CN116286960B - Genetic transformation and gene editing of maize haploid inducer lines - Google Patents
Genetic transformation and gene editing of maize haploid inducer lines Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular breeding, and particularly relates to genetic transformation and gene editing of a corn haploid induction line, and the invention firstly discloses a gene editing transformation vector which is based on a pCambia3301 vector skeleton and comprises three expression units ZmU pro from the right boundary of T-DNA, wherein the three expression units comprise AtU ter, zmUBI pro, spCas9 ter PsRbcS E9 ter and CaMV35Spro Bar, two BsaI restriction sites are reserved between ZmU pro and the gRNA skeleton, and are used for inserting gRNA recognition sequence fragments of any editing target genes by using a DNA ligase or Gibson cloning method after linearizing the vector. The invention discloses a method for directly transforming a corn haploid induction line, which comprises the steps of firstly transforming and editing a target gene of the haploid induction line to verify the function of a gene editing carrier, then inducing any corn hybridization or selfing material to generate haploids by using offspring of the transformed induction line, and forming a double haploid line for editing the same target gene after chromosome doubling, thereby thoroughly avoiding the bottleneck problem that the selfing line which restricts corn gene editing research and application is difficult to transform.
Description
Technical Field
The invention belongs to the technical field of molecular breeding, and particularly relates to genetic transformation and gene editing of a corn haploid induction line.
Background
Gene editing technology based on the acquired immune system CRISPR/Cas9 (Clustered Regularly Interspersed Short Palindromic Repeats) of Streptococcus pyogenes Streptococcus pyogenes bacteria was first realized in vivo in 2013. SpCas9 endonuclease forms a ribozyme complex with guide gRNA, and through the base pairing recognition target of the 20 bp long RNA sequence in gRNA and the edited DNA sequence, the specificity is high, the cleavage activity is strong, the experimental design is simple and easy, so that the CRISPR/Cas9 gene editing system can be rapidly and successfully applied to different animal and plant systems, and the knockout editing of a large number of genes of various plants, which is mainly based on Non-homologous end-linked pathways (Non-homologousend joining, NHEJ), and the accurate design editing mediated by few Homology-dependent repair (HDR), are realized. Based on the CRISPR/Cas9 gene editing system, a number of new technologies have been developed in recent years, including the SpG ubiquitin site editor, cytosine editor (Cytosine base editor, CBE), adeno glance sideways at-in editor (Adenine base editor, ABE), guide editor (PRIMEEDITING, PE), etc. (Chen et al, 2019,Annu Rev Plant Biol 70:667-697; mao et al, 2019,Natl Sci Rev 6:421-437). The final product of the genetically edited offspring can be free of any exogenous DNA after genetic separation, is identified as being equivalent to a conventional breeding product by a plurality of relevant national authorities, can avoid the supervision and approval process and the cost of complex and expensive transgenic products, and is suitable for rapid production and application.
However, gene editing has limited application in plants with difficult genetic transformation, such as corn and soybean, and only a few success reports （Char et al., 2017,Plant Biotechnol J 15: 257–268; Li et al., 2020,CropJ 8: 449-456; Svitashev et al., 2015,Plant Physiol 169: 931–945）, in corn establish a universal efficient genetic transformation system for these important crops as a necessary premise for gene editing application. The effective successful transformation of the current corn is limited to a few experimental hybrid materials such as HiII or inbred line B104 and the like （Frame et al.,2006,Plant Cell Rep 25: 1024-1034; Ishida et al., 2007,Nature Prot 2: 1614-1621; Ishida et al.,2020,Plant Biotechnol 37: 121-128; Zhao et al., 2001,Mol Breed 8: 323–333）., and the experimental inbred lines have relatively poor genetic characters, so that the experimental hybrid materials cannot be directly used for modern breeding, the transformation offspring of the experimental hybrid materials can be used for breeding after being subjected to hybridization and multi-generation backcross transformation and are introduced into the inbred lines with excellent characters, and the process severely restricts the application of gene editing.
The creation and screening of the inbred line is the core of corn crossbreeding, and the conventional hybridization and multi-round backcross method needs six generations of backcross and two generations of inbreds to obtain the stable inbred line with the homozygosity higher than 99 percent, which takes years, has high cost and long period. The haploid doubling technology is to hybridize a haploid inducer with a high-quality genetic resource material, and due to the gene defect of the inducer, all chromosomes from the parent of the inducer are completely lost in the process of zygote development, and about 10% of the generated offspring seeds are haploids and only carry one set of chromosomes of the induced material. By combining with the special color of the induction line, high oil and other phenotypic character screening, mature haploid seeds or young embryos can be selected, induced chromosomes are doubled by treatment of colchicine and other cell division inhibitors, double haploid (Doubled haploid, DH) inbred lines with the homozygosity of 100% are obtained in the current year, the creation process （Ren et al., 2017,Plant Biotechnol J 15: 1361–1370;Zhang et al., 2008,Plant Cell Rep 27: 1851–1860）. of the inbred lines is accelerated to the greatest extent, the male parent genes are expressed for a certain time and play a role because the haploids are gradually lost from the sperm chromosomes of the induction line which enter the zygotes after the fertilization of the ova, and therefore, the gene editing vectors carried by the male parent of the induction lines can also be expressed before the loss and edit the target genes of the female parent, and finally, the gene editing double haploid lines without any genetic materials of the induction lines are produced (KELLIHERET al, 2019,Nat Biotechnol 37:287-292; wang et al, 2019,Mol Plant 12:597-602).
Plant starch is mainly composed of linear glucopolysaccharide amylose and amylopectin with a large number of branches, and the ratio of the two starches determines the texture and mouthfeel of the grain. Corn kernels have a starch content of about 73% by weight, three quarters of which are amylopectin, and genetic and biochemical studies have identified that more than 20 genes are involved in starch biosynthesis, where starch synthase gene wall is a key enzyme (Granule-bound starch synthase 1, GBSS1,GRMZM2G024993;https://phytozome.jgi.doe.gov/pz/portal.html), for amylose synthesis in the endosperm whose activity level directly affects the amylose content, and where mutation of the gene results in amylose loss, can be used to create high amylopectin varieties.
Disclosure of Invention
In order to establish a universal corn gene editing genetic transformation system, the invention selects a corn haploid induction system as a transformation receptor material, adopts a promoter, a gene, a terminator and the like which are efficiently expressed in monocotyledonous plants, takes a corn starch synthase gene as a target gene, designs and constructs a knockout editing transformation vector, establishes the corn haploid induction system transformation system, simultaneously tries gene editing, successfully uses the gene editing vector to directly transform the corn haploid induction system for the first time, and realizes the effective knockout editing of the corn starch synthase gene. The target gene of the haploid induction system is directly transformed and edited, then the offspring of the transformed induction system is used for inducing any maize hybrid or selfing material to generate haploid, a doubled haploid system for editing the same target gene can be formed after the chromosome is doubled, the bottleneck problem that the selfing line which restricts the research and application of maize gene editing is difficult to transform is thoroughly avoided, the genetic transformation system of one haploid induction system is only required to be established, the gene editing of any maize material including excellent selfing line can be indirectly realized by combining with the doubled haploid technology, and the edited offspring is obtained to be the homozygous doubled haploid system which can be directly used for breeding and screening.
The invention mainly relates to a direct genetic transformation and gene editing method of a corn haploid inducer and application thereof. The method uses maize haploid induction line reclamation mutagenesis 8556 (KY 8556) and a derivative line thereof as transformation receptors, uses starch synthase wall gene GRMZM2G024993 as an editing target, and covers the complete process from the design and construction of an editing vector to the identification of gene editing of a transformed plant offspring. The direct transformation and gene editing of the corn haploid induction system are realized for the first time, and the double haploid system for gene editing is created by using the transgene induction system carrying the editing carrier, so that the editing improvement of the same target gene in any corn material can be realized. The major innovation breakthroughs are direct genetic transformation and gene editing of haploid inducer lines.
The specific technical scheme of the invention is as follows:
the invention provides a gene editing transformation vector, which is an agrobacterium expression vector, and comprises three expression units ZmU pro: gRNA AtU ter, zmUBI pro: spCas9: psRbcS E9 ter and CaMV35S pro: bar: caMV35S ter from the right boundary of T-DNA based on a pCambia3301 vector skeleton.
Further, the SpCas9 gene alters or adds other gene expression units or components, forming a dedicated SpG pan-site editor for corn gene editing, a cytosine editor, a adeno glance sideways at-in editor, a guide editor, and other modifications that can promote corn transformation.
The invention also provides a construction method of the gene editing transformation vector, which comprises the steps of reserving two BsaI enzyme cutting sites between ZmU pro and gRNA skeleton of the transformation vector, linearizing the vector, and inserting any gRNA recognition sequence fragment of editing target genes by adopting a DNA ligase or Gibson cloning method.
As a specific example, the target gene is a maize starch synthase wall gene, and the tRNA-ZmWx-G1-gRNA-tRNA-ZmWx-G2-gRNA tandem DNA fragments are artificially synthesized by the interactive tandem of tRNA multiple gRNA expression technology; the BsaI site was inserted by Gibson cloning using NEBuilder kit to construct a transformation vector for editing two sites of the maize starch synthase wall gene simultaneously, the nucleotide sequence of which is shown in SEQ ID NO 1.
The invention also provides a genetic transformation method of the corn haploid induction system, namely, a transformation vector is introduced into cells of a corn haploid induction system plant, the obtained regenerated plant is self-matured, and then, the separated offspring plant which can be inherited, transgenic and non-transgenic and stably edited is obtained through screening.
Further, the genetic transformation method comprises the steps of:
(1) Genetic transformation is carried out on the haploid induction line maize acceptor plant to obtain a regenerated plant;
(2) Detecting whether gene editing of the regenerated plant is successful or not;
(3) And after germination of the successfully edited regenerated plant offspring seeds, screening to obtain the offspring plants which can be inherited, transgenic and non-transgenic and stably edited.
