WO2019062895A1 - Use of maize gene zmabcg20 in regulating crop male fertility and dna molecular markers associated with maize male fertility and use thereof - Google Patents

Abstract

The application of the maize gene ZmABCG20 in regulating male fertility of crops, the genomic DNA sequence of ZmABCG20 in maize variety B73 is shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 3. The mutant zmabcg20-1 of the gene ZmABCG20 and its use, the mutated gene sequence is shown in SEQ ID NO: 7, and a molecular marker identification method for the mutated gene is also provided. a DNA molecular marker associated with male fertility in maize, the DNA molecular marker located at bases 326-329 after the start codon of the maize gene ZmABCG20 nucleic acid sequence, the sequence is TGCA, and the 4 base deleted maize line exhibits Recessive male sterility traits. The DNA molecular marker of the present invention can be used to identify or breed male sterile maize germplasm resources and the like.

Description

Application of Maize Gene ZmABCG20 in Regulating Crop Male Fertility and DNA Molecular Markers Related to Maize Male Fertility and Its Application

cross reference

This application claims the patent application entitled "The Application of Maize Gene ZmABCG20 in Regulating Crop Male Fertility", filed on September 30, 2017, and the patent name filed on September 30, 2017 is "with corn" The priority of the Chinese Patent Application No. 201710919712.7, the entire disclosure of which is incorporated herein by reference.

Technical field

The invention belongs to the field of genetic engineering and molecular breeding, and in particular relates to the application of the maize gene ZmABCG20 in regulating male fertility of crops and the DNA molecular markers associated with male fertility of corn and applications thereof.

Background technique

Plant male sterility mutations are a very common phenomenon in nature, and male sterility mutants have been found in at least 617 species of 43 families and 162 genera. In genetics, male sterility is divided into three categories: nuclear male sterility, cytoplasmic male sterility and nuclear cytoplasmic interaction. 1) Nuclear male sterility is caused by nuclear gene mutation, with dominant and recessive mutations. , sporozoite gene mutations and gametophytic gene mutations. Dominant mutations and gametophytic gene mutations can only be inherited by female gametes, which can be inherited either by female gametes or by male gametes, and follow Mendel's law. Some sporozoite recessive nuclear sterility genes have been cloned, such as ms2 of Arabidopsis thaliana, ms45 of maize and mil1 of rice (Aarts et al., 1997, The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes, Plant Journal, 12: 615-623; Albertsen, 2006, Male tissue-preferred regulatory sequences of MS45gene and method of using same, Patent No.: US7154024B2; Hong et al, 2012, Somatic and reproductive cell development in rice anther is regulated by a putative Glutaredoxin, Plant Cell, 24:577-588); some gametophytic recessive genic male sterility genes have also been cloned, such as the two microspores of Arabidopsis thaliana mitotic abnormalities sidecar pollen and gemini pollen (Oh et al, 2010, The SIDECAR POLLEN gene encodes a microspore-specific LOB/AS2domain protein required for the correct timing and orientation of asymmetric cell division, Plant Journal, 64: 839-50; Park et al, 1998, The Arabidopsis thaliana gametophytic mu Ttation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate, Development, 125:3789-99); a sporophytic dominant sterility gene MS44 was also cloned on maize (Cigan and Albertsen, 1998, Reversible nuclear genetic system for Male sterility in transgenic plants, US5750868); 2) cytoplasmic male sterility is controlled by cytoplasmic genes, and there is no corresponding nuclear restorer gene, which is maternal inheritance; 3) cytoplasmic cytoplasmic interaction male sterility by cytoplasmic gene and nuclear gene Joint control, the essence of which is the result of disharmony between cytoplasm and nuclear genetic material. Sterile cytoplasm is caused by mutant mitochondrial genes, but has a corresponding nuclear restorer gene that inhibits sterile cytoplasmic genes. Sterile cytoplasmic genes produce a new protein that affects normal mitochondrial function (Chen and Liu, 2014, Male sterility and fertility restoration in crops, Annu Rev Plant Biol, 65:5.1-5.28). In terms of fertility restorer genes, genes such as Rf-1, Rf-2, Rf-4, and Rf-5 have been cloned in rice (Komori et al., 2004, Map-based cloning of a fertility restorer gene, Rf-1, In rice(Oryza sativa L.), Plant Journal, 37: 315-325; Itabashi et al, 2011, The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein, Plant Journal , 65: 359-367; Tang et al, 2014, The rice restorer Rf4 for wild-abortive cytoplasmic male sterility encodes a PPR protein that functions in reduction of WA352transcripts, Molecular Plant, 7: 1497-500; Hu et al, 2012, The rice pentatricopeptide Repeat protein RF5restorers fertility in Hong-Lian Cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162, Plant Cell, 24: 109-22).

Maize has become the world's largest food crop in the world and is an important raw material for feed, food processing and bio-energy. It is also one of the most consumed vegetables in foreign countries. At present, almost all corn grown in China is hybrid corn. Maize hybrid pollination is mainly carried out by artificial emasculation, which requires a large amount of labor and is costly; and because the emasculation damages the top leaves of the corn, it will cause loss of seed production. The use of male sterile lines for seed production can solve the problems caused by artificial emasculation. However, the cytoplasmic male sterile lines used in maize have some defects: firstly, because the cytoplasmic male sterile line needs specific recovery genes to restore fertility, the utilization rate of germplasm resources is very low, which limits the breeding efficiency of excellent varieties. Secondly, the sterile line of the sterile line is unstable, and the fertility can be restored under certain conditions, which affects the purity of the hybrid; finally, due to the single cytoplasmic genotype, the corn leaf disease is outbreak, which directly leads to the cytoplasmic male sterility technology. qiut the market. Ordinary nuclear infertility can avoid these problems. For example, it can be used in corn, which not only saves the labor cost required for artificial emasculation, but also increases seed production.

The plant ABC protein family is a type of membrane transporter that localizes to the cell membrane and is responsible for the transmembrane transport of metabolites; the ABCG transporter is one of the largest subfamilies. ABCG proteins can be divided into two main types according to their structural features: full-size proteins contain two nucleotide binding domains and two transmembrane regions, which can form a complete transmembrane transport structure on their own, complete substrate transport; half-size proteins Only one nucleotide binding domain and one transmembrane domain need to bind to another half-size protein molecule to form a complete transport unit (Verrier et al., 2008, Plant ABC proteins–a unified nomenclature and updated inventory. Cell , Trends in Plant Science, 13(4): 151-159.). The AtABCG26 gene of Arabidopsis thaliana and the ortholog gene OsABCG15 in rice encode a transmembrane transporter of pollen wall component sporopollen precursor, expressed in the anther velvet layer, and the sporopollen precursor from the velvety cell Transfer to the anther chamber to synthesize sporopollenin on the pollen cell wall. The mutant atabcg26 showed extremely low pollen count and male fertility; the rice osabcg15 mutant was completely male sterile and had no pollen; in addition, the rice OsABCG26 mutation also showed male complete sterility, and the phenotype was similar to osabcg15 (Zhao et al. , 2016, ATP binding cassette G transporters and plant male reproduction. Plant Signal and Behavior, 11(3): e1136764.doi: 10.1080/15592324.2015.1136764). By genomic bioinformatics analysis, Pang et al. (Pang et al., 2013, Inventory and general analysis of the ATP-binding cassette (ABC) gene superfamily in maize (Zea May L.). Gene, 2013, 526(2): 411- 428) 54 ABCG genes were identified from maize varieties, but no genes related to male fertility were found.

