CN108034656A - SgRNA, CRISPR/Cas9 carrier related with rice bronzing glume character, vector construction, application - Google Patents

Abstract

The present invention discloses sgRNA, CRISPRCas9 carrier related with rice bronzing glume character, vector construction, application, overcomes needs to be all mutated the defects of just obtaining brown shell rice in two allele of OsCHI.The present invention：SgRNA fragments are provided, include the gRNA of selectively targeted the 5th extron of rice cinnamyl-alcohol dehydrogenase CAD genes.The sgRNA is specifically T1, T2, T3.The CRISPR/Cas9 gene editings carrier and its construction method for including above-mentioned sgRNA fragments, the carrier editable rice cinnamyl-alcohol dehydrogenase CAD genes are provided.Application of these fragments with carrier in obtaining with bronzing glume and stem knot character rice material is provided.The primer sequence for being used for detecting CRISPR/Cas9 CAD genetic recombination is provided.The present invention can reduce traditional breeding method cost, improve breeding of hybrid rice Mechanization Level, shorten breeding cycle.

Description

SgRNA, CRISPR/Cas9 carrier related with rice bronzing glume character, carrierStructure, application

Technical field

The present invention relates to a kind of sgRNA fragment related with bronzing glume character rice, CRISPR/Cas9 to edit loadBody, carrier construction method, and application.Belong to genetic engineering field.

Background technology

The breeding material of a small amount of different glume colors can be obtained using natural mutation and artificial mutation, in Rice Germplasm ResourcesMaterial.By glume different colours can in the rice mechanization production of hybrid seeds by the hybrid tied on sterile line with together being received with harvesterThe restorer seed obtained separates exactly, thus different glume colors have very high technological value in breeding of hybrid rice.But if do not changed using the method for traditional conventional hybridization, backcrossing by shell color character transformation to restorer, maintainer and originalThe characteristic of material, it is necessary to the time of last decade could realize.

Authorization Notice No. discloses a kind of orientation editor glume color for the Chinese invention patent of 104017821 B of CN and determinesDetermine the method that gene OsCHI formulates brown shell rice material.This method main contents are included in glume color and determine outside gene OsCHIAobvious sub-district chooses target fragments and builds plant CRISPR/Cas9 target practice recombinant vectors, by the recombinant vector Introduced into Rice cellAnd regenerate seedling, rice cell is sheared under the action of expression cassette in the carrier, triggering rice cell DNA selfRepair function.Again by the sequencing to regenerating strain genome target fragment, obtain two equipotential OsCHI genes of carrying and send out at the same timeThe strain of raw afunction mutation, is the brown shell rice material formulated by glume color identification.This method shortcomingIt is：Need all to be mutated in two allele of OsCHI and could produce brown shell rice, while to effect that two genes are mutatedRate is relatively low, it is difficult to obtains the Mutants homozygous of two gene mutations.

The content of the invention

The purpose of the present invention is aiming at the deficiencies in the prior art, there is provided one kind can quickly obtain bronzing glume characterThe technical solution of rice material.

To achieve the above object, present invention firstly provides a kind of sgRNA fragments, the sgRNA fragments can be directed to same baseBecause as follows into edlin, its technical solution：

A kind of sgRNA fragments, it is characterised in that：Include the 5th of selectively targeted rice cinnamyl-alcohol dehydrogenase CAD genesThe gRNA of a extron.

The gRNA that above-mentioned sgRNA fragments include is selectively targeted in rice cinnamyl-alcohol dehydrogenase CAD genes (OryzaSativa (indica cultivar-group) cinnamyl-alcohol dehydrogenase (CAD) gene, GenBank:DQ234272.1 the 5th extron), base sequence such as SEQ ID NO.6.

Above-mentioned sgRNA fragments, including three sgRNA fragments, are respectively：It is named as the sgRNA fragments of T1, base sequenceSuch as SEQ ID NO.1；It is named as the sgRNA fragments of T2, base sequence such as SEQ ID NO.2；The sgRNA fragments of T3 are named as,Base sequence such as SEQ ID NO.3.

Above three sgRNA fragments are selection rice cinnamyl-alcohol dehydrogenase CAD genes (Oryza sativa (japonicaCultivar-group) cinnamyl alcohol dehydrogenase (CAD) gene) on three sgRNA targeting positionPoint, with reference to http://cbi.hzau.edu.cn/cgi-bin/CRISPR target position point prediction and design, use http://Www.rgenome.net/cas-offinder/ network address analyzes undershooting-effect, and final choice determines.

Above-mentioned sgRNA fragments can be used for preparing rice cinnamyl-alcohol dehydrogenase gene C RISPR/Cas9 gene editing carriers.The present invention further provides technical solution：Above-mentioned sgRNA is preparing rice cinnamyl-alcohol dehydrogenase gene C RISPR/Cas9 genes volumeCollect the application in carrier.

Present invention simultaneously provides the CRISPR/Cas9 gene editing carriers comprising above-mentioned sgRNA fragments.

Above-mentioned CRISPR/Cas9 gene editings carrier is nuclease-mediated rice cinnamyl-alcohol dehydrogenase (CAD) bases of Cas9Because editing carrier, including three expression expressed respectively by OsU6a, OsU6b, OsU6c promoter regulation T1, T2, T3sgRNAFrame, the expression cassette expressed by maize ubiquitin Ubiquitin (Ubi) promoter regulations Cas9.

Present invention simultaneously provides the construction method for including above-mentioned CRISPR/Cas9 gene editings carrier, its technical solution is such asUnder：

Above-mentioned CRISPR/Cas9 gene editings carrier construction method, it is characterised in that：By respectively connect promoter T1,T2, T3 are connected into pYLCRISPR/Cas 9P by Bsa I enzymes_ubi- H plasmids, structure obtain Pubi::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 expression cassettes, obtain pYLCRISPR/Pubi::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 edits the final carrier of gene.

Present invention simultaneously provides above-mentioned CRISPR/Cas9 gene editings carrier to be specifically directed to rice cinnamyl-alcohol dehydrogenase baseBecause carrying out the application of gene editing.

Further, the application process of above-mentioned CRISPR/Cas9 gene editings carrier, is that the carrier that will be built is transferred to agricultureBacillus EHA105 is used for agriculture bacillus mediated rice Nipponbare callus genetic transformation, and induction of callus is obtainedRegeneration plant.

Further, gained regeneration plant can obtain the bronzing glume character without transgenic fragment through selfing screeningRice strain/material；Also improvement restorer can be obtained through backcross transformation.

SgRNA fragments of the present invention, CRISPR/Cas9 gene editings carrier prepare bronzing glume character rice strain/Application in material；Rice strain/material of the bronzing glume character contains or does not contain SgRNA:Cas9:HPT turnsGenetic fragment.The application process is particular by structure SgRNA:Cas9:HPT fragments are applied to rice cinnamyl-alcohol dehydrogenase baseBecause being mutated rice strain.

To detect CRISPR/Cas9-CAD genetic recombination, the present invention also provides corresponding primer sequence, its technical solution is such asUnder：

Primer for detecting CRISPR/Cas9-CAD genetic recombination combines 1F, 1R, it is characterised in that 1F, 1R base sequenceRow are respectively as shown in SEQ ID NO.4, SEQ ID NO.5.

