CN102757486B - Plant development related protein GA20ox, and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a plant development related protein GA20ox, and an encoding gene and application thereof. The protein provided by the invention is a protein (a) or (b), wherein the protein (a) has an amino acid sequence shown in Sequence 1 in a sequence table, and the protein (b) is obtained through carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown in Sequence 1, related to plant development and derived from Sequence 1. In the invention, a recombinant plasmid containing a GA20ox gene is introduced to tobacco through an agrobacterium-mediated transformation method to obtain transgenic tobacco; and the development level of the transgenic tobacco is remarkably improved, which is mainly embodied in that the leaf area, the plant height and the node spacing are increased. The GA20ox gene provided by the invention has the function of promoting the growth and development of plants, and thus can be used for culturing transgenic plants of which the development levels are increased. The plant development related protein GA20ox has a great value for plant breeding.

Description

Plant development associated protein GA20ox and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant development associated protein GA20ox and encoding gene and application.

Background technology

In mid-term in last century, " Green Revolution ", by genetics means Crop Improvement, increases substantially whole world total grain output at short notice.Along with molecular biological development, " Green Revolution " gene is cloned out, and they are all relevant to Plant hormones regulators,gibberellins metabolism or signal transduction.

Plant hormones regulators,gibberellins (GA) is the important plant hormone of a class, have bioactive Plant hormones regulators,gibberellins all plays an important role in the whole life cycle of higher plant, it can promote the growth etc. of elongation, vane extension, trichome development, pollen tube growth, flower and the fruit of seed germination, stem and root, and plant also can be by regulating the biosynthesizing of endogenous gibberellins mediate self replying environmental factor.

Active Plant hormones regulators,gibberellins is by synthetic from Mang ox geranylpyrophosphate (geranylggeranyl diphpsphate GGDP), there are three kinds of different enzymes to participate in the synthetic of active Plant hormones regulators,gibberellins, they are respectively terpenes synthetic enzyme (TPSs), the dioxygenase (2ODDs) that cytochrome P 450 monooxygenases (P450s) and 2-oxoglutarate rely on.First GGDP is by Nei Gen-Ke Ba pyrophosphate synthetase (ent-copalyl diphosphate synthase, CPS) be cyclized into Nei Gen-Ke Ba tetra-sodium (ent-copalyl diphosphate, CDP), CDP is further cyclized into Nei Gen-kaurene (ent-kaurene) by Nei Gen-kaurene synthetic enzyme (ent-kaurene synthase, KS).Then Nei Gen-kaurene is by two cytochrome P 450 monooxygenases) Nei Gen-ent-kaurene oxidase (ent-kaurene oxidase, KO) and Nei Gen-kaurene acid oxidase (ent-kuarenoic acid oxidase, KAO) be converted into GA12-aldehyde.Final step has been participated in by the oxydase of two solubilities.GA12-aldehyde and its 13-hydroxylation product GA53 carry out three times continuous oxidation by GA20-oxydase (gibberellin 20-oxidase, GA20ox) on the C-20 position of GA12 and GA53, generate GA9 and GA20.3 beta-hydroxy groups of GA3-oxydase (gibberellin 3-oxidase, GA3ox) mediation are upper to GA9 and GA20, generate and have bioactive Plant hormones regulators,gibberellins, GA1 and GA4.Due to the downstream of GA20ox in Plant hormones regulators,gibberellins route of synthesis, its activity directly has influence on the synthetic of active Plant hormones regulators,gibberellins.Therefore gene GA20ox has obtained clone and functional verification in different species in recent years, and these researchs all show that GA20ox can promote the synthetic of active Plant hormones regulators,gibberellins, and then promote growing of plant.

Festuca Arundinacea is named again alta fascue, reed shape fox thatch, is cold ground type turfgrass, belongs to Gramineae festuca per nnial herb.The advantages such as nutritious, resistance to trample that Festuca Arundinacea has, cold-resistant, disease-resistant, drought resisting, widespread use in the planting lawn of the many North Cities of China.

