CN106868036A - A kind of method of rite-directed mutagenesis initiative corn compact plant germplasm and its application - Google Patents
A kind of method of rite-directed mutagenesis initiative corn compact plant germplasm and its application Download PDFInfo
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Abstract
Method and its application the invention discloses a kind of rite-directed mutagenesis initiative corn compact plant germplasm.The method for cultivating compact plant corn of the invention, including to the carrier of Maize genome editor is imported in purpose corn, compact plant corn is obtained, compared with purpose corn, Leaf angle diminishes compact plant corn；The carrier of Maize genome editor contains the promoter of Cas9 GFPs, sgRNA encoding genes and RNA polymerase III identification.Gene editing to corn can be realized using the method for the present invention, the compact corn with vertical property is obtained, with breeding very high and cultivating value.
Description
Technical field
The method of corn compact plant germplasm is formulated the present invention relates to a kind of rite-directed mutagenesis in genetic engineering field and its answer
With.
Background technology
Corn (Zea mays L.) is one of Three major grain crops important in the world, and yield needs constantly to increase to meet
The growing demand of current and future.Planting density is the key factor for influenceing yield per unit area, the jade of many decades
Rice yield data shows that the contribution that the increase of planting density increases to total output is far above the increase of single plant yield.Therefore,
The planting density for improving corn unit area is one of feasible method for increasing the total yield (Brekke et al., 2011).
Corn can make upright blade, Basic leaf angle diminish without tip of a leaf proterties, photosynthetic area becomes big, the efficiency of light energy utilization is improved, to hair
Open up resistance to close cropping pattern and improve population yield and all play an important roll.The light quantity of the catch of unit area and the close phase of yield
Close, and upright blade corn has more light quantity of the catch (Lambert and in planting density and unit area higher
Johnson, 1978), also there is same discovery (Sinclair and Sheehy, 1999) in paddy rice.Upright blade
Proterties existing a large amount of report (Duvick, 2005 in the document that corn hybrid seed develops；Hammer et al.,2009).
Therefore, upright blade proterties has breeding and cultivating value very high.
The corn tip of a leaf be blade with leaf sheath intersection on the inside of membranaceous projection, auricle is a pair of tip of a leaf both sides from blade base
The protrusion of portion's Elongation of Edge such as ear of summary out.Used as blade and the joint portion of leaf sheath, auricle and the tip of a leaf make blade
Shape has a certain degree between stalk.Auricle tip of a leaf deletion mutant (liguleless mutants) has lacked leaf
Ear and the tip of a leaf (Becraft and Freeling, 1991；Fowler and Freeling,1996；Moon et al.,
2013), the cenospecies of the mutant shown in field test yield potential (Lambert and Johnson,
1978).The rise of China's compact corn breeding is influenceed by the international breeding of high photosynthetic efficiency and Ideotype Breeding, plant type
Improvement can be to significantly improve photosynthetic efficiency, the combination of Plant-type Breeding and Yield Breeding, the actually breeding of high photosynthetic efficiency
With the combination of ideal type breeding.Compact corn plant leaf is rushed on tiltedly lifting, and fringe position above Leaf angle is less than 15 °, one
As translucency it is good, leaf area index is high, and biological yield is high, suitable dense planting, is China's corn with high yield, superelevation
The evolutionary path of product.Corn lg1 mutant plants upright blades, plant type is compact, and belongs to single-gene recessive mutation,
There is phenotype as homozygote, do not find other bad proterties, therefore be the excellent material for developing compact corn.
CRISPR/Cas9 is that the short palindrome in interval based on bacterium or archeobacteria rule cluster repeats CRISPR (clustered
Regularly interspaced short palindromic repeats) mediation acquired immune system derive and
The gene editing technology come.The technology recognizes DNA by RNA base pair complementarities, instructs Cas9 nucleic acid cleavage to know
Other double-stranded DNA, induces homologous recombination (HDR, homologous directed repair) or nonhomologous end chain
(NHEJ, non-homologous end-joining) is met, and then realizes target DNA editor.Based on bacterium II types
The gene editing technology of immunologic mechanism exploitation has great application prospect on plant particularly crop genetic improvement.Should
One of basic demand of technology is exactly recipient cell intracellular expression single molecular recognition RNA (sgRNA, single guiding
RNA), the molecule is responsible for recognizing specific gene editing site.Then mediate and cut with reference to Cas9 albumen enforcement DNA enzymatic
Activity, DNA double strand breaks are introduced in the site of design, and repairing approach by the NHEJ or HDR of intracellular introduces prominent
Become.Therefore, the expression of sgRNA is the important component of the technology.