Compared with prior art, the beneficial effects of the invention are as follows：(1) the present invention provides a kind of sgRNA fragments, it is wrappedSelectively targeted the 5th extron (SEQ IDNO.6) in rice cinnamyl-alcohol dehydrogenase CAD genes of gRNA contained.The sgRNAFragment specifically includes T1 (the 156th~176 of SEQ IDNO.6), T2 (SEQ IDNO.6 the 226th~246), T3 (SEQIDNO.6 the 516th~536) three sgRNA fragments.(2) the CRISPR/Cas9 gene editings of sgRNA fragments of the present invention are includedCarrier can carry out gene editing to rice cinnamyl-alcohol dehydrogenase (CAD) gene (SEQ IDNO.7).(3) sgRNA fragments of the present inventionIt is applied to obtain the rice material with bronzing glume and stem knot character with CRISPR/Cas9 gene editings carrier.(4) originallyThe primer that invention provides for detecting CRISPR/Cas9-CAD genetic recombination combines.(5) the method for the present invention only needs induction oneThe mutation of a allele just can obtain bronzing glume and stem knot character rice material, without obtaining Mutants homozygous, methodIt is efficient.(6) mutant that the present invention obtains can be by hybridizing and being selfed separation SgRNA:Cas9:HPT transgenic fragments, a sideFace is to remove the transgenic fragment so as to obtain the gene editing mutant of no transgene component；On the other hand can be by SgRNA:Cas9:HPT transgenic fragments are imported in new rice sterile line or restorer, can continue to carry out CAD genes to new acceptorEditor, so as to obtain the new sterile line with bronzing glume, restorer.The present invention is applied to agricultural production, can save biographyThe manpower and time cost of system breeding, improve the Mechanization Level of breeding of hybrid rice, shorten breeding cycle.

Brief description of the drawings

Fig. 1 is gene editing vector construction figure.

Fig. 2 transgenic lines are identified.

Fig. 3 gene editings site citing (T2 and T3 are two sgRNA editing sites of design respectively, with Fig. 1 a).

Fig. 4 is phenotype after CAD gene editings (WT is Nipponbare, and cad is the mutant after CAD genes are undergone mutation).

Fig. 5 is rice paddy seed phenotype (WT is Nipponbare, and cad is the mutant after CAD gene mutations).

Fig. 6 is that the selfing of Crispr/Cas9 gene editings strain removes transgene component analysis chart.

Fig. 7 is backcross transformation improvement restorer analysis chart.

Fig. 8 is rice mechanization production of hybrid seeds brown glume screening technique line map.

Embodiment

Below in conjunction with the accompanying drawings, the preferred embodiment of the present invention is further described.

Embodiment one

As shown in Figure 1a, sgRNA fragments design of the present invention.

Genetic material：Rice cinnamyl-alcohol dehydrogenase (CAD) gene, the gene entitled Oryza sativa (japonica entirelyCultivar-group) cinnamyl alcohol dehydrogenase (CAD) gene, GenBank:BK003969.1, is shown inhttp://www.ncbi.nlm.nih.gov/nuccore/64519465.Gene coding region base sequence such as SEQ ID NO.7.

Three sgRNA target sites on rice cinnamyl-alcohol dehydrogenase (CAD) gene are selected, with reference to http://Cbi.hzau.edu.cn/cgi-bin/CRISPR target position point prediction and design, use http://www.rgenome.net/cas-Offinder/ network address analyzes undershooting-effect.It finally have selected three sgRNA and be respectively designated as T1, T2, T3 (Fig. 1 a).T1、T2、The base sequence of T3 is respectively as shown in SEQ ID NO.1,2,3.

The 5th of the selectively targeted rice cinnamyl-alcohol dehydrogenase CAD genes of the gRNA that three sgRNA fragments includeExtron (SEQ IDNO.6).T1 is the 156th~176 of SEQ IDNO.6, T2 be SEQ IDNO.6 the 226th~246,T3 is SEQ IDNO.6 the 516th~536.

Embodiment two

As shown in Figure 1, structure includes the CRISPR/Cas9 gene editing carriers of sgRNA fragments of the present invention.

Three sgRNA fragments of the gained of embodiment one are connected into rice Os U6a (1~485 in SEQ ID NO.8 respectivelyPosition), OsU6b (1~371 in SEQ ID NO.13), under three promoters of OsU6c (1~780 in SEQ ID NO.18)Trip, behind respectively immediately following Ploy-T terminators (505~628 in SEQ ID NO.8,391~515 in SEQ ID NO.13800~923 in position, SEQ ID NO.18).Concrete operations are by taking T1 as an example, first respectively with the primer SEQ ID containing T1 sequencesThe primer and SEQ ID NO.11 and SEQ IDNO.12 of NO.10 and SEQ IDNO.9 carries out first round PCR, obtains two PCRProduct, then again using the two PCR products as template, SEQ IDNO.9 and SEQ IDNO.12 carry out the second wheel PCR for primer,Obtain OsU6a::T1(SEQ ID NO.8).Similarly OsU6b can be obtained by two-wheeled PCR::T2(SEQ ID NO.13)、OsU6c::T3(SEQ ID NO.18).Expression cassette OsU6a is obtained after sequencing is correct::T1(SEQ ID NO.8)、OsU6b::T2(SEQ ID NO.13)、OsU6c::T3 (SEQ ID NO.18).There are the Bsa I that end is matched two-by-two at three expression cassette both endsRestriction enzyme site.

Gained expression cassette is connected into plasmid pYLCRISPR/Cas9Pubi-H's (referring to reference paper 1) by Bsa I enzymesOn Bsa I restriction enzyme sites (6471~6176 positions and 7148~7152 positions in SEQ ID NO.23), Pubi is formed::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 expression cassettes, obtain pYLCRISPR/Pubi::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 edits the final carrier (Fig. 1) of gene.Cas9 (SEQ in plasmid pYLCRISPR/Cas9Pubi-H1973~6175 positions in ID NO.23) gene is by maize ubiquitin promoter Ubiqutin (1~1972 in SEQ ID NO.23Put) driving.The different cohesive ends produced after same Bsa I digestions are shown in Fig. 1 b.

Reference paper 1：Ma,X.,Zhang,Q.,Zhu,Q.,Liu,W.,Chen,Y.,Qiu,R.,Wang,B.,Yang,Z.,Li,H.,Lin,Y.,Xie,Y.,Shen,R.,Chen,S.,Wang,Z.,Chen,Y.,Guo,J.,Chen,L.,Zhao,X.,Dong,Z.and Liu,Y.G.(2015)A Robust CRISPR/Cas9System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant8,1274-1284.

Embodiment three

CRISPR/Cas9 gene editings carrier of the present invention is specifically carrying out gene volume for rice cinnamyl-alcohol dehydrogenase geneThe application collected.

