CN110128518A - The method for downgrading material using gene editing technology initiative corn - Google Patents

Abstract

The present invention provides a kind of method for downgrading material using gene editing technology initiative corn, including designing the sgRNA based on CRISPR/Cas9 for the target gene ZmGA20ox3/ZmGA20ox5 in corn, it will be connected in the carrier for carrying Cas containing the DNA fragmentation for encoding the sgRNA, with the carrier maize transformation of building, it realizes the rite-directed mutagenesis to gene ZmGA20ox3/ZmGA20ox5, and then obtains the transgenic corn plant of corresponding gene afunction.The invention firstly discloses the biological functions of corn ZmGA20ox3/ZmGA20ox5 gene, gene editing is carried out to corn ZmGA20ox3/ZmGA20ox5 gene by CRISPR/Cas9 technology, and further screening obtains the mutant material for being free of transgenic insert, these corns, which downgrade material, has important Breeding value.

Description

The method for downgrading material using gene editing technology initiative corn

Technical field

The present invention relates to biotechnologys and genetic breeding field, specifically, being related to a kind of utilization gene editing technology wound The method that corn processed downgrades material.

Background technique

The raising of twentieth century corn yield relies primarily on planting density (the Duvick et for improving corn in unit area Al., 2010), but density is excessively high it is followed by that the risk to lodge, may reduce yield, therefore to a certain degree Upper reduction corn plant height helps to improve corn yield.The end of the sixties in last century, well-known " green revolution " have started half The overbearing tide of yield can be improved in dwarfed plant.Half high product for downgrading rice and wheat has successfully been formulated during " green revolution " Kind.Many corn Dwarf Mutants are identified in the past few decades, but are not applied in corn breeding completely.

Gibberellin (GAs) is a kind of natural tetracyclic diterpene carboxylic acid, in the shape of plant seed germination, the elongation of stem and flower It plays an important role at waiting in growth and development processes.Presently found gibberellin type has had more than 130 kinds, wherein active Gibberellin there are four types of, be GA1, GA3, GA4 and GA7 (Hedden et al., 2000) respectively.In plant there are two types of GA Existence form, one kind exist in the form of free state, participate in plant growth metabolic pathway, and another kind exists in the form of reference state, Be do not have it is active, the normal content of GA is maintained by the dynamic equilibrium of reference state GA and free state GA in plant (Kawaide.,2006).The biosynthesis of GA including the following steps: 1) the window ox base window ox base two in protoplast Phosphoric acid synthesizes Nei Gen-kaurene；2) GA12 is generated in the online Nei Gen of endoplasm-kaurene oxidation, GA12 is further converted into GA53；3) GA12 and GA53 are entered in cytoplasm, and active GA points are generated under the action of a series of gibberellin oxidizing ferment Son (Teng et al., 2012).

CRISPR/Cas9 technology can accurately edit the privileged site in genome, and gene both may be implemented Knockout also can produce the insertion of specific fragment, be successfully applied to the genome editor of different plants, accelerate gene function and grind Study carefully and crop molecular genetic manipulation.Corn Dwarf Mutant material is obtained by CRISPR/Cas9 technology, is expected to be applied to corn In breeding work.

Summary of the invention

The object of the present invention is to provide a kind of methods for downgrading material using gene editing technology initiative corn.

In order to achieve the object of the present invention, in a first aspect, the present invention provides the gene of control plant plant height, including corn ZmGA20ox3 and/or ZmGA20ox5 gene, wherein corn ZmGA20ox3 gene is the following protein (a) of coding or (b) Gene:

(a) protein that the amino acid sequence shown in SEQ ID NO:1 forms；

(b) sequence shown in SEQ ID NO:1 is substituted, lacks or adds one or several amino acid and has same function The protein as derived from (a).

Corn ZmGA20ox5 gene is the following protein (a ') of coding or the gene of (b '):

The protein that (a ') amino acid sequence shown in SEQ ID NO:2 forms；

Sequence shown in (b ') SEQ ID NO:2 is substituted, lacks or adds one or several amino acid and has same function The protein as derived from (a ') of energy.

