CN110257406A - The Plant Nme2Cas9 gene of codon vegetalization transformation and its application - Google Patents

Abstract

The present invention relates to biotechnologys and field of plant genetic project technology, disclose Plant Nme2Cas9 gene and its application of the transformation of codon vegetalization.The Plant Nme2Cas9 gene of the codon vegetalization transformation has nucleotide sequence shown in SEQ ID No:1.The Plant Nme2Cas9 gene of codon vegetalization provided by the invention transformation is to be transformed based on crop rice codon to obtain in mode, i.e. under the premise of maintaining encoding amino acid sequence constant, original codon is replaced using the codon that inventor is filtered out from paddy gene corresponding codons, the Plant Nme2Cas9 gene of vegetalization transformation is obtained, using chemical synthesis.Shear efficiency can be significantly improved using gene of the invention.

Description

The Plant Nme2Cas9 gene of codon vegetalization transformation and its application

Technical field

The present invention relates to biotechnologys and field of plant genetic project technology.Specifically, the present invention relates to a kind of codons The Plant Nme2Cas9 gene of vegetalization transformation, the table of the Plant Nme2Cas9 gene containing codon vegetalization transformation Up to box, expression vector, targeting vector, transgenic cell and their application.

Background technique

CRISPR- nucleic acid zymotechnic is a kind of eucaryote specific site gene editing technology developed in recent years.It should Nuclease involved in technology is presently found mainly two major classes, and applied to gene editing technology typically from second Class.There are mainly three types of types for second class CRISPR- nuclease: TypeII, TypeV and TypeVI.And NmeCas9 belongs to TypeII-C type, main function mode are specific cleavage DNA double chains with the help of sgRNA and PAM, introduce the double of DNA Chain fracture, to be edited to specific site.Wherein NmeCas9 is divided into Nme1Cas9, Nme2Cas9 and Nme3Cas9 again.Carefully Born of the same parents' experiment has been proven that Nme1Cas9 and Nme2Cas9 can play the function of nuclease.And Nme2Cas9 and Nme1Cas9 phase Than following advantages: (1) can efficient cutting DNA double-strand；(2) PAM that can rely on N4CC carries out target identification, extends The editable range of CRISPR system.

But Nme2Cas9 albumen used in cell and zoopery is separated from diplococcus meningitidis, and former There are different codon preferences and base compositions for core biology and eucaryote.Such as it is planted by the unifacial leaf of representative of rice Object, compared with species such as bacterium and dicotyledons, codon preference is strong, and G/C content is high.Therefore directly use is without artificial The Nme2Cas9 of optimization design will affect its expression efficiency in eukaryocyte, imitate to influence it to the cutting of DNA double chain Rate.Further, since Nme2Cas9 may adversely affect eukaryon transformation receptor genome, it is also possible to draw from bacterium Send out the worry to its safety.

Summary of the invention

The purpose of the invention is to overcome drawbacks described above of the existing technology, a kind of codon vegetalization transformation is provided Plant Nme2Cas9 gene and its application in Plant Genome editor,

To achieve the goals above, one aspect of the present invention provides a kind of Plant Nme2Cas9 of codon vegetalization transformation The Plant Nme2Cas9 gene of gene, the codon vegetalization transformation has nucleotide sequence shown in SEQ ID No:1.

Second aspect of the present invention provides a kind of expression cassette, and the expression cassette contains codon vegetalization transformation as described above Plant Nme2Cas9 gene.

Third aspect present invention provides a kind of expression vector, and the expression vector is inserted with codon plant as described above Change the Plant Nme2Cas9 gene or expression cassette as described above of transformation.

Fourth aspect present invention provides a kind of targeting vector, and the targeting vector is inserted with codon vegetalization as described above The Plant Nme2Cas9 gene or expression cassette as described above and target site sequence of transformation.

Fifth aspect present invention provides a kind of transgenic cell, and the transgenic cell has been transferred to codon as described above Plant Nme2Cas9 gene, expression cassette as described above, the expression vector as described above or as described above of vegetalization transformation Targeting vector.

Sixth aspect present invention provides the Plant Nme2Cas9 gene, as above of codon vegetalization as described above transformation Expression cassette, expression vector as described above, targeting vector as described above or the transgenic cell as described above is being planted Application in object genome editor, wherein the Plant Genome editor includes shearing to Plant Genome, is contained The genetically modified plants in mutational site or plant part.

Seventh aspect present invention provides a kind of method for obtaining transgenic paddy rice or rice containing mutation sites part, should Method includes:

(1) targeting vector as described above is converted into Agrobacterium tumefaciems, obtains the transgenic cell of Agrobacterium tumefaciems；

(2) EMBRYO IN RICE is induced on callus inducing medium, to obtain secondary callus；

(3) transgenic cell of the Agrobacterium tumefaciems is contacted and is infected with the secondary callus, knot will be infected Secondary callus after beam is co-cultured in Agrobacterium tumefaciems culture medium, with the secondary callus group after being co-cultured It knits；

(4) the secondary callus is successively screened, seedling and rooting treatment.

The Plant Nme2Cas9 gene of codon vegetalization provided by the invention transformation is crop rice password in mode Based on son transformation obtain, i.e., maintain encoding amino acid sequence it is constant under the premise of, using inventor from paddy gene The codon filtered out in corresponding codons replaces original codon, obtains the Plant Nme2Cas9 base of vegetalization transformation Because using chemical synthesis.The Plant Nme2Cas9 base that the vegetalization of acquisition is transformed is integrated into expression and carried by the present invention In body, corresponding targeting vector is constructed on this basis, is then realized by Genetic Transformation in Higher Plants to plant specific gene editor, Shear efficiency can be significantly improved.