Summary of the invention

The object of the present invention is to provide a use of the maize gene ZmABCG20 for regulating crop male fertility.

Another object of the present invention is to provide a mutant zmabcg20-1 of the maize gene ZmABCG20 and uses thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides the use of the maize gene ZmABCG20 for regulating crop male fertility, wherein the cDNA sequence of the gene ZmABCG20 is:

i) the nucleotide sequence shown in SEQ ID NO: 2;

Ii) a nucleotide sequence in which the nucleotide sequence shown by SEQ ID NO: 2 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein;

Iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 2 and which expresses the same functional protein, which is 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC containing 0.1% SDS. In solution, hybridize at 65 ° C and wash the membrane with the solution; or

Iv) a nucleotide sequence having more than 85% homology to the nucleotide sequence of i), ii) or iii) and expressing the same functional protein.

In the foregoing application, the regulation refers to making the crop malely fertile. The applications include:

1) causing the crop to contain the ZmABCG20 gene; or

2) The crop is expressed with a protein encoded by the ZmABCG20 gene.

The invention firstly performs cobalt 60 radiation mutagenesis treatment on the corn variety Jingkejing 2000 seeds (M ₀ generation), the planted seeds are obtained from the M ₁ generation plants; the M ₁ generation plants are selfed to produce seeds (for the M ₂ generation), planting M ₂ generation plants were subjected to morphological, histological and genetic identification of M ₂ generation plants, and sterile plants were screened; then the sterile plants were subjected to gene sequencing and DNA sequence analysis, and verified at the molecular level. Finally, homozygous sterile plants were obtained and used for cross breeding and biotechnology research.

The maize ZmABCG20 gene (pollen development control gene) provided by the present invention exhibits complete male sterility after mutation. Its nucleotide sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 4; the DNA sequence of its coding region is shown as SEQ ID NO: 2 or SEQ ID NO: 5; the encoded protein sequence is SEQ ID NO: : 3 or SEQ ID NO: 6.

In a second aspect, the invention provides the use of the maize gene ZmABCG20 in the preparation of a transgenic plant. For example, a recombinant expression vector carrying the gene ZmABCG20 cDNA or genomic sequence is transferred into wild type maize callus, and the transformed material is subjected to co-culture-screening-differentiation-rooting-transgenic seedling training and transplanting, and the transgenic plant is screened. Then, the transgenic corn is crossed with the male sterile maize to restore the fertility of the male sterile maize.

In a third aspect, the invention provides the use of the maize gene ZmABCG20 for restoring fertility in a male sterile plant, wherein the male sterility trait is caused by the genetic mutant.

In a fourth aspect, the present invention provides a method for preparing a male sterility transgenic maize by inhibiting the activity of a maize ZmABCG20 gene, which utilizes gene silencing, gene suppression, gene knockout or directed gene mutation to transcribe the ZmABCG20 gene in maize. The level of protein activity after translation or translation is reduced, and male genic sterile GM maize is obtained.

For example, an RNAi sequence carrying a cDNA sequence against the gene ZmABCG20 can be operably linked to a constitutive promoter or a floral organ-specific expression promoter, and transferred into a plant callus, and the transformed material is subjected to co-culture-screening-differentiation. - Rooting-transgenic seedlings were exercised and transplanted, and male sterility GM maize was screened.

In a specific embodiment of the invention, the target DNA sequence for RNAi action is set forth in SEQ ID NO:23.

In a fifth aspect, the present invention provides the use of the biological material obtained by the above method in crop improved breeding and seed production.

In a sixth aspect, the present invention provides the use of the maize gene ZmABCG20 in crop improved breeding and seed production.

In the foregoing application, a plant containing or expressing the ZmABCG20 gene, or a plant inactivated according to the above method or the ZmABCG20 gene, is hybridized with the same crop having excellent agronomic traits.

In the present invention, the crop is a self-pollinating or cross-pollinated crop, including but not limited to corn, wheat or rice, etc., preferably corn.

In the present invention, the excellent agronomic traits include, but are not limited to, increased yield, improved quality, resistance to pests and diseases, stress resistance, lodging resistance, and the like.

In a seventh aspect, the present invention provides an inhibitor for inhibiting the activity of a ZmABCG20 gene selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular weight compound, peptide, antibody, and the like. One.

In an eighth aspect, the invention provides an expression cassette, expression vector or cloning vector comprising an inhibitor encoding the nucleic acid molecule described above.

In a ninth aspect, the present invention provides a mutant zmabcg20-1 gene of the maize gene ZmABCG20, the nucleic acid sequence of which is:

i) a mutant gene formed by a 4 base TGCA deletion at bases 326-329 after the start codon of the maize gene ZmABCG2 nucleic acid sequence;

Ii) the nucleotide sequence shown in SEQ ID NO:7;

Iii) a nucleotide sequence in which the sequence shown in i) or ii) is substituted, deleted and/or added to one or more nucleotides and expresses the same functional protein, and comprises 4 bases at an equivalent position to the gene ZmABCG20 TGCA is missing;

Iv) a nucleotide sequence which hybridizes under stringent conditions to the sequence shown in i) or ii) and which expresses the same functional protein, and which comprises a 4-base TGCA deletion at the equivalent position to the gene ZmABCG20; Hybridization at 65 ° C in 0.1 × SSPE containing 0.1% SDS or 0.1 × SSC solution containing 0.1% SDS, and washing the membrane with the solution; or

v) A nucleotide sequence having more than 85% homology to the nucleotide sequence of i) or ii) and expressing the same functional protein, and comprising a 4 base TGCA deletion at an equivalent position to the gene ZmABCG20.

The coding region DNA sequence of the maize gene zmabcg20-1 is shown in SEQ ID NO: 8. A protein encoded by the maize gene zmabcg20-1, the amino acid sequence of which is set forth in SEQ ID NO: 9, or an amino acid sequence of the same sequence formed by substitution, deletion or addition of one or several amino acids.

In a tenth aspect, the present invention provides the use of the gene zmabcg20-1 for regulating corn fertility, the application comprising:

1) causing the crop to contain the zmabcg20-1 gene; or

2) The crop is expressed by a protein encoded by the zmabcg20-1 gene.

In the foregoing application, maize containing or expressing the mutant zmabcg20-1 gene exhibits recessive male sterility.

In an eleventh aspect, the present invention provides the use of the gene zmabcg20-1 in improved breeding and seed production of maize.

In the foregoing application, corn comprising or expressing the mutant zmabcg20-1 gene is crossed with corn having excellent agronomic traits.

In a twelfth aspect, the invention provides an expression cassette, expression vector or cloning vector comprising a nucleic acid sequence comprising the gene zmabcg20-1.

In a thirteenth aspect, the present invention provides an engineered bacteria, a host cell, or a transgenic cell line comprising the gene zmabcg20-1, or the expression cassette, expression vector or cloning vector.

In a fourteenth aspect, the invention provides the use of a biomaterial comprising or expressing the gene zmabcg20-1 for the preparation of transgenic corn.

In a fifteenth aspect, the present invention provides a plant panicle-specific promoter of the male flower or the male and female, the promoter being:

i) the nucleotide sequence shown in SEQ ID NO: 12;

Ii) a nucleotide sequence in which the nucleotide sequence shown by SEQ ID NO: 12 is substituted, deleted and/or increased by one or more nucleotides and has the same function;

Iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 12 and which has the same function, in stringent conditions of 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC solution containing 0.1% SDS Medium, hybridizing at 65 ° C, and washing the membrane with the solution; or

Iv) a nucleotide sequence having more than 85% homology to the nucleotide sequence of i), ii) or iii) and having the same function.

