CN106701803B - Maize female parent haploid main induced gene and its application - Google Patents
Maize female parent haploid main induced gene and its application Download PDFInfo
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Abstract
The invention discloses a major induction gene of a corn female parent haploid and application thereof, and also discloses a major gene for inducing and producing the corn female parent haploid and application thereof in Double Haploid (DH) breeding. The gene codes Phospholipase (PLA), and the nucleotide sequence of the gene is sequence 1. The gene has female parent haploid inducting capacity in the process of selfing or crossing with other corn materials as a male parent after mutation in a coding region. The invention obtains the serial allelic mutation of the gene for the first time, and proves the female parent haploid induction function through self-crossing and hybridization. The obtaining of the maternal haploidy has important theoretical and practical significance for haploid breeding (DH) and related researches thereof.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to corn female parent monoploid main effect induced gene and application.
Background technique
Corn is the first big crop in the world, has the versatiles such as edible, feeding and industrial processes.The volume increase of corn
It is had a very important significance for supplying current edible, feeding and industrial processes demand.It is gradually decreased in current cultivated area
In the case where, cultivate high yield, how anti-and extensively suitable corn hybrid seed is crucial.The incubation of corn hybrid seed depends on excellent selfing
The breeding of system.The method of traditional breeding self-mating system is time-consuming and laborious, usually needs that a stable selfing more than generation can be bred as by 7
System.In recent years, haploid breeding technology is short, high-efficient with breeding cycle, is easy to binding molecule marker-assisted breeding method etc.
Advantage has been increasingly becoming the major technique of selecting and breeding corn self-mating system.Currently, to be mainly derived from corn orphan female for monoploid in corn
The induction of reproduction induction system, i.e. Stock6 or induction system derived from it are used as male parent, generate after hybridizing with other materials.Due to
Most of induction systems have imported R1-nj label, thus embryo and the Endosperm Color label progress haploid identification of corn can be used.
Thus substantially increase the efficiency of Haploid Breeding of Maize.
Due to induction system by produce single-female generation generate maternal haploid method be with a wide range of applications with
Value, therefore, global more R&D institutions for Stock6 and its Derivative line induction generate maternal haploid hereditary basis and
Basic of Biology has carried out a large amount of research.The result shows that corn single-female generation induction can generate this property of corn monoploid
Shape is heritable, and the control by multiple genetic locus.(1999) etc. 2 control inductivity characters are detected
Genetic locus is located at No. 1 chromosome and No. 2 chromosomes.It can explain about 17% phenotypic variation.Barrant etc.
(2008) it also detects that the genetic locus positioned at No. 1 chromosome, demonstrates the result of forefathers' research.Prigge etc. (2012) is utilized
Multiple groups carry out genome-wide screening, the genetic locus of 8 control inductivities are found altogether, including positioned at No. 1 chromosome
The main effect genetic locus of 1.04bin, and it is named as qhir1.It therefore, is that multiple control haploid-inductions are related positioned at qhir1
The QTL that effect in QTL is maximum, function is mostly important.Dong Xin etc. (2014) has carried out finely positioning to qhir1, and successfully will
Positioning section is contracted to the range of 243Kb.The candidate gene of research qhir1 lures the breeding and single-female generation of novel induction system
Leading is to induce the science of heredity of Haploid production and Biological Mechanism particularly important, in view of haploid breeding skill in current breeding industry
The popularity that art utilizes, the invention are widely used space and market prospects.
Summary of the invention
It is an object of the present invention to provide corn female parent monoploid main effect induced gene, ZmPLA mutated genes.
ZmPLA mutated gene provided by the invention, nucleotides sequence are classified as wild ZmPLA gene nucleotide series are enterprising
Row insertion or/and missing or/and Substitution, obtain sequence；
The wild ZmPLA gene nucleotide series are sequence 1.
In said gene, the nucleotides sequence of the ZmPLA mutated gene is classified as following 1) -4) in it is any (corresponding below
ZmPLA mutated gene ZmHIR1-1, ZmHIR1-2, ZmHIR1-3, ZmHIR1-Stock6 in embodiment):
It 1) is that T base is inserted between wild ZmPLA gene nucleotide series the 280th and 281, other bases are constant,
Obtained sequence；
It 2) is the 271st -281 bit bases missing of wild ZmPLA gene nucleotide series, other bases are constant, obtain
Sequence；
3) it is lacked for wild the 281st bit base G of ZmPLA gene nucleotide series, other bases are constant, obtained sequence；
4) to be inserted into CGAG after wild ZmPLA gene nucleotide series the 1569th, and the 409th C sports T, the
421 C sport G, and the 441st T sports C, and the 887th T sports G, and the 1210th G sports C, and the 1306th
The T of position sports C, and the 1435th G sports A, and the 1471st C sports A, and the 1541st A sports C, and the 1588th
The T of position sports C, and the 1591st C sports A, DNA molecular shown in obtained sequence.1687th bit base A sports C,
1691st bit base G sports A, and the 1706th bit base T sports C, and the 1708th bit base G sports C, 45-46 base position
It lacks two bases TA, 65-67 and CAA is replaced with by TCG for base, two base TC are inserted between 67-68 base position, the
80-81 bit base replaces with CG, 499-503 bit base GTAC missing by TT, and 524 bit base C sport G, and 530 bit base G are prominent
Becoming T, 553-560 bit base GCATGCAT missing, 806-809 bit base GTAC is lacked, and the 1741st bit base G sports A,
1781st bit base C sports T, and the 1787th bit base A sports T, other bases are constant, obtained sequence.