Summary of the invention

The object of this invention is to provide a kind of plant development associated protein GA20ox and encoding gene and application.

Protein provided by the invention, name is called GA20ox albumen, derives from Festuca Arundinacea (Festuca arundinacea), is following (a) or (b):

(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;

(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to development of plants through one or several amino-acid residue by the aminoacid sequence of sequence 1.

In order to make the protein in (a) be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Protein in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

The gene of encoding said proteins (GA20ox gene) also belongs to protection scope of the present invention.

Described gene can be following 1) to 4) in arbitrary described DNA molecular:

1) sequence 2 of sequence table is from the DNA molecular shown in the 178th to 1278 Nucleotide of 5 ' end;

2) DNA molecular shown in sequence 2 in sequence table;

3) under stringent condition with 1) or 2) the DNA sequence dna hybridization that limits and the DNA molecular of coded plant development associated protein;

4) with 1) or 2) DNA sequence dna limiting has the DNA molecular of 90% above homology and coded plant development associated protein.

Above-mentioned stringent condition can be at 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridizes and wash film under 65 ℃ of conditions.

The recombinant expression vector that contains described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.

The recombinant expression vector that available existing plant expression vector construction contains described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described recombinant expression vector specifically can be the recombinant plasmid that the multiple clone site of described gene insertion vector pCAMBIA1302 is obtained.

The total length of described gene that increases or the primer pair of its arbitrary fragment also belong to protection scope of the present invention.

The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in object plant, obtains plant development level higher than the transgenic plant of described object plant.Described gene specifically can import in described object plant by described recombinant expression vector.Carry the expression vector of described gene can be by using conventional biological method transformed plant cells or the tissue such as Ti-plasmids, Bi plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, agriculture bacillus mediated, particle gun, and the plant tissue of conversion is cultivated into plant.Described plant development level is high is embodied as that leaf area is large and/or plant height is high and/or interval is large.Described interval specifically can be plant and counts from the bottom up the length between second section.Described object plant can be both that monocotyledons can be also dicotyledons.Described dicotyledons specifically can be tobacco (as Nicotiana tabacum).

In the present invention, the recombinant plasmid that contains GA20ox gene is imported to tobacco by Agrobacterium-mediated Transformation method, obtained transgene tobacco, the developmental level of this transgene tobacco significantly improves, and major embodiment is that leaf area, plant height and interval increase.GA20ox gene provided by the invention has the function that promotes vine growth and development, therefore can be used for cultivating the transgenic plant that developmental level increases.On the other hand, by suppressing the expression of GA20ox gene, may cultivate to obtain the transgenic plant of dwarfing.The present invention has great value for plant breeding.

Accompanying drawing explanation

Fig. 1 is the structure schema of recombinant plasmid pCAMBIA1302-GA20ox.

Fig. 2 is the electrophorogram that pcr amplification obtains total length GA20ox gene.

Fig. 3 is T ₀pCR for transgene tobacco identifies electrophorogram; 1 is 1kb DNA ladder, is respectively from down to up 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp; 2 positive contrasts (recombinant plasmid pCAMBIA1302-GA20ox); 3 negative contrasts (wild-type tobacco); 4-14 is followed successively by T ₀for transgene tobacco L1 to L11.

Fig. 4 is T ₀rT-PCR for transgene tobacco identifies electrophorogram; The negative contrast of WT (wild-type tobacco); L3, L6 and L7 are followed successively by transgene tobacco L3, L6 and L7.

Fig. 5 is T ₀fluorescence microscope photo for transgene tobacco; A is the photo of wild-type tobacco under natural light; B is the photo of wild-type tobacco under green fluorescence; C is the photo of transgene tobacco L3 under natural light; D is the photo of transgene tobacco L3 under green fluorescence.

