CN104004074A

CN104004074A - Application of protein UCH677 and coding gene thereof in regulation and control of growth and development of plants

Info

Publication number: CN104004074A
Application number: CN201410228344.8A
Authority: CN
Inventors: 王东辉; 白书农; 许智宏
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2014-05-27
Filing date: 2014-05-27
Publication date: 2014-08-27
Anticipated expiration: 2034-05-27
Also published as: CN104004074B

Abstract

The invention discloses application of protein UCH677 and a coding gene thereof in regulation and control of growth and development of plants. The application means application of protein, which is formed by an amino acid sequence represented by a sequence 1 in a sequence listing, or a coding gene thereof in regulation and control of growth and development, namely one of (1) to (10), of the plants: (1) seed weight, (2) seed volume, (3) seed grain length, (4) seed grain width, (5) seed grain thickness, (6) number of seeds per ear, (7) plant height, (8) plant leaf length, (9) plant leaf width and (10) plant ear length. Shown by experiments, during the development of paddy rice, through increasing the expression level of the protein UCH677 in the paddy rice, compared with wild-type rice seeds, rice seeds can have the following phenotypes that the weight and volume of the seeds are increased, and meanwhile, the number of the seeds per ear is increased; in addition, in the aspect of plant phenotype, the height of over-expressed plants is higher than that of the wild-type plants. According to the application, a foundation for finding out ideas and methods for more simply creating the high-yield traits of crops is laid.

Description

The application in regulating growth of plants of UCH677 albumen and encoding gene thereof

Technical field

The invention belongs to molecular biology of plants technical field, relate to a kind of UCH677 albumen and the application of encoding gene in regulating growth of plants thereof.

Background technology

Since the seventies in last century, heterosis utilization is that China's Rice Production has been made great contribution.Face China's Economic development proposes " high yield, high-quality, efficient, safety, ecology " new great demand to agriculture production, can further excavate heterotic application potential with reply, just become the severe challenge of pendulum in face of contemporary science man.

The combination of heterotic formation based on two different parents.To on the basis of existing application, further excavate its potentiality, form machine-processed research except strengthening hybrid vigour, also be badly in need of setting up effective means and create male-sterile character, to effectively expand screening and the outstanding application being combined in production of cross combination.

At present, in breeding work and production, the male-sterile character of widespread use comes from spontaneous mutation and proterties transformation strain thereof more.The source of male-sterile character is very limited, is to expand the particularly serious restrictive factor of application of cross combination screening.According to the rule of the current current international practice, all innovations with application potential are all subject to intellectual property protection.Therefore, find thinking and the method for initiative Artificial Control crop seed size new, that there is independent intellectual property right and output, become and wished to hold one of cannot avoiding of excavating that the countries and regions of hybrid vigour application potential initiative face, key issue urgently to be resolved hurrily.

For a long time, people utilize the method seed selection hybrid crop of conventional breeding always, and these are long in method cycles, it is slow to take effect, can not meet production development in the urgent need to.Gene engineering method is compared traditional method and is had Some features: the seed selection cycle shortens, and fertility is relatively stable, affected by environment little, genotype is relied on few, low in the pollution of the environment.

Summary of the invention

The object of this invention is to provide a kind of UCH677 albumen and the application of encoding gene in regulating growth of plants thereof.

Application provided by the present invention, is specially the protein (called after UCH677 albumen) that is made up of the aminoacid sequence shown in sequence in sequence table 1 or its encoding gene (called after UCH677 gene) at regulating plant following 1)-10) in apply at least one:

1) seed weight;

2) seed volume;

3) seed grain length;

4) seed grain is wide;

5) seed grain is thick;

6) single fringe seed number;

7) plant plant height;

8) Plant Leaf is long;

9) Plant Leaf is wide;

10) plant spike length.

Above-mentioned application is embodied in: the expression amount of the described UCH677 albumen being made up of the aminoacid sequence shown in sequence in sequence table 1 in described plant is higher, and the seed weight of described plant is heavier and/or seed volume is larger and/or seed grain length is longer and/or seed grain wide and/or seed grain is thick and/or single fringe seed number is more and/or plant plant height is higher and/or Plant Leaf longer and/or Plant Leaf is wide and/or plant spike length is longer; The expression amount of the described UCH677 albumen being made up of the aminoacid sequence shown in sequence in sequence table 1 in described plant is lower, the seed weight of described plant is lighter and/or seed volume is less and/or seed grain length is shorter and/or the thick thinner and/or single fringe seed number of the wide narrower and/or seed grain of seed grain less and/or plant plant height is shorter and/or the long shorter and/or Plant Leaf of Plant Leaf is wide narrower and/or plant spike length is shorter.

