CN116769796B - ZmENR1 and application of coded protein thereof in corn fertility control - Google Patents

Abstract

The invention discloses cornZmENR1The application of the gene and the coding protein thereof in corn fertility control. The invention relates to a male sterile mutant obtained by EMS mutagenesisenr1Identifying a maize male flower controlling plantZmENR1The gene has a nucleotide sequence shown as SEQ ID NO.1 and an amino acid sequence shown as SEQ ID NO.2. Furthermore, the CRISPR/Cas9 gene editing technology is used for carrying out site-directed mutation on the gene in wild corn, so that three allelic mutants with complete male flower abortion are created, and the method has important significance for corn fertility control and hybrid seed production. The invention is also directed toenr1Functional molecular markers are designed for the three allelic male sterile mutants, and the method has important application value in corn male sterile line cultivation, sterile hybridization seed production and molecular marker auxiliary selection.

Description

ZmENR1 and application of coded protein thereof in corn fertility control

Technical Field

The invention belongs to the field of plant biotechnology breeding, and relates to a male sterile mutant geneenr1A kind of electronic deviceZmENR1And the application of the coded protein thereof in corn fertility control.

Background

Corn (corn)Zea mays L.) Is an important source of feed and biomass fuel. Since the corn varieties used in current production are almost all hybrid varieties, the cultivation and production of excellent hybrid varieties are particularly important. In the conventional hybrid seed production process, a manual emasculation process is needed, so that a large amount of manpower, material resources and financial resources are consumed, mechanical damage is easily caused to plants, and false hybrids appear in the process of emasculation. The male sterile line of the corn can omit the link of manual emasculation, greatly reduce the seed production cost and improve the corn yield. Male sterile materials are important materials for researching maize pollen and anther development, crop heterosis utilization and hybrid seed production. Crossbreeding and seed production are carried out by taking male sterile line as female parent, so that the breeding speed of new variety is accelerated, and the seed production yield are improvedPurity, and seed production cost saving. The new stable corn male sterile material is continuously created and the new male sterile gene is identified, so that the method can promote the wide application of the corn male sterile breeding seed production in production, and finally the improvement of the corn yield and the cost saving and synergy are realized.

The factors responsible for male sterility of plants are diverse, and therefore there are different criteria for classifying male sterility of plants. According to the theory of three types, male sterility can be divided into three categories: the first is Cytoplasmic Male Sterility (CMS), where the genes controlling sterility are located in mitochondria in the cytoplasm; the second is nuclear male sterility (GMS), where the genes controlling sterility are located in the chromosomes of the nucleus; the third category is nuclear-cytoplasmic-interaction male sterility, the trait of which is commonly controlled by chromosomal and mitochondrial genes. Plant male sterility is classified into two types, namely, nuclear male sterility and nuclear-cytoplasmic-interaction male sterility according to the theory of type II, and researchers refer to nuclear-cytoplasmic-interaction male sterility as cytoplasmic male sterility in order to correspond to nuclear male sterility. CMS is controlled by mitochondrial genes and nuclear genes, and although the CMS is applied to corn hybrid production in a small area, the CMS sterility of corn cannot be effectively applied to production like rice CMS because of single sterile cytoplasm, outstanding susceptibility and other problems. The GMS is controlled by nuclear gene alone, which can overcome CMS defect, but it is difficult to mass reproduce homozygous sterile line by conventional breeding method. In recent years, with the progress of biotechnology, the problems of maintenance and propagation of a recessive nuclear male sterile line of corn can be effectively solved by a corn multi-control sterile technology and a plant universal dominant sterile technology which are created by combining genetic engineering and molecular design breeding. An important premise for realizing the application of the technology is to obtain a large number of GMS genes and corresponding male sterile materials which have definite functions and control the male development of corn.

The invention relates to a maize male sterile mutant obtained by EMS mutagenesisenr1As a material, a maize male flower growth control gene was identified by map cloningZmENR1Genes and 3 kinds of genes are obtained through CRISPR/Cas9 technologyenr1Allelic male sterile mutant for resolving molecular mechanism of maize male flower developmentProvides genetic material of great value and provides excellent gene and key germplasm resource for improving the utilization of maize heterosis.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provideZmENR1And the application of the coded protein in corn fertility control can be used for creating male sterile lines, can be applied to corn cross breeding and seed production, and can improve the utilization efficiency of corn hybrid vigor.

To achieve the above object, the present invention provides a maize male fertility geneZmENR1Sterile mutant genes thereofenr1Characterized in that the maize male fertility geneZmENR1The nucleotide sequence is SEQ ID NO.1, and the coded protein ZmENR1 sequence is SEQ ID NO.2; maize male sterility mutant geneenr1Characterized in that the mutation is from wild typeZmENR1The deletion of 2 bases (GG) at positions +84 and +85 of exon 1 of the gene results in premature termination of translation of ZmENR1 protein, with a protein length of only 29 amino acids, which results in a nucleotide sequence containing the mutationenr1Although the mutant can normally draw out the male, the mutant can not normally bloom, and the anther is obviously smaller and blushing and shrinking, and finally the mutant is in a complete male sterile phenotype; the mutant geneenr1The specific sequences of the full-length DNA and the amino acid of the DNA are shown as SEQ ID NO.3 and SEQ ID NO.4.

In another aspect, the present invention also providesZmENR1The application of the gene and ZmENR1 protein in cultivating male sterile plants.