Above-mentioned mutated gene or the wild ZmPLA gene nucleotide series are in induction generation corn or other plant list
Times body or the application in Doubled haploid line (Double Haploid, DH) breeding are also the scope of protection of the invention.
Silencing inhibits the expression of ZmPLA gene in purpose Plant Genome or knocks out ZmPLA gene in production plant list
Application in times body is also the scope of protection of the invention；
Or, the expression of ZmPLA gene or the substance of knockout ZmPLA gene are in life in silencing or inhibition purpose Plant Genome
Producing the application in plant female parent monoploid is also the scope of protection of the invention.
Above-mentioned application is that the expression of ZmPLA gene, obtains transgenosis in silencing or inhibition or knockout purpose Plant Genome
Plant, then the genetically modified plants are used to hybridize or be selfed, obtain maternal monoploid.
In above-mentioned application, the silencing or expression or the knockout ZmPLA base for inhibiting ZmPLA gene in purpose Plant Genome
Because ZmPLA gene expression amount in purpose Plant Genome is made to reduce or occur missing or insertion mutation；
It is described that ZmPLA gene in purpose Plant Genome is made to occur to make described in missing or insertion mutation in above-mentioned application
In purpose Plant Genome outside ZmPLA gene First Exon and/or Second Exon and/or third exon and/or the 4th
Missing or insertion mutation occur for aobvious son；
Or the mode CRISPR/ for making ZmPLA gene in purpose Plant Genome that missing or insertion mutation occur
Cas9 and/or TELLEN technology and/or T-DNA insertion and/or EMS mutagenesis.
It is described to make ZmPLA gene First Exon in purpose Plant Genome that missing occur or be inserted into prominent in above-mentioned application
The mode of change is CRISPR/Cas9；
Or the substance of the expression of ZmPLA gene or knockout ZmPLA gene in the silencing or inhibition purpose Plant Genome
To make the substance of ZmPLA gene First Exon generation missing or insertion mutation in purpose Plant Genome；
It is described to make the substance of ZmPLA gene First Exon generation missing or insertion mutation in purpose Plant Genome
CRISPR/Cas9 system；
The target sequence of the CRISPR/Cas9 system is 264-286 bit base in the 1st exon shown in sequence 3；
The sgRNA sequence of the CRISPR/Cas9 system is sequence 4.
Silencing inhibits the expression of ZmPLA gene in purpose Plant Genome or knocks out ZmPLA gene in Doubled haploid line
Application in (Double Haploid, DH) breeding or breeding hybridized based on DH system is also the scope of protection of the invention.
Silencing or inhibit purpose Plant Genome in ZmPLA gene expression or knock out ZmPLA gene substance double
Application in monoploid system's (Double Haploid, DH) breeding or breeding hybridized based on DH system is also of the invention protects
Range.
Above-mentioned purpose plant is corn or other plant.
Another object of the present invention is to provide silencing or inhibits the expression or knockout of ZmPLA gene in purpose Plant Genome
The substance of ZmPLA gene.
Substance provided by the invention, including CRISPR/Cas9 system, the target sequence of the CRISPR/Cas9 system are sequence
264-286 bit base in 1st exon shown in column 3.
In above-mentioned substance, the sgRNA sequence of the CRISPR/Cas9 system is sequence 4.
Technical scheme is as follows: being predicted by candidate gene, a coding phosphatide is obtained in the section qhir1
Enzyme gene (PLA) is named as ZmPLA, and gene is had successfully been obtained by CRISPR/Cas9 site-directed mutagenesis technique and Transgenic studies
The mutant material of target gene, it is miscellaneous to other corn materials using heterozygous genotypes mutant and homozygous genotype mutant
It hands over, the material after demonstrating ZmPLA mutation can induce the maternal haploid function of generation as male parent, will not have after series jump
Functional ZmPLA unnamed gene is ZmHIR1.The artificial rite-directed mutagenesis of gene ZmPLA is pinpointed using CRISPR/Cas9
Mutating technology modifies the First Exon of ZmPLA gene so that First Exon base occur replacement, missing and/
Or insertion obtains.Modification shot design length is 20bp when CRISPR/Cas9 is modified, and is located at the in the 1st exon of ZmPLA
264-286 bit base, target site sequence are as follows: GCTGCAGGAGCTGGACGGACCGG.
The artificial directed mutants of ZmPLA that the CRISPR/Cas9 site-directed mutagenesis technique is generated in target site body, feature
It is, the CRISPR/Cas9 gene modification technology causes 1bpT base between 280-281 bit base position to insert in modification target site
Enter, obtain ZmPLA gene mutation body, the First Exon sequence after insertion base, the unnamed gene after being inserted into base is
ZmHIR1-1 can generate about 1%~2% corn female parent monoploid in the Mutant progeny
The artificial directed mutants of ZmPLA that the CRISPR/Cas9 site-directed mutagenesis technique is generated in target site body, feature
It is, the CRISPR/Cas9 gene modification technology causes to lack between 271-281 bit base position in modification target site
GAGCTGGACGG obtains ZmPLA gene mutation body, the First Exon sequence after lacking base, the gene life after lacking base
Entitled ZmHIR1-2 can generate about 1%~2% corn female parent monoploid in the Mutant progeny
The artificial directed mutants of ZmPLA that the CRISPR/Cas9 site-directed mutagenesis technique is generated in target site body, feature
It is, the CRISPR/Cas9 gene modification technology causes the 281st bit base G to lack in modification target site, obtains ZmPLA base
Because of mutant, First Exon sequence after lacking base, the unnamed gene after lacking base is ZmHIR1-3, after the mutant
About 1%~2% corn female parent monoploid can be generated in generation
The present invention also provides the mutant gene sequences of known corn female parent haploid inducing line Stock6 a kind of, and by its
It is named as ZmHIR1-Stock6, characteristic Z mHIR1-Stock6 leads to the offspring for being selfed or hybridizing as male parent with other materials
There is monoploid.The sequence is that the present invention predicts and be sequenced by candidate gene to obtain, and demonstrates this by Transgenic studies
The function forfeiture of gene results in the maternal haploid generation of corn.
