Method for expanding propagation of corn cell nucleus male sterile line by using seed morphological markTechnical Field
The invention belongs to the field of plant genetic breeding and seed production, and in particular relates to a method for expanding propagation of corn cell nucleus male sterile lines by using a seed morphological mark.
Background
Plants in nature are of three types, namely self-pollination, cross-pollination and frequent cross-pollination, wherein the self-pollination refers to the phenomenon that pollen of one plant pollinates pistils of the same individual. In plants with amphoteric flowers, it can be divided into homoflower pollination (phaseolus) where stamens and pistils of the same flower pollinate, adjacent flower pollination where pollination is performed between different flowers in one inflorescence (individual), and homoplant cross pollination where pollination is performed between different flowers of the same plant. Some plant stamens and pistils do not grow in the same flower, even on the same plant, and cannot self-pollinate, and their pistils can only obtain pollen of other flowers, which is called cross-pollination. One type of crop that has a natural crossing rate of greater than 50% and is self-declining is classified as a common cross pollinated crop, such as corn.
Corn is a hermaphroditic plant, female flowers and male flowers are positioned at different parts of the plant, the corn can be bred and descended through self-pollination or cross-pollination, and under natural conditions, natural pollination is completed when wind blows pollen from tassel to filaments of female ears.
Due to the existence of heterosis, the biomass, disease and pest resistance and stress resistance (drought, high temperature, low temperature, saline-alkali and the like) of the hybrid seeds are improved compared with the parents, for example, the yield of hybrid corn and hybrid rice is far higher than that of the homozygous parents. The method for producing the hybrid is generally that female parent and male parent are planted together, the tassel of the female parent is removed, the tassel of the male parent is reserved, and the seeds harvested by the female parent are the hybrid.
In maize breeding, homozygous maize inbred lines should first be developed, then the two inbred lines are crossed, and the yield, stress resistance, etc. of the crossed progeny are evaluated to determine if they have commercial potential. Wherein each inbred line may have one or more elite traits lacking in the other inbred line or complements one or more undesirable traits of the other inbred line. The first generation seeds of the two inbred lines are F1 generation seeds, F1 generation plants are obtained after the F1 generation seeds germinate, and the F1 generation plants are more robust than the two inbred line parents (parent-child) and have more biomass.
Hybrid can be produced by artificially emasculating the female parent, i.e., the loose-powder female parent (which can be sown with the male parent in the field at intervals, e.g., 5 rows of female parent, one row of male parent) tassel is removed, and the male parent tassel is retained. Then, only the external corn pollen is isolated, female parent female ears only can receive pollen of male parent, and the obtained seeds are hybrid seeds (F1), and the hybrid seeds can be used for agricultural production.
In the process of producing hybrid seeds, plants are also tasseled after emasculation is finished or emasculation is incomplete due to environmental changes, the two conditions can lead to female parent self-pollination, so that seeds of female parent self-bred lines are mixed in the produced hybrid seeds, the yield of the female parent self-bred lines is far lower than that of the hybrid seeds, the seeds are unqualified products, the income of peasants can be influenced, the reputation of seed production companies can be influenced, and the seed production companies can be seriously caused to bear corresponding compensation responsibility.
The female parent can also be emasculated by a machine, which is substantially the same as manual emasculation, but faster and less costly. However, most emasculation machines cause greater damage to plants than manual emasculation, and so there is currently no fully satisfactory emasculation method, and alternatives with lower costs and more thorough emasculation are still being sought.
The stable male sterility system provides a simple and efficient means by which heavy emasculation can be avoided in some genotypes by using nuclear-cytoplasmic interactive male sterile (CMS) inbreds. The means comprises three main materials, namely a sterile line, namely a male sterile material, a maintainer line, wherein pollen can be provided for the sterile line, so that the offspring of the sterile line is still the sterile line, and a restorer line can restore the fertility of the sterile line. The sterile line and the restorer line are hybridized to generate F1, namely the hybrid seed for agricultural production. More specifically, the nuclear-cytoplasmic interaction sterility manifests itself as genetic nuclear-cytoplasmic interaction. Not only is the cytoplasmic sterility gene S required, but also the homozygous sterility gene (rfrf) is required in the nucleus, and both exist at the same time, so that the plant can be rendered male sterile. If the cytoplasmic gene is fertility N, the nuclear gene is rendered male-fertile, whether it is fertility (RfRf) or sterile (rfrf). Similarly, if the fertility gene (RfRf) or (Rfrf) is present in the nucleus, the cytoplasmic gene is male-fertile, regardless of whether it is fertility N or sterility S. The male sterile line formed by nuclear-plasma interaction has a genetic composition of S (rfrf) and cannot generate normal pollen, but can serve as a hybrid female parent. The F1 produced by crossing the maintainer line N (RfRf) [ with the sterile line can still keep male sterility, namely S (RfRf) ×N (RfRf) →S (RfRf) (sterile) ] and can accept the restorer line S (RfRf) or N (Rf) [ with the sterile line, the F1 produced by crossing the maintainer line N (RfRf) [ with the sterile line ] is fertile, namely S (RfRf) ×S (Rf) →S (Rfrf) (F1) (fertility), or S (RfRf) ×N (RfRf) →S (Rfrf) (fertility) ] pollen, so that F1 is restored to male fertility, F1 plants are selfed to produce F2, and the method can be widely applied to agricultural production. The male sterile line can eliminate manual emasculation, save labor, reduce seed cost and ensure seed purity. At present, crops such as rice, corn, sorghum, onion, castor, beet, rape and the like have been produced by utilizing nuclear-cytoplasmic-interaction male sterility, and the nuclear-cytoplasmic-interaction male sterility lines of other crops are also being widely studied.