Vegetable material：Rice Nipponbare (Oryza.Sativa L.spp.japonica)

It is using the final carrier conversion bacillus coli DH 5 alpha of the gained of embodiment two, the sequencing of picking monoclonal that sequencing is correctBacterial strain expansion is numerous, and extraction plasmid is transferred in agrobacterium strains EHA105, obtains the bacterium solution for disseminating.

Using Nipponbare mature seed as material, seed is sterilized with 1 ‰ mercuric chloride, it is sterile washing five times after culture in MS culture mediumsOn, evoked callus.30 DEG C of illumination cultivation fortnights obtain rice Nipponbare mature embryo callus.Callus is cut intoThe particle of 2mm~3mm sizes, is incubated on fresh MS culture mediums.

After 4 days, rice Nipponbare mature embryo callus particle is disseminated with EHA105 bacterium solutions, it is specific to disseminate operating method ginsengSee reference file 2.

Culture terminates to obtain 60 plants of transgenic lines.Transgenic line genomic DNA is extracted, with primer 1F (SEQ IDNO.4), 1R (SEQ ID NO.5) amplifications include the sequence (long 580bp) in sgRNA mutational sites, carry out transgenic line identification.

It was found from Fig. 2, Fig. 3, purpose fragment has deletion mutation and insertion icon.By Fig. 2 obtain containing mutational siteFragment rubber tapping sequencing, with DSDecodeM (http://skl.scau.edu.cn/home/) analysis 60 transgenic lines volumeThe situation of collecting, wherein there is a strain not edit, remaining has different degrees of editor (Fig. 3).Wherein No. 7 and No. 55 areThere occurs the Mutants homozygous of about 200bp missings.

Qualification result shows that, using CRISPR/Cas9 gene editings carrier of the present invention, it is right in rice Nipponbare to succeedPurpose rice cinnamyl-alcohol dehydrogenase (CAD) gene has been inserted into or has been lacked into edlin, editor's target site of the geneMutation.

Fig. 4 is that (WT is Nipponbare to phenotype after rice cinnamyl-alcohol dehydrogenase (CAD) gene editing, and cad is that CAD genes occurMutant after mutation), Fig. 5 be rice paddy seed phenotype (WT is Nipponbare, and cad is the mutant after CAD gene mutations).Fig. 4,Fig. 5 is shown, after producer editor, the stem knot and glume of rice Nipponbare are all bronzing.Phenotype after the gene mutation is simultaneouslyDo not influence the normal growth and yield of plant, thus can in the case where not influencing other characters, by the gene it is other notIt is to be mutated with restorer to educate, so as to be used in breeding.Glume face can be especially used in the paddy machinery production of hybrid seedsColor is to be applied in selection markers.

Reference paper 2：Agrobacterium-mediated transformation of Japonicacv.Nipponbare callus tissue and recovery of transgenic rice plants. http://ptf.agron.iastate.edu/protocol/Rice.PDF

Application examples one

Fig. 6 is that the selfing of Crispr/Cas9 gene editings strain removes transgene component analysis chart.Analysis shows, the present inventionThe restructuring rice strain that editor is completed to target gene obtained can be filtered out by selfing does not contain SgRNA:Cas9:HPT turnsThe plant of genetic fragment.

Application examples two

Fig. 7 is backcross transformation improvement restorer analysis chart.Analysis shows, can be by SgRNA:Cas9:HPT transgenic fragmentsThe editor completed to target gene rice cinnamyl-alcohol dehydrogenase (CAD) gene of new material is imported in new material.

Application examples three

SgRNA fragments of the present invention, CRISPR/Cas9 edit carrier and are applied to the paddy machinery production of hybrid seeds, itself and application examples one, twoSomething in common is not repeated.

As shown in figure 8, in the paddy machinery production of hybrid seeds, genetic transformation is carried out to maintainer with the inventive technique scheme, is importedCRISPR/Cas9 edits carrier, to target gene rice cinnamyl-alcohol dehydrogenase (CAD) gene in maintainer into edlin；EditorAfter the completion of by selfing isolate homozygosis cad mutant strains, and retain contain SgRNA:Cas9:HPT transgenic fragment strainsSystem；During hybridizing with sterile line, edit rice cinnamyl-alcohol dehydrogenase (CAD) gene in sterile line, and fromIn filter out the mutant of homozygosis, while abandon containing SgRNA:Cas9:The strain of HPT transgenic fragments, so that it is obtainedBronzing glume character, and do not contain transgene component.

The sterile line and restorer Mixed plant improved with this, mechanical harvesting can be removed extensive by the color of glume afterwardsThe seed of multiple system's selfing, what is left is all hybrid seeding F1 generation.

The maximum difficult point that the rice mechanization production of hybrid seeds at present faces is how the hybrid tied on sterile line is received with together usingThe restorer seed of cutting mill harvest separates exactly.The method of the present invention is used to be carried to solve this problem in the mechanical production of hybrid seedsFor effective means, mechanism seed production efficiency and seed purity are greatly improved.

Sequence table

SEQ ID NO.1：

gctgctgtgcgccgggctga

SEQ ID NO.2：

cgcggcggcgtcctggggct

SEQ ID NO.3：

gctgagcttcatctcgccca

SEQ ID NO.4：

5’-atccacctgcctaatctgaaag-3’

SEQ ID NO.5：

5’-agttgagcacctcctccgtct-3’

SEQ ID NO.6：

aatccacctgcctaatctgaaagttgtcacgtcttaaaaagattaaaatatttggttagtgagatggtaagaaactagtaaaaaaagtaatgaatttgtgtgcaggaagtttgtggtgaagatcccggcggggctagcgccggagcaggcggcgccgctgctgtgcgccgggctgacggtgtacagcccactgaagcacttcgggctgatgtcgccaggtctccgcggcggcgtcctggggctcggcggcgtggggcacatgggcgtgaaggtggccaagtcgatggggcaccacgtgacggtgatcagctcgtcggcgaggaagcgcggcgaggccatggacgacctgggcgccgacgcctacctcgtcagctccgacgcggcggcgatggcggccgccggcgactcgctggactacatcatcgacaccgtgccggtgcaccacccgctggagccgtacctggcgctgctgaagctggacgggaagctgatcctgatgggggtgatcaaccagccgctgagcttcatctcgcccatggtgatgctcggccggaaggccatcaccggcagcttcatcgggagcatggccgagacggaggaggtgctcaacttctgcgtcgacaaggggctcacctcccagatcgaggtcgtcaagatggactacgtcaaccaggccctcgagcgcctcgagcgcaacgacgtccgctaccgcttcgtcgtcgacgtcgccggcagcaacatcgacgacgccgacgcgccgcccgcctga

