CN102757487A

CN102757487A - Plant dwarfing related protein GA2ox, and encoding gene and application thereof

Info

Publication number: CN102757487A
Application number: CN2011101069000A
Authority: CN
Inventors: 王涛; 刘松虎; 徐光远; 马磊; 董江丽
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2011-04-27
Filing date: 2011-04-27
Publication date: 2012-10-31
Anticipated expiration: 2031-04-27
Also published as: CN102757487B

Abstract

The invention discloses a plant dwarfing related protein GA2ox, 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 dwarfing and derived from Sequence 1. In the invention, a recombinant plasmid containing a GA2ox gene is introduced to tobacco through an agrobacterium-mediated transformation method to obtain transgenic tobacco which has an obvious dwarfing phenotype. The plant dwarfing related protein GA2ox has a great value for plant dwarfing breeding.

Description

Dwarfing plants GAP-associated protein GAP GA2ox and encoding sox thereof and application

Technical field

The present invention relates to a kind of dwarfing plants GAP-associated protein GAP GA2ox and encoding sox and application.

Background technology

Breeding wheat for semidwarfness is significant aspect the crop quality improving." Green Revolution " of eighties of last century introduced dwarfted varieties in paddy rice and wheat, the output of paddy rice and wheat is had increased significantly.Research shows that the gene that causes the dwarfing proterties is relevant with Plant hormones regulators,gibberellins metabolism or signal transduction.

Plant hormones regulators,gibberellins (GA) is one type of important plant hormone; The Plant hormones regulators,gibberellins of biologically active all plays an important role in the whole life 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 mediate self replying environmental factor through the biosynthesizing of regulating endogenous gibberellins.(gibberellin 2-oxidase, under mediation GA2ox), the C-2 position of active Plant hormones regulators,gibberellins oh group of interpolation becomes does not have active GA8 and GA34 at the GA2-oxydase.Therefore the activity of GA2ox directly has influence on the content of active Plant hormones regulators,gibberellins.

Festuca Arundinacea is named alta fascue, reed shape fox thatch again, is cold ground type turfgrass, belongs to Gramineae festuca per nnial herb.Advantages such as nutritious, anti-trample that Festuca Arundinacea has, cold-resistant, disease-resistant, drought resisting, widespread use in the planting lawn in the many northern cities of China.But because Festuca Arundinacea vertical-growth amount is big, also caused a series of problems, wherein the most outstanding performance is: prune the frequency height, the lawn quality is difficult to guarantee.The pruning of overfrequency not only makes the turf management cost improve, and fancy points descends, and also quickens the scarcity of cursive script nutrition, causes early ageing.

Obtaining to downgrade phenotype through traditional breeding technique is the comparison difficulty because the conventional breeding method to Festuca Arundinacea carry out the germplasm improvement cycle long, effect is low.Along with the develop rapidly of Protocols in Molecular Biology, transgenic technology becomes a kind of good selection in recent years.Therefore clone in the Festuca Arundinacea key gene in the Plant hormones regulators,gibberellins pathways metabolism and carry out functional verification, can lay a solid foundation for the breeding wheat for semidwarfness of Festuca Arundinacea.

Summary of the invention

The purpose of this invention is to provide a kind of dwarfing plants GAP-associated protein GAP GA2ox and encoding sox and application.

Protein provided by the invention, name is called GA2ox albumen, derives from Festuca Arundinacea (Festuca arundinacea), is (a) or (b) as follows:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with dwarfing plants by sequence 1 deutero-protein.

In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table 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

Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The gene of encoding said proteins (GA2ox gene) also belongs to protection scope of the present invention.

Said 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 5 ' terminal the 148th to 1173 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant dwarf-related protein;

4) with 1) or 2) dna sequence dna that limits has the dna molecular of 90% above homology and coded plant dwarf-related protein.

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

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

Available existing plant expression vector construction contains the recombinant expression vector of said gene.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination with other plant promoter; In addition; When using gene constructed plant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones 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 said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Said recombinant expression vector specifically can be the recombinant plasmid that the MCS with said gene insertion vector pCAMBIA1302 obtains.