CMS also has the disadvantages that firstly, individual CMS materials are easy to be infected, secondly, the restorer line is difficult to find, thirdly, the sterile line is easy to be influenced by the environment, the individual single plants or individual anthers can be fertile, and fourthly, the fertility of the produced hybrid seeds is easy to be influenced by the environment, and the large-area sterile condition can be generated. These problems have prevented the wide application of CMS systems in seed production.
One type of genetic sterility is disclosed in U.S. Pat. nos. 4654465 and 4727219 to Brar et al. However, this type of genetic sterility requires the maintenance of a corresponding genotype at a number of different loci within the genome, requiring molecular marker-tracking detection of these loci per generation. Patterson also describes a potentially useful chromosomal translocation gene system, but this system is more complex (see U.S. Pat. Nos. 3861709 and 3710511).
Attempts have been made to optimize the male sterility system, for example, fabijanski et al developed methods for rendering plants male sterile (EPO 89/3010153.8 publication No. 329308 and PCT application PCT/CA90/00037 published as WO 90/08828). The method is mainly to inhibit male fertility of plants by connecting a promoter specifically expressed by male tissues with a cytotoxin gene and transferring the promoter into the plants, so that male flowers cannot normally scatter powder and other characters are not influenced, and the method is to interfere cloned genes for controlling male fertility of the plants by a transgenic method through a gene interference method, so that the male flowers cannot normally function. There are also means for inhibiting gene expression by means of several gene regulatory elements, thereby affecting plant fertility (WO 90/08829).
In most cases, only nuclear recessive homozygous (msms) plants that control male sterility will appear male sterile, and since the male sterile plants cannot be selfed, male sterile plants (msms) will only be obtained by crossing them with heterozygous plants (Msms). And male sterile seeds (msms) and fertile heterozygous seeds (Msms) exist on the same cluster at the same time, which are sterile seeds can not be distinguished through the seeds, which are fertile seeds, can be distinguished only after sowing and when plants are scattered, and the time for emasculation is not longer.
In recent years, there have been also means for maintaining sterility of male sterile plants by means of transgenesis (US 6743968). The method constructs pollen lethal gene and male fertility restoring gene in a carrier, introduces the pollen lethal gene and the male fertility restoring gene into a male sterile plant, and generates pollen which does not contain restoring gene after transgene representing is fertility. When such plants are crossed with male sterile plants, the homozygous recessive state of the recessive sterile plants is maintained. Firstly, constructing a transgenic vector which contains a pollen cell lethal gene and a dominant gene for restoring plant fertility. The vector is transferred into a male sterile plant, the vector exists in a heterozygous state in the transgenic plant, the plant is fertile due to the existence of the fertility restoration gene, when the vector hybridizes with the male sterile plant, pollen is aborted due to the fact that pollen (Msms) containing the fertility restoration gene contains the lethal gene, therefore, only pollen (ms) without the fertility restoration gene hybridizes with female gametes (ms) of the male sterile plant, and offspring are recessive homozygous individuals (msms). The inventors used the gene mn1 controlling grain size for marking (ZL 201210406155.6) earlier, but mn1 was different in different genetic backgrounds, and some backgrounds could not be effectively used.
As described above, an important problem in many efforts to produce seeds using the male sterile system is how to use the male sterile gene and distinguish between male sterile seeds and breedable seeds, and also how to maintain sterility of the sterile individuals.
Disclosure of Invention
The invention aims to solve the technical problems of how to use male sterile genes and distinguish male sterile seeds and breedable seeds, and how to keep the sterility of sterile individuals.
The inventor of the application obtains a method for producing a maintainer line plant capable of maintaining the sterility of a male sterile line plant through careful design and a large number of experiments, and the maintainer line plant can obtain distinguishable sterile lines and maintainer line offspring simultaneously when being hybridized with the male sterile line plant, so that the propagation efficiency and the breeding efficiency of the sterile line plant are obviously improved.
Thus, in a first aspect of the present application, there is provided a method for seed morphology marker expansion of maize nuclear male sterile line comprising the steps of:
a) Providing a maintainer line B which is an inbred line comprising a homozygous recessive male sterile gene identical to that in the sterile line A and comprising a nucleic acid molecule in heterozygous state, wherein the nucleic acid molecule comprises a first polynucleotide comprising a restorer gene capable of restoring male fertility of a plant which is male sterile due to the recessive male sterile gene and a second polynucleotide comprising a screening gene capable of regulating grain shape comprising seed size, length, width or/and thickness and comprising endosperm dent, shrunken or/and powdery, wherein the grain shape comprises endosperm dent, shrunken or/and powdery;
The screening gene silences the Su1 gene, the Su1 gene codes for a Su1 protein, and the Su1 protein is a protein of C1, C2 or C3 as follows:
C1. the amino acid sequence is a protein with the amino acid sequence shown in SEQ ID No. 10;
C2. A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.10, has more than 80% of identity with the protein shown in C1) and has the same function;
C3. Fusion proteins obtained by ligating protein tags at the N-terminal or/and the C-terminal of C1) or C2);
b) And (3) fertilizing the male gametes of the maintainer line B and the female gametes of the sterile line A, and dividing the obtained seeds into the seeds of the sterile line A and the seeds of the maintainer line B according to the external characters of the screening genes to finish the propagation of the male sterile line and the corresponding maintainer line.
In the propagation method, the sterile line is a plant sterile line, and the maintainer line is a plant maintainer line.