SEQ ID NO.7：

atgggcagcctcgccgccgagaagaccgtcaccgggtgggccgccagggacgcctccggccacctcaccccctacaactacaccctcaggaagactgggcctgaagatgtggtggtgaaggttttgtattgtggtatctgccatactgacatccaccaggccaagaaccaccttggtgcttccaagtaccccatggtccctggccatgaggtggtcggcgaggtggtggaggtcgggccggaggtgaccaagtacagcgccggcgacgtcgtcggcgtcggcgtcatcgtcggctgctgccgcgagtgccatccgtgcaaggccaatgtggagcagtactgcaacaagaggatttggtcctacaacgacgtctacaccgacggccggccaacccagggcggcttcgcctccgccatggtcgtcgaccagaagtttgtggtgaagatcccggcggggctagcgccggagcaggcggcgccgctgctgtgcgccgggctgacggtgtacagcccactgaagcacttcgggctgatgtcgccaggtctccgcggcggcgtcctggggctcggcggcgtggggcacatgggcgtgaaggtggccaagtcgatggggcaccacgtgacggtgatcagctcgtcggcgaggaagcgcggcgaggccatggacgacctgggcgccgacgcctacctcgtcagctccgacgcggcggcgatggcggccgccggcgactcgctggactacatcatcgacaccgtgccggtgcaccacccgctggagccgtacctggcgctgctgaagctggacgggaagctgatcctgatgggggtgatcaaccagccgctgagcttcatctcgcccatggtgatgctcggccggaaggccatcaccggcagcttcatcgggagcatggccgagacggaggaggtgctcaacttctgcgtcgacaaggggctcacctcccagatcgaggtcgtcaagatggactacgtcaaccaggccctcgagcgcctcgagcgcaacgacgtccgctaccgcttcgtcgtcgacgtcgccggcagcaacatcgacgacgccgacgcgccgcccgcctga

SEQ ID NO.8：

Ttcagaggtctctctcgaactggaatcggcagcaaaggattttttcctgtagttttcccacaaccattttttaccatccgaatgataggataggaaaaatatccaagtgaacagtattcctataaaattcccgtaaaaagcctgcaatccgaatgagccctgaagtctgaactagccggtcacctgtacaggctatcgagatgccatacaagagacggtagtaggaactaggaagacgatggttgattcgtcaggcgaaatcgtcgtcctgcagtcgcatctatgggcctggacggaataggggaaaaagttggccggataggagggaaaggcccaggtgcttacgtgcgaggtaggcctgggctctcagcacttcgattcgttggcaccggggtaggatgcaatagagagcaacgtttagtaccacctcgcttagctagagcaaactggactgccttatatgcgcgggtgctggcttggctgccgctgctgtgcgccgggctgagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttcaagagcttggagtggatggtgtgttgcgagacccacgct

SEQ ID NO.9：

5’gcgcggtctctctcgaactggaatcggcagcaaagga 3’

SEQ ID NO.10：

5’tcagcccggcgcacagcagcgaccaatgttgctccctc3’

SEQ ID NO.11：

5’gctgctgtgcgccgggctgagttttagagctagaaatag 3’

SEQ ID NO.12：

5’gcgcggtctcgcaacacaccatccactccaagctc 3’

SEQ ID NO.13：

Ttcagaggtctctgttgaactggaatcggcagcaaaggatgcaagaacgaactaagccggacaaaaaaaaaaggagcacatatacaaaccggttttattcatgaatggtcacgatggatgatggggctcagacttgagctacgaggccgcaggcgagagaagcctagtgtgctctctgcttgtttgggccgtaacggaggatacggccgacgagcgtgtactaccgcgcgggatgccgctgggcgctgcgggggccgttggatggggatcggtgggtcgcgggagcgttgaggggagacaggtttagtaccacctcgcctaccgaacaatgaagaacccaccttataaccccgcgcgctgccgcttgtgttggcggcgtcctggggctcgggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttcaagagcttggagtggatggtgttcagcgagacccacgct

SEQ ID NO.14：

5’gcgcggtctctgttgaactggaatcggcagcaaagga 3’

SEQ ID NO.15：

5’ccgagccccaggacgccgccaacacaagcggcagcgc 3’

SEQ ID NO.16：

5’ggcggcgtcctggggctcgggttttagagctagaaatag3’

SEQ ID NO.17：

5’gcgcggtctcgctgaacaccatccactccaagctc 3’

SEQ ID NO.18：

ttcagaggtctcttcagaactggaatcggcagcaaaggactcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaa accaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatac ttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacatt atccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaat aatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgaca ccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtac aatggaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacg gcccacttttctccgtggtggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaacaacagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcagctgagcttcatctcgcccagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttcaagagcttggagtggatggtgtcggtcgagacccacgct

SEQ ID NO.19：

5’gcgcggtctcttcagaactggaatcggcagcaaagga 3’

SEQ ID NO.20：

5’tgggcgacctcagctgagcctcagcgcagcag 3’

SEQ ID NO.21：

5’gctgagcttcatctcgcccagttttagagctagaaatag 3’

SEQ ID NO.22：

5’gcgcggtctcgaccgacaccatccactccaagctc 3’

