CN112680459A - Male sterile gene ZmTGA10 and application thereof in creating male sterile line of corn - Google Patents

Abstract

The present invention discloses a male sterility geneZmTGA10The gene has a nucleotide sequence shown in SEQ ID NO.1, and the protein coded by the gene has an amino acid sequence shown in SEQ ID NO. 2. The gene is subjected to site-specific mutation in wild corn by a CRISPR/Cas9 gene editing technology, althoughHowever, pollen can be normally formed, but anthers are not cracked at all, and the discovery shows thatZmTGA10The gene has the function of regulating and controlling the male reproductive development of the corn. Through progeny screening, mutants without transgenic components can be obtained, a stable sterile line is created, and the method has important significance for researching the development of maize anthers. The invention is also directed to the obtainedtga10The male sterile mutant designs a functional molecular marker, and has important application value in the cultivation of a corn male sterile line, sterile hybrid seed production and molecular marker-assisted selection.

Description

Male sterility geneZmTGA10And application thereof in creating male sterile line of corn

Technical Field

The invention belongs to the field of plant biotechnology breeding, and particularly relates to a male sterility geneZmTGA10And the application thereof in creating a male sterile line of the corn.

Background

Corn is the first large grain crop in China, the perennial sowing area is more than 5.5 hundred million acres, and the healthy development of the corn seed industry has great strategic significance for guaranteeing national grain safety. Meanwhile, the corn breeding industry is also the breeding industry field with the largest market value, the highest commercialization degree and the highest technological content in the world and is a strategic place for global breeding competition. Compared with the international leading level, the corn planting industry in China still has a huge gap in aspects such as scientific and technological innovation, industrial modes and the like. Firstly, the method is limited by the factors of fundamental breakthrough and the like of basic research of corn male sterility, so that the intellectual property protection of a corn inbred line is difficult, the follow-up and imitation breeding phenomena exist in the corn seed industry of China for a long time in recent years, and the breeding efficiency of a great new variety is slow. Secondly, the corn seed production industry is still in a labor intensive stage mainly relying on manual castration, the cost is high, the resource consumption is huge, and the seed quality is difficult to guarantee.

Corn is one of the most successful crops for heterosis utilization, a male sterile line is an important material for heterosis utilization and hybrid seed production of crops, and the two conditions are included according to the characteristics of stamen development: firstly, normal pollen cannot be produced, and secondly, pollen fertility is normal but anther dehiscence is abnormal and pollen cannot be scattered in time. Depending on the gene classification controlled, male sterility mainly includes Cytoplasmic Male Sterility (CMS) and nuclear male sterility (GMS). CMS is controlled by both mitochondrial gene and nuclear gene, and has been applied to maize breeding and hybrid production, but has the problems of low resource utilization rate, single cytoplasm of sterile line, susceptibility to diseases and the like. GMS is controlled by nuclear genes independently, so that the defect of CMS can be overcome, but the homozygous sterile line is difficult to propagate in a large amount by a conventional breeding method. In recent years, with the progress of biotechnology, the problems of maintenance and propagation of a maize recessive nucleus male sterile line can be effectively solved by a maize multi-control sterile technology created by combining genetic engineering and molecular design breeding and a plant universal dominant sterile technology. The important premise for realizing the application of the technology is to obtain a large amount of GMS genes with definite functions for controlling the male development of the corn and corresponding male sterile materials.

Compared with the model plants Arabidopsis thaliana and rice, the cloned and identified GMS gene and the created male sterile material in maize are relatively few. The CRISPR/Cas9 (structured, Short palindromic repeats-associated Endonuclease 9) gene editing technology has the characteristics of low cost, simplicity in operation, high mutation induction rate and the like, is increasingly and widely applied to the aspects of plant gene function research, crop genetic improvement, breeding and the like, and has a very wide application prospect. The CRISPR/Cas9 technology is used for mining and identifying the candidate gene of the corn male sterility and creating a male sterile material, and can quickly enrich the resources of the corn GMS gene and the sterile material, thereby promoting the popularization and the application of the corn sterile breeding and seed production, and finally effectively solving the bottleneck problems of the lack of stable corn sterile lines and breakthrough large varieties in the corn seed industry in China for a long time.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a male sterility geneZmTGA10And the application thereof in creating the male sterile line of the corn, can be used for creating the male sterile line of the corn, and is further applied to corn crossbreeding and seed production.

To achieve the above object, the present invention providesZmTGA10The application of the gene in controlling the male reproductive development of the corn is characterized in that the amino acid sequence of the gene is shown as SEQ ID NO. 2. It is generally expected that these homologous genes from different plants or different maize material will have the same or similar function, and thus the agronomic traits of the plants may be modified using these genes as well. Further, even if the function of these genes cannot be predicted, one of ordinary skill in the art can determine whether they have the function of controlling male fertility of plants based on the methods provided by the present invention and the prior art.

