CN106676130A

CN106676130A - Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology

Info

Publication number: CN106676130A
Application number: CN201611265175.0A
Authority: CN
Inventors: 沈倩; 李继明; 徐冉
Original assignee: Huazhi Rice Bio-Tech Co Ltd
Current assignee: Huazhi Rice Bio-Tech Co Ltd
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2017-05-17

Abstract

The invention provides a paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology. According to the fact that a paddy rice BADH2 gene is designed based on a sgRNA sequence of CRISPR/Cas9, a DNA fragment with the sgRNA sequence coded is connected to a carrier carrying CRISPR/Cas, and paddy rice is transformed, thereby achieving site-directed mutagenesis of the paddy rice BADH2 gene. The nucleotide sequence of a sgRNA acting site is shown as SEQ ID NO:1. The paddy rice endogenous gene BADH2 is edited through CRISPR-CAS9 technology to obtain a BADH2 mutant, which is convenient and efficient in creation of jasmine rice germplasm resources.

Description

Using method of the CRISPR-CAS9 technologies to Oryza sativa L. BADH2 site-directed point mutations

Technical field

The present invention relates to technical field of plant transgene and field of crop genetic breeding, specifically, are related to a kind of utilization Method of the CRISPR-CAS9 technologies to Oryza sativa L. BADH2 site-directed point mutations.

Background technology

Fragrant rice is used as the Special member in Oryza sativa L. family, and its cultivation history is quite long, in the world each Rice Production state or Area nearly all has fragrant seed rice to plant.Fragrance is fine quality rice key character, and fragrant rice breeding has become the important of Genetic and breeding in rice One of content.Bradbury etc. (2005) has found a gene-BADH2 that there is sequence difference between scented rice and common rice, This gene code glycine betaine aldehyde dehydrogenase (betaine aldehyde dehydrogenase), thus it is speculated that may be with meter Xiang's Synthesis is relevant.Chen etc. (2008) by transgene complementation test to demonstrating BADH2 Gene Handling rice perfume, scented rice varieties There is natural mutation in second of BADH2 genes or the 7th exon, the BADH2 transcripts without total length, 2AP (2- One of acetyl-1-pyrroline, Oryza sativa L. scented rice fragrance ingredient) a large amount of accumulation；And only there is total length in common rice BADH2 transcripts, wherein 2AP contents are extremely low.By proceeding to total length BADH2 gene in scented rice varieties, its 2AP content significantly drops Low, fragrance is lost.Prove that BADH2 is the major gene resistance for controlling meter Xiang.

At present, the cultivation of scented rice varieties is mostly scented rice gene to be proceeded in common rice using Crossing system, although can be with Assisted Selection is carried out by molecular marker, but this process Complicated Periodic is longer.With ZFN (zinc-finger nucleases) With the sequence-specific core that TALEN (transcription activator-like effector nucleases) is representative Sour zymotechnic can be realized pinpointing genome editor.Realize that knocking out BADH2 using TALEN technologies obtains Oryza sativa L. in Oryza sativa L. Fragrance.But TALEN technology modules assembling process is loaded down with trivial details, a large amount of examining orders are needed, it is relatively costly and with cytotoxicity.ZFN Upstream and downstream sequence is depended in Technology design, miss rate is high, equally with cytotoxicity.

CRISPR/Cas technologies have been broken away from and have synthesized and assembled the loaded down with trivial details behaviour with specific DNA identification ability module Make, the design of its gRNA and synthetic work amount are far smaller than the building process of the DNA identification modules of TALEN and ZFN technologies, and poison Property has the advantages that targeting is accurate, miss rate is low, cytotoxicity is low, cheap easy well below ZFN technologies.CRISPR/Cas9 Technology since the advent of the world, just there is other unrivaled advantages of gene editing technology, after technology is updated, is more considered to It is enough it is most effective in living cells, most easily " edit " any gene.

The content of the invention

It is an object of the invention to provide a kind of side of utilization CRISPR-CAS9 technologies to Oryza sativa L. BADH2 site-directed point mutations Method.

In order to realize the object of the invention, the utilization CRISPR-CAS9 technologies that the present invention is provided are gene site-directed to Oryza sativa L. BADH2 The method of mutation, for sgRNA sequence of the Oryza sativa L. BADH2 gene design based on CRISPR/Cas9, will be described containing coding The DNA fragmentation of sgRNA sequences is connected in the carrier for carrying CRISPR/Cas, and rice transformation is realized to Oryza sativa L. BADH2 genes Rite-directed mutagenesises.

Wherein, the nucleotides sequence of sgRNA action sites is classified as 5 '-GAGTGGCGCGCCCCCGCGCT-3 '.(SEQ ID NO:1)

Aforesaid method, carrier construction pHZ1-CAS9-gRNA-BADH2, using Agrobacterium-mediated transformation Oryza sativa L., screening Positive transgenic plant.

The construction method of the carrier pHZ1-CAS9-gRNA-BADH2 is as follows：

1) structure of OsU6-BADH2-gRNA-polyT fragments

First round PCR is with Cris-GL3 plasmids (by Inst. of Genetics and Development Biology, CAS's doctor's Xu Ran favour Give) it is template, expand the 20bp target sequences of OsU6 promoteres and BADH2 genes, clip size with primer CRI-F1 and SQ3-R1 329bp；Equally with Cris-GL3 plasmids as template, with primer SQ3-F2 and CRI-R2 the 20bp target sequences of BADH2 genes are expanded With gRNA-polyT, clip size 180bp；Second wheel PCR be with first round PCR primer as template, with primer CRI-F1 and CRI-R2 amplifications obtain final product OsU6-BADH2-gRNA-polyT fragments, clip size 489bp, sequence such as SEQ ID NO:Shown in 2；

2) structure of carrier pHZ1-CAS9-gRNA-BADH2

OsU6-BADH2-gRNA-polyT fragments are entered respectively with the PMDC99-Cas9 carriers Jing after Hind III digestions Row agarose gel electrophoresis, cut and carry out after glue reclaim seamless clone, based on the principle of homologous recombination, OsU6-BADH2-gRNA- PolyT fragments are fused to the Hind III sites of PMDC99-CAS9 carriers by the method for Infusion, that is, build and obtain PHZ1-CAS9-gRNA-BADH2 carriers.

Wherein, step 1) used in primer sequence it is as follows：

CRI-F1：5’-CGACGGCCAGTGCCAAGCTTCTCGGATCCACTAGTAACGGC-3’

SQ3-R1：5’-AGCGCGGGGGCGCGCCACTCACACAAGCGACAGCGCGCGGG-3’

SQ3-F2：5’-GAGTGGCGCGCCCCCGCGCTGTTTTAGAGCTAGAAATAGCA-3’

CRI-R2:5’-ACCTGCAGGCATGCAAGCTTCGACCTCGAGCGGCCGCCAGT-3’

The nucleotide sequence such as SEQ ID NO of the pHZ1-CAS9-gRNA-BADH2 carriers of the present invention:Shown in 3.

The present invention also provides application of the methods described in Oryza sativa L. BADH2 site-directed point mutation breedings.

Comprise the following steps：

(1) Oryza sativa L. ' Japan is fine ' mature embryo callus induction；

(2) invade in the Agrobacterium bacterium solution that the calluss immersion of induction is carried pHZ1-CAS9-gRNA-BADH2 carriers Dye；Agrobacterium strains are preferably used for EHA105；

(3) calluss are moved to and is cultivated in co-cultivation culture medium, washed away and move to after Agrobacterium training in prescreening culture medium Support, then move to carry out resistant calli screening in screening culture medium；

(4) resistant calli is moved to and is cultivated in regeneration culture, form vegetative seedling；

(5) seedling exercising, transplanting, obtain transgenic paddy rice after vegetative seedling is taken root on root media；

(6) primer is designed according to the nucleotide sequence of sgRNA action sites, identifies that rice plant is mutated position by PCR methods Point.