SEQ ID NO.23：

gcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagt actacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtatttt gacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataata cttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttat tctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaa ataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagta gataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgt cgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccac cgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcag gcggcctcctcctcctctcacggcacggcagctacgggggattcctttcccaccgctccttcgctttcccttcct cgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacaca cacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccc cccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgtt agatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgat tgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatg attttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcat cttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgttt caaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgat ggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgtt cgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaact acctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaa tatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattc atatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatga tggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtc gatgctcaccctgttgtttggtgttacttctgcagatggctcctaagaagaagcggaaggttggtattcacgggg tgcctgcggctgacaagaagtactccatcggcctcgacatcggcaccaacagcgtcggctgggcggtgatc accgacgagtacaaggtcccgtccaagaagttcaaggtcctgggcaacaccgaccgccactccatcaagaa gaacctcatcggcgccctcctcttcgactccggcgagacggcggaggcgacccgcctcaagcgcaccgcc cgccgccgctacacccgccgcaagaaccgcatctgctacctccaggagatcttctccaacgagatggcgaa ggtcgacgactccttcttccaccgcctcgaggagtccttcctcgtggaggaggacaagaagcacgagcgccaccccatcttcggcaacatcgtcgacgaggtcgcctaccacgagaagtaccccactatctaccaccttcgtaag aagcttgttgactctactgataaggctgatcttcgtctcatctaccttgctctcgctcacatgatcaagttccgtggt cacttccttatcgagggtgaccttaaccctgataactccgacgtggacaagctcttcatccagctcgtccagacc tacaaccagctcttcgaggagaaccctatcaacgcttccggtgtcgacgctaaggcgatcctttccgctaggct ctccaagtccaggcgtctcgagaacctcatcgcccagctccctggtgagaagaagaacggtcttttcggtaac ctcatcgctctctccctcggtctgacccctaacttcaagtccaacttcgacctcgctgaggacgctaagcttcag ctctccaaggatacctacgacgatgatctcgacaacctcctcgctcagattggagatcagtacgctgatctcttc cttgctgctaagaacctctccgatgctatcctcctttcggatatccttagggttaacactgagatcactaaggctcc tctttctgcttccatgatcaagcgctacgacgagcaccaccaggacctcaccctcctcaaggctcttgttcgtca gcagctccccgagaagtacaaggagatcttcttcgaccagtccaagaacggctacgccggttacattgacggt ggagctagccaggaggagttctacaagttcatcaagccaatccttgagaagatggatggtactgaggagcttc tcgttaagcttaaccgtgaggacctccttaggaagcagaggactttcgataacggctctatccctcaccagatc caccttggtgagcttcacgccatccttcgtaggcaggaggacttctaccctttcctcaaggacaaccgtgagaagatcgagaagatccttactttccgtattccttactacgttggtcctcttgctcgtggtaactcccgtttcgcttggat gactaggaagtccgaggagactatcaccccttggaacttcgaggaggttgttgacaagggtgcttccgcccagtccttcatcgagcgcatgaccaacttcgacaagaacctccccaacgagaaggtcctccccaagcactccctcctctacgagtacttcacggtctacaacgagctcaccaaggtcaagtacgtcaccgagggtatgcgcaagcctgccttcctctccggcgagcagaagaaggctatcgttgacctcctcttcaagaccaaccgcaaggtcaccgtcaagcagctcaaggaggactacttcaagaagatcgagtgcttcgactccgtcgagatcagcggcgttgaggaccgtttcaacgcttctctcggtacctaccacgatctcctcaagatcatcaaggacaaggacttcctcgacaacgaggagaacgaggacatcctcgaggacatcgtcctcactcttactctcttcgaggatagggagatgatcgaggagaggctcaagacttacgctcatctcttcgatgacaaggttatgaagcagctcaagcgtcgccgttacaccggttggggtaggctctcccgcaagctcatcaacggtatcagggataagcagagcggcaagactatcctcgacttcctcaagtctgatggtttcgctaacaggaacttcatgcagctcatccacgatgactctcttaccttcaaggaggatattcagaaggctcaggtgtccggtcagggcgactctctccacgagcacattgctaaccttgctggttcccctgctatcaagaagggcatccttcagactgttaaggttgtcgatgagcttgtcaaggttatgggtcgtcacaagcctgagaacatcgtcatcgagatggctcgtgagaaccagactacccagaagggtcagaagaactcgagggagcgcatgaagaggattgaggagggtatcaaggagcttggttctcagatccttaaggagcaccctgtcgagaacacccagctccagaacgagaagctctacctctactacctccagaacggtagggatatgtacgttgaccaggagctcgacatcaacaggctttctgactacgacgtcgaccacattgttcctcagtctttccttaaggatgactccatcgacaacaaggtcctcacgaggtccgacaagaacaggggtaagtcggacaacgtcccttccgaggaggttgtcaagaagatgaagaactactggaggcagcttctcaacgctaagctcattacccagaggaagttcgacaacctcacgaaggctgagaggggtggcctttccgagcttgacaaggctggtttcatcaagaggcagcttgttgagacgaggcagattaccaagcacgttgctcagatcctcgattctaggatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtcaaggtgatcaccctcaagtccaagctcgtctccgacttccgcaaggacttccagttctacaaggtccgcgagatcaacaactaccaccacgctcacgatgcttaccttaacgctgtcgttggtaccgctcttatcaagaagtaccctaagcttgagtccgagttcgtctacggtgactacaaggtctacgacgttcgtaagatgatcgccaagtccgagcaggagatcggcaaggccaccgccaagtacttcttctactccaacatcatgaacttcttcaagaccgagatcaccctcgccaacggcgagatccgcaagcgccctcttatcgagacgaacggtgagactggtgagatcgtttgggacaagggtcgcgacttcgctactgttcgcaaggtcctttctatgcctcaggttaacatcgtcaagaagaccgaggtccagaccggtggcttctccaaggagtctatccttccaaagagaaactcggacaagctcatcgctaggaagaaggattgggaccctaagaagtacggtggtttcgactcccctactgtcgcctactccgtcctcgtggtcgccaaggtggagaagggtaagtcgaagaagctcaagtccgtcaaggagctcctcggcatcaccatcatggagcgctcctccttcgagaagaacccgatcgacttcctcgaggccaagggctacaaggaggtcaagaaggacctcatcatcaagctccccaagtactctcttttcgagctcgagaacggtcgtaagaggatgctggcttccgctggtgagctccagaagggtaacgagcttgctcttccttccaagtacgtgaacttcctctacctcgcctcccactacgagaagctcaagggttcccctgaggataacgagcagaagcagctcttcgtggagcagcacaagcactacctcgacgagatcatcgagcagatctccgagttctccaagcgcgtcatcctcgctgacgctaacctcgacaaggtcctctccgcctacaacaagcaccgcgacaagcccatccgcgagcaggccgagaacatcatccacctcttcacgctcacgaacctcggcgcccctgctgctttcaagtacttcgacaccaccatcgacaggaagcgttacacgtccaccaaggaggttctcgacgctactctcatccaccagtccatcaccggtctttacgagactcgtatcgacctttcccagcttggtggtgataagcgtcctgctgccaccaaaaaggccggacaggctaagaaaaagaagtaggatcctcccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacg ttaagcatgtaataattaacatgtaatgcatgacgttatttatgaggtgggtttttatgattagagtcccgcaattata catttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgtt actagatcgggagcaccggtaaggcgcgccgtagtgctcgagagacctctgaagtggccgattcattaatgc agctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaagaaacagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctatgcatcaagctcaatgggtctagtctgtagatacccatcacactggcgaccgctcgaacatcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgcctgtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgagcgtgggtctcgcggtatcattg g

Sequence table

<110>Institute of Nuclear and Biotechnology, Sichuan Academy of Agriculture Science

<120>SgRNA, CRISPR/Cas9 carrier related with rice bronzing glume character, vector construction, application

<130> 20170816

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213>" artificial sequence " ()

<400> 1

gctgctgtgc gccgggctga 20

<210> 2

<211> 20

<212> DNA

<213>" artificial sequence " ()

<400> 2

cgcggcggcg tcctggggct 20

<210> 3

<211> 20

<212> DNA

<213>" artificial sequence " ()

<400> 3

gctgagcttc atctcgccca 20

<210> 4

<211> 22

<212> DNA

<213>" artificial sequence " ()

<400> 4

atccacctgc ctaatctgaa ag 22

<210> 5

<211> 21

<212> DNA

<213>" artificial sequence " ()

<400> 5

agttgagcac ctcctccgtc t 21

<210> 6

<211> 769

<212> DNA

<213> Oryza sativa (japonica cultivar-group)

<400> 6

aatccacctg cctaatctga aagttgtcac gtcttaaaaa gattaaaata tttggttagt 60

gagatggtaa gaaactagta aaaaaagtaa tgaatttgtg tgcaggaagt ttgtggtgaa 120

gatcccggcg gggctagcgc cggagcaggc ggcgccgctg ctgtgcgccg ggctgacggt 180

gtacagccca ctgaagcact tcgggctgat gtcgccaggt ctccgcggcg gcgtcctggg 240

gctcggcggc gtggggcaca tgggcgtgaa ggtggccaag tcgatggggc accacgtgac 300

ggtgatcagc tcgtcggcga ggaagcgcgg cgaggccatg gacgacctgg gcgccgacgc 360

ctacctcgtc agctccgacg cggcggcgat ggcggccgcc ggcgactcgc tggactacat 420

catcgacacc gtgccggtgc accacccgct ggagccgtac ctggcgctgc tgaagctgga 480

cgggaagctg atcctgatgg gggtgatcaa ccagccgctg agcttcatct cgcccatggt 540

gatgctcggc cggaaggcca tcaccggcag cttcatcggg agcatggccg agacggagga 600

ggtgctcaac ttctgcgtcg acaaggggct cacctcccag atcgaggtcg tcaagatgga 660

ctacgtcaac caggccctcg agcgcctcga gcgcaacgac gtccgctacc gcttcgtcgt 720

cgacgtcgcc ggcagcaaca tcgacgacgc cgacgcgccg cccgcctga 769

<210> 7

<211> 1092

<212> DNA

<213> Oryza sativa (japonica cultivar-group)

