CN106701777A - Application of rice OsTOE1 gene in increasing of rice ear grain number and decreasing of rice ear length - Google Patents

Abstract

The invention relates to the technical field of plant gene engineering, in particular to cloning of a rice OsTOE1 gene, and an application thereof in increasing of rice ear grain number and decreasing of rice ear length. A nucleotide sequence of the OsTOE1 gene is SEQ ID NO: 1 in a sequence table; the nucleotide sequence of a protein encoding area is SEQ ID NO: 2 in the sequence table; an encoded protein sequence is SEQ ID NO: 3 in the sequence table. The complete protein encoding area sequence of the OsTOE1 gene is controlled by a constitutive promoter 35S to excessively express in the rice, the transgenic plant obviously increases the rice ear grain number and decreases the rice ear length, and the potential utilization value of the OsTOE1 gene in the genetic improvement of rice is indicated. The application has the advantage that the action of the OsTOE1 gene in increasing of rice ear grain number and decreasing of rice ear length is realized. The invention also provides a rice improving method.

Description

Application of the rice Os TOE1 genes in increasing grain number per spike and reducing spike length

Technical field

The present invention relates to field of plant genetic.Specifically related to application of the rice Os TOE1 genes in increasing grain number per spike and reducing spike length. By transgene method, the protein coding region of overexpression OsTOE1 genes is a significant increase grain number per spike and reduces spike length in paddy rice, Show that OsTOE1 genes have important value in rice genetic improvement.

Background technology

With the continuous growth and the change of human consumption's mode of population in the world, the crisis in food in the whole world is increasingly serious.Paddy rice is that the whole world is most important One of cereal crops, China has more than the population of half with paddy as staple food grain, therefore improves the yield of paddy rice to ensureing that it is heavy that the grain security of country has The strategic importance wanted.

The single plant yield of paddy rice is together decided on again by tiller number, grains per panicle and grain, wherein the number of grains per panicle depending on fringe type shape Into process.The fringe of paddy rice is differentiated after reproductive growth is entered by inflorescence meristem, before floral organ is ultimately formed, the fringe of paddy rice Portion can gradually produce Primary branch separate living tissue, Secondary branch separate living tissue and small ear separate living tissue, the time changed between these different separate living tissues Largely determine fringe type (Kyozuka et al., the Control of grass inflorescence form by the fine-tuning of of paddy rice meristem phase change.Curr Opin Plant Biol,2014,17:110-115).If the differentiation of small ear separate living tissue is postponed, then may be used Even three times branch stalks of more Secondary branch can be produced, such that it is able to improve final grain number per spike.In view of having important exploitation value in agricultural production Value, the research of relevant fringe type is all for a long time the popular domain of botany research, and scientist separates and cloned multiple ginsengs by various means The gene for determining is formed with fringe type, and some genes are applied (Zhang and Yuan, Molecular control of in the practices of breeding grass inflorescence development.Annu Rev Plant Biol,2014,65:553-578).From the point of view of the gene being separated at present, transcription because Son account for a very big part wherein, show that formation of the gene transcription regulation to fringe type has important decisive action.Applicant passes through analyzing rice It has also been found that transcription factor expression pattern is very big in different developmental stage difference, this also enters for the full-length genome transcript profile of the young fringes of multiple continuous stages of development One step implies that transcription factor plays effect (Wang et al., the A dynamic gene expression atlas covering the of core in fringe type is modified entire life cycle of rice.Plant J,2010,61:752-766)。