As described in the above applicationZmENR1The gene is a DNA molecule shown as SEQ ID NO.1 in a sequence table, and the ZmENR1 protein is a protein consisting of an amino acid sequence shown as SEQ ID NO.2 in the sequence table. Such applications include, but are not limited to, the following: (1) By inhibiting or reducingZmENR1Expressing the gene to obtain a maize male sterile line; (2) The corn male sterile line is obtained by inhibiting the activity and/or content of ZmENR1 protein.

In another aspect, the present invention also provides a method of creating cornenr1A method of allelic male sterility mutant, characterized in that the method is CRIS-basedPR/Cas9 gene editing method for corn genomeZmENR1The gene is subjected to site-directed mutagenesis, so that fertility functions of the maize male sterile line are lost, and maize male sterile lines with different mutation types are obtained.

The CRISPR-Cas9 system comprises a gene for expressing Cas9 protein and a gene for sgRNA, wherein target sequences of the sgRNA are shown as SEQ ID NO.5 and SEQ ID NO.6 and are positioned in the following wayZmENR1The 1 st exon region of the gene.

Further the invention provides cornenr1Is characterized in that,enr1allelic mutants includeZmENR1-Cas9-1，ZmENR1-Cas9-2AndZmENR1-Cas9-3the method comprises the steps of carrying out a first treatment on the surface of the Wherein,ZmENR1-Cas9-113 bases GTGCAGATGGTGG deleted at exons 25 bp-37 bp and 1 base G deleted at 85 bp;ZmENR1- Cas9-26 bases ATGGTG at exons 31 bp-36 bp and 1 base G at 85 bp;ZmENR1-Cas9-33 bases (AGA) were deleted at exons 29 bp-31 bp and 1 base G was deleted at 85 bp.

In another aspect, the present invention also provides an obtaining ofenr1A method for producing a male sterile line, characterized in that the obtainedenr1The male sterile line is hybridized and backcrossed with the target material, so that the target material is obtainedenr1Male sterility traits and genetic mutations. Obtained byenr1The male sterile line can be applied to crossbreeding and seed production.

The invention also provides cornenr1The allelic male sterile mutant is applied to a molecular marker assisted selection method in maize sterile breeding and seed production; the method comprises the following 3 groups of functional markers:

(1) Male sterility mutant for cornZmENR1-Cas9-1The functional marker is characterized in that the sequences of the functional molecular marker primers ZmENR1-F1 and ZmENR1-R1 are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and the functional marker can simultaneously distinguish wild typeENR1Genes and mutationsenr1 -Cas9-1A gene;

(2) Male sterility mutant for cornZmENR1-Cas9-2Functional indicia of (c), characterized in thatThe sequence of the functional molecular marker primers ZmENR1-F2 and ZmENR1-R2 is shown as SEQ ID NO.9 and SEQ ID NO.10 respectively, and the functional marker can simultaneously distinguish wild typeENR1Genes and mutationsenr1 -Cas9-2A gene;

(3) Maize male sterile mutantZmENR1-Cas9-3The functional marker is characterized in that the sequences of the functional molecular marker primers ZmENR1-F3 and ZmENR1-R3 are respectively shown as SEQ ID NO.11 and SEQ ID NO.12, and the functional marker can simultaneously distinguish wild typeENR1Genes and mutationsenr1 -Cas9-3And (3) a gene.

The invention has the advantages and beneficial effects as follows:ZmENR1and the use of the encoded proteins in corn fertility control have not been previously reported. The invention adopts a map-based cloning method, firstly, the obtained maize male sterile mutant material is preparedenr1In the method, a novel gene for regulating and controlling the development of male flowers of corn is isolatedZmENR1Found thatZmENR1The mutation causes failure to bloom normally, and the anther is significantly smaller and blunted, eventually showing complete male sterility without pollen. The cloning of the gene provides resources and ways for artificially creating male sterile lines. The invention uses CRISPR/Cas9 gene editing method to mutate ZmENR1 protein coding gene in fixed point, which can be used for corn fertility control and hybrid seed production. Three obtained after editing for CRISPR/Cas9enr1The functional molecular marker developed by the allelic male sterile mutant can be applied to the identification of alleles, the screening of target single plants, the identification of seed purity and the like in maize sterile line breeding and seed production. Therefore, the invention has important significance in sterile breeding and seed production of corn, and can provide important biological resources for increasing the yield of corn.

Drawings

FIG. 1 is a maize Wild Type (WT) and mutantenr1Phenotype of (2)

A and B: WT and mutantenr1Is a tassel phenotype; c and D: WT and mutantenr1An anther phenotype of (a); e and F: WT and mutantenr1Anther I of (A) ₂ -KI staining phenotype.

Scale = 5cm (A, B); 2mm (C, D); 100 μm (E, F).

FIG. 2 is cornZmENR1Fine localization and map cloning of genes

A, sterilityenr1The separation ratio of polymorphic molecular markers of population DNA to fertile population DNA; b, F ₂ 48 male sterile plants and 48 male fertile plants in the population for maizeZmENR1Initial localization of Gene, preliminary toZmENR1The gene is positioned between the chromosome 4 SSR markers umc1926 and umc 1902; c, the step of setting the position of the base plate,ZmENR1the gene is finely positioned between SSR markers P2-2 and P2-4, about 111.45 kb interval; d, finely positioning 4 gene models predicted in the interval; e, carrying out expression profile analysis on 4 candidate genes in the interval through maize anther RNA-Seq data, wherein Zm00001d049975 has an expression peak in anthers in the S7-S9 period, and other genes have low expression quantity or have no obvious expression peak; f, wild type andenr1in mutantsZmENR1And (5) analyzing the gene structure and sequencing.

