CN109486830A - Rice SNB gene and application, the method for regulating and controlling seed size - Google Patents

Abstract

The present invention relates to a kind of utilizations and transformation riceSNBGene regulates and controls seed size, wherein the nucleotide sequence of SNB gene is by changing or being transformed as shown in the SEQ ID NO.1 using pointSNBThe expression of gene regulates and controls seed size, specifically: overexpression overall lengthSNBGene such as SEQ ID NO.1, mutationSNBm2Gene such as SEQ ID NO.5, reaches and is substantially reduced seed size；By CRISP/CAS9 technological means, insertion and deletion causes gene to be terminated in advance such as SEQ ID NO.7 or overexpression mutation in 5 ' the end areas 400bp-500bp of First Exon (EXON1) sequenceSNBm1Gene such as SEQ ID NO.3, reaches and dramatically increases seed size, to show important function of the protein in adjusting and controlling rice seed size of SNB coding, while also providing a kind of method of new raising rice yield genetic improvement.

Description

Rice SNB gene and application, the method for regulating and controlling seed size

Technical field

The invention belongs to field of plant genetic project technology, and in particular to rice SNB gene is answered regulation seed size With.

Background technique

Rice is one of cereal crops important in the world, and there are 122 national rice cultivations in the whole world, supports the whole world to be more than 23% population.China is a large agricultural country and populous nation, and not only paddy is the staple food grain in China, and the production of paddy is also China Main Agricultural activity, therefore improve rice yield to ensure China's grain security be of great significance.

Grain weight, grain number per spike and number of productive ear are rice yield three elements.Three elements one of of the grain weight as yield are One important yield traits, it is that grain is long, grain is wide and the overall target of grain thickness, is generally indicated with mass of 1000 kernel.Grain weight and paddy Grain size is closely related, and is a particularly important quality trait.Grain weight or grain size are on cereal crop is evolved Also have great importance, since small-sized seed viability is lower, mechanical harvest difficulty is big.The people during domestication of rice It is intended to that the kind of big grain is selected to be cultivated, has gradually formed the cultivar shape bigger than normal than their wild relatives seed Condition.International Rice Research Institute, Philippine can increase production 30% or more (Ma Lilian etc., rice big grain kind it also holds that improving grain weight simultaneously Matter resource and genetic analysis, BULLETIN OF BOTANY Vol., 2006,23 (4): 395-401), cultivating Large grain rice kind is also current water One important directions of rice breeding.

Rice grain shape size character is complicated quantitative character, and the effort of QTL genetic dissection is carried out to grain shape quantitative character It is continued above 30 years, with the progress of modern age molecular engineering, is taken in the clone of adjusting and controlling rice grain shape major gene resistance and functional analysis Obtained greater advance.The gene for influencing grain shape is roughly divided into 3 classes, and first kind phenotype is substantially that seed shortens, and plant becomes short, and leaf inclines Angle becomes smaller, such as D1, D2, D11；Second class phenotype is small and circle grain shape, and plant becomes short, main distribution and japonica rice, as SRS1, SRS2, SRS5 etc.；Third class is mainly in fringe portion expression, such as GS3, GW2, GW5 (Huang et al. Genetic bases of rice grain shape: so many genes, so little known. Trands in plant science, 2013,18 (4): 218-226).Preceding two class is affected to vine growth and development, and utility value is affected in production, the Three classes are main to influence seed size and character since the growth and development influence on entire plant is small, are considered Breeding Application potentiality It is larger.

Seed properties are further decomposed, can be analyzed to that grain is long, grain is wide with thick 3 indexs of grain, wherein the thick hereditary shadow of grain Very little is rung, particle shape is mainly by grain length and the wide control of grain.Finding GS3, qGL3/gl3.1, TGW6, PGL1/2 after decomposition is regulation The major gene resistance of grain length, and GW2, GW5/qSW5, GW8, GS5, GS6, HGW are to regulate and control the wide major gene resistance of grain, it has been found that simultaneously Grain length and the wide major gene resistance of grain, such as SMG1, GL7/GW7 and GS2/GL2(Zhu Ye treasured are influenced, rice grain shape controlling gene Interaction and its Advance of Domesticating.Fujian Journal of Agricultural Sciench, 2016,31(1): 95-101).The existing transcription of these gene coded proteins The factor also has kinases, phosphatase, E3 ubiquitin ligase and other organized enzymes etc., and there is also complicated phases between these genes Mutual regulation relationship shows that the grain shape regulation of rice is related to multiple gene regulations, is typical complex character.Although in grain shape research On the research that achieves these progress, but think to grain shape there are still many unknown factors to wait understanding, while being conducive to strain The new gene that type improvement improves yield still needs to be excavated.

It further include grain number per spike and number of productive ear in the influence factor of rice single plant yield.Supernumerary Bract (SNB) gene discovery plays the part of important function (Lee et al, The rice heterochronic in Rice Panicle development gene SUPERNUMERARY BRACT regulates the transition from spikelet meristem to Floral meristem. The Plant Journal, 2006,49 (1): 64-78), control floral meristem foundation With inflorescence structure (Lee et al, Two AP2 family genes, SUPERNUMERARY BRACT (SNB) and OsINDETERMINATE SPIKELET 1 (OsIDS1), synergistically control inflorescence architecture and floral meristem establishment in rice. The Plant Journal, 2011,69 (3): 445-461).Meanwhile the regulatory mechanism of SNB is also found, which is the target gene of microRNA172, If binding site is mutated, plant height can significantly reduce, increase grain number per spike, which is applying for a patent a kind of (utilization rice SNB Gene is come the method for increasing grain number per spike and reducing plant height, and number of patent application: 201510786256.4) being described in detail, but such as SNB gene knockout or inhibition are often resulted in the bad character of flower development (Wang et al, Coordinated regulation by fruit of vegetative and reproductive branching in rice. Proc Natl Acad Sci U S A. 2015,112 (50): 15504-15509).We are by Rice Germplasm Resources the study found that SNB gene can also influence water Rice size, and by gene editing and transformation, increase or reduce seed size, can be used for the accurate breeding of rice grain, It lays the foundation for the high yield of rice.

