, and it comprises eucalyptus PGEF10 gene as above 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 eucalyptus PGEF10 gene as above or its fragment or the plant expression vector as above described in any one.

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 a kind of gene as above, plant expression vector or host cell in the application promoting vegetation growth of plant and improve in phytomass, specifically described gene or its fragment and/or plant expression vector are transformed into plant, or with described host cell infected plant.

In one embodiment, by means commonly known in the art, such as Agrobacterium tumefaciens mediated conversion method, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 is transformed into plant, the transformation generation of this plant is compared with the control plant of non-quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10, and the growth of the vegetative organ such as blade is promoted, and shows as the number of blade and increases, plant diameter increases, and side by side upper part increment is improved.

In second embodiment, by means commonly known in the art, such as Agrobacterium tumefaciens mediated conversion method, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 is transformed into plant, the transformation generation of this plant is compared with the control plant of non-quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10, the growth of root is promoted, and the length showing as root increases.

In the 3rd embodiment, by means commonly known in the art, such as Agrobacterium tumefaciens mediated conversion method, gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10 is transformed into plant, after the conversion of this plant compared with the control plant of non-quiding gene PGEF10 or recombinant vectors pCAMBIA1301-PGEF10, nourish and grow under condition of tissue culture and be promoted, increment is improved.

By adopting technique scheme, the present invention has following beneficial effect: confirm by experiment, and the polypeptide of the PGEF10 gene and coding thereof that come from eucalyptus provided by the invention has the function promoting vegetation growth of plant and improve phytomass.Specifically, the expression amount improving 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 the output improving 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 embryo occurs, extend to the whole growth cycle of plant always, comprise that seed germination and seedling are formed, the prolongation of root and stem and branch, the formation of blade and extension, 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)).

Accompanying drawing explanation

Fig. 1 is that the aminoacid sequence of polypeptide of PGEF10 coding utilizes the CD-Search of NCBI website to retrieve albumen to guard the result that regional data base (CDD v3.05-42589 PSSMs) exports.

Fig. 2 is that unconverted Col-0 Arabidopis thaliana and process LAN PGEF10 Arabidopsis plant contrast photo in the transplanting aerial growth situation of latter 28 days.

Fig. 3 is that unconverted Col-0 Arabidopis thaliana and process LAN PGEF10 Arabidopsis plant add up histogram at the transplanting lotus throne diameter of latter 28 days and the number of blade.

Fig. 4 is the photo of unconverted Col-0 Arabidopis thaliana root growth situation.

Fig. 5 is the photo of process LAN PGEF10 Arabidopsis plant root growth situation.

Fig. 6 is the root long statistics histogram of unconverted Col-0 Arabidopis thaliana and process LAN PGEF10 Arabidopsis thaliana Seedlings.

Embodiment

Below by specific embodiment, also with reference to accompanying drawing, the present invention will be described in more detail.It should be understood that the embodiment of the following stated be only for illustration of instead of restriction the present invention.

One, the cloned and sequenced of PGEF10 gene

1. the preparation of eucalyptus cDNA

Choose fresh eucalyptus ( eucalyptus) blade, such as eucalyptus belongs to tail alpine ash kind DH32-29( eucalyptus urophylla × grandiscv DH32-29) blade, put into rapidly liquid nitrogen freezing, then put into-80 ° of C Ultralow Temperature Freezers (SANYO, MDF-382E) and save backup.

According to existing hexadecyl trimethyl ammonium bromide (Cetyltrimethyl Ammonium Bromide, CTAB) extraction method people such as (, Plant Molecular Biology Reporter 11 (2): 113-116 (1993)) Chang extracts eucalyptus blade total serum IgE.Use the RNA that do not extract containing DNA enzymatic (Takara, the catalog number (Cat.No.) D2270A) process of RNA enzyme with mix inside removing genomic dna.

Get the eucalyptus blade total tissue RNA of about 5 μ g, adopt Reverse Transcription box, SuperScript III First-Strand Synthesis System for RT-PCR(catalog number (Cat.No.) 18080-051 as Invitrogen company), synthesis eucalyptus cDNA(DNA sequence is as shown in SEQ ID NO:1).The cDNA of synthesis 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.) as invitrogen: 11708-039) or the KOD FX(catalog number (Cat.No.) of TOYOBO: KFX-101X5), design with the primer containing restriction enzyme site joint, the sequence that described primer pair is answered is SEQ ID NO:3 and SEQ ID NO:4, with above-mentioned eucalyptus cDNA for template, clone PGEF10 gene.

PCR primer can cut object band after 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 reclaimed uses flat end Cloning Kit to be connected to cloning vector, such as pEASY-Blunt Cloning Kit(Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.): CB101), after transformation of E. coli, send Sangon Biotech (Shanghai) Co., Ltd. to check order positive bacterium colony.The single bacterium colony choosing order-checking correct is preserved.