FIG. 3 is Wild Type (WT) andenr1in mutantsZm00001d049975Sequence alignment of genes

FIG. 4 is a diagram ofZmENR1Analysis of Gene expression Pattern in different stages of maize anther development

S5, spore forming cell stage; s6, microsporocyte stage; s7, meiosis starting period; s8a, meiosis I, binary phase; s8b, meiosis II, tetrad stage; s9, a single-core microspore period; s10, a microspore cavitation period; s11, the microspores are subjected to unequal mitosis for the first time, and the two-core microspores are subjected to period; s12, microspore second mitosis and trinuclear microspore stage.

FIG. 5 is a schematic view of a displaypCas9-ZmENR1Physical map of site-directed mutagenesis expression vector

pCas9-ZmENR1: from the left border to the right border of the T-DNA are herbicide resistance genes, respectivelyBarIs a gene expression cassette; nuclease encoding geneCas9Is a gene expression cassette;ZmENR1an expression cassette for gene target 2 (MT 2); expression cassette of target 1 (MT 1).

FIG. 6 is a wild typeZmENR1And (3) withZmENR1-Cas9Gene structure and DNA sequence analysis of sterile mutants

Wild typeZmENR1（WT- ZmENR1): full length 3970 bp of the gene, comprising 11 exons and 10 introns;enr1mutantZmENR1-Cas9-1: 13 bases (GTGCAGATGGTGG) deleted at exons 25 bp-37 bp and 1 base G deleted at 85 bp; mutantZmENR1-Cas9-2: deletion of 6 bases (ATGGTG) at exons 31 bp-36 bp and 1 base G at 85 bp; mutantZmENR1-Cas9-3:3 bases (AGA) and 1 base G at 85 bp are deleted at exons 129 bp-31 bp.

FIG. 7 is Wild Type (WT) andenr1tassel, anther and pollen grain phenotyping of three allelic homozygous mutants

The first row is corn WT andZmENR1-Cas9-1、ZmENR1-Cas9-2andZmENR1-Cas9-3phenotype comparison of homozygous mutant tassel; the second row is WT andZmENR1-Cas9-1、ZmENR1-Cas9-2andZmENR1-Cas9-3phenotype comparison of homozygous mutant anthers; the third row is WT andZmENR1-Cas9-1、ZmENR1-Cas9-2andZmENR1-Cas9-3homozygous mutant pollen grain I ₂ KI staining comparison.

FIG. 8 is a schematic representation of the use of co-segregating tag pairsZmENR1-Cas9-1F of sterile mutant ₂ Genotyping of the plants of the generation

Co-segregation marker ZmENR1-F1/R1 pair 13 strainsZmENR1-Cas9-1Sterile mutant F ₂ PCR and polyacrylamide gel electrophoresis (PAGE) identification of the generation plants: amplifying 136 bp band in homozygous wild type (AA) plants; at the position ofENR1/ enr1-Cas9-1Two bands 136 bp and 123 bp are amplified in heterozygous (Aa) plants; at the position ofenr1-Cas9-1/enr1- Cas9-1The 123 bp band was amplified in homozygous mutant (aa) plants.

FIG. 9 is a schematic representation of the use of co-segregating tag pairsZmENR1-Cas9-2F of sterile mutant ₂ Genotyping of the plants of the generation

Co-segregation markers ZmENR1-F2/R2 pair 11 strainsZmENR1-Cas9-2Sterile mutant F ₂ PCR and polyacrylamide gel electrophoresis (PAGE) identification of the generation plants: amplifying a 135 bp band in a homozygous wild type (AA) plant; at the position ofENR1/ enr1-Cas9-2Two bands, 135 bp and 129 bp, were amplified in heterozygous (Aa) plants; at the position ofenr1-Cas9-2/enr1- Cas9-2A129 bp band was amplified in homozygous mutant (aa) plants.

FIG. 10 shows the use of co-segregating tag pairsZmENR1-Cas9-3F of sterile mutant ₂ Genotyping of the plants of the generation

Co-segregation markers ZmENR1-F3/R3 vs 10 strainsZmENR1-Cas9-3Sterile mutant F ₂ PCR and polyacrylamide gel electrophoresis (PAGE) identification of the generation plants: amplifying a 135 bp band in a homozygous wild type (AA) plant; at the position ofENR1/ enr1-Cas9-3Two bands 135 bp and 132 bp are amplified in heterozygous (Aa) plants; at the position ofenr1-Cas9-3/enr1- Cas9-3A132 bp band was amplified in homozygous mutant (aa) plants.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. Unless otherwise specified, the synthesis and sequencing of the primers and genes used in the examples were performed by the company Shanghai, inc. of Biotechnology. Other biochemical reagents are not particularly noted as being conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Embodiment one: maize male sterile mutantenr1Is obtained by (a)

A completely male sterile mutant designated as Zheng 58 mutant was obtained by screening the library of maize inbred line Zheng 58 mutants induced by ethyl methylsulfonate (Ethyl Methane Sulfonate, EMS) in the laboratoryenr1. The male sterile character of the mutant can be stably inherited in Beijing and Hainan three-pass hybridization with wild multiple generations, and is not influenced by environment. The mutant and the wild type plant form in the whole growth cycle are observed and compared under the conditions of Beijing university of science and technology test fields, hainan three and the like, no obvious difference is found, and only the male sterility character is shown.

Embodiment two: plant phenotype identification and pollen fertility observation

enr1The mutant has no difference compared with the wild type in nutrition growth and female ear development; in the aspect of tassel development, wild type can normally perform tassel, anthers can normally crack and scatter powder, and can normally set after selfingenr1Although the mutant can normally draw out the male, the mutant can not bloom normally, the anther glume is not cracked, the anther is obviously smaller, and the white and shrunken anther is not exposed (figure 1); further performing I on wild type and mutant pollen ₂ KI staining, found that wild pollen developed normally, pollen grains were black after staining, but mutants were not pollen grains formed (figure 1).

