CN103665127A

CN103665127A - Rice tillering relevant protein, encoding gene thereof and application of rice tillering relevant protein and encoding gene thereof

Info

Publication number: CN103665127A
Application number: CN201310669443.5A
Authority: CN
Inventors: 万建民; 周峰; 林启冰; 朱立宏; 江玲; 张欣; 郭秀萍; 任玉龙; 周坤能
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2013-12-10
Filing date: 2013-12-10
Publication date: 2014-03-26

Abstract

The invention discloses a rice tillering relevant protein, an encoding gene thereof and application of the rice tillering relevant protein and the encoding gene thereof. The rice tillering relevant protein disclosed by the invention is a protein consisting of an amino acid sequence shown as SEQ ID NO.1, or is a protein which is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown as SEQ ID NO.1, is relative to plant tillering and is derived from the amino acid sequence shown as SEQ ID NO.1. By introducing the encoding gene of the protein into breeding materials of different backgrounds, transgenic plants of which the branching numbers are increased at certain degrees can be cultivated. The protein and the encoding gene thereof can be applied to genetic improvement of plants.

Description

A kind of rice tillering associated protein and encoding gene thereof and application

Technical field

The invention belongs to genetic breeding field, relate to a kind of rice tillering associated protein and encoding gene thereof and application.

Background technology

Infant farming originates from the exploitation to many floristic domestications and natural plant resource.The mankind have experienced nearly 10,000 years just makes global food total amount reach annual 1000000000 tons in nineteen sixty, but in the time of short 40 years subsequently, by 2000, this numeral has just reached annual 2000000000 tons.Huge like this achievement should give the credit to numerous breeding men and plant geneticist at the genetic improvement of adhering to constantly carrying out crop, has improved the output of crop, thereby has been met population growth's continuous demand.But recent two decades comes, and the situation fluctuating has appearred in the rice yield of China.Tracing it to its cause, is to be interbreed breeding in subspecies because front twice breeding breaks through to a great extent, and the germ plasm resource in subspecies and Breeding Potential have all obtained utilizing fully and excavating, and the difficulty that the interior breeding of subspecies produces new breakthrough is larger.Therefore, want to obtain new important breakthrough on rice yield, must look for another way.

In recent years, in order further to improve rice yield, the concept of " ideotype " was mentioned by most breeding men and rice genetic scholar.This is new approaches that further improve rice yield, and its breeding objective is to cultivate the new rice variety of the high biological yield of few tiller, thick bar, large fringe.The formation of plant type of rice depends primarily on the factors such as plant height, tillering number, tillering angle and fringe portion form, and suitable ability for tillering can improve colonial organism output, and tiller is also the basis that output forms simultaneously.A large number of experiments show that a little less than ability for tillering is crossed that often ability adjustment is poor automatically, be difficult in early days forming the high colony of biological yield, cause biological yield low, the raising of restriction economic yield; And ability for tillering is excessively strong, easily cause stem stalk thin and delicate, lodging tolerance reduces, also unfavorable to producing, and this just requires ability for tillering appropriateness.Therefore to the control of rice tillering, be, the important prerequisite that realizes " ideotype ".

In the recent period, research to a plurality of rice dwarf More-tiller mutants shows, a kind of new plant hormone---witchweed lactone plays a part very important in the process that suppresses rice tillering growth, but also very few to the understanding of this functions of hormones mechanism, therefore further investigate this hormone at regulating plant the mechanism of action aspect tillering, will provide prospect widely for its application in agriculture production.

Summary of the invention

The object of this invention is to provide a kind of rice tillering associated protein.

Another object of the present invention is to provide the encoding gene of this albumen.

Another object of the present invention is to provide the application of this albumen and encoding gene thereof.