The content of the invention
The technical problems to be solved by the invention are how to cultivate compact plant corn.
In order to solve the above technical problems, present invention firstly provides cultivation compact plant corn (Leaf angle change Corn)
Method.
The method for cultivating compact plant corn (Leaf angle change Corn) provided by the present invention, including to purpose corn
The middle carrier for importing Maize genome editor, obtains compact plant corn, and the compact plant corn is beautiful with the purpose
Rice is compared, and Leaf angle diminishes；
The carrier of the Maize genome editor contains Cas9 GFPs, sgRNA encoding genes and RNA polymerase III
The promoter of identification；The promoter of the identification of the RNA polymerase III starts the transcription of the sgRNA encoding genes；
The target DNA that the sgRNA is recognized in corn is the DNA fragmentation for encoding LG1 albumen, the LG1 albumen
It is the protein of a1 or a2：
A1, amino acid sequence are the protein of SEQ ID No.1；
A2, in the amino acid sequence shown in SEQ ID No.1 by substitution and/or missing and/or addition one or several
The protein as derived from a1 that individual amino acid residue is obtained.
In the above method, the LG1 albumen is the amino acid sequence shown in SEQ ID No.1, contains 399 amino
Acid.
In the above method, the cDNA genes of the LG1 albumen, nucleotide sequence as shown in SEQ ID No.2, SEQ
248-1447 of ID No.2 is the coded sequence of the LG1 albumen.
In the above method, the target site sequence of the sgRNA identifications is the DNA molecular shown in SEQ ID No.3.
In the above method, the sgRNA encoding genes are the DNA shown in 10087-10106 of SEQ ID No.4
Molecule.
In the above method, the promoter of the identification of the RNA polymerase III can be the ca99 of promoter ZmPol III, described to open
The ca99 of mover ZmPol III are following DNA fragmentations a) or b) or c)：
A) DNA molecular shown in SEQ ID No.5；
B) DNA sequence dna for and a) limiting has 75% or more than 75% homogeneity, and the DNA with promoter function
Molecule；
C) under strict conditions with the DNA sequence dna hybridization for a) or b) limiting, and the DNA with promoter function points
Son.
In the above method, the Leaf angle diminishes as fringe position above Leaf angle diminishes.
In order to solve the above technical problems, present invention also offers the carrier of Maize genome editor.
The carrier of Maize genome editor provided by the present invention, containing Cas9 GFPs, sgRNA encoding genes and
The promoter of the identification of RNA polymerase III；The promoter of the identification of the RNA polymerase III starts the sgRNA codings base
The transcription of cause；
The target DNA that the sgRNA is recognized in corn is the DNA fragmentation for encoding LG1 albumen, the LG1 albumen
Be the protein of a1 or a2：
A1, amino acid sequence are the protein of SEQ ID No.1；
A2, in the amino acid sequence shown in SEQ ID No.1 by substitution and/or missing and/or addition one or several
The protein as derived from a1 that individual amino acid residue is obtained.
Above-mentioned carrier also includes starting the promoter (such as enhanced promoters of CaMV 35s) of Cas9 GFPs transcription,
Terminate the terminator (such as NOS terminator) of Cas9 GFPs transcription, (such as kanamycins resists for replicon and resistant gene
Property gene).
In above-mentioned carrier, the target site sequence of the sgRNA identifications is the DNA molecular shown in SEQ ID No.3.
In above-mentioned carrier, the sgRNA encoding genes are the DNA shown in 10087-10106 of SEQ ID No.4
Molecule.
In above-mentioned carrier, the promoter of the identification of the RNA polymerase III can be the ca99 of promoter ZmPol III, described to open
The ca99 of mover ZmPol III are following DNA fragmentations a) or b) or c)：
A) DNA molecular shown in SEQ ID No.5；
B) DNA sequence dna for and a) limiting has 75% or more than 75% homogeneity, and the DNA with promoter function
Molecule；
C) under strict conditions with the DNA sequence dna hybridization for a) or b) limiting, and the DNA with promoter function points
Son.