<400> 7

atgggcagcc tcgccgccga gaagaccgtc accgggtggg ccgccaggga cgcctccggc 60

cacctcaccc cctacaacta caccctcagg aagactgggc ctgaagatgt ggtggtgaag 120

gttttgtatt gtggtatctg ccatactgac atccaccagg ccaagaacca ccttggtgct 180

tccaagtacc ccatggtccc tggccatgag gtggtcggcg aggtggtgga ggtcgggccg 240

gaggtgacca agtacagcgc cggcgacgtc gtcggcgtcg gcgtcatcgt cggctgctgc 300

cgcgagtgcc atccgtgcaa ggccaatgtg gagcagtact gcaacaagag gatttggtcc 360

tacaacgacg tctacaccga cggccggcca acccagggcg gcttcgcctc cgccatggtc 420

gtcgaccaga agtttgtggt gaagatcccg gcggggctag cgccggagca ggcggcgccg 480

ctgctgtgcg ccgggctgac ggtgtacagc ccactgaagc acttcgggct gatgtcgcca 540

ggtctccgcg gcggcgtcct ggggctcggc ggcgtggggc acatgggcgt gaaggtggcc 600

aagtcgatgg ggcaccacgt gacggtgatc agctcgtcgg cgaggaagcg cggcgaggcc 660

atggacgacc tgggcgccga cgcctacctc gtcagctccg acgcggcggc gatggcggcc 720

gccggcgact cgctggacta catcatcgac accgtgccgg tgcaccaccc gctggagccg 780

tacctggcgc tgctgaagct ggacgggaag ctgatcctga tgggggtgat caaccagccg 840

ctgagcttca tctcgcccat ggtgatgctc ggccggaagg ccatcaccgg cagcttcatc 900

gggagcatgg ccgagacgga ggaggtgctc aacttctgcg tcgacaaggg gctcacctcc 960

cagatcgagg tcgtcaagat ggactacgtc aaccaggccc tcgagcgcct cgagcgcaac 1020

gacgtccgct accgcttcgt cgtcgacgtc gccggcagca acatcgacga cgccgacgcg 1080

ccgcccgcct ga 1092

<210> 8

<211> 628

<212> DNA

<213> Oryza sativa

<400> 8

ttcagaggtc tctctcgaac tggaatcggc agcaaaggat tttttcctgt agttttccca 60

caaccatttt ttaccatccg aatgatagga taggaaaaat atccaagtga acagtattcc 120

tataaaattc ccgtaaaaag cctgcaatcc gaatgagccc tgaagtctga actagccggt 180

cacctgtaca ggctatcgag atgccataca agagacggta gtaggaacta ggaagacgat 240

ggttgattcg tcaggcgaaa tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata 300

ggggaaaaag ttggccggat aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct 360

gggctctcag cacttcgatt cgttggcacc ggggtaggat gcaatagaga gcaacgttta 420

gtaccacctc gcttagctag agcaaactgg actgccttat atgcgcgggt gctggcttgg 480

ctgccgctgc tgtgcgccgg gctgagtttt agagctagaa atagcaagtt aaaataaggc 540

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttca agagcttgga 600

gtggatggtg tgttgcgaga cccacgct 628

<210> 9

<211> 37

<212> DNA

<213>Artificial sequence ()

<400> 9

gcgcggtctc tctcgaactg gaatcggcag caaagga 37

<210> 10

<211> 38

<212> DNA

<213>Artificial sequence ()

<400> 10

tcagcccggc gcacagcagc gaccaatgtt gctccctc 38

<210> 11

<211> 39

<212> DNA

<213>Artificial sequence ()

<400> 11

gctgctgtgc gccgggctga gttttagagc tagaaatag 39

<210> 12

<211> 35

<212> DNA

<213>Artificial sequence ()

<400> 12

gcgcggtctc gcaacacacc atccactcca agctc 35

<210> 13

<211> 514

<212> DNA

<213>Artificial sequence ()

<400> 13

ttcagaggtc tctgttgaac tggaatcggc agcaaaggat gcaagaacga actaagccgg 60

acaaaaaaaa aaggagcaca tatacaaacc ggttttattc atgaatggtc acgatggatg 120

atggggctca gacttgagct acgaggccgc aggcgagaga agcctagtgt gctctctgct 180

tgtttgggcc gtaacggagg atacggccga cgagcgtgta ctaccgcgcg ggatgccgct 240

gggcgctgcg ggggccgttg gatggggatc ggtgggtcgc gggagcgttg aggggagaca 300

ggtttagtac cacctcgcct accgaacaat gaagaaccca ccttataacc ccgcgcgctg 360

ccgcttgtgt tggcggcgtc ctggggctcg ggttttagag ctagaaatag caagttaaaa 420

taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttcaagag 480

cttggagtgg atggtgttca gcgagaccca cgct 514

<210> 14

<211> 37

<212> DNA

<213>Artificial sequence ()

<400> 14

gcgcggtctc tgttgaactg gaatcggcag caaagga 37

<210> 15

<211> 37

<212> DNA

<213>Artificial sequence ()

<400> 15

ccgagcccca ggacgccgcc aacacaagcg gcagcgc 37

<210> 16

<211> 39

<212> DNA

<213>Artificial sequence ()

<400> 16

ggcggcgtcc tggggctcgg gttttagagc tagaaatag 39

<210> 17

<211> 35

<212> DNA

<213>Artificial sequence ()

<400> 17

gcgcggtctc gctgaacacc atccactcca agctc 35

<210> 18

<211> 923

<212> DNA

<213>Artificial sequence ()

<400> 18

ttcagaggtc tcttcagaac tggaatcggc agcaaaggac tcattagcgg tatgcatgtt 60

ggtagaagtc ggagatgtaa ataattttca ttatataaaa aaggtacttc gagaaaaata 120

aatgcatacg aattaattct ttttatgttt tttaaaccaa gtatatagaa tttattgatg 180

gttaaaattt caaaaatatg acgagagaaa ggttaaacgt acggcatata cttctgaaca 240

gagagggaat atggggtttt tgttgctccc aacaattctt aagcacgtaa aggaaaaaag 300

cacattatcc acattgtact tccagagata tgtacagcat tacgtaggta cgttttcttt 360

ttcttcccgg agagatgata caataatcat gtaaacccag aatttaaaaa atattcttta 420

ctataaaaat tttaattagg gaacgtatta ttttttacat gacacctttt gagaaagagg 480

gacttgtaat atgggacaaa tgaacaattt ctaagaaatg ggcatatgac tctcagtaca 540

atggaccaaa ttccctccag tcggcccagc aatacaaagg gaaagaaatg agggggccca 600

caggccacgg cccacttttc tccgtggtgg ggagatccag ctagaggtcc ggcccacaag 660

tggcccttgc cccgtgggac ggtgggattg cagagcgcgt gggcggaaac aacagtttag 720

taccacctcg ctcacgcaac gacgcgacca cttgcttata agctgctgcg ctgaggctca 780

gctgagcttc atctcgccca gttttagagc tagaaatagc aagttaaaat aaggctagtc 840

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttcaagagc ttggagtgga 900

tggtgtcggt cgagacccac gct 923

<210> 19

<211> 37

<212> DNA

<213>Artificial sequence ()