Object of the present invention can be achieved through the following technical solutions:

Rice tillering associated protein provided by the invention, derives from Oryza paddy rice (No. 8, Oryza sativa var. agricultural), is following (a) or (b) described protein:

(a) protein being formed by the aminoacid sequence shown in SEQ ID NO.1;

(b) by the aminoacid sequence of SEQ ID NO.1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to plant tillering by the derivative protein of SEQ ID NO.1.

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

The sequence of table 1 protein fusion label

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

Sequence in above-mentioned (a) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain; The encoding gene of the albumen in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in SEQ ID NO.2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

The gene D53 of above-mentioned rice tillering associated protein of encoding also belongs to protection scope of the present invention.

Described gene is preferably as follows 1) or 2) or 3) or 4) DNA molecular:

1) DNA molecular shown in SEQ ID NO.2;

2) DNA molecular shown in SEQ ID NO.3;

3) under stringent condition with 1) or 2) the DNA sequence dna hybridization that limits and the DNA molecular of encoding said proteins;

4) with 1) or 2) or 3) DNA sequence dna that limits has 90% above homology, and the DNA molecular of coded plant tillering associated protein.

4, the recombinant expression vector that contains above arbitrary described gene also belongs to protection scope of the present invention.

The recombinant expression vector that available existing plant expression vector construction contains described gene.

Described plant expression vector comprises double base agrobacterium vector or can be used for plant instantaneous conversion carrier etc.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, and the non-translational region of transcribing as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean stores protein gene) 3 ' end all has similar functions.

While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, as Actin muscle (Actin1) promotor of the ubiquitin of the 35S promoter of cauliflower mosaic virus (CaMV), corn (Ubiquitin) promotor and paddy rice etc., they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.

For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor (beta-Glucuronidase gus gene, luciferase LUC gene or green fluorescent protein GFP gene etc.), have the antibiotic marker thing (ammonia benzyl mycin Amp marker or kantlex Kan marker etc.) of resistance or anti-chemical reagent marker gene (as herbicide-resistant gene Bar) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described recombinant expression vector preferably inserts the recombinant plasmid that gene D53 of the present invention obtains between the restriction enzyme site PstI of pCUbi1390 carrier and BamHI.Described recombinant expression vector is specially pCUbi1390-D53.The expression cassette that contains the above gene (D53), transgenic cell line and recombinant bacterium all belong to protection scope of the present invention.

The primer pair of described gene D53 total length or the arbitrary fragment of increasing also belongs to protection scope of the present invention.

Albumen of the present invention, described gene D53, any one in described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium cultivated the application of tillering in paddy rice more.

Utilize any carrier that can guide foreign gene to express in plant, by the gene transfered plant cell of encoding said proteins, can obtain transgenic cell line and transfer-gen plant.Carry described gene expression vector can by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be 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.The plant host being converted can be both monocotyledons, can be also dicotyledons, as: tobacco, Root or stem of Littleleaf Indianmulberry, Arabidopis thaliana, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.

Utilizing transgenic technology to increase a novel method for rice tillering, is that described gene D53 is imported in the plant of different background, obtains the transgenic plant that branch amount increases to a certain extent; Preferably described gene D53 is imported in the plant of different background by described recombinant expression vector.

Utilizing transgenic technology to reduce a novel method for rice tillering, is the expression that suppresses gene D53 described in object plant, obtains the transgenic plant that branch amount reduces to a certain extent; Described object plant is the plant of carrying described gene D53.The expression that suppresses gene D53 described in object plant can adopt RNAi technology, by building, for the interference carrier of D53, realizes.

Beneficial effect:

The invention provides a new rice tillering associated protein and encoding gene thereof, the regulation and control of this rice tillering associated protein involved in plant witchweed lactone hormone signal.The expression that suppresses this protein coding gene can cause the minimizing of rice tillering number, thereby can cultivate the transgenic plant of low branch amount, high bar, large fringe.The encoding gene of described albumen is imported in the breeding material of different background, can cultivate branch amount and obtain the transgenic plant that increase to a certain extent.Described albumen and encoding gene thereof can be applied to genetic modification of plants.