Above-mentioned carrier concretely ca99-sgRNA-Cas9 of recombinant vector ZmPol III, its nucleotides sequence is classified as SEQ ID
Shown in No.4.Wherein, the nucleotides sequence shown in 12309-16409 of SEQ ID No.4 is classified as Cas9 albumen bases
Cause, the nucleotides sequence shown in 208-884 of SEQ ID No.4 is classified as the enhanced promoters of CaMV 35s, SEQ ID
Nucleotides sequence shown in 10087-10106 of No.4 is classified as the encoding gene of sgRNA, the of SEQ ID No.4
Nucleotides sequence shown in 9691-10086 is classified as the 16477-16729 of promoter ZmPol III ca99, SEQ ID No.4
Nucleotides sequence shown in position is classified as NOS terminator, the nucleotides sequence shown in 4407-4995 of SEQ ID No.4
It is classified as the nucleotides sequence shown in 2188-2982 of replicon ori, SEQ ID No.4 and is classified as kalamycin resistance base
Cause.
Application of the carrier of above-mentioned Maize genome editor in compact plant corn is cultivated falls within the model of present invention protection
Enclose.
The above-mentioned ca99-sgRNA-Cas9 of recombinant vector ZmPol III can be by using agriculture bacillus mediated, Ti-plasmids, Ri matter
The conventional biology methods such as grain, plant viral vector, directly delivered DNA, microinjection, conductance, particle gun are converted
Plant cell or tissue, and the plant cell that will convert or tissue cultivating are into plant.
It is demonstrated experimentally that being planted using the recombinational agrobacterium maize transformation containing the ca99-sgRNA-Cas9 of recombinant vector ZmPol III
Strain, can enter edlin to LG1 genes, obtain plant (the double equipotential lg1 frameshit bases for producing lg1 frameshit gene pures
The sequence of cause is identical) with the plant of lg1 frameshit genetic heterozygosis (sequence of double equipotential lg1 frameshit genes is different), with nothing
The phenotype of auricle, and Leaf angle is substantially reduced compared with wild type group, and upright blade, leaf is significantly improved to value, is
Compact corn plant.Gene editing to corn can be realized using the method for the present invention, obtaining has vertical property
Compact corn, with breeding very high and cultivating value.
Brief description of the drawings
Fig. 1 is the structural representation of the ca99-sgRNA-Cas9 of recombinant vector ZmPol III.
Fig. 2 is the agarose gel electrophoresis of the pcr amplification product of corn compact mutant strain.Wherein, M is DNA points
Son amount Marker, wt represent wild-type corn self-mating system CA335, and positive control is recombinant vector ZmPol III
Ca99-sgRNA-Cas9,1-22 is experimental group corn.
Fig. 3 is the mode of appearance for having phenotype (without auricle) maize mutant type plant.Wherein, A is wild type seedling stage, B
It it is saltant type seedling stage, C is the wild-type mature phase, and D is the saltant type maturity period.
Fig. 4 is the SfcI restriction enzyme site schematic diagrames of sgRNA.
Fig. 5 is the digestion identification of gene mutation strain.Wherein, wt is the wild-type corn self-mating system without Sfc1 digestions
The pcr amplified fragment of CA335 plant, 1-5 is through the wild-type corn self-mating system CA335 plant of Sfc1 digestions
Pcr amplified fragment, 6-10 is the pcr amplified fragment through the homozygous mutant plants of Sfc1 digestions, 11-16 be through
The pcr amplified fragment of the heterozygous mutation body plant of Sfc1 digestions.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given only for
The present invention is illustrated, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, unless otherwise specified, is conventional method.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Corn inbred line-Zheng 58 in following embodiments, is the mother of Henan Agricultural Sciences institute improved variety Zheng Dan 958
This, is common used material on domestic corn breeding, and this material takes from country of Institute of Crop Science, Chinese Academy of Agricultural Science
Germplasm resource bank.
Trans5 α Competents cell, pEASY-Uni Seamless Cloning and in following embodiments
Assembly Kit are the product of Beijing Quanshijin Biotechnology Co., Ltd.
PEASY-blunt simple vector in following embodiments are Beijing Quanshijin Biotechnology Co., Ltd
Product, Trans T1 competent cells are the product of Beijing Quanshijin Biotechnology Co., Ltd.
Ago-Gel QIAquick Gel Extraction Kit in following embodiments is the product of TIANGEN Biotech (Beijing) Co., Ltd.,
Catalog number is DP209.
Corn inbred line CA335 (Chuanxiao Xie, Shihuang Zhang_, Minshun in following embodiments
Li,Xinhai Li,Zhuanfang Hao,Li Bai,Degui Zhang,Yehong Liang.Inferring
Genome Ancestry and Estimating Molecular Relatedness Among 187Chinese Maize
Inbred Lines.Journal of Genetics and Genomics(Formerly Acta Genetica Sinica).
2007,34(8):738_748) public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science (i.e. applicant)
, the biomaterial is only attached most importance to again used by related experiment of the invention, can not be used as other purposes.