MicroRNA172 is the highly conserved non-coding tiny RNA in different plants of a class, and it is played by adjusting the transcription factor of AP2 classes Effect.AP2 is the A genoids for participating in floral organ pattern formation certified earliest, therefore the regulating and controlling effect between microRNA172 and AP2 There is important adjustment effect (Chen, A microRNA as a translational repressor of APETALA2in to the morphogenesis of floral organ Arabidopsis flower development.Science,2004,303:2022-2025).Additionally, the AP2 genoids adjusted by microRNA172 Also played a role in plant multiple growth course, such as the Sex Determination process of corn is participated in, also with the cleistogamy and the formation of dense cluster of barley Closely related (Chuck et al., the The maize tasselseed4microRNA controls sex determination and meristem cell fate by of journey targeting Tasselseed6/indeterminate spikelet1.Nat Genet,2007,39:1517-1521；Houston et al.,Variation in the interaction between alleles of HvAPETALA2and microRNA172determines the density of grains on the barley inflorescence.Proc Natl Acad Sci U S A,2013,110:16675-16680；Nair et al.,Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2mRNA cleavage.Proc Natl Acad Sci U S A,2010, 107:490-495).MicroRNA172 can regulate and control 5 target genes of coding AP2 class transcription factors in paddy rice, wherein 3 work(of target gene Can be disclosed and report (Lee and An, Two AP2family genes, SUPERNUMERARY BRACT (SNB) and OsINDETERMINATE SPIKELET 1(OsIDS1),synergistically control inflorescence architecture and floral meristem establishment in rice.Plant J,2012,69:445-461；Zhou et al.,Genetic control of seed shattering in rice by the APETALA2transcription factor SHATTERING ABORTION1.Plant Cell,2012,24:1034-1048).Wherein SNB Development of floral organs is primarily involved in OsIDS1, and SHAT1 is then the seed holding for participating in regulation paddy rice.Received disclosure sets forth another The function of the gene OsTOE1 of microRNA172 regulation and control.The present invention is significantly increased by the protein coding region of overexpression OsTOE1 genes Paddy rice grain number per spike and reduce the spike length of paddy rice, in agricultural production paddy rice is carried out using OsTOE1 genes genetic improvement provide it is new Method.

The content of the invention

It is the application for providing a kind of OsTOE1 genes in increasing paddy rice grain number per spike and reducing spike length that the purpose of the present invention is.Of the invention other one Individual purpose there is provided a kind of genetic transformation carrier and method for increasing paddy rice grain number per spike and reduction spike length by OsTOE1 genes.OsTOE1 bases Because having such as SEQ ID NO:Nucleotide sequence shown in 1, also including with SEQ ID NO:DNA sequence dna shown in 1 has more than 90% homology Gene order, also mutant allele or derivative including being produced because inserting, substitute or lacking one or more bases.Present invention additionally comprises The protein of OsTOE1 gene codes, its sequence such as SEQ ID NO:Shown in 3, also including with SEQ ID NO:Sequence shown in 3 have 90% with The protein sequence of upper homology, the albumen or protide for also changing including the function of being produced because inserting, substitute or lacking one or more amino acid Like thing.The present invention is a significant increase the grain number per spike of paddy rice and reduces the spike length of paddy rice by the protein coding region of overexpression OsTOE1 genes, These results show that OsTOE1 genes may have important value in rice breeding.The sequence of the protein coding region of OsTOE1 genes Such as the SEQ ID NO in sequence table:Shown in 2.The OsTOE1 genes that the present invention is provided can be used for other controlling elements, such as constitutive promoter (such as CaMV35S promoters) or organ specific promoters construct expression vector；Also can by transgenic technology, antisense RNA, RNAi, Zinc finger nuclease (zinc-finger nucleases, ZFN), activating transcription factor sample effector nuclease (transcription activator-like Effector nucleases, TALEN) and the skill such as CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 Art is manipulated with the grain number per spike and spike length of adjusting and controlling rice to it.

Realize that concrete technical scheme of the invention is as follows：

1st, using PCR method from rice leaf RNA reverse transcriptions and come cDNA in expand OsTOE1 genes protein coding region；Specifically Method has a detailed description in embodiment 1.

2nd, the protein coding region of OsTOE1 genes is connected on pCAMBIA1301S carriers, builds overexpression carrier 35S:OsTOE1OE； Specific method has a detailed description in example 2.