(the UCH677 albumen) being made up of the aminoacid sequence shown in sequence in sequence table 1 or its encoding gene (UCH677 gene) have following I in seed selection)-X) application in object proterties at least one plant variety also belongs to protection scope of the present invention:

I) seed weight increases or reduces;

II) seed volume increases or reduces;

III) seed grain length increases or shortens;

IV) the wide broadening of seed grain or narrow;

V) seed grain is thick thickens or attenuation;

VI) single fringe seed number increases or reduces;

VII) plant plant height increases or becomes short;

VIII) Plant Leaf is long increases or shortens;

IX) the wide broadening of Plant Leaf or narrow;

X) plant spike length increases or shortens.

In actual applications, when the plant variety of institute's seed selection be that seed weight increases and/or seed volume increases and/or seed grain length increases and/or the wide broadening of seed grain and/or seed grain thick thicken and/or single fringe seed number increases and/or) plant plant height increases and/or Plant Leaf is long increases and/or when plant variety that the wide broadening of Plant Leaf and/or plant spike length increase, the higher plant of described UCH677 expressing quantity need be hybridized as parent.When the plant variety of institute's seed selection is that seed weight reduces and/or seed volume reduces and/or seed grain length shortens and/or seed grain is wide narrows and/or the thick attenuation of seed grain and/or single fringe seed number reduce and/or plant plant height becomes short and/or Plant Leaf length shortens and/or Plant Leaf is wide narrows and/or when plant variety that plant spike length shortens, the lower plant of described UCH677 expressing quantity need be hybridized as parent.

A further object of the present invention is to provide a kind of method of cultivating transgenic plant.

The method of cultivation provided by the present invention transgenic plant, specifically can be following (A) or (B):

(A) cultivate and there is following b1)-b10) method of the transgenic plant of at least one in object proterties, comprise the steps:

A), to the encoding gene that imports the protein being formed by the aminoacid sequence shown in sequence in sequence table 1 in object plant, obtain expressing the transgenic plant of described encoding gene;

B) a) gained transgenic plant, obtain, compared with described object plant, thering is following b1 from step)-b10) at least one transgenic plant in object proterties:

B1) seed weight increases;

B2) seed volume increases;

B3) seed grain length increases;

B4) the wide broadening of seed grain;

B5) seed grain is thick thickens;

B6) single fringe seed number increases;

B7) plant plant height increases;

B8) Plant Leaf is long increases;

B9) the wide broadening of Plant Leaf;

B10) plant spike length increases;

(B) cultivate and there is following d1)-d10) method of the transgenic plant of at least one in object proterties, comprise the steps:

The encoding gene of the protein c) in object plant, the aminoacid sequence shown in sequence 1 in by sequence table being formed suppresses to express, and obtains transgenic plant;

D) c) gained transgenic plant, obtain, compared with described object plant, thering is following d1 from step)-d10) at least one transgenic plant in object proterties:

D1) seed weight reduces;

D2) seed volume reduces;

D3) seed grain length shortens;

D4) seed grain is wide narrows;

D5) the thick attenuation of seed grain;

D6) single fringe seed number reduces;

D7) plant plant height becomes short;

D8) Plant Leaf length shortens;

D9) Plant Leaf is wide narrows;

D10) plant spike length shortens.

In above-mentioned application or method, the encoding gene (UCH677 gene) of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 (UCH677 albumen) is arbitrary described DNA molecular in following (1) to (3):

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

(2) the protein DNA molecule of the aminoacid sequence composition in the DNA molecule hybridize limiting with (1) under stringent condition and coding sequence table shown in sequence 1;

(3) DNA molecular limiting with (1) or (2) has the protein DNA molecule that in 90% above homology and coding sequence table, the aminoacid sequence shown in sequence 1 forms.

Above-mentioned stringent condition can be with 6 × SSC, the solution of 0.5%SDS, and at 65 DEG C, hybridization, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.

Wherein, sequence 2 is made up of 534 Nucleotide, is the encoding sequence (ORF) of described UCH677 gene; Protein shown in sequence 1 in sequence 2 code sequence lists, sequence 1 is made up of 177 amino-acid residues.