Increase total length or its arbitrary segmental primer of said gene to also belonging to protection scope of the present invention.

The present invention also protects a kind of method of cultivating transgenic plant, is said gene is imported in the purpose plant, obtains plant height and is lower than said purpose plant and/or internode apart from the transgenic plant less than said purpose plant.Said gene specifically can import in the said purpose plant through said recombinant expression vector.Carry said expression carrier can be through using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated, particle gun, and the plant transformed tissue cultivating become plant.Said purpose plant both can be that monocotyledons also can be a dicotyledons.Said dicotyledons specifically can be tobacco (like Nicotiana tabacum).Said internode is apart from the length that specifically can be several from the bottom up second internodes of plant.

Among the present invention, the recombinant plasmid that will contain the GA2ox gene imports tobacco through the Agrobacterium-mediated Transformation method, has obtained transgene tobacco, and this transgene tobacco has the phenotype of remarkable dwarfing.The GA2ox gene is imported Festuca Arundinacea, and the Festuca Arundinacea kind that can obtain to downgrade in theory realizes reducing and prunes frequency, and is difficult for the target of lodging.The present invention has important value for the breeding wheat for semidwarfness of plant.

Description of drawings

Fig. 1 is the structure schema of recombinant plasmid pCAMBIA1302-GA2ox.

Fig. 2 obtains the electrophorogram of total length GA2ox gene for pcr amplification.

Fig. 3 is T ₀PCR for transgene tobacco identifies electrophorogram; 1 is 1kb DNA ladder, is respectively 250bp from down to up, 500bp, 750bp, 1000bp, 1500bp, 2000bp; 2 positive contrasts (recombinant plasmid pCAMBIA1302-GA2ox); 3 negative contrasts (wild-type tobacco); 4-13 is followed successively by T ₀For transgene tobacco L1 to L10.

Fig. 4 is T ₀RT-PCR for transgene tobacco identifies electrophorogram; The negative contrast of WT (wild-type tobacco); L3, L4 and L6 are followed successively by transgene tobacco L3, L4 and L6.

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; The negative contrast of WT (wild-type tobacco); L3, L4 and L6 are followed successively by transgene tobacco L3, L4 and L6.

Fig. 7 is T ₀For the internode of transgene tobacco apart from statistics; WT is the MV of 15 strain wild-type tobacco internode distances; GA2ox is 15 strain T ₀MV for plant (transgene tobacco) internode distance.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Festuca Arundinacea (Festuca arundinacea): Chinese is the person of pursuing, and English name is Quest, purchases the grass cultivation company in east.Tobacco (Nicotiana tabacum): purchase grass cultivation company, as set out plant and the wild-type tobacco contrast of embodiment in east.Agrobacterium LBA4404: purchase root biochemical technology ltd in the sky.

The discovery of embodiment 1, GA2ox albumen and encoding sox thereof

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

2, be template with good in integrity, free of contamination RNA, reverse transcription RNA is cDNA with M-MLV enzyme (available from Promage company).

3, the clone of intermediate segment

Homology conserved sequence design primer (F1 and R1 according to GA2ox; M=A/C, S=G/C V=A/G/C), is that template is carried out pcr amplification with the cDNA of Festuca Arundinacea, product obtains the intermediate segment of 244bp through reclaiming order-checking.

F1 (5 ' primer): 5 '-MTS GGG TGG VTC GAG TAC-3 ';

R1 (3 ' primer): 5 '-GGT AGT GGT TCA SSC GGA-3 '.

4,3 '-terminal clone

According to the intermediate results of sequencing of the primer was designed 3'GSP? I (gene-specific? Primer), the primer sequence 5'-CAC? GTC? CAC? CGC? GCT? GAG? AGA? C-3 ', in order to improve the product-specific resistance, the GSP? I redesign a Testis type downstream primer 3'GSPII, primer sequence is 5'-GCG? TGC? TGG? CCG? ACA? TGG? TGA? C-3 '.