The present invention also provides artificial directed mutants the answering in Haploid Breeding of Maize of the gene ZmPLA
With.
Basic principle of the invention is as follows: being directed to candidate gene ZmPLA, designs target position on first exon of gene
First exon of ZmPLA gene is carried out screen mutation, is obtained by point sequence by the method for CRISPR/Cas9 rite-directed mutagenesis
Obtain the genetically modified mutant of ZmPLA gene lacks functionality.Will after single plant that success is mutated is selfed, the T1 of acquisition for seed,
Plant again, and with T1 for the pollen of plant Mutants homozygous and Heterozygous mutants to two corn hybrid seed Zheng Dan 958 and Jing Ke
968 hybridization, obtain offspring.It is thin according to the growing way in offspring's single plant field, molecular labeling and streaming by the filial generation kind in field
Whether the verifying of the methods of born of the same parents' Ploidy Identification wherein there is maternal monoploid.
The experiment proves that the mutation of ZmPLA can result in the maternal haploid generation of corn, for disclosing corn
The science of heredity and Biological Mechanism that maternal monoploid generates have established important basis.Meanwhile utilizing this experiment or this method institute
The mutation single plant of acquisition, the haploid induction ability with corn female parent, the induction system novel for breeding further increase and lure
Conductance, and have great importance in terms of improving Haploid Breeding of Maize efficiency.
Detailed description of the invention
Fig. 1 is the setting of ZmPLA gene structure display and the target site using CRISPR/Cas9 technology.
Fig. 2 is the ZmPLA site-directed point mutation and sequencing result mediated using PCR and Sanger sequencing detection CRISPER.
Fig. 3 is ZmPLA after hybridizing with cenospecies Zheng Dan 958, capital section 968, the monoploid photo of appearance.
Fig. 4 is field monoploid blade ploidy identification result.
Fig. 5 is field monoploid molecular markers for identification result.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, induction generate the maternal haploid method of corn
One, the positioning of corn female parent monoploid phenotype correlation gene
By being positioned to the inductivity correlation QTL in the derivative induction system of corn female parent monoploid Stock6, obtain
Main effect QTL-the qhir1 for controlling haploid induction, by pre- to the gene progress functional annotation and candidate gene that position in section
It surveys, a candidate gene ZmPLA has finally been determined.
Two, corn female parent haploid induction ability is obtained after knocking out corn ZmPLA gene
1, CRISPR/Cas9 system knocks out corn ZmPLA gene
1) selection of sgRNA sequence
Fig. 1 is gene structure and target site schematic diagram.
The genome sequence of corn ZmPLA gene is as shown in sequence table 1.The sequence of the First Exon of corn ZmPLA gene
Column (sequence 2 sequence 1 91-450) as shown in sequence table 2.
Target site sequence is designed in the First Exon sequence of corn ZmPLA gene, length 21bp is located at outside first
Show the 264-286 base position of son.
Target site sequence is GCTGCAGGAGCTGGACGGACCGG (sequence 3).
Target position point design sgRNA sequence is GCUGCAGGAGCUGGACGGACCGG (sequence 4), the coding DNA of the sgRNA
Molecule is sequence 3.
2), the building of CRISPR/Cas9 carrier
CRISPR/Cas9 carrier is that the non-coding DNA molecules of sgRNA shown in sequence 3 in sequence table will be inserted into pBUN411
Carrier (is recorded in the following literature: Xing H L, Dong L, Wang Z P, et al.A CRISPR/Cas9 toolkit
For multiplex genome editing in plants [J] .BMC plant biology, 2014,14 (1): 1.)
The carrier arrived.
3), the acquisition of transgenic corns
CRISPR/Cas9 carrier is gone into Agrobacterium competent cell EHA105 by heat-shock transformed, obtains recombinant bacterium
EHA105/CRISPR/Cas9 carrier.
Agrobacterium EHA105 competent cell is purchased from Hua Yue ocean Biotechnology Co., Ltd, and the public can be obtained by purchase
By recombinant bacterium EHA105/CRISPR/Cas9 carrier, using Agrobacterium infestation method, (recombinational agrobacterium carries out 28 DEG C of expansions again
It is numerous, using expand it is numerous after bacterium solution maize immature embryos are infected) maize transformation Xu178 (and record in the following literature: Xiang Yan, Wu
Big strong, Jiang Haiyang waits foundation [J] laser biology journal of Plant Regeneration System from Mature Embryos of Maize Elite Inbred Lines, 2007,16 (5):
649-654., the public can obtain from country, China Agricultural University corn improvement center) rataria, by screening, breaks up and takes root
T0 is obtained afterwards for transgenic corn plant.