In aforesaid method (A), the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 specifically imports in described object plant by the form of recombinant expression vector.

Described recombinant expression vector can be used existing plant expression vector construction.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.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 expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other plant promoter; In addition, while using gene constructed recombinant 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 ensure 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 recombinant 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 antibiotic marker thing or the anti-chemical reagent marker gene etc. of resistance.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.

In an embodiment of the present invention, the promotor that starts the genetic transcription of described UCH677 gene in described recombinant expression vector is specially Actin promotor.

More specifically, described recombinant expression vector is that described UCH677 gene gene is inserted into the recombinant plasmid obtaining after the multiple clone site of pCAM23A carrier; In one embodiment of the invention, described multiple clone site is specially Xba I and Sal I.

In aforesaid method (B), described in object plant to the encoding gene of described UCH677 albumen suppress express, can be any method that reduces the expression of UCH677 gene described in described object plant.

In the method (A) and method (B) of above-mentioned cultivation transgenic plant, to carry the described recombinant expression vector of described UCH677 gene or the described rnai expression carrier of described UCH677 gene imports described object plant, specifically can be: by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity are led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.

In above-mentioned each application or each method, described plant can be monocotyledons, also can be dicotyledons.In the present invention, described plant is specially monocotyledon rice, as spent 11 in rice varieties.

In above-mentioned each application or each method, described leaf is grown and the wide leaf that is all specially boot leaf of described leaf is grown and leaf is wide.

Experimental results show that, in the growth course of paddy rice, the present invention is by gene overexpression technology, UCH677 albumen in paddy rice and encoding gene expression level thereof are raised, can cause rice paddy seed to show following phenotype: to compare than wild-type rice paddy seed, seed weight increases, volume increases (grain length and width, thick all increases), and single fringe seed number increases simultaneously; In addition, plant phenotype, compared with wild-type, cross and express that the plant height of plant is higher, leaf is long, leaf is wide and spike length all increases.The present invention finds out thinking and the method for more simply formulating high crop yield proterties to lay a good foundation.

Brief description of the drawings

Fig. 1 is T ₁in generation, proceeds to the PCR qualification result of the transgenic paddy rice of Overexpression vector pCAM23A-UCH677ox.Wherein, swimming lane M is DNA molecular amount standard, and each band is followed successively by 5000,3000,2000,1000,750,500,300,200bp from big to small; Swimming lane 1-7 is the positive plant of qualification, and 8 for spending 11 in not genetically modified wild-type paddy rice.

Fig. 2 is T ₁the real-time fluorescence quantitative PCR that generation proceeds to UCH677 gene in the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox detects.Wherein, WT represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox.In not genetically modified wild-type paddy rice, spend the expression amount of UCH677 gene in 11 as 1.* represent significant difference compared with WT (P<0.05).

Fig. 3 is the each genetic stocks rice plant of UCH677 gene phenotype.Wherein, WT is expressed as in not genetically modified wild-type paddy rice and spends 11; 677ox represents to identify positive T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox.

Fig. 4 is the plant height of the each genetic stocks rice plant of UCH677 gene, the statistics that leaf is long and leaf is wide of boot leaf.Wherein, A is plant height; B and C are respectively that the leaf of boot leaf is long and leaf is wide.In A-C, WT represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox, and * represents significant difference compared with WT (P<0.05).

Fig. 5 is the each genetic stocks paddy rice of UCH677 gene spike of rice phenotype.Wherein, WT represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox.

Fig. 6 is the single fringe seed number of the each genetic stocks paddy rice of UCH677 gene.Wherein, ZH11 represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox, and * represents significant difference compared with ZH11 (P<0.05).

Fig. 7 is the each genetic stocks rice grain of UCH677 gene phenotype.Wherein, WT represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox.

Fig. 8 is the grain length of the each genetic stocks rice grain of UCH677 gene, the statistics that grain is thick and grain is wide.Wherein, A, B and C are respectively grain length, grain is wide and grain is thick.In A-C, WT represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox, and * represents significant difference compared with WT (P<0.05).

Fig. 9 is the thousand seed weight of the each genetic stocks rice paddy seed of UCH677 gene.Wherein, ZH11 represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox, and * represents significant difference compared with ZH11 (P<0.05).

Figure 10 is the setting percentage of the each genetic stocks rice paddy seed of UCH677 gene.Wherein, ZH11 represents to spend 11 in not genetically modified wild-type paddy rice; 677ox represents T ₁in generation, proceeds to the transgenic rice plant of Overexpression vector pCAM23A-UCH677ox.