The universal primer sequence of utilizing is following:

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.

With the second PCR product electrophoresis detection of taking turns, glue reclaims, and sequencing analysis obtains the 3 ' terminal fragment of 923bp.

5,5 '-terminal clone

CDNA3 ' end is carried out 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 following:

AAP(abridged?anchor?primer)：5’-GGC?CAC?GCG?TCG?ACT?AGT?ACG?GGI?IGG?GIIGGG?IIG-3’。

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

According to results of the intermediate fragment sequencing primers designed 5'GSP? I (gene-specific? Primer), primer sequences: 5'-TGG? TTC? AGC? CGG? AAC? ACC? TGG? TCG? CTC-3 ', in order improve product specificity, in GSPI upstream redesign a Testis-type primer GSP? II, primer sequences: 5'-GCA? CGG? GAG? GCC? TTT? TCG? GA-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.

With the second PCR product electrophoresis detection of taking turns, glue reclaims, and sequencing analysis obtains the 5 ' terminal fragment of 530bp.

6, the acquisition of full length sequence

CDNA with Festuca Arundinacea is a template, utilizes primers F 2 and R2 to carry out pcr amplification.Pcr amplification product is checked order, and sequencing result shows that the nucleotide sequence of pcr amplification product is shown in the sequence 2 of sequence table.

F2 (5 ' primer): 5 '-TGC ACC ACC GAA GCT CGA GCT-3 ';

R2 (3 ' primer): 5 '-CTT AGC AAA CAC AAG ACT CCC ATA AAT AGA G-3 '.

With the protein called after GA2ox albumen (forming) shown in the sequence 1 of sequence table by 341 amino-acid residues.With the proteic encoding sox called after of GA2ox GA2ox gene, its full length sequence is shown in the sequence 2 of sequence table, and ORFs is that the sequence 2 of sequence table is from 5 ' terminal the 148th to 1173 Nucleotide.

Acquisition of embodiment 2, transgenic plant and evaluation

One, the structure of recombinant plasmid pCAMBIA1302-GA2ox

Make up schema and see Fig. 1.

1, extract total RNA of Festuca Arundinacea, reverse transcription is cDNA.

2, the cDNA with step 1 is a template, and the primer of using GA2ox_5 ' and GA2ox_3 ' composition obtains pcr amplification product (target sequence is for removing the GA2ox gene ORFs of terminator codon) to carrying out pcr amplification.

GA2ox_5’：5’-ACT?AGT?ATG?GTG?GTG?CTC?GCG?AAG-3’；

GA2ox_3’：5’-ACT?AGT?GGA?CCG?GTG?GTG?GTG?GCC-3’。

1.0% agarose gel electrophoresis figure of pcr amplification product sees Fig. 2.

3, the pcr amplification product with step 2 inserts pMD18-T simple carrier (available from TaKaRa bio-engineering corporation), obtains recombinant plasmid pMD18-Tsimple-GA2ox.

4, recombinant plasmid pMD18-Tsimple-GA2ox is checked order; Sequencing result shows; The two ends of pcr amplification product are respectively Spe I restriction endonuclease recognition sequence, and the sequence 2 that between the restriction endonuclease recognition sequence is sequence table is from the DNA shown in 5 ' terminal the 148th to 1170 Nucleotide.

5,, reclaim small segment (about 1035bp) with restriction enzyme Spe I single endonuclease digestion recombinant plasmid pMD18-Tsimple-GA2ox.

6, with restriction enzyme Spe I single endonuclease digestion carrier pCAMBIA1302 (Center for the Application of Molecular Biology to International Agriclture, Www.cambia.org), reclaim carrier framework (about 10549bp).

7, the small segment of step 5 and the carrier framework of step 6 are connected, obtain recombinant plasmid pCAMBIA1302-GA2ox (GA2ox composing type efficient expression vector).