Detailed description of the invention

The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:

Fig. 1 is PHUN7B11 (inserted with plant Nme2Cas9) vector plasmid schematic diagram.

Fig. 2 is that the PCR of transgenic plant detects electrophoretogram；Wherein, amplified fragments are the portion of plant Nme2Cas9 gene Point, clip size 697bp；M is DL2kbmarker；NC is negative control；1-11 is the transgenic plant selected at random.

Specific embodiment

The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more New numberical range, these numberical ranges should be considered as specific open herein.

In a first aspect, the present invention provides a kind of Plant Nme2Cas9 gene of codon vegetalization transformation, the password The Plant Nme2Cas9 gene of sub- vegetalization transformation has nucleotide sequence shown in SEQ ID No:1.

The present invention should be noted " have SEQ ID No:1 shown in nucleotide sequence " of the present invention and Do not mean that it is arbitrary containing the nucleotide sequence shown in the SEQ ID No:1 in addition to nucleotide or nucleotide sequence, but Refer to according to the conventional means of those skilled in the art, for the ease of or be conducive to nucleotide sequence shown in SEQ ID No:1 The core for not influencing its Function for being operated or being realized duplication of nucleotide sequence shown in SEQ ID No:1 etc. and contain Thuja acid or nucleotide sequence, for example, restriction enzyme site, marker gene, screening-gene, promoter, enhancer etc..Therefore, of the invention Described refers to " with nucleotide sequence shown in SEQ ID No:1 " with nucleotide sequence shown in SEQ ID No:1, but It still is able to realize the sequence of nucleotide sequence function shown in SEQ ID No:1.

A kind of specific embodiment according to the present invention, the nucleotide sequence such as SEQ of the plant Nme2Cas9 gene Shown in ID No:1.

According to the present invention, the both ends of the plant Nme2Cas9 gene can connect NotI/SacI restriction enzyme site, example It such as, can be in 5 ' end connection NotI restriction enzyme sites of plant Nme2Cas9 gene, in 3 ' end connection SacI restriction enzyme sites.

According to the present invention, in order to enable plant Nme2Cas9 gene provided by the invention can more efficiently enter cell Core, preferably plant Nme2Cas9 gene 3 ' end series connection it is multiple enter nuclear signal, for example, can be 3 NLS signals, core Acid sequence is as shown in SEQ ID No:2.

A kind of preferred embodiment according to the present invention, 5 ' end connection NotI digestions of the plant Nme2Cas9 gene Site, in 3 ' end connection SacI restriction enzyme sites and 3 NLS signals, wherein 3 NLS signals are connected to SacI restriction enzyme site Upstream, particular sequence is as shown in SEQ ID No:3.

Second aspect, the present invention also provides a kind of expression cassette, the expression cassette contains codon plant as described above Change the Plant Nme2Cas9 gene of transformation.

The third aspect, the present invention also provides a kind of expression vector, the expression vector is inserted with password as described above The Plant Nme2Cas9 gene or expression cassette as described above of sub- vegetalization transformation.

According to the present invention, the construction method of the expression vector can be carried out according to the method for this field routine, for example, making Digestion is carried out to plant Nme2Cas9 gene and the carrier being inserted into identical restriction enzyme, then reuses connection Plant Nme2Cas9 gene is connected in carrier by enzyme, obtains expression vector of the invention.

Wherein, the restriction enzyme can be carried out according to the restriction enzyme site being introduced into plant Nme2Cas9 gene Specific selection, for example, can be NotI/SacI restriction enzyme.

Wherein, the various companies that two kinds of nucleic acid fragments can be attached that the ligase can be commonly used in the art Enzyme is connect, for example, can be T4 ligase.

Wherein, the carrier can be various carriers commonly used in the art, it is preferred that the carrier can be PHUN600, pHUN611, PUC57-AMP, pHUN400 or pHUN900.A kind of preferred embodiment according to the present invention, it is described Carrier is pHUN600, further, can use NotI/SacI restriction enzyme site, with NotI/SacI digestion pHUN600 carrier and Plant Nme2Cas9 gene simultaneously recycles, and plant Nme2Cas9 gene is connected to pHUN600 using T4 ligase later and is carried Body obtains plant expression vector pHUN-plant Nme2Cas9 (abbreviation pHUN 7B11).

Fourth aspect, the present invention also provides a kind of targeting vector, which plants inserted with codon as described above The Plant Nme2Cas9 gene or expression cassette as described above and target site sequence of materialization transformation.

According to the present invention, depending on the target site sequence can carry out the sequence of genome editor according to actual needs, but It is the form that the target site sequence is 5 '-(N) X-NNCC-3 ', wherein sequence based on X, NNCC are characterized sequence.According to this A kind of specific embodiment is invented, the target site sequence is nucleotide sequence (rice PDS base shown in SEQ ID No:8 Because of 1381-1402 in (LOC_Os03g0184000) nucleotide sequencesGGCACCATGATATTTGCCATGCCAAA, under Dashed part is the part TGCC in described 5 '-(N) X-NNCC-3 ' structures).

According to the present invention, the targeting vector only need to simply be annealed on the basis of expression vector, digestion connects Effect can be obtained.