In another aspect, the invention also providesZmTGA10The application of the gene in controlling the male reproductive development of the corn is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

In another aspect, the invention also provides a method of creating a male sterile line in maize, characterized by inhibiting the growth of the male sterile line in maizeZmTGA10Expression and/or activity of the gene, selecting a male sterile plant of maize.

In some embodiments, the above-described method of inhibiting gene expression and/or activity comprises any one of gene editing, RNA interference, T-DNA insertion.

In some embodiments, the above gene editing employs the CRISPR/Cas9 method.

In some embodiments, the CRISPR/Cas9 method comprises: designing CRISPR/Cas9 vector targets at a fourth exon, an 8 th intron and a ninth exon of the gene, wherein the DNA sequence of the target is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

In another aspect, the present invention also provides a method of obtainingtga10Method for male sterile line, male sterile line obtained by the methodtga10Hybridizing and backcrossing the male sterile line with the target material to obtain the target materialtga10Traits of male sterility and genetic mutations.

The invention also includes a process obtained by any of the above processestga10The application of the male sterile line in cross breeding and seed production. The application in cross breeding and seed production refers to the steps oftga10Hybridizing the male sterile line as female parent with other male parents, or obtainingtga10Hybridizing and backcrossing the male sterile line with other target materials to obtain the target materialstga10Traits of male sterility and genetic mutations.

Furthermore, the invention also provides two corn male sterile linestga10The primer ZmTGA10-F1 and ZmTGA10-R1 are respectively shown in SEQ ID NO.7 and SEQ ID NO. 8; the sequences of the primers ZmTGA10-F2 and ZmTGA10-R2 are shown as SEQ ID NO.9 and SEQ ID NO.10 respectively.

The invention has the following advantages and beneficial effects:ZmTGA10（Zm00001d020938) The regulation of male reproductive development in maize by genes and encoded proteins has not been previously reported. The invention utilizes CRISPR/Cas9 method to mutate corn geneTGA10（Zm00001d020938) To discoverZmTGA10（Zm00001d020938) The gene has the function of regulating and controlling the development of the maize anther. The method for editing CRISPR/Cas9 gene and the male sterile mutant obtained after editing can be used for creating a corn male sterile line, thereby being applied to corn cross breeding and seed production. For three kindstga10The male sterile line of the method develops the coseparation molecular marker, and can be used for the fertility allele identification of plants, the screening of target single plants in molecular marker assisted breeding, the seed purity identification and the like.

Drawings

FIG. 1 is a drawing ofZmTGA10Analysis of expression pattern of gene in anther of corn in different development stages

S5, sporogenic cell stage; s6, microsporocyte stage; s7, meiosis onset period; s8a, meiosis I, dyad period; s8b, meiosis II, tetrad period; s8b-9, tetrad-monocyte microspore period; s9, the monocytic microspore stage; s9-10, during the vacuolation period of the monocytic microspores and microspores; s10, microspore vacuolization period; s11, the first non-uniform mitosis of microspore, the second nucleus microspore period; s12, microspore second mitosis, trilaryotic microspore stage.

FIG. 2 ispCas9-ZmTGA10Physical map of site-directed mutagenesis expression vector

pCas9-ZmTGA10-A: from the left border to the right border of the T-DNA are the herbicide resistance genes, respectivelyBarThe expression cassette of (1); nuclease encoding geneCas9The expression cassette of (1);ZmTGA10an expression cassette for gene target 2 (MT 2); expression cassette of target 1 (MT 1).pCas9-ZmTGA10-B: from the left border to the right border of the T-DNA are the herbicide resistance genes, respectivelyBarThe expression cassette of (1); nuclease encoding geneCas9The expression cassette of (1);ZmTGA10an expression cassette for gene target 4 (MT 4); expression cassette of target 3 (MT 3).

FIG. 3 shows the wild typeZmTGA10Gene structure and DNA sequence analysis of sterile mutant

Wild typeZmTGA10（WT- ZmTGA10): the gene has the full length of 9447 bp, and comprises 10 exons and 9 introns;TGA10mutantsZmTGA10-Cas9-1: 167 bases are deleted at the 8 th intron and the 9 th exon 4368 bp-4534 bp; mutantsZmTGA10-Cas9-2: 162 bases are deleted between 8 th intron and 9 th exon 4373 bp-4534 bp; mutantsZmTGA10-Cas9-3:1 base (C) at exon 4bp and 1 base (C) at 3246 bp.