Embodiment 1, ca99 (the hereinafter referred promoters of promoter ZmPol III containing the identification of corn RNA polymerase III
The ca99 of ZmPol III) CRISPR/Cas9 gene editing carriers structure
First, the clone of the ca99 of promoter ZmPol III
Corn inbred line-Zheng 58 seed 3-4 is taken, after sterilization seed soaking, training in the quartzy sand table of humidity is put it into
Educate, blade to be grown, extract blade genome standby.With blade genome as template, using primer pair ZmPol
III ca99-F (5 '-AATTGGCCCTTACAAAATAG-3 ') and ca99-R (the 5 '-GGAGCGGTGGTC of ZmPol III
GCAGCTG-3 ') enter performing PCR amplification, obtain pcr amplified fragment.Gained pcr amplified fragment is utilized
PEASY-Blunt Cloning Kit are subcloned, and experimental implementation is stated with reference to explanation and carried out, and obtains recombinant vector
1.The recombinant vector 1 of acquisition is transformed into Trans5 α Competent cells, overnight incubation, picking Dan Ke
Grand, Amplification Culture is simultaneously sequenced, and as a result shows to contain the promoter ZmPol shown in SEQ ID No.5 in recombinant vector 1
III ca99 sequences, the ca99 subcloning vectors of ZmPol III are named as by the recombinant vector 1.
2nd, the structure of the CRISPR/Cas9 gene editing carriers containing the ca99 of promoter ZmPol III
CRISPR/Cas9 gene editing carriers containing the ca99 of promoter ZmPol III, its nucleotides sequence is classified as SEQ ID
No.4.Wherein, the nucleotides sequence shown in 12309-16409 of SEQ ID No.4 is classified as Cas9 GFPs,
Nucleotides sequence shown in 208-884 of SEQ ID No.4 is classified as the enhanced promoters of CaMV 35s, SEQ ID No.4
10087-10106 shown in nucleotides sequence be classified as the encoding gene of sgRNA, the of SEQ ID No.4
Nucleotides sequence shown in 9691-10086 is classified as the 16477-16729 of promoter ZmPol III ca99, SEQ ID No.4
Nucleotides sequence shown in position is classified as NOS terminator, the nucleotides sequence shown in 4407-4995 of SEQ ID No.4
It is classified as the nucleotides sequence shown in 2188-2982 of replicon ori, SEQ ID No.4 and is classified as kalamycin resistance base
Cause.
By the digestion with restriction enzyme CPB carriers of Hind III, linear carrier is obtained.Using primer pair ZmPol III
Ca99 (the HindIII)-F and ca99 of ZmPol III (Seed)-R expand from the ca99 subcloning vectors of ZmPol III of step one
Increase the ca99 fragments of promoter ZmPol III, and the ca99 fragments upstreams of promoter ZmPol III is carried and linearized vector one
Nucleotide fragments of the end with 15bp homologous sequences, the ca99 fragments downstreams of promoter ZmPol III carry Cas9 target sequences
GCGGAGACTAAGTGGCTGTA fragments in row, obtain the ca99 fragments 1 of promoter ZmPol III.By round pcr profit
The upstream of the sgRNA scaffold templates of synthesis is added with primer sgRNA (LG)-F and sgRNA (HindIII)-R
GCGGAGACTAAGTGGCTGTA sequences, downstream increases the nucleotides piece for having 15bp homologous sequences with linearized vector
Section, obtains the encoding gene segment 1 of sgRNA.By pEASY-Uni Seamless Cloning and Assembly
The encoding gene segment 1 of linear carrier, the ca99 fragments 1 of promoter ZmPol III and sgRNA is realized seamless company by Kit
Clone is met, the ca99-sgRNA-Cas9 of recombinant vector ZmPol III are obtained.By recombinant vector ZmPol III
Ca99-sgRNA-Cas9 is converted into Trans5 α competent cells, overnight incubation, picking monoclonal, Amplification Culture
And be sequenced.Sequencing result shows that the nucleotides sequence of the ca99-sgRNA-Cas9 of recombinant vector ZmPol III is classified as SEQ ID
No.4, CRISPR/Cas9 gene editings carrier (Fig. 1) as containing the ca99 of promoter ZmPol III.