3rd, using agriculture bacillus mediated transgenic method (Lin and Zhang, the Optimising the tissue culture conditions for for optimizing high efficiency transformation of indica rice.Plant Cell Rep,2005,23:540-548) by carrier 35S:OsTOE1OE imports water 11 (Institute of Crop Science, Chinese Academy of Agricultural Science) are spent in rice acceptor, transformed plant is obtained；Specific method has a detailed description in embodiment 3.

4th, analyze and identify positive transgenic plant by the method for PCR and isolate detection, and phenotypic evaluation and statistical are carried out for individual plant to T1 Analysis；Specific method has a detailed description in example 4.

Compared with prior art, advantages of the present invention is as follows：

1st, the function disclosure sets forth rice Os TOE1 genes in increasing paddy rice grain number per spike and reducing spike length；

2nd, the protein coding region Introduced into Rice of OsTOE1 genes is realized the genetic improvement to paddy rice grain number per spike and spike length by the present invention；

3rd, present invention finds a kind of method that genetic improvement is carried out to paddy rice using OsTOE1 genes.

Brief description of the drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of OsTOE1 genes.

Sequence table SEQ ID NO:2 is the CDS sequences of the coded protein of OsTOE1 genes.

Sequence table SEQ ID NO:3 is the protein sequence of OsTOE1 gene codes.

Fig. 1：The oligonucleotide ligand of OsTOE1 and microRNA172 (miR172) is to schematic diagram.

Fig. 2：Expression vector 35S:OsTOE1OE building process schematic diagrames.Description of reference numerals：

A figures in Fig. 2：Insertion conversion carrier 35S:The DNA fragmentation schematic diagram for including OsTOE1 protein coding regions of OsTOE1OE.

B figures in Fig. 2：The structural representation of empty carrier pCAMBIA1301S.Fragment shown in A figures in Fig. 2 is passed through into KpnI and BamHI The whole carrier 35S of conversion is formed in the middle of KpnI the and BamHI restriction enzyme sites of double digestion insertion vector pCAMBIA1301S:OsTOE1OE.

Fig. 3：The expression vector 35S that the present invention builds:The structural representation of OsTOE1OE.

Fig. 4：35S:Genotype detections of the OsTOE1OE transgenosis T1 for individual plant.Description of reference numerals：

A figures in Fig. 4：35S:First T1 of family of OsTOE1OE transgenosis is for plant genotype detection.Primer used is The product of OsTOE1OEJCF and OsTOE1OEJCR, 752bp size is this pair of genomic DNA fragment of primer amplification, 219bp sizes Product is this pair of fragment of the protein coding region of the OsTOE1 genes being transferred to of primer amplification.The individual plant of amplified production for 219bp sizes occur is Transgenic positive individual plant.First swimming lane is DNA marker.

B figures in Fig. 4：35S:Second T1 of family of OsTOE1OE transgenosis is for plant genotype detection.Primer used is The product of OsTOE1OEJCF and OsTOE1OEJCR, 752bp size is this pair of genomic DNA fragment of primer amplification, 219bp sizes Product is this pair of fragment of the protein coding region of the OsTOE1 genes being transferred to of primer amplification.The individual plant of amplified production for 219bp sizes occur is Transgenic positive individual plant.First swimming lane is DNA marker.

Fig. 5：11 (WT) and 35S are spent in wild type:The phenotype of the plant of OsTOE1OE transgenosis (OsTOE1OE) compares.Reference is said It is bright：

A figures in Fig. 5：11 (WT) and 35S are spent in wild type:The form of the plant of OsTOE1OE transgenosis (OsTOE1OE) compares.

B figures in Fig. 5：11 (WT) and 35S are spent in wild type:The fringe type of the plant of OsTOE1OE transgenosis (OsTOE1OE) compares.

C figures in Fig. 5：11 (WT) and 35S are spent in wild type:The Primary branch shape of the plant of OsTOE1OE transgenosis (OsTOE1OE) State compares.