FIG. 4 shows the wild type andtga10analysis of tassel, anther and pollen grain phenotype of homozygous mutants

At the upper row of Wild Type (WT) andZmTGA10-Cas9-1、ZmTGA10-Cas9-2、ZmTGA10-Cas9-3comparing the phenotypes of the mutant tassels; the second row is WT andZmTGA10-Cas9-1、ZmTGA10-Cas9-2、ZmTGA10-Cas9-3phenotype comparison of mutant anthers; is arranged at the lower row asWT andZmTGA10-Cas9-1、ZmTGA10-Cas9-2、ZmTGA10-Cas9-3i of mutant pollen grains₂KI staining comparison.

FIG. 5 shows wild type andtga10scanning Electron Microscope (SEM) analysis of anthers of homozygous mutants

From left to right are: wild Type (WT) anther whole;tga10integrating anthers; WT after peeling off andtga10(lower) anthers; mature pollen grains of WT (top) andtga10pollen grains (bottom) could not be scanned; WT (upper) andtga10(lower) the outer cuticle layer of anthers; WT (upper) andtga10(below) the inner epidermis of the anther, Usnea.

FIG. 6 shows the use of co-separation mark pairsZmTGA10-Cas9-1F of sterile line₂Genotyping plants

Coseparation marker ZmTGA10-F1/R1 for 8 strainsZmTGA10-Cas9-1Sterile line F₂And (3) identifying the result of PCR and agarose gel electrophoresis of the generation plant: a 445bp strip is amplified in a homozygous wild type (AA) plant; in thatTGA10/ tga10A 445bp band and a 278 bp band are amplified in a heterozygous (Aa) plant; in thattga10/ tga10278 bp bands are amplified in homozygous mutant (aa) plants.

FIG. 7 shows the use of co-separation marker pairsZmTGA10-Cas9-2F of sterile line₂Genotyping plants

Coseparation marker ZmTGA10-F1/R1 for 6 strainsZmTGA10-Cas9-2Sterile line F₂And (3) identifying the result of PCR and agarose gel electrophoresis of the generation plant: a 445bp strip is amplified in a homozygous wild type (AA) plant; in thatTGA10/ tga10A 445bp band and a 283 bp band are amplified in a heterozygous (Aa) plant; in thattga10/ tga10283 bp bands are amplified in homozygous mutant type (aa) plants.

FIG. 8 shows the use of co-separation mark pairsZmTGA10-Cas9-3F of sterile line₂Genotyping plants

Coseparation marker ZmTGA10-F2/R2 for 5 strainsZmTGA10-Cas9-3Sterile line F₂The PCR and polyacrylamide gel electrophoresis (PAGE) electrophoresis identification results of the generation plants are as follows: in homozygous wild type (AA) plantsAmplifying a 74bp band; in thatTGA10/ tga10Two bands of 74bp and 75 bp are amplified in a heterozygous (Aa) plant; in thattga10/ tga1075 bp bands are amplified in homozygous mutant (aa) plants.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the synthesis and sequencing of the primers and genes used in the examples were carried out by Biotechnology engineering (Shanghai) Ltd. Other biochemical reagents are not specifically noted but are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Example one cornTGA10（Zm00001d020938) Gene sequence and expression Pattern analysis

In the mailedDB library (https:// www.maizegdb.org /), maize was queriedTGA10（Zm00001d020938，GRMZM2G006578) The nucleic acid sequence of the gene in B73 is shown in SEQ ID NO.1, and the functional annotation of the gene isbZIP7A transcription factor (bZIP-transcription factor 7,bZIP7)the coded protein comprises 486 amino acids, and the sequence is shown as SEQ ID NO. 2.

bZIP transcription factors are involved in the regulation of numerous physiological processes in plants due toZmTGA10The actual functions of the gene in the corn have no published research data, and in order to research the relationship between the gene and the male reproductive development of the corn, the invention firstly analyzes the expression modes of the gene at different stages of the development of the corn anther by utilizing qRT-PCR. The method comprises the following specific steps:

1. sampling and development period identification of corn anther

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

The fixed anthers for the resin sections were dehydrated with gradient ethanol (50%, 70%, 90%, 100%) for 15-30 minutes each step. The anther can be stored in 70% ethanol for a long time in the dehydration process; in order to facilitate later embedding, 0.1% of eosin can be added into 90% ethanol to dye the material; to ensure complete dehydration, the material must be dehydrated in absolute ethanol for 2-3 times. And then performing resin replacement, namely sequentially placing the anthers in liquids with the volume ratio of ethanol to Spurr resin of 3:1, 1:1 and 1:3 for 2-4 hours, and finally placing the anthers in pure resin overnight. After the resin replacement was completed, the anthers were placed in a mold, 200 μ L of Spurr resin was added, 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 picked up with tweezers and placed in sterile water in the center of the slide and spread overnight at 42 ℃. Immersing the glass slide fixed with the sample into 0.1% toluidine blue dye solution, dyeing for 1 minute, then washing with deionized water, then placing on a slide-developing table, and drying to be used for microscopic observation; or sealing and storing for a long time. The resin sections were analyzed and the specific developmental Stage of each sample was determined based on the cytological characteristics of 14 different developmental stages of maize (Stage 1-Stage 14: S1-S14).