According to sequencing result, it is following that recombinant plasmid pCAMBIA1302-GA2ox is carried out structrual description: the sequence 2 of having inserted sequence table at the Spe of carrier pCAMBIA1302 I restriction enzyme site is from the DNA shown in 5 ' terminal the 148th to 1170 Nucleotide (removing the GA2ox gene and the formation of the GFP gene on carrier fusion gene of terminator codon).

Two, recombinant plasmid pCAMBIA1302-GA2ox imports Agrobacterium

1, the preparation of Agrobacterium competent cell

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

(2) the bacterium liquid with step (1) changes aseptic centrifuge tube over to, and the centrifugal 5min of 5000rpm removes supernatant, deposition is added the 0.15M CaCl of 10ml precooling ₂The aqueous solution, suspension cell is placed 20min on ice gently;

(3) with the suspension-s of step (2) centrifugal (4 ℃, 5000rpm) 5min, remove supernatant, deposition is added the 0.15M CaCl that contains 10% (volumn concentration) glycerine of 4ml precooling ₂The aqueous solution suspends gently;

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

2, recombinant plasmid transformed Agrobacterium

Get 1 μ g recombinant plasmid pCAMBIA1302-GA2ox and join in the 200 μ l Agrobacterium LBA4404 competent cells mixing, static 5min; Quick-frozen 1min in the 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 deposition adds 0.1mlYEB liquid nutrient medium, suspension cell again; Coat on the YEB solid plate that contains 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 used primer (primer is to the skeleton carrier design, and target fragment is about 1035bp) as follows:

5 ' primer: 5 '-ACT AGT ATG GTG GTG CTC GCG AAG-3 ';

3 ' primer: 5 '-ACT AGT GGA CCG GTG GTG GTG GCC-3 '.

Qualification result shows: recombinant plasmid pCAMBIA1302-GA2ox successfully changes among the Agrobacterium LBA4404, has obtained the reorganization Agrobacterium.

Three, transformation of tobacco

1, the reorganization Agrobacterium that step 2 is obtained is inoculated in the 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, the rifampicin resistance gene is from Agrobacterium LBA4404), shaking culture (28 ℃, 220rpm) is to OD ₆₀₀Be 0.6-0.8, centrifugal 15 minutes of 4000rpm removes supernatant, and deposition 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 edge and main vein, be cut into 0.4 * 0.6cm ²The explant of size.

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

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

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

6, treat that resistant buds grows to 2-3cm when high, downcut budlet and change root induction in the root media (MS minimum medium+10mg/L Totomycin+200mg/L Pyocianil) over to.

7, treat that root grows to 10-20cm long (about 4 weeks), and have some amount and can remove and seal film, in group training chamber, took exercise 2-3 days, transplant then in flowerpot and at room temperature take exercise 2-3 week, transplant plantation at last, be T to the land for growing field crops ₀For plant.

Carrier pCAMBIA1302 is imported Agrobacterium LBA4404, obtain contrasting Agrobacterium; Replace the reorganization Agrobacterium to carry out step 1 with the contrast Agrobacterium, obtain adjoining tree to 7.

Four, the evaluation of plant

1, PCR identifies

Respectively with T ₀For the genomic dna of plant and adjoining tree is template (recombinant plasmid pCAMBIA1302-GA2ox as positive control, wild-type tobacco as negative control), identifies carrying out PCR with the primer that F3 (corresponding GA2ox gene) and R3 (corresponding GFP gene) form.

F3 (5 ' primer): 5 '-CGA CTC CCT CCA GGT GCT GAC GAA T-3 ';

R3 (3 ' primer): 5 '-GGC GGG TCT TGA AGT TGG CTT TGA T-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 ℃ of renaturation 50sec, 72 ℃ are extended 70s, 30 circulations; Third round: 72 ℃ are extended 10min.

After reaction finished, pcr amplification product carried out 1.0% agarose gel electrophoresis, and target stripe is about 800bp.T ₀For there being 25 strains to be accredited as the positive in the plant.The qualification result of part PCR positive plant (L1 to L10) is seen Fig. 3.