Embodiment

The experimental technique using in following embodiment if no special instructions, is ordinary method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

PCAM23A carrier: Beijing DingGuo ChangSheng Biology Technology Co., Ltd.Be recorded in " Chi Zhengchang. the OsSG01 functional study of paddy rice meiotic gene and analysis. Yangzhou University,, Master's thesis in 2010 " in a literary composition.The promotor that is positioned at Xba I upstream carrying on pCAM23A carrier is Actin promotor.

In rice varieties, spend No. 11: purchased from crop investigations institute of the Chinese Academy of Agricultural Sciences; Within 1979, spent training with No. five/Te Tepu/good fortune of capital wind brocade by the Chinese Academy of Agricultural Sciences crop.Be recorded in " Ni Pichong. Anther Culture new variety-in spend No. 11. Crop Germplasm Resources, 04 phase in 1989 ".

Agrobacterium EHA105: Beijing Quan Shijin biotechnology company limited.

In following embodiment, obtain substratum related in the process of transgenic plant as follows:

1, rice callus induction and subculture medium (japonica rice) NB minimum medium

2, AAM substratum

3, be total to culture medium

4, resistance screening substratum 1L, is poured on disposable flat board about 40-50

5, paddy rice division culture medium (japonica rice) 1L is poured on Boiling tube, approximately 20 left and right

6, paddy rice root media (japonica rice)

1/4MS inorganic salt	?
		MS VITAMIN	?

NAA	0.5mg/L
		Paclobutrazol	1m?g/L
Plant gel	2.6g/L
		pH	5.8

The related hormone mother liquor compound method of each substratum above:

(1) 0.5mg/ml2,4-D mother liquor join method: take 100mg2,4-D, is placed in small beaker; Adding a small amount of dehydrated alcohol makes it to dissolve completely; 2,4-D spirituous solution is slowly added in the water on magnetic stirring apparatus, if there is precipitation, need to reconfigure; Water is settled to 200ml, 4 DEG C of preservations.

(2) 0.5mg/ml α-NAA mother liquor join method: take 100mgNAA and be placed in small beaker; With the KOH solution dissolving NAA of 1M; Water is settled to 200ml, 4 DEG C of preservations.

(3) 0.5mg/ml6-BA mother liquor join method: take 100mg6-BA and be placed in small beaker; Add a small amount of concentrated hydrochloric acid, grind to form pasty state with glass rod, then add a small amount of concentrated hydrochloric acid, make it to dissolve completely; Dilute with water is also settled to 200ml, 4 DEG C of preservations.

(4) preparation of 100mM Syringylethanone (As): take 196.2mg As, directly dissolve with 5ml DMSO, and be settled to 10ml, be distributed into aseptic tubule ,-20 DEG C of stored frozen.

(5) 5mg/ml KT join method: take 100mg kinetin Kinetin, dissolve with a small amount of 1M KOH, dilute with water is settled to 20ml.After filtration sterilization, be distributed in aseptic tubule-20 DEG C of stored frozen.

Acquisition and the qualification of embodiment 1, UCH677 overexpression transgenic rice plant

UCH677 gene source related in the present embodiment is in paddy rice (Oryza.sativa L.), and its cDNA sequence is as shown in sequence in sequence table 2, and sequence 2 is made up of 534 Nucleotide; Protein (UCH677 albumen) in sequence 2 code sequence lists shown in sequence 1, sequence 1 is made up of 177 amino-acid residues.

One, the structure of recombinant expression vector pCAM23A-UCH677ox

Design following primer sequence according to sequence in sequence table 2:

23A677-F:5 '-CC tCT AGAaTGCTACGTTGTGGCGCA-3 ' (underscore place is the recognition sequence of restriction enzyme site Xba I, is thereafter the 1-18 position of sequence 2);

23A677-R:5 '-CC gTC GACtCA CTG CTT TGA AAG AGC-3 ' (underscore place is the recognition sequence of restriction enzyme site Sal I, is thereafter the reverse complemental of the 517-534 position of sequence 2).