The primer：
sgRNA(LG)-F:GCGGAGACTAAGTGGCTGTAGTTTTAGAGCTAGAAATAGCA
sgRNA(HindⅢ)-R:TGCACTGCACAAGCTTAAAAAAAGCACCGACTCG
ZmPolⅢca99(HindIII)-F:GGCCAGTGCCAAGCTTAATTGGCCCTTACAAAATAGCTA；
ZmPolⅢca99(Seed)-R:TACAGCCACTTAGTCTCCGCGGAGCGGTGGTCGCAGCTGA。
Embodiment 2, cultivation corn compact mutant strain and its checking
First, the structure of recombinational agrobacterium
The ca99-sgRNA-Cas9 of the recombinant vector ZmPol III conversion Agrobacterium EHA105 competence that embodiment 1 is obtained
Cell, obtains recombinational agrobacterium.Recombinational agrobacterium is extracted into plasmid, is sequenced, sequencing result shows recombinant vector
The ca99-sgRNA-Cas9 of ZmPol III are successfully transferred in Agrobacterium EHA105 competent cells, and recombinational agrobacterium builds just
Really.
2nd, corn compact mutant strain is cultivated
The recombinational agrobacterium that step one is built is entered in corn inbred line CA335 ratarias through sieving by Induction Transformation
Choosing, dedifferentiation and break up again, obtain and complete break up plant again.Comprise the following steps that：
1st, it is inoculated with
After pollination in 9-15d, when rataria grows to 1.8-2.2mm, whole fruit ear is fetched into interior, use 70% wine
Essence is sterilized to fruit ear, often goes 1 layer of skin, 70% alcohol swab to sterilize 1 time, after removing the peel completely, with 0.1% mercuric chloride
Sterilizing about 10min.Pruned line by line from top to bottom kind of skin and endosperm with scalpel again, chosen from seed middle and upper part with tweezers
Rataria, the recombinational agrobacterium bacterium solution for adding the step of resuspended good OD values are 0.5-0.6 in right amount to prepare soaks 15-20
Min, shake that around here will be frequently.After having soaked, bacterium solution is abandoned, unnecessary bacterium solution is blotted with aseptic filter paper.By scultellum to
On be placed in and filled in the culture dish of N6 inducing cultures through autoclaving, being placed in 26 DEG C of incubators carries out light culture, about
The young shoot and root for growing carefully are removed during 3-5d, continues to cultivate.
2nd, subculture
The embryo callus that subculture is 3-4 times are most suitable to make transgenic acceptor, every 3 weeks subcultures 1 time, subculture altogether
3 times.Select embryo callus within the 11st week after inoculation, embryo callus are caught broken into the small of 2.5-3mm sizes
Block, in the way of every bottle of 3 pieces of callus being transferred to differential medium carries out dedifferentiation culture.
3rd, break up
Embryo callus are transferred to after redifferential medium daily l2-16h illumination cultivations at the same temperature, and illumination is strong
Degree is about 2000lx.Transformation and selection and degerming, while regeneration induction plant, two weeks subcultures once, obtain seedling, i.e.,
To recombinate the milpa seedling of Agrobacterium-mediated Transformation.
3rd, the identification of corn compact mutant strain
1st, PCR augmentation detections
1) when the milpa seedling that the recombinational agrobacterium of step 2 is converted grows to 3-4 leaves, obtain recombinational agrobacterium and turn
The milpa of change, the milpa to recombinating Agrobacterium-mediated Transformation extracts DNA, takes 2cm2Fresh blade is put into clean
In mortar, mortar need to add Liquid nitrogen precooler, and the blade fetched is put into liquid nitrogen, is then smashed blade with pestle.
2) with 65 DEG C of CTAB buffer solutions of preheating of 800 μ L of liquid-transfering gun addition, (CTAB for preparing 500mL extracts slow
Fliud flushing：It is 1M to add 50mL concentration, and the Tris solution of pH7.5,70mL concentration is the NaCl solution of 5M, 50mL
Concentration is 0.5M, and the EDTA solution of pH8.0,5.0g CTAB, 5.0mL concentration is 14M beta -mercaptoethanol solution,
325mL ddH2O, matching while using), rapid acutely vibration makes ground sample immerse in extract.65 DEG C of heating water baths
15min, will overturn mixing several times during water-bath.After water-bath, centrifuge tube is taken out, be cooled to room temperature.
3) chloroform of 800 μ L is added under fume hood：Isoamyl alcohol (v：V=24：1) mixed liquor, gently reverses shake 10min
12000rpm, 10min are centrifuged afterwards, during supernatant gone into a new 2mL centrifuge tubes.
4) the RNase solution of 3 μ L, warm bath 0.5-1h at room temperature or at 37 DEG C are added.
5) 3 steps are repeated, supernatant is transferred in the centrifuge tube of new 1.5mL.