Accompanying drawing explanation

No. 8, Fig. 1 wild-type kind agricultural and the phenotype comparison of downgrading More-tiller mutant d53.

A. No. 8, wild-type agricultural and the comparison of d53 mutant phenotype in seedling stage; B. No. 8, wild-type agricultural and the comparison of d53 mutant ripening stage phenotype; C. wild-type and d53 mutant tiller number Dynamic comparison; D. wild-type and d53 mutant plant height constituent ratio are.

The semidominance effect of Fig. 2 d53 mutator gene.A. wild-type, heterozygosis F ₁with the overall phenotype comparison of d53 mutant plant; B. wild-type, heterozygosis F ₁with the comparison of d53 mutant plant sword-like leave phenotype; C. wild-type, heterozygosis F ₁and stem stalk crosscut phenotype comparison between d53 mutant plant first segment; D. wild-type, heterozygosis F ₁with the comparison of the main fringe phenotype of d53 mutant plant; E. wild-type, heterozygosis F ₁with d53 mutant plant plant height quantitative comparison; F. wild-type, heterozygosis F ₁count quantitative comparison with d53 mutant plant tillering; G. wild-type, heterozygosis F ₁with d53 mutant plant the 3rd stem stalk diameter quantitative comparison; H. wild-type agricultural are hybridized F with d53 mutant No. 8 ₂population genetic compartment analysis.

Fig. 3 d53 mutant is insensitive to witchweed lactone HORMONE TREATMENT.A. wild-type, d53, d14 and d27 mutant are through the phenotype comparison after 2 weeks of witchweed lactone HORMONE TREATMENT; B. wild-type, d53, d14 and d27 mutant are through the phenotype comparison after 5 weeks of witchweed lactone HORMONE TREATMENT; C. wild-type, d53 and the endogenous witchweed lactone of d27 mutant hormone-content are measured relatively; D. wild-type, d53, d14 and d27 mutant are through witchweed lactone HORMONE TREATMENT lateral bud quantitative-length statistical after 2 weeks; E. wild-type, d53, d14 and d27 mutant are through the tiller number quantitative statistics comparison after 5 weeks of witchweed lactone HORMONE TREATMENT.

Fine Mapping, map based cloning and the expression analysis of Fig. 4 D53 gene.A.D53 gene Fine Mapping figure; B. No. 8, wild-type agricultural and d53 mutant D53 gene nucleotide and the comparison of amino acid difference; Quantitative comparison is expressed by c.D53 gene organization; D. witchweed lactone hormone induction D53 genetic expression temporal evolution is analyzed.

Fig. 5 D53 gene overexpression transfer-gen plant phenotypic evaluation.A. wild-type background descended to express normal D53 albumen and the comparison of sudden change d53 protein transgene plant phenotype; B. wild-type background descended to express normal D53 albumen and sudden change d53 protein transgene plant D53 genetic expression comparison; C. wild-type background was descended to express normal D53 albumen and was counted statistical with sudden change d53 protein transgene plant tillering.

Fig. 6 disturbs D53 gene transgenic plant phenotypic evaluation.Under a.d53 mutant background, disturb the comparison of D53 genetic expression transfer-gen plant phenotype; Under b.d53 mutant background, disturb D53 genetic expression transfer-gen plant tiller number statistical.

Fig. 7 binary vector pCUbi1390 collection of illustrative plates

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

The discovery of embodiment 1, plant tillering associated protein and encoding gene thereof

One, the phenotypic evaluation of rice dwarf More-tiller mutant d53 and genetic analysis

1.d53 the source of mutant

Original mutating strain series KL908 be by Japanese scholars from No. 8, agricultural through gamma Rays mutagenesis, and introduce to domestic from Kyushu University in 1988 Zhu Lihong of Nian You Agricultural University Of Nanjing professors, through many generation screenings, obtain homozygous mutation body, rear called after dwarf53(d53).