Embodiment III:ZmENR1localization, cloning and mutation site analysis of genes

Inbred line B73 as male parent and mutantenr1Hybridization, F ₁ Normal fertility, F ₂ The generation shows fertility segregation, as shown in Table 1, F ₂ The separation of the normal fertile strain (F) and the sterile strain (S) in the population accords with the separation ratio of single gene 3:1, namely the male sterile phenotype of the mutant shows obvious single-gene recessive inheritance.

Table 1 cornenr1Genetic analysis of mutation sports isolation

F 2 Group of people

Total number of plants

Number of fertile plants

Number of sterile plants

F/S ratio

X 2 Value of

Significance test, P>0.05

enr1×B73

1276

934

342

2.73:1

2.21(X 2 0.05 =3.84)

ns*

Selectingenr1×B73 F ₂ 48 male sterile plants and 48 male fertile plants in the population for cornZmENR1Initial localization study of genes. The SSR markers used are shown in Table 2, and the gene mapping is shown in FIG. 2.

Table 2 forZmENR1Gene-localized SSR markers

Primer name	Primer sequence (5 '-3')
		umc1926-F	ATGCCAGCATTCTTCATCCTACAT
umc1926-R	TGAGGCTTGGTCCACTAAAGAAAG
		umc123-F	CACAAAGAGCAGCCCACTTT
umc123-R	AAGTTGCTGACATCGATCCA
		P1-1-F	AGAGAGAAGAAAGCGGGTGC
P1-1-R	ACCTGTCGGCGCTGCCGCTTC
		P1-2-F	AGCAGCAGGCAGGCAGATGG
P1-2-R	AACTGCTGCTGCTGCTGAGG
		P1-3-F	TTCGACGCGCTCTGAGCTCG
P1-3-R	CAATGGGGAAGGCAACCGAG
		P2-1-F	TATCGTCGGCGCGCAACTCCTC
P2-1-R	ACGGTACGTAGCGATGCCGAC
		P2-2-F	AAGGCTCCCACTCCAAGTTC
P2-2-R	CTTTCCGACACGTCGGATTG
		P2-3-F	TTGACTTGACTTATATGGTG
P2-3-R	TTAATGCGGTTAGTGAGTGC
		P2-4-F	TAAGCAATGATAACACGGCTAG
P2-4-R	AGCCCATTGAGAATGGTAATC
		P2-5-F	ACGTGTTCGACGCGCTCTGAG
P2-5-R	AATGGGGAAGGCAACCGAGC

The results show that the method has the advantages of,ZmENR1gene was initially mapped between chromosome 4 SSR markers umc1926 and umc1902 (FIG. 2B), further pinpointing to the region of about 111.45 kb between markers P2-2 and P2-4 (FIG. 2C), where 4 candidate genes were predicted (FIG. 2D); expression profile analysis of genes using anther RNA-Seq data from different stages of maize development showedZm00001d049975There was an expression peak at the S7-S9 stage of anther development, and other genes were expressed with little or no expression peak (fig. 2E). For a pair ofZmENR1（Zm00001d049975) Cloning and sequencing analysis of the mutant gene, foundenr1The mutant lacks 2 bases (GG) at the positions ranging from +84 to +85 (fig. 2F and 3), so that the amino acid frame shift mutation is caused, and the protein translation is terminated in advance (fig. 2F).ZmENR1The gene contains 3970 nucleotides, including 11 exons, encoding an acyl carrier protein reductase (Enoyl-acyl-carrier-protein reductase) and is therefore designated by the present inventionZmENR1。

Implementation of the embodimentsExample four:ZmENR1space-time expression analysis of (2)

In order to study the relation between the gene and the male reproductive development of corn, the invention firstly utilizes qPCR to analyze the expression mode of the gene at different stages of the anther development of corn. The method comprises the following specific steps:

1. sampling and developmental stage identification of maize anthers

Anther samples with different lengths are collected from tassels of the maize inbred line B73 at different development stages according to the lengths of the anthers; each sample was collected with 20 fresh anthers of similar length, 3 of which were immobilized in FAA solution (Coolaber, china) and the specific developmental stage was determined by resin semi-thin slice experiments, the remaining 17 anthers were immediately frozen in liquid nitrogen for RNA extraction.

The immobilized anthers used for resin sections were dehydrated with gradient ethanol (50%, 70%, 90%, 100%) for 15-30 minutes per step. The anther can be stored in 70% ethanol for a long time during dehydration; to facilitate later embedding, 0.1% eosin can be added into 90% ethanol to dye the material; in order to ensure thorough dehydration, the material must be dehydrated 2-3 times in absolute ethanol. Then resin replacement is carried out, anthers are sequentially placed in liquid with the volume ratio of ethanol to Spurr resin of 3:1, 1:1 and 1:3 for 2-4 hours, and finally placed in pure resin overnight. After the resin replacement was completed, the anther was placed in a mold, 200 μl of sprr resin was added, and the mixture was placed in an oven and polymerized overnight at 70 ℃. Then trimming, and then slicing by using a German lycra slicer, wherein the slicing thickness is 2 mu m; the cut pieces were grasped with forceps and placed in sterile water in the center of the slide and the pieces were spread overnight at 42 ℃. Immersing the glass slide fixed with the sample into 0.1% toluidine blue dye solution, dyeing for 1 min, washing with deionized water, placing on a slide spreading table, and drying for microscopic observation; can also be stored for a long time after sealing. The results of the resin sections were analyzed to determine the specific developmental Stage of each sample based on the cytological characteristics of the maize 14 different developmental stages (Stage 1-Stage14: S1-S14).

