CN106367433B

CN106367433B - Plant is improved to the method and its application of gibberellin inhibitor sensitiveness

Info

Publication number: CN106367433B
Application number: CN201510434881.2A
Authority: CN
Inventors: 李召虎; 张娟; 李芳军; 张明才; 杜明伟; 田晓莉
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2015-07-22
Filing date: 2015-07-22
Publication date: 2019-06-25
Anticipated expiration: 2035-07-22
Also published as: CN106367433A

Abstract

The invention discloses improve plant to the method and its application of gibberellin inhibitor sensitiveness.It is provided by the present invention to improve plant to the method for gibberellin inhibitor sensitiveness, include the steps that the encoding gene that protein is imported into recipient plant obtains genetically modified plants；Genetically modified plants gibberellin inhibitor sensitiveness compared with the recipient plant increases；The protein is following protein a) or b) or c): a) amino acid sequence is the protein of the 1-821 amino acids of sequence 1；B) amino acid sequence is the protein of sequence 1；C) in N-terminal or/and the obtained fused protein of C-terminal connection label a) or b).It is demonstrated experimentally that the method for raising plant gibberellin inhibitor sensitiveness of the invention can be used to improve plant to the sensibility of gibberellin inhibitor, and the plant height of final height-related protein HRP regulation plant of the invention can be used.

Description

Plant is improved to the method and its application of gibberellin inhibitor sensitiveness

Technical field

The present invention relates to plant is improved in field of biotechnology to the method and its application of gibberellin inhibitor sensitiveness.

Background technique

Casing coupling pacifies (N, N-dimethylpiperidinium chloride, DPC), and its chemical name is 1,1- dimethyl Piperidines father-in-law's chloride, is a kind of plant growth regulator of suppressive, has retarding action to vegetation growth of plant, capable of inhibiting cell Elongation weakens plant terminal bud growing way, controls it and grow in length and breadth.After cotton applies DPC, thickening vanes, leaf color is deepened, and chlorophyll contains Amount increases, and Photosynthetic Rate raising (He Zhong pendant etc., 1991；Li Piming etc., 1991；Tian Xiaoli etc., 2004), thus be conducive to Field light transmission, enhances the illumination of cotton plant lower part, and can inhibit the growth of cotton plant stem, makes cotton plant shortened internodes, and plant type is compact, from And cotton plant prosperous growth is prevented, postpone its envelope departure date.DPC also can be improved cotton plant root activity (Tian Xiaoli etc., 2006), while DPC The stability of cell membrane can also be improved, increased cotton plant resistance (Shao Li is equal, 2004).

Application of the DPC on national cotton accounts for 80% of cultivated area or more, to cotton under the environmental condition that can be grown in the field Flower is regulated and controled, and can be inhibited the growth of cotton stem by shortening internode and reducing internode number, be reduced plant height, make its plant type It is compact, effectively control the prosperous growth of cotton, mould ideal plant type to optimize its economic characters (He Zhong wears etc., 1991；Reddy et al.,1992).During tomato cultivation, tomato plant excessive growth is able to suppress using DPC, increases seedling chlorophyll and can The content of dissolubility sugar, reduce relative conductivity, significantly improve big fruit tomato yield (Mao Xiujie etc., 1999；Wang Mei etc., 2012).But DPC is not significant to gramineous crop such as corn regulating effect, handles corn Zheng Dan 958 using 1000mg/L DPC, Plant height and stem thickness all do not differ significantly (Chen Yin, 2012) with compareing.Activities of Some Plants is insensitive to DPC, has limited to DPC and has made Application in object production.

Summary of the invention

The technical problem to be solved by the present invention is to how improve the gibberellin inhibitor sensitiveness of plant.

In order to solve the above technical problems, present invention firstly provides improve plant to the side of gibberellin inhibitor sensitiveness Method.

It is provided by the present invention to improve plant and imported to the method for gibberellin inhibitor sensitiveness, including into recipient plant The step of encoding gene of protein obtains genetically modified plants；The genetically modified plants are described red mould compared with the recipient plant Plain inhibitor sensitiveness increases；

The entitled final height-related protein (HRP) of the protein is following protein a) or b) or c):

A) amino acid sequence is the protein of the 1-821 amino acids of sequence 1；

B) amino acid sequence is the protein of sequence 1；

C) in N-terminal or/and the obtained fused protein of C-terminal connection label a) or b).

Wherein, the 1-821 amino acids of sequence 1 are the amino acid sequence of final height-related protein HRP, by the of sequence 2 The coding of final height-related protein HRP gene shown in 1-2463 nucleotide；The 824-996 amino acids of sequence 1 are the ammonia of GFP Base acid sequence, the coding of the DNA molecular as shown in 2470-2720 nucleotide of sequence 2.