2.d53 the phenotypic evaluation of mutant

Tiller number dynamic measurement: sow after 1 week and counted once every 1 week, 30 strains that at every turn count, continuous counter 10 weeks; Tiller number static measurement: in the ripening stage, whole strain paddy rice is extracted, cleaned basal internode mud, primary tillering, secondary are tillered, tillered for three times and tiller for four times and count respectively; Tiller bud linear measure: the rice seedling phase divests each leaf sheath, exposes the tiller bud that is wrapped in axil place, under body formula mirror, measures its length; Plant height is measured: the length of measuring the extremely Gao Suiding in ground in the ripening stage.

This mutant shows as lateral bud growth at Seedling Stage to be accelerated.4 leaves are during the phase, d53 shows height of seedling and downgrades, incomplete leaf place axillalry bud forms first and tillers, the 1st leaf place axillalry bud also forms normally and tillers, and incomplete leaf axillalry bud may be because the reasons such as nutrient distribution stop growing in No. 8, wild-type agricultural, be tending towards degenerating, can not form normally and tiller as seen, but tillering of the 1st leaf place forms that (Fig. 1 a).During the ripening stage, d53 mutant is downgraded (be about wild-type 60%), the increase of tillering (be about wild-type 3 times) (Fig. 1 b) compared with wild-type plant performance plant height.From after planting 1 week, start total tiller number of wild-type and mutant to carry out finding dynamic measurement, As time goes on, the tiller number of d53 mutant is always higher than No. 8, wild-type agricultural (Fig. 1 c).From plant height, form, d53 spike length and each panel length all shorten (Fig. 1 d).Therefore, in general, d53 mutant main manifestations is that tiller bud dormancy is disengaged, and flushes, and the speed of growth is accelerated.

3.d53 the genetic analysis of sudden change

By d53 mutant and No. 8 hybridization of its wild-type parent agricultural, F ₁in generation, obtains F after selfing ₂for colony.By investigating F ₂for each individual plant plant height in segregating population, tiller, find after spike length and stem stalk diameter isophenous, d53 mutant phenotype is semidominant inheritance (Fig. 2).

4.d53 mutant is processed insensitive to witchweed lactone

Because d53 mutant and the d having been found that in paddy rice are that mutant has the similar dwarfing phenotype of tillering more, so we infer that d53 may also participate in witchweed lactone approach.Utilizing concentration is 1 μ M GR24(witchweed lactone synthetic analogue) mill water culture nutrient solution (Chiralix) cultivates rice seedling, after rice seedling being grown in mill water culture nutrient solution 1 week, be transferred in the mill water culture nutrient solution that contains 1 μ M GR24, change weekly nutritive medium 1 time, in cultured continuously 2-5 week, finally tiller bud or tiller number are counted.Using do not add 1 μ M GR24's and the Rooted Cuttings under isometric growth condition contrasting as processing with signal deletion mutant d27 and d14 with witchweed lactone hormone is synthetic.

Water planting plantation: plant with ciltivating process for measuring endogenous witchweed lactone hormone-content and hormone-treated seedling.Paddy rice is cultivated in the warm and humid controlled incubator of light (Ruihua).Light application time: 8 up to 22 o'clock, intensity of illumination: 250 μ mol.m ^-2.s ^-1, temperature: 30 ℃ of daytimes, 25 ℃ of nights, humidity: 70%.Mill water culture nutrient solution mother liquor formula is as follows:

1000 times (1ml/l) of mother liquor dilution of every kind of numbering, pH value is within the scope of 5.8-6.2, and nutrient solution is changed once weekly.