In the propagation method, the plant is a monocotyledonous plant or a dicotyledonous plant. The plant may be maize (Zea mays), canola (Brassica napus), rice (Oryza sativa), arabidopsis (Arabidopsis thaliana), barley (Hordeumvulgare), wheat (Triticumaestivum), sorghum (Sorghum bicolor), soybean (Glycine max), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypiumhirsutum), sunflower (Helianthus annuus), or sugarcane (Saccharum officinarum). In an embodiment of the invention, the plant is maize (Zea mays).
In the propagation method, the recessive male sterile gene leads to male sterility of plants in a homozygous state. The recessive male sterile gene is selected from at least one of ms1,ms2,ms3,ms4,ms5,ms6,ms7,ms8,ms9,ms10,ms11,ms12,ms13,ms14,ms15,ms16,ms17,ms18,ms19,ms20,ms21,ms22,ms23,ms24,ms25,ms26,ms27,ms28,ms29,ms30,ms31,ms32,ms33,ms34,ms35,ms36,ms37,ms38,ms43,ms45,ms47,ms48,ms49,ms50 and ms 52. In an embodiment of the invention, the male sterile gene is ms45.
In the propagation method, the restoring gene can restore the male fertility of the plant with male sterility caused by the recessive male sterility gene. The restorer gene is selected from at least one of Ms1,Ms2,Ms3,Ms4,Ms5,Ms6,Ms7,Ms8,Ms9,Ms10,Ms11,Ms12,Ms13,Ms14,Ms15,Ms16,Ms17,Ms18,Ms19,Ms20,Ms21,Ms22,Ms23,Ms24,Ms25,Ms26,Ms27,Ms28,Ms29,Ms30,Ms31,Ms32,Ms33,Ms34,Ms35,Ms36,Ms37,Ms38,Ms43,Ms45,Ms47,Ms48,Ms49,Ms50 and Ms 52. In an embodiment of the invention, the restorer gene is Ms45.
It will be readily appreciated that in the nucleic acid molecules of the application, the restorer gene and the male sterility gene should be corresponding so that it is able to rescue the sterility trait caused by the recessive male sterility gene. In an embodiment of the application, the recessive male sterile gene is Ms45, and the restorer gene is Ms45.
Specifically, the restorer gene Ms45 encodes an Ms45 protein, which Ms45 protein is a protein of A1, A2 or A3 as follows:
a1, the amino acid sequence is the protein of the amino acid sequence shown in SEQ ID No. 2;
A2, the protein which is obtained by substituting and/or deleting and/or adding the amino acid residues in the amino acid sequence shown in SEQ ID No.2, has more than 80 percent of identity with the protein shown in A1) and has the same function;
a3, N-terminal or/and C-terminal of A1) or A2).
In the propagation method, the restoring gene Ms45 may be specifically a nucleic acid molecule with a coding sequence of SEQ ID No. 1.
In the above propagation method, the first polynucleotide further comprises an expression regulatory element, such as a promoter and an enhancer, operably linked to the nucleotide sequence of the restorer gene. In certain embodiments, the expression regulatory element is selected from the group consisting of a promoter, an enhancer, a regulatory sequence, an inducible element, and any combination thereof. In the nucleic acid molecule, the promoter is selected from the group consisting of constitutive promoters, inducible promoters, tissue-preferred promoters, tissue-specific promoters, and growth phase-preferred promoters. Promoters useful in the present application are not limited to the promoters listed above. It is to be readily understood that any one of the promoters known to those skilled in the art may be used in the above nucleic acid molecules according to actual needs.
In the propagation method, the first polynucleotide sequence may be SEQ ID No.3.
In the above propagation method, the Su1 gene may specifically be a nucleic acid molecule whose coding sequence of the coding strand is a nucleotide sequence of SEQ ID No. 9.
In the propagation method, the screening gene comprises a forward interference fragment, a reverse interference fragment and an intron forming a hairpin structure between the forward interference fragment and the reverse interference fragment, wherein the sequence of the forward interference fragment is SEQ ID No.4, the sequence of the reverse interference fragment is SEQ ID No.5, and the sequence of the intron forming the hairpin structure is SEQ ID No.6.
In the above propagation method, the second polynucleotide further comprises an expression regulatory element, such as a promoter and an enhancer, operably linked to the nucleotide sequence of the screening gene. In certain embodiments, the expression regulatory element is selected from the group consisting of a promoter, an enhancer, a regulatory sequence, an inducible element, and any combination thereof. The promoter is selected from the group consisting of constitutive promoters, inducible promoters, tissue-preferred promoters, tissue-specific promoters, growth phase-preferred promoters. Promoters useful in the present application are not limited to the promoters listed above. It is to be readily understood that any one of promoters known to those skilled in the art may be used in the second polynucleotide according to actual needs.
In the propagation method, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.
In the propagation method, the 80% or more identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
In the propagation method, the first polynucleotide and the second polynucleotide are genetically linked. For example, the first polynucleotide and the second polynucleotide are covalently linked. The covalent linkage may or may not be through the linking nucleotide. The length of the linked nucleotide is not more than 10kb, not more than 5kb, not more than 1kb, not more than 500bp, not more than 100bp, not more than 50bp, not more than 10bp, not more than 5bp, or shorter.
In a second aspect of the present application, there is provided a method of constructing a maintainer line, the method comprising introducing the nucleic acid molecule described above into a sterile line comprising a homozygous recessive male sterile gene to obtain a maintainer line in which the nucleic acid molecule exists in a heterozygous state and which is homozygous for the recessive male sterile gene, which maintainer line is maintainer line B for use in the propagation method described above.
In a third aspect of the application, there is provided an isolated nucleic acid molecule comprising a first polynucleotide comprising a restorer gene capable of restoring male fertility (male fertility) in a plant that is male sterile due to a recessive male sterile gene and a second polynucleotide comprising a screening gene capable of regulating grain shape comprising seed size, length, width or/and thickness and/or endosperm development morphology comprising endosperm dent or not, shrunken or not or/and being powdery.