Further, the step (1) includes:
(1) Planting a transformation acceptor material, wherein the transformation acceptor material is maize haploid induction line reclamation induction 8556 (KY 8556) and a derivative line thereof;
(2) Preparing and picking up corn young embryo explants;
(3) Preparing agrobacterium and infecting young embryo to obtain transformed cell;
(4) Callus induction and screening;
(5) Callus differentiation and seedling regeneration;
(6) Rooting and culturing regenerated seedlings;
(7) After domestication and transplanting of the resistant regenerated plants, screening to obtain edited regenerated plants.
Furthermore, the invention also provides a method for creating the maize editing double haploid line, which comprises the steps of establishing direct transformation and gene editing of a maize haploid induction line, screening transgenic induction line offspring carrying a gene editing carrier and having editing function as male parent, hybridizing with maize female parent materials of any genetic background, inducing haploids and carrying out chromosome doubling treatment to obtain the homozygous double haploid line for finishing gene editing.
In a specific embodiment, the gene editing transformation vector, genetic transformation method and double haploid line creation process are as follows:
1) Construction of maize gene editing transformation vector:
Gene editing based on a streptococcus Streptococcus pyogenes CRISPR endonuclease Cas9-gRNA system has been realized in various plants, and has wide application prospect in carrying out accurate gene editing improvement on special characters in crop genetic breeding. The vectors for the transformation of maize-specific gene editing agrobacteria are composed of three gene expression units ZmU pro: gRNA: atU6-26 term,ZmUBI1 pro:SpCas9:PsE9 term and 35S pro:Bar:35S term (FIG. 1), two BsaI cleavage sites are left between ZmU pro and the gRNA backbone, and the vectors can be linearized to facilitate insertion of the gRNA recognition sequence of any editing target gene by DNA ligase or Gibson cloning, using the monocot-specific promoter maize ubiquitin gene GRMZM2G409726 promoter ZmUBI pro and one U6 snRNA gene promoter ZmU pro downstream of the unknown gene GRMZM2G148773, respectively. The above gene editing transformation vectors can be further modified, for example, by altering the SpCas9 gene therein or adding other gene expression units or components, to form a dedicated SpG pan-site editor for corn gene editing, cytosine editor (Cytosinebase editor, CBE), adeno glance sideways at-in editor (Adenine base editor, ABE), guide editor (PRIME EDITING, PE), and other modifications that can promote corn transformation or increase gene editing efficiency, among others.
The maize starch synthase wall gene GRMZM2G024993 consists of 14 exons and 13 introns, the protein coding sequence starts from the second exon (fig. 2 a), and the mutant inactivation of this gene results in a significant reduction of amylose content in maize kernels. In order to knock out the mutant gene by using a gene editing method, gRNA ZmWx-G1 and ZmWx-G2 sequences are designed at the 2 nd and 4 th exons positioned at the upstream of the gene respectively (figure 2 b), and a corresponding gene editing vector KF77 is constructed by taking the special maize gene editing agrobacterium transformation vector as a framework. The two gRNAs ZmWx-G1 and ZmWx-G2 are constructed in the same carrier through the tRNA multiple gRNA expression technology in an interactive connection mode, and meanwhile, the maize haploid induction system is transformed into a maize haploid induction system reclamation lure 8556 (KY 8556) and a derivative system thereof.
2) Genetic transformation of maize haploid inducer lines:
agrobacterium-mediated maize genetic transformation is used to obtain regenerated plants by infecting young embryos, inducing callus to differentiate in somatic embryos or direct shoots. In addition to the necessity of batch-wise planting of transformation recipient material in a greenhouse to provide young embryo explants for transformation throughout the year, transformation experiments are simple to operate, do not require expensive and complex experimental equipment, and are easy to implement, but only a few experimental inbreds can be successfully transformed, and most breeding inbreds with excellent properties are extremely difficult to successfully transform. In order to develop the transformation of the corn haploid induction line, we refer to the published various transformation technologies and culture medium formulas （Frame et al., 2006,Plant Cell Rep 25: 1024-1034; Ishida etal., 2007,Nature Prot 2: 1614-1621; Cho et al., 2014,Plant Cell Rep 33: 1767–1777;Lowe et al.,2018,In Vitro Cell Dev Biol-Plant 54: 240–252）,, firstly, tissue culture screening is carried out on tens of inbred lines for breeding and haploid induction lines KY8556 and derivative lines thereof, materials with good tissue culture reaction and difficult browning and death are selected, agrobacterium transformation screening is carried out on the materials, the best inbred lines are selected for repeated transformation, experimental conditions and culture medium composition are adjusted and optimized, and the like, so that successful transformation is realized firstly, then, the transformation efficiency is improved by further perfecting the technology, and the genetic transformation method of the corn haploid induction lines KY8556 and the derivative lines thereof is formed. The main operation steps are as follows:
(1) And (3) planting a transformation receptor material: sowing corn inbred line materials in a Harbin greenhouse every 2-3 weeks, bagging before heading and spinning, pollinating for 10-13 days, taking the ears for sterilization and disinfection, and selecting young embryo with the length of about 2.0 mm for tissue culture screening. And repeatedly planting the inbred line with better performance, and carrying out agrobacterium infection and transformation screening. Preferably, a few inbred lines which perform well in two rounds of screening and have a successful transformation tendency comprise a haploid inducer line KY8556 which is continuously planted in batches in a greenhouse, and about 30 seeds are planted every 2-3 weeks, so that about 15 clusters can be picked every week to pick young embryos for transformation experiments.
(2) Preparation and picking of corn young embryo explants: removing surface bract, cutting off top silk, spraying 75% alcohol, and storing in a refrigerator at 4deg.C for 1-3 days in dark place. Before picking embryo, removing bract layer by layer, spraying alcohol for disinfection on each layer, cleaning bract and filament, spraying Shi Guosui surface with 75% alcohol, and soaking in 75% alcohol for disinfection for 10 min. The ears are placed in a culture dish with the length of 150 multiplied by 15 mm, the upper part of the seeds is scraped by a sharp scalpel blade for about 1-2 mm, a scalpel tip is inserted between endosperm and seed coat at one end from top to bottom, young embryo at the base of the seeds is picked out, and the young embryo is collected in a 2ml centrifuge tube containing infection culture medium for transformation infection.
(3) Agrobacterium preparation and embryo infection: and taking out the agrobacterium glycerol preservation tube carrying the gene editing vector from the low-temperature refrigerator at the temperature of minus 80 ℃, taking a small amount of agrobacterium to streak and inoculate on a solid YEP culture medium plate containing the corresponding antibiotics, and carrying out dark culture at the temperature of 28 ℃ for 2-3 days. Single colonies were picked and streaked on a YEP medium plate, streaked, and then inverted dark cultured in a 28℃incubator for 1-2 days. And inoculating 5 ml of YEP liquid culture medium, culturing at 28 ℃ on a 200-rpm rotary shaking table for overnight, taking a small amount of bacterial liquid to extract plasmid DNA for carrier-specific PCR detection, and confirming that the strain carries a target gene editing carrier. The agrobacterium plate is rinsed with infection medium during infection, bacterial liquid is collected in a 100 ml triangular flask and placed on a rotary shaking table at 28 ℃, and activated and cultured for about 2 hours at 200 revolutions per minute. The OD600 value is sampled and measured, and the OD600 value of agrobacterium tumefaciens is 0.6-0.8 by using an infection medium and then the agrobacterium tumefaciens is used for embryo infection.
Young embryos are picked up and collected in a 2 ml centrifuge tube containing 1 ml of infection medium, washed once with the same infection medium, 1 ml of fresh agrobacterium suspension with OD600 of about 0.6 is added, mixed upside down, the young embryos are fully immersed in the suspension, and placed in the dark for static infection for 10 minutes. Carefully sucking out the bacterial liquid by using a sterilizing pipetting gun head, transferring the young embryo to the surface of a co-culture medium by using tweezers, sucking out residual agrobacterium suspension, placing young embryo scutellum upwards at uniform intervals, sealing a culture dish by using a sealing film, and co-culturing in the dark at 20 ℃ for three days.
(4) Callus induction and resistant callus screening: transferring the infected young embryo into a 15 ml sterile centrifuge tube, and cleaning with sterile water for several times until the water is clear and transparent. Transferring young embryo into culture dish, sucking surface water with sterile filter paper, sub-packaging, placing on recovery culture medium, and dark culturing in constant temperature incubator at 28deg.C for 1 week. Root buds growing on young embryos are excised one by one, and are directly transferred to a screening culture medium for dark culture for 2 weeks to induce callus generation, and the fresh screening culture medium is replaced once, so that the dark screening culture is continued for 2 weeks. Immature embryo explants will expand and develop irregular callus-like tissue, non-transformed cells will stop growing and die gradually, and successfully transformed cells will develop resistant callus and continue cell division expansion.
(5) Tissue differentiation and seedling regeneration: and (3) transferring the explant of the healthy growth-resistant callus to a regeneration culture medium, continuously carrying out dark culture at 28 ℃ for 2-4 weeks, differentiating a granular embryo-like structure during which light yellow leaves and buds grow and elongation occurs, and rooting part of the callus to form regenerated seedlings with normal morphology.
(6) Rooting and culturing regenerated seedlings: and (3) selecting differentiated seedlings with normal morphology, transferring the differentiated seedlings into a rooting culture medium bottle, continuously culturing the seedlings for 3-4 weeks by using visible light at 28 ℃, and growing green stems and leaves, and taking out some differentiated roots to obtain healthy developed root systems.