The endogenous witchweed lactone hormone-content of rice root secretion is measured: method, with reference to Yoneyama et al. (2008), is slightly revised.(1) collect the water-culturing rice nutritive medium of different varieties rice seedling (each 30 strains) equivalent; (2) be extracted with ethyl acetate extracting 2 times; (3) ethyl acetate is used 0.2M K mutually ₂hPO ₄rinsing, uses anhydrous Na ₂sO ₄dry, and concentrated in a vacuum; (4) extract is dissolved in the acetonitrile of 50% (v/v), and by LC/MS-MS(triple quadrupole bar series connection liquid chromatography mass technology) analyze.

Process after 2 weeks and find, the first leaf axil tiller bud growth of wild-type is subject to obvious inhibition; In d27 mutant, because exogenous hormone makes mutant return to the phenotype of wild-type to the restraining effect of tiller bud, its incomplete leaf and the first leaf axillary bud growth are all subject to obvious inhibition; But after processing, there is not such inhibition phenotype (Fig. 1 a, d) in d14 and d53 mutant.In addition, the rice plant of processing after 5 weeks is added up to rear discovery, d27 mutant is at tiller number or all returned to the phenotype of wild-type at plant height, and d14 and d53 still keep mutant phenotype (Fig. 1 b, e).We further carry out quantitative assay to the endogenous witchweed lactone hormone of root system secretion in No. 8, wild-type agricultural and d53 mutant.Result shows that endogenous witchweed lactone hormone accumulates in a large number in d53 mutant, is 7.4 times of wild-type, and in contrast witchweed lactone hormone synthesis mutant d27, we almost can't detect the content (Fig. 1 c) of this endogenous hormones.Comprehensively these results show, d53 is a witchweed lactone signal pathway defect mutant.

The Fine Mapping of embodiment 2, D53 gene, map based cloning and expression analysis

The Fine Mapping of 1.D53 gene and map based cloning: utilize the F obtaining after d53 mutant and Java kind Ketan Nangka hybridization ₂for segregating population, D53 gene is carried out to Fine Mapping.First we have screened and between d53 and Ketan Nangka, have had polymorphic SSR mark from the existing consensus primer in laboratory, by interval between polymorphic mark, be not less than the standard of 20cM, selected 242 polymorphism marks that are uniformly distributed on 12 karyomit(e)s and carried out Preliminary Linkage Analysis.We utilize F ₂in colony, 20 high bars phenotype individual plant of tillering less carries out tentatively chainly, and d53 and Ketan Nangka are in contrast.If 20 extreme individual plants of recessiveness (high bar tiller less plant) all performance or the mostly banding pattern of expression mutation body d53, minority individual plant shows as heterozygosis banding pattern, and we just think that this mark and goal gene exist linkage relationship.Result finds on the 11st the short arm of a chromosome end that by analyzing us two SSR mark W1(primer sequences are as SEQ ID NO.4 and SEQ ID NO.5) and W2(primer sequence as SEQ ID NO.6 and SEQ IDNO.7) and goal gene between there is linkage relationship.Subsequently, we have designed respectively again some and between two parents, have had the polymorphism mark of banding pattern difference on 3 BAC clones between these two marks.Finally utilize 2893 extremists (high bar tiller less plant) that goal gene is positioned to mark Z7(primer sequence as SEQ ID NO.8 and SEQ ID NO.9) and Z3(primer sequence as SEQ ID NO.10 and SEQ ID NO.11) between, the individual plant that exchange occurs for two marks has respectively 4 strains and 2 strains, and (Fig. 4 is a).

PCR reaction system and condition: PCR reaction system (10 μ l): DNA (20ng/ul) 1ul, upstream primer (2pmol/ul) 1ul, downstream primer (2pmol/ul) 1ul, 10 * Buffer (MgCl ₂free) 1ul, dNTP (10mM) 0.2ul, MgCl ₂(25mM) 0.6ul, rTaq (5u/ul) 0.1ul, ddH ₂o5.1ul, altogether 10ul.PCR response procedures: 94.0 ℃ of sex change 5min; 94.0 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 1min, circulate 35 times altogether; 72 ℃ are extended 7min; 10 ℃ of preservations.PCR reaction is carried out in MJ Research PTC-225 thermal cycler.