4) the ZmPLA gene transgenic corn identification, to mutate
T0 is acquired for transgenic corn plant blade, and extracts genomic DNA as template, carries out PCR with following primer
Amplification, obtains the pcr amplification product of different strains.
ZmPLA mutant nucleotide sequence detection primer:
1240F:CCCUCGACGAGUAUCUAUAGC
1240R:GAAGAUGAUAGGCUGCAGC.
The pcr amplification product of different strains is subjected to Sanger sequencing, according to sequencing result and wild-type corn ZmPLA base
The First Exon (sequence 2) of cause is compared, and whether identification T0 dashes forward for ZmPLA gene in transgenic corns difference strain
Become.
As a result as follows: 21 plants of T0 are in transgenic corn plant, and the ZmPLA gene in 8 plants mutates, specific mutant form
Formula is as follows, and part is as shown in Figure 2:
ZmPLA mutated gene ZmHIR1-1 is that T alkali is inserted between ZmPLA gene nucleotide series 1 the 280th -281
Base, DNA molecular shown in obtained sequence；
ZmPLA mutated gene ZmHIR1-2 is the 271st -281 11 base deletions of ZmPLA gene nucleotide series 1,
DNA molecular shown in obtained sequence.
ZmPLA mutated gene ZmHIR1-3 is the 281st bit base G of ZmPLA gene nucleotide series 1 missing, obtained sequence
DNA molecular shown in column；
The plant that ZmPLA gene mutates is denoted as positive T0 for transgenic corns.
5) genotype identification for the transgenic corns that T1 mutates for ZmPLA gene
The positive T0 that above-mentioned 1 is obtained sows again after harvesting seed for transgenic corns, obtains T1 for transgenic corns.
Identify T1 for transgenic corns ZmPLA gene whether be mutation genotype, it is specific as follows: T1 for transgenosis jade
The genomic DNA of rice is used as template, utilizes ZmPLA mutant nucleotide sequence detection primer: 1240F:CCCTCGACGAGTATCTATAGC with
1240R:GAAGATGATAGGCTGCAGC is expanded, and PCR product is carried out Sanger sequencing, according to sequencing result to T1 generation
The genotype of transgenic corns is classified.
Then it is heterozygous genotypes with the sequence of double-peak feature from target site sequence in sequencing result, then turns for T1 generation
Gene corn heterozygous ZmPLA gene mutation (the middle ZmPLA gene mutation of 1 of homologue, another 1 of homologue
Middle ZmPLA gene is unmutated)；
There is the sequence of special unimodal feature, the First Exon (sequence with corn ZmPLA gene from target site sequence
2) it compares, if equally, for wild type, there is no mutation, lower surface analysis does not consider；If there is mutation, for T0 for plant from
The homozygous mutation obtained after friendship, then it is homozygous (in 2 of homologue for transgenic corns ZmPLA gene mutation for T1
ZmPLA gene mutates).T1 has ZmHIR1-1, ZmHIR1- for transgenic corns heterozygous ZmPLA gene mutation strain
2, and the mutation type of each strain is as follows:
T1 contains for 1 in homologue in transgenic corns ZmPLA gene mutation heterozygous strain ZmHIR1-1
ZmPLA mutated gene, the mutated gene are insertion T base between ZmPLA gene nucleotide series 1 the 280th -281, and its
DNA molecular shown in the constant obtained sequence of his base, another contains wild type ZmPLA gene；
T1 contains for 1 in homologue in transgenic corns ZmPLA gene mutation heterozygous strain ZmHIR1-2
ZmPLA mutated gene, the mutated gene are the 271st -281 missing GAGCTGGACGG alkali of ZmPLA gene nucleotide series 1
Base, and DNA molecular shown in the constant obtained sequence of other bases, another contains wild type ZmPLA gene；
T1 in two homologues in the homozygous strain ZmHIR1-3 of transgenic corns ZmPLA gene mutation for containing
ZmPLA mutated gene, which is the 281st missing G base of ZmPLA gene nucleotide series 1, and other bases are constant
DNA molecular shown in obtained sequence.
2, CRISPR/Cas9 system knocks out the identification of the haploid induction ability of the obtained mutant of corn ZmPLA gene
1) T1 is identified for heterozygous genotypes transgenic corns ZmPLA gene mutation single plant haploid induction ability
(1) field phenotypic evaluation
T1 is authorized respectively for the pollen of transgenic corns ZmPLA genetic heterozygosis mutating strain series ZmHIR1-1, ZmHIR1-2
Cenospecies Zheng Dan 958 (blocks up pure letter, Cao Chunjing, Cao Qing wait the breeding of corn hybrid seed Zheng Dan 958 and apply [J] corn section
Learn, 2006,14 (6): 43-45 is obtained from Rui Jin Zhong Ye difficult to understand limited liability company) and (cenospecies capital section, cenospecies capital section 968
968 are purchased from Beijing Tun Yuzhong industry Co., Ltd, and article No. is the capital Tun Yu section 968, and the public can collect beautiful kind of an industry by Beijing to be had
Responsible company's purchase is limited to obtain), obtain filial generation；
T1 is selfed for transgenic corns ZmPLA genetic heterozygosis mutating strain series ZmHIR1-2, obtains self progeny.