Summary of the invention

The present invention is based on a part from riceOsSNBThe discovery of gene regulation rice grain development.The present invention Be designed to provide one kindSNBThe application of gene alteration rice paddy seed size.

For this purpose, the present invention provides one kind to pass through overexpressionSNBGene is come the method that reduces rice paddy seed size.Simultaneously originally Invention additionally provides a kind of by overexpression artificial mutationSNBGene increases the method for rice paddy seed size.

The present invention also provides one kind simultaneously makes generation shift to an earlier date termination codon by human-edited's rice genome to increase The method of rice paddy seed size.The present invention also includes using SNB base obtained from other method transformation 5 ' terminal sequences of code area The allele or derivative of cause.The present invention mentioned transformation SNB gene and forms mutation allele or be overexpressed artificial mutation base Cause dramatically increases rice paddy seed size, these are the result shows that SNB gene applies valence with important in rice yield breeding Value.By accurately improveing SNB gene, plant expression vector appropriate is constructed, can expand in current Plant Biotechnology can answer For changing the gene of rice yield, accurate breeding technique is provided to improve the Main Agronomic Characters of rice.

The nucleotide sequence of SNB gene provided by the invention is as shown in SEQ ID NO.1, the site SNB gene EAR It carries out artificial mutation and forms SNBm1 gene, the nucleotide sequence of the SNBm1 gene is as shown in SEQ ID NO.3.

The preferred embodiments of the invention are that the application of the SNBm1 gene of the SNB gene EAR site artificial mutation is It is that template carries out PCR amplification, and recycles PCR production using the gene coding region SNB cDNA with primer SNBm1F and SNBR combination Object, in which:

SNBm1F:ATGGTGATGGATATCAATGTGGAGTCGCC；

SNBR: TCAGGCGGTCGGGGGGAAGTAG。

The preferred embodiments of the invention are that SNBm2 full length gene code cDNA is that interception SNB gene 3 ' holds 1071bp long The mutated gene of degree, nucleotide sequence is as shown in SEQ ID NO.5.

The preferred embodiments of the invention are the applications of the SNBm2 gene for the artificial mutation that the interception SNB gene 3 ' is held It is with primer SNBm2F and SNBR combination, is that template carries out PCR amplification, and recycles PCR production using the gene coding region SNB cDNA Object, in which:

SNBm2F:ATGCCGATGCCGGCGGCGGCTGGG

SNBR: TCAGGCGGTCGGGGGGAAGTAG。

The preferred embodiments of the invention are, the SNB gene First Exon region the sequence 400-500bp insertion or Missing, the mutation terminate gene translation in advance, form the mutant knSNB gene of SNB gene, the knSNB gene First Exon nucleotide sequence is as shown in SEQ ID NO.7.

On the other hand, the riceSNBGene guide RNA target point sequence primer editor rice genome in application be WithOsSNBThe nucleotides sequence of gene induction RNA is classified as SNB-sgRNA:GCTCTTCCCTTCGCCGTCTGCGG.

On the other hand, the application includes that the method for adding connector PCR amplification constructs above-mentioned guidance RNA target point sequence The sgRNA expression cassette of column；

SgRNA expression cassette is loaded on CRISPR/Cas9 carrier, the CRISPR/Cas9-sgRNA containing target sequence is obtained and carries Body；

By target spot CRISPR/Cas9-sgRNA carrier rice transformation callus, rice is obtainedSNBGene mutation body knSNB plants Strain.

The invention discloses a kind of isolated DNA moleculars of polynucleotides containing code nucleic acid in prepare transgenosis rice Purposes in kind, separating rice OsSNB gene coding region cDNA, described in nucleotide sequence as shown in SEQ ID NO.2.

On the other hand, the invention discloses the methods of artificial reconstructed mutation SNBm1 gene, using above-mentioned cDNA as template, Obtain the SNBm1 gene of EAR site mutation by PCR amplification, described in nucleotide sequence as shown in SEQ ID NO.3.

On the other hand, the invention discloses the methods of artificial reconstructed mutation SNBm2 gene, using above-mentioned cDNA as template, 3 ' end 1071bp segments of interception OsSNB full length gene code cDNA, nucleotide sequence such as SEQ ID are obtained by PCR amplification Shown in NO.5.

On the other hand, the invention discloses SNB genes in a kind of rice genome obtained by way of gene editing The method of allele knSNB gene, it is characterised in that CRISP/CAS9 carrier of the building containing SNB-sgRNA expression cassette, benefit The rice of insertion or missing is generated in the SNB gene First Exon region sequence 400-500bp with rice transformation Mutant, First Exon nucleotide sequence is as shown in SEQ ID NO.7.

On the other hand, the invention discloses the plant expression that separated DNA sequence dna is connected to composition promoter driving Carrier, OsSNB gene, mutation SNBm1 gene and mutation SNBm2 gene can be overexpressed by being obtained using rice transformation method Transgenic Rice Plants.

The invention also discloses a kind of rice paddy seedsOsSNBGene, wherein describedOsSNBGene is used for adjusting and controlling rice The size of seed.

The invention also discloses one kindOsSNBThe protein of gene mutation body coding, wherein describedOsSNBGene mutation The controllable rice paddy seed of protein of body coding obtains size.

The present invention also provides a kind of methods of genetically modified plants for producing the change of rice paddy seed size, it is characterised in that:

Method includes the following steps:

1) above-mentioned SNB gene, mutated gene and its gene editing are operably connected to plant expression regulation sequence, are formed Plant expression vector；

2) the resulting plant expression vector of step 1) is transferred to plant cell；

3) transformed cells obtained through screening, are regenerated as plant and its offspring, the plant includes plant cell, plant tissue Or vegetable seeds.

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

1. SNB gene and SNB gene mutation body that the present invention clones, which have, increases or decreases rice paddy seed size.

2. the present invention will overexpress the protein-coding region Introduced into Rice of SNB mutated gene, it is big to realize increase rice paddy seed It is small.