2. qRT-PCR analysis

Extracting the above-identified total RNA of maize anthers at different developmental stages (S5-S12) with Trizol reagent (Invitrogen, USA); cDNA was then synthesized using 5X All-in-One RT Master Mix (ABM, Canada); quantitative reverse transcription polymerase chain reaction detection is carried out on a QuantStaudio 5 Real-Time PCR System (ABI, USA) by adopting a TB Green (TB Green) PreMix Ex Taq (TaKaRa, Japan) and amplification primers are as follows: qTGA10-F (5'-TCCGCCTCTTGTTCCTTGTC-3') and qTGA10-R (5'-GACCTAGGCTGCTTGGATCC-3');ZmActin1as a reference gene, the amplification primers are as follows: actin1-F (5'-AAATGACGCAGATTATGTTTGA-3') and Actin1-R (5'-GCTCGTAGTGAGGGAGTACC-3'); each developmental stage included three biological replicates, with three technical replicates per sample; for data2^-ΔΔCtThe method was analyzed and the quantitative results are given as mean ± standard deviation (Means ± SD).

ZmTGA10The gene presents a pattern of specific expression during anther development: there was higher expression early in maize anther development, such as during S5, followed by a decline and then little expression to the mid-late stage of anther development (S9) (fig. 1).

Example two cornTGA10（Zm00001d020938) Gene function and method for creating maize male sterile line by using CRISPR/Cas9 method

To clarify cornTGA10（Zm00001d020938) The function in corn, the invention adopts CRISPR/Cas9 gene editing method to mutateZm00001d020938Gene sequence, knocking out the function of the gene in corn. The invention selects the maize hybrid Hi II as the receptor material for gene editing. The invention respectively selects the sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 of the gene conservation region as the target region for CRISPR/Cas9 gene editing.

1、ZmTGA10Construction of CRISPR/Cas9 Gene editing vector

The gene editing vector of the present invention ispBUE411-MT1T2-Cas9The basic vector of the vector ispBUE411- Cas9The intermediate carrier ispCBCmT1T2A gRNA is provided. The invention designs a target spot on a primer, obtains MT-sgRNA through PCR and then connects the MT-sgRNA to a basic vector through enzyme digestion, and the specific construction process is as follows:

(1) design of target gRNA. Will be provided withZmTGA10（Zm00001d020938) The gene sequence of (a) is inputted with http:// criprp. hzau.edu.cn/cgi-bin/CRISPR2/CRISPR for target design. The DNA sequences of the four target regions selected by the invention are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6. sgRNA framework sequences of the invention from intermediate vectorspCBCmT1T2Obtained by direct amplification.

(2) MT-sgRNA was obtained by designing the target on the primers and then PCR-amplifying. Intermediate vector for amplification of primer ZmTGA10-MT1-F and primer ZmTGA10-MT2-RpCBCmT1T2For obtaining a target comprising a first and a second targetThe same procedure used for amplification of target sgRNA fragments using primers ZmTGA10-MT3-F and ZmTGA10-MT4-R yielded sgRNA fragments containing the third and fourth targets, each 891 bp in length. The PCR system and conditions were as follows: template DNA (intermediate vector)pCBCmT1T2Not less than 30 ng/muL) 1.2 muL; primer F/R: 1.2. mu.L each; sterilization ddH₂O: 11.4 mu L; 2X MCLAB enzyme (product No.: I5 HM-200): 15 μ L. The temperature program for PCR was as follows: firstly, the temperature is 98 ℃ for 2 minutes; ② 10 seconds at 98 ℃; ③ 30 seconds at 58 ℃; fourthly, 30 seconds at 72 ℃; fifthly, circulating for 34 times from the second to the fourth; sixthly, 5 minutes at 72 ℃; seventhly, 25 ℃ for 10 minutes. And finally, recovering the PCR product. The primer sequences required for vector construction are as follows:

ZmTGA10-MT1-F:5’-ATATATGGTCTCTGGCGAGTCCTGTCCCCGGCGCCGGGTTTTAGAGCTAGAAATAGCAA-3’