Above-mentioned gained offspring is seeded in field, observes offspring's single plant phenotype, monoploid has plant short and small, blade compared with
Narrow, and upper punching, plant type is compact, the features such as male sterility, and diploid then shows as that plant is tall and big, and blade is roomy, hangs down loosely, fertility is just
Often.
It is compareed with wild-type corn (ZmPLA gene is unmutated) with the offspring of cenospecies.Each strain amount detection is such as
Shown in table 1.
Statistical result is as shown in table 1 and Fig. 3:
T1 hybridize for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-1 with cenospecies Zheng Dan 958 54
1 is obtained in a offspring and shows as monoploid character single plant, is drafted as haplobiont；
T1 hybridize for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-1 with cenospecies capital section 968 50
1 is obtained in a offspring and shows as monoploid character single plant, is drafted as haplobiont；
T1 hybridize for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-2 with cenospecies Zheng Dan 958 93
2 are obtained in a offspring and shows as monoploid character single plant, are drafted as haplobiont；
T1 hybridize for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-2 with cenospecies capital section 968 57
2 are obtained in a offspring and shows as monoploid character single plant, are drafted as haplobiont；
1 is obtained in 27 offsprings that T1 is selfed for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-2
It is a to show as monoploid character single plant, it drafts as haplobiont.
(2) FCM analysis blade ploidy
By identification in above-mentioned (1) ZmHIR1-1 and cenospecies offspring obtain totally 2 show as monoploid character plant,
In ZmHIR1-2 and cenospecies offspring identification obtain totally 4 show as monoploid character plant, in ZmHIR1-2 self progeny
1 of identification acquisition shows as monoploid character plant and carries out FCM analysis, the method is as follows:
The nucleus for extracting plant young leaflet tablet to be measured, using diploid maize leaf as control；Flow cytometer is used again
Device detects signal, first detection diploid cell nuclear signal, and diploid cell nuclear signal peak position is set as 100 (due to two times
Inhereditary material in body cell is twice of inhereditary material in haploid cell, and therefore, haploid cell nuclear signal peak position is 50
Nearby occur)；If the signal peak of plant to be measured appears near 100, then it is assumed that it is enriched with diploid cell nuclear signal intensity
Position is identical, which is diploid.If plant cell nuclear signal to be measured peak appears near 50, then it is assumed that the plant to be measured
Strain is haplobiont.
Each strain amount detection is as shown in table 1.
As a result as shown in figure 4, upper figure is wild-type corn FCM analysis as a result, the following figure is T1 for transgenic corns
ZmPLA gene mutation heterozygous strain FCM analysis result；
As a result as follows:
The pseudohaploids that ZmHIR1-1 goes out with 2 in cenospecies filial generation through phenotypic evaluation are through flow cytomery
Afterwards, ploidy is monoploid, is denoted as T1 for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-1 pseudohaploid
Plant.
The pseudohaploids that ZmHIR1-2 goes out with 4 in cenospecies filial generation through phenotypic evaluation are through flow cytomery
Afterwards, ploidy is monoploid, is denoted as T1 for transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-2 pseudohaploid
Plant.
For the pseudohaploid that 1 phenotypic evaluation goes out in ZmHIR1-2 self progeny after flow cytomery, ploidy is equal
For monoploid, T1 is denoted as transgenic corns ZmPLA heterozygous gene mutation strain ZmHIR1-2 pseudohaploid plant.
(3) molecular markers for identification
Random Design 30 utilizes transgenic line Xu178 (Xiang Yan, Wu great Qiang, Jiang Hai to molecular labeling in the genome
Foundation [J] laser biology journal of Plant Regeneration System from Mature Embryos of Maize Elite Inbred Lines is waited, 2007,16 (5) in ocean: 649-654.,
The public can obtain from country, China Agricultural University corn improvement center) and cenospecies Zheng Dan 958, capital section 968 genomic DNA
As template, amplification and polymorphic molecular marker screening are carried out, final to obtain a pair of of molecular labeling, PCR product is in Xu178
For 500bp, and it is 300bp in cenospecies Zheng Dan 958 and the product length of cenospecies capital section 968, there is larger difference, Ke Yili
It is differentiated with agarose gel electrophoresis, Xu178PCR product is larger, and electrophoretic velocity is slow, and cenospecies Zheng Dan 958 and cenospecies
The PCR product segment of capital section 968 is smaller, and electrophoretic velocity is fast, and therefore, the band of Xu178 is located at cenospecies Zheng Dan 958 and cenospecies
The top of 968 band of capital section.(Fig. 5,3,4 swimming lanes are respectively cenospecies Zheng Dan 958,968 banding pattern of cenospecies capital section, and 5 swimming lanes are
Xu178 banding pattern)
To above-mentioned T1 for 2 quasi-simple times occurred in heterozygous gene mutation strain ZmHIR1-1 and cenospecies filial generation
Body plant and T1 are for the 4 pseudohaploid plant occurred in heterozygous gene mutation strain ZmHIR1-2 and cenospecies filial generation
Extracting genome DNA, PCR and the detection of agarose banding pattern are carried out, if single plant to be measured only has the band (Fig. 5,1 swimming lane) of Zheng Dan 958,
Then think that the banding pattern of male parent material is not present in the single plant, therefore is maternal monoploid.If being existed simultaneously in filial generation single plant
The band (Fig. 5,2 swimming lanes) of 958/ capital section 968 of Xu178 and Zheng Dan, then it is assumed that the single plant is the offspring of normal hybridisation, is two times
Body.