In a fourth aspect of the present application, there is provided a biological material associated with the nucleic acid molecule described above, which is any one of the following B1) to B8):
B1 An expression cassette comprising said nucleic acid molecule;
B2 A recombinant vector comprising said nucleic acid molecule, or a recombinant vector comprising said expression cassette of B1);
b3 A recombinant microorganism comprising said nucleic acid molecule, or a recombinant microorganism comprising B1) said expression cassette, or a recombinant microorganism comprising B2) said recombinant vector;
B4 A transgenic plant cell line comprising said nucleic acid molecule, or a transgenic plant cell line comprising B1) said expression cassette;
b5 A transgenic plant tissue comprising said nucleic acid molecule, or a transgenic plant tissue comprising said expression cassette of B1);
b6 A transgenic plant organ comprising said nucleic acid molecule, or a transgenic plant organ comprising said expression cassette of B1).
The recombinant expression vector of B2) can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pCAMBIA3301、pAHC25、pWMB123、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene which confers resistance to kanamycin and related antibiotics), bialaphos resistance gene (bar gene) which confers resistance to the herbicide phosphinothricin, hph gene which confers resistance to the antibiotic hygromycin, dhfr gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene which provides mannose metabolism, neomycin resistance gene (such as gene which encodes neomycin phosphotransferase), hygromycin resistance gene (such as gene which encodes hygromycin phosphotransferase), chloramphenicol resistance gene, streptomycin resistance gene, spectinomycin resistance gene, lymycin resistance gene, sulfamide resistance gene, bromelain resistance gene, glyphosate resistance gene, and megamycetin resistance gene, etc. In an embodiment of the invention, the selectable marker gene is a herbicide resistance gene, in particular the herbicide resistance gene is the bar gene. The vector is capable of expressing a nucleic acid molecule as described above in a plant cell (e.g., maize).
In the above biological material, the recombinant microorganism of B3) may be specifically yeast, bacteria, algae and fungi.
In the above biological material, B4) the plant cell line is a monocotyledonous plant or dicotyledonous plant cell line.
Of the above biological materials, B4) the plant cell line is a cell line of a plant selected from the group consisting of maize (Zea mays), canola (Brassica napus), rice (Oryza sativa), arabidopsis thaliana (Arabidopsis thaliana), barley (Hordeumvulgare), wheat (Triticumaestivum), sorghum (Sorghum bicolor), soybean (Glycine max), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypiumhirsutum), sunflower (Helianthus annuus) and sugarcane (Saccharum officinarum).
In a fifth aspect of the application, there is provided the use of the above-described propagation method, and/or the above-described construction method, and/or the above-described nucleic acid molecule, and/or the above-described biological material in plant hybrid seed production.
The beneficial effects are that:
the application provides a propagation method of a male sterile line and a corresponding maintainer line and a nucleic acid molecule used by the same. After crossing with the male sterile plant, the maintainer line plant provided by the application can be used for harvesting simultaneously and effectively distinguishing the progeny of the male sterile plant and the maintainer line plant. Therefore, the method and the maintainer line plant provided by the application can realize efficient propagation of the male sterile plant, and improve the breeding efficiency.
In the invention, the inventor utilizes a technique of controlling plant male fertility genes, seed morphology marker genes and transgenes to invent a novel method for efficiently propagating plant male sterile lines. In one embodiment of the invention, the seed morphology marker gene is a screening gene that regulates seed size (size, length, width, or/and thickness) and/or endosperm development morphology (whether endosperm is depressed, shrunken, or/and is floury). The invention transfers the wild nucleotide sequence for controlling male fertility and the seed morphology marker gene sequence into the conventional corn, then backcross the corn to the homozygous recessive male sterile line plant, and a large amount of sterile line and maintainer line seeds can be obtained simultaneously after the hybridization of the obtained transgenic plant and the homozygous recessive sterile line. Due to the effect of controlling the grain shape or endosperm development morphology nucleotide sequence, sterile lines and maintainer lines can be distinguished by the grain shape or endosperm development morphology. Wherein the seeds with normal shape or normal endosperm development are sterile lines (without transgene sequences), and the seeds with abnormal shape (such as the changes of the size, length, width, thickness and the like of the seeds) or abnormal endosperm development (such as the recession, shrinkage, floury endosperm and the like of the endosperm) are maintainer lines.
In one embodiment of the invention, the inventors constructed a plant transformation vector comprising an expression element for restoring male fertility genes and a length of interfering sequences controlling grain shape and/or endosperm development morphology, transferred the vector into HiIIA x HiIIB maize hybrids, and then backcrossed the resulting transgenic plants with male sterile plants to introduce nucleotide sequences controlling plant male fertility, grain shape (e.g., size, length, width, thickness, etc.), or endosperm development morphology (e.g., dent, shrunken, floury endosperm, etc.) into male sterile plants. The plant appears to be fertile due to the presence of the restorer gene. When a transgenic heterozygous plant (Msmsms) hybridizes to a male sterile plant (msms), it produces progeny, one of which is a normal grain male sterile grain (sterile line, genotype msms) that can be restored to fertility by either wild-type plant, and the other of which is an abnormal grain fertile grain (maintainer line, genotype Msmsms) that is recessively homozygous at the locus of control male fertility, the plant exhibits fertility due to the presence of the complementarily transgenic sequence, and the seed has a grain shape (e.g., size, length, width, thickness, etc.) or endosperm development morphology (e.g., dent, shrunken, floury endosperm, etc.) that differs from wild-type.