Fig. 6 is T ₀phenotype photo for transgene tobacco; (a) being blade photo, is (b) plant photo; The negative contrast of WT (wild-type tobacco); L3, L6 and L7 are followed successively by transgene tobacco L3, L6 and L7.

Fig. 7 is T ₀leaf area statistics for transgene tobacco; WT is the mean value of 15 strain wild-type tobacco leaf areas; GA20ox is 15 strain T ₀mean value for plant (transgene tobacco) leaf area.

Fig. 8 is T ₀interval statistics for transgene tobacco; WT is the mean value of 15 strain wild-type tobacco intervals; GA20ox is 15 strain T ₀mean value for plant (transgene tobacco) interval.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.Festuca Arundinacea (Festuca arundinacea): Chinese is the person of pursuing, and English name is Quest, is purchased from east grass cultivation company.Tobacco (Nicotiana tabacum): be purchased from east grass cultivation company, as set out plant and the wild-type tobacco contrast of embodiment.Agrobacterium LBA4404: be purchased from Tian Gen biochemical technology company limited.

The discovery of embodiment 1, GA20ox albumen and encoding gene thereof

1, utilize SDS method to extract the RNA of Festuca Arundinacea.

2, take good, the free of contamination RNA of integrity is template, and with M-MLV enzyme (purchased from Promage company), reverse transcription RNA is cDNA.

3, the clone of intermediate segment

According to homology conserved sequence design primer (F1 and the R1 of GA20ox; (S=G/C)), the cDNA of Festuca Arundinacea of take carries out pcr amplification as template, and product, through reclaiming order-checking, obtains the intermediate segment of 506bp.

F1 (5 ' primer): 5 '-CTG TCG CTG GAG ATT ATG GAG GTG-3 ';

R1 (3 ' primer): 5 '-GTC TTC ATG TCG GCS CGG TAG TG-3 '.

4, the clone of 3 '-end

According to sequencing result design primer 3 ' the GSP I (gene-specific primer) of intermediate segment, primer sequence is 5 '-GAC ACC TTC ATG GCG CTC TCC AAC G-3 ', in order to improve product specificity, in GSPI downstream, design Yi Testis formula primer 3 ' GSPII, primer sequence is 5 '-GTT CTT CCT GTG CCC GGA GATGGA C-3 ' again.

The universal primer sequence of utilizing is as follows:

AP(adapter primer)：5’-ATT CTA GAG GCC GAG GCG GCC GAC ATG TTT TTT TTTTTT TTT TTT TTT TTT TTT TTT V-3’(V＝A/G/C)。

AUAP(abridged universal amplification primer)：5’-ATT CTA GAG GCC GAGGCG GCC G-3’。

First round PCR reaction system (50 μ l): 10 * PCR buffer, 5 μ l, dNTP Mix (2.5mM each) 4 μ l, 3 ' GSP I (10 μ M), 1 μ l, AP (10 μ M) 1 μ l, cDNA 2 μ l, Ex taq archaeal dna polymerase 0.25 μ l, ddH ₂o 36.75 μ l.

Second takes turns PCR reaction system (50 μ l): 10 * PCR buffer, 5 μ l, dNTP Mix (2.5mM each) 4 μ l, 3 ' GSP II (10 μ M), 1 μ l, AUAP (10 μ M) 1 μ l, 10 times of 2 μ l of PCR product dilution, Extaq archaeal dna polymerase 0.25 μ l, ddH ₂o 36.75 μ l.

By the second PCR product electrophoresis detection of taking turns, glue reclaims, sequencing analysis, the 3 ' terminal fragment of acquisition 326bp.

5, the clone of 5 '-end

CDNA3 ' end is carried out to tailing, reaction system (50 μ l; NEB company provides): terminal enzyme (DNA) TdT0.5 μ l, 10 * buffer, 5 μ l, dCTP (10mM) 1 μ l, CoCl ₂5 μ l, reverse transcription product 20 μ l, ddH ₂o 18.5 μ l.