Second aspect, the present invention provide corn ZmGA20ox3 and/or ZmGA20ox5 gene and downgrade material breeding in corn In application, be that rite-directed mutagenesis is carried out to corn ZmGA20ox3 and/or ZmGA20ox5 gene using genetic engineering means, So that corn ZmGA20ox3 and/or ZmGA20ox5 gene lacks functionality, to realize that the genetic improvement to corn plant height (reduces strain It is high).

The third aspect, the present invention provides the method for downgrading material using gene editing technology initiative corn, in corn Target gene ZmGA20ox3 and/or ZmGA20ox5 design the sgRNA sequence based on CRISPR/Cas9, will containing coding institute The DNA fragmentation for stating sgRNA sequence is connected in the carrier for carrying Cas, with carrier maize transformation (such as mediated by agriculture bacillus of building Method), realize to the rite-directed mutagenesis of gene ZmGA20ox3 and/or ZmGA20ox5, and then obtain ZmGA20ox3 and/or The transgenic corn plant of ZmGA20ox5 gene lacks functionality.

Preferably, for gene ZmGA20ox3, the nucleotides sequence of sgRNA action site is classified as 5 '- GGAGCCATTCCTGTGGCCGC-3 ' and 5 '-CTGTCCTTCGGCTTCCACGA-3 '.(SEQ ID NO:3-4)

Preferably, for gene ZmGA20ox5, the nucleotides sequence of sgRNA action site is classified as 5 '- AGATCCCCGCGCCATTCCTG-3 ' and 5 '-CTGTCGTTCGGCTACCACGA-3 '.(SEQ ID NO:5-6)

It is highly preferred that two sgRNA action sites are connected in series on same gene editor's carrier by different expression cassettes.

In the present invention, the carrier for carrying Cas9 is pBUE411.Built corn transformation carrier is pBUE411-2gR- GA20ox3 and pBUE411-2gR-GA20ox5.

Optionally, the corn is selfing assemblage 31.

Fourth aspect, the present invention provide a kind of method that initiative corn downgrades material, according to the method described above prepare transgenosis Plant, then transgenic corn plant is hybridized, is returned, is selfed or vegetative propagation, so that formulating corn downgrades material.

Preferably, the corn formulated is downgraded in material without external source Insert Fragment.

The albumen that NOD gene encodes in corn has the function of regulating cell quantity, and nod mutant is in growth and development and carefully Born of the same parents' differentiation etc. is defective, shows that blade is narrow and plant height extremely downgrades (Rosa et al., 2017).Corn Blh12/blh14 double-mutant panel length serious curtailment causes the phenotype downgraded, and the mutant tassel developmental deformity causes Infertility (Tsuda et al., 2017) completely.Corn gif1 mutant blade narrows, internode is irregularly bent and plant becomes Short, the long branch of female inflorescence increases, and male inflorescence branch reduces (Zhang et al., 2018).The corn that the present invention formulates downgrades material Material belongs to half dwarfing material, and internode development is normal and can be normal solid, with downgrade it is serious, downgrade after infertility and downgrading send out between deutomerite It educates irregular corn material to compare, is more suitably applied to hybrid maize production.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

The invention firstly discloses the biological functions of corn ZmGA20ox3 and/or ZmGA20ox5 gene, pass through CRISPR/Cas9 technology carries out gene editing to corn ZmGA20ox3 and/or ZmGA20ox5 gene, and further screening obtains Mutant material without transgenic insert, these corns, which downgrade material, has important Breeding value.

Detailed description of the invention

Fig. 1 is the analysis result of Mutants homozygous target site mutant nucleotide sequence in the embodiment of the present invention 5.

Fig. 2 is ZmGA20ox3 gene editing plant plant height comparing result in the embodiment of the present invention 5.Wherein, it A: edits Heterozygous mutants and Mutants homozygous material compared with the height of wild-type corn plant；B: the column diagram of plant height.