6) 0.7 times of isopropanol of volume precooling (- 80 DEG C) is added, is gently mixed to DNA and is separated out aggegation, in 12000rpm
Under the conditions of 10min is centrifuged, abandon supernatant (careful DNA is poured out).
7) 70% ethanol of 700 μ L is added, is washed 1-2 times.
8) ddH of 50 μ L is added after room temperature is dried2O dissolving DNAs.
9) DNA sample of 2% Ago-Gel Detection and Extraction is prepared, the milpa that as recombinational agrobacterium is converted
DNA。
10) DNA of the milpa of the recombinational agrobacterium conversion obtained with step 9 is template, with ZmPol III
The ca99-Ubi-F1 and ca99-Ubi-R1 of ZmPol III carries out pcr amplification reaction, detection recombinational agrobacterium conversion for primer
Milpa in whether contain Cas9 GFP fragments, be designated as experimental group.Wild-type corn self-mating system CA335
The DNA extraction method of milpa that is converted with recombinational agrobacterium of DNA extraction method, with recombinant vector ZmPol III
Ca99-sgRNA-Cas9 is positive control.
Table 1, design of primers
Table 2, the configuration of PCR reaction systems
PCR reaction conditions
Pcr amplification product detects with 2% Ago-Gel, and with ultraviolet gel imaging instrument image checking.
Result as shown in Fig. 2 in 22 plants of pcr amplification products of the milpa of the recombinational agrobacterium conversion of experimental group
Purpose fragment containing 421bp, wild-type corn self-mating system CA335 does not contain the purpose fragment of 421bp, shows weight
The ca99-sgRNA-Cas9 successful conversions of group carrier ZmPol III enter the milpa of recombinational agrobacterium conversion.
2nd, the Molecular Detection of mutation type
To analyze the mutation type of target sequence, with LG1 gene C as9 target sequences as core, turned with recombinational agrobacterium
The DNA of the milpa of change is template, and using Primer Premier 6.0, primers F-lg1-E1 is designed in downstream thereon
And R-lg1-E1, PCR primer is obtained, built by PEASY-blunt simple vector and be subcloned, will connect
Good subcloning vector is converted into Trans T1 competent cells, overnight incubation, picking monoclonal, and Amplification Culture is simultaneously
Sequencing, counts mutation type.The pcr amplification product of wild-type corn self-mating system CA335 contains SEQ ID No.2's
DNA fragmentation shown in 563-585, is LG1 genes by the corresponding unnamed gene of the amplified fragments；Restructuring agriculture bar
Pcr amplification product does not contain the DNA pieces shown in 563-585 of SEQ ID No.2 in the milpa of bacterium conversion
The plant of section is mutant plants, is lg1 genes by the corresponding unnamed gene of the amplified fragments.Used by Molecular Detection
Primer：
F-lg1-E1:GCCAACACCGCCGTATTAG
R-lg1-E1:GATCCAGTGATGACCGAGTG
Event number=(homozygous mutation strain number × 2+ heterozygosis is dashed forward for mutation rate=be totally converted event mutator sum/be totally converted
Become strain number)/it is totally converted event number × 100%.
113 plants of the milpa that detection recombinational agrobacterium is converted altogether, wherein homozygous mutation strain number are 104 plants, and heterozygosis is dashed forward
It is 1 plant to become strain number, and mutation rate is 92.48%.Homozygous mutation strain and heterozygous mutant strain are designated as mutant.
Maize genome is dliploid, and lg1 genes are recessive gene, and LG1 genes are dominant gene.LG1 genes are passed through
After CRISPR/Cas9 albumen editors, the lg1 genes of frameshift mutation and non-frameshift mutation can be produced, frameshit will be produced to dash forward
The lg1 unnamed genes of change are lg1 frameshit genes, and the lg1 unnamed genes that will produce non-frameshift mutation are the non-frameshit of lg1
Gene.The result of this experiment shows, in the milpa of 113 plant weight group Agrobacterium-mediated Transformations, there is 15 plants of lg1 frameshit bases
Because of the plant (sequence of double equipotential lg1 frameshit genes is identical) of homozygosis, without auricle；There are 9 plants to contain LG1 genes
Plant, there is auricle；There is 3 plants of plant of the non-frameshit gene pures of lg1 (sequence phase of double non-frameshit genes of equipotential lg1
Together), there is auricle；There are 46 plants of plant of lg1 frameshit genetic heterozygosis (sequence of double equipotential lg1 frameshit genes is different),
Without auricle；There is the heterozygous plant of 40 plants of lg1 frameshit genes and the non-frameshit genes of lg1, there is auricle.Maize leaves
Piece can cause Leaf angle to diminish without auricle, upright blade.