In aforementioned applications, step (1) is specially：Shell after mature seed threshing, washed 2 minutes with 75% ethanol, then immerse In liquor natrii hypochloritises' (active chlorine 5.5%), 30min is shaken on shaking table, washed 5 times, during NBi culture medium is placed in after seed treatment For callus induction, the dark 10h of 26 DEG C of illumination 14h/ is cultivated 15-20 days；Calluss are stripped, is moved in new NBi culture medium, 26 DEG C light culture is used to infect after 4 days.

The preparation method of the Agrobacterium bacterium solution of the carrying pHZ1-CAS9-gRNA-BADH2 carriers is as follows：From -80 DEG C of ice Agrobacterium competence being taken in case to thaw on ice, adding 3-5 μ L plasmids pHZ1-CAS9-gRNA-BADH2 to mix, ice puts 30min, 1min in liquid nitrogen is put into, then heat shock 1min in 37 DEG C of water-baths, correspondence adds 1mL YEP culture medium in aseptic operating platform, 2-4h is shaken on 28 DEG C of shaking tables, is taken out, 8000rpm/min centrifugation 1min outwell part supernatant in aseptic operating platform, use spreader Equably remaining suspension is mixed and is coated on the YEP flat boards added with rifampicin and kanamycin, be put into after sealed membrane sealing 28 DEG C of incubator cultures.Picking single bacterium colony after 2d, 28 DEG C are shaken after bacterium 24-36h, carry out bacterium colony PCR, obtain positive bacterium colony.

With liquid-transfering gun, from 100mL agrobacterium suspensions, (formula of the agrobacterium suspension is calculated as by 1L：20 × N6 mother solutions 50mL, 100 × EDTA-Fe mother solution 10mL, 100 × B5 trace element mother solution 10mL, 100 × B5 vitamin stock solution 10mL, CEH 0.3g, inositol 2g, sucrose 30g, 3 ', 5 '-dimethoxy-4 ' ' 100 μM of-hydroxy acetophenone, 800 μ L are drawn in pH 5.2~5.3) Flushing Agrobacterium bacterium plate three to four times, draws bacterium solution 3~5 and drips in suspension, and slightly muddiness can (i.e. bacterial concentration reaches OD₆₀₀For 0.3 or so), calluss tweezers are sandwiched in suspension, timing 15min rocks once every three four minutes, makes It is fully contacted.Bacterium solution is gone, the calluss for infecting is poured on aseptic filter paper, blot the bacterium solution of its excess surface, Wound healing is moved to into NBco after 10min to co-culture on base, is put between tissue culture 26 DEG C, 2~3d of illumination cultivation, until wound healing group Knit visible Agrobacterium around to grow.

The wound healing for infecting is put in the empty triangular flask of sterilizing, with aseptic water washing for several times, till water becomes limpid, 200 μ L cephalos (cef, concentration 0.25g/mL, final concentration 500mg/L) are added in the sterilized water of 100mL sterilizings, by wound healing tweezer Sub-folder enters triangular flask, is put on 28 DEG C of shaking tables and shakes 15min, and the water that will be equipped with cephalo is poured out, and wound healing is pushed and fill on aseptic filter paper Divide and dry up, regular turnover wound healing is put into wound healing in NBps prescreening culture medium after 1-2 hours, light culture 7d at 26 DEG C.

Wound healing in NBps prescreening culture medium is moved on in NBS screening culture medium.After 1 month, the resistance for growing is healed Hinder number consecutively to be placed on regeneration culture medium, the dark 10h cultures 3-4 weeks left and right of illumination 14h/ at 26 DEG C.

Kanamycin-resistant callus tissue development in culture medium to be regenerated is grown after 2-3 centimetre of seedling, you can gently pressed from both sides out with tweezers, Be placed on sterilized empty culture dish, remove Seedling around and root culture medium and old wound healing, be then gently placed with 3.5cm Taking root in pipe for high root media, often puts a Seedling in pipe, number consistent with regenerating tube numbering.Illumination 14h/ is black at 26 DEG C Dark 10h cultures 3-4 is all, after seedling grow it is sturdy and after the root system of prosperity by uncap seedling exercising culture, periodically add water in pipe.It is little Seedling length continues to be put into and takes root in pipe and add water to upright blade and during well developed root system after seedling root culture medium is cleaned, and treats Plant in soil after seedling is sufficiently robust.

In the present invention, the formula for co-culturing culture medium is calculated as by 2L：

20 × N6 mother solution 100mL, 100 × EDTA-Fe mother solution 20mL, 100 × B5 trace element mother solution 20mL, 100 × B5 Vitamin stock solution 20mL, glutamine 1g, proline 1g, CEH 0.6g, inositol 0.2g, sucrose 60g, 2,4-D 4mg, AS is (i.e. 3 ', 5 '-dimethoxy-4 ' '-hydroxy acetophenone) 100 μM, plant gum 5.2g, pH 5.2~5.3.

The prescreening culture medium prescription is：

NBi culture medium containing 500mg/L cephamycins, pH=5.8.

The formula of the screening culture medium is：

The NBi culture medium of the cephamycin of the hygromycin containing final concentration of 40mg/L and final concentration of 500mg/L, pH 5.8。

It is described regeneration culture formula be：

NBi culture medium without 2,4-D, the 6-BA containing final concentration of 3mg/L, the NAA of final concentration of 0.5mg/L and end Concentration for 30mg/L hygromycin, pH 5.8.

The formula of the root media is calculated as by 2L：

20 × MS a great number of elements mother solution 50mL, 100 × EDTA-Fe mother solution 10mL, 100 × MS trace element mother solution 10mL, 100 × B5 vitamin stock solution 20mL, inositol 0.2g, glucose 14g, sucrose 6g, plant gum 5.2g, hygromycin final concentration 40mg/ L, pH 5.8.

Wherein, the formula of the NBi culture medium is calculated as by 2L：

20 × N6 mother solution 100mL, 100 × EDTA-Fe mother solution 20mL, 100 × B5 trace element mother solution 20mL, 100 × B5 Vitamin stock solution 20mL, glutamine 1g, proline 1g, CEH 0.6g, inositol 0.2g, sucrose 60g, 2,4-D 4mg, plant gum 5.2g, pH 5.8.

The formula of 20 × N6 mother solutions is calculated as by 1L：

KNO₃：56.6g, (NH₄)₂SO₄：9.26g, KH₂PO₄：8.00g, MgSO₄·7H₂O：3.70g, CaCl₂·2H₂O： 3.32g。

The formula of 100 × EDTA-Fe mother solutions is calculated as by 1L：

FeSO₄·7H₂O 2.78g, Na₂-EDTA 3.73g。

The formula of 100 × B5 trace element mother solution is calculated as by 1L：

MnSO₄·H₂O 1.0g, ZnSO₄·7H₂O 0.2g, H₃BO₃0.3g, CuSO₄·5H₂O 2.5mg, Na₂MoO₄· 2H₂O 25.0mg, CoCl₂·6H₂O 2.5mg, KI 75.0mg.

The formula of 100 × B5 vitamin stock solutions is calculated as by 500mL：

Thiamine hydrochloride VB1 500mg, pyridoxine hydrochloride VB6 50mg, nicotinic acid VB5 50mg.

The formula of 20 × MS a great number of elements mother solution is calculated as by 1L：

KNO₃38.0g, NH₄NO₃33.0g, KH₂PO₄3.4g, MgSO₄·7H₂O 7.4g, CaCl₂·2H₂O 8.8g。

The formula of 100 × MS trace element mother solution is calculated as by 1L：

MnSO₄·H₂O 1.56g, ZnSO₄·7H₂O 0.86g, H₃BO₃0.62g, CuSO₄·5H₂O 2.5mg, Na₂MoO₄·2H₂O 25.0mg, CoCl₂·6H₂O 2.5mg, KI 83.0mg.

In aforementioned applications, step (6) chooses each 200bp-400bp sequences of target site upstream and downstream, designs primer, enters performing PCR Amplification, amplified fragments send sequencing, judge whether target site undergos mutation by peak figure.The primer sequence of identification BADH2 mutation effects Arrange as follows, clip size 494bp.