As a result as shown in figure 5, M:Marker, 5 be male parent Xu178 banding pattern, and 4 be female parent 958 banding pattern of Zheng Dan, and 3 be maternal capital
968 banding pattern of section, 1 is monoploid banding pattern in offspring, and 2 be heterozygous diploid banding pattern in offspring.
Molecular markers for identification result is as follows:
The molecular markers for identification result table of 2 ZmHIR1-1 and the pseudohaploid gone out in cenospecies offspring through phenotypic evaluation
It is bright, it is maternal haplobiont.
The molecular markers for identification result table of 4 ZmHIR1-2 and the pseudohaploid gone out in cenospecies offspring through phenotypic evaluation
It is bright, it is maternal haplobiont.
Therefore, the offspring's single plant or Heterozygous transgenic strain self progeny's single plant of Heterozygous transgenic strain and cenospecies
In, if being accredited as monoploid according to any method in above-mentioned 3 kinds of method qualification results, which is or candidate is corn mother
This monoploid；If above-mentioned 3 kinds of method qualification results are not monoploid, which is not or candidate is not maternal single times of corn
Body.
It is as shown in table 1 to count above-mentioned qualification result, haploid-induction (%)=(haploid number/test total strain number) *
100, it can be seen that hybridize after ZmPLA gene mutation with other materials, can get corn female parent monoploid in offspring.
The frequency of occurrences of haplobiont in 1 heterozygous mutant strain test progeny of table
Note: control is the offspring obtained after being pollinated with wild type Xu178 material and cenospecies Zheng Dan 958 and capital section 968.
2) T1 is identified for homozygous genotype transgenic corns ZmPLA gene mutation single plant haploid induction ability
(1) field phenotypic evaluation
T1 is authorized to cenospecies Zheng Dan 958 for the pollen of transgenic corns ZmPLA homozygous mutation strain ZmHIR1-3,
Obtain filial generation；
T1 is selfed for transgenic corns ZmPLA genetic heterozygosis mutating strain series ZmHIR1-3, obtains self progeny.
Above-mentioned gained offspring is seeded in field, observes offspring's single plant phenotype, monoploid has plant short and small, blade compared with
Narrow, and upper punching, plant type is compact, the features such as male sterility, and diploid then shows as that plant is tall and big, and blade is roomy, hangs down loosely, fertility is just
Often.
As a result as follows:
T1 is miscellaneous for 256 of transgenic corns ZmPLA homozygous gene mutating strain series ZmHIR1-3 and cenospecies Zheng Dan 958
4 are obtained in friendship offspring and shows as monoploid character single plant, are drafted as haplobiont；
T1 has obtained 2 in 30 self progenies of transgenic corns ZmPLA homozygous gene mutating strain series ZmHIR1-3
A single plant for showing as monoploid character, is drafted as haplobiont.
(2) FCM analysis blade ploidy
After T1 is hybridized for transgenic corns ZmPLA homozygous gene mutating strain series ZmHIR1-3 with cenospecies Zheng Dan 958
2 pseudohaploid single plants carry out fluidic cell in 4 pseudohaploids and ZmHIR1-3 homozygous mutation self progeny in offspring
Detection, the method is as follows:
The nucleus of plant young leaflet tablet to be measured is extracted, with wild-type corn (ZmPLA gene is unmutated, diploid) blade
As control；Signal is detected with fluidic cell instrument again, first detection diploid cell nuclear signal, and diploid cell core is believed
Number peak position be set as 100 (since the inhereditary material in diploid cell is twice of inhereditary material in haploid cell, it is single
Times somatic cell nuclear signal peak position occurs near 50)；If the signal peak of plant to be measured appears near 100, then it is assumed that it is with two
Times somatic cell nuclear signal strength enrichment positions are identical, which is diploid.If plant to be measured carefully each strain testing number
Amount is as shown in table 2.
As a result as shown in figure 4, upper figure is wild-type corn FCM analysis as a result, the following figure is T1 for transgenic corns
ZmHIR1-3 homozygous lines offspring's pseudohaploid FCM analysis result；
As a result as follows:
4 pseudohaploids occurred in ZmHIR1-3 and 958 filial generation of Zheng Dan are after flow cytomery, ploidy
It is monoploid.
2 pseudohaploid plant that the self progeny of ZmHIR1-3 homozygous mutation material generates are through flow cytomery
Afterwards, ploidy is monoploid.
(3) molecular markers for identification
Random Design 30 marks agarose molecules in the genome, utilizes transgenic line Xu178 and cenospecies Zheng Dan
958, the genomic DNA of capital section 968 carries out amplification and polymorphic molecular marker screening as template, obtains a pair of of molecular labeling,
Its PCR product is 500bp in Xu178, and is 300bp in cenospecies Zheng Dan 958 and the product length of cenospecies capital section 968,
With larger difference, can use can be differentiated using agarose gel electrophoresis, and Xu178PCR product is larger, and electrophoretic velocity is slow,
And the PCR product segment of cenospecies Zheng Dan 958 and cenospecies capital section 968 is smaller, electrophoretic velocity is fast, therefore, the band position of Xu178
In the top of 968 band of cenospecies Zheng Dan 958 and cenospecies capital section.(Fig. 5,3,4 swimming lanes are respectively cenospecies Zheng Dan 958, hybridization
968 banding pattern of Zhong Jing section, 5 swimming lanes are Xu178 banding pattern)
The ZmHIR1-3T1 selected to field is for transgenic corns homozygous gene mutating strain series and 958 filial generation of Zheng Dan
In 4 pseudohaploid plant carry out extracting genome DNA, PCR and agarose banding pattern detection, if single plant to be measured only has Zheng Dan
958 band (Fig. 5,1 swimming lane), then it is assumed that the banding pattern of male parent material is not present in the single plant, therefore is maternal monoploid.If hybridization
The band (Fig. 5,2 swimming lanes) of Xu178 and Zheng Dan 958 is existed simultaneously in offspring's single plant, then it is assumed that after the single plant is normal hybridisation
Generation is diploid.