3. the method that the present invention establishes four kinds using SNB gene to carry out genetic improvement to rice paddy seed size.

Detailed description of the invention

Fig. 1 OsSNB protein structure figure

Using BLAST software (http://blast.ncbi.nlm.nih.gov/) in NCBI to SEQ ID NO.2 protein sequence Analysis and correlation deliver document and draw OsSNB protein structure figure.ATG is translation initiation site, and TGA is terminator codon, The site EAR be amphiphilic repression (EAR) motif, nuclear location indicate nuclear localization signal site, miR172 Point indicates microRNA172 binding site.Arrow m1 indicates that SNBm1 gene mutation site, arrow m2 indicate that SNBm2 gene starts Translation initiation site, arrow kn indicate insertion and deletion mutational site in knSNB gene.

Fig. 2 SNBm1 gene mutation site figure

SNBm1 gene increases overstriking in SNB gene 5 ' end EAR site mutation, mutated nucleotides and its corresponding mutating acid Display.

The building schematic diagram of Fig. 3 expression vector pCBH04-OsSNB；

The building schematic diagram of Fig. 4 expression vector pCBH04-SNBm1；

The building schematic diagram of Fig. 5 expression vector pCBH04-SNBm2；

Fig. 6 CRISP/Cas9 plant conversion carrier constructs schematic diagram

Fig. 7 overexpresses transgenic plant relative expression quantity schematic diagram

OE indicates that overexpression strain, OryzasativaLcv.Nipponbare indicate non-transgenic receptor parent plant.Using quantifying PCR method 2^-△△ctIt calculates Relative expression quantity, reference are non-transgenic receptor parent OryzasativaLcv.Nipponbare.

Fig. 8 CRISP/Cas9 converts the sequence verification result of plant after knocking out

OsSNB indicates OryzasativaLcv.Nipponbare Partial Fragment sequence, and knSNB indicates to knock out strain Partial Fragment sequence.

Fig. 9 overexpression and the seed size statistics for knocking out transgenic plant

WT indicates parent's OryzasativaLcv.Nipponbare, and * * * indicates that t-test analyzes p < 0.001.

Figure 10 overexpresses the seed size statistics of SNBm1 and SNBm2 transgenic plant

WT indicates parent's OryzasativaLcv.Nipponbare, and * * indicates that t-test analyzes p < 0.01, and * indicates p < 0.05.

Specific embodiment

Herein, term " separation ", " purifying " DNA refer to, the DNA or segment are located at it under native state It is separated in the sequence of two sides, also refers to that the DNA or segment are separated with the component under native state with nucleic acid, Er Qieyi Through being separated in cell with its protein.

Polynucleotides (DNA or RNA), carrier, transformant and life can be separated and purified by methods known in the art Object.

It can be for carrier of the invention such as bacteriophage, plasmid, clay, minichromosome, virus or retrovirus Carrier.The carrier that can be used for cloning and/or expressing polynucleotides of the invention can need to replicate and/or express polynucleotides The carrier of polynucleotides is replicated and/or expressed in host cell.It is, in general, that carrying the recombinant expression of nucleic acid sequence of the invention Ti-plasmids, plant viral vector can be used in carrier, and the standard biologics technical method such as directly delivered DNA, microinjection, electroporation is led Enter plant cell (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

A variety of methods have been developed to be connected for operate polynucleotides can with carrier via complementary cohesive end.Example Such as, complementary homopolymer sequence fragment can be added in the DNA section in carrier DNA to be inserted into.Then by complementary with poly- Hydrogen bond connection carrier and DNA section between body tail is to form recombinant DNA molecule.

Synthetic linker containing one or more restriction sites provides the side of another connection DNA section and carrier Method.It is digested with bacteriophage T4 DNA polymerase or e. coli dna polymerase I processing by endonuclease restriction The DNA section of generation, its 3' of two kinds of polymerases, 5 '-exonucleolytic activities remove γ-single stranded end outstanding, And with its polymerization activity 3 '-female end of filling-in.Therefore, these it is active combine produce flush end DNA section, then can urge The enzyme for changing the connection of flush end DNA molecule, as in the presence of bacteriophage T4 DNA ligase by flush end section and molar excess Linkers are kept the temperature together.Therefore, reaction product is the DNA section that end carries polylinker sequence, then with appropriate Restriction enzyme cracks these DNA sections, and is connected in the expression vector for having used enzymatic lysis, the enzyme can generate with it is described The compatible end of DNA section.The synthetic linker containing multiple restriction endonuclease sites can be bought from multiple businessmans.

The technology of other new developments utilizes methods of homologous recombination, will carry particular sequence connector or homologous sequence connector Polynucleotides and carrier carry out homologous recombination, are intended to the DNA section being inserted into carrier DNA and equally carry particular sequence Or the carrier of homologous sequence forms recombinant DNA molecules by the effect of recombinase.

Polynucleotides insert should be operably connected to expression polynucleotides host cell it is compatible suitably open On mover, promoter can be strong promoter and/or inducible promoter.The example for some promoters enumerated includes bacteriophage PL promoter, Escherichia coli lac, trP, phoA, tac promoter, the early and late promoter of SV40 and reverse transcription disease Malicious LTR promoter；Other appropriate promoters are known to the skilled in the art.Expression recombinant vector further contains transcription Starting, termination site, and contain the ribosome bind site for translation in transcriptional domain.The volume of the transcript of recombinant vector expression Code part may include being located at the translation initiation codon of starting point and being suitably positioned at the termination codon for being translated the end of polypeptide Sub (UAA, UGA or UAG).

As described above, expression vector may include at least one selected marker.The label includes the resistance of coding antibiotic Gene, such as: neomycin phosphotransferase (Neomycin phosphotransferase) gene npt II, hygromycin phosphoric acid turn Move enzyme (Hygromycin phosphotransferase) gene hpt and dihyrofolate reductase (Dihydrofolate Reductase) gene dhfr；Another kind of is encoding herbicide resistance gene, for example, glufosinate transacetylase (Phosphinothricin acetyltransferase) Bar gene, 5- enolpyruvyl acyl oxalic acid -3- phosphate synthase (5- Enoylpyruvate shikimatr-3-phosphate) gene epsps.The representative example of appropriate host includes but unlimited In: protoplasm somatocyte and plant cell.The appropriate culture medium and condition of culture of above-mentioned host cell are known in the art.