JDSQ3F1：5’-ACCTATCGCTTTCCACCTCA-3’

JDSQ3R1：5’-CGGTTCCTCTTCAGCGCCTC-3’

The present invention further provides for the CRISPR-CAS9 carriers of Oryza sativa L. BADH2 site-directed point mutations, the carrier Nucleotide sequence such as SEQ ID NO:Shown in 3.

The present invention enters edlin by CRISPR-CAS9 technologies to Oryza sativa L. endogenous gene BADH2, obtains BADH2 mutation Body, the more convenient and efficient in initiative scented rice germ plasm resource.The fixed point to Oryza sativa L. BADH2 genes is successfully realized using the method Mutation, the method have experimental period it is short, it is easy to operate the features such as, new method is provided for rice modification breeding, for improvement Rice Characters have important practical significance.

Description of the drawings

Fig. 1 is the structure figure of OsU6-BADH2-gRNA-polyT fragments in the embodiment of the present invention 1.

Fig. 2 is the double digestion electrophoresis result of pHZ1-CAS9-gRNA-BADH2 carriers in the embodiment of the present invention 1.

Fig. 3 is the plasmid map of support C ris-GL3 of the present invention.

Fig. 4 is the plasmid map of carrier PMDC99-CAS9 of the present invention.

Fig. 5 is the plasmid map of carrier pHZ1-CAS9-gRNA-BADH2 of the present invention.

Fig. 6 is the PCR testing results of transgenic paddy rice BADH2 gene knockouts in the embodiment of the present invention 2.

Fig. 7 is transgenic rice plant FZCX3-1 (A) and FZCX3-2 (B) sequencings peak figure in the embodiment of the present invention 2.

Fig. 8 is the pre- of transgenic rice plant (T0 generations) FZCX3-1 and FZCX3-2 mutation types in the embodiment of the present invention 2 Survey result.By analyzing the set peak situation that FZCX3-1, FZCX3-2 plant is sequenced in peak figure, predict the outcome with reference to DSDecode, The mutation type for confirming BADH2 in FZCX3-1, FZCX3-2 plant is all the disappearance (deletion sites one that a chain has 4 bases Cause), another chain has the mutation (two strain base mutation positions are inconsistent) of 5 bases.

Specific embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's description suggestion.

The structure of the carrier pHZ1-CAS9-gRNA-BADH2 of embodiment 1

1st, in Oryza sativa L. BADH2 (LOC_Os08g32870) gene target site selection

Log in plant transcription factor data base (http://rice.plantbiology.msu.edu/analyses_ Search_locus.shtml), Oryza sativa L. BADH2 genes (No. LOC are inquired about：LOC_Os08g32870 sequence), design is based on The sgRNA sequences of CRISPR/Cas9.

The nucleotides sequence of sgRNA action sites is classified as 5 '-GAGTGGCGCGCCCCCGCGCT-3 '.

2nd, the structure of carrier pHZ1-CAS9-gRNA-BADH2

1) structure of OsU6-BADH2-gRNA-polyT fragments

First round PCR with Cris-GL3 plasmids as template, with primer CRI-F1 and SQ3-R1 amplification OsU6 promoteres with The 20bp target sequences of BADH2 genes, clip size 329bp；It is same with Cris-GL3 plasmids as template, with primer SQ3-F2 and The 20bp target sequences and gRNA-polyT of CRI-R2 amplification BADH2 genes, clip size 180bp；Second wheel PCR is with the first round PCR primer is template, and with primer CRI-F1 and CRI-R2 amplification OsU6-BADH2-gRNA-polyT fragments, clip size are obtained final product 489bp, sequence such as SEQ ID NO:Shown in 2.(Fig. 1)

2) structure of carrier pHZ1-CAS9-gRNA-BADH2

OsU6-BADH2-gRNA-polyT fragments are entered respectively with the PMDC99-Cas9 carriers Jing after Hind III digestions Row agarose gel electrophoresis, cut and carry out after glue reclaim seamless clone, based on the principle of homologous recombination, OsU6-BADH2-gRNA- PolyT fragments are fused to the Hind III sites of PMDC99-CAS9 carriers by the method for Infusion, that is, build and obtain PHZ1-CAS9-gRNA-BADH2 carriers (sequence such as SEQ ID NO:Shown in 3).

Wherein, step 1) used in primer sequence it is as follows：

CRI-F1：5’-CGACGGCCAGTGCCAAGCTTCTCGGATCCACTAGTAACGGC-3’

SQ3-R1：5’-AGCGCGGGGGCGCGCCACTCACACAAGCGACAGCGCGCGGG-3’

SQ3-F2：5’-GAGTGGCGCGCCCCCGCGCTGTTTTAGAGCTAGAAATAGCA-3’

CRI-R2:5’-ACCTGCAGGCATGCAAGCTTCGACCTCGAGCGGCCGCCAGT-3’。

The checking of carrier pHZ1-CAS9-gRNA-BADH2：With BamH I/Hind III double digestions.Enzyme action result is shown in Fig. 2, 1st swimming lane represents pHZ1-CAS9-gRNA-BADH2 carrier enzyme action results, can obtain tri- bars of 9205bp, 5180bp, 447bp Band.2nd swimming lane represents PMDC99-CAS9 control vector enzyme action results, can obtain two bands of 9197bp, 5180bp, and M is 2kb Marker, stripe size is respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

The plasmid map difference of Cris-GL3 plasmids, PMDC99-CAS9 carriers and carrier pHZ1-CAS9-gRNA-BADH2 See Fig. 3-Fig. 5.

The preparation method of the Oryza sativa L. BADH2 site-directed point mutation plant of embodiment 2

1st, Agrobacterium-mediated Transformation and bacterium colony PCR are identified

Agrobacterium competence is taken from -80 DEG C of refrigerators to thaw on ice, adds 3-5 μ L plasmid pHZ1-CAS9-gRNA- BADH2 is mixed, and ice puts 30min, is put into 1min in liquid nitrogen, and then heat shock 1min in 37 DEG C of water-baths, right in aseptic operating platform 1mL YEP culture medium should be added, on 28 DEG C of shaking tables 2-4h is shaken, be taken out, 8000rpm/min centrifugation 1min, in aseptic operating platform Part supernatant is outwelled, equably remaining suspension is mixed with spreader the YEP being coated in added with rifampicin and kanamycin and is put down On plate, after sealed membrane sealing 28 DEG C of incubator cultures are put into.Picking single bacterium colony after 2d, 28 DEG C are shaken after bacterium 24-36h, carry out bacterium colony PCR, obtains positive bacterium colony.

Bacterium colony PCR：The preparation of template：The bacterium solution of 200 μ L is drawn from test tube in 1.5mL centrifuge tubes, first 8000rpm/ Min centrifugation 1min outwell supernatant, are separately added into the ddH of 50 μ L₂O suspends and precipitates, and is put in boiling water and boils 2min, 12000rpm/min is centrifuged 5min, draws supernatant as template.Positive control：To dilute 20-50 times of μ L of plasmid 1 as template. Negative control：With 1 μ L ddH₂O is template.

2nd, agriculture bacillus mediated rice conversion

(1) mature embryo callus induction

Need to shell after Oryza sativa L. ' Japan is fine ' mature seed threshing, washed 2 minutes with 75% ethanol, then immerse liquor natrii hypochloritises In (active chlorine 5.5%), 30min (can shake twice according to practical situation, each 20min) is shaken on shaking table, washed 5 times, seed Being placed on after process in NBi culture medium is used for callus induction, the dark 10h cultures of illumination 14h/ at 26 DEG C.

(2) calluss are stripped

Mature embryo callus induction needs grow for 15-20 days, in super-clean bench on aseptic filter paper, with tweezers and hook wound healing is stripped Tissue, moves in new NBi culture medium, light culture at 26 DEG C.Wound healing is cultivated in NBi culture medium after peeling and can be used for for 4 days Infect, that what is not infected answers the periodically successive transfer culture in new NBi culture medium, and infection rate can decline after long-time is cultivated.