In order to make protein in a) convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 1 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of table 1, label

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

It is above-mentioned b) in HRP can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.It is above-mentioned B) encoding gene of the HRP in can by by missing one in DNA sequence dna shown in 1-2463 of sequence 2 in sequence table or The codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or at its 5 ' end and/or The coded sequence that 3 ' ends connect label shown in table 1 obtains.

It is above-mentioned d) in HRP can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.It is above-mentioned D) encoding gene of the HRP in can be residual by will lack one or several amino acid in DNA sequence dna shown in sequence 2 in sequence table The codon of base, and/or the missense mutation of one or several base-pairs is carried out, and/or connect table 1 at its 5 ' end and/or 3 ' ends Shown in the coded sequence of label obtain.

In the above method, the encoding gene of the HRP be it is following 1) or 2) or 3) or 4) or 5) or 6) shown in gene:

1) nucleotide sequence is the cDNA molecule or DNA molecular of 1-2463 nucleotide of sequence 2 in sequence table；

2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes cDNA points of the HRP Son or genomic DNA molecule；

3) nucleotide sequence hybridization with 1) restriction, and the cDNA molecule or gene of the coding HRP under strict conditions Group DNA molecular；

4) nucleotide sequence is the cDNA molecule or DNA molecular of sequence 2 in sequence table；

5) there is 75% or 75% or more identity with the nucleotide sequence 4) limited, and encodes cDNA points of the HRP Son or genomic DNA molecule；

6) nucleotide sequence hybridization with 4) restriction, and the cDNA molecule or gene of the coding HRP under strict conditions Group DNA molecular.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

Wherein, amino acid sequence shown in the nucleotide sequence coded sequence 1 of sequence 2.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding HRP of the invention.Those have and separate with the present invention by manually modified The nucleotide sequence 75% of obtained HRP or the nucleotide of higher identity are as long as encoding HRP and having the function of HRP Derived from nucleotide sequence of the invention and it is equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair The nucleotide sequence of protein shown in the 1-821 amino acids of bright coded sequence 1 or sequence 1 has 75% or higher, Or 85% or higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or meter Calculation machine software is evaluated.Using computer software, the identity between two or more sequences can use percentage (%) table Show, can be used to evaluate the identity between correlated series.

In the above method, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, each 5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min； Or, hybridizing under the conditions of 65 DEG C in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS and washing film.

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

In an embodiment of the present invention, the encoding gene of the HRP is (i.e. shown in 1-821 nucleotide of sequence 2 DNA molecular) it is imported in purpose plant by the HRP gene recombinant vectors containing HRP expression casette.

In the above method, wherein the HRP gene can be modified first as follows, then import in receptor seed plant, to reach To better expression effect:

1) it modifies and optimizes according to actual needs, so that gene efficient expression；For example, can be according to recipient plant institute partially The codon of love, it is inclined to meet plant while keeping the amino acid sequence of HRP gene of the present invention to change its codon Love property；In optimization process, it is desirable that certain G/C content is kept in the coded sequence after optimization, to be best implemented in plant The high level expression of quiding gene, wherein G/C content can be 35%, be more than 45%, more than 50% or more than about 60%；

2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant The effective sequence known is modified；

3) it is connect with the promoter of various plants expression, in favor of its expression in plant；The promoter may include Composing type, induction type, timing adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter Selection will need with expression time and space and be changed, and also depend on target kind；Such as the specificity of tissue or organ Promoter is expressed, receptor as needed is depending on what period of development；Although demonstrating many from dicotyledon Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for Expression in dicotyledon, monocotyledonous promoter is for the expression in monocotyledon；

4) it is connect with suitable transcription terminator, can also be improved the expression efficiency of gene of the present invention；Such as from The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention Gene is attached；

5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence (such as from TMV, MCMV and AMV).

The HRP gene recombinant vectors can be by using Ti-plasmids, plant virus carrying agent, directly delivered DNA, micro- note It penetrates, the standard biologics technical method such as electroporation imports plant cell (Weissbach, 1998, Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463；Geiserson and Corey, 1998,Plant Molecular Biology(2nd Edition).)。

In the above method, the genetically modified plants are interpreted as not only comprising obtaining the HRP genetic transformation purpose plant First generation genetically modified plants, also include its filial generation.For genetically modified plants, the gene can be bred in the species, it can also The gene transfer is entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described to turn base Because plant includes seed, callus, intact plant and cell.