5th aspect, the present invention also provides a kind of transgenic cell, the transgenic cell has been transferred to as described above Plant Nme2Cas9 gene, expression cassette as described above, the expression vector as described above or such as of codon vegetalization transformation The upper targeting vector.

According to the present invention, the selection of the cell can be determined according to the stage locating for gene targeting, specifically, for example, Before carrying out targeting vector building, when needing to realize the amplification of plant Nme2Cas9 gene, it can be made using Escherichia coli Agrobacterium tumefaciems can be used when needing for targeting vector to be transformed into plant cell for host cell (Agrobacterium tumefaciens) is used as host cell.

6th aspect, the present invention also provides the Plant Nme2Cas9 bases of codon vegetalization as described above transformation Cause, expression cassette as described above, expression vector as described above, targeting vector as described above or transgenosis as described above are thin Application of the born of the same parents in Plant Genome editor, wherein the Plant Genome editor includes shearing to Plant Genome, is obtained Obtain genetically modified plants containing mutation sites or plant part.

The present invention utilizes DNA double in the achievable plant of plant Nme2Cas9 gene of codon vegetalization transformation The shearing of chain, and under the action of itself repair system, obtain the genetically modified plants with mutational site or plant part.

According to the present invention, the plant is preferably monocotyledon, more preferably rice, further preferably japonica rice, then Further preferably japonica rice OryzasativaLcv.Nipponbare.

7th aspect, the present invention also provides a kind of sides for obtaining transgenic paddy rice or rice containing mutation sites part Method, this method comprises:

(1) targeting vector as described above is converted into Agrobacterium tumefaciems, obtains the transgenic cell of Agrobacterium tumefaciems；

(2) EMBRYO IN RICE is induced on callus inducing medium, to obtain secondary callus；

(3) transgenic cell of the Agrobacterium tumefaciems is contacted and is infected with the secondary callus, then will invaded Secondary callus after dye is co-cultured in Agrobacterium tumefaciems culture medium, with the secondary callus after being co-cultured Tissue；

(4) the secondary callus is successively screened, seedling differentiation and rooting treatment.

According to the present invention, in step (1), the Agrobacterium tumefaciems can be Agrobacterium tumefaciems commonly used in the art, For example, Agrobacterium tumefaciems (Agrobacterium can be saved for Paddy Rice Inst., Anhui Agriculture Science Academy Tumefaciens) EHA105 bacterial strain.

It wherein, can be according to this field conventional technology using the method for targeting vector conversion Agrobacterium tumefaciems It is operated, such as can be freeze-thaw method.After conversion, the Agrobacterium tumefaciems containing the targeting vector can contained The flat lining out of the LB of 40-60mg/L kanamycins (ingredient is shown in Table 1), 25-35 DEG C of dark culturing use aseptic inoculation after 20-30h The Agrobacterium tumefaciems of activation is seeded on the fresh LB plate containing 40-60mg/L kanamycins by ring, carries out second and lives Change, 25-35 DEG C of dark culturing is stayed overnight.Agrobacterium suspension medium (ingredient is shown in Table 1) is added in sterile centrifugation tube, uses oese The Agrobacterium tumefaciems for activating 2 times is scraped, adjusts OD660 (Optical density660nm, 660nm light absorption value) to about It is spare to be stored at room temperature 30min or more by 0.10-0.25.

In step (2), the preparation method of the EMBRYO IN RICE can be carried out according to the means of this field routine, for example, will be at Embryo is separated after the decladding of boiled water rice, sterilizing.Specifically, it is normal, clean to be chosen appearance for mature rice paddy seed decladding Seed without mildew, successively cleans seed disinfection with alcohol and liquor natrii hypochloritis.Sodium hypochlorite is outwelled later, it is sterile to be washed to Without sodium hypochlorite smell, it is eventually adding sterile water, soaked overnight separates embryo along aleurone with knife blade.

Wherein, the callus inducing medium can be callus inducing medium commonly used in the art, It is specific as shown in table 1.The scultellum of isolated embryo is placed on callus inducing medium 28-32 DEG C of dark culture upward to lure Lead callus.

The time of induction, which is subject to, there is spherical, coarse, lurid secondary callus, can carry out preculture later Operation, i.e., go to secondary callus on new callus inducing medium, continues 28-32 DEG C dark culture preculture 3-8 days.In advance After culture, little particle in good condition, that division is vigorous is collected with spoon into sterile centrifugation tube, is invaded for Agrobacterium tumefaciems Dye.

In step (3), the transgenic cell of the Agrobacterium tumefaciems is contacted and infected with the secondary callus Method may include: to add step (1) ready Agrobacterium suspension into the callus that step (2) obtain, and impregnate 10-20min is shaked gently frequently therebetween, to realize the contact and infect.

Wherein, the step of co-cultivation may include: and outwell liquid after immersion as above (as far as possible to drip liquid Only), the extra Agrobacterium bacterium solution on callus surface, sterile wind drying are sucked.The aseptic filter paper on sterile petri dish pad, adds Enter suitable Agrobacterium tumefaciems suspension medium, callus is dispersed on filter paper, 20-25 DEG C of dark co-cultivation 45- 50h。

In step (4), the screening may include preceding screening and formal screening, that is, by the callus through co-culturing Intersperse among in preceding screening and culturing medium (ingredient is shown in Table 1), 25-32 DEG C dark culturing 4-6 days.After preceding screening and culturing, by callus Tissue is gone on screening and culturing medium (ingredient is shown in Table 1), 25-32 DEG C of dark culturing, and after 2-3 weeks, resistant calli growth is obvious, It can carry out differentiation and regeneration operation.