Drawings
FIG. 1 is a male sterile mutant and its wild type male flower phenotype. The left panel of FIG. 1 shows the male-floral phenotype of the male sterile mutant (containing the homozygous Ms45 gene) and the right panel of FIG. 1 shows the male-floral phenotype of the wild type (containing the Ms45 gene).
FIG. 2 shows grain phenotype of mutant and wild type grain size control gene su 1. 1 is a mutant grain containing a homozygous Su1 gene, and 2 is a wild type grain containing a Su1 gene.
FIG. 3 is a plant expression vector pMs-Su 1RNAi containing the male fertility gene Ms45 expression element and the grain size control gene Su1 interference fragment expression element.
FIG. 4 shows the male flowers and grain phenotype of plants transformed with the plant expression vector of the male fertility gene Ms45 expression element and the grain size control gene Su1 interference fragment expression element. The right panel of FIG. 4 shows the grain size determination fertility results, wherein B is the hybrid progeny grain containing the transgene element Ms45-Su1RNAi, and A is the hybrid progeny grain without the transgene element Ms45-Su1 RNAi. The left graph of fig. 4 shows the male flowers of the plant after the growth of the B grains in the right graph of fig. 4.
FIG. 5 is a roadmap for seed production using nuclear male sterility genes, genes controlling grain size or endosperm development morphology, and transgenic techniques.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Corn variety B73 comes from the national center for corn improvement at the university of china agriculture. The public is available from the applicant to repeat experiments.
Maize varieties HiIIA and HiIIB are both described in the following literature ："Armstrong C L,Green C E and Phillips R L.Development and availability of germplasm with high Type II culture formation response.Maize Genetics Cooperation News Letter,1991,65:92-93". publicly available from applicant to repeat the present experiment.
Maize ms45 male sterile material 905I is a maize genetic partnership center (Maize Genetics Cooperation Stock Center) product.
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise indicated, the techniques used or referred to herein are standard techniques, materials, methods, and examples which are recognized by one of ordinary skill in the art by way of illustration only and not limitation.
The following more detailed description is provided by way of illustration and description and is not intended to limit the scope of the invention.
Example 1
Nuclear male sterility is the result of mutations, inhibition, or other effects on key genes in the microsporogenesis process, which are collectively referred to as male sterility genes. The pollen development pathway is controlled by many genes, so that mutation of many genes eventually leads to male sterility, and a large number of male sterile mutants are currently identified in maize (as shown in table 1), each male sterile gene has its specific restorer gene, i.e., each male sterile mutant can only be restored by its wild type allele.
TABLE 1 Male sterile mutant caused by nuclear Gene
These genes have been cloned successively, such as ms45 (Albertsen et al 1993) and ms26 (PTC/US 2006/024973), while some male sterility genes have been cloned successively, such as dpw (jin Shi et al 2011) and some male sterility genes identified in Arabidopsis thaliana, such as (Aats, et al 1993).
The genes are integrated with the marker genes related to the invention, so that the sterile line and the maintainer line can be effectively separated.
The invention takes one male sterile mutant in table 1 as an example, for example, a mutant male flower containing a homozygous Ms45 gene cannot normally scatter (as shown in fig. 1, the left graph of fig. 1 is the male flower phenotype of the male sterile mutant, containing the homozygous Ms45 gene; the right graph of fig. 1 is the male flower phenotype of the wild type, containing the Ms45 gene), and the mutant fertility can be restored by the wild type plant.
The Su1 (Sugary 1) gene encodes a maize isoamylase, which after inactivation, the kernel became wrinkled and translucent, the kernel shrunken smaller, and the kernel became smaller (as shown in fig. 2, 1 of fig. 2 is a mutant kernel containing the homozygous Su1 gene, and 2 of fig. 2 is a wild type kernel containing the Su1 gene). The Su1 mutant obtained under natural conditions or by artificial mutagenesis is mostly a recessive mutant, and the inventor interferes with the wild-type Su1 gene at the mRNA level by a transgenic technology, so that the grain containing the transformed interfering nucleotide fragment becomes smaller. Thus, when the transformed fragment exists in a heterozygous state in the kernel, the inventors can rapidly distinguish the kernel containing the transformed fragment by the kernel size. Similarly, other genes affecting the development of the grain shape (such as size, length, width, thickness, etc.) can be subjected to the same operation by the same method, so as to obtain the transgenic grain which is convenient to distinguish. The Su1 gene RNAi interfering sequence in the present invention is derived from B73, but is not limited to inbred line B73, and can be derived from any other wild-type maize inbred line, or homologous genes of other species, or synthetic nucleotide sequences.
The key point of the invention is that the interference fragments of the male restorer gene Ms45 and the grain marker gene Su1 are constructed in a carrier, the carrier can restore the fertility of the male sterile mutant Ms45, and simultaneously, the grain containing the transgene sequence is reduced, namely, the grain containing the restorer gene is marked so as to distinguish the fertile grain (maintainer line) and the sterile grain (sterile line). The invention provides a high-efficiency seed marking method which is not only suitable for corn (Zea mays), but also suitable for crops such as rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticum aestivum), soybean (Glycine max), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) and the like.
The method of the invention is as follows:
1. Construction of plant transformation vector pMs-Su 1RNAi containing DNA fragment (DNA construct) to control male fertility and control corn kernel size in corn
1. Synthesis of Ms45 wild-type allele (Ms 45 expression element) that restores the male fertility of maize male sterile mutant Ms45
The male sterility restoring gene Ms45 is derived from an inbred line B73, and a Ms45 expression element is synthesized by taking genomic DNA of the B73 as a reference, and comprises a promoter, a coding region and a terminator, wherein an EcoRI enzyme recognition sequence is added at the 5 'end, and a BstEII enzyme recognition sequence is added at the 3' end, as shown in SEQ ID No. 3. The coding sequence (CDS) of the Ms45 gene is shown as SEQ ID No.1, and the amino acid sequence of the encoded Ms45 protein is shown as SEQ ID No. 2. After the SEQ ID No.3 sequence was transferred into the ms45 male sterile mutant, the mutant plant became fertile.