In a sixteenth aspect, the invention provides an expression cassette, expression vector or cloning vector comprising a nucleic acid comprising the sequence set forth in SEQ ID NO: 12.

In a seventeenth aspect, the invention provides an engineered bacterial, transgenic cell line comprising the specific promoter, or the expression cassette or vector.

In a eighteenth aspect, the invention provides the use of the specific promoter for regulating downstream gene expression.

In a nineteenth aspect, the invention provides the use of the specific promoter in the preparation of a transgenic plant.

For example, a promoter sequence is operably linked to a gene of interest, and the resulting construct is used to transform a target plant, and the promoter drives the gene of interest to be specifically expressed in the young ears of the male flower or the male and female.

In a twentieth aspect, the present invention provides a DNA molecular marker associated with male fertility in maize, the DNA molecule marker being located at bases 326-329 after the start codon of the maize gene ZmABCG20 nucleic acid sequence, the sequence being TGCA, The 4 base deleted maize line showed recessive male sterility.

In a twenty first aspect, the invention provides a primer for specifically amplifying a marker of the DNA molecule, comprising:

Upstream primer 3326_F1: 5'-CCAGACGAGGGCAGACCAG-3' (SEQ ID NO: 10)

Downstream primer 3326_R1: 5'-GATCTCGCCAGGGTCCACA-3' SEQ ID NO: 11)

In a twenty second aspect, the invention provides a detection reagent or kit comprising the primers 3326_F1 and 3326_R1.

In a twenty-third aspect, the present invention provides the use of the DNA molecule marker, the primer or the detection reagent or kit in maize molecular marker-assisted breeding.

In a twenty-fourth aspect, the present invention provides the use of the DNA molecular marker, the primer or the detection reagent or kit for identifying or breeding a male sterile maize germplasm resource. The specific method is as follows:

The genomic DNA of the tested corn is extracted, and the PCR amplification reaction is carried out by using primers 3326_F1 and 3326_R1, and the amplified product is detected by electrophoresis. If a characteristic band of 79 bp is present in the amplified product, the fertility of the corn to be tested is normal, corresponding The genotype of ZmABCG20 is wild type; if a characteristic band of 75 bp is present in the amplified product, the maize to be tested is male sterile, and the corresponding ZmABCG20 genotype is zmabcg20-1 mutant; if the amplification product is 79 bp and The two-band type of 75 bp, the corn to be tested is a heterozygous genotype.

In a twenty-fifth aspect, the present invention provides the use of the DNA molecule marker, the primer or the detection reagent or kit in the classification of the maize gene ZmABCG20.

The advantages of the ZmABCG20 gene provided by the present invention are as follows:

1) It was first discovered that the ZmABCG20 mutation can cause the male sterility phenotype of maize, which is of great significance for the study of the utilization and function of this gene and the regulation mechanism of male fertility in maize.

2) ZmABCG20 mutation only affects male fertility, which can cause male complete sterility, but has no effect on male and female fertility and other agronomic traits, and is suitable for industrial application such as cross breeding, seed production and production.

3) ZmABCG20 is only expressed in the shoots of male flowers, and has strong time and tissue specificity. Its promoter can be used to drive specific expression of any gene in the young flower spikes.

The advantages of the mutant zmabcg20-1 provided by the present invention are as follows:

1) zmabcg20-1 is the first reported ZmABCG20 mutant, which is of great significance for the utilization and function of this gene.

2) The mutant only affects male fertility, resulting in male complete infertility, but has no effect on male and female fertility and other agronomic traits.

3) The mutant is a gene deletion mutation caused by a 4 base deletion, and there is no potential risk of fertility recovery, and there is no risk of genetic instability.

4) The mutant is a 4-base deletion in the gene and does not affect the function of adjacent genes on both sides of ZmABCG20.

5) The mutant is a 4 base deletion mutation, and the Indel label can be designed to perform high-throughput detection by ordinary PCR and electrophoresis; and can also be designed as a gene chip detection marker.

6) The genetic background of the mutant is a contemporary Chinese main variety, which can be directly used for the selection of Chinese corn varieties without a long process of improvement.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing the mutant wild type and the male flower of zmabcg20-1 and the ear of zmabcg20-1 in Example 2 of the present invention.

Figure 2 is a graph showing the results of anther and pollen I ₂ -KI staining of wild type and mutant zmabcg20-1 in Example 3 of the present invention.

3 is a real-time quantitative PCR result of ZmABCG20 gene expression in young ears and different tissues of Jingkejing 2000 in Example 7 of the present invention.

Figure 4 is a schematic diagram showing the ZmABCG20 gene structure and the mutation site of zmabcg20-1 identified in Examples 6 and 8 of the present invention.

5 is an electrophoresis result of molecular marker identification of a mutant of a self-crossing F ₂ progeny and a wild type plant ZmABCG20 gene after open pollination of the zmabcg20-1 mutant in Example 9 of the present invention.

Figure 6 is a schematic flow chart showing the construction of the RNAi vector of the ZmABCG20 gene in Example 10 of the present invention.

Figure 7 is a result of pollen iodine staining of control and RNAi male sterile plants in Example 11 of the present invention.

Figure 8 is a technical route diagram for the hybridization of the zmabcg20-1 sterile gene described in Example 13 of the present invention.

Detailed ways

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are based on routine experimental conditions, such as the Sambrook J&Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or as suggested by the manufacturer's instructions.

Example 1 Cobalt 60 Radiation Mutagenesis Mutant Library

In September 2015, in Changsha, cobalt 60 ( ⁶⁰ Co) was used to irradiate Jingke 糯2000 seeds (M ₀ generation) 3 kg, and the radiation dose was 250 Gy. The seeds of the radiation were planted in the field of Yacheng District, Sanya City, Hainan Province in October 2015. The individual plants were strictly selfed and the M ₁ generation seeds were harvested.

5,400 strains of M ₁ seed were selected, and 50 plants per plant were planted. In February 2016, they were planted in Lin Gaotian, Hainan. At the seedling stage, heading stage, flowering stage, and filling stage, the field traits were carefully observed, and various types of mutants such as plant type, panicle type, fertility and yield were screened, and each type of mutant was collected and preserved.

Example 2 M ₂ Generation Planting and Character Observation

During the heading and flowering period of M ₂ generation, the anthers were observed in the field, and anthers with abnormal color, small shape and small amount of pollen were selected for further microscopic examination under the microscope. In the family numbered 3326, 9 plants with abnormal fertility were found, which could not be loosely powdered, but were firm and normal (Fig. 1). The mutant anther was smaller than the wild type, the color was light yellow, and there was no visible pollen. However, there was no significant difference between the vegetative growth, heading stage and panicle type and the wild type, which was selected as the candidate mutant material for further study. Based on the finally identified mutant gene (see Examples 6, 7, 8), the mutant was named zmabcg20-1.

Example 3 Pollen microscopy and genetic analysis of sterile mutants

Pollen fertility was counted by iodine staining and non-coloring pollen ratio. The morphology of zmabcg20-1 male flowers was observed under a stereo microscope. The anthers were smaller than the wild type and the color was lighter (Fig. 2). In the field, flowering florets were collected, and the anthers were removed with tweezers. The anthers were gently squeezed in iodine-potassium iodide solution (0.6% KI, 0.3% I ₂ , w/w), dropped on glass slides, and covered with coverslips. The pollen iodine staining was observed under a microscope and photographed. Wild-type pollen is mostly stained blue-black, while mutants do not see pollen grains (Fig. 2).