The universal primer sequence of utilizing is as follows:

AAP(abridged anchor primer)：5’-CTC TCC CCG AGC ACC TCC ATG ATC TCC-3’。

AUAP(abridged universal amplification primer)：5’-GGC CAC GCG TCG ACTAGT AC-3’。

According to sequencing result design primer 5 ' the GSP I (gene-specific primer) of intermediate segment, primer sequence is: 5 ' GAC GAC GAA GGC GTC GGC CCT GGG CCG-3 ', and in order to improve product specificity, at GSP

I upstream is designed Yi Testis formula primer GSP II again, and primer sequence is: 5 '-GTC GTT GCC CTC GAA GAA GCGCCG-3 '.

First round PCR reaction system (50 μ l): 10 * PCR buffer, 5 μ l, dNTP Mix (2.5mM each) 4 μ l, 5 ' GSP I (10 μ M), 1 μ l, AAP (10 μ M) 1 μ l, cDNA tailing product 2 μ l, Ex taq archaeal dna polymerase 0.25 μ l, ddH ₂o 36.75 μ l.

Second takes turns PCR reaction system (50 μ l): 10 * PCR buffer, 5 μ l, dNTP Mix (2.5mM each) 4 μ l, 5 ' GSP II (10 μ M), 1 μ l, AUAP (10 μ M) 1 μ l, 10 times of 2 μ l of PCR product dilution, EXtaq archaeal dna polymerase 0.25 μ l, ddH ₂o 36.75 μ l.

By the second PCR product electrophoresis detection of taking turns, glue reclaims, sequencing analysis, the 5 ' terminal fragment of acquisition 780bp.

6, the acquisition of full length sequence

The cDNA of Festuca Arundinacea of take is template, utilizes primers F 2 and R2 to carry out pcr amplification.Pcr amplification product is checked order, and sequencing result shows, the nucleotide sequence of pcr amplification product is as shown in the sequence 2 of sequence table.

F2 (5 ' primer): 5 '-AGA GCT AAG TCA CCA CCT TCC TCC ACT GC-3 ';

R2 (3 ' primer): 5 '-GAC AAC CGA TCG CTC GTA TCT CAG CCT CAG-3 '.

By the protein called after GA20ox albumen (being formed by 366 amino-acid residues) shown in the sequence of sequence table 1.By the encoding gene called after GA20ox gene of GA20ox albumen, its full length sequence is as shown in the sequence 2 of sequence table, and the sequence 2 that open reading frame is sequence table is from the 178th to 1278 Nucleotide of 5 ' end.

The acquisition of embodiment 2, transgenic plant and evaluation

One, the structure of recombinant plasmid pCAMBIA1302-GA20ox

Build schema and see Fig. 1.

1, the total RNA that extracts Festuca Arundinacea, reverse transcription is cDNA.

2, take the cDNA of step 1 is template, with the primer pair that GA20ox_5 ' and GA20ox_3 ' form, carries out pcr amplification, obtains pcr amplification product (target sequence is for removing the GA20ox gene open reading frame of initiator codon and terminator codon).

GA20ox_5’：5’-CCA TGG CAG TGC AGC CTG TCT TCG AC-3’；

GA20ox_3’：5’-ACT AGT TGT CGT CGT CGT GGC GGC-3’。

1.0% agarose gel electrophoresis figure of pcr amplification product is shown in Fig. 2.

3, the pcr amplification product of step 2 is inserted to pMD18-T simple carrier (purchased from TaKaRa bio-engineering corporation), obtain recombinant plasmid pMD18-Tsimple-GA20ox.

4, recombinant plasmid pMD18-Tsimple-GA20ox is checked order, sequencing result shows, the two ends of pcr amplification product are respectively Nco I and Spe I restriction endonuclease recognition sequence, are that the sequence 2 of sequence table is from the DNA shown in the 181st to 1275 Nucleotide of 5 ' end between restriction endonuclease recognition sequence.