Taking the sequence 2 in sequence table as template, carry out pcr amplification with primer 2 3A677-F and 23A677-R.After cutting PCR product with restriction enzyme Xba I and Sal I enzyme, reclaim object fragment, the pCAM23A carrier framework large fragment after it is cut with Sal I enzyme with restriction enzyme Xba I is connected, and obtains recombinant plasmid.The recombinant plasmid called after pCAM23A-UCH677ox of DNA fragmentation shown in sequence 2 in insertion sequence table will be shown through order-checking between the restriction enzyme site Xba I of pCAM23A carrier and Sal I.In recombinant expression vector pCAM23A-UCH677ox, the promotor that in initiating sequence table, the DNA fragmentation shown in sequence 2 is transcribed is Actin promotor.

Two, the acquisition of UCH677 overexpression transgenic rice plant and qualification

1, the acquisition of UCH677 overexpression transgenic rice plant

(1) preparation of rice conversion acceptor

A. the inducing culture of Rice Young Embryo callus

Take away the young fringe threshing of spending No. 11 in the rice varieties of spending about rear 12-15 days, float blighted grain with clear water, with 70% alcohol immersion 1-2 minute, then soak 90 minutes with 1.25% the aqueous sodium hypochlorite solution (active chlorine content is 1.25% (w/v)) that is added with 1% (v/v) Tween20, carry out surface sterilization.(when sterilizing, will often stir) uses aseptic water washing 3-4 time, drains water for subsequent use.On aseptic filter paper, extrude Rice Young Embryo with tweezers and dental scaler and be placed in solid inducing culture (NB minimum medium) above, 26 DEG C of dark evoked callus of cultivating.After about 5-7 days, peel callus, proceed to freshly prepared subculture medium (NB minimum medium) upper, succeeding transfer culture is about 5 days, for common cultivation under the same conditions.

B. the inducing culture of Mature Embryos of Rice callus

The Mature seed of rice shelling is first used 70% alcohol immersion 1-2 minute, then use the aqueous sodium hypochlorite solution (active chlorine content is 30%-40% (w/v)) of 30%-40% to soak 30 minutes, carry out surface sterilization (be preferably on shaking table and carry out), aseptic water washing 3-4 time, again seed is placed on aseptic filter paper after suck dry moisture, be placed on mature embryo calli induction media (NB minimum medium), 26 DEG C of dark cultivations (can cultivate by light, light is cultivated and looked fast).After approximately 20 days, peel the callus that mature embryo scultellum grows, proceed to mature embryo subculture medium (NB minimum medium) upper, succeeding transfer culture under the same conditions.Every two weeks succeeding transfer culture once later.Select succeeding transfer culture 4-5 days, the yellowish granular callus of color and luster cultivates altogether.

(2) conversion of Agrobacterium and cultivation

A. extracting and purifying plasmid

The bacillus coli DH 5 alpha bacterial classification of the recombinant expression vector pCAM23A-UCH677ox that contains step 1 structure and pCAM23A empty carrier is inoculated in respectively in 5ml LB (containing kantlex 50mg/L) liquid nutrient medium, 37 DEG C, 200rpm shakes overnight incubation.The plasmid extraction kit of pressing V-GENE company extracts recombinant plasmid.

B. cup soaked in absolute ethyl alcohol is hit in power taking, dries.

C. Agrobacterium EHA105 electric shock preliminary treatment

I. Agrobacterium EHA105 is inoculated in 5ml YEP (containing Streptomycin sulphate Sm50mg/L) liquid nutrient medium, and 28 DEG C, 200rpm concussion overnight incubation to OD600 value is 0.4.

In II.1.5ml centrifuge tube, collect 1ml bacterium liquid, 4 DEG C, 8000rpm, centrifugal 30s.

III. remove raffinate, 200 μ l ddH for precipitation ₂o fully suspends, and 4 DEG C, 8000rpm, centrifugal 30s.

IV. repeating step III tri-times.

V. remove raffinate, precipitation ddH ₂o fully suspends, and is electric shock Agrobacterium EHA105 competence.Add 200 μ l sterile glycerols to mix to be placed on-80 DEG C for subsequent use.

D. electric shock

I., to 200 μ l EHA105 competence, tip-tap mixes to get plasmid (recombinant expression vector pCAM23A-UCH677ox or pCAM23A empty carrier that step 1 builds), is then transferred in electric shock cup, puts on ice.

II. be ready to electric shock device (BioRad), voltage is 2.5V, pins shock button with hand, until a acoustic-electric hits complete.

III. room temperature adds YEP liquid medium after leaving standstill 2min, 28 DEG C of standing 1h, and then 28 DEG C, 200rpm cultivates 2h.