2. qPCR analysis

Extraction of the above with Trizol reagent (Invitrogen, USA)Identifying total RNA of the maize anther at different developmental stages (S5-S12); cDNA was then synthesized using 5 Xall-in-One RT Master Mix (ABM, canada); quantitative reverse transcription polymerase chain reaction detection was performed on a Quantum studio5 Real-Time PCR System (ABI, USA) using TB Green ™ Premix Ex Taq ™ (TaKaRa, japan), the amplification primers were: qENR1-F (5'-CAATCCAGCCAACCTTACC-3') and qENR1-R (5'-CCATAGACAGCAGGAACCAA-3');ZmUbiqutinas a reference gene, the amplification primers are: ubiqutin-F (5'-CGACAACGTGAAGGCGAAGA-3') and Ubiqutin-R (5'-ACGCAGATACCCAGGTACAGC-3'); each developmental stage included three biological replicates, with three technical replicates for each sample; data 2 ^-ΔΔCt The method was analyzed and quantitative results were given as mean ± standard deviation (Means ± SD).

ZmENR1The gene exhibits a pattern of anther development period specific expression: there was lower expression in early stages of maize anther development, such as S5 and S6, followed by an increase from S7, the highest expression level in S9, followed by a decrease from S10 (fig. 4).

Fifth embodiment:ZmENR1functional verification of genes and creation of maize male sterile mutants by CRISPR/Cas9 method

To clarify the cornZmENR1Function in maize, the invention adopts CRISPR/Cas9 gene editing method to mutateZm00001d049975Gene sequence, knock out the function of the gene in corn. The invention selects maize hybrid Hi II as a receptor material for gene editing. The sequences shown as SEQ ID NO.5 and SEQ ID NO.6 of the gene conservation region are respectively selected as target regions for CRISPR/Cas9 gene editing.

1、ZmENR1Construction of CRISPR/Cas9 Gene editing vector

The gene editing carrier of the invention ispBUE411-MT1T2-Cas9The basic carrier of the carrier ispBUE411- Cas9The intermediate carrier ispCBCmT1T2Providing the gRNA. The invention designs target spots on the primers, obtains MT-sgRNA by PCR and connects the MT-sgRNA to a basic vector by enzyme digestion, and the specific construction process is as followsThe following steps:

(1) Design of target gRNA. Will beZmENR1（Zm00001d049975) Is input into http:// CRISPR. Hzau. Edu. Cn/cgi-bin/CRISPR2/CRISPR for target design. The DNA sequences of the two target areas selected by the invention are shown as SEQ ID NO.5 and SEQ ID NO. 6. The sgRNA framework sequences of the invention are derived from intermediate vectorspCBCmT1T2And directly amplifying to obtain the target.

(2) MT-sgRNA was obtained by designing targets on the primers and then PCR amplification. Primer ZmENR1-MT1-F and primer ZmENR1-MT2-R amplification intermediate vectorpCBCmT1T2Fragments for obtaining sgrnas comprising the first and second targets were 965 bp in product length. The PCR system and conditions were as follows: template DNA (intermediate vector)pCBCmT1T2Not less than 30 ng/. Mu.L) 1.2. Mu.L; primer F/R: 1.2. Mu.L each; sterilizing ddH ₂ O:11.4 Mu L;2 XMCLAB enzyme (product number: I5 HM-200): 15. mu L. The temperature program of PCR was as follows: (1) 98 ℃ for 2 minutes; (2) 98 ℃ for 10 seconds; (3) 58 ℃ for 30 seconds; (4) 30 seconds at 72 ℃; (5) cycling 34 times from (2) - (4); (6) 72 ℃ for 5 minutes; (7) 25℃for 10 minutes. Finally, the PCR product is recovered. The primer sequences required for vector construction are as follows:

ZmENR1-MT1-F: 5’-ATATATGGTCTCTGGCGAGCTGCTGGTGTGCAGATGGGTTTTAGAGCTAGAAATAGCAA-3’

ZmENR1-MT2-R: 5’-ATTATTGGTCTCTAAACCACGGGTTTCTAGGACTGATGCTTCTTGGTGCCGC-3’

(3) Constructed to backbone vectors by enzyme cleavage ligation. Will bepBUE411-Cas9Vector and method for recovering sgRNA fragment with targetBsaIDigestion, while adding T4 ligase, ligates the vector and sgRNA fragments. 10. mu.L of the cleavage ligation system is as follows, sgRNA fragments: 1. mu L, pBUE411-Cas9 vector (. Gtoreq.60 ng/. Mu.L): 1. mu L,10 XNEB Buffer: 1. the concentration of the solution in mu.L,BsaIendoenzymes (product number: #R3733S): 0.5 mu.L, T4 ligase (product number: #M0202M): 0.25 Mu L, sterilized ddH ₂ O：6.25 μL。

FIG. 5 shows the target geneZmENR1（Zm00001d049975) The marker genes Cas9 and bar and the backbone vector (corresponding to the first and second targets)pBUE411-Cas9Constructed expression vectorpCas9-ZmENR1。