5, with restriction enzyme Nco I and the two single endonuclease digestion recombinant plasmid pMD18-Tsimple-GA20ox of Spe I, reclaim small segment (about 1109bp).

6, with restriction enzyme Nco I and Spe I double digestion carrier pCAMBIA1302 (Center for the Application of Molecular Biology to International Agriclture, www.cambia.org), reclaim carrier framework (about 10535bp).

7, the carrier framework of the small segment of step 5 and step 6 is connected, obtains recombinant plasmid pCAMBIA1302-GA20ox (GA20ox composing type efficient expression vector).

According to sequencing result, recombinant plasmid pCAMBIA1302-GA20ox is carried out to structrual description as follows: between the Nco of carrier pCAMBIA1302 I and Spe I restriction enzyme site, inserted the sequence 2 of sequence table from the DNA shown in the 181st to 1275 Nucleotide of 5 ' end (GA20ox gene and the GFP gene on carrier of removing initiator codon and terminator codon form fusion gene).

Two, recombinant plasmid pCAMBIA1302-GA20ox transforms Agrobacterium

1, the preparation of Agrobacterium competent cell

(1) picking Agrobacterium LBA4404 list colony inoculation is in 100ml YEB liquid nutrient medium, and shaking culture (220rpm, 28 ℃) is to OD ₆₀₀=0.6;

(2) the bacterium liquid of step (1) is proceeded to aseptic centrifuge tube, the centrifugal 5min of 5000rpm, removes supernatant, precipitation is added to the 0.15M CaCl of 10ml precooling ₂the aqueous solution, suspension cell, places 20min on ice gently;

(3) by the suspension of step (2) centrifugal (4 ℃, 5000rpm) 5min, remove supernatant, precipitation is added to the 0.15M CaCl containing 10% (volumn concentration) glycerine of 4ml precooling ₂the aqueous solution, suspends gently;

(4) suspension of step (3) is sub-packed in sterile eppendorf tubes, every pipe 200 μ l, quick-frozen 1min in liquid nitrogen, frozen in-70 ℃.

2, recombinant plasmid transformed Agrobacterium

Get 1 μ g recombinant plasmid pCAMBIA1302-GA20ox and join in 200 μ l Agrobacterium LBA4404 competent cells, mix static 5min; Quick-frozen 1min in liquid nitrogen, 37 ℃ of water-bath 5min, ice bath 5min, adds 1ml YEB liquid nutrient medium, shaking culture (28 ℃, 150rpm) 4h; The centrifugal 3min of 5000rpm, abandons supernatant, and precipitation adds 0.1mlYEB liquid nutrient medium, Eddy diffusion cell; Coat on the YEB solid plate containing 50 μ g/ml kantlex and 75 μ g/ml Rifampins, be inverted for 28 ℃ and cultivate about 48h.

3, PCR identifies positive colony

Identify primer used following (primer is for skeleton carrier design, and target fragment is 1109bp):

5 ' primer: 5 '-CCA TGG CAG TGC AGC CTG TCT TCG AC-3 ';

3 ' primer: 5 '-ACT AGT TGT CGT CGT CGT GGC GGC-3 '.

Qualification result shows: recombinant plasmid pCAMBIA1302-GA20ox successfully proceeds in Agrobacterium LBA4404, has obtained restructuring Agrobacterium.

Three, transformation of tobacco

1, restructuring Agrobacterium step 2 being obtained is inoculated in YEB liquid medium and (contains 100 μ g/ml kantlex, 100 μ g/ml Streptomycin sulphates and 75 μ g/m Rifampins; Kalamycin resistance gene is from carrier pCAMBIA1302, the helper plasmid pBA4404 that streptomycin resistance gene carries from Agrobacterium LBA4404, rifampicin resistance gene is from Agrobacterium LBA4404), shaking culture (28 ℃, 220rpm) is to OD ₆₀₀for 0.6-0.8, centrifugal 15 minutes of 4000rpm, removes supernatant, and precipitation suspends with the MS liquid nutrient medium (pH5.8) of 5-10 times of volume, and Agrobacterium bacterium liquid obtains recombinating.