In order to solve the above technical problems, the present invention also provides the products of following M1 or M2:

M1, the protein；

M2, biomaterial relevant to the protein are following B1) any one of to B20):

B1) the nucleic acid molecules of code for said proteins；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules；

B4) contain B2) recombinant vector of the expression cassette；

B5) contain B1) recombinant microorganisms of the nucleic acid molecules；

B6) contain B2) recombinant microorganism of the expression cassette；

B7) contain B3) recombinant microorganism of the recombinant vector；

B8) contain B4) recombinant microorganism of the recombinant vector；

B9) contain B1) the transgenic plant cells systems of the nucleic acid molecules；

B10) contain B2) the transgenic plant cells system of the expression cassette；

B11) contain B3) the transgenic plant cells system of the recombinant vector；

B12) contain B4) the transgenic plant cells system of the recombinant vector；

B13) contain B1) Transgenic plant tissues of the nucleic acid molecules；

B14) contain B2) Transgenic plant tissue of the expression cassette；

B15) contain B3) Transgenic plant tissue of the recombinant vector；

B16) contain B4) Transgenic plant tissue of the recombinant vector；

B17) contain B1) the genetically modified plants organs of the nucleic acid molecules；

B18) contain B2) the genetically modified plants organ of the expression cassette；

B19) contain B3) the genetically modified plants organ of the recombinant vector；

B20) contain B4) the genetically modified plants organ of the recombinant vector.

In the said goods, B2) described in the nucleic acid molecules containing coding HRP expression cassette (HRP expression casette), refer to The DNA of HRP can be expressed in host cell, which may include not only the promoter for starting HRP genetic transcription, may also include Terminate the terminator of HRP genetic transcription.Further, the expression cassette may also include enhancer sequence.It is for use in the present invention to open Mover includes but is not limited to: constitutive promoter, organizes, the promoter and inducible promoter that organ and development are special.Starting The example of son includes but is not limited to: the constitutive promoter 35S of cauliflower mosaic virus: the Wound-inducible from tomato opens Mover, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol 120:979-992)；From tobacco Chemical inducible promoter, pathogenesis correlation 1 (PR1) is (by salicylic acid and BTH (diazosulfide -7- carbothioic acid S- first Ester) induction)；Tomato protease inhibitors II promoter (PIN2) or LAP promoter (available methyl jasmonate induction)； Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed is special Specific Promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), The special promoter of seed storage protein matter is (for example, phaseolin, napin, oleosin and soybean beta conglycin Promoter (Beachy et al. (1985) EMBO is J.4:3047-3053)).They can be used alone or open with other plants Mover is used in combination.All references cited herein is cited in full text.Suitable transcription terminator includes but is not limited to: agriculture Bacillus nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea RbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g.: Odell et al. (I⁹⁸⁵)Nature 313:810；Rosenberg et al. (1987) Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262: 141；Proudfoot(1991)Cell,64:671；Sanfacon et al. Genes Dev., 5:141；Mogen et al. (1990) Plant Cell,2:1261；Munroe et al. (1990) Gene, 91:151；Ballad et al. (1989) Nucleic Acids Res.17:7891；Joshi et al. (1987) Nucleic Acid Res., 15:9627).

The recombinant vector of the HRP expression casette can be contained with existing expression vector establishment.The plant expression carries Body includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.As pAHC25, pBin438, PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions. When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used, These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive, Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants Add any selected marker, transformed plant is directly screened with adverse circumstance.

In the said goods, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.

In the said goods, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.

In the said goods, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are not wrapped Include propagation material.

In an embodiment of the invention, (i.e. DNA shown in 1-2463 of sequence 2 points of the encoding gene of HRP Son) it is imported in Agrobacterium tumefaciems GV3101 by the recombinant vector of the expression cassette of the encoding gene containing HRP.The recombinant vector Between Xba I and Kpn I the identification sequence for replacing carrier pSuper1300 for the DNA molecular shown in 1-2463 of sequence 2 The obtained recombinant vector pSuper1300-HRP of DNA fragmentation, the difference of the pSuper1300-HRP and the pSuper1300 It is not only that and Xba I and the Kpn I of the pSuper1300-HRP is identified to the DNA fragmentation between sequence replaces with sequence 2 the DNA molecular shown in 1-2463 nucleotide.Protein shown in the recombinant vector pSuper1300-HRP expressed sequence 1.

In the said goods, B1) nucleic acid molecules can for it is described 1) or it is described 2) it is described 3) or it is described 4) or it is described 5) Or it is described 6) shown in gene.

In order to solve the above technical problems, the present invention also provides any applications in following N1-N4:

N1, the method are in the application for regulating and controlling plant strain senior middle school；

N2, the product are in the application for regulating and controlling plant strain senior middle school；

N3, the product are cultivating the application increased gibberellin inhibitor sensitiveness in plant；

N4, the product are cultivating the application in plant height increase plant.

In above-mentioned application, the plant is genetically modified plants.

In above-mentioned application, using including importing the encoding gene of the HRP into recipient plant to obtain transgenosis described in N4 The step of plant；Genetically modified plants plant height compared with the recipient plant increases.

In above-mentioned application, using including S1 described in N1 or N2) and S2):

S1 the encoding gene that institute HRP) is imported into recipient plant obtains genetically modified plants；

S2 the gibberellin inhibitor) is applied to the genetically modified plants and obtains the plant height drop compared with the genetically modified plants Low plant.