ZmTGA10-MT2-R:5’-ATTATTGGTCTCTAAACCTGCTGAGCCAGCAGCACCTGCTTCTTGGTGCCGC-3’

ZmTGA10-MT3-F:5’-ATATATGGTCTCTGGCGATACGTCGGCCATATATACGGTTTTAGAGCTAGAAATAGCAA-3’

ZmTGA10-MT4-R:5’-ATTATTGGTCTCTAAACCCATCTCGTCGTGGTGAGCTGCTTCTTGGTGCCGC-3’

(3) constructing the skeleton vector by enzyme digestion connection. Will be provided withpBUE411-Cas9Vectors and recovered sgRNA fragments with targetsBsaIDigestion was performed while T4 ligase was added to ligate the vector and sgRNA fragment. The sgRNA fragment was prepared as follows using 15 μ L of the enzyme ligation system: 2 μ L, pBUE411-Cas9 vector (. gtoreq.60 ng/. mu.L): 2 μ L, 10 × NEB Buffer: 1.5 mu L of the suspension liquid,BsaIendonuclease (product No. # R3733S): 1 μ L, T4 ligase (product code: # M0202M): 1 μ L, sterilized ddH₂O：6 μL。

FIG. 2 shows the genes of interestZmTGA10（Zm00001d020938) The double targets (corresponding to the first target and the second target), the marker geneCas9Andbarwith a skeletal carrierpBUE411-Cas9Constructed expression vectorpCas9-ZmTGA10-A(ii) a Final expression vectors corresponding to the third and fourth targetspCas9-ZmTGA10-BExcept for the difference in target sequence, the other sequences arepCas9-ZmTGA10-AThe same is true.

2. Agrobacterium-mediated genetic transformation of maize

Constructed as described abovepCas9-ZmTGA10-AAndpCas9-ZmTGA10-Bthe vectors are respectively transferred into agrobacterium tumefaciens EHA105 by a heat shock method, and PCR is carried out for identification; then, agrobacterium containing two knockout vectors respectively is mixed at a concentration of 1:1, and after mixing, glycerol is added to preserve the bacterial liquid at-80 ℃. Taking freshly stripped young embryos of the corn hybrid Hi II with the size of about 1.5 mm as a receptor material, putting the stripped young embryos into 2 mL plastic centrifuge tubes containing 1.8 mL of suspension, and placing for no more than 1 hour, wherein about 100 young embryos are placed into each centrifuge tube; the suspension was aspirated off and the embryos were washed 2 times with fresh suspension, leaving a small amount of suspension on the bottom of the tube that could sink the embryos, then heat shocked at 43 ℃ for 2 minutes, then iced again for 1 minute, the remaining wash on the bottom of the tube was aspirated up with a pipette gun and 1.0 mL of Agrobacterium-infected solution was added, shaken gently for 30 seconds, and then left to stand in the dark for 8 minutes. And then pouring the young embryos and the infection liquid in the centrifugal tube onto a co-culture medium, uniformly shaking, sucking out the redundant infection liquid by using a liquid transfer gun, enabling the scutellum of all the young embryos to face upwards, and carrying out dark co-culture at 23 ℃ for 3 days. After the co-culture is finished, transferring the young embryo to a recovery culture medium by using sterile forceps, culturing for 7-14 days at 28 ℃, and taking care to remove young buds growing on the young embryo in time in the middle process. After the recovery culture is finished, the immature embryos are placed on a screening culture medium containing 1.5 mg/L of Bialaphos for screening and culturing for 3 rounds, each round of screening is carried out for 2 weeks, then the immature embryos are transferred to a screening culture medium containing 2 mg/L of Bialaphos for screening and culturing for 2 rounds, and each round of screening is carried out for 2 weeks. Transferring the resistant callus to a propagation culture medium, and culturing at 28 ℃ in the dark for 2 weeks. The expanded resistant callus was then transferred to induction medium and cultured in dark at 28 ℃ for 2 weeks. Then transferred to a differentiation medium, and cultured at 25 ℃ and 5000 lx under light for 2 weeks. After the culture is finished, separating a single seedling from the differentiated seedling cluster, placing the single seedling in a rooting culture medium, and carrying out illumination culture at 25 ℃ and 5000 lx until the single seedling is rooted; transferring the plantlets into a small nutrition pot for growth, transplanting the plantlets into a greenhouse after the plantlets grow alive, and harvesting progeny seeds after 3-4 months.