(3) infect

With liquid-transfering gun, from 100mL agrobacterium suspensions, (formula of the agrobacterium suspension is calculated as by 1L：20 × N6 mother solutions 50mL, 100 × EDTA-Fe mother solution 10mL, 100 × B5 trace element mother solution 10mL, 100 × B5 vitamin stock solution 10mL, CEH 100 μM of 0.3g, inositol 2g, sucrose 30g, AS, pH 5.2~5.3) 800 μ L of middle absorption flushing Agrobacterium bacterium plate three to four times, inhale Take bacterium solution 3~5 to drip in suspension, slightly muddiness can (i.e. bacterial concentration reaches OD₆₀₀For 0.3 or so), calluss are used Tweezers are sandwiched in suspension, timing 15min, are rocked once every three four minutes so as to be fully contacted.Bacterium solution is gone, will be infected The calluss crossed are poured on aseptic filter paper, blot the bacterium solution of its excess surface, wound healing is moved to into NBco after 10min and co-cultures base On, put between tissue culture 26 DEG C, 2~3d of illumination cultivation, visible Agrobacterium grows around calluss.

(4) wound healing and prescreening are cleaned

(5) screen

Wound healing in NBps prescreening culture medium is moved on in NBS screening culture medium.

(6) regeneration culture

After 1 month, the kanamycin-resistant callus tissue number consecutively for growing is placed on regeneration culture medium, the dark 10h of illumination 14h/ at 26 DEG C Culture 3-4 week left and right.

(7) root culture

Kanamycin-resistant callus tissue development in culture medium to be regenerated is grown after 2-3 centimetre of seedling, you can gently pressed from both sides out with tweezers, Be placed on sterilized empty culture dish, remove Seedling around and root culture medium and old wound healing, be then gently placed with 3.5cm Taking root in pipe for high root media, often puts a Seedling in pipe, number consistent with regenerating tube numbering.Illumination 14h/ is black at 26 DEG C Dark 10h cultures 3-4 is all, after seedling grow it is sturdy and after the root system of prosperity by uncap seedling exercising culture, periodically to Guan Zhongjia originally Water.

(8) seedling

Seedling length continues to be put into and takes root in pipe simultaneously to upright blade and during well developed root system after seedling root culture medium is cleaned Tap water is added, is planted in soil after seedling is sufficiently robust.

3rd, BADH2 knocks out effect detection

The each 200bp-400bp sequences of target site upstream and downstream are chosen, primer is designed, enters performing PCR amplification, amplified fragments send survey Sequence, judges whether target site undergos mutation by peak figure.The primer sequence of identification BADH2 mutation effects is as follows, clip size 494bp。

JDSQ3F1：5’-ACCTATCGCTTTCCACCTCA-3’

JDSQ3R1：5’-CGGTTCCTCTTCAGCGCCTC-3’

Amplification is shown in Fig. 6, and the 1st swimming lane represents that the 1st Seedling T0 of No. 107 strain is for plant in BADH2 transfer-gen plants, FZCX3-1 is named as, the 2nd swimming lane represents that the 2nd Seedling T0 of No. 132 strain is for plant in BADH2 transfer-gen plants, by it It is named as FZCX3-2；+ Japanese fine genome positive control is represented ,-represent ddH₂O negative controls, M be 2kb Marker, stripe size Respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Sequencing is sent by the PCR fragment of above-mentioned BADH2, sequencing primer is JDSQ3F1, FZCX3-1, FZCX3-2 plant (T0 Generation) sequencing result show BADH2 occur near PAM sites (i.e. CGG sequences) mutation.

FZCX3-1 and FZCX3-2 plant sequencing peak figure is shown in respectively Fig. 7 A and Fig. 7 B.By sequencing result in DSDecode websites (http://dsdecode.scgene.com/home/) in carry out the prediction of mutation type, as a result see Fig. 8.

It follows that BADH2 is compound heterozygote mutation in FZCX3-1 plant.Wherein one chain lacks 4 bases, separately One chain is the mutation of 5 bases；BADH2 is also compound heterozygote mutation in FZCX3-2 plant.Wherein one chain lacks 4 In base, deletion sites and FZCX3-1, another chain is the mutation of 5 bases, and mutated site part is different.

The disappearance of 4 bases causes frameshit, translation to terminate in advance in FZCX3-1 and FZCX3-2, base sequence and derives ammonia Base acid sequence is as follows：

ATGGCCACGGCGATCCCGCAGCGGCAGCTCTTCGTCGCCGGCGAGTGGCGCGCCCGCTCGGCCGCCGCC TCCCCGTCGTCAACCCCGCCACCGAGTCCCCCATCGGCGAGATCCCGGCGGGCACGGCGGAGGACGTGGACGCGGCG GTGGCGGCGGCGCGGGAGGCGCTGAAGAGGAACCGGGGCCGCGACTGGGCGCGCGCGCCGGGCGCCGTCCGGGCCAA GTACCTCCGCGCAATCGCGGCCAAGATAATCGAGAGGAAATCTGAGCTGGCTAGACTAGAGACGCTTGATTGTGGGA AGCCTCTTGATGAAGCAGCATGGGACATGGACGATGTTGCTGGATGCTTTGAGTACTTTGCAGATCTTGCAGAATCC TTGGACAAAAGGCAAAATGCACCTGTCTCTCTTCCAATGGAAAACTTTAAATGCTATCTTCGGAAAGAGCCTATCGG TGTAGTTGGGTTGATCACACCTTGGAACTATCCTCTCCTGATGGCAACATGGAAGGTAGCTCCTGCCCTGGCTGCTG GCTGTACAGCTGTACTAAAACCATCTGAATTGGCTTCCGTGACTTGTTTGGAGCTTGCTGATGTGTGTAAAGAGGTT GGTCTTCCTTCAGGTGTGCTAAACATAGTGACTGGATTAGGTTCTGAAGCCGGTGCTCCTTTGTCATCACACCCTGG TGTAGACAAGGTTGCATTTACTGGGAGTTATGAAACTGGTAAAAAGATTATGGCTTCAGCTGCTCCTATGGTTAAGC CTGTTTCACTGGAACTTGGTGGAAAAAGTCCTATAGTGGTGTTTGATGATGTTGATGTTGAAAAAGCTGTTGAGTGG ACTCTCTTTGGTTGCTTTTGGACCAATGGCCAGATTTGCAGTGCAACATCGCGTCTTATTCTTCATAAAAAAATCGC TAAAGAATTTCAAGAAAGGATGGTTGCATGGGCCAAAAATATTAAGGTGTCAGATCCACTTGAAGAGGGTTGCAGGC TTGGGCCCGTTGTTAGTGAAGGACAGTATGAGAAGATTAAGCAATTTGTATCTACCGCCAAAAGCCAAGGTGCTACC ATTCTGACTGGTGGGGTTAGACCCAAGCATCTGGAGAAAGGTTTCTATATTGAACCCACAATCATTACTGATGTCGA TACATCAATGCAAATTTGGAGGGAAGAAGTTTTTGGTCCAGTGCTCTGTGTGAAAGAATTTAGCACTGAAGAAGAAG CCATTGAATTGGCCAACGATACTCATTATGGTCTGGCTGGTGCTGTGCTTTCCGGTGACCGCGAGCGATGCCAGAGA TTAACTGAGGAGATCGATGCCGGAATTATCTGGGTGAACTGCTCGCAACCCTGCTTCTGCCAAGCTCCATGGGGCGG GAACAAGCGCAGCGGCTTTGGACGCGAGCTCGGAGAAGGGGGCATTGACAACTACCTAAGCGTCAAGCAAGTGACGG AGTACGCCTCCGATGAGCCGTGGGGATGGTACAAATCCCCTTCCAAGCTGTAA