Specific embodiment

Embodiment 1：The protein coding region clone of rice Os TOE1 genes

A gene for having 5 AP2 classes is regulated and controled by microRNA172 in paddy rice, and wherein the gene number of logging in is LOC_Os05g03040's Function is not set forth also.According to the result (http that rice genome is annotated://rice.plantbiology.msu.edu/), the nucleotide sequence of the gene Such as the SEQ ID NO in sequence table:Shown in 1, the SEQ ID NO in its protein coding region sequence such as sequence table:Shown in 2, the protein sequence of coding The SEQ ID NO that row are shown in sequence table:3.The protein sequence of the gene is compared with the protein of arabidopsis, it is found that the gene is arabidopsis Bloom the ortholog of repressor TOE1 genes, therefore it is OsTOE1 that the gene is named in the present invention.OsTOE1 genes and microRNA172 Sequence pair relationhip see Fig. 1.

It is template by the cDNA that reverse transcription is obtained that the present invention uses the RNA of rice leaf, using the special primer of OsTOE1 genes, is passed through The method of PCR separates the protein coding region of clone's OsTOE1 genes, and specific operation is as follows：

(1) rice leaf RNA is extracted

The RNA of 11 plant tillering phase blades is spent in extracting rice varieties, the reagent of RNA extractings is Trizol of the purchase from Invitrogen companies Extraction agent box (concrete operation step is shown in kit specification).

(2) the reverse transcription synthesis chains of cDNA first

Step is as follows：

1) the μ g of total serum IgE 2 of extracting are taken, the μ l of 1 μ l, 10xDNaseI buffer of DNaseI 1, plus DEPC (pyrocarbonic acid diethyl ester, RNA is added The strong inhibition agent of enzyme, working concentration is 0.01%) treated water to 10 μ l, mixes and places 15min to remove the gene of residual after room temperature Group DNA；

2) the μ l of 0.2M EDTA 1 are added after 15min, and 10min is incubated in 65 DEG C of water-baths to remove the activity of DNaseI；

3) 1 μ l primers oligo (dT) is added₁₅, and 10min is incubated in 65 DEG C of water-baths to destroy the secondary structure of RNA, then place on ice 5min；

4) the μ l of 4 μ l, 0.1M DTT (mercaptoethanol) of 5x first strand buffer, 2 μ l, 10mM dNTP mixture 1, reverse transcriptase 1 are added μ l, are placed in warm bath 1.5h in 42 DEG C of water-baths after mixing；

5) reverse transcription product is placed in 90 DEG C of dry bath 3min to inactivate reverse transcriptase by reaction after terminating；

6) to 120 μ l water are added in reverse transcription product, -20 DEG C preserve reaction final product after mixing.The reagent used in reaction is all purchased from Invitrogen companies.

(3) protein coding region of OsTOE1 genes is expanded

In order to expand OsTOE1 gene proteins code area, following primer is designed：

OsTOE1OEF (forward primer):5'-GGTACCATGGAGTTGGATCTGAACAACGTGG-3'(underlined sequences are known for KpnI Other site)；

OsTOE1OER (reverse primer):5'-GGATCCTCAATGGTGGTGGTGATGGCG-3'(underlined sequences are that BamHI recognizes position Point)；

This pair of primer can be amplified such as sequence table SEQ ID NO:Genetic fragment shown in 2.

The cDNA come with step (2) reverse transcription is entered performing PCR and expanded as template, with primer OsTOE1OEF and OsTOE1OER.PCR Reaction cumulative volume is 20 μ l, comprising μ l, the 10mM primers OsTOE1OEF of 1 μ l, 10xPCR buffer of cDNA templates, 2 μ l, 10mM dNTP 2 0.3 μ l, ExTaq enzyme 0.2 μ l each with OsTOE1OER, plus deionized water to 20 μ l (used PCR buffer, dNTP, rTaq enzymes etc. Purchased from precious bioengineering Dalian Co., Ltd).