(7) Domestication and transplanting of the resistant regenerated plants: when the main root system of the seedling grows about 5 cm and is stronger, opening a sealing film of a culture bottle, adding a small amount of distilled water to keep humidity, after hardening the seedling for 24 hours, carefully clamping the plant out by using forceps to avoid damaging stems and roots, flushing a culture medium attached to the root by using clear water, transplanting the plant into a seedling tray containing vermiculite nutrient soil, covering the seedling tray to keep humidity, culturing in a 28 ℃/24 ℃ artificial climate chamber or a greenhouse with 16/8 hour illumination for about 1 week until new leaves grow out, transplanting the seedling into a larger flowerpot to be cultured, and applying a proper amount of fertilizer until flowers and fruits are obtained. During this period, leaves of these T0 plants were sampled, and DNA was extracted for transgene identification and gene editing analysis.
3) Identification of Gene editing molecules of transformed plants:
Resistant regenerated plants must be tested by molecular biological methods to confirm whether the genetic transformation was successful. A common simple and reliable detection method is PCR amplification. Designing a vector specific primer according to a transformation vector DNA sequence, sampling regenerated plant leaves, extracting a small amount of genome DNA, and carrying out PCR amplification, wherein the plant with the obtained specific amplified fragment is transformation positive, and the editing effect of the target gene can be further detected.
The target gene editing condition can be rapidly analyzed by combining PCR amplification with DNA sequencing, and has high efficiency, low cost, simplicity and reliability. Based on the target gene DNA sequence and the expected Cas9 cleavage site position, PCR primers are designed that can cover about 300 bp length each upstream and downstream of the cleavage site, and the target gene fragment of about 400-800 bp is amplified from the genomic DNA from the regenerated plant leaf or other tissue source, and after purification, sequenced directly with the same upstream or downstream primers. If the DNA fragment sequence of the regenerated plant is not different from the wild type sequence, no gene editing is carried out; if the detected DNA sequence shows a sleeve peak near the expected Cas9 cleavage site, the sequenced PCR fragment is a mixed template, and the regenerated plant is heterozygous edited; if the regenerated plant DNA has a definite sequence difference with the wild type, the homozygous gene is edited. The PCR fragment of the target gene of the heterozygous editing plant can be cloned by using a PCR fragment cloning kit, at least 3 cloning vectors are selected for specific primer sequencing, and the specific editing change of the target gene sequence is confirmed.
4) Genetic isolation and molecular characterization of gene editing offspring:
The outstanding advantage of gene editing breeding is that the target gene can be preselected for editing, and as the target gene is not linked with an editing tool Cas9-gRNA transformed into cells, the gene editing offspring without carrying any exogenous gene can be obtained through simple genetic separation of offspring, and the method is not different from the traditional mutation breeding, and is accurate directional mutation breeding. The T0 plant regenerated through transformation is artificially bagged, self-pollinated for propagation, the T1 seed generated by natural setting is subjected to genetic separation, and if a single target gene locus of the T0 plant is edited, the T1 seed can be subjected to homozygous editing, heterozygous editing and non-editing wild type three offspring according to Mendelian genetic separation. The transformed gene editing vector will also segregate independently according to mendelian inheritance.
Harvesting the T1 seeds for timely sowing and germination, and due to the recombination and separation of two sites of a target gene and a transformation vector inserted into a genome, namely a Cas9 editing tool, sowing and analysis of at least 16T 1 plants are required. Sampling the leaf, extracting DNA and carrying out PCR amplification analysis. The plant with the specific amplified fragment obtained by amplifying the DNA primer pair with the transformation vector still carries the transformation vector and can be reserved as a transformed haploid inducer, wherein heterozygous and homozygous separated plants are included. The method comprises the steps of respectively designing a PCR primer pair with specificity of a gene editing transformation vector and specificity of a corn endogenous gene such as alcohol dehydrogenase (AF 123535, https:// www.ncbi.nlm.nih.gov /), analyzing the copy number of the gene editing vector contained in each plant by using a fluorescent green quantitative PCR method, and identifying and selecting single copy homozygous plants as a transformation haploid induction system with a gene editing function for haploid induction and creation of a gene editing doubled haploid system.
The plants which can not obtain the specific PCR fragment of the gene editing transformation vector are non-transgenic separation T1 offspring, and the editing condition can be further confirmed by target gene specific PCR analysis. Amplifying the non-transgenic offspring by using a pair of primers positioned at about 300 bp parts of the upper and lower stream of the cleavage point of the target gene Cas9, and directly sequencing the obtained PCR fragment by using the same upper or lower primers to confirm the DNA sequence change of the edited target gene, wherein the DNA fragment sequence of the unedited plant is not different from the wild type sequence; the DNA fragment sequence of the heterozygous editing plant has a cover peak near the expected cutting site of Cas9, and the fragment sequence can be determined by sequencing after cloning; the DNA fragment sequence of the homozygous editing plant has obvious differences of deletion, insertion, base substitution and the like compared with the wild type sequence.
5) Gene editing haploid induction and creation of doubled haploid lines:
The single copy homozygous transgenic haploid induction line can be used as a male parent to be hybridized with any female parent including hybridization materials or inbred lines to induce haploids, and after the chromosome is doubled, brand new or improved double haploid inbred lines for gene editing are created respectively, and the inbred lines for the production of the improved seed production of the gene editing can quickly and accurately improve the defects of the existing variety or enhance a certain special character, so that the inbred line has more realistic breeding significance. Sowing transgenic haploid induction line and inbred line to be improved by gene editing in good time, regulating error period to ensure that male parent and female parent meet in flowering period, manually bagging, taking out spike after hybridization pollination by using transgenic induction line as male parent for about 15 days, sterilizing, taking young embryo whose size is about 3 mm, placing the young embryo on a culture dish with simple culture medium, and after illumination, displaying purple embryo as hybridization diploid, and if it is not developed, obtaining haploid embryo. Selecting haploid embryo, treating with colchicine to promote chromosome doubling, transferring into culture flask, germinating into seedling (China patent application No. 202110889973.5), and timely transplanting into soil for growth and development.
6) Gene editing identification of doubled haploid lines:
Leaves can be sampled during the growth of doubled haploid seedlings, and genomic DNA extracted for amplification analysis using the same PCR method as described above. Detecting whether the doubled haploid plant carries the gene editing vector by the gene editing vector specificity PCR, and ensuring that no gene pollution of transgene induction line male parent residue exists; and detecting and revealing the editing condition of the target gene in the double haploid plant by combining target gene specificity PCR amplification with DNA sequencing. Selecting plants with homozygous gene editing, manually bagging the plants before heading and spinning, automatically pollinating the plants, and normally setting the plants and harvesting seeds to form a double haploid line with the gene editing.
The invention has the advantages that:
1) The efficient genetic transformation of the maize haploid induction line KY8556 and the derived line thereof is realized for the first time, the transformed regeneration plant is directly obtained from the young embryo explant through a somatic embryogenesis way, the subculture process of amplifying embryogenic callus is not needed, and the method is fast and efficient.
2) The special CRISPR-Cas9 gene editing vector for corn agrobacterium transformation is designed and constructed, the corn haploid induction line is successfully transformed, and the direct site-directed editing of the corn haploid induction line gene is realized for the first time. By altering the SpCas9 gene of the basic vector or adding other gene expression units or components, a SpG universal site editor, a cytosine editor CBE, a adeno glance sideways at-inch editor ABE, a guide editor PE special for corn gene editing, other improved modifications including promotion of corn transformation or improvement of gene editing efficiency and the like can be formed.
3) A simple and rapid analysis method for identifying the editing effect of the gene is developed, and the heterozygous or homozygous editing can be effectively identified by combining the editing sequence specificity PCR analysis and the simple DNA sequencing, so that the edited DNA sequence is obtained. Single copy transgenic inducible lines can be identified by quantitative PCR analysis.
4) The haploid induction line carrying the gene editing carrier can be used as a male parent to be hybridized with any other inbred line to generate haploids, wherein the corresponding target genes of part of haploids are edited, and the double haploid inbred line with the gene editing is obtained after chromosome doubling, so that the gene editing improvement of the original inbred line is realized.
5) The haploid induction line carrying the gene editing carrier can be used as a male parent to be hybridized with other corn materials to generate haploids, wherein the corresponding target genes of partial haploids are edited, and the doubled haploid inbred line for gene editing is obtained after chromosome doubling, so that the creation of the doubled haploid line and the gene editing improvement of the original corn materials are synchronously realized.
6) The haploid induction line KY8556 and the derivative line thereof can also be used as a common genetic transformation receptor for transformation, the obtained haploid induction line carrying the transgene is hybridized with other excellent inbred lines and backcrossed for a plurality of times, and the transgene is introduced into the excellent inbred lines for transgenic breeding.
Drawings
FIG. 1 is a schematic diagram of a maize-specific gene editing Agrobacterium transformation vector; (in the figure, the corn gene editing basic vector uses a pCambia3301 vector skeleton, and comprises three expression units ZmU pro from the right boundary of T-DNA, wherein the three expression units ZmU pro comprise gRNA AtU, 6 term, zmUBI1 pro comprise SpCas9 comprise PsRbcS E9 term and CaMV35S pro comprise Bar comprise CaMV35S term, the first expression unit of the vector KF77 simultaneously expresses two gRNAs comprising ZmWx-G1 and ZmWx-G2 by using tRNA tandem technology, the corn starch synthase gene GRMZM2G024993 can be edited at two corresponding sites, zmU pro comprises a corn U6snRNA gene promoter, atU-26 term comprises an Arabidopsis U6snRNA gene terminator, zmUBI pro comprises a corn ubiquitin gene promoter, cas9 comprises a streptococcus pyogenes 9 gene, psE9 term comprises pea nuclear carbohydrate-1, 5-bisphosphate carboxylase/oxygenase small subunit E9 gene terminator comprises Castor, and cauliflower mosaic virus 35S 35 comprises cauliflower gene terminator, and the cauliflower mosaic virus 35S 35 comprises cauliflower gene terminator.