MATAIPQRQLFVAGEWRARSAAASPSSTPPPSPPSARSRRARRRTWTRRWRRRGRR.RGTGAATGRARRAPSGPSTS AQSRPR.SRGNLSWLD.RRLIVGSLLMKQHGTWTMLLDALSTLQILQNPWTKGKMHLSLFQWKTLNAIFGKSLSV.L G.SHLGTILS.WQHGR.LLPWLLAVQLY.NHLNWLP.LVWSLLMCVKRLVFLQVC.T..LD.VLKPVLLCHHTLV.T RLHLLGVMKLVKRLWLQLLLWLSLFHWNLVEKVL.WCLMMLMLKKLLSGLSLVAFGPMARFAVQHRVLFFIKKSLKN FKKGWLHGPKILRCQIHLKRVAGLGPLLVKDSMRRLSNLYLPPKAKVLPF.LVGLDPSIWRKVSILNPQSLLMSIHQ CKFGGKKFLVQCSV.KNLALKKKPLNWPTILIMVWLVLCFPVTASDARD.LRRSMPELSG.TARNPASAKLHGAGTS AAALDASSEKGALTTT.ASSK.RSTPPMSRGDGTNPLPSC

The mutation of 5 bases in FZCX3-1 causes 3 continuous amino acid mutation, its base sequence and derivation aminoacid sequence Row are as follows：

ATGGCCACGGCGATCCCGCAGCGGCAGCTCTTCGTCGCCGGCGAGTGGCGCGCCCCCGGGCGGCGGCGC CGCCTCCCCGTCGTCAACCCCGCCACCGAGTCCCCCATCGGCGAGATCCCGGCGGGCACGGCGGAGGACGTGGACGC GGCGGTGGCGGCGGCGCGGGAGGCGCTGAAGAGGAACCGGGGCCGCGACTGGGCGCGCGCGCCGGGCGCCGTCCGGG CCAAGTACCTCCGCGCAATCGCGGCCAAGATAATCGAGAGGAAATCTGAGCTGGCTAGACTAGAGACGCTTGATTGT GGGAAGCCTCTTGATGAAGCAGCATGGGACATGGACGATGTTGCTGGATGCTTTGAGTACTTTGCAGATCTTGCAGA ATCCTTGGACAAAAGGCAAAATGCACCTGTCTCTCTTCCAATGGAAAACTTTAAATGCTATCTTCGGAAAGAGCCTA TCGGTGTAGTTGGGTTGATCACACCTTGGAACTATCCTCTCCTGATGGCAACATGGAAGGTAGCTCCTGCCCTGGCT GCTGGCTGTACAGCTGTACTAAAACCATCTGAATTGGCTTCCGTGACTTGTTTGGAGCTTGCTGATGTGTGTAAAGA GGTTGGTCTTCCTTCAGGTGTGCTAAACATAGTGACTGGATTAGGTTCTGAAGCCGGTGCTCCTTTGTCATCACACC CTGGTGTAGACAAGGTTGCATTTACTGGGAGTTATGAAACTGGTAAAAAGATTATGGCTTCAGCTGCTCCTATGGTT AAGCCTGTTTCACTGGAACTTGGTGGAAAAAGTCCTATAGTGGTGTTTGATGATGTTGATGTTGAAAAAGCTGTTGA GTGGACTCTCTTTGGTTGCTTTTGGACCAATGGCCAGATTTGCAGTGCAACATCGCGTCTTATTCTTCATAAAAAAA TCGCTAAAGAATTTCAAGAAAGGATGGTTGCATGGGCCAAAAATATTAAGGTGTCAGATCCACTTGAAGAGGGTTGC AGGCTTGGGCCCGTTGTTAGTGAAGGACAGTATGAGAAGATTAAGCAATTTGTATCTACCGCCAAAAGCCAAGGTGC TACCATTCTGACTGGTGGGGTTAGACCCAAGCATCTGGAGAAAGGTTTCTATATTGAACCCACAATCATTACTGATG TCGATACATCAATGCAAATTTGGAGGGAAGAAGTTTTTGGTCCAGTGCTCTGTGTGAAAGAATTTAGCACTGAAGAA GAAGCCATTGAATTGGCCAACGATACTCATTATGGTCTGGCTGGTGCTGTGCTTTCCGGTGACCGCGAGCGATGCCA GAGATTAACTGAGGAGATCGATGCCGGAATTATCTGGGTGAACTGCTCGCAACCCTGCTTCTGCCAAGCTCCATGGG GCGGGAACAAGCGCAGCGGCTTTGGACGCGAGCTCGGAGAAGGGGGCATTGACAACTACCTAAGCGTCAAGCAAGTG ACGGAGTACGCCTCCGATGAGCCGTGGGGATGGTACAAATCCCCTTCCAAGCTGTAA

MATAIPQRQLFVAGEWRAPGRRRRLPVVNPATESPIGEIPAGTAEDVDAAVAAAREALKRNRGRDWARA PGAVRAKYLRAIAAKIIERKSELARLETLDCGKPLDEAAWDMDDVAGCFEYFADLAESLDKRQNAPVSLPMENFKCY LRKEPIGVVGLITPWNYPLLMATWKVAPALAAGCTAVLKPSELASVTCLELADVCKEVGLPSGVLNIVTGLGSEAGA PLSSHPGVDKVAFTGSYETGKKIMASAAPMVKPVSLELGGKSPIVVFDDVDVEKAVEWTLFGCFWTNGQICSATSRL ILHKKIAKEFQERMVAWAKNIKVSDPLEEGCRLGPVVSEGQYEKIKQFVSTAKSQGATILTGGVRPKHLEKGFYIEP TIITDVDTSMQIWREEVFGPVLCVKEFSTEEEAIELANDTHYGLAGAVLSGDRERCQRLTEEIDAGIIWVNCSQPCF CQAPWGGNKRSGFGRELGEGGIDNYLSVKQVTEYASDEPWGWYKSPSKL

The mutation of 5 bases in FZCX3-2 causes 2 amino acid mutations, and its base sequence and derivation aminoacid sequence are such as Under：

ATGGCCACGGCGATCCCGCAGCGGCAGCTCTTCGTCGCCGGCGAGTGGCGCGCCCCGGGGCTGCGGCGC CGCCTCCCCGTCGTCAACCCCGCCACCGAGTCCCCCATCGGCGAGATCCCGGCGGGCACGGCGGAGGACGTGGACGC GGCGGTGGCGGCGGCGCGGGAGGCGCTGAAGAGGAACCGGGGCCGCGACTGGGCGCGCGCGCCGGGCGCCGTCCGGG CCAAGTACCTCCGCGCAATCGCGGCCAAGATAATCGAGAGGAAATCTGAGCTGGCTAGACTAGAGACGCTTGATTGT GGGAAGCCTCTTGATGAAGCAGCATGGGACATGGACGATGTTGCTGGATGCTTTGAGTACTTTGCAGATCTTGCAGA ATCCTTGGACAAAAGGCAAAATGCACCTGTCTCTCTTCCAATGGAAAACTTTAAATGCTATCTTCGGAAAGAGCCTA TCGGTGTAGTTGGGTTGATCACACCTTGGAACTATCCTCTCCTGATGGCAACATGGAAGGTAGCTCCTGCCCTGGCT GCTGGCTGTACAGCTGTACTAAAACCATCTGAATTGGCTTCCGTGACTTGTTTGGAGCTTGCTGATGTGTGTAAAGA GGTTGGTCTTCCTTCAGGTGTGCTAAACATAGTGACTGGATTAGGTTCTGAAGCCGGTGCTCCTTTGTCATCACACC CTGGTGTAGACAAGGTTGCATTTACTGGGAGTTATGAAACTGGTAAAAAGATTATGGCTTCAGCTGCTCCTATGGTT AAGCCTGTTTCACTGGAACTTGGTGGAAAAAGTCCTATAGTGGTGTTTGATGATGTTGATGTTGAAAAAGCTGTTGA GTGGACTCTCTTTGGTTGCTTTTGGACCAATGGCCAGATTTGCAGTGCAACATCGCGTCTTATTCTTCATAAAAAAA TCGCTAAAGAATTTCAAGAAAGGATGGTTGCATGGGCCAAAAATATTAAGGTGTCAGATCCACTTGAAGAGGGTTGC AGGCTTGGGCCCGTTGTTAGTGAAGGACAGTATGAGAAGATTAAGCAATTTGTATCTACCGCCAAAAGCCAAGGTGC TACCATTCTGACTGGTGGGGTTAGACCCAAGCATCTGGAGAAAGGTTTCTATATTGAACCCACAATCATTACTGATG TCGATACATCAATGCAAATTTGGAGGGAAGAAGTTTTTGGTCCAGTGCTCTGTGTGAAAGAATTTAGCACTGAAGAA GAAGCCATTGAATTGGCCAACGATACTCATTATGGTCTGGCTGGTGCTGTGCTTTCCGGTGACCGCGAGCGATGCCA GAGATTAACTGAGGAGATCGATGCCGGAATTATCTGGGTGAACTGCTCGCAACCCTGCTTCTGCCAAGCTCCATGGG GCGGGAACAAGCGCAGCGGCTTTGGACGCGAGCTCGGAGAAGGGGGCATTGACAACTACCTAAGCGTCAAGCAAGTG ACGGAGTACGCCTCCGATGAGCCGTGGGGATGGTACAAATCCCCTTCCAAGCTGTAA