3. PGEF10 gene conserved regions 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 see NIH (National Institutes of Health, NIH) state-run medical library (National Library of Medicine, NLM) NCBI (National Center for Biotechnology Information, NCBI) to the explanation of CD-Search and conserved structure regional data base (Conserved Domain Database) on World Wide Web Site) and so on analysis tool, conserved structure domain analysis is carried out to the peptide sequence (aminoacid sequence is SEQ ID NO:2) of PGEF10 coding.Fig. 1 illustrates and utilizes CD-Search with the result of a kind of algorithm at CDD lane database region retrieval.

Two, PGEF10 crosses the table acquisition of Arabidopsis plant and ground segment thereof and to nourish and grow the observation of state

1. the structure of recombinant vectors pCAMBIA1301-PGEF10

By using, toolenzyme carries out plasmid double digestion, object fragment reclaims and be connected, and PGEF10 is cloned into plant expression vector, as pCAMBIA1301, makes this gene be positioned at promotor as under the control of CaMV 35S promoter.

Implement according to following working method, detailed step can be modified according to method known to those skilled in the art: use SanPrep pillar plasmid extraction agent box (Sangon Biotech (Shanghai) Co., Ltd. in a small amount, catalog number (Cat.No.): SK8192) from the intestinal bacteria of above-mentioned conversion, extract the recombinant plasmid containing PGEF10, use restriction enzyme bgliI(NEB, catalog number (Cat.No.): R0144) and pmli(NEB, catalog number (Cat.No.): R0532) carry out double digestion; Separately get pCAMBIA1301 plasmid, use bgliI and pmli carries out double digestion.Digestion products carries out 1% agarose gel electrophoresis, cut size respectively and be about the pCAMBIA1301 skeleton band that the PGEF10 of 800 bp and size be about 10000 bp, and use MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0(Takara, catalog number (Cat.No.): D823A) reclaim.By above-mentioned two fragments according to the mol ratio of PGEF10:pCAMBIA1301 be 3:1 mixing, use T4 DNA ligase (Fermentas, catalog number (Cat.No.): #EL0011) carry out connecting rear transformation of E. coli, send Sangon Biotech (Shanghai) Co., Ltd. to check order positive bacterium colony.Choose the correct single bacterium colony of order-checking and use SanPrep pillar plasmid extraction agent box extraction plasmid in a small amount, the plasmid of gained is recombinant vectors pCAMBIA1301-PGEF10.

2. the genetic transformation of Arabidopis thaliana and the screening of transformation generation

2.1 preparations of agrobacterium tumefaciens GV3101 cell containing recombinant vectors pCAMBIA1301-PGEF10

According to the method well known to those skilled in the art (people such as Wang, Agrobacterium Protocols, Human Press, 2nd edition (2006)), the competent cell of preparation agrobacterium tumefaciens GV3101, and use heat-shock transformed method by recombinant vectors pCAMBIA1301-PGEF10 transform Agrobacterium tumefaciens, the agrobacterium tumefaciens GV3101 cell of preparation containing pCAMBIA1301-PGEF10.

The genetic transformation of 2.2 Arabidopis thalianas and the screening of transformation generation

Get Arabidopis thaliana (Arabidopsis thaliana) environmental Columbia-0(Col-0) 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 with existing and screen positive plant from offspring.According to the method, after transforming, the seed of contemporary results is that T1 is for transgenic seed.Get Arabidopis thaliana T1 and contain 50 mg/L hygromycin B (Hygromycin B), Murashige and Skoog substratum (the MS substratum of 1% sucrose and 0.8 g/L for after transgenic seed sterilization; The people such as Murashige, Physiologia plantarum 15 (3): 473-497 (1962)) go up screening and culturing 12 days, the seedling that energy normal growth is grown is transgenic positive T1 for plant.Carefully transferred on compost for plant shoots by transgenic positive T1, cultivate in above-mentioned culture condition, the seed that plant maturation is gathered in the crops afterwards is T2 for transgenic seed.

In order to confirm positive transgenic plant further, PCR method people such as (, Cold Spring Harbor symposia on quantitative biology 51:263-273 (1986)) Mullis can be used to carry out Molecular Identification to screening the positive plant obtained.After seedling grows 8-10 sheet blade, get the long blade of 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, hpt-R3 as sequence number to be the hpt-F3 of SEQ ID NO:5 and sequence number be SEQ ID NO:6 increases to hpt gene, can be defined as positive transgenic plant by the Successful amplification plant that goes out the sample of about 750 bp bands corresponding.