FIG. 2 shows the structure and selected gene editing sites of the maize starch synthase wall gene GRMZM2G 024993; (in the figure, (a) the wall gene consists of 14 exons and 13 introns, one site is selected from exons 2 and 4 to design gRNA ZmWx-G1 and ZmWx-G2 respectively, (b) the recognition sites of ZmWx-G1 and ZmWx-G2 and nearby DNA sequences, and the bold NGG base is a PAM sequence).
FIG. 3. Agrobacterium transformation flow diagram of haploid inducer line; the method comprises the steps of (a) carrying out young embryo on a co-culture medium after infection, (b) recovering callus induction on the culture medium, (c) differentiating embryoid bodies from resistant callus on a regeneration medium I, (d) differentiating the embryoid bodies on a regeneration medium II to be green, (e) germinating embryoid bodies on the regeneration medium II to form seedlings, and (f) carrying out rooting on regenerated seedlings on a rooting medium.
FIG. 4 shows a representation of PCR identification of haploid inducer KY8556 transformed regenerated T0 plants; (in the figure, (a) regenerated seedling leaf genomic DNA is extracted as a template, the expected 540 bp fragment is amplified by PCR using Cas9 gene specific primers Cas9-F1 and Cas9-R1, and unsuccessful amplification is a non-transgenic event.) the same genomic DNA is used as a template, the expected 728 bp fragment is amplified by PCR using starch synthase gene, wax GRMZM2G024993 specific primers ZmWx-F1 and ZmWx-R1, and the editing of the ZmWx-G1 and ZmWx-G1 sites is analyzed by sequencing the marker DNA fragment lengths of 2000, 1000, 750, 500, 250, and 100 bp, respectively.
FIG. 5 shows a representation of transgene identification in haploid inducer KY8556 transformed T0 plants; (in the figure, the leaf of the transformation-screened resistant regenerated T0 seedling is taken to extract crude protein, the BAR protein is detected by using a transgenic PAT/Bar rapid detection test strip, and samples with both control and Bar specific bands developed are transgenic positive).
FIG. 6 shows a graph of gene editing identification of haploid inducer KY8556 transformed regenerated T0 plants; (in the figure, the expected 728 bp fragment is amplified by PCR by using GRMZM2G024993 gene specific primers ZmWx-F1 and ZmWx-R1, the same PCR primer is used for direct sequencing, overlapping sequences appear at the expected enzyme digestion point of Cas9 and can be regarded as editing events, the homozygote editing of clear DNA sequence deletion, insertion and base conversion difference appears, 7 representing events in the figure realize homozygote editing at two editing sites, (a) the sequence editing of the ZmWx-G1 site and (b) the sequence editing of the ZmWx-G2 site).
FIG. 7 shows a conventional breeding scheme for early materials of maize haploid inducer line.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited to the scope of the examples.
Experimental methods without specific conditions noted in the following examples were selected according to conventional methods and conditions, or according to the commodity specifications; the reagents and materials used in the present invention are commercially available.
The maize haploid inducer KY8556 and its derivatives used in the present invention are obtained by the following methods (specific screening methods can also be referred to as CN 2022109290076):
The breeding method of the haploid induction line KY8556 comprises the following steps: hybridization is carried out by taking Longzhong No. 4 as female parent Stock6 as male parent to obtain F1 generation, taking F1 generation as female parent, stock6 as male parent to obtain backcross BC1F1 generation, selecting 3-5 plants with optimal disease resistance, early maturity and purple embryo in the same generation plants, obtaining haploid induction plant KY8556 strain after 6 generation selfing.
Breeding of haploid induction line KY8556 and derivative line thereof: the derived offspring of haploid inducer Stock6 are widely used resource materials for haploid breeding of maize, available to the public from a variety of sources such as the Jilin province institute of agriculture, the U.S. corn genetic collaboration germplasm resource center. Dragon reclamation No. 4 is a hybrid corn product of North Da reclamation abundant seed industry stock limited company, has good comprehensive properties of early ripening, disease resistance and the like, and can be obtained from North Da reclamation abundant seed industry.
In spring 2012, F1 generation is obtained by hybridization of the Harbin with the Dragon reclamation No. 4 as a female parent and the Stock6 material as a male parent, F1 generation of the Dragon reclamation No. 4/Stock 6 is used as a female parent in winter in the Hainan, and after the Stock6 is used as a male parent, backcross first generation BC1F1 is obtained, 6 generation selfing and pedigree breeding are carried out. In spring 2013, BC1F1 purple seeds are sown in Harbin, 3-5 single plants are selected for each generation, each single plant line is 5m long and 20 holes are single-row, a dragon-cultivated No. 4 female parent and a Stock6 material male parent are synchronously sown as controls, the conditions of natural field infection with leaf spot and head smut of different single plants are observed and compared, and disease-resistant single plants are selected; observing and comparing the scattered powder and the spinning time of different single plants and comparing the scattered powder and the spinning time with those of control plants, and selecting early-matured single plants; observing and comparing the seed colors of different single plants with those of a control plant, and selecting an embryo purple single plant; and finally, selecting 3-5 single plants with good performance in each generation, selecting 3-5 single plants with good disease resistance, precocity and purple embryo according to the same process and method in the next generation, and eliminating the strains with non-ideal performance. The haploid inductivity of the selected single plant is tested from each generation after BC1F3, namely, pollen of the single plant of the selected induction line is hybridized with a female parent test variety, more than 5 materials are used for each plant, each combination is subjected to test cross for at least 3 ears, haploids are judged according to purple performances of endosperm and embryo in seeds after seeds are harvested, the endosperm and embryo are purple seeds are hybridized diploids, the endosperm is purple, the embryo is purple-free seed is haploid derived from the female parent, the haploid inductivity reaches more than 6% and preferably the highest 3-5 single plants are subjected to next generation selection, 6 generation selfing breeding is performed in total as shown in fig. 7, the plant with the highest inductivity is named KY8556 and is used as a haploid induction line, and other plants are the derivative lines of the same generation.
During the test use period of KY8556 strain, the material is found to have the advantages of early ripening, disease resistance, large powder scattering amount, high induction rate and the like. In the haploid induction experiment of the Harbin area in 2016, different breeding materials are selected and planted in 55 cells, each cell consists of 25 hole rows with the length of 1-3 meters according to the requirements of the different materials, male spikes and female spikes are respectively bagged before the spikes are produced and the silking, pollen of KY8556 is used for pollination manually, the haploid induction condition is counted after the mature of the ears, endosperm is purple, embryo is purple, the endosperm and embryo are purple, the haploid induction rate is up to 12.83%, the lowest is 3.95%, and the average is 9.43%.
The method comprises the steps of establishing direct transformation and gene editing of a corn haploid induction line, screening transgenic induction line offspring carrying a gene editing carrier and having editing functions as male parents, hybridizing with corn female parent materials of any genetic background, inducing haploids and carrying out chromosome doubling treatment to obtain a homozygous doubled haploid line for finishing gene editing, thereby creating a gene editing inbred line of any genetic background. The specific implementation includes 1) constructing a gene editing carrier, and constructing a specific endonuclease and other gene editing molecular tools into an agrobacterium transformation carrier; 2) Genetic transformation of a corn haploid induction line, and placing the gene editing carrier into corn cells to play a role; 3) Molecular biological analysis of the transformed T0 generation regenerated plant, detecting the state of a target gene editing site, and confirming whether the gene editing is successful or not; 4) T1 offspring analysis of transformed plants, screening transgenic induction system offspring carrying a gene editing vector and having editing function; 5) The offspring of the transgenic induction line carrying the gene editing carrier is used as a male parent to hybridize with the female parent material of corn with any genetic background to induce and generate haploid; 6) Selecting haploid immature embryo or mature seed grain, and performing chromosome doubling treatment to obtain a doubled haploid line; 7) Molecular biological analysis of the double haploid line, confirming the editing state of a target gene, and selecting the homozygous double haploid line with the gene editing as an inbred line for cross breeding.
Example 1
1) Constructing a corn gene editing basic transformation vector:
The corn transformant is screened by using a monocotyledonous plant special promoter corn ubiquitin gene GRMZM2G409726 promoter ZmUBI pro and a U6 snRNA gene promoter ZmU pro positioned at the downstream of an unknown gene GRMZM2G148773, respectively expressing Cas9 and gRNA genes with high efficiency, using a 35S pro promoter to express a streptothricin acetyltransferase gene (Phosphinothricinacetyltransferase, bar) of streptothricin (Streptomyces hygroscopicus), screening the transformed plant, and screening corn transformant by anti-Bialaphos (biamphos) or glufosinate (Glufosinate ammonium) herbicide, thus constructing an agrobacterium transformation vector KF76 special for corn gene editing, which consists of three gene expression units ZmU pro: gRNA: atU-26 term,ZmUBI1 pro:SpCas9:PsE9 term, and 35Spro:Bar:35S term (figure 1). Two BsaI cleavage sites are reserved between ZmU pro and the gRNA skeleton, the vector can be linearized to facilitate insertion of any gRNA recognition sequence fragment of the edited target gene by using a DNA ligase or Gibson cloning method, and the maize gene editing and transformation full-function vector can be constructed by only one cloning step. According to the needs of different gene editing experiments, the SpCas9 genes in the vector can be changed or other gene expression units or components can be added to form a SpG universal site editor, a cytosine editor (Cytosine base editor, CBE), a adeno glance sideways at (Adenine baseeditor, ABE), a guidance editor (PRIME EDITING, PE) special for corn gene editing and other improved modifications for promoting corn transformation.