3、T₀Mutation result detection of CRISPR/Cas9 of generation plant

To determine T₀The CRISPR/Cas9 mutation result of the generation plants is obtained by the following stepsCarrying out the following steps:

the invention firstly adopts a CTAB method to extract the DNA of the corn leaf, and the specific method is as follows: shearing seedling leaves with the length of about 2 centimeters, and putting the seedling leaves into a 2 mL centrifuge tube filled with steel balls; immersing the centrifugal tube with the blades in liquid nitrogen for 5 minutes, and then breaking the blade samples by using a grinder; adding 700 μ L CTAB extraction buffer solution (containing 1% of beta-mercaptoethanol) into a centrifuge tube, shaking with force, mixing, preheating in a constant temperature water bath at 65 deg.C for 20-30 min (taking out, reversing for 1-2 times, and paying attention to the corresponding number of the experimental sample); the centrifuge tube was cooled to room temperature and 700 μ L chloroform was added: shaking the isoamyl alcohol (24: 1) extract for 30s, and standing at room temperature for a while; centrifuging at 12000 rpm for 5 min at 4 deg.C, and placing 500 μ l of supernatant in a new 1.5 mL centrifuge tube; adding isopropanol with the same volume into a centrifuge tube containing the supernatant, gently shaking and uniformly mixing, and standing for about 10 min at room temperature; then placing the centrifuge tube containing the sample into a 4 ℃ centrifuge, centrifuging for 10 min at 12000 rpm, gently sucking the supernatant, removing the supernatant, and keeping the precipitate; adding 800 μ L75% ethanol, washing the precipitate twice, centrifuging at 10000 rpm for 5 min, and discarding the supernatant; and (3) naturally drying the sample at room temperature for 2-4 hours to obtain DNA precipitate, adding a proper amount of sterile water to dissolve, and slightly shaking to fully dissolve the DNA. DNA samples were stored at-20 ℃. The DNA concentration was measured by Nanodrop and diluted to 10 ng/L for use as a PCR template.

Then according toZmTGA10（Zm00001d020938) The gene sequence was used to design PCR primers.

(1) Detecting a target: MT1 and MT 2; the size of the product is as follows: 435 bp; the primer sequences are as follows:

ZmTGA10-T-F1:5’-CAGATTTGTTCAAGCACTCGATGTC-3’；

ZmTGA10-T-R1:5’-CAGCAATGGTCAAATCGAGAGAG-3’。

(2) detecting a target: MT3 and MT 4; the size of the product is as follows: 445 bp; the primer sequences are as follows:

ZmTGA10-T-F2:5’-CGGCCTCAGCTCAGGTACAA-3’；

ZmTGA10-T-R2:5’-GTAAGGGAGGGAGGGACACGT-3’。

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

reaction system: 15 μ L MIX conventional PCR System, 0.5 μ L forward primer, 0.5 μ L reverse primer, 1 μ L DNA, 5.5 μ L sterile ddH₂O, 7.5. mu.L of 2 × taq mix (product No. 10103 ES).

Reaction procedure: conventional PCR: annealing at 58 ℃, extending for 1 minute and cycling for 32 rounds.

The PCR product was then recovered and ligated into T-vector sequencing by sequencing multiple T-vectors₀The DNA sequence of the independent positive transformation event target region is substituted, whether the target region is subjected to gene editing is determined, and finally 3T genes are found₀The sequences of the target regions of the transformation events were changed and were all homologous mutations, and the sequences before and after editing were shown in FIG. 3, corresponding to 3TGA10Mutant:ZmTGA10- Cas9-1、ZmTGA10-Cas9-2、ZmTGA10-Cas9-3. The alignment with the wild type sequence showsZmTGA10-Cas9-1Deletion mutations occurred at targets 3 and 4,ZmTGA10-Cas9-2deletion mutations also occurred at targets 3 and 4, andZmTGA10- Cas9-3insertional mutations occurred at targets 1 and 2.

For 3 piecestga10The amino acid sequences in the mutants were compared and analyzed to find that the lines after mutation were compared to unedited WTZmTGA10-Cas9-1AndZmTGA10-Cas9-2the deletion of the coded nucleotide at the target 3 or 4 partially occurs in the 8 th intron, and the 147 bp base deletion is carried out on the 9 th exon, so that 47 amino acids are lost. In addition, theZmTGA10-Cas9-3Insertions and deletions of the nucleotides encoded in the lines at targets 1 and 2 also cause a frame shift of the amino acids and lead to premature termination of amino acid translation. Thus, the function of the Zm00001d020938 protein appears to be absent in these transformants.

4、F₁Genotyping of surrogate plants

Maize T due to growth in the greenhouse₀Generation of plants, often with uncoordinated development of the female and male ears, and also when the edited genes are involved in male development, fertility is affected, and therefore for propagation of T₀Plants are generated and the resulting gene editing types are inheritedNext, the present invention uses the wild type pollen of Zheng 58 of the maize inbred line as obtained aboveZmTGA10-Cas9-1、ZmTGA10-Cas9-2、ZmTGA10-Cas9-3T of₀Pollinating the plant to obtain F₁Seed generation, growing plant F₁And (5) plant generation.