2, aseptic tobacco leaf is cut to edge and main vein, be cut into 0.4 * 0.6cm ²the explant of size.

3, the explant of step 2 is soaked 5 minutes in the restructuring Agrobacterium bacterium liquid of step 1.

4, with aseptic filter paper, blot the bacterium liquid on vegetable material surface, proceed to the MS minimum medium of upper berth one deck filter paper, 25 ℃ of dark cultivations.

5,, after three days, vegetable material is forwarded to contain in antibiotic division culture medium (MS minimum medium+3mg/L 6-benzyl aminopurine+0.2mg/L a-naphthylacetic acid+10mg/L Totomycin+500mg/L Pyocianil) and cultivate.

6, until resistant buds, grow to 2-3cm when high, cut budlet and proceed to root induction in root media (MS minimum medium+10mg/L Totomycin+200mg/L Pyocianil).

7, treat that root grows to 10-20cm long (approximately 4 weeks), and there is some amount and can remove sealed membrane, in group training chamber, temper 2-3 days, then transplant in flowerpot and at room temperature take exercise 2-3 week, finally transplant to land for growing field crops plantation, be T ₀for plant.

Carrier pCAMBIA1302 is imported to Agrobacterium LBA4404, obtain contrasting Agrobacterium; With contrast Agrobacterium, replace restructuring Agrobacterium to carry out step 1 to 7, obtain adjoining tree.

Four, the evaluation of plant

1, PCR identifies

Respectively with T ₀for the genomic dna of plant and adjoining tree, be template (recombinant plasmid pCAMBIA1302-GA20ox as positive control, wild-type tobacco as negative control), with the primer pair that F3 (corresponding GA20ox gene) and R3 (corresponding GFP gene) form, carry out PCR evaluation.

F3 (5 ' primer): 5 '-CGG AGA TGG ACA AGG TGG TG-3 ';

R3 (3 ' primer): 5 '-TCA CAC GTG GTG GTG GTG-3 '.

PCR reaction system (50 μ l): ddH ₂o 37 μ l, 10 * PCR Buffer, 5 μ l, dNTP (25mM) 4 μ l, 5 ' primer (5pmol/ μ l), 1 μ l, 3 ' primer (5pmol/ μ l), 1 μ l, rTaq enzyme (5U/ μ l) 1 μ l, template (1 μ g/ μ l) 1 μ l.

PCR response procedures: the first round: 94 ℃ of sex change 5min; Second takes turns: 94 ℃ of sex change 50sec, and 66.8 ℃ of renaturation 50sec, 72 ℃ are extended 1min10s, 30 circulations; Third round: 72 ℃ are extended 10min.

After reaction finishes, pcr amplification product carries out 1.0% agarose gel electrophoresis, and target stripe is 959bp left and right.T ₀for having 20 strains in plant, be accredited as the positive.The qualification result of part PCR positive plant (L1 to L11) is shown in Fig. 3.

2, RT-PCR identifies

PCR is accredited as to three strains (L3, L6 and L7) in 20 positive strain plant and RT-PCR detection that wild-type tobacco carries out respectively step 2.

(1) Trizol method is extracted respectively total RNA of plant L3, L6 and L7 and wild-type tobacco.

(2) take good, the free of contamination total RNA of integrity is template, and with M-MLV enzyme (purchased from Promage company), reverse transcription RNA is cDNA.