PCR reaction conditions are as follows：1. 94 DEG C of 4min, 2. 94 DEG C of 40s, 3. 58 DEG C of 40s, 4. 72 DEG C of 2min, 5. from 2. -4. circulate 38 It is secondary, 6. 72 DEG C of 7min, 7. 4 DEG C of preservations.PCR primer electrophoresis detection on the TBE Ago-Gels of 1% (mass/volume), reclaims length It is the DNA fragmentation of 1551bp (the target dna section of 1539bp is plus the two restriction enzyme site 12bp added on primer).And will The PCR primer of recovery is connected on T-A cloning vectors pGEMT-vector (purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e., beautiful Promega companies of state), carry out sequence verification using T7 and SP6 primer pairs PCR primer on carrier.By the restructuring matter comprising the PCR primer Grain is named as plasmid TA-OsTOE1.

Embodiment 2：The overexpression vector construction of OsTOE1 genes

With KpnI and BamHI double digestion plasmids TA-OsTOE1 (described KpnI and BamHI are bought from precious bioengineering Dalian Co., Ltd, Detailed directions refer to the specification of the said firm's corresponding product with consumption, and plasmid TA-OsTOE1 comes from embodiment 1, as shown in Figure 2 A), reclaim bag The DNA fragmentation of the 1551bp of the protein coding region containing OsTOE1 genes, then with the vector plasmid by KpnI and BamHI double digestions [carrier is disclosed report to pCAMBIA1301S:Zhou et al.,Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds.Theor Appl Genet,2009, 118:1381-1390；Its basic framework originates from Australian CAMBIA laboratories (http://www.cambia.org/daisy/cambia/materials/overview.html) pCAMBIA1301, by adding 35S promoter reality Now to the expression regulation of transformed gene, its structure is as shown in Figure 2 B] (it is purchased from Promega companies, detailed directions and use using T4DNA ligases Specification of the amount with reference to the said firm's product) it is attached.Connection product by electricity conversion method (electric conversion instrument be eppendorf Products, Applied voltage of the present invention is 1800V, operation instructions of the concrete operations with reference to the instrument) Escherichia coli DH10B is imported (purchased from Promega public affairs Department) in, plus 400 μ l LB culture medium recovery 45min, it is applied on the LA culture medium flat plates of the kanamycins containing 50mg/L, 37 DEG C of incubator trainings (LA and LB is formulated reference to support 14-16h：Pehanorm Brooker,《Molecular Cloning:A Laboratory guide》The third edition, Science Press, 2002).Choose Monoclonal is taken, Amplification Culture simultaneously extracts plasmid, by KpnI and BamHI double digestion screening positive clones, and the expression vector of gained is named as 35S:OsTOE1OE。

Embodiment 3：The acquisition of transgenic paddy rice

By expression vector 35S:OsTOE1OE (coming from embodiment 2) in agrobcterium-mediated transformation Introduced into Rice kind by spending 11 Callus, by preculture, infect, co-culture, screen with hygromycin (for screen the positive transgenosis callus a kind of antibiotic, purchase Buy the Co., Ltd of Roche Group from Denmark) callus of resistance, break up, take root and acclimatization and transplantses crop field, obtain transfer-gen plant.Agrobacterium The method of genetic transformation and reagent used and formula are to optimize (Hiei et al., Efficient transformation of according to the report of Hiei et al. rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J, 1994,6:271-282；Lin and Zhang,Optimising the tissue culture conditions for high efficiency transformation of indica rice.Plant Cell Rep,2005,23:540-548)。

The Agrobacterium-mediated genetic transformation reagent and formula that the present invention relates to are as follows：

(1) reagent and solution are abridged

6-BA (6-BenzylaminoPurine, 6-benzyladenine)；KT (Kinetin, kinetin)；NAA (Napthalene acetic acid, Methyl α-naphthyl acetate)；IAA (Indole-3-acetic acid, heteroauxin)；2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4- dichloro-benzenes Fluoroacetic acid)；AS (Acetosringone, acetosyringone)；CH (Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)；DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO))；N6max (a large amount of ingredient solutions of N6)； N6min (N6 a small amount of ingredient solution)；MSmax (a large amount of ingredient solutions of MS)；MSmin (MS a small amount of ingredient solution)

(2) main solution formula

1) N6max mother liquors [10 times of concentrates (10X)]

Dissolve one by one, 1000ml is then settled at room temperature.