The maize starch synthase wall gene GRMZM2G024993 consists of 14 exons and 13 introns, the protein coding sequence starts from the second exon and is 1830 bp long, the protein (figure 2a,SEQ ID NO 5,6,7) with 609 amino acids long is coded, and mutation inactivation of the gene can lead to significant reduction of amylose content in maize kernels and increase of amylopectin content, so that the maize variety is obtained. To knock out the mutant gene by gene editing, two gRNA sequences of ZmWx-G1 and ZmWx-G2 were designed at the 2 nd and 4 th exons upstream of the gene, respectively (FIG. 2b, SEQ ID NO 2, 3), and the two gRNAs were cross-linked by tRNA multiplex gRNA expression techniques (Xie et al, 2015,Proc Natl Acad Sci USA 112:3570-3575). Artificially synthesizing a tRNA-ZmWx-G1-gRNA-tRNA-ZmWx-G2-gRNA tandem DNA fragment (SEQ ID NO 4), wherein two ends respectively comprise ZmU pro homologous sequences with the BsaI restriction site of the corn gene editing vector KF76 and 20 bp homologous sequences with the downstream gRNA skeleton, respectively, inserting the BsaI site of the KF76 by using a NEBuilder (NEW ENGLAND Biolabs) kit through a Gibson cloning method, constructing an agrobacterium transformation vector KF77 (SEQ ID NO 1, figure 1) capable of editing two sites of a corn starch synthase wax gene simultaneously, sequencing and confirming the sequence by using ZmU6-F2 primers (SEQ ID NO 8), and transferring the sequence into a corn transformation agrobacterium strain EHA105 for later use at a refrigerator of-80 ℃.
2) Genetic transformation of maize haploid inducer lines:
The gene editing can be realized by transforming the gene editing vector into cells for expression, and the establishment of a high-efficiency maize inbred line transformation system is a necessary premise for developing maize gene editing and breeding. In order to establish a transformation system of an excellent corn inbred line, firstly, about 30 inbred lines respectively representing hard stalk (Stiff-stalk, SS) and Non-hard stalk (Non-stinff-stalk, NSS) groups are selected, a haploid induction line KY8556 or a derivative line thereof and common transformation receptor materials such as B104, hiII are used as controls for tissue culture screening, different culture medium formulas are tested, and a plurality of materials which are difficult to brown, have good induction and regeneration capacity are selected for test transformation by agrobacterium carrying a gene editing vector such as KF 77. Through screening tests for many years and batches, successful transformation of a plurality of inbred lines including a haploid inducer is finally realized, a transformation system of a haploid inducer KY8556 is mainly established, gene editing is synchronously realized, and the formulas of various culture media used for transformation and tissue culture are listed in Table 1, and representative pictures of each stage of the transformation process are shown in FIG. 3.
(1) And (3) planting a transformation receptor material: the transformed material is planted in batches in a sunlight greenhouse or a phytotron every year, wherein the transformed material comprises a haploid induction line KY8556, 30 plants are planted every 2-3 weeks, and potted plants are illuminated at 28 ℃/24 ℃ and 16/8 hours every day, so that young embryos are supplied every week. The male flowers and female ears are respectively covered before the corn heading and silk spitting, the ears are harvested after 10-13 days of artificial pollination, young embryos with the length of about 2.0 mm are picked up and used as explants for genetic transformation.
(2) Preparation and picking of corn young embryo explants: removing surface bract, cutting off top silk, spraying 75% alcohol, and storing in a refrigerator at 4deg.C for 1-3 days in dark place. Removing bracts layer by layer, spraying alcohol on each layer for disinfection, cleaning the bracts and filaments, spraying Shi Guosui% alcohol on the surface of the clean bench, and then soaking in 75% alcohol for disinfection for 10 minutes. The ears are placed in a culture dish with the thickness of 150 multiplied by 15 mm, the upper part of the seeds is scraped by a surgical knife for about 1-2 mm, the knife tip is inserted between endosperm and seed coat at the embryo end from top to bottom, young embryos at the base of the seeds are picked out, and the young embryos are collected in a2 ml centrifuge tube containing an infection culture medium and are infected by agrobacterium.
(3) Agrobacterium preparation and embryo infection: the agrobacterium glycerol storage tube carrying the gene editing transformation vector such as KF77 is removed from the-80 ℃ low temperature refrigerator, a small amount of agrobacterium is streaked and inoculated on a solid YEP culture medium plate containing 50 mg/l kanamycin for activation, and the incubator is inverted and cultured in the dark at 28 ℃ for 2-3 days. Single colonies are picked and streaked on a YEP culture medium plate, and after streaking, the single colonies are inversely cultured in a 28 ℃ incubator for 1-2 days. And inoculating 5ml of YEP liquid culture medium, culturing overnight on a rotary shaking table at 28 ℃ at 200 rpm, taking a small amount of bacterial liquid to extract plasmid DNA, and carrying out carrier-specific PCR detection to confirm that the strain carries a target gene editing carrier. The agrobacterium plate is rinsed with an infection medium during infection, bacterial liquid is collected in a 100 ml triangular flask and placed on a rotary shaking table at 28 ℃, and the bacterial liquid is activated and cultured for 2 hours at 200 revolutions per minute. The OD600 value is sampled and measured, and the OD600 value of agrobacterium tumefaciens is 0.6-0.8 by using an infection medium and then the agrobacterium tumefaciens is used for embryo infection.
Picking and collecting young embryo, placing the young embryo into a 2ml centrifuge tube containing 1 ml of infection culture medium, cleaning once with the same infection culture medium after embryo taking, adding 1 ml of agrobacterium suspension with OD600 of about 0.6, inversely mixing, completely immersing the young embryo into the suspension, and standing in dark for infection for 10 minutes. Carefully sucking out the bacterial liquid by using a sterilizing pipetting gun head, transferring the young embryo to the surface of a co-culture medium by using tweezers, sucking out residual agrobacterium liquid, placing young embryo scutellum upwards at uniform intervals, sealing a culture dish by using a sealing film, and co-culturing in the dark at 20 ℃ for three days.
(4) Callus induction and screening: transfer the infested chick to a 15 ml sterile centrifuge tube, and wash with sterile water several times until the water is clear. Transferring young embryo into culture dish, sucking surface water with sterile filter paper, sub-packaging, placing on recovery culture medium, and culturing in a constant temperature incubator at 28deg.C for 1 week in the dark, wherein young embryo can grow root bud (fig. 3a, b). The elongated root buds on the young embryos are excised one by one, and the young embryos are transferred to a screening medium I and are cultured in the dark for 2 weeks to induce callus generation. The immature embryos from which callus grows are transferred to screening medium II and the screening culture is continued in the dark for 2 weeks. Non-resistant calli will stop growing and gradually brown to die, and successfully transformed cells will grow into resistant calli and continue cell division and expand.
(5) Callus differentiation and seedling regeneration: healthy resistant calli were picked and stripped, transferred to regeneration medium I, and continued to culture in the dark at 28 ℃ for 2-3 weeks, during which time most of the explants differentiated into white granular protruding structures (fig. 3c, red arrow), judged to be embryoid structures by microscopic observation, and part elongated to pale yellow leaves in the dark. Transferring the callus with good embryoid growth to a culture flask containing regeneration medium II, culturing at 28deg.C under light for 2-3 weeks, and turning most embryoid into leaf structure (figure 3 d), partially elongating into green leaf and bud, and elongating to form regenerated seedling with root, stem and leaf and relatively normal morphology (figure 3 e).
(6) Rooting and culturing regenerated seedlings: regenerated seedlings with a normal morphology and a height of about 2-5 cm are selected and transferred into glass bottles containing rooting medium, and strong seedlings are cultivated for 2-3 weeks under illumination at 28 ℃ until healthy developed root systems are differentiated (figure 3 f).
(7) Domestication and transplanting of the resistant regenerated plants: when the main root system of the seedling grows about 5 cm and is stronger, opening a sealing film of a culture bottle, adding a small amount of distilled water to keep humidity, after hardening the seedling for 24 hours, carefully clamping the plant out by forceps to avoid damaging stems and roots, flushing a culture medium attached to the roots by clear water, transplanting the plant into a seedling tray containing vermiculite nutrient soil, covering the seedling tray to keep humidity, culturing in a 28 ℃/24 ℃ artificial climate chamber or a greenhouse with 16/8 hours of illumination for about 1 week until new leaves grow out, transplanting the seedling, culturing in a larger flowerpot, applying a proper amount of fertilizer, bagging before flowering, and performing artificial self-pollination until the seed is mature. Leaves of these T0 plants can be sampled during the seedling stage, and DNA extracted for transgene identification and gene editing analysis.