Reverse transcription reaction system (15.0 μ l): RNA (1.0 μ g/ μ l) 2.0 μ l, Oligd T 2.0 μ l, RNfreeH ₂o 11.0 μ l.After said mixture is mixed, of short duration centrifugal by it be collected in pipe the end, 72 ℃ of incubation 5min, be placed in immediately again 5min on ice, add again following composition (cumulative volume 25.0 μ l): 5 * M-MLV Buffer, 5.0 μ l, dNTP (25mM) 1.5 μ l, RTase M-MLV (200U/ μ l) 1.0 μ l, HPR (40U/ μ l) 0.65 μ l, RNfreeH ₂o 1.85 μ l.Said mixture is mixed, of short durationly centrifugal it is collected in to the pipe end, at 42 ℃ of incubation 60min, 70 ℃ of reaction 15min, take out and to be placed on ice, are stored in-20 ℃ after centrifugal.

(3) take the reverse transcription product (cDNA) of 0.5 μ l step (2) carries out pcr amplification as template, with the primer pair that F3 and R3 form, carries out PCR evaluation.

PCR reaction system and PCR response procedures are with step 1.

Using NtActin as reference gene, PCR identifies that the primer pair of reference gene is as follows:

F4：5’-GGA ATA GTA AGC AAC TGG GAC GT-3’；

R4：5’-CTG AGC CAA TGG TAA TGA CCT G-3’。

The results are shown in Figure 4.L3, L6 and L7 have all obtained and have expected and the fragment of about 959bp show that GA20ox gene has carried out the expression of rna level in tobacco.

3, fluorescence microscopy Microscopic observation GFP

GA20ox gene and the GFP gene on carrier of removing initiator codon and terminator codon form fusion gene, therefore can pass through fluorescence microscopy Microscopic observation transgene tobacco root Green fluorescin, carry out the evaluation of GA20ox gene.

PCR is accredited as 20 positive strain T ₀for plant, under fluorescent microscope, all show green fluorescence, wild-type tobacco can not be observed green fluorescence.Fig. 5 is shown in by L3 and the wild-type tobacco photo under fluorescent microscope.

4, Phenotypic Observation and proterties statistical study

PCR is accredited as to 15 positive strain T ₀for plant, turn empty carrier plant (15 strain) and wild-type tobacco (15 strain) and take pictures and measure the leaf area (representing leaf area with the vertical footpath of blade * transverse diameter) of the blade of full expand and count from the bottom up the length (interval) between second sections in the ripening stage.Result is all got the mean value of 15 strains.

PCR is accredited as 15 positive strain T ₀leaf area and interval for plant are significantly greater than wild-type plant.Turn the leaf area of empty carrier plant and interval and wild-type plant and there is no significant difference.Fig. 6 is shown in by part plant photo.Leaf area the results are shown in Table 2 and Fig. 7.Interval the results are shown in Table 3 and Fig. 8.

Leaf area the results are shown in Table 2 and Fig. 7.Panel length measuring result is in Table 3 and Fig. 8.

The leaf area of table 2 plant

	Leaf area (cm 2)
		WT	14.96±0.773
Transfer-gen plant	19.10±1.762

The panel length of table 3 plant

	Interval (cm)
		WT	0.69±0.0446
Transfer-gen plant	0.97±0.1010

Result shows, it is large that the blade area of transgene tobacco and panel length obviously become, and is that GA20ox gene has promoted plant development.

Claims (2)

1. cultivating a method for transgenic plant, is that the encoding gene of the protein of the aminoacid sequence composition shown in sequence in sequence table 1 is imported in object plant, obtains plant development level higher than the transgenic plant of described object plant;

Described plant is tobacco;

Described developmental level is high is presented as that blade face area is large and/or plant height is high and/or interval is large.

2. method according to claim 1, is characterized in that: described encoding gene is following 1) or 2) shown in:

1) sequence 2 of sequence table is from the DNA molecular shown in the 178th to 1278 Nucleotide of 5 ' end;

2) DNA molecular shown in sequence 2 in sequence table.