The mutant was normally robust under open pollination (Fig. 1), indicating that the mutant was a male sterile mutant and the fertility of the ear was not affected. The zmabcg20-1 open pollination seed (F ₁ ) was harvested and sown. After the heading, the powder could be normally scattered. The bagging self-crossing could be normal and the F ₂ seeds were harvested from the single ear. A single-eared F ₂ seed ear was sown, and the fertility was identified after heading. Among them, 125 pollen were normal iodine dyed and 38 were pollen-free, which was consistent with 3:1 separation (χ ² =0.30), indicating the sterility trait. Controlled by a single recessive gene.

Example 4 Leaf Sampling and DNA Extraction

In this study, CTAB method was used to extract corn leaf DNA. The specific method was as follows: weigh about 0.1g of leaves, put into a centrifuge tube, add 600μL CTAB extraction buffer, 5μL RNase A, shake and disperse, and circulate at 65°C for 0.5hr. 2-3 times; add equal volume of chloroform / Tris-saturated phenol (1:1, v / v), mix, gently shake for 10min; centrifuge at 10,000 rpm for 20min at 4 ° C; transfer the supernatant to a new tube, add 1/10 volume 3M sodium acetate (pH 5.2), 0.6-1 volume of cold isopropanol; lightly shake and mix until flocculation occurs; centrifuge at 10,000 rpm for 10 min at 4 ° C; discard the supernatant, use 70% by volume of ethanol The precipitate was washed twice; air-dried, 50 μL of 1×TE was added to dissolve the precipitate, and stored at -20 °C. The DNA concentration was measured with Nanodrop 2000 and diluted to 10 ng/L for use as a PCR template.

Example 5 Preliminary Positioning of Male Sterility Candidate Gene Chromosomes

The genetic map IBM2 2008 (www.maizegdb.org) and screened Indel SSR markers on each chromosome maize uniform distribution, selected polymorphic marker is present between the 2000 Beijing Branch waxy parent of Beijing Branch Waxy and M ₂ in 2000 Nine strains of zmabcg20-1 were genotyped. The PCR procedure consists of 1 μL of 10× reaction buffer, 0.25 μL of dNTP, 0.25 μL of forward primer and 0.25 μL of reverse primer, 0.5 U of Taq enzyme, 1 μL of 10 ng/μL of template DNA, and total volume of ultrapure water. Make up to 10μL. The PCR reaction procedure was: denaturation at 94-98 ° C for 1-3 min, and then the following cycles were performed: denaturation at 95 ° C for 20 s, renaturation at 53-58 ° C for 20 s, extension at 72 ° C for 30 s, 30-40 cycles.

The reaction product was electrophoretically separated on a 6% polyacrylamide gel. The polyacrylamide gel electrophoresis method is as follows: (1) Preparation of polyacrylamide gel: 6% PA gel 80 mL, 10% ammonium persulfate 250 μL (winter) / 125 μL (summer), tetramethylethylenediamine (TEMED) 80 μL . Shake well and mix. Wipe the glass plate repeatedly with detergent, wipe it with alcohol, and dry it. After applying the 2% Repel Silane to the gravure in a fume hood, wipe it with alcohol, dry it, and apply another plate to 0.5% Bingding Silane 1.5 mL (add 7.5 μL Binding Silane to a 1.5 ml centrifuge tube and 7.5 μL of glacial acetic acid to make up 95% ethanol to 1.5 mL). During the operation, the two glass plates are prevented from being polluted by each other, and after thorough drying, the glass plates are assembled and filled. (2) Pre-electrophoresis: After the gel is solidified, remove the comb and wash off the upper gel, especially pay attention to the seam to be cleaned. First, a 1×TBE electrode buffer was placed in the lower tank (cathode) of the electrophoresis tank, and the polymerized gel plate was placed in an electrophoresis tank, and 0.5×TBE electrode buffer was injected into the upper tank. Constant power 40W-65W, pre-electrophoresis for about 30min. Use a pipette to remove the urea and bubbles deposited on the rubber surface and insert the comb. (3) Electrophoresis: Add 5 μl of 5×Loading Buffer to the amplification product and mix at 95 °C for 5 minutes. Immediately transfer to ice for cooling, and add 1.5-3 μl to the sample well; constant power 40W-65W for electrophoresis to bromophenol Blue reaches the end of the bottom of the electrophoresis tank. The electrophoresis time was adjusted depending on the molecular weight of the SSR amplification product and the discriminability of the difference band type. (4) Silver dyeing color, a piece of glass plate with glue is placed in 10% glacial acetic acid fixing solution, shaking at 65r/min for about 30min until all the xylonitrile is decolorized; distilled water is washed twice for 5min each time; The washed rubber plate is placed in a freshly prepared dyeing solution (2 g of silver nitrate, 3 mL of 37% formaldehyde) in a solution of 65 g/min for 30 min; the dyed rubber plate is rinsed in distilled water for 5 s, and immediately taken out for development; The rubber plate was quickly transferred to a 4 ° C pre-cooled developing solution (30 g of sodium hydroxide in 2 L of water, 10 ml of 37% formaldehyde) and gently shaken until the band appeared; the rubber plate was placed in a 10% glacial acetic acid fixing solution until no bubbles Rinse twice with distilled water for 2 min each time; after drying at room temperature, photograph and save the image.

In the identification marker, the Indel marker IDP8150 located on chromosome 9 (forward primer: 5'-TGCTCGCAGGAATAGAAAGC-3'; reverse primer: 5'-GACGCAATCGACAGAGTACG-3'), the amplification band in Jingkejing 2000 is heterozygous The conjugated type, and 9 strains of zmabcg20-1 are all homozygous, indicating that the mutated gene controlling fertility is linked to IDP8150 and is located on chromosome 9. Analysis of the male fertility control genes of the cloned plants revealed that the maize Ms45 (GRMZM2G307906), the Arabidopsis AtABCG26 and the rice OsABCG15 ortholog ZmABCG20 (GRMZM2G076526/Zm00001d046537) were located on chromosome 9. We used these two genes as target genes for sequencing analysis.