2, RT-PCR identifies

PCR is accredited as the RT-PCR that three strains (L3, L4 and L6) and wild-type tobacco in the male 25 strain plant carry out step 2 respectively to be detected.

(1) the Trizol method is extracted total RNA of plant L3, L4 and L6 and wild-type tobacco respectively.

(2) be template with good in integrity, free of contamination total RNA, reverse transcription RNA is cDNA with M-MLV enzyme (available from Promage company).

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.Behind the said mixture mixing, of short duration centrifugal it is collected in managed the end, 72 ℃ of incubation 5min; Place 5min on ice more immediately; Add following composition (TV 25.0 μ l) again: 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.With the said mixture mixing, of short durationly centrifugal it is collected in the pipe end, at 42 ℃ of incubation 60min, 70 ℃ of reaction 15min take out and to place on ice, are stored in-20 ℃ after centrifugal.

(3) reverse transcription product (cDNA) with 0.5 μ l step (2) is that template is carried out pcr amplification, identifies carrying out PCR with the primer that F3 and R3 form.

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

As internal control gene, the primer that PCR identifies internal control gene is to as follows with NtActin:

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

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

The result sees Fig. 4.L3, L4 and L6 have all obtained the fragment of the about 800bp of expection, show that the GA2ox gene has carried out the expression of rna level in tobacco.

3, fluorescent microscope is observed GFP down

GA2ox gene and the GFP gene on the carrier of removing terminator codon form fusion gene, therefore can observe green fluorescent protein in the transgene tobacco root down through fluorescent microscope, carry out the evaluation of GA2ox gene.

PCR is accredited as male 25 strain T ₀Under fluorescent microscope, all show green fluorescence for plant, wild-type tobacco can not be observed green fluorescence.L3 and the wild-type tobacco photo under fluorescent microscope is seen Fig. 5.

4, phenotype is observed and the proterties statistical study

PCR is accredited as male 15 strain T ₀For plant, change empty carrier plant (15 strain) and wild-type tobacco (15 strain) and take pictures and measure the length (internode distance) of several from the bottom up second internodes in the ripening stage.The result all gets the MV of 15 strains.

PCR is accredited as male 15 strain T ₀The remarkable internode distance of internode distance for plant less than the wild-type plant.The internode distance of commentaries on classics empty carrier plant does not have significant difference with the internode distance of wild-type plant.Part plant photo is seen Fig. 6.Internode is seen table 2 and Fig. 7 apart from the result.

The panel length of table 2 plant

	Internode is apart from (cm)
		Wild-type tobacco	0.69±0.0446
PCR is accredited as male T ₀For plant	0.2±0.049

The result shows: PCR is accredited as male 15 strain T ₀Obviously shorten for plant (transgene tobacco) panel length, promptly the GA2ox gene has caused the plant dwarfing.

Claims

1. protein is (a) or (b) as follows:

2. coding claim 1 said proteic gene.

3. gene as claimed in claim 2 is characterized in that: said gene is following 1) to 4) in arbitrary described dna molecular:

2) dna molecular shown in the sequence 2 in the sequence table;

4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.

5. recombinant expression vector as claimed in claim 4 is characterized in that: the recombinant plasmid that said recombinant expression vector obtains for the MCS with claim 2 or 3 said gene insertion vector pCAMBIA1302.

6. total length or its arbitrary segmental primer of amplification claim 2 or 3 said genes are right.

7. method of cultivating transgenic plant is that claim 2 or 3 said genes are imported in the purpose plants, obtains plant height and is lower than said purpose plant and/or internode apart from the transgenic plant less than said purpose plant.

8. method as claimed in claim 7 is characterized in that: claim 2 or 3 said genes import in the said purpose plant through claim 4 or 5 said recombinant expression vectors.

9. like claim 7 or 8 described methods, it is characterized in that: said purpose plant is monocotyledons or dicotyledons.