Table 1: culture medium for transformation of corn haploid induction line
| Culture medium | Composition of components |
| YEP Medium | Yeast extract 5 g/l, peptone 10 g/l, sodium chloride 5 g/l, screening gene according to strain resistance adding appropriate amount of antibiotic such as 50 mg/l kanamycin, solid medium adding agar 15 g/l for solidification, pH=7.0 |
| Infection medium | MS (Murashige & Skoog) minimal medium 4.3 g/L, B5 medium organic 1.0 ml/L, asparagine 0.2 g/L, 2,4-D (2, 4-Dichlorophenoxyacetic acid) 2 mg/L, proline 0.7 g/L, sucrose 68.4 g/L, glucose 36 g/L, acetosyringone 200 mmol, pH=5.2 |
| Co-culture medium | MS minimal medium 4.3 g/L, B5 medium organic composition 1.0 ml/L, asparagine 0.2 g/L, 2, 4-D2 mg/L, proline 0.7 g/L, sucrose 30 g/L, copper sulfate 1.25 mg/L, plant gel 3.0 g/L, silver nitrate 0.85 mg/L, cysteine 400 mg/L, acetosyringone 200 mmol, pH=5.8 |
| Recovery medium | MS minimal medium 4.3 g/L, B5 medium organic component 1.0 ml/L, asparagine 0.2 g/L, 2, 4-D2.0 mg/L, hydrolyzed casein 0.5 g/L, proline 0.69 g/L, sucrose 30 g/L, MES (4-Morpholineethanesulfonic acid) 0.5 g/L, plant gel 4.0 g/L, silver nitrate 0.85 mg/L, carbenicillin 100 mg/L, cephalosporin 100 mg/L, pH=5.8 |
| Screening Medium I | MS minimal medium 4.3 g/L, B5 medium organic component 1.0 ml/L, asparagine 0.2 g/L, 2, 4-D2.0 mg/L, hydrolyzed casein 0.5 g/L, proline 0.7 g/L, sucrose 30 g/L, MES 0.5 g/L, plant gel 4.0 g/L, silver nitrate 0.85 mg/L, cephalosporin 100 mg/L, carbenicillin 100 mg/L, bialaphos 1.5 mg/L, pH=5.8 |
| Screening Medium II | MS minimal medium 4.3 g/L, B5 medium organic component 1.0 ml/L, asparagine 0.2 g/L, 2, 4-D2.0 mg/L, hydrolyzed casein 0.5 g/L, proline 0.7 g/L, sucrose 30 g/L, MES 0.5 g/L, plant gel 4.0 g/L, silver nitrate 0.85 mg/L, cephalosporin 100 mg/L, carbenicillin 100 mg/L, bialaphos 3 mg/L, pH=5.8 |
| Regeneration Medium I | MS minimal medium 4.3 g/L, B5 medium organic component 1.0 ml/L, hydrolyzed casein 0.5 g/L, proline 0.7 g/L, asparagine 0.2 g/L, sucrose 30 g/L, vegetable gel 4.0 g/L, 6-BA (6-Benzylaminopurine) 1.0 mg/L, ZT (Zeatin) 0.5 mg/L, pH=5.8 |
| Regeneration Medium II | MS minimal medium 4.3 g/L, MS medium organic component 1.0 ml/L, inositol 0.1 g/L, sucrose-30 g/L, plant gel-4 g/L, pH=5.8. |
| Rooting culture medium | MS minimal medium 2.15 g/L, B5 medium organic component 1.0 ml/L, hydrolyzed casein 0.5 g/L, proline 0.7 g/L, asparagine 0.2 g/L, sucrose-30 g/L, vegetable gel 4.0 g/L, IBA (Indole-3-butyric acid) 1.0 mg/L, NAA (1-NAPHTHALENEACETICACID) 0.5 mg/L, pH=5.8 |
Gene editing identification of KY8556 regenerated T0 plants:
(1) Identification of transformation-positive plants by vector-specific PCR: sampling and regenerating T0 plant leaves, extracting genome DNA by using a SDS (Sodium dodecyl sulphate) -based DNA rapid extraction method, and carrying out Cas9 gene-specific PCR to detect whether the transformation of the gene editing vector is successful. The PCR reaction system adopts a Quick Taq HS DyeMix kit (TOYOBO LIFE SCIENCE), and the 20 microliter PCR reaction system comprises 10 mu l 2X Quick Taq HS DyeMix, 1.0 mu l 10 pmol/mu l primer Cas9-F1 (SEQ ID NO 9) and 1.0 mu l 10 pmol/mu l primer Cas9-R1 (SEQ ID NO 10), 6.0 mu l sterile water and finally 2.0 mu l genomic DNA of 50 ng/mu l sample. The PCR reaction conditions were 95℃for 5 minutes, then 95℃for 30 seconds, 60℃for 1 minute, 72℃for 1 minute for 35 cycles, and finally 72℃for 7 minutes and maintained at 4 ℃. The PCR amplified product was separated by 1% agarose gel electrophoresis and samples successfully amplified a 540bp (SEQ ID NO 11) long fragment specific for the Cas9 gene were judged to be transgene positive (FIG. 4 a). Identification of transgenic Positive plants Bar Gene-specific PCR detection was also performed with the primers Bar-F1 (SEQ ID NO 12) and Bar-R1 (SEQ ID NO 13), with the expected amplified fragment being 429 bp long (SEQ ID NO 14).
(2) Identification of transgenic expression of haploid induction line KY8556 transformed T0 plants. The PCR positive transformed plants can further be used for confirming Bar gene expression by using a transgenic detection test strip. The leaves of resistant regenerated T0 seedlings from transformation screening were taken, crude protein was extracted, and the transgenic PAT/Bar rapid test strips were used for detection (Envirologix, portland, USA), and samples developed for both control and Bar-specific bands were transgenic positive (FIG. 5).
(3) Editing conditions of target gene specific PCR detection positive plants: using the same general PCR amplification method as described above and using the maize wall gene GRMZM2G024993 specific primers ZmWx-F1 and ZmWx-R1 (SEQ ID NO 15, 16), target fragments (FIG. 2a, FIG. 4B, SEQ ID NO 22, 23) that were 728 bp long (B73 inbred) or 732 bp long (KY 8556 inducible) near the editing site of the ZmWx-G1 and ZmWx-G2 genes could be obtained from the same genomic DNA of the Cas9 positive samples, and were unidirectionally sequenced using the ZmWx-F1 or ZmWx-R1 primers, compared to the wild-type fragment sequences, and if the PCR fragment sequencing had defined sequence variations at the expected site, such as small fragment deletions, insertions or base changes, it was judged that homozygous editing occurred at that site (FIG. 6). If a set of peaks exists near the expected editing sites of ZmWx-G1 and ZmWx-G2 gRNA, even if downstream DNA cannot be accurately sequenced, judging that the DNA sequence heterozygous editing occurs at the sites. The PCR fragments TOPO of the edited samples were cloned into pCR2.1 vector (Thermo FisherScientific), and 3 cloning vector specific primers were used to sequence each sample to accurately analyze specific DNA editing sequence changes. Alignment of DNA sequences, DNA vector, PCR primer, CRISPR GRNA design, etc. were all accomplished using Geneious Prime software (Biomatters ltd.).
13 Batches of 9862 infection transformed haploid inducer KY8556 young embryos (Table 2) were analyzed as described above, with genetic transformation rates ranging from zero to 3%, average 0.6%, gene editing efficiencies ranging from 50% to 100%, average 82%, and a total of 101 regenerants obtained representing 61 independent transgenic events, of which 81 seedlings represented 50 independent editing events. Wherein 7 events achieved homozygous editing at both ZmWx-G1 and ZmWx-G2 sites, including single base insertions, single base or multiple base deletions (table 3, fig. 6), and 43 additional events were heterozygous editing. The sequences of the ZmWx-F1 and ZmWx-R1 fragments of the 7 events homozygously edited were (SEQ ID NO 24-30), respectively. The invention benefits from the efficient editing efficiency of the gene editing vector, and successfully realizes the effective gene editing of the practical haploid inducer KY 8556.
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TABLE 2 genetic transformation of haploid inducer KY8556 and efficiency of wax gene editing
| Date of infection | Invasion of young embryo | Resistant callus | Induction rate% * | Regenerated seedling | PCR positive seedlings | PCR Positive event | Percent conversion% | Editing seedling | Editing events | Gene editing rate% |
| 2021.7.1 | 1115 | 23 | 2.1% | 24 | 24 | 12 | 1.1% | 23 | 12 | 100.0% |
| 2021.7.5 | 917 | 22 | 2.4% | 3 | 1 | 2 | 0.2% | 1 | 1 | 50.0% |
| 2021.7.12 | 962 | 20 | 2.1% | 2 | 2 | 0 | 0.0% | 0 | 0 | |
| 2021.7.15 | 474 | 8 | 1.7% | 1 | 0 | 0 | 0.0% | 0 | 0 | |
| 2021.7.26 | 962 | 2 | 0.2% | 2 | 0 | 0 | 0.0% | 0 | 0 | |
| 2021.8.9 | 380 | 13 | 3.4% | 4 | 4 | 3 | 0.8% | 4 | 3 | 100.0% |
| 2021.8.16 | 757 | 30 | 4.0% | 38 | 21 | 15 | 2.0% | 17 | 13 | 86.7% |
| 2021.6.28 | 760 | 51 | 6.7% | 34 | 13 | 6 | 0.8% | 8 | 4 | 66.7% |
| 2021.7.5 | 935 | 66 | 7.1% | 0 | 0 | 0 | 0.0% | 0 | 0 | |
| 2021.7.15 | 637 | 24 | 3.8% | 18 | 13 | 6 | 0.9% | 10 | 4 | 66.7% |
| 2021.7.19 | 1218 | 13 | 1.1% | 1 | 0 | 0 | 0.0% | 0 | 0 | |
| 2021.8.9 | 266 | 72 | 27.1% | 46 | 12 | 8 | 3.0% | 7 | 4 | 50.0% |
| 2021.8.16 | 479 | 19 | 4.0% | 25 | 11 | 9 | 1.9% | 11 | 9 | 100.0% |
| Average of the total number | 9862 | 363 | 3.7% | 198 | 101 | 61 | 0.6% | 81 | 50 | 82.0% |
* Induction% = resistant callus/infected chick embryo; percent conversion =pcr positive event/infection of young embryo; gene editing rate% = editing event/PCR positive event.
TABLE 3 DNA sequence variation of target Gene loci of homozygous Gene-edited T0 plants of haploid inducer
| Event(s) | ZmWx-G1 site editing * | ZmWx-G2 site editing |
| M1-2 | Insertion 18A | ACAGG replaces 44 bp including the entire ZmWx-G2 |
| M2-1 | Deletion of 17T | Insertion 17T |
| M2-2 | Insertion 18A | Deletion of 17G |
| M4-1 | Insertion 18A | Insertion 17T |
| M4-2 | Insertion 16T | Deletion of 16-18TGT |
| M9-2 | Insertion 18A | Deletion of 18-19TG |
| M9-9 | Insertion 18A | Deletion of 17G |
* The editing site position is first counted from the terminal base far away from PAM; the ZmWx-G2 site editing sequence in the table is the complementary strand sequence shown in FIG. 6 b. Two independent events M2-2 and M9-2 have the same edits.