3. PGEF10 crosses the observation of table Arabidopsis plant ground segment growth conditions

Get above-mentioned T2 for transgenic seed, containing 25 mg/L hygromycin B, screening and culturing 12 days on the MS substratum of 1% sucrose and 0.8 g/L agar after sterilization, the seedling that energy normal growth is grown is transgenic positive T2 for plant.Separately get after the seed disinfection of non-transgenic Col-0 Arabidopis thaliana identical but do not cultivate 12 days containing on the MS substratum of hygromycin B in nutritive ingredient, in contrast.Choose the normal positive transgenic seedling of form and contrast seedling and carefully transfer to respectively on compost, cultivate in aforesaid culture condition.Transplant and add up plant lotus throne diameter and the number of blade (sample size >15) afterwards in 28 days, and take the photo of upgrowth situation, as shown in Figure 2.Statistics according to Fig. 3, transformation of Arabidopsis thaliana offspring (PGEF10-T2) plant of process LAN PGEF10 is compared with unconverted Col-0 plant, lotus throne diameter and the number of blade all have remarkable increase, and wherein lotus throne diameter PGEF10-T2 is 7.7 ± 1.6 cm, 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 on this two indices.

Three, PGEF10 process LAN Arabidopsis plant root growth phenotype

The Arabidopis thaliana transgenic seed of process LAN PGEF10 can obtain according to above-described method.And vertical flat plate can be used to detect the Arabidopsis plant root growth phenotype analyzing process LAN PGEF10.

Get above-mentioned T2 for transgenic seed, after sterilization, point has been sowed at 25 mg/L hygromycin B, and 0.5% sucrose is with on the vertical MS culture medium flat plate of 0.8 ~ 1% plant gel (Phytagel), and 10 seeds put by each flat board.After separately getting the seed disinfection of non-transgenic Col-0 Arabidopis thaliana, point is sowed at identical nutritive ingredient but does not contain on the vertical MS culture medium flat plate of hygromycin B, each flat board point 10 seeds, in contrast.Postvaccinal flat board is placed in temperature 20-23oC, humidity 70-85%, illumination 16 h/ days, cultivates in the environment of intensity of illumination 2000 Lux.Cultivate the root long (sample size >15) adding up seedling for 12 days afterwards, and take the photo of upgrowth situation respectively, and as shown in Figures 4 and 5, the statistics shown in composition graphs 6, PGEF10-T2 seedling root length is 5.1 ± 0.4 cm, Col-0 seedling root length is 3.2 ± 0.6 cm.In t test, P value <0.01, illustrating that the root of the more unconverted plant shoots of PGEF10-T2 plant is long has remarkable increase.

Four, the growth conditions under PGEF10 process LAN Arabidopsis plant sterile culture condition

The Arabidopis thaliana transgenic seed of process LAN PGEF10 can obtain according to above-described method.

Get above-mentioned T2 for transgenic seed, containing 25 mg/L hygromycin B, screening and culturing 12 days on the MS substratum of 1% sucrose and 0.8 g/L agar after sterilization, the seedling that energy normal growth is grown is transgenic positive T2 for plant.Separately get after the seed disinfection of non-transgenic Col-0 Arabidopis thaliana identical but do not cultivate 12 days containing on the MS substratum of hygromycin B in nutritive ingredient, in contrast.Choose the normal positive transgenic seedling of form and contrast seedling and carefully transfer to respectively in culturing bottle, described culturing bottle is the glass jar of diameter 8 cm, high 12 cm, inside there are 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, humidity 70-85%, illumination 16 h/ days, cultivates in the environment of intensity of illumination 3500 Lux.Cultivate the fresh weight (sample size >15) adding up plant for 35 days afterwards.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, illustrate that the more unconverted plant of PGEF10-T2 plant is under condition of tissue culture, biomass (fresh weight) has remarkable increase.

SEQUENCE LISTING

 

<110> Prime Green Energy (Shenzhen) Co., Ltd.

 

<120> eucalyptus PGEF10 gene and plant expression vector, host cell and application

 

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Claims (7)

1. an eucalyptus PGEF10 gene, is characterized in that, its cDNA sequence is the DNA sequence dna shown in SEQ ID NO:1.

2. a plant expression vector, is characterized in that: it comprises eucalyptus PGEF10 gene as claimed in claim 1.

3. a host cell, is characterized in that: comprise eucalyptus PGEF10 gene or plant expression vector as claimed in claim 2 as claimed in claim 1.

4. host cell according to claim 3, is characterized in that: described host cell is selected from intestinal bacteria, Agrobacterium or vegetable cell.

5. gene as claimed in claim 1 is in the application promoting vegetation growth of plant and improve in phytomass, it is characterized in that, described gene transformation is entered plant.

6. plant expression vector as claimed in claim 2 is in the application promoting vegetation growth of plant and improve in phytomass, it is characterized in that, described plant expression vector is transformed into plant.

7. the host cell as described in claim 3 or 4, in the application promoting vegetation growth of plant and improve in phytomass, is characterized in that, with described host cell infected plant.