2. Synthesis of interfering fragments of genes related to regulation of grain shape and endosperm development morphology
Synthesis of Su1 interference fragment expression element
The invention silences the Su1 gene by RNAi technology, and the used Su1 interference fragment (used as screening gene) is named Su1 RNAi. The coding sequence (CDS) of the Su1 gene is shown as SEQ ID No.9, and the corresponding Su1 protein sequence is shown as SEQ ID No. 10. Su1RNAi comprises a forward interference fragment SEQ ID No.4, a reverse interference fragment SEQ ID No.5, and an intron SEQ ID No.6 forming a hairpin structure between the forward interference fragment and the reverse interference fragment. The SEQ ID No.4, the SEQ ID No.5 and the SEQ ID No.6 are synthesized artificially by an artificial synthesis method, and simultaneously a promoter SEQ ID No.7 for promoting the expression of Su1RNAi and a terminator SEQ ID No.8 for stopping the expression of Su1RNAi are synthesized to form a Su1 interference fragment expression element, and BstEII enzyme cutting sites are respectively added at the 5 'end and the 3' end of the Su1 interference fragment expression element.
3. Construction of plant expression vectors comprising expression elements for Male fertility Gene Ms45 and expression elements for interfering fragments of genes related to grain shape and endosperm development morphology
The plasmid pCAMBIA3301 (product of International center for application of agricultural molecular biology, australia) is used as backbone DNA, and the vector contains a selection marker gene bar. The bar gene coding sequence (CDS) is shown as SEQ ID No. 11.
Constructing the following plant expression vectors by using the vector skeleton:
Plant expression vector pMs-Su 1RNAi the synthesized Ms45 wild type allele expression element and Su1 interference fragment expression element are integrated into pCAMBIA3301 vector by EcoRI, bstEII cleavage and BstEII single cleavage, respectively, to obtain pMs45-Su1RNAi, which contains the selection marker gene bar itself. Plant transformation vector pMs-Su 1RNAi is shown in figure 3, which contains DNA fragments (DNA constructs) and selectable marker genes that control male fertility and control corn kernel size in corn. Wherein, the DNA fragment for controlling male fertility of corn and controlling grain size of corn is named Ms45-Su1RNAi and is the DNA fragment between LB and RB of pMs-Su 1 RNAi. Ms45-Su1RNAi comprises the Ms45 expression element of SEQ ID No. 3 (first polynucleotide) and an expression element silencing the Su1 gene (second polynucleotide). The expression element for silencing the Su1 gene is formed by connecting a promoter (Su 1 promoter) of the Su1, a Su1 interference fragment Su1RNAi (serving as a screening gene) and a terminator. The Ms45 expression element is closely linked to the expression element silencing the Su1 gene, and when pMs-Su 1RNAi is transferred into a plant, both expression elements are present in the plant at the same time.
2. Transformation of maize with plant transformation vectors to obtain transgenic maize
1. Transformation of maize with plant transformation vectors
The recipients used in the transgenic process in the laboratory are the hybrid F1 generation of the inbred lines HiIIA and HiIIB.
Plant transformation vector pMs-Su 1RNAi is used for transforming agrobacterium EHA105, and recombinant agrobacterium EHA105/pMs45-Su1RNAi containing transgenic elements is obtained.
The invention obtains transgenic plants by a method of infecting maize immature embryo with agrobacterium. Firstly, planting maize inbred lines HiIIA and HiIIB in fields, respectively bagging the inbred lines until the inbred lines are scattered, then preparing pollination, wherein two pollination modes are adopted, namely HiIIA is used as a female parent, hiIIB is used as a male parent, hiIIA is used as a male parent, hiIIB is used as a female parent, immature hybrid immature embryos on pollinated ear kernels are taken 9-11 days after pollination, then in-house infection by recombinant agrobacterium EHA105/pMs45-Su1RNAi is carried out, the immature embryos invaded by the recombinant agrobacterium EHA105/pMs45-Su1RNAi are placed on a selection medium, after screening by herbicide dipropyl amine, resistant callus is obtained, and the resistant callus is regenerated into seedlings to obtain transgenic T0 generation plants. After the transgenic T0 generation was obtained, pollen from the T0 generation transgenic plants was used to hybridize some of the female parent and Ms45 male sterile material, and the phenotype was observed.
The specific method comprises the following steps:
Peeling off the young embryo
1) Removing the bract. About 1cm of the top end of the hybrid F1 generation cluster of HiIIA and HiIIB is cut off, and the cluster is inserted from the top end by using tweezers, so that the tweezers can be used as handles, the operation is facilitated, then the clusters are placed into a beaker containing disinfectant, and 4-6 clusters can be placed in the same beaker according to actual needs.
2) About 700ml of sterilizing liquid (50% bleaching agent or 5.25% sodium hypochlorite and a drop of Tween 20) is added into the beaker to soak the ears, the ears are rotated from time to time in the process of sterilizing for 20 minutes while the beaker is gently beaten to remove bubbles on the surfaces of the seeds, so that the best sterilizing effect is achieved, and after the sterilization is finished, the ears are taken out and put into the beaker filled with sterilizing water, washed 3 times in the water, and then the embryo peeling is prepared.
3) The sterilized ears are placed at one end on a large petri dish, and the tops (1.5-1.8 mm) of the kernels are shaved off with a large scalpel, during which time the tools used for sterilization, such as surgical blades, petri dishes, embryo strippers, etc., are handled.