The method for transformation of target gene or polynucleotide of interest: one kind is carrier mediated method for transformation, i.e., by purpose base Because being inserted on the carrier molecules such as the plasmid of Agrobacterium or the DNA of virus, target gene is led with the transfer of carrier DNA Enter into Plant Genome；Mediated by agriculture bacillus and virus-mediated methods just belong to this method.Second class is gene direct guiding method, Refer to and is directly imported external source target gene by method physically or chemically in the genome of plant.Physical method includes gene Rifle conversion method, Electroporation conversion, supercritical ultrasonics technology, microinjection and laser microbeam method etc.；Chemical method has the mediated transformation side PEG Method and liposome method etc..Third class is germplasm systems approach, this includes pollen tube passage method, reproduction cell dip method, blastular and son Room injection method etc..

In the present invention, the term " transformant " (transformant) used has the host cell of heterologous DNA molecule Or organism.

The invention also includes contain nucleotide sequence of the invention host cell, the nucleotide sequence through this field The technology known can be operated with one or more heterologous control regions (such as promoter and/or enhancer) to be connected.Can choose be adjustable it is slotting The expression of the gene order entered, or the host strain with processed gene product can be modified according to required particular form.Certain In the presence of inducer, the expression of certain promoter startings can be increased.

It can be identified by widely-known technique by the cell of successful conversion, that is, contain nucleotides sequence of the present invention The cell of the recombinant vector of column or organism.

Below in conjunction with specific embodiment, the present invention is furture elucidated.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.

Embodiment 1: separation cloneOsSNBGene

1.1 OsSNBGene cloning

Clip rice leaf, using TRIzol reagent (GIBCO BRL, USA) extracted total RNA.Utilize reverse transcriptase MLV (Tiangen, China) is by its reverse transcription at cDNA.With primer SNBRF (5 '-ATGGTGCTGGATCTCAATGTGG -3 ') and Primer SNBR(5 '-TCAGGCGGTCGGGGGGAAGTAG-3 '), amplify the overall length code cDNA of gene.PCR reaction condition Are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 60sec are recycled for 35 totally；72 DEG C of extension 5min.It will amplification The PCR product of acquisition is connected into pGEM-T carrier (Promega, USA), and screening positive clone is simultaneously sequenced, and is obtainedOsSNBGene CDNA sequence (SEQ ID NO. 1).OsSNBThe protein sequence of supposition carries out the comparison analysis of homologous gene, it is found that the gene is compiled Code albumen is the transcription factor (see figure 1) comprising 2 AP2 domain.Further analyze the gene nucleotide series and protein Sequence signature, the gene 3 ' end includes a microRNA172 binding site, separately includes one in protein N terminal and centre EAR motif (see figure 1).

1.2 OsSNBMutated gene clone

SNBm1 mutated gene clone, withOsSNBGene order is template, uses primer SNBM1F (5 '-respectively ATGGTGATGGATATCAATGTGGAGTCGCC-3 ') and SNBR(5 '-TCAGGCGGTCGGGGGGAAGTAG-3 '), amplification is prominent Become gene SNBm1, PCR condition is same as above described in 1.1.The mutated gene will be located at protein N terminal EAR motif in 2 amino acid into It has gone mutation, LDLN has been changed into MDIN, Fig. 2 is seen in specific mutational site.

SNBm2 mutated gene clone, withOsSNBGene order is template, uses primer SNBM2F (5 '-respectively ATGCCGATGCCGGCGGCGGCTGGG-3 ') and SNBR(5 '-TCAGGCGGTCGGGGGGAAGTAG-3 '), amplification mutation base Because of SNBm2, PCR condition is same as above described in 1.1.The mutated gene amputates 80 amino acid sequence of protein N terminal, and specific sequence is long Degree is shown in Fig. 1, i.e., terminates since m2 arrow locations to the position TGA.

Embodiment 2:OsSNBThe building of gene-correlation expression vector

The building of 2.1 overexpression vectors containing target gene:

According toOsSNBThe full length sequence (SEQ ID NO. 1) of gene designs and amplifies the complete primer for encoding reading frame, and Adapter-primer is added respectively in upstream primer and downstream primer, so as to construction of expression vector.It is produced with the amplification obtained in embodiment 1 Object is template, will after carrying out PCR amplification through high-fidelity Taq enzyme pfu enzyme (Tiangen, China)OsSNBGene cDNA clone To intermediate vector (such as pDONR207), bacillus coli DH 5 alpha is further converted, is identified under the premise of guaranteeing that reading frame is correct Then intermediate vector extracts plasmid, then use LR Clonase recombinase and include promoter and terminator protein plant table Recombining reaction is carried out up to carrier vector pCBH04, constitutes a complete expression unit (see figure 3), converts Agrobacterium EHA105 finally carries out Rice Callus transformation experiment.Its overexpression transgenic plant obtained is named as OsSNB.

With above-mentioned carrier construction method, SNBm1 and SNBm2 mutated gene is packed into plant expression vector pCBH04 respectively In, carrier schematic diagram is shown in Fig. 4 and Fig. 5, converts Agrobacterium EHA105, finally carries out Rice Callus transformation experiment.It is obtained The transgenic plant obtained is named as SNBm1 and SNBm2.

Plant conversion carrier building

2.2.1, the selection of guidance RNA target point sequence and design of primers

According to the genome sequence (LOC_Os07g13170) of control paddy gene OsSNB, the sgRNA of gene OsSNB is designed. The nucleotide sgRNA target sequence of 20nt is designed according to 5 '-N20-NGG-3 ' sequence, while designing target sequence primer GRT+ and OsU6aT-, 3 ' ends have 15-17 nt to match respectively with sgRNA and U6a promoter.Specific target spot nucleotide sequence It is as follows, see Fig. 1.