In above-mentioned application, the coded sequence of the encoding gene of the HRP is the DNA molecular of sequence 2 in sequence table.

In above-mentioned application, wherein the HRP gene can be modified first as follows, then import in receptor seed plant, to reach To better expression effect:

In above-mentioned application, the genetically modified plants are interpreted as not only comprising obtaining the HRP genetic transformation purpose plant First generation genetically modified plants, also include its filial generation.For genetically modified plants, the gene can be bred in the species, it can also The gene transfer is entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described to turn base Because plant includes seed, callus, intact plant and cell.

In the present invention, the gibberellin inhibitor can be gibberellin synthetic inhibitor, such as gibberellin biological synthetic inhibitor. Concretely casing coupling pacifies DPC to the gibberellin inhibitor.Concretely moisten the casing coupling peace company, agrochemical Co., Ltd in Jiangsu Product, article No. HG/T2856-1997.

In the present invention, the plant can be dicotyledon or monocotyledon.The dicotyledon can be cruciate flower Section plant, such as arabidopsis (Arabidopsis thaliana).

In the present invention, the gibberellin inhibitor sensitiveness increase is embodied in be increased in identical gibberellin inhibitor concentration Under amount, the plant height reduced rate of genetically modified plants is higher than recipient plant.Such as, what 30mg/L mepiquat chloride was handled turns HRP gene plant The plant height reduced rate for the plant height for turning HRP gene plant that plant height is handled relative to 0mg/L mepiquat chloride is 33.92%, 30mg/L contracting The plant height reduced rate of the plant height for the recipient plant that the plant height of the recipient plant of section amine processing is handled relative to 0mg/L mepiquat chloride is 13.60%.

It is demonstrated experimentally that plant can be improved to gibberellin in the method for raising plant gibberellin inhibitor sensitiveness of the invention The method of the sensibility of inhibitor, the final height-related protein in the method for raising plant gibberellin inhibitor sensitiveness of the invention HRP and DPC can regulate and control the plant height of arabidopsis, and the plant height of transgenic arabidopsis is reduced with the increase of DPC concentration.DPC The plant height reduced rate of the HRP::ga1-1 of processing be much higher than the processing of corresponding DPC concentration ZmCPS::ga1-1, The plant height reduced rate of the HRP::ga1-1 of pSuper1300::ga1-1 and ga1-1:30mg/L DPC processing is the 2.49 of ga1-1 Times；The plant height reduced rate of the HRP::ga1-1 of 500mg/L DPC processing is 3.47 times of ga1-1.The HRP::ga1-5 of DPC processing Plant height reduced rate be much higher than ZmCPS::ga1-1, pSuper1300::ga1-5 and ga1-5 of the processing of corresponding DPC concentration: The plant height reduced rate of the HRP::ga1-5 of 30mg/L DPC processing is 4.68 times of ga1-5；The HRP: of 500mg/L DPC processing: The plant height reduced rate of ga1-5 is 6.66 times of ga1-5.

It is demonstrated experimentally that the plant height of arabidopsis: untreated HRP: can be improved in final height-related protein HRP of the invention: The plant height of the HRP::ga1-1 of the HRP::ga1-1 and 500mg/L DPC processing of ga1-1,30mg/L DPC processing is respectively corresponding 3.54 times, 2.71 times and 1.71 times of the ga1-1 plant height of processing；Untreated HRP::ga1-5,30mg/L DPC processing The plant height of the HRP::ga1-5 of HRP::ga1-5 and 500mg/L DPC processing is respectively the 2.26 of respective treated ga1-5 plant height Again, 1.65 times and 1.00 times.

It is demonstrated experimentally that the method for raising plant gibberellin inhibitor sensitiveness of the invention can be used to improve plant to red The sensibility of mycin inhibitor, and the plant height of final height-related protein HRP regulation plant of the invention can be used.

Detailed description of the invention

Fig. 1 is the plant height for turning HRP and ZmCPS gene arabidopsis before DPC processing.Wherein, A is the strain of ZmCPS::ga1-1 Height, B are the plant height of HRP::ga1-1, and C is the plant height of ZmCPS::ga1-5, and D is the plant height of HRP::ga1-5.

Fig. 2 is the plant height of the arabidopsis of different disposal.Wherein, A is the ZmCPS::ga1-1 of the DPC processing of various concentration Plant height, B are the plant height for the HRP::ga1-1 that the DPC of various concentration is handled, and C is the ZmCPS::ga1- that the DPC of various concentration is handled 5 plant height, D are the plant height for the HRP::ga1-5 that the DPC of various concentration is handled.

Fig. 3 is the plant height of the HRP::ga1-1 and HRP::ga1-5 of various concentration DPC processing.Wherein, A is various concentration The plant height of the HRP::ga1-1 of DPC processing；B is the plant height of the HRP::ga1-5 of various concentration DPC processing.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Carrier pSuper1300 in following embodiments is document (3 as a of Lipid transfer protein Target of MYB96 mediates freezing and drought stress in Arabidopsis, Journal Of Experimental Botany, Guo et al., Vol.64, No.6, pp.1755-1767,2013) in PSuper1300::GFP, the public can obtain the biomaterial from applicant, which only attaches most importance to the correlation of duplicate invention Used in experiment, it not can be used as other purposes and use.