Fig. 3 is in the embodiment of the present invention 6 without transgenic fragment mutant plants the selection result.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Embodiment 1 designs the editing sites based on CRISPR/Cas9

Pass through CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/) website design target site, corn Target gene ZmGA20ox3, ZmGA20ox5 and relevant information from database MaizeGDB (https: // Www.maizegdb.org/ it) downloads.The exon sequence of target gene is uploaded into the website CRISPR-P, selection refers to gene Group is Maize genome, obtains the potential editing sites for closing on 5 '-NGG.Each gene designs two target sites, and is located at gene First exon on, two target sites (being located at different expression cassettes) are connected in series on the same gene editing carrier. ZmGA20ox3 target site sequence is 5 '-GGAGCCATTCCTGTGGCCGC-3 ' and 5 '-CTGTCCTTCGGCTTCCACGA-3 ', ZmGA20ox5 target site sequence is 5 '-AGATCCCCGCGCCATTCCTG-3 ' and 5 '-CTGTCGTTCGGCTACCACGA-3 ',

The building of 2 gene editing carrier of embodiment

It is set according to the restriction enzyme site at selected editing sites and carrier pCBC-MT1T2 and pBUE411 multiple cloning sites Primer is counted, primer information is shown in Table 1:

1 amplimer of table

With two-wheeled PCR method expand target fragment, the first round PCR reaction using pair of primers MT1-F0/MT2-R0 with PCBC-MT1T2 plasmid is template amplification, and the second wheel PCR reaction is expanded using second couple of primer MT1-BsF/MT2-BsR with the first round Volume increase object is template amplification, and two target sites are together in series, target fragment is obtained.

First round reaction system are as follows:

Second wheel reaction system are as follows:

Two-wheeled pcr amplification reaction program are as follows: 94 DEG C of 2min；98 DEG C of 10s, 58 DEG C of 30s, 68 DEG C of 1min, 35 circulations；68℃ 7min, 25 DEG C of ∞.

Then, the target fragment and pBUE411 carrier purified with restriction enzyme BsaI-HF single endonuclease digestion, after digestion 0.2 37 DEG C of processing 30min of μ L dephosphorylation enzyme CPI of linear carrier prevent carrier after digestion from connecting certainly.

Single endonuclease digestion system:

Digestion condition: 37 DEG C of water-bath 2h.

Target fragment is connect with expression vector by T4 ligase:

Linked system:

Room temperature connects 1h or 16 DEG C of connection overnight.Building obtains gene ZmGA20ox3, ZmGA20ox5 and edits load respectively Body.

3 gene editing carrier of embodiment is transferred to Agrobacterium LBA4404

Gene ZmGA20ox3, ZmGA20ox5 that embodiment 2 constructs are edited into carrier and are transferred to Agrobacterium LBA4404 respectively, Specific step is as follows:

(1) 5 μ L plasmids is taken to be added in 200 μ L Agrobacterium competent cells；

(2) ice bath 30min；

(3) it is taken out from ice, is immediately placed in quick-frozen 5min in liquid nitrogen；

(4) it is removed from liquid nitrogen, incubates 5min in 37 DEG C of water-baths；

(5) 5min on ice is placed again after taking out；

(6) 800 μ L blank YEB culture mediums are added, restore 4-5h in 28 DEG C of shaking table 200rpm；

(7) 4000rpm room temperature is centrifuged 5min；

(8) part supernatant is abandoned, remaining about 200 μ L supernatants suspend precipitating, are coated in the YEB solid culture plate containing corresponding resistant On, 28 DEG C of inversion dark culture 36h；

(9) single colonie grown on picking plate is inoculated in the YEB liquid medium containing corresponding resistant, 28 DEG C 200rpm shaken cultivation is stayed overnight；PCR amplification identification is carried out by template of bacterium solution.

4 Agrobacterium-mediated Transformation corn of embodiment

Picking single colonie is inoculated into YEB fluid nutrient medium of the 5-10mL containing corresponding antibiotic, in 200rpm, 28 DEG C of oscillations Cultivate 8h；The bacterium solution after shake culture is inoculated by the fresh YEB Liquid Culture containing corresponding antibiotic with the ratio of 1:100 again In base, in 200rpm, 28 DEG C of shaken overnights.Next day, the bacterium solution that shake culture is stayed overnight are dispensed into 2mL centrifuge tube, in 5000rpm, centrifugation 5min；It is resuspended with the culture medium that infects containing 200 μM of acetosyringones, adjusts OD₆₀₀=0.3, it is spare.