(4) Vector specific fluorescent quantitative qPCR (quantitative polymerase chain reaction) detection of copy number of transformation vector in positive plants: fluorescent quantitative qPCR allows the estimation of the copy number of the transformation vector contained in the positive plants described above, preferably single copy transformation plants for subsequent studies. Firstly, preparing a standard curve, taking genomic DNA of a transgenic corn plant known to contain a single copy target gene Cas9 as a standard sample, obtaining diluted standard samples with different concentrations through 5-time serial dilution, respectively carrying out fluorescent green PCR amplification by using primers specific to the target gene Cas9 and an endogenous reference gene corn alcohol dehydrogenase (Alcohol dehydrogenase Adh, AF 123535), establishing respective standard curves, evaluating and confirming that the PCR amplification efficiency of each primer pair meets the requirement, and then carrying out copy number detection on an unknown sample by using the same method. The PCR reaction system used was SYBR GREEN REALTIME PCR MASTER Mix kit (TOYOBO LIFE SCIENCE), and the 20. Mu.l PCR reaction system contained: 10. mu.l 2X Master Mix, 0.5 mu.l 10 pmol/mu.l primer Cas9-F1 (SEQ ID NO 9) and 0.5 mu.l 10 pmol/mu.l primer Cas9-R6 (SEQ ID NO 20), 8.0 mu.l sterile water and finally 1.0 mu.l 30 ng/mu.l genomic DNA of the sample are added. The PCR reaction conditions were 95℃for 5 minutes, then 95℃for 10 seconds, 66℃for 10 seconds, and 72℃for 15 seconds for 40 cycles, and the PCR amplified DNA fragment was 123 bp (SEQ ID NO 21) long. The PCR amplified DNA fragment was 131 long bp (SEQ ID NO 19) using maize ethanol dehydrogenase specific primers ZmAdh-F3 and ZmAdh-R3 as endogenous reference gene controls (SEQ ID NO 17, 18). In the real-time quantitative qPCR reaction, the PCR reaction of the standard sample and the sample to be detected is carried out on the same plate, each sample is set for 3 times of repetition, and the copy number is calculated by taking the average value. Event breeding offspring with single copy transformation and gene editing are reserved preferentially and used as haploid induction lines with the function of gene editing for creation of doubled haploid lines.
4) Analysis and identification of KY8556 gene editing event T1 offspring plants: because maize self-pollination follows mendelian genetic segregation, the transformed editing tool Cas9 gene vector and the edited wall gene GRMZM2G024993 are located on different chromosomes, each as a single gene according to 1:2:1 are separated independently. Part of T1 plants generated by selfing continue to carry homozygous or heterozygous gene editing vectors; part of T1 plants no longer contain any editing vector DNA and are non-transgenic offspring, wherein the wall genes of part of the plants can reach a homozygously edited state, so that stable non-transgenic homozygously edited plants can be obtained in the T1 generation.
(1) Identifying and screening non-transgenic gene editing offspring: transplanting the T0 plants subjected to gene editing to a greenhouse for continuous culture after rooting, harvesting T1 generation seeds, sowing in the greenhouse at proper time, taking a small amount of leaf samples of the T1 plants to extract genome DNA when the seedlings grow into about three leaves, detecting and identifying whether the T0 plants are transgenic offspring by using carrier specific PCR (polymerase chain reaction), and detecting the editing condition of the target genes by combining DNA sequencing. The target gene sequence is changed at the expected enzyme cutting point of Cas9 to be a homozygous editing offspring, the overlapping sequence is a heterozygous editing offspring, and the sequence is unchanged (i.e. the same as the wild type) to be an unedited offspring. Selecting non-transgenic homozygous editing offspring, harvesting T2 generation seeds, and expanding analysis and identification experiments to evaluate the detailed genotype and phenotype of the target genes such as GRMZM2G024993 editing offspring.
(2) Identification and selection of inducible lines carrying the gene editing vector: the specific common PCR of the vector can only identify whether the T1 plant carries the transformation vector, and can not distinguish heterozygous or homozygous transformation plants. The copy number of the transformation vector in the T1 plant can be detected by carrying out the same analysis as the vector specific fluorescence quantitative qPCR used in the T0 plant analysis, the homozygous transgenic T1 plant can be screened out, the gene editing vector with the editing function is verified to be carried stably, and the T2 generation seed can be used as a transgenic improved haploid induction system with the gene editing function after harvesting the T2 generation seed for haploid induction and synchronous gene editing experiments of any corn material.
5) Creating haploids by using a gene editing induction system: the improved haploid induction line carrying the gene editing carrier is used as a male parent, hybridized with corn inbred line or hybridization material with any genetic background to induce haploid, and the improved or brand new double haploid line for gene editing is created after chromosome doubling. The existing seed-producing excellent inbred line can quickly and accurately improve the defects of the existing variety or enhance a special character, and has more realistic breeding significance. Timely sowing an induction line carrying a gene editing vector and an inbred line to be subjected to gene editing improvement, adjusting the staggered period to ensure that the flowering periods of a father parent and a mother parent meet, manually bagging, taking the gene editing induction line as the father parent, cross pollination for about 13 days, sampling, and selecting haploid young embryos for chromosome tissue culture doubling treatment.
6) Doubling haploid tissue culture to create a Doubled Haploid (DH) line: the chromosome of the haploid can be doubled by two methods of bud cutting or tissue culture, and the latter directly doubles the haploid embryo without waiting for seed maturation, so that one growing season can be saved. Selecting young spikes after pollination for about 13 days, sterilizing, picking young embryos with the size of about 3mm, placing the young embryos on a culture dish containing a simple culture medium, and obtaining purple embryos which are hybridized diploid after illumination and haploid embryos which are not developed. And selecting haploid young embryos, carrying out chromosome doubling treatment through colchicine, germinating the young embryos into seedlings, and obtaining a doubled haploid line through self-pollination and fructification. The formulations of the various media used for tissue culture chromosome doubling are listed in Table 4.
(1) Disinfecting young embryo, and obtaining materials: taking young corn ears with young embryo length of about 3 mm after pollination for 13 days, sterilizing for 5 minutes by using 75% secondary alcohol, transferring into an ultra-clean workbench, sterilizing for 15 minutes by using 75% new alcohol, and washing twice by using sterile distilled water.
(2) Doubling of haploid chick embryos: picking young embryo in ultra clean bench with surgical knife, placing scutellum upwards in induction culture medium, and performing light treatment at 28deg.C for 24 hr, wherein the hybridized young embryo turns purple, selecting colorless young embryo, placing on doubling culture medium, and performing dark doubling treatment for 24 hr.
(3) And (3) regenerative culture of young embryo: and (3) cleaning the doubled young embryo for 3 times by using sterile water, transferring the young embryo into a growth culture medium, and culturing for 1-2 weeks under illumination, wherein the young embryo germinates into seedlings until the root development is complete. And transferring the seedlings with the height of about 5 cm and developed root systems into plug trays, and transferring the seedlings to greenhouse potting or field cultivation after hardening the seedlings for one week.
(4) Selfing and setting double haploid lines: the plants generated after the haploid doubling treatment can be derived from haploid embryos which fail to be doubled, hybrid embryos and chromosome doubled haploid embryos, the three are easy to distinguish in morphological performance, the haploid plants are weak and can not be loose and firm, the hybrid plants are high and grow vigorously, and both can be removed in time. The double haploid plant is smaller as the inbred line, most of the plants can normally scatter powder, the plants need to be bagged in time, the artificial pollination is carried out for selfing and setting, and the offspring is the double haploid (Doubled Haploid, DH) line.
Table 4: culture medium for doubling corn haploid chromosome tissue culture
| Culture medium | Composition of components |
| Induction medium | MS (Murashige and Skoog) minimal medium 3.0 g/l, sucrose 30 g/l, plant gel 4.0 g/l, pH=6.0 |
| Doubling medium | MS minimal medium 3.0 g/l, sucrose 30 g/l, colchicine 0.005-0.05%, dimethyl sulfoxide (Dimethylsulfoxide, DMSO) 0.5%, plant gel 4.0 g/l, pH=6.0 |
| Growth medium | MS minimal medium 3.0 g/L, sucrose 30 g/L, timentin 200 mg/L, carbenicillin 200 mg/L, plant gel 4.0 g/L, pH=6.0 |
7) Molecular biological identification of the Gene editing double haploid line (DH): confirming that there is no genetic material pollution of the induction system by carrier specific PCR; analyzing the editing state of the target gene by combining target gene specific PCR with DNA sequencing, and selecting a homozygous doubled haploid line which completes gene editing as a germplasm resource created by gene editing for cross breeding.
(1) Vector-specific PCR confirms non-transgenic doubled haploid lines: sampling plant leaves of double haploid lines, extracting genome DNA by using a SDS (Sodium dodecyl sulphate) -based DNA rapid extraction method, and carrying out Cas9 gene-specific PCR to detect whether the transformation of the gene editing vector is successful. The PCR reaction system adopts a Quick Taq HS DyeMix kit (TOYOBO LIFE SCIENCE), and the 20 microliter PCR reaction system comprises 10 mu l 2X Quick Taq HS DyeMix, 1.0 mu l 10 pmol/mu l primer Cas9-F1 (SEQ ID NO 9) and 1.0 mu l 10 pmol/mu l primer Cas9-R1 (SEQ ID NO 10), 6.0 mu l sterile water and finally 2.0 mu l genomic DNA of 50 ng/mu l sample. The PCR reaction conditions were 95℃for 5 minutes, then 95℃for 30 seconds, 60℃for 1 minute, 72℃for 1 minute for 35 cycles, and finally 72℃for 7 minutes and maintained at 4 ℃. The PCR amplified product was separated by 1% agarose gel electrophoresis, and samples that failed to successfully amplify the Cas9 gene-specific 540 bp long fragment (SEQ ID NO 11) were judged to be non-transgenic, i.e., the detected doubled haploid line was not contaminated with the gene editing inducible line and did not carry the gene of the inducible line. Identification of non-transgenic double haploid lines the primers Bar-F1 (SEQ ID NO 12) and Bar-R1 (SEQ ID NO 13) can also be used for the detection of the PCR specific for the Bar gene on the vector, the expected amplified fragment is 429 bp long (SEQ ID NO 14) and samples that failed to amplify this fragment are judged to be non-transgenic.