Example 6 Candidate Gene Sequencing

The primers were designed according to the Ms45 and ZmABCG20 gene sequences of maize inbred line B73, and the genomic DNA of wild type Jingke 糯2000 and zmabcg20-1 were amplified. The amplified products were sequenced and spliced out the complete sequence. The primer pair for amplifying maize ZmABCG20 is ZmABCG20_1~3, and the primer pair for amplifying Ms45 is Ms45_1~Ms45_4; the sequence is shown in Table 1:

Table 1 Primer pair sequences for amplification of maize ZmABCG20 and Ms45

Primer pair name Forward primer (5'-3') Reverse primer (5'-3') ZmABCG20_1 CCATCTTTCTTGCTCTAGCCTT CAGGCGAAAACTCAAATTCCC ZmABCG20_2 GACAGGGTAGACGCCATCATC CGCATCTTGTCCAGGTAGTCG ZmABCG20_3 GGGCGCATAACAAGGACCG TGCGCCCCAATTTAATTAGGTG Ms45_1 GCCGAATTTGGATTGTCACG GCACAGCTTGCACTCGTAGCT Ms45_2 CTATTTCATGCGCAACCACCTC ACACCTGATTCAAAATTCGAGCTTAAG Ms45_3 CTAATGACATTATACCTCATGATTCGCA GGTCTTGGCTTGAAGTACAGTTGC Ms45_4 TCTTCTCTTACTGGGATCCTGACAAG AGTCTAGAGACCATGTAAGCCTATGA ZmABCG20_T1 CAACAACATTACAACAAAGTAAGCAT CACCGTCAGCTGTGGGAAG ZmABCG20_T2 AAAAGGAGGATCGGATTTGTGACTC CCGCATCTTGTCCAGGTAGTCG ZmABCG20_T3 CGCATCCGACTACCTGGACAA CTGGCACAGTTGTACGTGAGGT ZmABCG20_T4 TCATGCACTACGGCTTCAACC AGGAACGGCTAGCATAAACACTT

The PCR reaction system consisted of 1 μL of 10× reaction buffer, 0.25 μL of dNTP, 0.25 μL of forward primer and 0.25 μL of reverse primer, 0.5 U of Taq enzyme, and 1 μL of 10 ng/μL of template DNA, and the total volume was supplemented to 10 μL with ultrapure water. . The PCR reaction procedure was: denaturation at 94-98 ° C for 1-3 min, and then the following cycles were performed: denaturation at 95 ° C for 20 s, renaturation at 53-58 ° C for 20 s, extension at 72 ° C for 30 s, 30-40 cycles. After the end of the cycle, the extension was extended at 72 ° C for 3-10 min to terminate the reaction. A 1.5% agarose gel was placed and electrophoresed under an electric field of 5 V/cm for 30 min; the PCR product was recovered using a commercially available DNA gel recovery kit.

The PCR product DNA of the wild type and mutant obtained was sequenced using an ABI3730 sequencer, and the sequencing primers used a forward primer and a reverse primer, respectively. The bidirectional sequencing results were spliced using the common DNA sequence analysis software DNAman6.0. The analysis showed that the Ms45 gene sequence of the mutant zmabcg20-1 was identical to the wild type Jingke 糯2000, and no mutation occurred. The full-length nucleotide sequence of the ZmABCG20 gene of the mutant zmabcg20-1 is shown in SEQ ID NO: 7, and is deleted by 4 bases from the genus 京2000.

ZmABCG20 is an orthologous gene with OsABCG15 in rice and AtABCG26 in Arabidopsis, while the latter two mutant phenotypes also show male sterility, and the rice mutant osabcg15 also has no mature pollen.

Example 7 Tissue Specificity of ZmABCG20 Expression

The flower spikes of Jingkejing 2000 from different periods were selected, from V7 (forming of corn tassels) to V18 (maize of tassel ears) (How a Corn Plant Develops. Special Report No. 48. Iowa State University of Science and Technology, Cooperative Extension Servce, Ames, Iowa. Reprinted 2/1996), and roots, stems, leaves, male flowers, gems, and ears; liquid nitrogen transport, storage at -80 ° C; extraction reagent with TRIzol RNA The above tissue RNA was extracted from the cassette (Invitrogen, USA), and the RNA was reverse-transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Dalian) according to the instructions.

Quantitative PCR using ^{PowerUp TM SYBR TM Green Master Mix (} Thermo Fisher USA), amplification and detection of the fluorescence PikoReal 96 quantitative PCR instrument (Thermo Fisher, USA). The maize Actin1 gene was selected as the internal reference gene, the amplification primers were actinI-F and actinI-R (SEQ ID NO: 15-16), and the amplification primers for ZmABCG20 fluorescence quantification were ABCG-2F and ABCG-2R (SEQ ID NO). :17-18).

The real-time PCR reaction system was as follows: SYBR Green Mix 5 μL, Forward Primer 0.5 μL, Reverse Primer 0.5 μL, cDNA 1 μL, and ultrapure water 3 μL. The PCR reaction procedure was: denaturation at 95 ° C for 5 min; denaturation at 95 ° C for 15 s, annealing at 60 ° C for 1 min, cycle 40 times; 60 ° C for 30 s. The dissolution curve was started at a temperature of 60 ° C; the final temperature was 95 ° C; the holding time was 1 s; and the temperature was increased by 0.2 ° C.

The results of real-time quantitative PCR were shown in Figure 3. The ZmABCG20 gene was expressed only in the spikelets of V10-V15, and the expression was sharply increased in V12, only slightly expressed in other periods; in roots, stems, leaves, ears, The expression of ZmABCG20 was not detected in other tissues such as Nei Ying and Wai Ying. The V12 phase corresponds to the pollen mononuclear phase, and its outer wall is forming. This expression organization and period are consistent with the functions of Arabidopsis and rice homologous genes.

Example 8 Sequence Analysis of ZmABCG20 Transcript

In the Gramene database, the ZmABCG20 gene has two gene annotation numbers, GRMZM2G076526 and Zm00001d046537, for a total of 8 predicted transcripts. In order to determine the coding region of the gene, cDNA amplification obtained from the spikes of Kyosuke 2000 male flowers was amplified with primers covering the full length of the ZmABCG20 coding region, ZmABCG20_T1 to T4 (see Table 1 for the sequence), and the product was as in Example 6. The method was isolated and sequenced. Sequencing Results The ZmABCG20 coding region is set forth in SEQ ID NO: 5 and is identical to GRMZM2G076526-T001 (SEQ ID NO: 2).

Comparing the mutant genome, the Jingke 糯2000 genome and cDNA sequence, it was found that zmabcg20-1 is located at the 246th base of the coding region, ie, the 326th base of the start codon in the genomic sequence, and is located in the second explicit The deletion of the 4 base TGCA of the subunit results in a frameshift mutation after the 82nd amino acid residue in the translated protein, and the translation is terminated early after translation to the 100th amino acid residue. The coding region sequence of the mutant gene zmabcg20-1 is shown in SEQ ID NO: 8, and the encoded protein sequence is shown in SEQ ID NO: 9. The ZmABCG20 genomic sequence, coding region sequence and protein sequence of B73 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the ZmABCG20 genomic sequence, coding region sequence and protein sequence of Jingke 糯 2000 are shown in SEQ, respectively. ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The ZmABCG20 gene structure and the zmabcg20-1 mutation site are shown in Figure 4.

Example 9 Functional marker identification of F ₂ population ZmABCG20 genotype

A pair of gene-specific primers were designed according to the sequence flanking the mutation site obtained in Example 6: forward primer 3326_F1, nucleotide sequence shown as SEQ ID NO: 10; reverse primer 3326_R1, nucleotide sequence thereof As shown in SEQ ID NO:11.

If the size of the product amplified by the above primer pair is 79 bp, it indicates that the genotype of the plant to be tested is wild type; if the size of the amplified product is 75 bp, it indicates that the plant to be tested is a zmabcg20-1 mutant; The size of the amplified product was 79 bp and 75 bp, indicating that the ZmABCG20 gene of the plant to be tested was a heterozygous genotype of the wild type and zmabcg20-1 mutant.