As a result as shown in figure 5, M:Marker, 5 be Xu178 banding pattern, and 4 be 958 banding pattern of cenospecies Zheng Dan, and 3 be cenospecies capital
968 banding pattern of section, 1 is monoploid banding pattern in offspring, and 2 be zygoid banding pattern in offspring；
As a result as follows:
T1 is for 4 obtained in the filial generation of transgenic corns ZmHIR1-3 homozygous mutation strain and Zheng Dan 958
Pseudohaploid shows as maternal monoploid after molecular markers for identification.
Therefore, the offspring's single plant or Transgenic wheat line self progeny's single plant of Transgenic wheat line and cenospecies
In, if being accredited as monoploid according to any method in above-mentioned 3 kinds of method qualification results, which is or candidate is corn mother
This monoploid；If above-mentioned 3 kinds of method qualification results are not monoploid, which is not or candidate is not maternal single times of corn
Body.
The results are shown in Table 2, inductivity (%)=(monoploid strain number/test total strain number) * 100, it can be seen that ZmPLA
Hybridize after gene mutation with other materials, can get corn female parent monoploid in offspring.
The frequency of occurrences of haplobiont in 2 heterozygous mutant strain test progeny of table
Three, the genotype identification of corn female parent monoploid Stock6
Stock6 is that capable of inducing of reporting for the first time generates maternal haploid special material (Coe EH (1959) A of corn
Line of maize with high haploid frequency.Am Nat 93:381-382) by inductivity main effect
The finely positioning and candidate gene of QTL is predicted, is found compared with B73, and it is prominent that there are many places SNP on the gene ZmPLA of Stock6
Change and the insertion (table 3) of a 4bp, so that the gene loses normal function.Using Crisper technology to wild-type corn
After the ZmPLA gene of material carries out rite-directed mutagenesis, it was demonstrated that it pollinates after the gene mutation as male parent and other materials, energy in offspring
There is the monoploid of certain frequency.ZmPLA gene in Stock6 genome is by the generation of gene ZmPLA shown in sequence 1
Obtained mutant nucleotide sequence after following mutation, is named as ZmHIR-Stock6.
The exons mutation form of 3 gene ZmPLA of table
5 ' the UTR regions of gene ZmPLA are mutated are as follows: 45-46 base position lacks two base TA, and 65-67 is base
CAA is replaced with by TCG, is inserted into two base TC between 67-68 base position, 80-81 bit base replaces with CG by TT
The introne region mutagenesis of gene ZmPLA are as follows: 499-503 bit base GTAC missing, 524 bit base C sport G,
530 bit base G sport T, 553-560 bit base GCATGCAT missing, 806-809 bit base GTAC missing.
3 ' the UTR regions of gene ZmPLA are mutated are as follows: and the 1741st bit base G sports A, and the 1781st bit base C sports T,
1787th bit base A sports T.
Above-mentioned induction is that the ZmPLA mutated gene 4 in Stock6 is occurred compared to the ZmPLA wild type gene in B73
SNP and Insertion mutation, specific mutant form is as follows:
ZmHIR-Stock6 mutant nucleotide sequence is inserted into CGAG, and the 409th after being ZmPLA gene nucleotide series 1 the 1569th
The C of position sports T, the 421st C sports G, and the 441st T sports C, and the 887th T sports G, the 1210th
G sports C, and the 1306th T sports C, and the 1435th G sports A, and the 1471st C sports A, the 1541st A
C is sported, the 1588th T sports C, and the 1591st C sports A, DNA molecular shown in obtained sequence.1687th
Bit base A sports C, and the 1691st bit base G sports A, and the 1706th bit base T sports C, and the 1708th bit base G is sported
C, 45-46 base position lack two base TA, and 65-67 replaces with CAA by TCG for base, and 67-68 base position interleaves
Enter two base TC, 80-81 bit base replaces with CG, 499-503 bit base GTAC missing by TT, and 524 bit base C are sported
G, 530 bit base G sport T, 553-560 bit base GCATGCAT missing, and 806-809 bit base GTAC is lacked, and the 1741st
Bit base G sports A, and the 1781st bit base C sports T, and the 1787th bit base A sports T.
The above-mentioned CGAG insertion being located at after 1569 bases causes the gene that frameshift mutation has occurred.Positioned at 319 bases, 331
SNP variation at base and 1120 bases results in the variation of amino acid, also affects the function of protein.
From monoploid character, FCM analysis blade ploidy and molecular labeling in corn female parent monoploid Stock6 offspring
Identification proves there is monoploid.
Therefore, no matter corn ZmPLA gene which kind of mutation cause function forfeiture can form it into corn female parent monoploid.