2. Agrobacterium-mediated maize genetic transformation

By constructing the abovepCas9-ZmENR1Transferring the vectors into agrobacterium EHA105 by a heat shock method, and performing PCR identification; the bacterial solution was then stored by mixing 1:1 volumes of Agrobacterium and glycerol at-80 ℃. Taking young embryo of freshly stripped corn hybrid Hi II of about 1.8 and mm as a receptor material, placing the stripped corn embryo into 2 mL plastic centrifuge tubes containing 1.8 and mL suspensions, and placing for no more than 1 hour, wherein about 100 young embryos are placed into each centrifuge tube; the suspension was aspirated and the young embryos were rinsed 2 times with fresh suspension, the bottom of the tube remained a small amount of suspension that could have passed through the young embryos, then heat shock was applied for 2 minutes at 43 ℃ followed by an additional ice bath for 1 minute, the bottom residual wash was aspirated with a pipette, and 1.0 mL of agrobacteria infested solution was added, gently shaken for 30 seconds, and then allowed to stand in the dark for 8 minutes. Pouring the young embryo and the infection liquid in the centrifuge tube into a co-culture medium, shaking uniformly, sucking out excessive infection liquid by using a pipetting gun, and co-culturing in darkness at 23 ℃ for 3 days with scutellum of all young embryos facing upwards. After the co-cultivation is finished, the young embryo is transferred to a recovery culture medium by sterile forceps, and is cultivated for 7-14 days at 28 ℃, and the young embryo growing on the young embryo needs to be removed in time in the middle process. After the recovery culture, the young embryo is placed on 1.5 mg/L biamap screening medium for screening and culturing for 3 rounds, each round of screening and culturing for 2 weeks, and then 2 rounds of screening and culturing for 2 weeks on 2 mg/L biamap screening medium are carried out. The resistant calli were transferred to expansion medium and dark cultured for 2 weeks at 28 ℃. The propagated resistant calli were then transferred to induction medium and incubated for 2 weeks at 28℃in the dark. Then transferred to a differentiation medium, cultured at 25℃under light for 2 weeks at 5000 lx. After the cultivation is finished, single seedlings are separated from the differentiated seedling clusters and placed in a rooting medium, and the temperature is 25 ℃, the temperature is 5000 and lx, and the seedlings are subjected to illumination cultivation until rooting; transferring the young seedling into a small nutrition pot for growth, transplanting the young seedling into a greenhouse after the young seedling survives growth, and harvesting offspring seeds after 3-4 months.

3、T ₀ CRISPR/Cas9 mutation result detection of generation plants

To determine T ₀ Plant generation CRISPR/Cas9 processAs a result, the following steps are taken:

the invention firstly adopts a CTAB method to extract corn leaf DNA, and the specific method is as follows: shearing seedling leaves with the length of about 2 cm, and placing the seedling leaves into a 2 mL centrifuge tube provided with steel balls; immersing a centrifugal tube with blades in liquid nitrogen for 5 minutes, and then crushing blade samples by using a grinder; adding 500 μl of CTAB extraction buffer (containing 1% beta-mercaptoethanol) into the centrifuge tube, shaking with force, mixing, preheating in 65deg.C constant temperature water bath for 20-30 min (taking out and reversing for 1-2 times, and paying attention to the corresponding number of experimental sample); after the tube cooled to room temperature, 500. Mu.L of chloroform was added: isoamyl alcohol (24:1) extract, shaking vigorously for 30s, and standing at room temperature for a moment; centrifuging at 12000 rpm for 5 min at 4deg.C, and collecting 500 μl supernatant in a new 1.5 mL centrifuge tube; adding an equal volume of isopropanol into a centrifuge tube containing supernatant, gently shaking and uniformly mixing, and standing for about 10 min at room temperature; then placing the centrifuge tube with the sample into a centrifuge at the temperature of 4 ℃, centrifuging for 10 min at the speed of 12000 rpm, gently sucking the supernatant, discarding the supernatant, and reserving the sediment; adding 800 μL of 75% ethanol, washing the precipitate twice, centrifuging at 10000 rpm for 5 min, and discarding the supernatant; naturally drying the sample at room temperature for 2-4 hours to obtain DNA precipitate, adding a proper amount of sterile water for dissolving, slightly shaking, and fully dissolving DNA. The DNA samples were stored at-20 ℃. The DNA concentration was measured using Nanodrop and diluted to 10 ng/L and used as a PCR template.

Then according toZmENR1（Zm00001d049975) The gene sequence was designed into PCR primers.

Detecting a target: MT1 and MT2; product size: 275 bp; the primer sequences were as follows:

ZmENR1-T-F1: 5’- AATGTTCTTGTAGCCGTAGTGA-3’；

ZmENR1-T-R1: 5’- TGCTCTTACAACAACATCATTGCG-3’。

genomic DNA was extracted and amplified according to the following PCR parameters:

the reaction system: 15. mu.L MIX conventional PCR system, 0.5. Mu.L forward primer, 0.5. Mu.L reverse primer, 1. Mu.L DNA, 5.5. Mu.L sterilized ddH ₂ O, 7.5. Mu.L of 2x taq mix (product number: 10103 ES).

The reaction procedure: conventional PCR: 58. annealing at the temperature, extending for 30s and 32 cycles.

The PCR product is then recovered and ligated to T vector sequencing by sequencing multiple T ₀ The DNA sequence of the target area of the generation independent positive transformation event is determined whether the target area is subjected to gene editing or not, and finally 3T are found ₀ The sequence of the target region of the transformation event is changed and is homozygously mutated, the sequences before and after editing are shown in figure 6, and the sequences correspond to 3enr1Allelic homozygous mutant:ZmENR1-Cas9-1、ZmENR1-Cas9-2andZmENR1-Cas9-3. An alignment with the wild-type sequence shows that,ZmENR1-Cas9- 1、ZmENR1-Cas9-2andZmENR1-Cas9-3deletion mutations occurred at both targets 1 and 2.