Among the F ₂ strains obtained in Example 3, wild-type and mutant phenotype plants were randomly selected, and leaf DNA was extracted and amplified with the above primer pairs together with the Jingke 糯2000 genomic DNA. The PCR reaction system consisted of 1 μL of 10× reaction buffer, 0.25 μL of dNTP, 0.25 μL of forward primer and 0.25 μL of reverse primer, 0.5 U of Taq enzyme, and 1 μL of 10 ng/μL of template DNA, and the total volume was supplemented to 10 μL with ultrapure water. . The PCR reaction procedure was: denaturation at 94-98 ° C for 1-3 min, and then the following cycles were performed: denaturation at 95 ° C for 20 s, renaturation at 53-58 ° C for 20 s, extension at 72 ° C for 30 s, 30-40 cycles. The amplified product was separated by 6% polyacrylamide gel electrophoresis, and electrophoresed under a 40 W constant power electric field for 1 hr. After silver nitrate staining, the electropherogram was photographed.

The results are shown in Figure 5. The size of the amplified product of the wild type control is 79 bp; the size of the amplified product of the sterile line in the F ₂ spike line is 75 bp; the size of the amplified product of all fertile plants is 79 bp, or 79 bp + 75 bp. Banded type, but not homozygous 75bp band type. This result indicates that the mutation site described in Example 6 is co-segregating with the recessive nuclear male sterility gene.

Example 10 Construction of RNAi Vector of ZmABCG20

To verify the function of the ZmABCG20 gene, this example constructs an RNAi vector of the gene. The carrier construction process is shown in Figure 6, and the specific method is as follows:

1. The high specificity cDNA fragment SEQ ID NO: 23 in ZmABCG20 was selected as the RNAi target sequence. From the V12 phase cDNA obtained in Example 7, a forward fragment 17N19-1 of the RNAi stem-loop structure was amplified with primer pairs 17N19-F1 (SEQ ID NO: 21) and 17N19-R1 (SEQ ID NO: 22); The reverse fragment 17N19-2 of the RNAi stem-loop structure was amplified with primer pairs 17N19-F2 (SEQ ID NO: 19) and 17N19-R2 (SEQ ID NO: 20).

2. The intermediate vector was provided by pBSK-RTM (provided by Chengdu Biotech Co., Ltd., and the vector pBSK-RTM was engineered from plasmid pBSK. It contains the intron of the Arabidopsis RTM1 gene as shown in SEQ ID NO: 24. As shown in Figure 6, the left side of the intron is the SacI and NotI restriction sites, and the right side is the XbaI and BamHI restriction sites). The pBSK-RTM and forward fragments were digested with SacI and NotI, ligated into E. coli, and 8 transformants were picked for PCR verification. Two positive transformants were picked and plasmids were extracted and sequenced to obtain pBSK-17N19-1 vector. .

3. The 17N19 gene reverse fragment 17N19-2 was cloned using pBSK-17N19-1 as a template.

The pBSK-17N19-1 and the reverse fragment 17N19-2 were digested with XbaI and BamHI, ligated into E. coli, and 8 transformants were picked for PCR verification. A positive transformant was picked and the plasmid was extracted and sequenced, and confirmed to be the target vector pBSK-17N19R.

The pBSK-17N19R vector was digested with BamHI and SacI, and the target fragment containing the forward fragment + RTM+ inverted fragment was recovered. The pCambia3301ky plasmid was digested with BamHI and SacI (provided by Chengdu Tuo Biotechnology Co., Ltd., in pCambia3301 plasmid). A 35S promoter was inserted upstream of the multiple cloning site to obtain pCambia3301ky). The target fragment and pCambia3301ky were ligated to transform E. coli. A PCR-positive transformant was picked and extracted with BamHI and SacI. The large fragment of the positive fragment (forward fragment + RTM + reverse fragment) and the pCambia3301ky plasmid skeleton band were electrophoresed. The results showed that the target fragment was correctly ligated to pCambia3301ky. In the cloning vector, the vector pCambia3301-17N19R, the RNAi vector was constructed.

Example 11 Genetic Transformation and Phenotypic Identification of Transformed Plants

The composition of MS and N6 medium is as follows:

The medium used in the remaining steps is as follows:

YEB culture solution: 5.0 g/L yeast, 10.0 g/L peptone, 5.0 g/L NaCl, 50.0 mg/L kanamycin and 25.0 mg/L rifampicin, pH 6.8;

Infecting solution: 2,4-D l.0mg/L, L-valine 700mg/L, hydrolyzed casein 100mg/L, inositol 120mg/L, sucrose 68g/L, glucose 36g / L, acetosyringone 100 μmol / L, pH 5.2;

Co-culture medium: N6 basic medium, adding 1.38 g/L of proline, 500 mg/L of hydrolyzed casein, 120 mg/L of inositol, 2,4-D 2.0 mg/L, 0.7% of agar, 3% of sucrose, acetyl Syringone 100μmol/L, cysteine 200mg/L, AgNO ₃ 0.85mg/L, pH6.0;

Recovery medium: N6 basic medium, adding 1.38g/L of proline, 500mg/L of casein, 120mg/L of inositol, 2,4-D 2.0mg/L, 0.7% of agar, 3% of sucrose, AgNO ₃ 0.85mg / L, cephalosporin 400mg / L, pH 5.8;

The first round of screening medium: 2,4-D l.0mg / L, L-valine 700mg / L, hydrolyzed casein 100mg / L, mannitol 20g / L, inositol 120mg /L, agar 0.7%, sucrose 3%, cephalosporin 400 mg / L, AgNO ₃ 0.85 mg / L, bialaphos 0.3 mg / L, pH 5.8;

The second round of screening medium: based on the first round of screening medium, the concentration of bialaphos was increased to 0.6 mg / L;

Differentiation medium: 1 mg/L kinetin was added to the basic medium MS, 100 mg/L hydrolyzed casein, 200 mg/L cephalosporin, 0.7% agar, 3% sucrose, pH 5.8;

Rooting medium: 1/2 MS basic medium was added with 100 mg/L hydrolyzed casein, 700 mg/L L-valine, 0.2 mg/L IBA, 0.7% agar, 3% sucrose, pH 5.8.

Follow the steps below to genetically transform and regenerate plants:

1) Preparation of infecting materials: Take corn inbred line B104 self-pollination 10-13d corn ear, pick young embryos, soak for 15s with 75% alcohol, soak for 2 minutes with 2.5% sodium hypochlorite, 3-5 times with distilled water .

2) Dyeing young embryos: Single colonies of genetically engineered Agrobacterium were picked from the plate and inoculated in YEB medium, cultured at 28 ° C, 220 rpm for 20 h - 36 h; when the bacteria reached logarithmic growth phase, centrifuged at 4 ° C, 3000 rpm After 10 min, the cells were collected by centrifugation and resuspended to OD ≈ 0.5 with the infecting solution to be used for infection. Immerse the 150 young embryos with the dip solution for 5 min - 10 min, and gently blot the dip solution with filter paper.

3) Co-cultivation and recovery culture: The immature embryos that had been digested were transferred to a common medium, and cultured at 22 ° C for 3 days, transferred to a recovery medium, and cultured at 28 ° C for 7 days.

4) Screening: The callus obtained after the step 3) treatment was transferred to a screening medium at 28 ° C for dark culture, and two rounds were screened for 3 weeks each.

5) Differentiation and rooting: the selected resistant calli were transferred to the differentiation medium and cultured at 25 ° C for 7 days; then cultured at 25 ° C, 16 h light (light intensity 2000 lux) - 8 h dark alternating cycle, wait for seedling When the length is about 5 cm, it is transferred to the rooting medium and cultured at 28 ° C for 15 days.

6) Refining seedling transplanting: transplanting the seedlings after rooting cultivation into small pots filled with nutrient soil, cultivating for 10 days at 28 °C; transplanting the seedlings to the greenhouse (natural light, daily temperature 32 ° C, night temperature 28 Culture in °C).