Wherein, the specific steps of the seedling differentiation may include: that each independent transformants are selected several growth shapes Good, the fresh little particle of state goes on differentiation and regeneration culture medium (ingredient is shown in Table 1) and carries out seedling differentiation.25-30 DEG C of illumination training It supports, periodicity of illumination is 12-18h illumination 6-10h dark, luminous intensity 3000-6000lx.

Wherein, the specific steps taken root may include: when the seedling of resistant calli differentiation it is long to about 2cm when, Each independent transformants take well-grown seedling, move on root media (ingredient is shown in Table 1), 25-30 DEG C of illumination cultivation, light light It is 12-18h illumination 6-10h dark, luminous intensity 3000-6000lx according to the period.After taking root, the seedling of well developed root system is selected, is used Culture medium is removed in washing, and transplanting is buried.

According to the present invention, the culture medium that various culture mediums as used above all can be commonly used in the art, preferably , culture medium listed in table 1 can be used, wherein the configuration of used culture medium can be with bibliography: Yongbo Duan,Chenguang Zhai,Hao Li,Juan Li,Wenqian Mei et al.An efficient and high- throughput protocol for Agrobacterium mediated transformation based on phosphomannose isomerase positive selection in Japonica rice(Oryza sativa L.) .Plant cell reports, 2012,31:1611-1624 are carried out.

Table 1

Note: " N6 a great number of elements " refers to [NO in the N6 a great number of elements₃ ^-]/[NH₄ ⁺]=40mM/10mM.

The present invention will be described in detail by way of examples below.

In the case where no other illustrate, the operation in following specific embodiments is all made of generally in the art Routine operation carries out.Those skilled in the art can be easily obtained from the prior art about such routine operation Introduction, such as it is referred to textbook Sambrook and David Russell, Molec μ lar Cloning:A Laboratory Manual,3rd ed.,Vols1,2；Charles Neal Stewart,Alisher Touraev,Vitaly Citovsky and Tzvi Tzfira, Plant Transformation Technologies etc..It is used in following embodiments Medicinal raw material, reagent, material etc., unless otherwise specified, be commercially available products.

Embodiment 1

The present embodiment is for illustrating the building containing plant Nme2Cas9 gene plant targeting vector

1, nucleotide sequence shown in entrusting shown in Suzhou Jin Weizhi Biotechnology Co., Ltd synthesis SEQ ID No:3 (including sequence shown in SEQ ID No:1, the end SEQ ID No:15 ' connection NotI restriction enzyme site, in 3 ' 3 NLS signals of end connection (shown in SEQ ID No:2) and SacI restriction enzyme site), it is connected on PUC57-AMP carrier, forms PUC57-AMP-plant Nme2Cas9 carrier, and be loaded into Escherichia coli XL-blue bacterial strain.

2, Axygen matter is used from the above-mentioned Escherichia coli XL-blue containing PUC57-AMP-plant Nme2Cas9 carrier Grain extracts kit extracts plasmid, with NotI/SacI digestion, recycles plant Nme2Cas9 segment.NotI/SacI is utilized simultaneously Enzyme carries out linearization process to pHUN600, pHUN600 is recycled, by above-mentioned plant Nme2Cas9 segment and pHUN600 segment It is attached with T4 ligase (being purchased from TaKaRa company), obtains plant expression vector pHUN600-plant Nme2Cas9 (figure 1), it is named as pHUN 7B11.

3,1381-1402 in rice PDS gene (LOC_Os03g0184000) nucleotide sequences are selectedGGCACCATGATATTTGCCATGCCAAA (underscore part is the part TGCC in 5 '-(N) X-NNCC-3 ' structures) is as target practice Site.Target site sequence is merged to form pHUN7B11-PDS with pHUN7B11.Plant expression vector is transferred to using freeze-thaw method (Paddy Rice Inst., Anhui Agriculture Science Academy in Agrobacterium tumefaciems (Agrobacterium tumefaciens) EHA105 bacterial strain Save), it is used for genetic transformation.

Embodiment 2

The present embodiment is for illustrating using pHUN7B11-PDS as the acquisition of the rice transformation of targeting vector and mutant.

1, the induction and preculture of mature embryo callus

By the mature seed decladding of OryzasativaLcv.Nipponbare (Paddy Rice Inst., Anhui Agriculture Science Academy's preservation), choose appearance it is normal, Seed of the cleaning without mildew rocks 90sec with 70% alcohol, outwells alcohol；It is (former with 50% sodium hypochlorite containing Tween20 again Liquid effective chlorine density is greater than 4%, wherein 50% sodium hypochlorite is the solution after stoste is diluted 1 times, and every 100 milliliters are added 1 drop Tween20) solution cleans seed, shakes 45min (180r/min) on shaking table.Outwell sodium hypochlorite, sterile washing 5-10 times To no sodium hypochlorite smell, it is eventually adding sterile water, 30 DEG C of soaked overnights.Embryo, scultellum court are separated along aleurone with knife blade On be placed on callus inducing medium (ingredient is shown in Table 1), 12/ware, 30 DEG C of dark cultures are with evoked callus.

Occur spherical, coarse, lurid secondary callus after two weeks, preculture operation can be carried out, i.e., it will be secondary Callus is gone on new callus inducing medium, 30 DEG C dark culture preculture 5 days.After preculture, by it is in good condition, It divides vigorous little particle to be collected with spoon into the sterile centrifugation tube of 50mL, be infected for Agrobacterium.