<400> 19

gcgcggtctc ttcagaactg gaatcggcag caaagga 37

<210> 20

<211> 32

<212> DNA

<213>Artificial sequence ()

<400> 20

tgggcgacct cagctgagcc tcagcgcagc ag 32

<210> 21

<211> 39

<212> DNA

<213>Artificial sequence ()

<400> 21

gctgagcttc atctcgccca gttttagagc tagaaatag 39

<210> 22

<211> 35

<212> DNA

<213>Artificial sequence ()

<400> 22

gcgcggtctc gaccgacacc atccactcca agctc 35

<210> 23

<211> 7176

<212> DNA

<213>Artificial sequence ()

<400> 23

gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta agttataaaa 60

aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta tctttataca 120

tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa tatcagtgtt 180

ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga gtattttgac 240

aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt ttttgcaaat 300

agcttcacct atataatact tcatccattt tattagtaca tccatttagg gtttagggtt 360

aatggttttt atagactaat ttttttagta catctatttt attctatttt agcctctaaa 420

ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata taaaatagaa 480

taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa aactaaggaa 540

acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga cgagtctaac 600

ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga cggcacggca 660

tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg acttgctccg 720

ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac ggcaggcggc 780

ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg ctccttcgct 840

ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt tccccaacct 900

cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac ccgtcggcac 960

ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct tctctagatc 1020

ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt tgtgttagat 1080

ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag 1140

acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg atggctctag 1200

ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata gggtttggtt 1260

tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca tcttttcatg 1320

cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct agatcggagt 1380

agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat gtgtgtgcca 1440

tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta ggataggtat 1500

acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc gcttggttgt 1560

gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag aatactgttt 1620

caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata catcttcata 1680

gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg ttgatgtggg 1740

ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct ctaaccttga 1800

gtacctatct attataataa acaagtatgt tttataatta ttttgatctt gatatacttg 1860

gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc atacgctatt 1920

tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg ttacttctgc 1980

agatggctcc taagaagaag cggaaggttg gtattcacgg ggtgcctgcg gctgacaaga 2040

agtactccat cggcctcgac atcggcacca acagcgtcgg ctgggcggtg atcaccgacg 2100

agtacaaggt cccgtccaag aagttcaagg tcctgggcaa caccgaccgc cactccatca 2160

agaagaacct catcggcgcc ctcctcttcg actccggcga gacggcggag gcgacccgcc 2220

tcaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc tacctccagg 2280

agatcttctc caacgagatg gcgaaggtcg acgactcctt cttccaccgc ctcgaggagt 2340

ccttcctcgt ggaggaggac aagaagcacg agcgccaccc catcttcggc aacatcgtcg 2400

acgaggtcgc ctaccacgag aagtacccca ctatctacca ccttcgtaag aagcttgttg 2460

actctactga taaggctgat cttcgtctca tctaccttgc tctcgctcac atgatcaagt 2520

tccgtggtca cttccttatc gagggtgacc ttaaccctga taactccgac gtggacaagc 2580

tcttcatcca gctcgtccag acctacaacc agctcttcga ggagaaccct atcaacgctt 2640

ccggtgtcga cgctaaggcg atcctttccg ctaggctctc caagtccagg cgtctcgaga 2700

acctcatcgc ccagctccct ggtgagaaga agaacggtct tttcggtaac ctcatcgctc 2760

tctccctcgg tctgacccct aacttcaagt ccaacttcga cctcgctgag gacgctaagc 2820

ttcagctctc caaggatacc tacgacgatg atctcgacaa cctcctcgct cagattggag 2880

atcagtacgc tgatctcttc cttgctgcta agaacctctc cgatgctatc ctcctttcgg 2940

atatccttag ggttaacact gagatcacta aggctcctct ttctgcttcc atgatcaagc 3000

gctacgacga gcaccaccag gacctcaccc tcctcaaggc tcttgttcgt cagcagctcc 3060

ccgagaagta caaggagatc ttcttcgacc agtccaagaa cggctacgcc ggttacattg 3120

acggtggagc tagccaggag gagttctaca agttcatcaa gccaatcctt gagaagatgg 3180

atggtactga ggagcttctc gttaagctta accgtgagga cctccttagg aagcagagga 3240

ctttcgataa cggctctatc cctcaccaga tccaccttgg tgagcttcac gccatccttc 3300

gtaggcagga ggacttctac cctttcctca aggacaaccg tgagaagatc gagaagatcc 3360

ttactttccg tattccttac tacgttggtc ctcttgctcg tggtaactcc cgtttcgctt 3420

ggatgactag gaagtccgag gagactatca ccccttggaa cttcgaggag gttgttgaca 3480

agggtgcttc cgcccagtcc ttcatcgagc gcatgaccaa cttcgacaag aacctcccca 3540

acgagaaggt cctccccaag cactccctcc tctacgagta cttcacggtc tacaacgagc 3600

tcaccaaggt caagtacgtc accgagggta tgcgcaagcc tgccttcctc tccggcgagc 3660

agaagaaggc tatcgttgac ctcctcttca agaccaaccg caaggtcacc gtcaagcagc 3720

tcaaggagga ctacttcaag aagatcgagt gcttcgactc cgtcgagatc agcggcgttg 3780

aggaccgttt caacgcttct ctcggtacct accacgatct cctcaagatc atcaaggaca 3840

aggacttcct cgacaacgag gagaacgagg acatcctcga ggacatcgtc ctcactctta 3900

ctctcttcga ggatagggag atgatcgagg agaggctcaa gacttacgct catctcttcg 3960

atgacaaggt tatgaagcag ctcaagcgtc gccgttacac cggttggggt aggctctccc 4020

gcaagctcat caacggtatc agggataagc agagcggcaa gactatcctc gacttcctca 4080

agtctgatgg tttcgctaac aggaacttca tgcagctcat ccacgatgac tctcttacct 4140

tcaaggagga tattcagaag gctcaggtgt ccggtcaggg cgactctctc cacgagcaca 4200

ttgctaacct tgctggttcc cctgctatca agaagggcat ccttcagact gttaaggttg 4260

tcgatgagct tgtcaaggtt atgggtcgtc acaagcctga gaacatcgtc atcgagatgg 4320

ctcgtgagaa ccagactacc cagaagggtc agaagaactc gagggagcgc atgaagagga 4380

ttgaggaggg tatcaaggag cttggttctc agatccttaa ggagcaccct gtcgagaaca 4440