2) N6min mother liquors [100 times of concentrates (100X)]

1000ml is dissolved and is settled at room temperature.

3)Fe₂- EDTA stores liquid (100X)

300ml distilled water and ferric sulfate (FeSO are added in a big triangular flask₄·7H₂O)2.78g

300ml distilled water is added in another big triangular flask and 70 DEG C are heated to, b diammonium disodium edta is subsequently adding (Na₂EDTA·2H₂O)3.73g

Mixed after they all dissolve, 2h is kept in 70 DEG C of water-baths, be settled to 1000ml, 4 DEG C save backup.

4) vitamins stock liquid (100X)

Add water and be settled to 1000ml, 4 DEG C save backup.

5) MSmax mother liquors (10X)

1000ml is dissolved and is settled at room temperature.

6) MSmin mother liquors (100X)

1000ml is dissolved and is settled at room temperature.

7) 2,4-D stores liquid (1mg/ml)

2,4-D 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

8) 6-BA stores liquid (1mg/ml)

6-BA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

9) NAA stores liquid (1mg/ml)

NAA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and 4 DEG C save backup.

10) IAA stores liquid (1mg/ml)

IAA 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, and 4 DEG C save backup.

11) glucose storage liquid (0.5g/ml)

Glucose 125g

Distillation water dissolves are settled to 250ml, and 4 DEG C save backup after sterilizing.

12) AS storages liquid

AS 0.392g

DMSO 10ml

In packing to 1.5ml centrifuge tubes, 4 DEG C save backup.

13) 1N potassium hydroxide storage liquid

Potassium hydroxide (KOH) 5.6g

Distillation water dissolves are settled to 100ml, and room temperature preservation is standby.

14) KT stores liquid (1mg/ml)

KT 100mg.

1ml 1N potassium hydroxide dissolves 5min, then plus after 10ml distillations water dissolves are complete is settled to 100ml, room temperature preservation.

(3) culture medium prescription

1) inducing culture

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml triangular flasks (25ml / bottle), sealing sterilizing.

2) subculture medium

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and be settled to 1000ml, is dispensed into 50ml triangular flasks (25ml / bottle), sealing sterilizing.

3) pre-culture medium

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.

Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, packing is poured into culture dish (25ml/ wares).

4) base is co-cultured

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, sealing sterilizing.

Using preceding heating for dissolving culture medium and 5ml glucose storages liquid and 250 μ l AS are added to store liquid, packing is poured into culture dish (25ml/ wares).

5) suspension medium

Plus distilled water, to 100ml, regulation pH value is dispensed into two triangular flasks of 100ml to 5.4, sealing sterilizing.

Liquid is stored using preceding addition 1ml glucose storages liquid and 100 μ lAS.

6) Selective agar medium

Plus distilled water, to 250ml, regulation pH value to 6.0, sealing sterilizes.

Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, (25ml/ is poured into culture dish in packing Ware).

7) pre- differential medium

Plus distilled water is to 250ml, 1N potassium hydroxide adjusts pH value to 5.9, sealing sterilizing.

Using preceding dissolving culture medium, the hygromycin and 400ppm cephalosporins of 250 μ l 50mg/ml are added, (25ml/ is poured into culture dish in packing Ware).

8) differential medium

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 6.0.

1000ml is boiled and be settled to, 100ml triangular flasks (50ml/ bottles), sealing sterilizing is dispensed into.

9) root media

Plus distilled water, to 900ml, 1N potassium hydroxide adjusts pH value to 5.8.

1000ml is boiled and be settled to, is dispensed into pipe of taking root (25ml/ pipes), sealing sterilizing.

10) LA culture mediums (LB culture mediums are free of agar powder)

Distillation water dissolves are settled to 250ml, and loaded on 500ml triangular flasks, room temperature is saved backup after sterilizing.