F₁The generation plants comprise 2 separation types, one isCas9Positive plants (transgenic plants), the other beingCas9Negative plants (non-transgenic plants), in order to avoid the complexity of mutation type by continuous editing of the wild-type allele of Zheng 58 introduced by cross pollination by sgRNA and Cas9, we need to genotype F from F₁The generation plants are selected to be free ofCas9Genes but containing T₀Generation of mutant plants, which after selfing can be obtained non-transgenic F₂And (4) generation. F₁The generation plant genotyping method comprises the following steps:

after extracting leaf DNA according to the CTAB method described above, the DNA was first usedCas9The gene specific primers Cas9-F (5'-CCCGGACAATAGCGATGT-3') and Cas9-R (5'-GAGTGGGCCGACGTAGTA-3') were PCR amplified. The PCR reaction system is the same as above; reaction procedure: conventional PCR: annealing at 58 ℃, extending for 1 minute and cycling for 32 rounds. After the PCR product was subjected to agarose gel electrophoresis, it was discriminated according to the resultCas9-positive plants andCas9negative plants.

Further aim atCas9Negative plants, PCR-amplified using the primers ZmTGA10-T-F1 and ZmTGA10-T-R1 for the detection of the MT1 and MT2 targets and the primers ZmTGA10-T-F2 and ZmTGA10-T-R2 for the detection of the MT3 and MT4 targets; after the PCR product is purified, connecting a T vector, and sequencing; determination of T from sequencing results₀Genetic profile of the generation mutation type.

EXAMPLE IIItga10Phenotypic analysis of sterile lines

None of the above examples identifiedCas9F of Gene₁Inbreeding of the plant generations to obtain F₂Generation of seed, threetga10Type of mutation (A)ZmTGA10-Cas9-1、ZmTGA10-Cas9-2AndZmTGA10-Cas9-3）respectively taking 1 selfing single ear to perform ear-row sowing, and sowing in the mature periodA phenotypic survey was conducted. Three kinds of F₂In the strain, the ratio of fertile plants to sterile plants is in accordance with 3:1 separation, further showing thattga10The sterile character of the sterile line is controlled by a single recessive gene and then aims at F₂Generation-derived stable non-transgenestga10Detailed phenotypic comparisons of sterile lines were made with wild type.

1. Observation of vigor of tassels, anthers and pollen

In terms of vegetative growth and development of the ear,tga10sterile line (ZmTGA10-Cas9-1、ZmTGA10-Cas9-2AndZmTGA10-Cas9-3）the plants of (a) are substantially non-different compared to the wild type; in terms of tassel development, wild type can be normally tasseled, anther can be normally cracked and scattered, and after selfing, it can be normally fruited, andTGA10although the sterile line can normally take out the male and can normally bloom, the anther can not crack at all and can not normally loose powder and self-copulate (figure 4); further subjecting the pollen of the wild type and the mutant to I₂KI staining, mutant and wild type pollen was found to develop normally, with pollen grains staining black (FIG. 4). This indicates thatZmTGA10（Zm00001d020938) The gene controls male development of corn by influencing anther dehiscence, is created by gene editing methodTGA10The sterile line is a sterile line with anther not cracking.

2. Scanning Electron Microscope (SEM) Observation of anthers

To deeply analyzetga10The inner and outer walls of wild-type and mutant anthers were analyzed by Scanning Electron Microscopy (SEM). Stripping wild type and mutant anthers in the mature stage (S13), and immediately fixing in FAA (Coolaber, China) solution, the volume of the fixing solution is not less than 20 times of the volume of the study material; for mutant anther, a dissecting needle can be used to perforate the anther wall to improve the permeation effect of the stationary liquid, or the vacuum pumping is repeated until the anther sinks to the bottom of the stationary liquid; after being fixed for 2 hours at room temperature, the material is stored at 4 ℃, or is dehydrated by being sequentially placed in 50%, 60%, 70%, 80%, 90% and 100% ethanol, and each gradient is kept for 15 minutes; the material can be placed in 70% ethanol overnight or stored. Drying the dehydrated sample at the critical point of carbon dioxideAnd plating gold to observe. Discoverytga10The pollen in the mutant anther is complete and full in shape, but partial residues are attached to the surface. The anther exodermis of the mutant can form a compact reticular stratum corneum structure as the wild type; tga10the inner epidermis of the mutant anther was also deposited with a small amount of residue, but granular Usnea small bodies were normally formed. The anther cuticle, which is an extracellular lipid layer covering the surface of anthers and protecting them from external abiotic stress, water loss from internal tissues and invasion by pathogens, is located in the Usnea on the inner wall of anthers and is considered as a transport vehicle for sporopouenin precursors from tapetum cells to microspores. The above results show thatZmTGA10（Zm00001d020938) After gene mutation, the formation of anther horny layer and the synthesis of sporulation pollen essence precursor substance in tapetum are not affected, but the dehiscence of anther is affected.