2, the culture of agrobacterium strains and suspension prepare

By the agrobacterium strains EHA105 containing pHUN7B11-PDS carrier in the LB solid containing 50mg/L kanamycins Cross (referring to table 1) on culture medium, 28 DEG C of dark culturings, for 24 hours afterwards with aseptic inoculation ring by the Agrobacterium inoculation of activation to fresh 50mg/L kanamycins LB plate on, carry out second and activate, 28 DEG C of dark culturings are stayed overnight.In the sterile centrifugation tube of 50mL The Agrobacterium for activating 2 times is scraped with oese, is adjusted by middle addition 20-30mL Agrobacterium suspension medium (ingredient is shown in Table 1) OD660 (Optical density 660nm, 660nm light absorption value) is stored at room temperature 30min or more to about 0.10-0.25.

3, it infects and co-cultures

(see step 1), add agrobacterium suspension into ready callus, impregnate 15min, gently shake frequently therebetween It is dynamic.Liquid (as far as possible dripping liquid net) is outwelled after immersion, and the extra agriculture bar on callus surface is sucked with aseptic filter paper Bacterium bacterium solution, and dried up in super-clean bench with sterile wind.Three sterile filters on the disposable sterilized culture dish pad of 100 × 25mm Paper is added 2.5mL Agrobacterium suspension medium, the callus after blotting is dispersed on filter paper, 23 DEG C of dark culturings 48h。

4, preceding screening and screening and culturing

After co-cultivation, the callus through co-culturing is dispersed evenly in preceding screening and culturing medium (ingredient is shown in Table 1), 30 DEG C dark culturing 5 days.After preceding screening and culturing, callus is gone on screening and culturing medium (ingredient is shown in Table 1), Mei Gepei Feeding ware connects 25 callus, and condition of culture is two kinds, a kind of are as follows: 30 DEG C of dark culturings, 45 days；Another kind for 12 hours 37 DEG C Dark culturing, 12 hours 30 DEG C of dark culturings, the period continuously cultivates 45 days at this temperature；After culture, kanamycin-resistant callus tissue group It is obvious to knit growth, differentiation and regeneration operation can be carried out.

5, differentiation and regeneration

Each independent transformants select the little particle that 2-3 growth conditions are good, fresh, go on differentiation and regeneration culture medium (ingredient is shown in Table 1).Every culture dish connects 5 independent transformants.28 DEG C of illumination cultivations, periodicity of illumination are 16h illumination 8h dark, light intensity Degree is 3000-6000lx.

6, it takes root and transplants

When the bud of resistant calli differentiation it is long to about 2cm when, each independent transformants only take one plant of well-grown seedling, It moves on root media (ingredient is shown in Table 1), 28 DEG C of illumination cultivations, periodicity of illumination is 16h illumination 8h dark, and luminous intensity is 3000-6000lx.After two weeks, 42 plants of seedlings for selecting well developed root system, are washed with water culture medium, transplanting is buried.

7, Molecular Identification

Before transplanting, rice leaf sample is taken, carries out that DNA is small to be mentioned with CTAB method.By obtained genomic DNA sample Product are analyzed for PCR.The PCR primer of plant Nme2Cas9 for expanding the transformation of codon vegetalization is SEQ ID No:4 (5 '-GGCCTACCACGCGATCTCCAGG-3 ') and SEQ ID No:5 (5 '-GTAGCCCTTCTCGTTGAGCCGC-3 ') is produced Growth degree is the segment of 697bp.By genomic DNA first 95 DEG C keep 5 minutes, then carry out 32 circulation: 94 DEG C 45 seconds, 56 DEG C 45 seconds, 72 DEG C 45 seconds, finally 72 DEG C extend 10 minutes.11 transgenic plants are selected at random, it is identified, it is sun Property, positive rate reaches 100% (see Fig. 2).

42 plant resulting to transgenosis all carry out leaf DNA extraction, and gained genome DNA sample is used for PCR Analysis.PCR primer for expanding PDS genetic fragment is SEQ ID No:6 (5 '-ACATATATGAATATGACAGATA-3 ') And SEQ ID No:7 (5 '-AACTTCACCTTCTCTGGCCAA-3 '), generate the segment that length is 553bp.By genomic DNA First 95 DEG C keep 5 minutes, then carry out 32 circulation: 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, finally prolong at 72 DEG C It stretches 10 minutes.PCR product is sequenced.Measured result is compared with wild-type sequence.In the 42 plants of transgenic plants surveyed There are 20 plants point mutation has occurred；Mutation efficiency is 47.6%.Illustrate that modified plant Nme2Cas9 can be to paddy gene Group is sheared, and has very high mutation efficiency.Similarly, it is equally constructed with original Plant Nme2Cas9 gene Targeting vector carries out rice transformation, but does not obtain positive editor plant.Plant after showing codon optimization Nme2Cas9 can realize efficient gene editor under the condition of culture of optimization in plant.

The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to Protection scope of the present invention.