The step of Agrobacterium-mediated genetic transformation that the present invention relates to, is as follows：

(1) callus induction

1) ripe rice paddy seed is shelled, then successively with 70% Ethanol Treatment 1min, 0.15% mercury chloride (HgCl₂)15min

2) sterilizing washing seed 4-5 times；

3) seed is placed on inducing culture；

4) it is placed at dark and cultivates 5 weeks, 26 ± 1 DEG C of temperature.

(2) callus subculture

The embryo callus subculture of glassy yellow, consolidation and relatively dry is selected, dark lower culture 2 weeks, 26 ± 1 DEG C of temperature is put on subculture medium.

(3) preculture

The embryo callus subculture of consolidation and relatively dry is selected, dark lower culture 4d, 26 ± 1 DEG C of temperature is put on pre-culture medium.

(4) Agrobacterium culture

1) Agrobacterium EHA1052d of the preculture containing structure good vector, 28 DEG C of temperature on the LA culture mediums with kanamycins；

2) Agrobacterium is transferred in suspension medium, 2-3h is cultivated on 28 DEG C of shaking tables.

(5) Agrobacterium is infected

1) callus of preculture is transferred in the bottle for having sterilized；

2) suspension of regulation Agrobacterium is to OD₆₀₀0.8-1.0；

3) callus is soaked into 30min in agrobacterium suspension；

4) blotted on transfer callus to the filter paper for having sterilized；It is then placed within co-culturing on base and cultivates 2d, 19-20 DEG C of temperature.

(6) callus washing and selection culture

1) sterilize water washing callus to invisible Agrobacterium；

2) it is immersed in 30min in the aqua sterilisa containing 400ppm cephalosporins；

3) blotted on transfer callus to the filter paper for having sterilized；

4) selected 2-3 times, every time 2 weeks on transfer callus to Selective agar medium.(first time cephalosporin screening concentration is 400ppm, second It is later 250ppm)

(7) break up

1) kanamycin-resistant callus tissue is transferred to and cultivates 5-7d on pre- differential medium at dark；

2) shift on callus to the differential medium of pre- differentiation culture, cultivated under illumination, 26 DEG C, 5-7 weeks of temperature.

(8) take root

1) young plant for having broken up is extracted, the root produced during differentiation is cut；

2) it is then transferred to be cultivated 2-3 weeks under illumination in root media, 26 DEG C of temperature.

(9) transplant

Wash the remaining medium on root off, the seedling with good root system is transferred to greenhouse, while keeping moisture moistening at initial several days.In greenhouse After hardening about 2 weeks or so, then transfer load to crop field.

Embodiment 4：The identification of transgenic paddy rice and Phenotypic Observation

To detect the genotype of transfer-gen plant, following primer is designed：

OsTOE1OEJCF (forward primer):5'-GGGCCAATTCCTTGGCAAGA-3'；

OsTOE1OEJCR (reverse primer):5'-TGTGAGGCTGCAGGCTGAGA-3'；

Because OsTOE1OEJCF is designed on the 2nd extron of OsTOE1 genes, and OsTOE1OEJCR is designed in OsTOE1 On 6th extron of gene, and genetic transformation be that the protein coding region of the OsTOE1 genes without introne is carried out, therefore make Transfer-gen plant is expanded with this pair of primer, a fragment of the 752bp from genomic DNA can be obtained, while if transgenic positive is planted If strain, a fragment of the 219bp from OsTOE1 gene proteins code area can also be obtained.According to this two strip-type it may determine that Whether PCR is successful and whether individual plant that detected is transgenic positive.