SNB-sgRNA: GCTCTTCCCTTCGCCGTCTGCGG

gRT+: 5’-GCTCTTCCCTTCGCCGTCTGgttttagagctagaaat-3’

OsU6aT-:5’-CAGACGGCGAAGGGAAGAGCggcagccaagccagca-3’

2.2.2, the building of target sequence sgRNA expression cassette

With reference to the method (Ma et al., 2015, Molecular Plant, 8 (8): 1274-1284) of Ma et al., 2-5 is taken Ng pYLgRNA-OsU6a/LacZ plasmid (1ul) is template, carries out PCR amplification respectively in two reaction systems: U-F and OsU6aT- is for expanding OsU6a- target spot segment, gR-R and gRT+ for expanding sgRNA- target spot segment.Generally use KOD Plus polymerase (TOYOBO), reaction system are 1ul plasmid template, and 2.5 uL 10 × Buffer, 0.5 uL KOD plus are poly- Synthase, the MgSO4 of 1ul 25mM, each 0.5 uL of the front and back primer of the dNTPs of 2.5 uL 2mM, 10 uM supplement dd H2O to 25 uL ；PCR amplification program are as follows: 95 DEG C of 2min, 98 DEG C of 10s, 58 DEG C of 15s, 68 DEG C of 20 s 25 circulation.Electrophoresis detection PCR product, OsU6a- target spot segment are 700bp or so, and gRNA- target spot segment is 131bp or so.Two PCR of the first round are taken to produce Each 1 μ l of object is template, carries out the second wheel PCR, 30 circulations by above step with primer U-GAL and Pgs-GAR.Primer size is 830bp or so.Detected through gel electrophoresis, gel extraction, this product are gene OsSNB target sequence-sgRNA expression cassette.

The primer of the Gibson assembly are as follows:

U-F: 5’- ctccgttttacctgtggaatcg -3’

gR-R: 5’- cggaggaaaattccatccac -3’

U-GAL: 5’-accggtaaggcgcgccgtagtgctcgactagtatggaatcggcagcaaagg-3’

Pgs-GAR: 5’-tagctcgagaggcgcgccaatgataccgacgcgtatccatccactccaagctcttg-3’

2.2.3, target spot CRISPR/Cas9-sgRNA vector construction

Target sequence sgRNA expression cassette is assembled to pYLCRISPR/Cas9Pubi-H carrier, only praising biotechnology with Nanjing promise has For the ClonExpress quick clone recombinase of limit company: 4 μ l 5 × CE II Buffer, 200 ng linearisations PYLCRISPR/Cas9Pubi-H plasmid, PCR product 200 ng, 2 μ l of the-sgRNA of target sequence containing OsSNB recycled above ExnaseTM II, finally plus water is to 20 μ l, 37 DEG C of warm bath 30min, 5 μ l reaction mixtures is converted Escherichia coli, coating contains The LB plate screening positive colony of kanamycins, next day picking positive monoclonal carry out sequence verification and extract plasmid preservation.Matter Carrier schematic diagram shown in grain is shown in Fig. 6, converts Agrobacterium EHA105, finally carries out Rice Callus transformation experiment.It is obtained Transgenic plant be named as knSNB.

Embodiment 3: rice transformation

3.1 seed disinfection

It is put into sterile triangular flask after mature OryzasativaLcv.Nipponbare rice paddy seed decladding, impregnates 1-2 min, sterile water punching with 75% alcohol It washes 2 times；30 min are sterilized with 30% NaClO again, need often to shake therebetween, then are washed 3-4 times with sterile, are blotted with aseptic filter paper Seed is inoculated on callus inducing medium (2.0 mg/L of MS+2,4-D), about 30, every ware by extra moisture, In 28 DEG C of dark cultures.

Squamous subculture

By the induction in nearly January, rice grows the callus that yellow is expanded, and removes its scultellum, and callus is gone to fresh callus Subculture is carried out on tissue induced medium (2.0 mg/L of MS+2,4-D).Subculture is primary every 2 weeks, can be obtained for subculture 2-4 times The bright yellow of suitable transgenosis, granular embryo callus.After squamous subculture 2 weeks, selects embryo particle and be used for heredity Conversion.

The culture of Agrobacterium

Picking single colonie is cultivated in 1ml Agrobacterium culture medium on conversion plate.In 50ml Agrobacterium culture medium (containing corresponding anti- Raw element) in the above-mentioned culture of 1ml, 200rpm is added, 28 DEG C of culture 5-6hr to OD600 are 0.6-1.0, and culture terminates preceding 2hr and adds Enter acetosyringone (AS, final concentration 100uM).Take above-mentioned bacterium solution at room temperature, 4000rpm, 10min abandon supernatant, and MS liquid is added Thallus is resuspended in body culture medium (100uM containing AS), is cultivating 2hr under the same conditions with upper, is making OD600=0.5-1 of bacterium solution, this When can be used to transformed calli.AS=acetosringone.

It co-cultures

Rice embryo callus is immersed into Agrobacterium bacterium solution 20-30min, then with sterile blotting paper suck dry moisture, by what is infected Callus is placed on co-culture medium (2.0 mg/L+AS of MS+2,4-D, 100 uM), 28 DEG C dark culture three days.

Wash bacterium

The callus of co-cultivation is first used aseptic water washing 3 times, then is immersed in the MS fluid nutrient medium of 400 mg/L containing Cef/CN After middle 20-30min, callus is transferred on aseptic filter paper and is blotted.

Selection culture

By the callus of suck dry moisture be inoculated in Selective agar medium (2.0 mg/L+Hyg of MS+2,4-D, 30 mg/L+ 400 mg/L of Cef) on.After 3 weeks, select the callus newly grown be inoculated in Selective agar medium (2.0 mg/L of MS+2,4-D+ 50 mg/L+Cef of Hyg, 250 mg/L) on, reselection 2 weeks.