Utilize NCBI annotation and RiceGAAS(http: //ricegaas.dna.affrc.go.jp/) database, we carry out predictive genes in interval to 34kb between mark Z7 and Z3, and (Fig. 4 is a) to found that 3 candidate genes of existence.After being checked order respectively, the genome of these 3 candidate genes and ORF find, having there is the replacement of 1 base and the disappearance of 15 bases in the genome sequence than one of them gene of wild-type, thereby causes the ORF of this gene that 1 amino acid whose change and 5 amino acid whose disappearances (Fig. 4 b) occur.Therefore, tentatively judge that this gene that amino acid change occurs is likely D53 gene.This ORF(SEQ ID NO.2) one of coding new structurally with the similar expressing protein of I class Clp ATPase (expression protein), sequence is as shown in SEQ ID NO.1.

The expression of 2.D53 gene:

Detect each tissue expression sampling of D53 gene: choose No. 8 different development stages of wild-type rice varieties agricultural each tissue (Seedling Stage root and bud, tillering phase leaf sheath and blade and heading stage young fringe, stem and stipes), after liquid nitrogen grinding, put into-80 ℃ of refrigerations standby.Result shows, D53 gene presents ubiquity in different rice tissue and expresses (Fig. 4 c).

HORMONE TREATMENT induction D53 genetic expression sampling: by No. 8 seedling of large wild-type agricultural in 1 week after witchweed lactone HORMONE TREATMENT, by different treatment time point, collect whole seedling plant, after liquid nitrogen grinding, put into-80 ℃ of refrigerations standby, wherein using do not add hormone processing as negative control.Result shows, D53 gene is subject to witchweed lactone hormone induction up-regulated expression (Fig. 4 d).

The extraction of RNA and real time fluorescent quantitative (qPCR) are analyzed:

(1) according to the cDNA sequence of D53 gene, use the special qPCR primer D53-qRT(primer sequence of Primer Premier5.0 software design as SEQ ID NO.12 and SEQ ID NO.13);

(2) use RNAprep pure plant total RNA extraction reagent box (Tiangen), extract wild-type and the RNA in different transfer-gen plant seedling stage or tillering phase;

(3), with DU800 spectrophotometer (Bechman Instrument Inc.USA), detect concentration and the quality of RNA;

(4) integrity of detected through gel electrophoresis RNA;

(5) get 2 μ g RNA as template, use PrimeScriptII1st Strand cDNA Synthesis Kit(TaKaRa), reverse transcription goes out cDNA;

(6) Real-time PCR expression analysis: configure reaction system, amplified reaction is at 7900HT Real-Time PCR(Applied Biosystems) instrument carries out.Reaction system is: 1.8 μ l cDNA templates, and 10 μ l SYBR Premix ExTaqII(2 *), 0.8 μ l D53-qRT Forward Primer(10 μ M), 0.8 μ l D53-qRT Reverse Primer(10 μ M), 6.8 μ l ddH ₂o.Response procedures is: 95 ℃ of denaturation 30s; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations; After reaction to be amplified finishes, use 7900HT Real-Time PCR(Applied Biosystems) the software analysis CT value that instrument carries, with the expression amount of OsUbiquitin gene, as internal reference, data analysis is pressed relative quantification method and is calculated (Livak.et al., 2001).

The acquisition of embodiment 2, transgenic plant and evaluation

One, recombinant expression vector builds

(1) take No. 8 cDNA of wild-type kind agricultural is template, utilize respectively gene-specific primer D53-cDNA-F(SEQ ID NO.14)/D53-cDNA-R(SEQ ID NO.15) and D53-RNAi-F(SEQ ID NO.16)/D53-RNAi-R(SEQ ID NO.17) carry out pcr amplification and obtain normal D53 full-length gene fragment and D53 gene interference fragment, and PCR product is reclaimed to purifying.The cDNA of d53 mutant of take is template, utilizes gene-specific primer D53-cDNA-F(SEQ ID NO.14)/D53-cDNA-R(SEQ ID NO.15) carry out pcr amplification and obtain sudden change d53 full-length gene fragment, and PCR product is reclaimed to purifying.