The seedlings transplanted through the above steps were identified by primer PCR, and it was confirmed that 5 strains were transform-positive strains, and the ZmABCG20 RNAi fragment designed in Example 9 was carried.

When the transformed plants were flowered, the anthers of the control and positive transformed plants were taken, and the pollen fertility was identified by the iodine dyeing method in Example 3. As a result, as shown in Fig. 7, the wild type inbred line B104 pollen was normally fertile, and one transformed plant numbered R02 showed male sterility, no pollen, and was consistent with the zmabcg20-1 mutant phenotype. This result indicates that knocking out ZmABCG20 does cause male sterility.

In combination with the results of Examples 1-10, the zmabcg20-1 mutant phenotype and mutant gene were identical to the ZmABCG20 homologous gene mutants in Arabidopsis and rice; the ZmABCG20 gene was specifically expressed in the young male spikelets, while in other periods and None of the tissues were expressed; the sterile phenotype was co-segregated with the zmabcg20-1 mutant gene; knocking out the ZmABCG20 gene resulted in a male sterility phenotype consistent with zmabcg20-1. The above results demonstrate that ZmABCG20 is an essential gene for male fertility development in maize; its lack of function can lead to a male sterility phenotype of maize; the male sterility phenotype of the zmabcg20-1 mutant is produced by the ZmABCG20 gene described in Example 6. Caused by point mutations.

Example 12 ZmABCG20 promoter clone

Amplification of maize genomic DNA using primers pZmABCG20_F (sequence as shown in SEQ ID NO: 13) and pZmABCG20_R (sequence as shown in SEQ ID NO: 14) can obtain a DNA fragment of 1653 bp in size, with a 1634 bp upstream of the initiation codon ATG. (SEQ ID NO: 12). The sequence was analyzed using the online transcription component analysis tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and found to be CAAT-box and TATA-box at +1237 and +1385 respectively. In addition, there are some hormone response components: abscisic acid response element ABRE (GCAACGTGTC, +1236); jasmonic acid response elements TGACG_motif (TGACG, +655) and CGTCA_motif (CGTCA, +765); gibberellin response element GRAE_motif (TCTGTTG, +513; AAACAGA, +1397). In +973 there is a circadian rhythm control element CAANNNNATC. A rich transcriptional and regulatory element indicates that this region is the promoter region of ZmABCG20.

Example 13 Hybridization of Mutant Genes

The mutant obtained by the present invention and the functional marker of the mutant gene described in Example 9 can be used for various molecular marker-assisted selection methods, and backcrossing is taken as an example, and the sterile gene zmabcg20-1 can be obtained according to the procedure of FIG. Through hybridization to other corn genetic backgrounds:

1 hybrid:

Using the zmabcg20-1 mutant as the female parent, the F ₁ seed was obtained by crossing the recipient maize material with the male parent;

2 first round of back:

After sowing F ₁ F ₁ plants obtained, the F ₁ plants hybridized with the recurrent parent to obtain BC ₁ seeds;

3BC ₁ sterile gene selection (foreground selection):

Seeding BC ₁ seeds, obtaining not less than 500 seedlings, collecting individual leaves in the seedling stage, extracting DNA according to the method described in Example 4, and performing amplification and electrophoresis using the primer pair (3326_F1, 3326_R1) in Example 9. Select a single plant with a genotype that is heterozygous to continue planting, and discard the homozygous wild-type individual plants;

4BC ₁ background selection:

A set of (eg, 100, or 200, etc.) molecular markers that are polymorphic between the mutant zmabcg20-1 and the recurrent parent and uniformly distributed across the genome (including but not limited to SSR, INDEL, SNP, EST , RFLP, AFLP, RAPD, SCAR and other types of markers), identify the individual plants selected in step 3, select materials with high similarity with the recurrent parent (for example, greater than 88% similarity, or 2% selection rate, etc.);

5 second round of backcrossing: using the single plant selected in step 4 as the male parent, pollinating the recurrent parent to obtain BC ₂ seeds;

6BC ₂ foreground and background selection: Repeat step 3 to step 4 for the selected material, and select BC with similarity to the recurrent parent than the selection criteria (such as similarity greater than 98%, or 2% selection rate, etc.) ₂ generation plants;

7 self-obtaining BC ₂ F ₂ seed: self-crossing the BC ₂ plants selected in step 6 to obtain BC ₂ F ₂ seeds;

Prospect selection of 8BC ₂ F ₂ : seeding BC ₂ F ₂ seeds obtained in step 7 to obtain more than 500 seedlings, collecting leaves at the seedling stage, extracting DNA according to the method described in Example 4, using the primer pair of Example 9. (3326_F1, 3326_R1) performing amplification and electrophoresis, selecting a single plant with a homozygous mutant and a heterozygous type to continue cultivation, and eliminating the homozygous wild type individual plants;

Background selection and application of 9BC ₂ F ₂ : The selected plants in step 8 were screened according to the method of step 4, and 100% background homozygous individual plants were selected. If the genotype of the selected plant is a homozygous mutant, the single plant is our final target material, which can be further mixed with the recurrent parent to preserve the material or hybridize with other corn materials. If the selected single plant is a heterozygous belt type, it can be directly used to preserve the germplasm, or obtain a sterile plant by cross-breeding for cross breeding or seed production.

Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Industrial applicability

The invention provides the application of the maize gene ZmABCG20 in regulating male fertility of crops. The genomic DNA sequence of ZmABCG20 in maize variety B73 is shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 3. The present invention also provides a mutant zmabcg20-1 of the gene ZmABCG20 and the use thereof, and the mutated gene sequence is shown as SEQ ID NO: 7; and a method for identifying a molecular marker of the mutated gene is also provided. The pollen development control gene, mutant and molecular marker thereof provided by the invention can be applied to crop cross breeding and hybrid seed production. The invention also provides the application of the maize gene ZmABCG20 in regulating male fertility of crops. The genomic DNA sequence of ZmABCG20 in maize variety B73 is shown in SEQ ID NO: 1, and the encoded protein sequence is shown in SEQ ID NO: 3. The present invention also provides a mutant zmabcg20-1 of the gene ZmABCG20 and the use thereof, and the mutated gene sequence is shown as SEQ ID NO: 7; and a method for identifying a molecular marker of the mutated gene is also provided. The pollen development control gene, the mutant and the molecular marker thereof provided by the invention can be applied to crop cross breeding and hybrid seed production, and have good economic value and application prospect.