T0 is for 35S for extracting:The STb gene of OsTOE1OE transformed plants (coming from embodiment 3) blade, DNA method for extracting is CTAB methods (Zhang et al.,Genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis.Theor Appl Genet,1992,83:495-499).With blade STb gene as template, enter performing PCR with primer OsTOE1OEJCF and OsTOE1OEJCR and expand Increase.PCR reaction cumulative volumes are 20 μ l, and comprising DNA profiling 100ng, 2 μ l, 10mM dNTP of 10xPCR buffer 2 μ l, 10mM draws Each μ l of 0.3 μ l, rTaq enzyme 0.2 of thing OsTOE1OEJCF and OsTOE1OEJCR, plus deionized water to 20 μ l (used PCR buffer, DNTP, rTaq enzyme etc. is purchased from precious bioengineering Dalian Co., Ltd).PCR reaction conditions are as follows：1. 94 DEG C of 4min, 2. 94 DEG C of 40s, 3. 58 DEG C 40s, 4. 72 DEG C of 1min, 5. from 2. -4. circulate 38 times, 6. 72 DEG C of 7min, 7. 4 DEG C of preservations.PCR primer is in 1% (mass/volume) TBE Ago-Gels on electrophoresis detection.

PCR testing results show, to carrier 35S:OsTOE1OE transgenosis and carry out positive plant for negative plant, all show as fringe Length shortens, constant per fringe Primary branch number, but all substantially increases with number of grain per ear per fringe Secondary branch number, and its phenotype is as shown in Figure 5.

In order to carry out phenotypic analysis, applicant is by 35S:2 independent transformations of OsTOE1OE and the seed (come from embodiment 3) of T0 plant that comes By being seeded in rice seedling bed after seed soaking, vernalization, transplanted after 20d to crop field and obtain T1 for transfer-gen plant.Planting density is 24 centimetres of 15 cm x, Plantation place is the experimental plot of Hongshan District Hua Zhong Agriculture University of Wuhan City of Hubei China province, the paddy rice under the conditions of having safety protection facility at routinely Implantation methods carry out field management.Enter the moon that performing PCR amplification separates individual plant to detect using primer OsTOE1OEJCF and OsTOE1OEJCR The positive, and carry out the detection that isolates of transgenic event and character mutation, and statistics phenotypic data.

Analysis result shows in T1 generations, the carrier 35S that the present invention builds:The all positive individual plant that OsTOE1OE transgenic progenies are separated is relative For negative individual plant, all show as spike length and shorten, it is constant per fringe Primary branch number, but all substantially increase with number of grain per ear per fringe Secondary branch number, Show 35S:The transgenic event of OsTOE1OE is (see the Fig. 4 and Fig. 5) for isolating with the change of these phenotypes.

The phenotypic data to record T1 generation related plant, including spike length, every fringe Primary branch number, every fringe Secondary branch number and number of grain per ear are investigated, And the negative individual plant separated with each family is control, carries out statistical analysis.

Table 135S:The phenotype statistical value of the T1 generations positive and negative individual plant of OsTOE1OE (OsTOE1OE) transgenosis.

Table 1 is illustrated：(+) and (-) in table 1 represents transgenic positive and negative individual plant respectively.Statistical analysis are carried out using t tests,^a,b,c P is represented respectively<0.05,0.01 the level of signifiance with 0.001.

Claims (4)

1. application of the rice Os TOE1 genes in increasing grain number per spike and reducing spike length, it is characterised in that the nucleotide sequence of OsTOE1 genes such as SEQ ID NO:Shown in 1.

2. application of the rice Os TOE1 genes in increasing grain number per spike and reducing spike length, it is characterised in that the protein sequence of OsTOE1 gene codes is such as SEQ ID NO:Shown in 3.

3. a kind of plant conversion carrier 35S:OsTOE1OE, it is characterised in that the carrier includes the OsTOE1 genes described in claim 1.

4. a kind of method for improveing paddy rice, including structure plant expression vector and Agrobacterium-mediated genetic transformation, it is characterised in that described method is bag Include by overexpression OsTOE1 genes, improvement includes increasing paddy rice grain number per spike and reduces spike length of rice proterties, or uses and OsTOE1 genes Gene with more than 90% homology, the mutant allele produced by inserting, substitute or lacking one or more bases or derivatives thereof Carry out rice transformation.