(2) adopt In-Fusion PCR Cloning System(TaKaRa) PCR product D 53 genes and sudden change d53 gene clone are arrived to binary vector pCUbi1390(Fig. 7) in, D53 gene interference fragment is cloned into carrier pLHRNAi(number of patent application: 201110055864.X).Recombining reaction system (5 μ l): the carrier after PCR product and enzyme are cut mixes (totally 4 μ l) in 1:1 ratio, adds after 1 μ l In-Fusion HD, 50 ℃ of PCR reaction 20min, and-20 ℃ of refrigerations are standby.

(3) heat shock method intestinal bacteria transform: by recombining reaction system of short duration centrifugal after, mix and the bacillus coli DH 5 alpha competent cell (Tiangen) melting on ice, after static 30min, system is placed in to 37 ℃ of water-bath heat shock 90s, then is placed in cooled on ice 2min.Add after non-resistant LB liquid nutrient medium, at 37 ℃ of shaking table 180rpm/min, cultivate after 1h, whole transformants are uniformly coated on the LB solid medium containing 50mg/l kantlex.Cultivate after 16h for 37 ℃, picking positive colony checks order.After having checked order, the recombinant expression vector of the pCUbi1390-D53 that contains correct sequence clone, pCUbi1390-d53 and pLHRNAi-D53 is preserved stand-by.

Two, the acquisition of restructuring Agrobacterium

By thermal shock method, by correct clone's recombinant plasmid transformed Agrobacterium EHA105 bacterial strain (Invitrogen) of above-mentioned pCUbi1390-D53, pCUbi1390-d53 and pLHRNAi-D53, obtain recombinant bacterial strain, extraction plasmid carries out PCR and enzyme is cut evaluation.PCR and enzyme are cut to recombinant bacterial strain called after EH-pCUbi1390-D53, EH-pCUbi1390-d53 and the EH-pLHRNAi-D53 that evaluation is correct.

Three, the acquisition of transgenic plant

Respectively by EH-pCUbi1390-D53 and EH-pCUbi1390-d53 and corresponding empty carrier contrast EH-pCUbi1390 conversion wild-type kind Kitaake; EH-pLHRNAi-D53 and empty carrier contrast EH-pLHRNAi transform d53 mutant concrete grammar and are:

Cultivate above-mentioned Agrobacterium recombinant bacterial strain 16h for (1) 28 ℃.Collect thalline, and be diluted to the N that contains 100 μ mol/l Syringylethanones ₆in liquid nutrient medium (Sigma) to concentration be OD ₆₀₀≈ 0.5, obtains bacterium liquid;

(2) will be cultured to the Kitaake Mature Embryos of Rice embryo callus of month and the bacterium liquid mixed infection 30min of step (1), filter paper proceeds to N after blotting bacterium liquid ₆solid is total in culture medium (Sigma), cultivates altogether 3d for 24 ℃;

(3) callus of step (2) is seeded in to the N that contains 100mg/l kantlex and Rifampin (Phyto Technology Laboratories) ₆screening (16d) for the first time in solid screening culture medium;

(4) the healthy callus of picking proceeds to the N that contains 100mg/l kantlex and Rifampin ₆programmed screening in solid screening culture medium, every 15d subculture is once;

(5) the healthy callus of picking proceeds to the N that contains 50mg/l kantlex and Rifampin ₆screening for the third time in solid screening culture medium, every 15d subculture is once;

(6) picking kanamycin-resistant callus tissue proceeds on division culture medium and breaks up; Obtain the T of seedling differentiation ₀for transfer-gen plant.