3rd, wild type and mutant leaf are determined to value
Maize leaves are to value：Parameter for representing corn leaf morphology.Its measuring method is：From 15 plants of step 2
7 plants are randomly selected in the plant (sequence of double equipotential lg1 frameshit genes is identical) of lg1 frameshit gene pures, is designated as dashing forward
Variant group, wild type group, every plant of measurement fringe position leaf above leaf numerical value are designated as by corn inbred line CA335.N is represented
The number of blade of measure, θ is the angle (degree) of blade and stalk vertical direction, and L is leaf (unit long：Centimetre), Lf
It is the distance (unit of phyllopodium to leaf peak：Centimetre), computational methods are：Lov (leaf is to value)=[∑ (90 °-
θ)(Lf/L)]/n。
The leaf of table 3, wild type and mutant is determined to value
As shown in table 3, the lov of mutant group is 67.78 to result, and the lov of wild type group is 48.75, mutant group
Leaf angle be substantially reduced compared with wild type group, upright blade, leaf is significantly improved to value, is compact corn plant
(B and D in Fig. 3).
4th, the digestion identification of corn lg1 gene mutation bodies
With reference to all G (N20) GG in LG1 gene orders search LG1 genes in B73 genomes (B73AGPv3.0)
Sequence, it is Cas9 target sites, the position that " GCGGAGACTAAGTGGCTGTA " sequence is finally chosen on the 1st extron
The C16-A20 and adjacent first bases G of PAM of point are the restriction enzyme site (Fig. 4) of restriction enzyme SfcI,
The restriction enzyme site contains the editing sites of Cas9 albumen simultaneously.The ca99 of promoter ZmPol III start initial base
G1, target site otch is T17, and PAM sequences are GGG, and Cas9 albumen pair is guided as sgRNA seed using this sequence
The 1st exon location in genome in LG1 genes carries out shearing and produces breach, is repaired through the NHEJ or HDR of intracellular
Approach introduces mutation, and mutation bonnet restriction enzyme site disappears, and purpose fragment lg1 can not be by restriction enzyme SfcI digestions.
The mutant plants and wild-type corn self-mating system CA335 plant leafs obtained with step 2 are experiment material,
Genomic DNA is extracted respectively.
Table 4, design of primers relevant information
PCR reaction systems
PCR reaction conditions
Pcr amplification product detects with 2% Ago-Gel, and with ultraviolet gel imaging instrument image checking；Use agarose
DNA QIAquick Gel Extraction Kits reclaim purpose fragment.
Digestion is identified
Digestion system：50μL
DNA mass：300ng
The digestion time：2h
Clip size：553bp
Digestion post-fragment size：340bp、213bp
As shown in figure 5, LG1 genes can be by Sfc1 digestions, digestion post-fragment size is 340bp and 213bp to result；
Lg1 genes cannot be by Sfc1 digestions, and clip size is still 553bp.Heterozygote because respectively contain LG1 and lg1 genes,
Therefore digestion post-fragment is three, i.e. 553bp, 340bp and 213bp.Can identify wild by Sfc1 digestions
Type corn inbred line CA335 plant (LG1LG1), homozygous mutation strain (lg1lg1) and heterozygous mutant strain (LG1lg1).
Claims (10)
1. the method for cultivating compact plant corn, including to the carrier of Maize genome editor is imported in purpose corn, obtain
To compact plant corn, compared with the purpose corn, Leaf angle diminishes the compact plant corn；
The carrier of the Maize genome editor contains Cas9 GFPs, sgRNA encoding genes and RNA polymerase III
The promoter of identification；The promoter of the identification of the RNA polymerase III starts the transcription of the sgRNA encoding genes；
The target DNA that the sgRNA is recognized in corn is the DNA fragmentation for encoding LG1 albumen, the LG1 albumen
It is the protein of a1 or a2：
A1, amino acid sequence are the protein of SEQ ID No.1；
A2, in the amino acid sequence shown in SEQ ID No.1 by substitution and/or missing and/or addition one or several
The protein as derived from a1 that individual amino acid residue is obtained.
2. method according to claim 1, it is characterised in that：The target site of the sgRNA identifications is SEQ ID
DNA molecular shown in No.3.
3. method according to claim 1 and 2, it is characterised in that：The sgRNA encoding genes are SEQ ID
DNA molecular shown in 10087-10106 of No.4.