SEQUENCE LISTING

<110>Paddy Rice Inst., Anhui Agriculture Science Academy

<120>the Plant Nme2Cas9 gene of codon vegetalization transformation and its application

<130> HFI00864-NYSD

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 3249

<212> DNA

<213>the Plant Nme2Cas9 gene of codon vegetalization transformation

<400> 1

atggcggcgt tcaagcctaa cccaatcaac tacatcctcg gtctggacat cggcatcgca 60

tcagtgggct gggccatggt cgagatcgac gaggaggaga atccaatcag gctgattgat 120

ctcggcgtcc gggtgttcga gagggcggag gtgcctaaga caggcgactc actggcaatg 180

gccaggaggc tggccaggtc tgtccggagg ctcacaagga ggagggccca caggctcctg 240

agggccagga ggctcctgaa gagggaggga gtgctccagg ccgcggactt cgatgagaat 300

ggcctgatca agtccctccc aaacacaccg tggcagctca gggctgccgc cctggatagg 360

aagctgaccc cgctggagtg gtccgccgtg ctcctgcacc tcattaagca tcgcggctac 420

ctgagccagc ggaagaacga gggagagaca gccgacaagg agctgggcgc gctcctgaag 480

ggagtcgcca acaatgccca tgcgctccag accggcgatt tcaggacacc ggccgagctg 540

gcgctgaata agttcgagaa ggagtccggc cacatccgga accagagggg cgactactcc 600

cataccttca gccgcaagga cctccaggcc gagctgattc tcctgttcga gaagcagaag 660

gagttcggca atccacatgt gtctggcggc ctcaaggagg gaatcgagac actcctgatg 720

actcagaggc cagcactctc aggcgacgcc gtccagaaga tgctgggaca ttgcaccttc 780

gagcctgccg agccaaaggc cgccaagaac acctacacag cggagaggtt catttggctg 840

acaaagctca acaatctgcg catcctggag cagggctctg agcggccact caccgacaca 900

gagagggcga ccctgatgga tgagccttac cggaagtcca agctcacata cgcacaggcc 960

aggaagctcc tgggcctgga ggacaccgcc ttcttcaagg gcctgcggta cggcaaggat 1020

aatgccgagg cgtccacact catggagatg aaggcctacc acgcgatctc cagggccctg 1080

gagaaggagg gcctgaagga caagaagagc ccgctcaacc tgtccagcga gcttcaggat 1140

gagattggca ccgcgttctc actgttcaag accgacgagg atattaccgg aaggctcaag 1200

gacagggtgc agccagagat tctggaggcc ctcctgaagc acatctcctt cgataagttc 1260

gtgcagatta gcctgaaggc gctcaggcgc atcgtccctc tcatggagca gggcaagcgc 1320

tacgatgagg cctgcgcgga gatctacggc gatcattacg gcaagaagaa cacagaggag 1380

aagatctacc tcccgccaat tccagccgac gagatcagga atcctgtggt cctgcgcgcc 1440

ctctcacagg ccaggaaggt catcaacggc gtggtccggc gctacggctc tcctgcgcgc 1500

atccatattg agacagcccg ggaggtcggc aagtccttca aggacaggaa ggagattgag 1560

aagcgccagg aggagaatcg caaggatcgg gagaaggctg ccgccaagtt ccgcgagtac 1620

ttccctaact tcgtgggcga gccgaagagc aaggacatcc tcaagctgcg gctctacgag 1680

cagcagcacg gcaagtgcct ctactcaggc aaggagatta atctggtgcg gctcaacgag 1740

aagggctacg tcgagatcga tcatgcgctg ccattctcca ggacctggga cgatagcttc 1800

aacaataagg tcctggtgct cggcagcgag aaccagaata agggcaatca gacaccttac 1860

gagtacttca atggcaagga taactcaagg gagtggcagg agttcaaggc ccgcgtggag 1920

acatccaggt tcccacgctc taagaagcag cgcatcctcc tccagaagtt cgacgaggat 1980

ggcttcaagg agtgcaacct caatgacacc cgctacgtga accgcttcct gtgccagttc 2040

gtcgccgatc acattctgct gacaggcaag ggcaagcgca gggtgttcgc gtctaatggc 2100

cagattacaa acctcctgag gggattctgg ggcctcagga aggtcagggc ggagaatgac 2160

cggcaccatg cgctcgatgc cgtggtggtg gcctgctcca cagtggccat gcagcagaag 2220

atcacacgct tcgtccggta caaggagatg aacgccttcg acggcaagac cattgataag 2280

gagacaggca aggtgctcca ccagaagaca catttcccac agccttggga gttcttcgcc 2340

caggaggtca tgatcagggt cttcggcaag ccagacggca agcctgagtt cgaggaggcg 2400

gatacaccag agaagctccg cacactcctg gcagagaagc tgtcatctcg gccagaggcg 2460

gtgcatgagt atgtgacccc gctcttcgtg tcacgggcgc caaataggaa gatgtctggc 2520

gcccacaagg acacactgcg ctcagcgaag cggttcgtca agcataacga gaagatttct 2580

gtgaagcgcg tctggctcac cgagatcaag ctggccgacc tggagaacat ggtgaattac 2640

aagaacggcc gggagatcga gctgtacgag gccctcaagg ccaggctgga ggcgtacggc 2700

ggaaatgcca agcaggcgtt cgacccgaag gataacccat tctacaagaa gggcggccag 2760

ctggtgaagg ccgtcagggt ggagaagacc caggagagcg gcgtcctcct gaataagaag 2820

aacgcctaca caattgcgga caacggcgat atggtccgcg tggacgtctt ctgcaaggtg 2880

gataagaagg gcaagaatca gtacttcatc gtgcctatct acgcctggca ggtcgcggag 2940

aacatcctcc cggacattga ttgcaagggc tacaggattg acgattcata cacattctgc 3000

ttctctctcc acaagtacga cctgatcgcc ttccagaagg atgagaagag caaggtcgag 3060

ttcgcgtact acattaattg cgactccagc aacggcaggt tctacctcgc ctggcatgat 3120

aagggctcca aggagcagca gttccgcatt agcacccaga atctggtgct catccagaag 3180

taccaggtca acgagctggg caaggagatc aggccttgcc gcctgaagaa gaggcctccg 3240

gtgcgctaa 3249

<210> 2

<211> 99

<212> DNA

<213>3 concatenated NLS signals

<400> 2

tccggcggct ccccgaagaa gaagaggaag gtgtccggcg gtagtccaaa gaagaagagg 60

aaggtgtcgg gaggtagccc aaagaagaag aggaaggtt 99

<210> 3

<211> 3362

<212> DNA

<213>+3 NLS signals of Plant Nme2Cas9+ restriction enzyme site of codon vegetalization transformation