Differentiation culture

Obtained resistant calli will be selected to be transferred to pre- differential medium (2.0 mg/L+NAA of N6+KT by 2 times 0.2 mg/L+6-BA, 2.0 mg/L+Hyg, 30 200 mg/L of mg/L+Cef+agar 9g/L+sucrose 45g/L) Upper dark culture 10 days or so, return again to differential medium (2.0 mg/L+NAA of N6+KT, 0.2 mg/L+6-BA 2.0 30 mg/L of mg/L+Hyg+agar 4.5g/L+30 g/L of sucrose) on illumination cultivation.

Culture of rootage

About 1-2 months, the high seedling of 2cm or so is gone into root media (15 mg/L of 1/2MS+Hyg+agar 4.5g/ L+sucrose 20g/L) on inducing adventitious root generation.

The transplanting of transgenic seedling

When seedling it is long to 10cm high when, seedling is taken out, the solid medium of attachment is cleaned with sterile water, is moved into soil, just Start to be removed cloche again after robust plant with cloche cover several days, and cultivated in greenhouse.

Embodiment 4:OsSNBExpression analysis of the gene in transgenic plant

4.1 materials prepare

After transgenosis T1 is for rice seed germination, transplant in fluid nutrient medium (tap water is configured to 1/5MS a great number of elements).Seedling After growing 15 d, clip blade quickly puts into Liquid nitrogen storage, the extracting for RNA.

It is prepared by the total serum IgE of no DNA

The a small amount of extraction agent box operation instructions extractings of leaves of plants RNA provided by Shanghai Quan Shijin Bioisystech Co., Ltd. RNA concentration is measured using 640 ultraviolet specrophotometer of Beckman Coulter DU.To remove the DNA remained in RNA, Each total serum IgE sample takes 5 μ g, and 1 μ L DNAase I (Invitrogen company, the U.S.) and 1 μ L10 × reaction buffering is added Liquid supplies volume to 10 μ L, 30 min of normal-temperature reaction, and then every pipe is added 1 μ L, 2 mmol L-1 EDTA and terminates reaction, most Inactivate DNAase I in 70 DEG C of 10 min of heating afterwards.

The synthesis of first chain cDNA

Above-mentioned RNA sample is respectively taken into 2 μ L, successively adds 4 μ L by the reagent that U.S.'s Promega company reverse transcription reagent box provides 25 mmol L-1 MgCl2,2 μ L10 × RT buffers, 2 μ L dNTP mixed liquors and 1 μ L oligo (dT) 15, add water to mend Sufficient volume is quickly cooled down to 18.5 μ L in 70 DEG C of 10 min of heat denatured on ice.Plus 0.5 μ L RNase then Inhibitor and 1 μ L AMVRTase heats 10 min at 42 DEG C of water-bath 60 min, 70 DEG C and terminates reaction.

Quantitative PCR

According to geneOsSNBSequence design specific primer QF:5 '-ctttgtgaacctcagggaagtacc -3 ', QR: 5 '-tcaggcggtcggggggaagtag -3 ' are used for quantitative fluorescent PCR, according to riceActinGene (GenBank Accession No. AY212324) cDNA sequence design specific primer AF:5 '-cttcctcatgccatcctgc-3 ', AR:5 '-gcaagcttctccttgatgtcc-3 ' is used for the quantitative fluorescent PCR of reference gene.PCR uses American AB I PRISM 7000 quantitative PCR apparatus, each PCR setting is primary to be repeated.Reaction system includes SYBR Premix Ex Taq (2 ×) 10 μ L, forward and reverse each 0.5 μ L of primer, the 1 μ L of cDNA template of various processing add water to supply volume to 25 μ L.Response procedures Are as follows: then 95 DEG C of 30 s is recycled 40 times at 95 DEG C of 10 s, 61 DEG C of 34 s, read when setting 60 DEG C of 34 s in each cycle Fluorescent value, while ROX value correction is carried out, the analysis of fluorescence PCR products melt curve analysis is finally added, other operations are detailed in instrument use Specification.In order to detect the pollution that whether there is DNA in RNA sample, 3 samples are randomly selected, respectively take 1 μ L RNA as mould Plate carries out PCR, and method is same as above.

Analysis method

Ct is that the fluorescence thresholding by 7000 system SDS Version1.2.3 softwares in PCR is determined as 0.2 by manual It generates afterwards, enters data into EXCEL and carry out calculating analysis.Data analysis uses method for 2^-ΔΔCT, then utilize EXCEL Table makees differential expression histogram.

Analyze result

Using blank non-transgenic OryzasativaLcv.Nipponbare kind as reference, 6 independent transgenic line T1-T6 are had detected respectively, and discovery turns Gene strain has stronger Enhanced expressing, and Enhanced expressing multiple illustrates the channel genes in 15-45 times or more (see figure 7) It is significantly overexpressed in blade after to rice, can be applied to further transgenic paddy rice research.

The identification of embodiment 5:knSNB plant

By the transgenic plant transplanting obtained of CRISPR/Cas9-sgRNA carrier rice transformation, 20mg blade is sheared, according to fast The illustration method that prompt type plant genome DNA extraction system (Tiangen, China) provides extracts leaf DNA, takes 1-2ul DNA is template, CasF:5 '-gggagagcggttggttagta-3 ', CasR:5 '-agaaataggagctcacccagg-3 ', After carrying out PCR amplification through Taq enzyme (Tiangen, China), recovery purifying PCR fragment submits company's sequencing, it is homozygous to find site The single plant of mutation.It is analyzed by sequence, discovery existsOsSNBThe topagnosis sequence in gene translation password initiation site downstream 178 Tcgccgtctgcgg (see figure 8), the sequence deletion cause protein translation to terminate in advance, make the gene lacks functionality, form function Deletion mutation plant knSNB can be knocked out.

Embodiment 6:OsSNBGene overexpression transgenosis, mutant plant and mutated gene transgenic plant seed size point Analysis

Respectively by transgenosis T1 obtained for plant OsSNB, knSNB, SNBm1 and SNBm2 in field planting, arrive plant strain growth Transgenosis T2 is harvested when mature for plant seed, and seed size is observed and measured.