cccagctcca gaacgagaag ctctacctct actacctcca gaacggtagg gatatgtacg 4500

ttgaccagga gctcgacatc aacaggcttt ctgactacga cgtcgaccac attgttcctc 4560

agtctttcct taaggatgac tccatcgaca acaaggtcct cacgaggtcc gacaagaaca 4620

ggggtaagtc ggacaacgtc ccttccgagg aggttgtcaa gaagatgaag aactactgga 4680

ggcagcttct caacgctaag ctcattaccc agaggaagtt cgacaacctc acgaaggctg 4740

agaggggtgg cctttccgag cttgacaagg ctggtttcat caagaggcag cttgttgaga 4800

cgaggcagat taccaagcac gttgctcaga tcctcgattc taggatgaac accaagtacg 4860

acgagaacga caagctcatc cgcgaggtca aggtgatcac cctcaagtcc aagctcgtct 4920

ccgacttccg caaggacttc cagttctaca aggtccgcga gatcaacaac taccaccacg 4980

ctcacgatgc ttaccttaac gctgtcgttg gtaccgctct tatcaagaag taccctaagc 5040

ttgagtccga gttcgtctac ggtgactaca aggtctacga cgttcgtaag atgatcgcca 5100

agtccgagca ggagatcggc aaggccaccg ccaagtactt cttctactcc aacatcatga 5160

acttcttcaa gaccgagatc accctcgcca acggcgagat ccgcaagcgc cctcttatcg 5220

agacgaacgg tgagactggt gagatcgttt gggacaaggg tcgcgacttc gctactgttc 5280

gcaaggtcct ttctatgcct caggttaaca tcgtcaagaa gaccgaggtc cagaccggtg 5340

gcttctccaa ggagtctatc cttccaaaga gaaactcgga caagctcatc gctaggaaga 5400

aggattggga ccctaagaag tacggtggtt tcgactcccc tactgtcgcc tactccgtcc 5460

tcgtggtcgc caaggtggag aagggtaagt cgaagaagct caagtccgtc aaggagctcc 5520

tcggcatcac catcatggag cgctcctcct tcgagaagaa cccgatcgac ttcctcgagg 5580

ccaagggcta caaggaggtc aagaaggacc tcatcatcaa gctccccaag tactctcttt 5640

tcgagctcga gaacggtcgt aagaggatgc tggcttccgc tggtgagctc cagaagggta 5700

acgagcttgc tcttccttcc aagtacgtga acttcctcta cctcgcctcc cactacgaga 5760

agctcaaggg ttcccctgag gataacgagc agaagcagct cttcgtggag cagcacaagc 5820

actacctcga cgagatcatc gagcagatct ccgagttctc caagcgcgtc atcctcgctg 5880

acgctaacct cgacaaggtc ctctccgcct acaacaagca ccgcgacaag cccatccgcg 5940

agcaggccga gaacatcatc cacctcttca cgctcacgaa cctcggcgcc cctgctgctt 6000

tcaagtactt cgacaccacc atcgacagga agcgttacac gtccaccaag gaggttctcg 6060

acgctactct catccaccag tccatcaccg gtctttacga gactcgtatc gacctttccc 6120

agcttggtgg tgataagcgt cctgctgcca ccaaaaaggc cggacaggct aagaaaaaga 6180

agtaggatcc tcccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 6240

ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 6300

ttaacatgta atgcatgacg ttatttatga ggtgggtttt tatgattaga gtcccgcaat 6360

tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 6420

gcgcggtgtc atctatgtta ctagatcggg agcaccggta aggcgcgccg tagtgctcga 6480

gagacctctg aagtggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 6540

agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 6600

tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 6660

cacaagaaac agctatgacc atgattacgc caagctattt aggtgacact atagaatact 6720

caagctatgc atcaagctca atgggtctag tctgtagata cccatcacac tggcgaccgc 6780

tcgaacatca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg 6840

atgtacagag tgatattatt gacacgccgg ggcgacggat ggtgatcccc ctggccagtg 6900

cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg 6960

aaagctggcg catgatgacc accgatatgg ccagtgtgcc tgtctccgtt atcggggaag 7020

aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct 7080

ggggaatata aatgtcaggc ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag 7140

cgcggcgggt gagcgtgggt ctcgcggtat cattgg 7176

Claims (10)

1.sgRNA fragments, it is characterised in that：It is aobvious outside the 5th comprising selectively targeted rice cinnamyl-alcohol dehydrogenase CAD genesThe gRNA of son.

2. sgRNA fragments according to claim 1, it is characterised in that：Including being named as T1, base sequence such as SEQ IDThe sgRNA of NO.1, is named as the sgRNA of T2, base sequence such as SEQ ID NO.2, is named as T3, base sequence such as SEQ IDThe sgRNA of NO.3.

3. include the CRISPR/Cas9 gene editing carriers of the sgRNA fragments described in claim 2.

4. CRISPR/Cas9 gene editings carrier according to claim 3, it is characterised in that：Including by OsU6a promotersRegulate and control the expression cassette OsU6a of T1 sgRNA fragment expressions::T1, the expression by OsU6b promoter regulation T2 sgRNA fragment expressionsFrame OsU6b::T2, the expression cassette OsU6c by OsU6c promoter regulation T3 sgRNA fragment expressions::T3, by maize ubiquitinThe expression cassette of Ubiqutin promoter regulations Cas9 expression.

5. the construction method of the CRISPR/Cas9 gene editing carriers described in claim 3 or 4, it is characterised in that：It will connect respectivelyConnect OsU6a, OsU6b, OsU6c promoter and T1, T2, T3 of Ploy-T terminators are connected into pYLCRISPR/Cas by Bsa I enzymes9P_ubi- H plasmids, structure obtain Pubi::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 expression cassettes, obtainpYLCRISPR/Pubi::Cas9-OsU6a::T1-OsU6b::T2-OsU6c::T3 edits the final carrier of gene.

6. the CRISPR/Cas9 gene editings carrier described in claim 3 or 4 is specifically being directed to rice cinnamyl-alcohol dehydrogenase geneCarry out the application of gene editing.

7. application process according to claim 6, it is characterised in that：The carrier built is transferred to Agrobacterium EHA105 to useIn agriculture bacillus mediated rice Nipponbare callus genetic transformation, and induction of callus is obtained into regeneration plant.

8. the CRISPR/Cas9 gene editings carrier described in sgRNA fragments, claim 4 described in claim 1 or 2 is being madeApplication in standby bronzing glume character rice material；The bronzing glume character rice material contains or does not containSgRNA:Cas9:HPT transgenic fragments.

9. the CRISPR/Cas9 gene editing carriers described in sgRNA fragments, claim 4 described in claim 1 or 2 are in waterApplication in the rice machinery production of hybrid seeds.

10. primer combination 1F, 1R for detecting CRISPR/Cas9-CAD genetic recombination, it is characterised in that 1F, 1R base sequenceRow are respectively as shown in SEQ ID NO.4, SEQ ID NO.5.