The centrifugal 30s of IV.8000rpm, collects bacterium liquid, precipitation ddH ₂o suspends, and is coated with and contains kantlex 50mg/L and the YEB solid medium flat board containing Streptomycin sulphate Sm50mg/L with glass stick, cultivates 48h for 28 DEG C.Scraping lawn Eddy diffusion is in YEB liquid nutrient medium, is cultured to logarithmic growth late period at 28 DEG C; Therefrom get again 0.5ml be forwarded in the same YEB liquid nutrient medium of 100ml after 2-3h to OD600 be 0.5 left and right, by centrifugal 10 minutes of cultured restructuring Agrobacterium 4000g, precipitation suspended into restructuring agrobacterium suspension with the AAM liquid nutrient medium of 100ml.

(3) the common cultivation of Rice Callus and Agrobacterium

Select the good subculture of the state obtaining in step 1 and put into the aseptic triangular flask of 100ml to the Rice Callus of certain hour (succeeding transfer culture 4-5 days, color and luster are yellowish, particulate state), then the restructuring agrobacterium suspension (at least ensuring enough bacterium liquid contacts with material) that adds appropriate step 2 to obtain, places 20min with 80-100r/min room temperature.Take out callus, on aseptic filter paper, suck unnecessary bacterium liquid, transfer to immediately in the common culture medium of solid that is covered with one deck aseptic filter paper, during by callus induction and succeeding transfer culture, being close to all the time the side of substratum still places down, callus should be put neatly, had better not stack each other 25 DEG C of dark culturing 3 days.

(4) screening of resistant calli

Callus after cultivating altogether is fully washed 4-6 time with sterilized water, becomes limpid until wash the aqueous solution of callus, then is aseptic washing 4-5 time of the cephamycin cef of 300mg/L with adding concentration, and 15-20min at every turn blots callus with aseptic filter paper.

Callus is placed in the screening culture medium that contains 25mg/L Totomycin Hygromycin and screens after 14 days and proceed in the screening culture medium that contains 50mg/L Totomycin Hygromycin and continue to screen.2 weeks generation.Most of callus is in latter 10 days brownization of left and right of screening, and milky resistant calli then regrows out at the edge of brownization tissue.Select general lasting 6-8 week.

(5) differentiation of resistant calli

From the resistant calli growing after the screening of two-three-wheel, the resistant calli of selecting milk yellow densification goes to the division culture medium that contains 50mg/L Totomycin Hygromycin and cultivates first dark cultivation 3 days, after forward again 16-20h/d to, intensity of illumination 100-120 μ molm ^-2s ^-1illumination condition under cultivate, after 30-40 days, further differentiate seedling.

(6) take root, strong sprout and transplanting

In the time that the bud of resistant calli differentiation grows to about 2-4cm, seedling is moved on on root media, cultivate about two weeks.Select the seedling of high about 10cm, well developed root system, wash away substratum with warm water, in greenhouse, transplant and bury.The water surface, not flood seedling as degree, if become a fine day, need to shade and survive (being as the criterion with guttation) to seedling.

By aforesaid operations, the final two kinds of transgenic seedlings with hygromycin resistance that obtain, proceed to the recombinant expression vector pCAM23A-UCH677ox of step 1 structure and the rice plant (T of pCAM23A empty carrier ₁generation).

2, the qualification of UCH677 overexpression transgenic rice plant

(1) PCR qualification

The T obtaining from step 1 ₁in generation, proceeds to the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox, and proceeds in the adjoining tree of pCAM23A empty carrier and extract respectively genomic dna.For the transgenic paddy rice that proceeds to recombinant expression vector pCAM23A-UCH677ox, carry out pcr amplification with primer 2 3A677-F and 23A677-R, obtain through qualification the plant that size is about 534bp object band and be the positive plant that proceeds to recombinant expression vector pCAM23A-UCH677ox.Because the endogenous UCH677 gene of paddy rice contains multiple introns, only have the successful plant of transgenosis can obtain the fragment of about 534bp by Plant Genome as template amplification, wild-type plant does not have this fragment.

The T that part steps 1 obtains ₁generation proceed to recombinant expression vector pCAM23A-UCH677ox transgenic paddy rice qualification result as shown in Figure 1.