(2) Target gene specific PCR detects gene editing condition of double haploid line: using the same general PCR amplification method as described above and using the maize wax gene GRMZM2G024993 specific primers ZmWx-F1 and ZmWx-R1 (SEQ ID NO 15, 16), target fragments (SEQ ID NO 22) of ZmWx-G1 and ZmWx-G2 near the gene editing site 728 bp long were obtained from the doubled haploid genome DNA, and these PCR fragments were sequenced unidirectionally and directly using either the ZmWx-F1 or ZmWx-R1 primers. Because the genome of the doubled haploid line is homozygous diploid, DNA sequence editing changes which occur near the expected ZmWx-G1 and ZmWx-G2 gene editing sites should also be homozygous, and the specific DNA editing sequence changing situation can be accurately analyzed by direct sequencing of PCR fragments, and the homozygous doubled haploid line with complete gene editing is selected as a novel germplasm resource created by gene editing for cross breeding.
Sequence description:
SEQ ID NO 1: KF77 genome editing vector sequence starting from RB T-DNA 16453 bp
SEQ ID NO 2: ZmWx-G1 gRNA 20 bp
SEQ ID NO 3: ZmWx-G2 gRNA 20 bp
SEQ ID NO 4: tRNA-ZmWx-G1-gRNA-tRNA-ZmWx-G2-gRNA in KF77 vector 386 bp
SEQ ID NO 5: Zea mays B73 waxy gene ZmWx GRMZM2G024993 3678 bp
SEQ ID NO 6: Zea mays B73 waxy gene ZmWx GRMZM2G024993 CDS 1830 bp
SEQ ID NO 7: Zea mays B73 waxy gene ZmWx GRMZM2G024993 protein 609 aa
SEQ ID NO 8: ZmU6-F2 oligonucleotide 22 bp
SEQ ID NO 9: Cas9-F1 oligonucleotide 24 bp
SEQ ID NO 10: Cas9-R1 oligonucleotide 20 bp
SEQ ID NO 11: Cas9-F1/Cas9-R1 PCR fragment 540 bp
SEQ ID NO 12: Bar-F1 oligonucleotide 20 bp
SEQ ID NO 13: Bar-R1 oligonucleotide 22 bp
SEQ ID NO 14: Bar-F1/Bar-R1 PCR fragment 429 bp
SEQ ID NO 15: ZmAdh-F3 oligonucleotide 22 bp
SEQ ID NO 16: ZmAdh-R3 oligonucleotide 22 bp
SEQ ID NO 17: ZmAdh-F3/ZmAdh-R3 PCR fragment 131 bp
SEQ ID NO 18: Cas9-R6 oligonucleotide 21 bp
SEQ ID NO 19: Cas9-F1/Cas9-R6 PCR fragment 123 bp
SEQ ID NO 20: ZmWx-F1 oligonucleotide 20 bp
SEQ ID NO 21: ZmWx-R1 oligonucleotide 21 bp
SEQ ID NO 22: Zea mays B73 waxy gene ZmWx-F1/ZmWx-R1 PCR fragment 728 bp
SEQ ID NO 23: Zea mays haploid inducer KY8556 waxy gene ZmWx-F1/ZmWx-R1 PCRfragment 732 bp
SEQ ID NO 24: Zea mays M1-2 event edited waxy gene PCR fragment 694 bp
SEQ ID NO 25: Zea mays M2-1 event edited waxy gene PCR fragment 732 bp
SEQ ID NO 26: Zea mays M2-2 event edited waxy gene PCR fragment 732 bp
SEQ ID NO 27: Zea mays M4-1 event edited waxy gene PCR fragment 734 bp
SEQ ID NO 28: Zea mays M4-2 event edited waxy gene PCR fragment 730 bp
SEQ ID NO 29: Zea mays M9-2 event edited waxy gene PCR fragment 731 bp
SEQ ID NO 30: Zea mays M9-9 event edited waxy gene PCR fragment 732 bp
Claims (11)
1. A genetic transformation method of a corn haploid induction system is characterized in that a gene editing transformation vector is introduced into cells of a corn haploid induction system plant, and then offspring plants capable of being inherited stably are obtained through screening; the gene editing transformation vector is an agrobacterium expression vector, the transformation vector is an agrobacterium expression vector, and based on a pCambia3301 vector skeleton, the transformation vector comprises three expression units from the right boundary of T-DNA
ZmU6pro gRNA AtU ter, zmUBI pro SpCas9 PsRbcS E9 ter and CaMV35S
pro:Bar:CaMV35S term；
The maize haploid inducer plant is reclamation lure 8556 (KY 8556);
The breeding method of the reclamation induction 8556 plant comprises the following steps: hybridization is carried out by taking Longzhong No. 4 as female parent and Stock6 as male parent to obtain F1 generation, taking F1 generation as female parent and Stock6 as male parent to obtain backcross BC1F1 generation, selecting 3-5 plants with optimal disease resistance, early maturity and purple embryo in the same generation plants, obtaining haploid induction plant reclamation 8556 (KY 8556) strain after 6 generation selfing.
2. The transformation method of claim 1, wherein the SpCas9 gene in the gene editing transformation vector alters or increases other gene expression units or components to form a SpG pan-site editor, cytosine editor, adeno glance sideways at-n editor, guide editor, and other modifications that can promote transformation of corn.
3. The transformation method according to claim 1, wherein the method for constructing the gene editing transformation vector is as follows: based on pCambia3301 vector skeleton, three expression units ZmU pro are included from the right boundary of T-DNA, wherein gRNA is AtU ter, zmUBI pro is SpCas9, psRbcS E9 ter and CaMV35Spro are Bar is CaMV35S ter, two BsaI cleavage sites are reserved between ZmU pro and gRNA skeleton of the transformation vector, and after linearization of the vector, any gRNA recognition sequence fragment for editing target genes is inserted by adopting a DNA ligase or Gibson cloning method.
4. The method according to claim 3, wherein the target gene is a maize starch synthase (wax) gene, the tRNA-ZmWx-G1-gRNA-tRNA-ZmWx-G2-gRNA tandem DNA fragments are artificially synthesized by the interactive tandem of tRNA multiple gRNA expression technology; the BsaI site is inserted by a Gibson cloning method by using a NEBuilder kit, and a transformation vector for simultaneously editing two sites of a corn starch synthase wall gene is constructed, wherein the nucleotide sequence of the transformation vector is shown as SEQ ID NO 1.
5. The conversion process according to claim 1, characterized by comprising the steps of:
(1) Genetic transformation is carried out on the haploid induction line maize acceptor plant to obtain a regenerated plant;
(2) Detecting whether gene editing of the regenerated plant is successful or not;
(3) The successfully edited regenerated plants are selfed and set to generate offspring seeds, and natural genetic separation of offspring is realized in the process;
(4) And after germination of the successfully edited regenerated plant offspring seeds, screening to obtain offspring plants which can be stably inherited and homozygous transgenes and offspring plants which are non-transgenes and stably inherited and homozygous edits.
6. The method of claim 5, wherein step (1) comprises:
(1) Planting a transformation acceptor material, wherein the transformation acceptor material is maize haploid induction line reclamation mutagenesis 8556 (KY 8556);
(2) Preparing and picking up corn young embryo explants;
(3) Preparing agrobacterium and infecting young embryo to obtain transformed cell;
(4) Callus induction and screening;
(5) Callus differentiation and seedling regeneration;
(6) Rooting and culturing regenerated seedlings;
(7) After domestication and transplanting of the resistant regenerated plants, screening to obtain edited regenerated plants.
7. The method of claim 6, wherein step (4) comprises: placing the infected transformed cells on a recovery medium, removing root buds after root buds grow, transferring the transformed cells to a screening medium I to induce callus generation, transferring young embryos with the grown callus to a screening medium II for continuous culture, wherein the screening medium II comprises MS+B5+asparagine+2, 4-D+hydrolyzed casein+proline+sucrose+MES+plant gel+silver nitrate+cephalosporin+carbenicillin+bialaphos.
8. The method of claim 6, wherein step (5) comprises: healthy resistant callus is selected and stripped, transferred to a regeneration culture medium I, and embryoid callus with good growth vigor is transferred to a culture flask containing a regeneration culture medium II, and regenerated seedlings are obtained after continuous culture, wherein the regeneration culture medium I comprises MS+B5+hydrolyzed casein+proline+asparagine+sucrose+plant gel+6-BA+ZT.
9. The method for obtaining the corn plant with the gene editing function and the transgenic improved haploid induction line is characterized in that the offspring plant T1 obtained by the transformation method of any one of claims 1-8 is obtained after screening homozygous transgenes, stably carrying the plant with the verified gene editing vector with the editing function, and the harvested seed T2 germinates.
10. A method for obtaining a diploid corn plant is characterized in that direct transformation and gene editing of a corn haploid induction system are established through the method of claim 9, a transgenic induction system offspring carrying a gene editing vector and having editing functions is screened as a male parent, hybridization is carried out on the transgenic induction system offspring and a corn female parent material with any genetic background, haploid is induced, chromosome doubling treatment is carried out, and a homozygous doubled haploid system for completing gene editing is obtained.
11. Use of the transformation method according to any one of claims 1 to 8, the method for obtaining maize plants of the haploid inducer line according to claim 9 or the method for obtaining diploid maize plants according to claim 10 in the field of haploid maize breeding.
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