Agrobacterium tumefaciems GV3101 in following embodiments is document (A Plasma Membrane Receptor Kinase,GHR1,Mediates Abscisic Acid-and Hydrogen Peroxide-Regulated Stomatal Movement in Arabidopsis,Hua et al.,The Plant Cell,Vol.24:2546–2561,June 2012) In Agrobacterium strain GV3101, the public can obtain the biomaterial from applicant, which is only It repeats used in related experiment of the invention, not can be used as other purposes and use.

Arabidopsis Mutants ga1-1 and ga1-5 in following embodiments are Ohio State University's arabidopsis living resources Hub products.

The DPC aqueous solution of 0mg/L in following embodiments is ultrapure water, and the DPC of the 30mg/L in following embodiments is water-soluble Liquid is the solution that 30mg DPC is added into 1L ultrapure water and obtains, and the DPC aqueous solution of the 500mg/L in following embodiments is to 1L The solution that 500mg DPC is obtained is added in ultrapure water.

The growth conditions of arabidopsis is equal in following embodiments are as follows: the photoperiod is that 16h light/8h is dark, and light intensity is 60 μm of ol/m²/ S, humidity 60%-70%, temperature are to cultivate in 22 DEG C of culturing room.

Mepiquat chloride in following embodiments is the moist agrochemical Co., Ltd Products in Jiangsu, article No. HG/T2856- 1997, casing coupling peace is gibberellin biological synthetic inhibitor.

Embodiment 1, the plant height for regulating and controlling arabidopsis using the reagent set of regulation plant plant height

The reagent set for regulating and controlling plant plant height pacifies (N, N- by final height-related protein and casing coupling Dimethylpiperidinium chloride, DPC, also referred to as 1,1- lupetidine father-in-law chloride) composition；The strain High GAP-associated protein GAP, entitled HRP, amino acid sequence are 1-821 of sequence 1 in sequence table.Final height-related protein (HRP) Gene is DNA molecular shown in 1-2463 of sequence 2.

HRP gene source is in Asiatic cotton (G.arboreum) Shi Xiya 1 (SXY1) (Li et al., Genome Sequence of the cultivated cotton Gossypium arboreum, Nature GenNetics VOLUME 46 NUMBER 6JUNE 2014).Final height-related protein HRP gene is DNA molecular shown in sequence 2 in sequence table.

1, the building of recombinant vector and recombinational agrobacterium

Xba I and the Kpn I of carrier pSuper1300 is identified that the segment between sequence replaces with 1-2463 of sequence 2 DNA molecular shown in nucleotide (i.e. HRP gene) obtains recombinant vector pSuper1300-HRP, pSuper1300-HRP with The difference of pSuper1300, which is only that, identifies that the DNA fragmentation between sequence replaces with for Xba I and the Kpn I of pSuper1300-HRP DNA molecular shown in 1-2463 nucleotide of sequence 2.Strain shown in recombinant vector pSuper1300-HRP expressed sequence 1 The fusion protein that high GAP-associated protein GAP HRP and GFP is formed.

Spe I and the Kpn I of carrier pSuper1300 is identified that the segment between sequence replaces with 1-2565 of sequence 3 Shown in DNA molecular obtain recombinant vector pSuper1300-ZmCPS, the difference of pSuper1300-ZmCPS and pSuper1300 It is only that and Spe I and the Kpn I of pSuper1300-ZmCPS is identified that the DNA fragmentation between sequence replaces with shown in sequence 3 DNA molecular.The 1-2565 coding albumen of recombinant vector pSuper1300-ZmCPS expressed sequence 3 merge egg with what GFP was formed It is white.

Wherein, 1-2565 of sequence 3 are the nucleotide sequence of ZmCPS gene, and ZmCPS gene source is in corn (B73)。

PSuper1300-HRP is imported in Agrobacterium tumefaciems GV3101, recombinant bacterium is obtained, which is named as GV3101-pSuper1300-HRP；PSuper1300-ZmCPS is imported in Agrobacterium tumefaciems GV3101, recombinant bacterium is obtained, it will The recombinant bacterium is named as GV3101-pSuper1300-ZmCPS；PSuper1300 is imported in Agrobacterium tumefaciems GV3101, is obtained The recombinant bacterium is named as GV3101-pSuper1300 by recombinant bacterium.