MATAIPQRQLFVAGEWRAPGLRRRLPVVNPATESPIGEIPAGTAEDVDAAVAAAREALKRNRGRDWARAPGAVRAKY LRAIAAKIIERKSELARLETLDCGKPLDEAAWDMDDVAGCFEYFADLAESLDKRQNAPVSLPMENFKCYLRKEPIGV VGLITPWNYPLLMATWKVAPALAAGCTAVLKPSELASVTCLELADVCKEVGLPSGVLNIVTGLGSEAGAPLSSHPGV DKVAFTGSYETGKKIMASAAPMVKPVSLELGGKSPIVVFDDVDVEKAVEWTLFGCFWTNGQICSATSRLILHKKIAK EFQERMVAWAKNIKVSDPLEEGCRLGPVVSEGQYEKIKQFVSTAKSQGATILTGGVRPKHLEKGFYIEPTIITDVDT SMQIWREEVFGPVLCVKEFSTEEEAIELANDTHYGLAGAVLSGDRERCQRLTEEIDAGIIWVNCSQPCFCQAPWGGN KRSGFGRELGEGGIDNYLSVKQVTEYASDEPWGWYKSPSKL.

The present invention successfully builds and obtains heterozygous BADH2 Mutant Rice plant FZCX3-1, FZCX3-2.Using carrier PHZ1-CAS9-gRNA-BADH2 is 50% to the efficiency of Oryza sativa L. BADH2 site-directed point mutations.

Although above with a general description of the specific embodiments the present invention is described in detail, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, without departing from theon the basis of the spirit of the present invention these modifications or improvements, belong to the scope of protection of present invention.

Sequence table

<110>Hua Zhi rice biologicals Technology Co., Ltd.

<120>Using method of the CRISPR-CAS9 technologies to Oryza sativa L. BADH2 site-directed point mutations

<130> KHP161117616.2

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213>Oryza sativa L.