<400> 3

gcggccgcat ggcggcgttc aagcctaacc caatcaacta catcctcggt ctggacatcg 60

gcatcgcatc agtgggctgg gccatggtcg agatcgacga ggaggagaat ccaatcaggc 120

tgattgatct cggcgtccgg gtgttcgaga gggcggaggt gcctaagaca ggcgactcac 180

tggcaatggc caggaggctg gccaggtctg tccggaggct cacaaggagg agggcccaca 240

ggctcctgag ggccaggagg ctcctgaaga gggagggagt gctccaggcc gcggacttcg 300

atgagaatgg cctgatcaag tccctcccaa acacaccgtg gcagctcagg gctgccgccc 360

tggataggaa gctgaccccg ctggagtggt ccgccgtgct cctgcacctc attaagcatc 420

gcggctacct gagccagcgg aagaacgagg gagagacagc cgacaaggag ctgggcgcgc 480

tcctgaaggg agtcgccaac aatgcccatg cgctccagac cggcgatttc aggacaccgg 540

ccgagctggc gctgaataag ttcgagaagg agtccggcca catccggaac cagaggggcg 600

actactccca taccttcagc cgcaaggacc tccaggccga gctgattctc ctgttcgaga 660

agcagaagga gttcggcaat ccacatgtgt ctggcggcct caaggaggga atcgagacac 720

tcctgatgac tcagaggcca gcactctcag gcgacgccgt ccagaagatg ctgggacatt 780

gcaccttcga gcctgccgag ccaaaggccg ccaagaacac ctacacagcg gagaggttca 840

tttggctgac aaagctcaac aatctgcgca tcctggagca gggctctgag cggccactca 900

ccgacacaga gagggcgacc ctgatggatg agccttaccg gaagtccaag ctcacatacg 960

cacaggccag gaagctcctg ggcctggagg acaccgcctt cttcaagggc ctgcggtacg 1020

gcaaggataa tgccgaggcg tccacactca tggagatgaa ggcctaccac gcgatctcca 1080

gggccctgga gaaggagggc ctgaaggaca agaagagccc gctcaacctg tccagcgagc 1140

ttcaggatga gattggcacc gcgttctcac tgttcaagac cgacgaggat attaccggaa 1200

ggctcaagga cagggtgcag ccagagattc tggaggccct cctgaagcac atctccttcg 1260

ataagttcgt gcagattagc ctgaaggcgc tcaggcgcat cgtccctctc atggagcagg 1320

gcaagcgcta cgatgaggcc tgcgcggaga tctacggcga tcattacggc aagaagaaca 1380

cagaggagaa gatctacctc ccgccaattc cagccgacga gatcaggaat cctgtggtcc 1440

tgcgcgccct ctcacaggcc aggaaggtca tcaacggcgt ggtccggcgc tacggctctc 1500

ctgcgcgcat ccatattgag acagcccggg aggtcggcaa gtccttcaag gacaggaagg 1560

agattgagaa gcgccaggag gagaatcgca aggatcggga gaaggctgcc gccaagttcc 1620

gcgagtactt ccctaacttc gtgggcgagc cgaagagcaa ggacatcctc aagctgcggc 1680

tctacgagca gcagcacggc aagtgcctct actcaggcaa ggagattaat ctggtgcggc 1740

tcaacgagaa gggctacgtc gagatcgatc atgcgctgcc attctccagg acctgggacg 1800

atagcttcaa caataaggtc ctggtgctcg gcagcgagaa ccagaataag ggcaatcaga 1860

caccttacga gtacttcaat ggcaaggata actcaaggga gtggcaggag ttcaaggccc 1920

gcgtggagac atccaggttc ccacgctcta agaagcagcg catcctcctc cagaagttcg 1980

acgaggatgg cttcaaggag tgcaacctca atgacacccg ctacgtgaac cgcttcctgt 2040

gccagttcgt cgccgatcac attctgctga caggcaaggg caagcgcagg gtgttcgcgt 2100

ctaatggcca gattacaaac ctcctgaggg gattctgggg cctcaggaag gtcagggcgg 2160

agaatgaccg gcaccatgcg ctcgatgccg tggtggtggc ctgctccaca gtggccatgc 2220

agcagaagat cacacgcttc gtccggtaca aggagatgaa cgccttcgac ggcaagacca 2280

ttgataagga gacaggcaag gtgctccacc agaagacaca tttcccacag ccttgggagt 2340

tcttcgccca ggaggtcatg atcagggtct tcggcaagcc agacggcaag cctgagttcg 2400

aggaggcgga tacaccagag aagctccgca cactcctggc agagaagctg tcatctcggc 2460

cagaggcggt gcatgagtat gtgaccccgc tcttcgtgtc acgggcgcca aataggaaga 2520

tgtctggcgc ccacaaggac acactgcgct cagcgaagcg gttcgtcaag cataacgaga 2580

agatttctgt gaagcgcgtc tggctcaccg agatcaagct ggccgacctg gagaacatgg 2640

tgaattacaa gaacggccgg gagatcgagc tgtacgaggc cctcaaggcc aggctggagg 2700

cgtacggcgg aaatgccaag caggcgttcg acccgaagga taacccattc tacaagaagg 2760

gcggccagct ggtgaaggcc gtcagggtgg agaagaccca ggagagcggc gtcctcctga 2820

ataagaagaa cgcctacaca attgcggaca acggcgatat ggtccgcgtg gacgtcttct 2880

gcaaggtgga taagaagggc aagaatcagt acttcatcgt gcctatctac gcctggcagg 2940

tcgcggagaa catcctcccg gacattgatt gcaagggcta caggattgac gattcataca 3000

cattctgctt ctctctccac aagtacgacc tgatcgcctt ccagaaggat gagaagagca 3060

aggtcgagtt cgcgtactac attaattgcg actccagcaa cggcaggttc tacctcgcct 3120

ggcatgataa gggctccaag gagcagcagt tccgcattag cacccagaat ctggtgctca 3180

tccagaagta ccaggtcaac gagctgggca aggagatcag gccttgccgc ctgaagaaga 3240