2, the building of transgenic arabidopsis

It is utilized respectively GV3101-pSuper1300-HRP, GV3101-pSuper1300-ZmCPS and GV3101- of step 1 PSuper1300 distinguishes arabidopsis thaliana transformation mutant ga1-1 and ga1-5, constructs transgenic arabidopsis.To Arabidopsis Mutants The method that ga1-1 turns HRP gene is as follows:

The culture of 2.1 arabidopsis: by mutant ga1-1 seed under the conditions of 4 DEG C vernalization 72h, dibbling is in gibberellin culture (gibberellin culture medium is that gibberellin (GA is added into MS culture medium to base₃) obtained GA₃Concentration is 10^-4The solid medium of M) In in 22 DEG C, 16h light/8h is dark, light intensity is 60 μm of ol/m²/ s, humidity 60%-70% culturing room in cultivate, grow into 4 it is true It is transplanted to when leaf in the culture pan that Nutrition Soil is mixed with vermiculite equal proportion, wherein mutant takes two days and sprays one time 10^-4M GA₃。

The preparation of 2.2 Agrobacterium bacterium solutions: the monoclonal GV3101-pSuper1300-HRP for detecting correct scribing line culture is taken The Agrobacterium inoculation of inoculation is in 5ml YEP fluid nutrient medium (contain antibiotic), and 28 DEG C, cultivate 30h under 220rpm, by volume Obtained bacterium solution is transferred in 50ml YEP (containing antibiotic) for 1:100,28 DEG C, 220rpm overnight incubation, until OD₆₀₀For 0.6-0.8；4 DEG C are centrifuged 15min in 6000g, and collected thallus is resuspended in 1/2MS+5% sucrose solution, are added before converting 0.02%-0.05%Silwet L-77 (a kind of surfactant is U.S. AMRESCO Products).

2.3 conversions: to Arabidopsis plant Post flowering, cutting off major branch top, promotes side shoot development.In 6 days after beta pruning, Ready Agrobacterium is dipped on the inflorescence that wet arabidopsis does not show money or valuables one carries unintentionally.By black plastic bag of the arabidopsis after conversion full of gas It encases, dark culture removes black plastic bag afterwards for 24 hours.Restore illumination by normal method culture plant to solid, harvests mature turn HRP gene ga1-1 (HRP::ga1-1) T₁For seed.

The pure lines plant of HRP::ga1-1 is obtained as follows: 1. by T₁Containing for the sowing of HRP::ga1-1 seed It is screened on the MS solid medium of hygromycin, by true leaf health, dark green, root extends the hygromycin into culture medium Positive plant move into soil in, harvest T₂For HRP::ga1-1 seed；2. by T₂Containing tide for the sowing of HRP::ga1-1 seed T is obtained on the MS solid medium of mycin₂It for HRP::ga1-1 plant, is screened using hygromycin, selects hygromycin: no Hygromycin=3:1 T₂It is moved into soil for HRP::ga1-1 plant and by hygromycin plant therein, harvests T₃Generation (hygromycin plant shows themselves in that true leaf health is dark green to HRP::ga1-1 seed, and root is extended into culture medium；Moisture resistance is not mould Plain plant shows themselves in that true leaf turns to be yellow, and root does not extend)；3. by T₃For the sowing of HRP::ga1-1 seed in the MS training containing hygromycin It supports on base, obtains T₃It for HRP::ga1-1 plant, is screened using hygromycin, selects the T of all hygromycins₃For HRP:: Ga1-1 plant is simultaneously transplanted in soil, and the pure lines plant of HRP::ga1-1 is obtained.

According to the method described above, ga1-1 being replaced with into ga1-5, other steps are constant, respectively obtain entitled HRP:: Ga1-1's turns HRP gene ga1-1 and its pure lines plant.

According to the method described above, GV3101-pSuper1300-HRP is replaced with into GV3101-pSuper1300-ZmCPS respectively And GV3101-pSuper1300, other steps are constant, and respectively obtain entitled ZmCPS::ga1-1 turns ZmCPS gene Ga1-1 and its pure lines plant and entitled pSuper1300::ga1-1 turn empty carrier ga1-1 and its pure lines plant.

According to the method described above, ga1-1 is replaced with into ga1-5, and GV3101-pSuper1300-HRP is replaced with respectively GV3101-pSuper1300-ZmCPS and GV3101-pSuper1300, other steps are constant, respectively obtain entitled ZmCPS::ga1-5 turn ZmCPS gene ga1-5 and its be sheerly plant and entitled pSuper1300::ga1-5 turns empty carrier Ga1-5 and its pure lines plant.