(2) real-time fluorescence quantitative PCR detects

Get step (1) and identify positive T ₁in generation, proceeds to the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox, proceed to the adjoining tree of pCAM23A empty carrier, and spend 11 in not genetically modified rice varieties, from blade, extract respectively total RNA, reverse transcription obtains cDNA, taking this cDNA as template, the cDNA of gene UCH677 is carried out to real-time fluorescence quantitative PCR amplification with primer 677RT-F and 677RT-R, taking GAPDH as internal reference, primer is GAPDH-RT-F and GAPDH-RT-R.

677RT-F:5 '-GCGTGCTTCGTTTCTTGAGG-3 ' (the 249-268 position of sequence 2);

677RT-R:5 '-ATCCTTGGCCTCTGTATCGC-3 ' (reverse complementary sequence of the 314-333 position of sequence 2);

GAPDH-RT-F：5’-AAGCCAGCATCCTATGATCAGATT-3’；

GAPDH-RT-R：5’-CGTAACCCAGAATACCCTTGAGTTT-3’。

The concrete Prism7000 of the ABI company type quantitative fluorescent PCR system that adopts, power SYBR Green I mix reagent box, carries out qPCR experiment.Real-time fluorescence quantitative PCR response procedures: 95 DEG C of denaturation 30s; 95 DEG C of 5s, 60 DEG C of 34s, 40 circulations.Adopt Power (2 ^{-△ △ Ct}) the template ratio calculated between different samples represents heterogeneic relative expression quantity.

Test in triplicate results averaged.

As shown in Figure 2, as seen, compared with spending 11 (WT) in turning the rice varieties of empty carrier gene, step (1) is identified positive T to result ₁the expression amount that generation proceeds to UCH677 in the transgenic paddy rice of recombinant expression vector pCAM23A-UCH677ox significantly improves.

Embodiment 2, UCH677 transgenic rice plant Function Identification

Identify positive T with embodiment 1 ₁generation proceed to recombinant expression vector pCAM23A-UCH677ox transgenic paddy rice transfer-gen plant, turn in the wild-type rice varieties of empty carrier gene and spend No. 11, and the adjoining tree that proceeds to pCAM23A empty carrier that embodiment 1 obtains is experiment material.The planting seed of each experiment material is carried out in culture dish to vernalization (every kind of experiment material sowing 80-100 grain), the seedling replanting after vernalization is emerged in flowerpot, then forwards large field, Beijing suburb to and grows.Carry out the Analysis and Identification of following several respects.Each genetic stocks is all chosen at random 10 strains and is tested, and for quantitative data, result is got the mean value of 10 strains.

1, rice plant phenotype

The plant height of each genetic stocks of observation and comparison contemporaneously, and to the leaf of plant plant height, boot leaf the long and wide quantitative statistics that carries out of leaf.

Result shows, and spend compared with No. 11 in not genetically modified wild-type rice varieties, embodiment 1 identifies positive T ₁in generation, proceeds to higher (Fig. 3 of plant height of the transgenic rice plant of recombinant expression vector pCAM23A-UCH677ox, A in Fig. 4, P<0.05), longer (the B in Fig. 4 of leaf of boot leaf, P<0.05), leaf wide (C, P<0.05 in Fig. 4).And the adjoining tree that proceeds to pCAM23A empty carrier obtaining for embodiment 1, the leaf of its plant height, boot leaf is long and leaf is wide, with in not genetically modified wild-type rice varieties, spend No. 11 basically identical, no difference of science of statistics.

2, spike of rice phenotype analytical

The spike length of each genetic stocks of observation and comparison contemporaneously, and carry out quantitative statistics to planting single fringe seed number.

Result shows, and spend compared with No. 11 in not genetically modified wild-type rice varieties, embodiment 1 identifies positive T ₁it is all fuller more greatly and spike length is longer (Fig. 5) that in generation, proceeds to the spike of rice of transgenic rice plant of recombinant expression vector pCAM23A-UCH677ox.In addition, and spend compared with No. 11 in not genetically modified wild-type rice varieties, embodiment 1 identifies positive T ₁in generation, proceeds to single fringe seed number more (Fig. 6 and table 1, P<0.05) of the transgenic rice plant of recombinant expression vector pCAM23A-UCH677ox.And the adjoining tree that proceeds to pCAM23A empty carrier obtaining for embodiment 1, its spike of rice phenotype and single fringe seed number, with in not genetically modified wild-type rice varieties, spend No. 11 basically identical, no difference of science of statistics.

The statistical study of table 1 UCH677ox transgenic rice plant list fringe seed number

?	Single fringe seed number	Error
			In spend No. 11	45	4.65
677ox	68	5.89

3, seed weight and the statistical study of seed volume

The seed phenotypes of each genetic stocks of observation and comparison contemporaneously, and seed volume (seed length, thickness and width) and thousand seed weight are carried out to quantitative statistical analysis.