<400> 1

gagtggcgcg cccccgcgct 20

<210> 2

<211> 347

<212> DNA

<213>OsU6-BADH2-gRNA-polyT fragments

<400> 2

ggatcatgaa ccaacggcct ggctgtattt ggtggttgtg tagggagatg gggagaagaa 60

aagcccgatt ctcttcgctg tgatgggctg gatgcatgcg ggggagcggg aggcccaagt 120

acgtgcacgg tgagcggccc acagggcgag tgtgagcgcg agaggcggga ggaacagttt 180

agtaccacat tgcccagcta actcgaacgc gaccaactta taaacccgcg cgctgtcgct 240

tgtgtgagtg gcgcgccccc gcgctgtttt agagctagaa atagcaagtt aaaataaggc 300

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttt 347

<210> 3

<211> 14832

<212> DNA

<213>PHZ1-CAS9-gRNA-BADH2 carriers

<400> 3

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 60

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 120

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 180

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattggctag agcagcttgc 240

caacatggtg gagcacgaca ctctcgtcta ctccaagaat atcaaagata cagtctcaga 300

agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc tcctcggatt 360

ccattgccca gctatctgtc acttcatcaa aaggacagta gaaaaggaag gtggcaccta 420

caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgcctctg ccgacagtgg 480

tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 540

gtcttcaaag caagtggatt gatgtgataa catggtggag cacgacactc tcgtctactc 600

caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 660

ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact tcatcaaaag 720

gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag gaaaggctat 780

cgttcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat 840

cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgatatctc 900

cactgacgta agggatgacg cacaatccca ctatccttcg caagaccttc ctctatataa 960

ggaagttcat ttcatttgga gaggacacgc tgaaatcacc agtctctctc tacaaatcta 1020

tctctctcga gctttcgcag atcccggggg gcaatgagat atgaaaaagc ctgaactcac 1080

cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 1140

gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt 1200

cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt 1260

tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggagttta gcgagagcct 1320

gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga 1380

actgcccgct gttctacaac cggtcgcgga ggctatggat gcgatcgctg cggccgatct 1440

tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg 1500

gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga 1560

cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga 1620

ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga 1680

caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata 1740

cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg 1800

ctacttcgag cggaggcatc cggagcttgc aggatcgcca cgactccggg cgtatatgct 1860

ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 1920

ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac 1980

acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga 2040

tagtggaaac cgacgcccca gcactcgtcc gagggcaaag aaatagagta gatgccgacc 2100

ggatctgtcg atcgacaagc tcgagtttct ccataataat gtgtgagtag ttcccagata 2160

agggaattag ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt 2220

atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat 2280

ccagtactaa aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg 2340

tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc 2400

caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag 2460

gcagcccatc agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc 2520

tcatgttacc gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat 2580

gatctcgcgg agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca 2640

ccgcggtttc aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt 2700

tgaaaaagct gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt 2760

cgtcttgtta taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa 2820

taataaatgg ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc 2880

tgcgtaaaag atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat 2940

gaaaacctat atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa 3000

cgggaaaagg acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac 3060

tttgaacggc atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc 3120

tcggaagagt atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc 3180

atcaggctct ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc 3240

cgcttagccg aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac 3300

tgggaagaag acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa 3360

aagcccgaag aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg 3420

aaagatggca aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg 3480

tatgacattg ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc 3540

gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt 3600

ttactggatg aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt 3660

tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 3720

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 3780

ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 3840

ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 3900

gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3960

aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4020

ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4080

agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4140

aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4200

aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4260

ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4320

cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4380

tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 4440

accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 4500

cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 4560

gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 4620

cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 4680

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 4740

catcaccgaa acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac 4800

ggcgcggctt gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg 4860

ccagcggccg cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg 4920

gtaggcgctt tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg 4980

cgggttttaa gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc 5040

tcttttatat cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca 5100

atgtacgggt tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca 5160

ggaaagagac cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc 5220

gtacattagg aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg 5280

atcgggccag cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg 5340

atcagcttgc gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg 5400

tagatcgtct tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg 5460

tagagaaaac ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg 5520

tccgggttct tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg 5580

tactccggcc gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc 5640

tcggataccg tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc 5700

gtggtgttta accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct 5760

cgccggcaga acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct 5820

cccttccctt cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc 5880

aggtcgtaat cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg 5940

tctggaagct cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa 6000

acggccacgt ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg 6060

aaaaaatctg gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc 6120

ggcggttgcc gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg 6180

cgtgcttctg cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc 6240

aggtcatcac ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgtcacgc 6300

cgattcctcg ggcttggggg ttccagtgcc attgcagggc cggcagacaa cccagccgct 6360

tacgcctggc caaccgcccg ttcctccaca catggggcat tccacggcgt cggtgcctgg 6420

ttgttcttga ttttccatgc cgcctccttt agccgctaaa attcatctac tcatttattc 6480

atttgctcat ttactctggt agctgcgcga tgtattcaga tagcagctcg gtaatggtct 6540

tgccttggcg taccgcgtac atcttcagct tggtgtgatc ctccgccggc aactgaaagt 6600

tgacccgctt catggctggc gtgtctgcca ggctggccaa cgttgcagcc ttgctgctgc 6660

gtgcgctcgg acggccggca cttagcgtgt ttgtgctttt gctcattttc tctttacctc 6720

attaactcaa atgagttttg atttaatttc agcggccagc gcctggacct cgcgggcagc 6780

gtcgccctcg ggttctgatt caagaacggt tgtgccggcg gcggcagtgc ctgggtagct 6840

cacgcgctgc gtgatacggg actcaagaat gggcagctcg tacccggcca gcgcctcggc 6900

aacctcaccg ccgatgcgcg tgcctttgat cgcccgcgac acgacaaagg ccgcttgtag 6960

ccttccatcc gtgacctcaa tgcgctgctt aaccagctcc accaggtcgg cggtggccca 7020

tatgtcgtaa gggcttggct gcaccggaat cagcacgaag tcggctgcct tgatcgcgga 7080

cacagccaag tccgccgcct ggggcgctcc gtcgatcact acgaagtcgc gccggccgat 7140

ggccttcacg tcgcggtcaa tcgtcgggcg gtcgatgccg acaacggtta gcggttgatc 7200

ttcccgcacg gccgcccaat cgcgggcact gccctgggga tcggaatcga ctaacagaac 7260

atcggccccg gcgagttgca gggcgcgggc tagatgggtt gcgatggtcg tcttgcctga 7320

cccgcctttc tggttaagta cagcgataac cttcatgcgt tccccttgcg tatttgttta 7380

tttactcatc gcatcatata cgcagcgacc gcatgacgca agctgtttta ctcaaataca 7440

catcaccttt ttagacggcg gcgctcggtt tcttcagcgg ccaagctggc cggccaggcc 7500

gccagcttgg catcagacaa accggccagg atttcatgca gccgcacggt tgagacgtgc 7560

gcgggcggct cgaacacgta cccggccgcg atcatctccg cctcgatctc ttcggtaatg 7620

aaaaacggtt cgtcctggcc gtcctggtgc ggtttcatgc ttgttcctct tggcgttcat 7680

tctcggcggc cgccagggcg tcggcctcgg tcaatgcgtc ctcacggaag gcaccgcgcc 7740

gcctggcctc ggtgggcgtc acttcctcgc tgcgctcaag tgcgcggtac agggtcgagc 7800

gatgcacgcc aagcagtgca gccgcctctt tcacggtgcg gccttcctgg tcgatcagct 7860

cgcgggcgtg cgcgatctgt gccggggtga gggtagggcg ggggccaaac ttcacgcctc 7920

gggccttggc ggcctcgcgc ccgctccggg tgcggtcgat gattagggaa cgctcgaact 7980

cggcaatgcc ggcgaacacg gtcaacacca tgcggccggc cggcgtggtg gtgtcggccc 8040

acggctctgc caggctacgc aggcccgcgc cggcctcctg gatgcgctcg gcaatgtcca 8100

gtaggtcgcg ggtgctgcgg gccaggcggt ctagcctggt cactgtcaca acgtcgccag 8160

ggcgtaggtg gtcaagcatc ctggccagct ccgggcggtc gcgcctggtg ccggtgatct 8220

tctcggaaaa cagcttggtg cagccggccg cgtgcagttc ggcccgttgg ttggtcaagt 8280

cctggtcgtc ggtgctgacg cgggcatagc ccagcaggcc agcggcggcg ctcttgttca 8340

tggcgtaatg tctccggttc tagtcgcaag tattctactt tatgcgacta aaacacgcga 8400

caagaaaacg ccaggaaaag ggcagggcgg cagcctgtcg cgtaacttag gacttgtgcg 8460

acatgtcgtt ttcagaagac ggctgcactg aacgtcagaa gccgactgca ctatagcagc 8520

ggaggggttg gatcaaagta ctttgatccc gaggggaacc ctgtggttgg catgcacata 8580

caaatggacg aacggataaa ccttttcacg cccttttaaa tatccgttat tctaataaac 8640

gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 8700

aaggcgggaa acgacaatct gatccaagct caagctgctc tagcattcgc cattcaggct 8760

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 8820

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 8880

ttgtaaaacg acggccagtg ccaagctctc ggatccacta gtaacggccg ccagtgtgct 8940

ggaattgccc ttggatcatg aaccaacggc ctggctgtat ttggtggttg tgtagggaga 9000

tggggagaag aaaagcccga ttctcttcgc tgtgatgggc tggatgcatg cgggggagcg 9060

ggaggcccaa gtacgtgcac ggtgagcggc ccacagggcg agtgtgagcg cgagaggcgg 9120

gaggaacagt ttagtaccac attgcccagc taactcgaac gcgaccaact tataaacccg 9180

cgcgctgtcg cttgtgtgag tggcgcgccc ccgcgctgtt ttagagctag aaatagcaag 9240

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 9300

gtcccttcga agggcaattc tgcagatatc catcacactg gcggccgctc gaggtcgaag 9360

cttgcatgcc tgcaggtcaa catggtggag cacgacacac ttgtctactc caaaaatatc 9420

aaagatacag tctcagaaga ccaaagggca attgagactt ttcaacaaag ggtaatatcc 9480

ggaaacctcc tcggattcca ttgcccagct atctgtcact ttattgtgaa gatagtggaa 9540

aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggccat cgttgaagat 9600

gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa 9660

gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgataacat ggtggagcac 9720

gacacacttg tctactccaa aaatatcaaa gatacagtct cagaagacca aagggcaatt 9780

gagacttttc aacaaagggt gatatccgga aacctcctcg gattccattg cccagctatc 9840

tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc 9900

gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc 9960

ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 10020

gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa 10080

gacccttcct ctatataagg aagttcattt catttggaga ggacctcgac ctcaacacaa 10140

catatacaaa acaaacgaat ctcaagcaat caagcattct acttctattg cagcaattta 10200

aatcatttct tttaaagcaa aagcaatttt ctgaaaattt tcaccattta cgaacgatac 10260

tcgagatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag 10320

acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag 10380

cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg 10440

tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc 10500

ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg 10560

aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct 10620

gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca 10680

gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg 10740

gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa 10800

agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc 10860

acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg 10920

acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc 10980

ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca 11040

gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa 11100

acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg 11160

aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg 11220

cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca 11280

tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct 11340

ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc 11400

ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg 11460

ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc 11520

tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc 11580

ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc 11640

acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga 11700

tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca 11760

gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg 11820

aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata 11880

agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg 11940

tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc 12000

tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga 12060

ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa 12120

tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa 12180

ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg 12240

tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg 12300

cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg 12360

gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc 12420

tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg 12480

acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc 12540

tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga 12600

cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg 12660

tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga 12720

gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc 12780

ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc 12840

gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc 12900

atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa 12960

gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga 13020

agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc 13080

tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac 13140

agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga 13200

acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt 13260

ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca 13320

actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa 13380

agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga 13440

agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca 13500

gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc 13560

ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt 13620

ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg 13680

tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga 13740

tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg 13800

cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg 13860

tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg 13920

actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta 13980

agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac 14040

tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca 14100

gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg 14160

aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag 14220

tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata 14280

agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag 14340

cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca 14400

aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga 14460

tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag gccggccagg 14520

caaaaaagaa aaagtaagga tcctgattga tcgatagagc tcgaatttcc ccgatcgttc 14580

aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 14640

catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt 14700

atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 14760

aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 14820

agatcgggaa tt 14832

<210> 4

<211> 41

<212> DNA

<213>Artificial sequence

<400> 4

cgacggccag tgccaagctt ctcggatcca ctagtaacgg c 41

<210> 5

<211> 41

<212> DNA

<213>Artificial sequence

<400> 5

agcgcggggg cgcgccactc acacaagcga cagcgcgcgg g 41

<210> 6

<211> 41

<212> DNA

<213>Artificial sequence

<400> 6

gagtggcgcg cccccgcgct gttttagagc tagaaatagc a 41

<210> 7

<211> 41

<212> DNA

<213>Artificial sequence

<400> 7

acctgcaggc atgcaagctt cgacctcgag cggccgccag t 41

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<400> 8

acctatcgct ttccacctca 20

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<400> 9

cggttcctct tcagcgcctc 20

Claims

1. method of the CRISPR-CAS9 technologies to Oryza sativa L. BADH2 site-directed point mutations is utilized, it is characterised in that for Oryza sativa L. SgRNA sequence of the BADH2 gene design based on CRISPR/Cas9, by the DNA fragmentation connection containing the coding sgRNA sequences To in the carrier for carrying CRISPR/Cas, rice transformation realizes the rite-directed mutagenesises to Oryza sativa L. BADH2 genes；

Wherein, the nucleotides sequence of sgRNA action sites is classified as 5 '-GAGTGGCGCGCCCCCGCGCT-3 '.