3, the identification of transgenic plant

The detection of HRP gene expression amount in the pure lines arabidopsis of 3.1 turns of HRP genes

It is planted using the pure lines plant of the HRP::ga1-1 of Real-Time PCR authentication step 2 and the pure lines of HRP::ga1-5 The pure lines plant of the pure lines plant and ZmCPS::ga1-5 of the ZmCPS::ga1-1 of the expression and step 2 of HRP gene in strain The expression of middle ZmCPS gene, the primer for detecting the expression of HRP gene is 5 '-ACCGAGGACTCGCAGAGTTA-3 ' With 5 '-ACCTTTAGCATTTGGCGATG-3 ', the primer for detecting the expression of ZmCPS gene is 5 '- TGCAGCCACTTATCGACCAG-3 ' and 5 '-AGGCGAGGGTGTTGATCATG-3 '.Internal reference is AtUbI gene, and internal reference draws Object is 5 '-ATTACCCGATGGGCAAGTCA-3 ' and 5 '-CACAAACGAGGGCTGGAACA-3 '.The results show that HRP::ga1- Express HRP gene in 1 pure lines plant and the pure lines plant of HRP::ga1-5, the pure lines plant of ZmCPS::ga1-1 and ZmCPS gene is expressed in the pure lines plant of ZmCPS::ga1-5.

The detection of HRP in the pure lines arabidopsis of 3.2 turns of HRP genes

Using Westernblot authentication step 2 HRP::ga1-1 pure lines plant, HRP::ga1-5 pure lines plant in HRP albumen and step 2 ZmCPS::ga1-1 pure lines plant and ZmCPS::ga1-5 pure lines plant in ZmCPS egg White, primary antibody is Anti-GFP Tag Rabbit (Roche product, article No. 14717400).The results show that HRP::ga1-1 Have a HRP protein expression in pure lines plant and the pure lines plant of HRP::ga1-5, the pure lines plant of ZmCPS::ga1-1 and There is ZmCPS protein expression in the pure lines plant of ZmCPS::ga1-5.

4, influence of the DPC to HRP gene arabidopsis plant height is turned

In triplicate, repeating test every time, specific step is as follows for experiment:

4.1DPC, which can be reduced, turns HRP gene arabidopsis plant height

30 plants of pure lines plant of the HRP::ga1-1 of step 2 are randomly selected, are randomly divided into three groups, every group 10 plants, This three groups of plant carry out the following processing respectively when sowing the 33rd day of (the sowing same day is denoted as sowing the 1st day) certainly: one group sprays water After (i.e. the DPC aqueous solution of 0mg/L), cultivates 12 days, obtain untreated HRP::ga1-1；One group of DPC water for spraying 30mg/L It after solution, cultivates 12 days, obtains the HRP::ga1-1 of 30mg/L DPC processing；After one group sprays the DPC aqueous solution of 500mg/L, Culture 12 days obtains the HRP::ga1-1 of 500mg/L DPC processing.

According to the method described above, by the pure lines plant of HRP::ga1-1 replace with respectively HRP::ga1-5 pure lines plant, ZmCPS::ga1-1 pure lines plant, ZmCPS::ga1-5 pure lines plant, PsSuper1300::ga1-1 pure lines plant and The pure lines plant of PsSuper1300::ga1-5, other steps are constant, respectively obtain untreated HRP::ga1-5,30mg/L HRP::ga1-5, untreated ZmCPS::ga1-1,30mg/L of HRP::ga1-5,500mg/L the DPC processing of DPC processing DPC processing ZmCPS::ga1-1,500mg/L DPC processing ZmCPS::ga1-1, untreated ZmCPS::ga1-5, The ZmCPS::ga1-5, untreated of ZmCPS::ga1-5,500mg/L the DPC processing of 30mg/L DPC processing PsSuper1300::ga1-1,500mg/L the DPC processing of PsSuper1300::ga1-1,30mg/L DPC processing The PsSuper1300: that PsSuper1300::ga1-1, untreated PsSuper1300::ga1-5,30mg/L DPC are handled: The PsSuper1300::ga1-5 of ga1-5 and 500mg/L DPC processing.

When ga1-1 is cultivated to from the 33rd day of sowing (sowing the same day be denoted as sowing the 1st day), 30 plants are taken, it is random It is divided into three groups, every group 10 plants, is carried out the following processing respectively in this three groups of plant: after one group sprays the DPC aqueous solution of 0mg/L, training It supports 12 days, obtains untreated ga1-1；After one group sprays the DPC aqueous solution of 30mg/L, cultivates 12 days, obtain 30mg/L DPC The ga1-1 of processing；It after one group sprays the DPC aqueous solution of 500mg/L, cultivates 12 days, obtains the ga1-1 of 500mg/L DPC processing.

According to the method described above, ga1-1 is replaced with into ga1-5, other steps are constant, respectively obtain untreated ga1-5, The ga1-5 of the ga1-5 and 500mg/L DPC processing of 30mg/L DPC processing.