Comprehensive 31 rataria of acceptor material corn inbred line is put into and is infected in the 1.5mL centrifuge tube of culture medium solution containing 1mL, 1h Sucked afterwards with pipettor infect culture medium and be added 1mL it is new infect culture medium, be mixed by inversion 1min, sufficiently cleaning maize immature embryos The endosperm on surface, then sucked with pipettor and infect culture medium solution, be added 0.2mL it is new infect culture medium solution.It will be equipped with jade The centrifuge tube of rice rataria is placed on heater (Hangzhou BIOER Technology Co., Ltd, model C HB-100) with 45 DEG C of heat shock 5min； It is sucked with pipettor and infects culture medium solution, the Agrobacterium bacterium solution of 1mL acetosyringone containing 40mg/L is added, and place 5min； Taking-up is blotted with sterilizing filter paper, is put into the co-culture medium of addition 300mg/L cysteine, under 23 DEG C of dark conditions altogether Culture 3 days.Rataria after having co-cultured is transferred in recovery media, is cultivated 7 days under dark condition.

After screening two-wheeled, kanamycin-resistant callus tissue is obtained, this callus is transferred to regeneration culture medium, makes plant regeneration under intense light irradiation. Condition of culture is 28 DEG C, illumination 16h, has regeneration seedling soon and occurs.It, can be by seedling when regenerated seedling grows to 3 leaves It is transplanted in root media, and cultivates indoors.After seedling grows young leaves and root, seedling is taken out from can, from Water washes down culture medium, transplants in the small flower for being mixed with Nutrition Soil and vermiculite (1:3, volume ratio).When again seedling grows 2-3 piece When young leaves, it can move it into crop field or big flowerpot.

Culture medium, co-culture medium, regeneration culture medium, root media are infected used in the present embodiment, it is specific to join See CN201710090814.2.

The identification for the transgenic corn plant that embodiment 5 is edited

The maize leaf survived after glufosinate-ammonium is screened extracts DNA using CTAB method, with ZmGA20ox3, ZmGA20ox5 Gene-specific primer carries out PCR identification, and amplified production is detected with 1% agarose gel electrophoresis.The primer sequence is shown in Table 2:

2 amplimer of table

Reaction system are as follows:

Response procedures are as follows: 94 DEG C of 2min；98 DEG C of 10s, 60 DEG C of 30s, 68 DEG C of 1min, 35 circulations；68 DEG C of extension 7min, 25 ℃∞.Primer size is about 1.1Kb, and the correct PCR product of amplified product band size sends to sequencing.Screen two The mutant strain (Fig. 1, A) of ZmGA20ox3 gene editing and 4 ZmGA20ox5 gene editings mutant strain (Fig. 1, B).The Heterozygous mutants and Mutants homozygous material edited are obviously (Fig. 2, A and B) shorter than wild-type corn plant.

The PCR detection of T-DNA segment in 6 Mutants homozygous of embodiment

According to the T-DNA sequence design 3 of insertion Maize genome to specific primer, with Mutants homozygous blade genome DNA is that template carries out PCR amplification.The sequence of 3 pairs of primers is shown in Table 3:

3 amplimer of table

Reaction system are as follows:

Response procedures are as follows: 95 DEG C of 5min；95 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72 DEG C of 7min, 25 DEG C ∞。

PCR amplification is carried out by template of the leaves genomic DNA of ga20ox3 Mutants homozygous mut1, amplified production is through 1% Agarose gel electrophoresis detection, discovery 2, No. 3, No. 7, No. 8 in the genome of totally 4 plants of transgenic corns without T-DNA insertion (figure 3).These corns, which downgrade material, has important Breeding value.

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

Bibliography

[1]Duvick D.N.,Smith J.S.C.,Cooper M.,et al.Long-term selection in a commercial hybrid maize breeding program.Plant Breeding,2010,24:109-151.

[2]Hedden P.,Phillips A.L..Gibberellin metabolism:new insights revealed by the genes.Trends in Plant Science,2000,5(12):523-530.