4. according to any described method in claim 1-3, it is characterised in that：The identification of the RNA polymerase III
Promoter is the ca99 of promoter ZmPol III, and the ca99 of promoter ZmPol III are following DNA a) or b) or c)
Fragment：
A) DNA molecular shown in SEQ ID No.5；
B) DNA sequence dna for and a) limiting has 75% or more than 75% homogeneity, and the DNA with promoter function
Molecule；
C) under strict conditions with the DNA sequence dna hybridization for a) or b) limiting, and the DNA with promoter function points
Son.
5. according to any described method in claim 1-4, it is characterised in that：The Leaf angle diminish for fringe position with
Upper Leaf angle diminishes.
6. the carrier of the Maize genome editor described in claim 1, encodes containing Cas9 GFPs, sgRNA
Gene and the promoter of the identification of RNA polymerase III；The promoter of the identification of the RNA polymerase III starts the sgRNA
The transcription of encoding gene；
The target DNA that the sgRNA is recognized in corn is the DNA fragmentation for encoding LG1 albumen, the LG1 albumen
It is the protein of a1 or a2：
A1, amino acid sequence are the protein of SEQ ID No.1；
A2, in the amino acid sequence shown in SEQ ID No.1 by substitution and/or missing and/or addition one or several
The protein as derived from a1 that individual amino acid residue is obtained.
7. carrier according to claim 6, it is characterised in that：The target site of the sgRNA identifications is SEQ ID
DNA molecular shown in No.3.
8. the carrier according to claim 6 or 7, it is characterised in that：The sgRNA encoding genes are SEQ ID
DNA molecular shown in 10087-10106 of No.4.
9. according to any described carrier in claim 6-8, it is characterised in that：The identification of the RNA polymerase III
Promoter is the ca99 of promoter ZmPol III, and the ca99 of promoter ZmPol III are following DNA a) or b) or c)
Fragment：
A) DNA molecular shown in SEQ ID No.5；
B) DNA sequence dna for and a) limiting has 75% or more than 75% homogeneity, and the DNA with promoter function
Molecule；
C) under strict conditions with the DNA sequence dna hybridization for a) or b) limiting, and the DNA with promoter function points
Son.
10. application of any described carrier in compact plant corn is cultivated in claim 6-9.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588270A (en) * | 2018-07-10 | 2018-09-28 | 北京市农林科学院 | Corn is without tip of a leaf character related SNP molecular markers development and its application |
| CN109022450A (en) * | 2018-08-15 | 2018-12-18 | 河南农业大学 | It is a kind of regulate and control corn Leaf angle ZmCLA2-1 gene and its application |
| CN112695041A (en) * | 2019-10-22 | 2021-04-23 | 华南农业大学 | Application of ZmSBP28 gene in regulation of corn plant type |
| CN116548300A (en) * | 2023-06-26 | 2023-08-08 | 中国农业科学院生物技术研究所 | Method for improving density-resistant yield of corn and application thereof |
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2015
- 2015-12-14 CN CN201510922653.XA patent/CN106868036B/en active Active
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| HUI-LI XING等: "A CRISPR/Cas9 toolkit for multiplex genome editing in plants", 《BMC PLANT BIOLOGY》 * |
| JIHYUN MOON等: "The Liguleless narrow mutation affects proximal-distal signaling and leaf growth", 《DEVELOPMENT》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588270A (en) * | 2018-07-10 | 2018-09-28 | 北京市农林科学院 | Corn is without tip of a leaf character related SNP molecular markers development and its application |
| CN108588270B (en) * | 2018-07-10 | 2021-06-29 | 北京市农林科学院 | Development and application of SNP molecular marker related to maize leafless tongue trait |
| CN109022450A (en) * | 2018-08-15 | 2018-12-18 | 河南农业大学 | It is a kind of regulate and control corn Leaf angle ZmCLA2-1 gene and its application |
| CN109022450B (en) * | 2018-08-15 | 2021-09-24 | 河南农业大学 | ZmCL 2-1 gene for regulating and controlling included angle of corn leaves and application thereof |
| CN112695041A (en) * | 2019-10-22 | 2021-04-23 | 华南农业大学 | Application of ZmSBP28 gene in regulation of corn plant type |
| CN112695041B (en) * | 2019-10-22 | 2024-03-26 | 华南农业大学 | Application of ZmSBP28 gene in regulation of corn plant type |
| CN116548300A (en) * | 2023-06-26 | 2023-08-08 | 中国农业科学院生物技术研究所 | Method for improving density-resistant yield of corn and application thereof |
| CN116548300B (en) * | 2023-06-26 | 2023-12-19 | 中国农业科学院生物技术研究所 | Method for improving density-resistant yield of corn and application thereof |
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