Plant height (Fig. 2 and table after measuring the plant height (Fig. 1) and different disposal of above-mentioned each arabidopsis before treatment respectively 2)。

Table 2, different disposal arabidopsis plant height and plant height reduced rate

The results show that pSuper1300::ga1-1 and the ga1-1 plant height after DPC is handled drop under identical DPC concentration Low rate difference is little, and DPC is equal on the plant height reduced rate of the ZmCPS::ga1-1 HRP::ga1-1 handled substantially without influence, DPC Much higher than pSuper1300::ga1-1 the and ga1-1:30mg/L DPC of the corresponding DPC concentration processing HRP::ga1-1's handled Plant height reduced rate is 2.49 times of ga1-1；The plant height reduced rate of the HRP::ga1-1 of 500mg/L DPC processing is ga1-1's 3.47 again.Under identical DPC concentration, plant height reduced rate difference is not after DPC is handled by pSuper1300::ga1-5 and ga1-5 Greatly, and DPC on ZmCPS::ga1-5 substantially without influence, the plant height reduced rate of the HRP::ga1-5 of DPC processing is much higher than accordingly The plant height reduced rate of the HRP::ga1-5 of the pSuper1300::ga1-5 and ga1-5:30mg/L DPC processing of DPC concentration processing It is 4.68 times of ga1-5；The plant height reduced rate of the HRP::ga1-5 of 500mg/L DPC processing is 6.66 times of ga1-5.Show Arabidopsis can be improved to the sensibility of DPC in HRP.

The results show that the plant height of arabidopsis: untreated HRP: can be improved in final height-related protein HRP of the invention: The plant height of the HRP::ga1-1 of the HRP::ga1-1 and 500mg/L DPC processing of ga1-1,30mg/L DPC processing is respectively corresponding 1.03 times, 0.68 times and 0.41 times of the ZmCPS::ga1-1 plant height of processing, the 3.54 of respectively respective treated ga1-1 plant height Again, 2.71 times and 1.71 times；At HRP::ga1-5 the and 500mg/L DPC of untreated HRP::ga1-5,30mg/LDPC processing The plant height of the HRP::ga1-5 of reason is respectively 0.95 times, 0.64 times and 0.38 times of respective treated ZmCPS::ga1-5 plant height, 2.26 times, 1.65 times and 1.00 times of respectively respective treated ga1-5 plant height.

Influence of the DPC of 4.2 various concentrations to HRP gene arabidopsis plant height is turned

70 plants of pure lines plant of the HRP::ga1-1 of step 2 are randomly selected, are randomly divided into seven groups, every group 10 plants, This seven groups of plant constantly carry out the following processing on the 33rd day respectively from sowing (the sowing same day is denoted as sowing the 1st day): one group sprays After water (i.e. the DPC aqueous solution of 0mg/L), cultivates 12 days, obtain untreated HRP::ga1-1；One group of DPC for spraying 30mg/L It after aqueous solution, cultivates 12 days, obtains the HRP::ga1-1 of 30mg/L DPC processing；After one group sprays the DPC aqueous solution of 50mg/L, Culture 12 days obtains the HRP::ga1-1 of 50mg/L DPC processing；After one group sprays the DPC aqueous solution of 100mg/L, culture 12 It, obtains the HRP::ga1-1 of 100mg/L DPC processing；After one group sprays the DPC aqueous solution of 300mg/L, cultivates 12 days, obtain The HRP::ga1-1 of 300mg/L DPC processing；After one group sprays the DPC aqueous solution of 500mg/L, cultivates 12 days, obtain 500mg/L After mono- group of HRP::ga1-1 of DPC processing sprays the DPC aqueous solution of 1000mg/L, cultivates 12 days, obtain at 1000mg/L DPC The HRP::ga1-1 of reason.

According to the method described above, the pure lines plant of HRP::ga1-1 is replaced with to the pure lines plant of HRP::ga1-5, other steps It is rapid constant, respectively obtain HRP::ga1-5,50mg/LDPC processing of untreated HRP::ga1-5,30mg/L DPC processing HRP::ga1-5,100mg/L DPC processing HRP::ga1-5,300mg/L DPC processing HRP::ga1-5,500mg/L The HRP::ga1-5 of the HRP::ga1-5 and 1000mg/L DPC processing of DPC processing.

The plant height (Fig. 3) of above-mentioned different disposal arabidopsis is measured respectively, and average plant height is shown in Table 3.

The results show that turn the plant height of HRP gene arabidopsis is influenced by the concentration of DPC, turn the plant height of HRP gene arabidopsis It is reduced with the increase of DPC concentration.

The plant height (cm) for turning HRP gene arabidopsis that table 3, various concentration DPC are handled

Claims

1. raising plant is to the method for gibberellin inhibitor sensitiveness, the encoding gene including importing protein into recipient plant The step of obtaining genetically modified plants；Genetically modified plants gibberellin inhibitor sensitiveness compared with the recipient plant increases Add；The gibberellin inhibitor is DPC；The plant is dicotyledon；

The protein is following protein a) or b):

A) amino acid sequence is the protein of the 1-821 amino acids of sequence 1；

B) amino acid sequence is the protein of sequence 1.

2. according to the method described in claim 1, it is characterized by: the nucleotide sequence of the encoding gene of the protein such as sequence In list shown in 1-2463 nucleotide of sequence 2.