4) The embryo is gently picked up by a small operation knife tip, the embryo is ensured not to be damaged, the embryo axis surface of the embryo is tightly attached to N6E culture medium with filter paper, and the density of the embryo is about 2X 2cm (30 pieces/dish).
5) The dishes were sealed with a sealing film and incubated at 28℃for 2-3 days in a dark place.
(II) Agrobacterium infection
1) Recombinant Agrobacterium EHA105/pMs45-Su1RNAi was cultured one week in advance on YEP (containing 33mg/L kanamycin and 100mg/L rifampicin) medium.
2) The recombinant Agrobacterium cultured as described above was transferred to fresh YEP (containing 33mg/L kanamycin and 50mg/L rifampicin) medium and cultured at 19℃for 3 days.
3) After 3 days, agrobacterium tumefaciens EHA105 was picked up and placed in a 50mL centrifuge tube containing 5mL of infection medium, AS (inf+AS) (solute AS in Table 2, water AS solvent) was added, and shaking was performed at 75rpm at room temperature (25 ℃) for 2-4 hours.
4) The young embryo is infected, the young embryo which is just stripped is placed into a centrifuge tube containing AS (inf+AS) liquid culture medium (2 ml), about 20-100 young embryos are washed for 2 times by using the culture medium, then 1-1.5ml of agrobacterium with specific concentration (OD550 =0.3-0.4) is added, the centrifuge tube is gently inverted for 20 times, and then the young embryo is vertically placed in a dark box for 5 minutes, so that the young embryo is completely soaked in agrobacterium liquid, and vortex oscillation is avoided in the whole process.
(III) Co-cultivation
1) After infection, the infected young embryos are transferred to a co-culture medium (solute as in Table 2, water is used as the solvent) and the embryonal axes of the young embryos are brought into contact with the surface of the medium, while removing excess Agrobacterium from the surface of the medium.
2) The dishes were sealed with a sealing film and dark-cultured at 20℃for 3 days.
(IV) resting culture
After 3 days of co-cultivation, the young embryos are transferred onto resting medium (solute as in Table 2, solvent water) while the dishes are sealed with sealing film and dark cultivated at 28℃for 7 days.
(Fifth) selection
1) After 7 days, all the young embryos are transferred onto a selection medium (35 young embryos per dish) containing 1.5mg/L of dipropylamine phosphorus (solute as in Table 2, solvent water) and incubated for two weeks, after which the concentration of the sub-incubated dipropylamine phosphorus can be raised to 3mg/L.
2) About 5 weeks after infection, cells containing the transformant will grow into visible type II calli.
Regeneration of transgenic plants
1) After 3 weeks on regeneration medium I (solute as shown in Table 2 and water as solvent), the maize was germinated on regeneration medium II (solute as shown in Table 2 and water as solvent) (in the light culture chamber) to obtain 100 plants T0 generation pMs-Su 1RNAi transformed maize.
2) Transferring the regenerated seedlings to a greenhouse when 3-4 leaves grow out, and pollinating the regenerated seedlings when the regenerated seedlings grow to a spinning and powder scattering period.
TABLE 2 solute and content of culture Medium
In Table 2, the MS salt was purchased from phyto Technology Laboratories under the trade designation M524.
2. Analysis of the obtained transgenic plants
The T0 generation pMs45-Su1RNAi transformed maize from step 1 and its progeny were evaluated for overall plant morphology and analyzed for pollen, plant and grain phenotype.
The T0 generation pMs-Su 1RNAi transformed corn is hybridized with ms45 homozygous recessive ms45 male sterile material 905I to obtain a filial generation.
1) Genotyping of hybrid offspring
The detection of Bar gene is used to determine if it contains transgenic element Ms45-Su1RNAi, and the Bar gene detection method includes PCR amplification of the filial generation with primer pair comprising Bar669F and Bar669R, if it contains 669bp target fragment, the filial generation is the filial generation containing transgenic element, if it does not contain 669bp target fragment, the filial generation is the filial generation not containing transgenic element.
The sequences of the primers Bar669F and Bar669R are as follows:
Bar669F:5'-TCTCGGTGACGGGCAGGAC-3';
Bar669R:5'-TGACGCACAATCCCACTATCCTT-3'。
The filial generation containing the transgene element Ms45-Su1RNAi is obtained.
2) Detection of phenotype of hybrid offspring
The field was observed for the filial generation containing the transgene element Ms45-Su1RNAi, and the results are shown in FIG. 4:
The fertility result of the grain size judgment is shown in the right graph of FIG. 4, B is the filial generation grain containing the transgene element Ms45-Su1RNAi, the grain is small (fertility detection), A is the filial generation grain without the transgene element Ms45-Su1RNAi, and the grain is normal (sterility detection);
The male flowers of the plants grown from the small grains in the right panel B of FIG. 4 are shown in the left panel of FIG. 4 as the filial generation containing the transgene element Ms45-Su1RNAi, and the plants are rendered fertile (detected fertility) due to the inclusion of Ms 45.
3. Large-scale propagation of male sterile lines using male sterile maintainer lines
The male sterile line was propagated on a large scale using a male sterile maintainer line according to the route of fig. 5, specifically as follows:
1. transformation of the Ms45Ms45 wild-type inbred into the Ms45Ms45 homozygous recessive inbred
The Ms45 homozygous recessive mutant (such as 905I) is used as a female parent, the female parent is hybridized with different inbred lines (such as Zheng58 (zheng 58), the crop of Henan national institute of science is used as a variety), the obtained F1 is further backcrossed with the corn inbred line Zheng58, genotype analysis is carried out on the obtained BC1 population, the plants with the Ms45 locus heterozygous are identified to be further backcrossed with Zheng58, after 5-6 generations of backcrossing, the Ms45 locus heterozygous is screened by using a molecular marker, and the single plants with the Zheng58 at other loci are self-crossed, so that the Ms45 homozygous recessive inbred line Zheng58 (called Zheng58 (Ms 45Ms 45)) is obtained, and the inbred line can be used as a sterile line and is called a first plant.