Wild rice, overexpression SNB and knockout knSNB seed are put together and are taken pictures and counted, it has been found that Rice paddy seed is set obviously to become smaller after overexpression SNB, grain length and grain is wide all obviously shortens, and knock out knSNB seed in obviously becoming larger, Especially significant changes (see figure 9) has occurred in grain length.

Wild rice, overexpression SNB and knockout knSNB seed are put together and are taken pictures and counted, it has been found that Overexpression SNBm1 makes seed become larger, and main contributions are that grain length is elongated, but overexpressing SNBm2 makes seed become smaller (see figure 10).

In conclusion overexpression wild type SNB gene and saltant type SNBm2 gene, make rice paddy seed become smaller, and knock out KnSNB and overexpression saltant type SNBm1 gene, make rice paddy seed become larger.

The scope of the present invention is not limited by the specific embodiments described, and the embodiment is only used as illustrating of the invention each The single example of a aspect further includes function equivalent method and component in the scope of the invention.In fact, in addition to as described herein Outside content, those skilled in the art can easily be grasped referring to description above and attached drawing to a variety of improvement of the invention.Institute Improvement is stated to also fall within the scope of the appended claims.Every bibliography mentioned above is all included in conduct herein in full With reference to.

Sequence table

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Pro Ser Ser Arg Pro His Pro Phe Pro Gly His His Gln Phe Tyr Phe

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Pro Pro Thr Ala

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Ala Arg Gly Ser Ser Lys Tyr Arg Gly Val Thr Leu His Lys Cys Gly

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Arg Trp Glu Ala Arg Met Gly Gln Leu Leu Gly Lys Lys Tyr Ile Tyr

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Leu Gly Leu Phe Asp Ser Glu Ile Glu Ala Ala Arg Ala Tyr Asp Arg

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Ala Ala Ile Arg Phe Asn Gly Arg Glu Ala Val Thr Asn Phe Asp Pro

260 265 270

Ser Ser Tyr Asp Gly Asp Val Leu Pro Glu Thr Asp Asn Glu Val Val

275 280 285

Asp Gly Asp Ile Ile Asp Leu Asn Leu Arg Ile Ser Gln Pro Asn Val

290 295 300

His Glu Leu Lys Ser Asp Gly Thr Leu Thr Gly Phe Gln Leu Asn Cys

305 310 315 320

Asp Ser Pro Glu Ala Ser Ser Ser Val Val Thr Gln Pro Ile Ser Pro

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Gln Trp Pro Val Leu Pro Gln Gly Thr Ser Met Ser Gln His Pro His

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Leu Tyr Ala Ser Pro Cys Pro Gly Phe Phe Val Asn Leu Arg Glu Val

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Pro Met Glu Lys Arg Pro Glu Leu Gly Pro Gln Ser Phe Pro Thr Ser

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Trp Ser Trp Gln Met Gln Gly Ser Pro Leu Pro Leu Leu Pro Thr Ala

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Ala Ser Ser Gly Phe Ser Thr Gly Thr Val Ala Asp Ala Ala Arg Ser

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Pro Ser Ser Arg Pro His Pro Phe Pro Gly His His Gln Phe Tyr Phe

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Pro Pro Thr Ala

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Gly Phe Ala Arg Gly Ser Ser Lys Tyr Arg Gly Val Thr Leu His Lys

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Ile Tyr Leu Gly Leu Phe Asp Ser Glu Ile Glu Ala Ala Arg Ala Tyr

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Asp Arg Ala Ala Ile Arg Phe Asn Gly Arg Glu Ala Val Thr Asn Phe

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gat cct agt tct tat gat gga gat gtt cta cct gaa acc gac aat gaa 624

Asp Pro Ser Ser Tyr Asp Gly Asp Val Leu Pro Glu Thr Asp Asn Glu

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aac gtt cat gag ctg aaa agt gat ggt acc cta act ggg ttc cag ttg 720

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Pro His Leu Tyr Ala Ser Pro Cys Pro Gly Phe Phe Val Asn Leu Arg

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Glu Val Pro Met Glu Lys Arg Pro Glu Leu Gly Pro Gln Ser Phe Pro

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Thr Ser Trp Ser Trp Gln Met Gln Gly Ser Pro Leu Pro Leu Leu Pro

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Thr Ala Ala Ser Ser Gly Phe Ser Thr Gly Thr Val Ala Asp Ala Ala

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Arg Ser Pro Ser Ser Arg Pro His Pro Phe Pro Gly His His Gln Phe

340 345 350

tac ttc ccc ccg acc gcc tga 1077

Tyr Phe Pro Pro Thr Ala

355

<210> 6

<211> 358

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Met Pro Met Pro Ala Ala Ala Gly Glu Gly Pro Trp Pro Arg Arg Ala

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Ala Asp Leu Gly Val Ala Gln Ser Gln Arg Ser Pro Ala Gly Gly Lys