Result shows, and spend compared with No. 11 in not genetically modified wild-type rice varieties, embodiment 1 identifies positive T ₁in generation, proceeds to larger (Fig. 7 of seed volume of the transgenic rice plant of recombinant expression vector pCAM23A-UCH677ox, P<0.05), be embodied as seed length, thickness and width and all increase (Fig. 8, P<0.05).In addition, and spend compared with No. 11 in not genetically modified wild-type rice varieties, embodiment 1 identifies positive T ₁in generation, proceeds to the thousand grain weigth heavier (Fig. 9, P<0.05) of the transgenic rice plant of recombinant expression vector pCAM23A-UCH677ox.And the adjoining tree that proceeds to pCAM23A empty carrier obtaining for embodiment 1, its seed volume (seed length, thickness and width) and thousand seed weight, with in not genetically modified wild-type rice varieties, spend No. 11 basically identical, no difference of science of statistics.

4, seed-setting rate statistical study

Seed on each genetic stocks plant spike of rice is carried out to setting percentage statistical study.Wherein, setting percentage=reality grain number/total number × 100%, real grain has referred to a meter seed, and total grain number is that a real grain number adds unfilled gtains number (without rice ghost).

Result demonstration, embodiment 1 identifies positive T ₁the seed-setting rate of transgenic rice plant that generation proceeds to recombinant expression vector pCAM23A-UCH677ox is spent No. 11 and is proceeded to not genetically modified wild-type rice varieties compared with the adjoining tree of pCAM23A empty carrier, basically identical, no difference of science of statistics (Figure 10, P>0.05).

Claims

1. the protein being formed by the aminoacid sequence shown in sequence in sequence table 1 or the application of its encoding gene in regulating growth of plants;

Described growth and development of plants, is embodied in following 1)-10) at least one:

1) seed weight;

2) seed volume;

3) seed grain length;

4) seed grain is wide;

5) seed grain is thick;

6) single fringe seed number;

7) plant plant height;

8) Plant Leaf is long;

9) Plant Leaf is wide;

10) plant spike length.

2. the protein or its encoding gene that are made up of the aminoacid sequence shown in sequence in sequence table 1 have following I in seed selection)-X) application in object proterties at least one plant variety:

I) seed weight increases or reduces;

II) seed volume increases or reduces;

III) seed grain length increases or shortens;

IV) the wide broadening of seed grain or narrow;

V) seed grain is thick thickens or attenuation;

VI) single fringe seed number increases or reduces;

VII) plant plant height increases or becomes short;

VIII) Plant Leaf is long increases or shortens;

IX) the wide broadening of Plant Leaf or narrow;

X) plant spike length increases or shortens.

3. cultivate the methods of transgenic plant, for following (A) or (B):

B1) seed weight increases;

B2) seed volume increases;

B3) seed grain length increases;

B4) the wide broadening of seed grain;

B5) seed grain is thick thickens;

B6) single fringe seed number increases;

B7) plant plant height increases;

B8) Plant Leaf is long increases;

B9) the wide broadening of Plant Leaf;

B10) plant spike length increases;

D1) seed weight reduces;

D2) seed volume reduces;

D3) seed grain length shortens;

D4) seed grain is wide narrows;

D5) the thick attenuation of seed grain;

D6) single fringe seed number reduces;

D7) plant plant height becomes short;

D8) Plant Leaf length shortens;

D9) Plant Leaf is wide narrows;

D10) plant spike length shortens.

4. according to arbitrary described application or method in claim 1-3, it is characterized in that: the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 is arbitrary described DNA molecular in following (1) to (3):

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

5. according to the method described in claim 3 or 4, it is characterized in that: in described (A), the encoding gene of the described protein being made up of the aminoacid sequence shown in sequence in sequence table 1 is to import in described object plant by the form of recombinant expression vector.

6. method according to claim 5, is characterized in that: in described recombinant expression vector, the promotor that the encoding gene of the protein being made up of the aminoacid sequence shown in sequence in sequence table 1 described in starting is transcribed is Actin promotor.

7. according to arbitrary described application or method in claim 1-6, it is characterized in that: described plant is monocotyledons or dicotyledons.

8. application according to claim 7 or method, is characterized in that: described monocotyledons is paddy rice.