[3]Kawaide H..Biochemical and molecular analyses of gibberellin biosynthesis in fungi.Journal of the Agricultural Chemical Society of Japan, 2006,70(3):583-590.

[4]Teng F.,Zhai L.,Liu R.,et al.ZmGA3ox2,a candidate gene for a major QTL,qPH3.1,for plant height in maize.The Plant Journal,2012,73(3):405-416.

[5]Rosa M.,Abraham J.,María J.,et al.The maize MID-COMPLEMENTING ACTIVITY homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF coordinates organ growth and tissue patterning.The Plant Cell,2017,29(3):474-490.

[6]Tsuda K.,Abraham-Juarez M.J.,Maeno A.,et al.KNOTTED1 cofactors, BLH12 and BLH14,regulate internode patterning and vein anastomosis in maize.The Plant Cell,2017,29(5):1105-1118.

[7]Zhang D.,Sun W.,Singh R.,et al.GRF-interacting factor1(gif1) regulates shoot architecture and meristem determinacy in Maize.The Plant Cell,2018 30(2):360-374.

Sequence table

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Claims (10)

1. controlling the gene of plant plant height, which is characterized in that including corn ZmGA20ox3 and/or ZmGA20ox5 gene, wherein Corn ZmGA20ox3 gene is the following protein (a) of coding or gene (b):

(a) protein that the amino acid sequence shown in SEQ ID NO:1 forms；

(b) sequence shown in SEQ ID NO:1 be substituted, lack or add one or several amino acid and with same function by (a) protein derived from；

Corn ZmGA20ox5 gene is the following protein (a ') of coding or the gene of (b '):

The protein that (a ') amino acid sequence shown in SEQ ID NO:2 forms；

Sequence shown in (b ') SEQ ID NO:2 is substituted, lacks or adds one or several amino acid and has same function The protein as derived from (a ').

2. gene described in claim 1 downgrades the application in material breeding in corn, which is characterized in that it is to utilize genetic engineering Means, to corn ZmGA20ox3 and/or ZmGA20ox5 gene carry out rite-directed mutagenesis so that corn ZmGA20ox3 and/or ZmGA20ox5 gene lacks functionality, to realize the genetic improvement to corn plant height.

3. the method for downgrading material using gene editing technology initiative corn, which is characterized in that for the target gene in corn ZmGA20ox3 and/or ZmGA20ox5 designs the sgRNA sequence based on CRISPR/Cas9, will contain the coding sgRNA sequence DNA fragmentation be connected in the carrier for carrying Cas, with the carrier maize transformation of building, realize to gene ZmGA20ox3 and/or The rite-directed mutagenesis of ZmGA20ox5, and then the transgenic corns for obtaining ZmGA20ox3 and/or ZmGA20ox5 gene lacks functionality are planted Strain.

4. according to the method described in claim 3, it is characterized in that, being directed to gene ZmGA20ox3, the core of sgRNA action site Nucleotide sequence is 5 '-GGAGCCATTCCTGTGGCCGC-3 ' and 5 '-CTGTCCTTCGGCTTCCACGA-3 '.

5. according to the method described in claim 3, it is characterized in that, being directed to gene ZmGA20ox5, the core of sgRNA action site Nucleotide sequence is 5 '-AGATCCCCGCGCCATTCCTG-3 ' and 5 '-CTGTCGTTCGGCTACCACGA-3 '.

6. method according to claim 4 or 5, which is characterized in that two sgRNA action sites are passed through different expression cassettes It is connected in series in identical carrier.

7. according to the method described in claim 3, it is characterized in that, the carrier for carrying Cas is pBUE411.

8. according to the method described in claim 3, it is characterized in that, passing through Agrobacterium-mediated transformation corn.

9. according to the method described in claim 3, it is characterized in that, the corn is selfing assemblage 31.

10. a kind of method that initiative corn downgrades material, which is characterized in that according to any one of claim 3-9 the method system Standby transgenic corn plant, then transgenic corn plant is hybridized, is returned, is selfed or vegetative propagation, to formulate corn Downgrade material；

Preferably, the corn formulated is downgraded in material without external source Insert Fragment.