ggcctccggt gcgctaatcc ggcggctccc cgaagaagaa gaggaaggtg tccggcggta 3300

gtccaaagaa gaagaggaag gtgtcgggag gtagcccaaa gaagaagagg aaggttgagc 3360

tc 3362

<210> 4

<211> 22

<212> DNA

<213>the PCR upstream primer of the plant Nme2Cas9 of amplification codon vegetalization transformation

<400> 4

ggcctaccac gcgatctcca gg 22

<210> 5

<211> 22

<212> DNA

<213>for expanding the PCR downstream primer of the plant Nme2Cas9 of codon vegetalization transformation

<400> 5

gtagcccttc tcgttgagcc gc 22

<210> 6

<211> 22

<212> DNA

<213>for expanding the PCR upstream primer of PDS genetic fragment

<400> 6

acatatatga atatgacaga ta 22

<210> 7

<211> 21

<212> DNA

<213>for expanding the PCR downstream primer of PDS genetic fragment

<400> 7

aacttcacct tctctggcca a 21

<210> 8

<211> 26

<212> DNA

<213>target site sequence

<400> 8

ggcaccatga tatttgccat gccaaa 26

Claims (10)

1. a kind of Plant Nme2Cas9 gene of codon vegetalization transformation, which is characterized in that the codon vegetalization changes The Plant Nme2Cas9 gene made has nucleotide sequence shown in SEQ ID No:1.

2. the Plant Nme2Cas9 gene of codon vegetalization transformation according to claim 1, wherein the codon The nucleotide sequence of the Plant Nme2Cas9 gene of vegetalization transformation is as shown in SEQ ID No:1.

3. a kind of expression cassette, which is characterized in that the expression cassette contains codon vegetalization transformation of any of claims 1 or 2 Plant Nme2Cas9 gene.

4. a kind of expression vector, which is characterized in that the expression vector is inserted with codon plant of any of claims 1 or 2 Change the Plant Nme2Cas9 gene or expression cassette as claimed in claim 3 of transformation.

5. expression vector according to claim 4, wherein the carrier be pHUN600, PUC57-AMP, pHUN400 or pHUN900。

6. a kind of targeting vector, which is characterized in that the targeting vector is inserted with codon vegetalization of any of claims 1 or 2 The Plant Nme2Cas9 gene or expression cassette as claimed in claim 3 and target site sequence of transformation.

7. a kind of transgenic cell, which is characterized in that the transgenic cell be transferred to have the right to require 1 or 2 described in codon The Plant Nme2Cas9 gene of vegetalization transformation, expression cassette as claimed in claim 3, expression described in claim 4 or 5 carry Body or targeting vector as claimed in claim 6.

8. the Plant Nme2Cas9 gene of codon vegetalization of any of claims 1 or 2 transformation, as claimed in claim 3 Expression cassette, expression vector described in claim 4 or 5, targeting vector as claimed in claim 6 or as claimed in claim 7 turn Application of the gene cell in Plant Genome editor, wherein the Plant Genome editor includes carrying out to Plant Genome Shearing, obtains genetically modified plants containing mutation sites or plant part.

9. application according to claim 8, wherein the plant is monocotyledon, preferably rice, more preferably round-grained rice Rice, further preferably japonica rice OryzasativaLcv.Nipponbare.

10. a kind of method for obtaining transgenic paddy rice or rice containing mutation sites part, which is characterized in that this method packet It includes:

(1) targeting vector as claimed in claim 6 is converted into Agrobacterium tumefaciems, obtains the transgenic cell of Agrobacterium tumefaciems；

(2) EMBRYO IN RICE is induced on callus inducing medium, to obtain secondary callus；

(3) transgenic cell of the Agrobacterium tumefaciems is contacted and is infected with the secondary callus, after infecting Secondary callus co-cultured in Agrobacterium tumefaciems culture medium, with the secondary callus after being co-cultured；

(4) the secondary callus is successively screened, seedling and rooting treatment.