For 3enr1Comparison of amino acid sequences in allelic mutants revealed that the mutated lines were compared to unedited WTZmENR1-Cas9-1、ZmENR1-Cas9-2AndZmENR1-Cas9-3deletion of the nucleotide encoding it at target 1 or 2 causes a frame shift mutation of its amino acid and premature termination of the subsequent amino acid. Thus, the Zm00001d049975 protein functions of these transformants were deleted.

4、F ₁ Genotyping of generation plants

Due to maize T grown in the greenhouse ₀ The generation of plants often has uncoordinated female and male spike development and also affects fertility when the edited gene is related to male development, thus in order to reproduce T ₀ The present invention uses the wild pollen of the maize inbred line Zheng 58 as the plant obtained above, and inherits the obtained gene editing typeZmENR1-Cas9-1、ZmENR1- Cas9-2AndZmENR1-Cas9-3t of (2) ₀ Pollinating the plants of the generation to obtain F ₁ Seed generation, the grown plant is F ₁ And (5) replacing plants.

F ₁ The plants of the generation comprise 2 isolated types, one isCas9Positive plants (transgenic plants), the other beingCas9Negative plants (non-transgenic plants) in order to avoid sgrnasAnd Cas9 continuous editing of the hybrid pollinated Zheng 58 wild type allele, thereby creating complexity of mutation type, we need to genotype from F ₁ Selecting plants of the generation not containingCas9Genes but containing T ₀ Plants of the mutant type, which, after selfing, give rise to F which is not transgenic ₂ And (3) replacing. F (F) ₁ The genotyping steps of the generation plants are as follows:

after extracting leaf DNA according to the CTAB method described above, first, use is made ofCas9Specific primers for the genes Cas9-F (5 ' -CCCGGACAATAGCGATGT-3) and Cas9-R (5'-GAGTGGGCCGACGTAGTA-3') were PCR amplified. The PCR reaction system is the same as that described above; the reaction procedure: conventional PCR: annealing at 58 deg.c, extending for 1 min, and 32 cycles. After agarose gel electrophoresis of the PCR products, the PCR products are distinguished according to the resultCas9-positive plantsCas9-negative plants.

Further aim atCas9-negative plants, PCR amplified using primers ZmENR1-T-F1 and ZmENR1-T-R1 described above for detection of MT1 and MT2 targets; after the PCR product is purified, connecting a T vector, and sequencing; determination of T from sequencing result analysis ₀ Genetic status of the generation mutation type.

Example six: cornZmENR1-Cas9Phenotypic analysis of male sterile mutants

The above example six identified noCas9F of Gene ₁ F is obtained after the selfing of the generation plants ₂ Seed generation, three mutation types%ZmENR1-Cas9-1、ZmENR1-Cas9-2AndZmENR1-Cas9-3) 1 selfing spike is taken for spike sowing, and phenotype investigation is carried out in the mature period. Three F ₂ In the strain, the ratio of the fertile strain to the sterile strain accords with 3:1 separation, further indicates thatZmENR1-Cas9The sterility of sterile mutant is controlled by single recessive gene and then directed against F ₂ Stable non-transgene obtained by generationZmENR1-Cas9Sterile mutants were subjected to detailed observations of tassel, anther and pollen viability with wild type.

In terms of vegetative growth and the development of the female ear,ZmENR1-Cas9-1、ZmENR1-Cas9-2andZmENR1-Cas9-3plants of sterile mutantsSubstantially no differences compared to the wild type; in the aspect of tassel development, wild type plants can normally perform tassel, anthers can normally crack and scatter powder, and can normally set after selfing, and three plants can normally setZmENR1-Cas9The sterile mutant can normally draw out the male, but cannot normally bloom, the anther glume is not cracked, the anther is obviously smaller, and the sterile mutant is whitish and shrunken and is not exposed (figure 7); further performing I on wild type and mutant pollen ₂ KI staining, found that wild pollen developed normally, pollen grains were black after staining, but mutants were not pollen grains formed (fig. 7). This indicatesZmENR1（Zm00001d049975) Gene control of maize male development, created by gene editing methodsZmENR1-Cas9The sterile mutant is a pollen-free sterile line and has the characteristic of complete abortion.

Embodiment seven:ZmENR1-Cas9co-separation functional molecular marker development and application for sterile body identification

1. Development of co-segregating molecular markers

In the present invention, the three obtained are aimed atZmENR1-Cas9The mutation site of the sterile mutant is subjected to Primer design by using Primer 5.0 software to develop three pairs of co-separation functional molecular markers: the ZmENR1-F1/R1, zmENR1-F2/R2 and ZmENR1-F3/R3 are combined with PCR and polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis detection methods, and the genotype of the mutant can be separated according to the obtained bands and sizes.

The co-separation molecular marker ZmENR1-F1/R1 comprises a first primer ZmENR1-F1 and a second primer ZmENR1-R1; the marker can specifically detect cornZmENR1-Cas9-1Mutant and mutant gene in maize sterile material transformed by sameenr1-Cas9-1And can simultaneously distinguish wild typeENR1Genes and mutationsenr1-Cas9-1A gene; against mutant genesenr1-Cas9-1The band of 123 bp was amplified in the middle, but for the wild typeENR1The gene amplified 136 bp band. The primer sequences were as follows:

ZmENR1-F1：5’- CTGGATAGGTTAGTTGGCATTGA -3’

ZmENR1-R1：5’- GAGCATGCAATCAGTCCTAGA -3’

co-separation molecular markersZmENR1-F2/R2 comprises a first primer ZmENR1-F2 and a second primer ZmENR1-R2, the tag being capable of specifically detecting maizeZmENR1-Cas9-2Mutant and mutant gene in maize sterile material transformed by sameenr1-Cas9-2And can simultaneously distinguish wild typeENR1Genes and mutationsenr1-Cas9-2A gene; against mutant genesenr1-Cas9-2The 129 bp band was amplified for wild typeENR1The gene amplified a band of 135 bp. The primer sequences were as follows:

ZmENR1-F2：5’- TGGATAGGTTAGTTGGCATTGA -3’

ZmENR1-R2：5’- GAGCATGCAATCAGTCCTAG -3’

co-separation molecular markers ZmENR1-F3/R3 comprising a first primer ZmENR1-F3 and a second primer ZmENR1-R3, the markers being capable of specifically detecting maizeZmENR1-Cas9-3Mutant and mutant gene in maize sterile material transformed by sameenr1-Cas9-3And can simultaneously distinguish wild typeENR1Genes and mutationsenr1-Cas9-3A gene; against mutant genesenr1-Cas9-3The 132 bp band was amplified for wild typeENR1The gene amplified a band of 135 bp. The primer sequences were as follows:

ZmENR1-F3：5’- TGGATAGGTTAGTTGGCATTG -3’

ZmENR1-R3：5’- GAGCATGCAATCAGTCCTA -3’

2. application of co-separation molecular marker

To verify the validity of the above-mentioned mark, F obtained in example six ₂ The strain is the material, and is carried outENR1Detection of alleles. The DNA extraction method, PCR amplification system and conditions are the same as in example two, and the PCR products are separated by PAGE or agarose gel electrophoresis.

In theory, zmENR1-F1/R1, zmENR1-F2/R2 and ZmENR1-F3/R3 areENR1/ ENR1Bands 136 bp, 135 bp and 135, respectively, can be amplified in homozygous wild type (AA) DNAenr1/ enr1Bands of 123 bp, 129 bp and 132 bp were amplified in homozygous mutant material (aa) DNA, respectivelyENR1/enr1In the hybrid (Aa) material, two corresponding bands can be amplified simultaneously. ZmENR1-F1/R1, zmENR1-F2/R2 and ZmENR1-F3/R3 minutesThe verification results of the sub-markers are shown in FIG. 8, FIG. 9 and FIG. 10, and the results show that the designed 3 functional molecular marker pairs F ₂ The detection result of the plant completely meets the expectation, inENR1/ ENR1Homozygous wild type (AA),ENR1/ enr1Hybrid (Aa) andenr1/ enr1the homozygous mutant material (aa) can be used as bands with corresponding sizes amplified respectivelyENR1，enr1Ideal markers for allele detection.

The molecular markers are favorable for determining the mutation genotype before flowering and pollination, so that hybridization and backcross breeding of male sterile lines can be carried out under different genetic backgrounds, and the molecular markers have important application value.

Claims (7)

1. Maize male sterile mutant geneenr1Characterized in that the maize male sterility mutant geneenr1Is prepared from male fertility gene of cornZmENR1Mutation acquisition，The method comprisesZmENR1The nucleotide sequence of the gene is SEQ ID NO.1, and the coding protein sequence is SEQ ID NO.2; the saidenr1The mutation site of the gene is located atZmENR1Exon 1 of the gene lacks 2 bases GG at positions +84 and +85; the mutant geneenr1The full-length DNA sequence of (2) is SEQ ID NO.3, and the coded amino acid sequence is SEQ ID NO.4.

2. A method for creating a maize male sterile line is characterized in that a gene editing method based on CRISPR/Cas9 is utilized to inhibit a maize male fertility geneZmENR1Selecting a maize male sterile plant; the saidZmENR1The nucleotide sequence of the gene is SEQ ID NO.1, and the coding protein sequence is SEQ ID NO.2.

3. The method of claim 2, wherein the CRISPR-Cas9 gene editing system comprises a gene expressing Cas9 protein and a gene for sgrnas whose target sequences are shown in SEQ ID No.5 and SEQ ID No.6, both locatedZmENR1The 1 st exon region of the gene.

4. The method of creating a maize male sterile line according to claim 2, wherein the maize male sterile line comprises ZmENR1-Cas9-1，ZmENR1-Cas9-2AndZmENR1-Cas9-3three maize allelic male sterility mutants; wherein,ZmENR1-Cas9-113 bases GTGCAGATGGTGG deleted at exons 25 bp-37 bp and 1 base G deleted at 85 bp;ZmENR1-Cas9-26 bases ATGGTG at exons 31 bp-36 bp and 1 base G at 85 bp;ZmENR1-Cas9-33 bases AGA were deleted at exons 29 bp-31 bp and 1 base G was deleted at 85 bp.

5. Corn obtainedenr1A method of male sterile line, characterized in that it is obtained by the method of claim 2enr1The male sterile line is hybridized and backcrossed with the target material, so that the target material is obtainedenr1Male sterility traits and genetic mutations.

6. Obtained by the method of claim 5enr1The application of the maize male sterile line in cross breeding and seed production.

7. The use according to claim 6, wherein the mutant allele is directed against maizeenr1Design of 3 sets of functional markers for application toenr1Molecular marker assisted selection in male sterile line crossbreeding and seed production: (1) Male sterility mutant for cornZmENR1-Cas9-1The sequences of the functional marker primers ZmENR1-F1 and ZmENR1-R1 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8; (2) Male sterility mutant for cornZmENR1-Cas9-2The sequences of the functional marker primers ZmENR1-F2 and ZmENR1-R2 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10; (3) Maize male sterile mutantZmENR1-Cas9-3The sequences of the functional marker primers ZmENR1-F3 and ZmENR1-R3 are shown as SEQ ID NO.11 and SEQ ID NO.12 respectively.