20 25 30

Lys Ser Arg Arg Gly Pro Arg Ser Arg Ser Ser Gln Tyr Arg Gly Val

35 40 45

Thr Phe Tyr Arg Arg Thr Gly Arg Trp Glu Ser His Ile Trp Asp Cys

50 55 60

Gly Lys Gln Val Tyr Leu Gly Gly Phe Asp Thr Ala His Ala Ala Ala

65 70 75 80

Arg Ala Tyr Asp Arg Ala Ala Ile Lys Phe Arg Gly Leu Asp Ala Asp

85 90 95

Ile Asn Phe Asn Leu Asn Asp Tyr Glu Asp Asp Leu Lys Gln Met Arg

100 105 110

Asn Trp Thr Lys Glu Glu Phe Val His Ile Leu Arg Arg Gln Ser Thr

115 120 125

Gly Phe Ala Arg Gly Ser Ser Lys Tyr Arg Gly Val Thr Leu His Lys

130 135 140

Cys Gly Arg Trp Glu Ala Arg Met Gly Gln Leu Leu Gly Lys Lys Tyr

145 150 155 160

Ile Tyr Leu Gly Leu Phe Asp Ser Glu Ile Glu Ala Ala Arg Ala Tyr

165 170 175

Asp Arg Ala Ala Ile Arg Phe Asn Gly Arg Glu Ala Val Thr Asn Phe

180 185 190

Asp Pro Ser Ser Tyr Asp Gly Asp Val Leu Pro Glu Thr Asp Asn Glu

195 200 205

Val Val Asp Gly Asp Ile Ile Asp Leu Asn Leu Arg Ile Ser Gln Pro

210 215 220

Asn Val His Glu Leu Lys Ser Asp Gly Thr Leu Thr Gly Phe Gln Leu

225 230 235 240

Asn Cys Asp Ser Pro Glu Ala Ser Ser Ser Val Val Thr Gln Pro Ile

245 250 255

Ser Pro Gln Trp Pro Val Leu Pro Gln Gly Thr Ser Met Ser Gln His

260 265 270

Pro His Leu Tyr Ala Ser Pro Cys Pro Gly Phe Phe Val Asn Leu Arg

275 280 285

Glu Val Pro Met Glu Lys Arg Pro Glu Leu Gly Pro Gln Ser Phe Pro

290 295 300

Thr Ser Trp Ser Trp Gln Met Gln Gly Ser Pro Leu Pro Leu Leu Pro

305 310 315 320

Thr Ala Ala Ser Ser Gly Phe Ser Thr Gly Thr Val Ala Asp Ala Ala

325 330 335

Arg Ser Pro Ser Ser Arg Pro His Pro Phe Pro Gly His His Gln Phe

340 345 350

Tyr Phe Pro Pro Thr Ala

355

<210> 7

<211> 406

<212> DNA

<213> Oryza sativa

<220>

<221> mutation

<222> (1)..(406)

<400> 7

atggtgctgg atctcaatgt ggagtcgccg ggtgggtcgg cggcgacgtc gagctcgtcc 60

acgccgccgc cgccgcccga cggtggcggc ggggggtact tccggttcga cctgctcggc 120

gggagccccg acgaggacgg gtgctcctcg cctgtcatga cgcgccagct cttcccttgg 180

tggcgctggc gggggacggg tcgtcgacgc caccgctgac gatgccgatg ccggcggcgg 240

ctggggaggg gccgtggccg cgccgcgcgg cggatctcgg ggtggcgcag agccagaggt 300

cccccgccgg cgggaagaag agccgccgcg gcccgaggtc tcggagctcc cagtacaggg 360

gcgtcacctt ctacaggagg accgggcgat gggagtcgca catctg 406

Claims (12)

1. a kind of application of the SNB gene of rice paddy seed, it is characterised in that: the nucleotide sequence of the SNB gene such as SEQ ID Shown in NO.1.

2. SNB gene according to claim 1, which is characterized in that the site gene EAR SNB carries out artificial mutation SNBm1 gene is formed, the nucleotide sequence of the SNBm1 gene is as shown in SEQ ID NO.3.

3. SNB gene according to claim 2, which is characterized in that the SNBm1 of the SNB gene EAR site artificial mutation The application of gene is with primer SNBm1F and SNBR combination, is template progress PCR amplification using the gene coding region SNB cDNA, and Recycle the PCR product, in which:

SNBm1F:ATGGTGATGGATATCAATGTGGAGTCGCC；

SNBR: TCAGGCGGTCGGGGGGAAGTAG。

4. SNB gene according to claim 1, which is characterized in that SNBm2 full length gene code cDNA is interception SNB base The mutated gene for holding 1071bp length because of 3 ', nucleotide sequence is as shown in SEQ ID NO.5.

5. SNB gene according to claim 4, which is characterized in that the artificial mutation that the interception SNB gene 3 ' is held The application of SNBm2 gene is with primer SNBm2F and SNBR combination, is that template carries out PCR expansion using the gene coding region SNB cDNA Increase, and recycle the PCR product, in which:

SNBm2F:ATGCCGATGCCGGCGGCGGCTGGG

SNBR: TCAGGCGGTCGGGGGGAAGTAG。

6. SNB gene according to claim 1, which is characterized in that the SNB gene First Exon sequence 400- The insertion of the region 500bp or missing, the mutation terminate gene translation in advance, form the mutant knSNB gene of SNB gene, institute The First Exon nucleotide sequence for the knSNB gene stated is as shown in SEQ ID NO.7.

7. the application of SNB gene according to claim 5, which is characterized in that the riceSNBGene guides RNA target point Aligning primer editor rice genome in application be withOsSNBThe nucleotides sequence of gene induction RNA is classified as SNB-sgRNA: GCTCTTCCCTTCGCCGTCTGCGG。

8. the application of SNB gene according to claim 5, which is characterized in that the application includes addition connector PCR The method of amplification constructs the sgRNA expression cassette of guidance RNA target point sequence as claimed in claim 4；

SgRNA expression cassette is loaded on CRISPR/Cas9 carrier, the CRISPR/Cas9-sgRNA containing target sequence is obtained and carries Body；

By target spot CRISPR/Cas9-sgRNA carrier rice transformation callus, rice is obtainedSNBGene mutation body knSNB plants Strain.

9. a kind of application comprising any SNB gene of claim 1-8, which is characterized in that the application can be used for adjusting The size of rice paddy seed.

10. a kind of rice paddy seedOsSNBGene, which is characterized in that describedOsSNBGene is big for adjusting and controlling rice seed It is small.

11. a kind of described in any one of claim 10OsSNBThe protein of gene mutation body coding, which is characterized in that describedOsSNBBase Because the controllable rice paddy seed of the protein of mutant code obtains size.

12. a kind of method for the genetically modified plants for producing the change of rice paddy seed size, it is characterised in that:

Method includes the following steps:

1) SNB gene, mutated gene and its gene editing any in claim 1,2,4 and 6 are operably connected In plant expression regulation sequence, plant expression vector is formed；

2) the resulting plant expression vector of step 1) is transferred to plant cell；

3) transformed cells obtained through screening, are regenerated as plant and its offspring, the plant includes plant cell, plant tissue Or vegetable seeds.