Four, the evaluation of transfer-gen plant

(1) hygromycin selection of rotaring gene plant blade is identified

It is (1 ‰ the 6-BA that adds concentration simultaneously and be is as preservation agent) in 1 ‰ Totomycin solution that rice plant of tillering stage rotaring gene plant blade (2-3cm) is immersed in to concentration, after 3 days, observes leaf morphology changing conditions.If blade still keeps emerald green, illustrate that this transfer-gen plant is positive; If blade occurs from edge to the old and feeble yellow isophenous at center, can assert that this transfer-gen plant is negative.With the known positive and negative rotaring gene plant blade, do parallel control simultaneously.Experimental result shows, our transgenic positive rate reaches 70% left and right.

(2) expression level of D53 gene is identified

Extract Transgenic Rice Plants blade RNA, utilize the quantitative primer D53-qRT of D53 gene specific to carry out quantitative fluorescent PCR and react to detect different transfer-gen plant D53 gene expression doses variations.Result shows, in the mistake that proceeds to pCUbi1390-D53 and pCUbi1390-d53, expressing the expression level of D53 and d53 gene in positive transfer-gen plant increases 5-30 doubly (Fig. 5 b) compared with the control.The method of RNA extraction and qPCR is the same.

(3) phenotypic evaluation of transfer-gen plant

Respectively by T ₀for transfer-gen plant and receptor parent Kitaake and d53 mutant, be planted in crop institute of Chinese Academy of Agricultural Sciences Changping Experimental Base.With turn empty carrier and contrast transfer-gen plant and compare with receptor parent Kitaake, turn pCUbi1390-D53 and pCUbi1390-d53 positive plant and all occur downgrading phenotype (Fig. 5 a of tillering more, c), illustrate that the phenotype of tillering all can appear downgrading in overexpression D53 gene and d53 gene more; Turn the mutant phenotype that pLHRNAi-D53 transfer-gen plant has recovered d53 mutant, i.e. plant height increase, the minimizing (Fig. 6) of tillering.Thereby verified that the mutant phenotype before transgenosis is caused by semidominance sudden change d53 gene, proved that D53 gene protein participates in growing of regulating plant branch (tiller) as inhibition of witchweed lactone signal pathway.

Claims

1. a rice tillering associated protein, is characterized in that:

(a) by the aminoacid sequence shown in SEQ ID NO.1, formed; Or

(b) by aminoacid sequence shown in SEQ ID NO.1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to plant tillering by the derivative protein of SEQ ID NO.1.

2. the gene of albumen described in the claim 1 of encoding.

3. gene according to claim 2, is characterized in that: described gene is following 1) or 2) or 3) or 4) DNA molecular:

1) DNA molecular shown in SEQ ID NO.2;

2) DNA molecular shown in SEQ ID NO.3;

4) with 1) or 2) or 3) DNA sequence dna that limits has 90% above homology, and the DNA molecular of the rice tillering associated protein of encoding.

4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain gene described in claim 2 or 3.

5. recombinant expression vector according to claim 4, is characterized in that: described recombinant expression vector is between the restriction enzyme site PstI of pCUbi1390 carrier and BamHI, to insert the recombinant plasmid that gene obtains described in claim 2 or 3.

6. the total length of gene and the primer pair of any fragment thereof described in the claim 2 or 3 that increases.

7. albumen described in claim 1, gene described in claim 2 or 3, any one described in claim 4 in recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium cultivated the application of tillering in paddy rice more.

8. utilizing transgenic technology to increase a novel method for rice tillering, is that gene described in claim 2 or 3 is imported in the plant of different background, obtains the transgenic plant that branch amount increases.

9. method according to claim 8, is characterized in that: described in claim 2 or 3, gene imports in the plant of different background by recombinant expression vector described in claim 4 or 5.