Sequence table
<110>China Agricultural University
<120>corn female parent monoploid main effect induced gene and application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1795
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
agttcatcac taatcacact tattgtgccc tcgacgagta tctatagcta gctcattaat 60
cgattcgggg gtgtgttgtc gaaggcggca atggcgagct actcgtcgcg gcgtccatgc 120
aatacctgta gcacgaaggc gatggccggg agcgtggtcg gcgagcccgt cgtgctgggg 180
cagagggtga cggtgctgac ggtggacggc ggcggcgtcc ggggtctcat cccgggaacc 240
atcctcgcct tcctggaggc caggctgcag gagctggacg gaccggaggc gaggctggcg 300
gactacttcg actacatcgc cggaaccagc accggcggtc tcatcaccgc catgctcacc 360
gcgcccggca aggacaagcg gcctctctac gctgccaagg acatcaacca cttttacatg 420
cagaactgcc cgcgcatctt tcctcagaag tgagtccgat gctgccgcca ttgttcttgc 480
atccatccag catcgtacgt acgtcctcta tacatctgcg gatcatcatg tgcgcatgtt 540
tgtggcatgc atgcatgcat gtgagcagga gcaggcttgc ggccgccatg tccgcgctga 600
ggaagccaaa gtacaacggc aagtgcatgc gcagcctgat taggagcatc ctcggcgaga 660
cgagggtaag cgagacgctg accaacgtca tcatccctgc cttcgacatc aggctgctgc 720
agcctatcat cttctctacc tacgacgtac gtacgtcgtc acgaatgatt catctgtacg 780
tcgtcgcatg cgaatggctg cctacgtacg ccgtgcgcta acatactcag ctctttccta 840
tctgctgcgc caatttgcag gccaagagca cgcctctgaa gaacgctctg ctctcggacg 900
tgtgcattgg cacgtccgcc gcgccgacct acctcccggc gcactacttc cagactgaag 960
acgccaacgg caaggagcgc gaatacaacc tcatcgacgg cggtgtggcg gccaacaacc 1020
cggtaactga ctagctaact ggaaaacgga cgcacagact ccatgtccat ggcggcccac 1080
aaggtcgatg ctaattgttg cttatgtatg tcgcccgatt gcacatgcgt agacgatggt 1140
tgcgatgacg cagatcacca aaaagatgct tgccagcaag gacaaggccg aggagctgta 1200
cccagtgaag ccgtcgaact gccgcaggtt cctggtgctg tccatcggga cggggtcgac 1260
gtccgagcag ggcctctaca cggcgcggca gtgctcccgg tggggtatct gccggtggct 1320
ccgcaacaac ggcatggccc ccatcatcga catcttcatg gcggccagct cggacctggt 1380
ggacatccac gtcgccgcga tgttccagtc gctccacagc gacggcgact acctgcgcat 1440
ccaggacaac tcgctccgtg gcgccgcggc caccgtggac gcggcgacgc cggagaacat 1500
gcggacgctc gtcgggatcg gggagcggat gctggcacag agggtgtcca gggtcaacgt 1560
ggagacaggg aggtacgaac cggtgactgg cgaaggaagc aatgccgatg ccctcggtgg 1620
gctcgctagg cagctctccg aggagaggag aacaaggctc gcgcgccgcg tctctgccat 1680
caacccaaga ggctctagat gtgcgtcgta cgatatctaa gacaagtggc tttactgtca 1740
gtcacatgct tgtaaataag tagactttat tttaataaaa cataaaaata tatat 1795
<210> 2
<211> 360
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
atggcgagct actcgtcgcg gcgtccatgc aatacctgta gcacgaaggc gatggccggg 60
agcgtggtcg gcgagcccgt cgtgctgggg cagagggtga cggtgctgac ggtggacggc 120
ggcggcgtcc ggggtctcat cccgggaacc atcctcgcct tcctggaggc caggctgcag 180
gagctggacg gaccggaggc gaggctggcg gactacttcg actacatcgc cggaaccagc 240
accggcggtc tcatcaccgc catgctcacc gcgcccggca aggacaagcg gcctctctac 300
gctgccaagg acatcaacca cttttacatg cagaactgcc cgcgcatctt tcctcagaag 360
<210> 3
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
gctgcaggag ctggacggac cgg 23
<210> 4
<211> 23
<212> RNA
<213>artificial sequence
<220>
<223>
<400> 4
gcugcaggag cuggacggac cgg 23
Claims (2)
1.ZmPLA mutated gene, nucleotides sequence, which is classified as, to be inserted into or/and be lacked on wild ZmPLA gene nucleotide series
Mutation is lost, ZmPLA mutated gene is obtained；
The wild ZmPLA gene nucleotide series are sequence 1；
The nucleotides sequence of the ZmPLA mutated gene is classified as following 1) -3) in it is any:
It 1) is that T base is inserted between wild ZmPLA gene nucleotide series the 280th and 281, other bases are constant, obtain
Sequence；
It 2) is the 271st -281 bit bases missing of wild ZmPLA gene nucleotide series, other bases are constant, obtained sequence；
3) it is lacked for wild the 281st bit base G of ZmPLA gene nucleotide series, other bases are constant, obtained sequence.
2. mutated gene described in claim 1 generates the application in the breeding of corn monoploid system in induction.
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| WO2020156406A1 (en) * | 2019-01-31 | 2020-08-06 | 中国农业大学 | Wheat haploid induced gene and application thereof |
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| CN114902957B (en) * | 2021-02-09 | 2023-07-07 | 中国农业大学 | A DNA Molecule for Improving the Induction Rate of Maize Haploid Inducer Lines |
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