Claims (30)

The application of the maize gene ZmABCG20 in regulating male fertility of crops is characterized in that the cDNA sequence of the gene ZmABCG20 is: i) the nucleotide sequence shown in SEQ ID NO: 2; Ii) a nucleotide sequence in which the nucleotide sequence shown by SEQ ID NO: 2 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein; Iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 2 and which expresses the same functional protein, which is 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC containing 0.1% SDS. In solution, hybridize at 65 ° C and wash the membrane with the solution; or Iv) a nucleotide sequence having more than 85% homology to the nucleotide sequence of i), ii) or iii) and expressing the same functional protein. The use according to claim 1, characterized in that said regulation means that the crop has male fertility. The application according to claim 2, wherein the application comprises: 1) causing the crop to contain the ZmABCG20 gene; or 2) The crop is expressed with a protein encoded by the ZmABCG20 gene. Application of the maize gene ZmABCG20 in the preparation of transgenic plants, wherein the definition of the gene ZmABCG20 is as described in claim 1. The use according to claim 4, wherein the recombinant expression vector carrying the gene ZmABCG20 cDNA or genomic sequence is transferred into wild type maize callus, and the transformed material is subjected to co-culture-screening-differentiation-rooting- The transgenic plants were trained and transplanted, and the transgenic plants were screened, and then the transgenic corn was crossed with the male sterile maize to restore the fertility of the male sterile maize. The use of the maize gene ZmABCG20 for restoring the fertility of a male sterile plant, wherein the male sterility trait is caused by the genetic mutant. A method for preparing male sterility transgenic maize by inhibiting the activity of maize ZmABCG20 gene, characterized in that the ZmABCG20 gene in maize is transcribed, translated or translated by gene silencing, gene suppression, gene knockout or directed gene mutation techniques The level of protein activity is reduced in terms of obtaining genetically modified maize with male sterility. The method according to claim 7, wherein the RNAi sequence carrying the cDNA sequence for the gene ZmABCG20 is operably linked to a constitutive promoter or a floral organ-specific expression promoter, and transferred into a plant callus. The transformed material was subjected to co-culture-screening-differentiation-rooting-transgenic seedlings for exercise and transplanting, and male sterility transgenic maize was screened. The method according to claim 8, wherein the target DNA sequence to which the RNAi acts is as shown in SEQ ID NO:23. The application of the maize gene ZmABCG20 in crop improved breeding and seed production, wherein the definition of the gene ZmABCG20 is as described in claim 1. Application of the biological material obtained by the method according to any one of claims 7-9 in crop improved breeding and seed production. The use according to claim 10 or 11, wherein the plant in which the ZmABCG20 gene is contained or expressed, or the plant inactivated by the ZmABCG20 gene obtained by the method of claim 7 or 8 is excellent in the same species Crops of agronomic traits are crossed. The use according to claim 12, wherein the crop is a self-pollinated or cross-pollinated crop, including corn, wheat or rice, preferably corn; said superior agronomic traits include increased yield, improved quality, resistance to pests and diseases , resistance, lodging resistance. The mutant zmabcg20-1 gene of the maize gene ZmABCG20 is characterized in that its nucleic acid sequence is: i) a mutant gene formed by a 4 base TGCA deletion at bases 326-329 after the start codon of the maize gene ZmABCG2 nucleic acid sequence; Ii) the nucleotide sequence shown in SEQ ID NO:7; Iii) a nucleotide sequence in which the sequence shown in i) or ii) is substituted, deleted and/or added to one or more nucleotides and expresses the same functional protein, and comprises 4 bases at an equivalent position to the gene ZmABCG20 TGCA is missing; Iv) a nucleotide sequence which hybridizes under stringent conditions to the sequence shown in i) or ii) and which expresses the same functional protein, and which comprises a 4-base TGCA deletion at the equivalent position to the gene ZmABCG20; Hybridization at 65 ° C in 0.1 × SSPE containing 0.1% SDS or 0.1 × SSC solution containing 0.1% SDS, and washing the membrane with the solution; or v) A nucleotide sequence having more than 85% homology to the nucleotide sequence of i) or ii) and expressing the same functional protein, and comprising a 4 base TGCA deletion at an equivalent position to the gene ZmABCG20. A protein encoded by the mutant zmabcg20-1 gene of claim 14, wherein the amino acid sequence is as shown in SEQ ID NO: 9, or the sequence is substituted by substitution, deletion or addition of one or several amino acids. Functional amino acid sequence. The use of the mutant zmabcg20-1 gene of claim 14 for regulating maize fertility, characterized in that said application comprises: 1) causing the crop to contain the zmabcg20-1 gene; or 2) The crop is expressed by a protein encoded by the zmabcg20-1 gene. The use according to claim 16, wherein the maize comprising or expressing the mutant zmabcg20-1 gene exhibits recessive male sterility. Use of a biomaterial comprising or expressing the mutant zmabcg20-1 gene of claim 14 in the preparation of transgenic maize. The use of the mutant zmabcg20-1 gene of claim 14 for improved breeding and seed production of maize. The use according to claim 19, characterized in that the maize comprising or expressing the mutant zmabcg20-1 gene is crossed with corn having excellent agronomic traits. The use according to claim 20, wherein said excellent agronomic traits include increased yield, improved quality, resistance to pests and diseases, stress resistance, and lodging resistance. An oryx-specific plant panicle-specific promoter, characterized in that the promoter is: i) the nucleotide sequence shown in SEQ ID NO: 12; Ii) a nucleotide sequence in which the nucleotide sequence shown by SEQ ID NO: 12 is substituted, deleted and/or increased by one or more nucleotides and has the same function; Iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 12 and which has the same function, in stringent conditions of 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC solution containing 0.1% SDS Medium, hybridizing at 65 ° C, and washing the membrane with the solution; or Iv) a nucleotide sequence having more than 85% homology to the nucleotide sequence of i), ii) or iii) and having the same function. The use of a specific promoter according to claim 22 for regulating expression of a downstream gene, characterized in that a promoter sequence is operably linked to a gene of interest, and the resulting construct is used to transform a target plant, and the promoter drives the gene of interest specifically. It is expressed in the young ears of male flowers or hermaphroditic plants. A DNA molecular marker associated with male fertility in maize, characterized in that the DNA molecular marker is located at bases 326-329 after the start codon of the maize gene ZmABCG20 nucleic acid sequence, the sequence is TGCA, and the four bases are deleted. Maize lines exhibit recessive male nuclear sterility traits; Among them, the cDNA sequence of the gene ZmABCG20 is: i) the nucleotide sequence shown in SEQ ID NO: 2; Ii) a nucleotide sequence in which the nucleotide sequence shown by SEQ ID NO: 2 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein; Iii) a nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 2 and which expresses the same functional protein, which is 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC containing 0.1% SDS. In solution, hybridize at 65 ° C and wash the membrane with the solution; or Iv) a nucleotide sequence having more than 85% homology to the nucleotide sequence of i), ii) or iii) and expressing the same functional protein. A primer for amplifying the DNA molecule marker of claim 24, comprising: Upstream primer 3326_F1: 5'-CCAGACGAGGGCAGACCAG-3' and Downstream primer 3326_R1: 5'-GATCTCGCCAGGGTCCACA-3'. A detection reagent or kit comprising the primer of claim 25. Use of the DNA molecular marker of claim 24, the primer of claim 25, or the detection reagent or kit of claim 26 in maize molecular marker-assisted breeding. Use of the DNA molecular marker of claim 24, the primer of claim 25, or the detection reagent or kit of claim 26 for identifying or breeding a male sterile maize germplasm resource. The application according to claim 28, wherein the genomic DNA of the corn to be tested is extracted, and the PCR amplification reaction is carried out by using primers 3326_F1 and 3326_R1, and the amplification product is detected by electrophoresis, and if the amplification product has a characteristic strip having a size of 79 bp. With the belt, the fertility of the tested corn is normal, and the corresponding ZmABCG20 genotype is wild type; if the amplified product has a characteristic band of 75 bp, the tested maize is male sterile, and the corresponding ZmABCG20 genotype is Mutant; if the amplification product is of the 79 bp and 75 bp bands, the maize to be tested is a heterozygous genotype. Use of the DNA molecular marker of claim 24, the primer of claim 25, or the detection reagent or kit of claim 26 in the genotyping of the maize gene ZmABCG20.

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