2. method according to claim 1, it is characterised in that carrier construction pHZ1-CAS9-gRNA-BADH2, using agriculture Bacillus mediated method rice transformation, screens positive transgenic plant；

The construction method of the carrier pHZ1-CAS9-gRNA-BADH2 is as follows：

1) structure of OsU6-BADH2-gRNA-polyT fragments

First round PCR expands OsU6 promoteres and BADH2 bases with Cris-GL3 plasmids as template with primer CRI-F1 and SQ3-R1 The 20bp target sequences of cause, clip size 329bp；Equally with Cris-GL3 plasmids as template, expanded with primer SQ3-F2 and CRI-R2 Increase the 20bp target sequences and gRNA-polyT of BADH2 genes, clip size 180bp；Second wheel PCR is with first round PCR primer For template, with primer CRI-F1 and CRI-R2 amplification OsU6-BADH2-gRNA-polyT fragments, clip size 489bp, sequence are obtained final product Row such as SEQ ID NO:Shown in 2；

2) structure of carrier pHZ1-CAS9-gRNA-BADH2

OsU6-BADH2-gRNA-polyT fragments are carried out into respectively fine jade with the PMDC99-Cas9 carriers Jing after Hind III digestions Sepharose electrophoresis, cuts and carry out after glue reclaim seamless clone, based on the principle of homologous recombination, OsU6-BADH2-gRNA-polyT Fragment is fused to the Hind III sites of PMDC99-CAS9 carriers by the method for Infusion, that is, build and obtain pHZ1- CAS9-gRNA-BADH2 carriers；

Wherein, step 1) used in primer sequence it is as follows：

CRI-F1：5’-CGACGGCCAGTGCCAAGCTTCTCGGATCCACTAGTAACGGC-3’

SQ3-R1：5’-AGCGCGGGGGCGCGCCACTCACACAAGCGACAGCGCGCGGG-3’

SQ3-F2：5’-GAGTGGCGCGCCCCCGCGCTGTTTTAGAGCTAGAAATAGCA-3’

CRI-R2:5’-ACCTGCAGGCATGCAAGCTTCGACCTCGAGCGGCCGCCAGT-3’。

3. method according to claim 2, it is characterised in that the nucleotide of the pHZ1-CAS9-gRNA-BADH2 carriers Sequence such as SEQ ID NO:Shown in 3.

4. application of any one of the claim 1-3 methods described in Oryza sativa L. BADH2 site-directed point mutation breedings.

5. application according to claim 4, it is characterised in that comprise the following steps：

(1) Oryza sativa L. ' Japan is fine ' mature embryo callus induction；

(2) infect in the Agrobacterium bacterium solution that the calluss immersion of induction is carried pHZ1-CAS9-gRNA-BADH2 carriers；It is excellent Choosing is EHA105 using agrobacterium strains；

(3) calluss are moved to and is cultivated in co-cultivation culture medium, washed away and move to after Agrobacterium culture in prescreening culture medium, then Move to and carry out in screening culture medium resistant calli screening；

(6) primer is designed according to the nucleotide sequence of sgRNA action sites, rice plant mutational site is identified by PCR methods.

6. application according to claim 5, it is characterised in that step (1) is specially：Shell after mature seed threshing, use 75% ethanol is washed 2 minutes, then is immersed in the liquor natrii hypochloritises of active chlorine content 5.5%, and 30min, washing 5 are shaken on shaking table Secondary, being placed in after seed treatment in NBi culture medium is used for callus induction, and the dark 10h of 26 DEG C of illumination 14h/ is cultivated 15-20 days；Strip more Injured tissue, moves in new NBi culture medium, and 26 DEG C of light cultures are used to infect after 4 days.

7. the application according to claim 5 or 6, it is characterised in that

The formula for co-culturing culture medium is calculated as by 2L：

20 × N6 mother solution 100mL, 100 × EDTA-Fe mother solution 20mL, 100 × B5 trace element mother solution 20mL, 100 × B5 dimension life Plain mother solution 20mL, glutamine 1g, proline 1g, CEH 0.6g, inositol 0.2g, sucrose 60g, 2,4-D 4mg, 3 ', 5 '-diformazan Epoxide -4 ' 100 μM of-hydroxy acetophenone, plant gum 5.2g, pH 5.2~5.3；

The prescreening culture medium prescription is：

NBi culture medium containing 500mg/L cephamycins, pH=5.8；

The formula of the screening culture medium is：

The NBi culture medium of the cephamycin of the hygromycin containing final concentration of 40mg/L and final concentration of 500mg/L, pH 5.8；

It is described regeneration culture formula be：

NBi culture medium without 2,4-D, the 6-BA containing final concentration of 3mg/L, the NAA of final concentration of 0.5mg/L and final concentration For the hygromycin of 30mg/L, pH 5.8；

The formula of the root media is calculated as by 2L：

20 × MS a great number of elements mother solution 50mL, 100 × EDTA-Fe mother solution 10mL, 100 × MS trace element mother solution 10mL, 100 × B5 vitamin stock solution 20mL, inositol 0.2g, glucose 14g, sucrose 6g, plant gum 5.2g, hygromycin final concentration 40mg/L, pH 5.8；

Wherein, the formula of the NBi culture medium is calculated as by 2L：

20 × N6 mother solution 100mL, 100 × EDTA-Fe mother solution 20mL, 100 × B5 trace element mother solution 20mL, 100 × B5 dimension life Plain mother solution 20mL, glutamine 1g, proline 1g, CEH 0.6g, inositol 0.2g, sucrose 60g, 2,4-D 4mg, plant gum 5.2g, pH5.8；

The formula of 20 × N6 mother solutions is calculated as by 1L：

KNO₃：56.6g, (NH₄)₂SO₄：9.26g, KH₂PO₄：8.00g, MgSO₄·7H₂O：3.70g, CaCl₂·2H₂O：3.32g；

The formula of 100 × EDTA-Fe mother solutions is calculated as by 1L：

FeSO₄·7H₂O 2.78g, Na₂-EDTA 3.73g；

MnSO₄·H₂O 1.0g, ZnSO₄·7H₂O 0.2g, H₃BO₃0.3g, CuSO₄·5H₂O 2.5mg, Na₂MoO₄·2H₂O 25.0mg, CoCl₂·6H₂O 2.5mg, KI 75.0mg；

The formula of 100 × B5 vitamin stock solutions is calculated as by 500mL：

Thiamine hydrochloride VB1 500mg, pyridoxine hydrochloride VB6 50mg, nicotinic acid VB5 50mg；

KNO₃38.0g, NH₄NO₃33.0g, KH₂PO₄3.4g, MgSO₄·7H₂O 7.4g, CaCl₂·2H₂O 8.8g；

MnSO₄·H₂O 1.56g, ZnSO₄·7H₂O 0.86g, H₃BO₃0.62g, CuSO₄·5H₂O 2.5mg, Na₂MoO₄· 2H₂O 25.0mg, CoCl₂·6H₂O 2.5mg, KI 83.0mg.

8. the application according to any one of claim 5-7, it is characterised in that the primer sequence of step (6) is as follows：

JDSQ3F1：5’-ACCTATCGCTTTCCACCTCA-3’

JDSQ3R1：5’-CGGTTCCTCTTCAGCGCCTC-3’.

9. the CRISPR-CAS9 carriers of Oryza sativa L. BADH2 site-directed point mutations are used for, it is characterised in that the nucleotide of the carrier Sequence such as SEQ ID NO:Shown in 3.