The above method for screening the genotype of Ms45 locus is carried out by PCR amplification of plant genome with the following primers Ms45F1 and Ms45R1 and sequencing of the amplification result. The size of the Ms45 target fragment is 859bp, and the size of the ms45 target fragment is 811bp. If the amplified product contains 859bp and 811bp target fragments, the genotype of the locus is heterozygous Ms45/Ms45, if the amplified product does not contain 811bp fragments, the genotype of the locus is dominant homozygosity of Ms45/Ms45, and if the amplified product does not contain 859bp target fragments, the genotype of the locus is recessive homozygosity of Ms45/Ms 45.
Ms45F1:5'-CTTGAGCGACAGCGGGAACT-3';
Ms45R1:5'-TGTTGTTTCTTGGCAAAGGTCAG-3'。
2. Preparation of the second plant
Preparation of second plants heterozygous for Ms45-Su1RNAi and homozygous for Ms45
Crossing the obtained Ms45 homozygous recessive inbred line Zheng 58 (Ms 45Ms 45)) (first plant, female parent) with the obtained T0 generation pMs45-Su1RNAi transformed corn (male parent), then carrying out multiple generation backcrossing by taking the Ms45 homozygous recessive inbred line Zheng 58 (Ms 45Ms 45)) (first plant) as recurrent parent, and converting the T0 generation pMs-Su 1RNAi transformed corn into an inbred line containing Ms45-Su1RNAi heterozygous and Ms45 locus as homozygous recessive, wherein the inbred line is a second plant with Ms45-Su1RNAi heterozygous and Ms45 homozygous, also called Zheng 58 (Ms 45-Su1RNAi heterozygous and Ms45 homozygous recessive).
The specific method comprises the following steps:
the T0 generation is transferred to pMs-Su 1RNAi corn as a male parent, the corn is hybridized with the obtained Ms45 homozygous recessive inbred line Zheng 58 (Ms 45Ms 45)) (female parent), seeds of small grains selected from the filial generation are sown into the field, 200mM dipropionate is sprayed, the surviving plants are further back-crossed with Zheng 58 (Ms 45Ms 45)), after 5-6 generations of back-crossing, small grain seed plants are always selected to hybridize with the first plants in the back-crossing process, and the small grain seeds or plants contain transgene sites (Ms 45-Su1 RNAi), so that the obtained small grain seeds or plant transgene sites (Ms 45-Su1 RNAi) in the filial generation are heterozygous. Pollinating the first plant by using pollen of the small grain plant, and if the obtained normal grains (large grain) are sterile, providing pollen, wherein the transgenic locus Ms45-Su1RNAi of the plant is heterozygous and the locus Ms45 is stealthy homozygous, namely the second plant.
3. Acquisition of the maintainer line
The second plant Zheng 58 (Ms 45-Su1RNAi heterozygous and Ms45 homozygous recessive) obtained was used as a male parent, crossed with the first plant Ms45 homozygous recessive inbred line Zheng 58 (Ms 45Ms 45)) (female parent), and the offspring produced had not only Zheng 58 (Ms 45Ms 45) but also the maintainer line Zheng 58 of male sterile Zheng 58 (Ms 45-Su1RNAi heterozygous Ms45Ms 45). And Zheng 58 (Ms 45Ms 45) is normal, while its maintainer line Zheng 58 (Ms 45-Su1RNAi heterozygous Ms45Ms 45) is small (as shown in right panel B of FIG. 4).
4. Large-scale propagation of ms45 male sterile inbred lines using male sterile maintainer lines
Taking Zheng 58 inbred line as an example, the male sterile line Zheng 58 (Ms 45Ms 45) and the male sterile maintainer line Zheng 58 (Ms 45-Su1RNAi heterozygous Ms45Ms 45) are sown in the field, two materials are sown at intervals, each 1 line of maintainer line is sown with 5 lines of sterile lines correspondingly, no other corn sowing is ensured in 300 meters around seed reproduction, and the sterile lines and the maintainer line field are naturally pollinated. The maintainer line can only accept pollen of the maintainer line, and seeds containing homozygous transgenic components in the generated offspring cannot be distinguished from heterozygous seeds, so that the seeds are discarded, and the seeds (large seeds) with normal sizes can be used as sterile lines. Sterile line material received pollen from the maintainer line, normal size grain in the offspring was sterile line without transgenic components, while small grain was maintainer line with transgenic components. The maintainer line is used for the next year of propagation of sterile line and maintainer line, while most of sterile line is used for the manufacturer variety, and the rest is used for the next year of propagation of sterile line and maintainer line, and the specific production flow is shown in figure 5.
4. Large-scale production of hybrid seeds using male sterile lines
By selecting an inbred line with high mating force with male sterile (ms 45ms 45), such as male sterile Zheng 58 (ms 45ms 45), such as Chang 7-2, the hybrid with excellent agronomic characteristics can be produced. In order to achieve the purpose, the inventor sows the male sterile inbred line and the wild type inbred line in the field in an interlaced manner, ensures that no other corn is sown in 300 meters around seed reproduction, and the ears of the sterile line can only accept pollen of the wild type inbred line and the wild type inbred line can only be inbred. Thus, seeds produced on the sterile line ears are hybrid seeds.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.