Example fourtga10Development and application of coseparation molecular marker for sterile line identification

1. Development of co-segregating molecular markers

In the present invention, the three obtained are aimed attga10And (3) designing a primer for the mutation site of the sterile line by using Primer5.0 software to develop two pairs of coseparation molecular markers: ZmTGA10-F1/R1 and ZmTGA10-F2/R2, and the genotype of the mutant can be separated according to the obtained band and size by combining the PCR and polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis detection method.

The coseparation molecular marker ZmTGA10-F1/R1 comprises a first primer ZmTGA10-F1 and a second primer ZmTGA 10-R1; the marker can specifically detect cornZmTGA10-Cas9-1Mutant and mutant gene in corn sterile material transformed by sametga10And can simultaneously distinguish wild typeTGA10Gene and mutanttga10A gene; against mutant genestga10A 278 bp band is amplified in the wild typeTGA10A 445bp band is amplified from the gene; the marker also enables specific detection of maizeZmTGA10-Cas9-2Mutant and mutant gene in corn sterile material transferred by the mutant and can simultaneously distinguish wild typeTGA10Gene and mutanttga10A gene; against mutant genestga10The 283 bp band is amplified, while the wild type is amplifiedTGA10The 445bp band primer sequences obtained by gene amplification are as follows:

ZmTGA10-F1：5’-CGGCCTCAGCTCAGGTACAA-3’

ZmTGA10-R1：5’-GTAAGGGAGGGAGGGACACGT-3’

the coseparation molecular marker ZmTGA10-F2/R2 comprises a first primer ZmTGA10-F2 and a second primer ZmTGA10-R2, and the marker can specifically detect cornZmTGA10-Cas9-3Mutant and mutant gene in corn sterile material transformed by sametga10And can simultaneously distinguish wild typeTGA10Gene and mutanttga10A gene; against mutant genestga10A 75 bp band is amplified in the wild typeTGA10The gene amplified a 74bp band. The primer sequences are as follows:

ZmTGA10-F2：5’-TGGTAGGGGGAGATTAGTTTAGT-3’

ZmTGA10-R2：5’-TGATGGTCCGGTGACCTAG-3’

2. application of coseparation molecular marker

To verify the validity of the above-mentioned mark, F obtained in example III₂The strain is taken as a material, andTGA10detection of alleles. The DNA extraction method, PCR amplification system and conditions were the same as in example two, and the PCR products were separated by PAGE or agarose gel electrophoresis.

Theoretically, ZmTGA10-F1/R1 and ZmTGA10-F2/R2 are inTGA10/ TGA10The homozygous wild type (AA) DNA can respectively amplify bands of 445bp, 445bp and 74bptga10/ tga10The DNA of homozygous mutant material (aa) has 278 bp, 283 bp and 75 bp bands amplified separatelyTGA10/ tga10In the hybrid (Aa) material, two bands are amplified simultaneously. The results of the verification of the molecular markers ZmTGA10-F1/R1 and ZmTGA10-F2/R2 are shown in FIG. 6, FIG. 7 and FIG. 8, and show that 2 pairs of functional molecular marker pairs F are designed₂The detection result of the plant completely meets the expectation thatTGA10/ TGA10Homozygous wild-type (AA),TGA10/ tga10Heterozygote type (Aa) andtga10/ tga10bands with corresponding sizes are respectively amplified in the homozygous mutant type material (aa), and can be used asTGA10Ideal markers for allele detection。

The molecular markers are helpful for determining mutant genotypes before flowering and pollination so as to carry out hybridization and backcross breeding on male sterile lines under different genetic backgrounds, and have important application value.

Sequence listing

<110> Beijing Chujiali Hua Koch Co., Ltd

Xinyang head Jiali Hua agriculture science and technology limited

<120> male sterility gene ZmTGA10 and application thereof in creating male sterility line of corn

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9447

<212> DNA

<213> corn (Zea mays)

<400> 1

atggtgagcg gcggcagcac caaggagcag cagcacgaga tgaacatctc cttcgggatg 60

atgaaccagc accatcagcc tccgtcctcc tcctcttcct cgtccatgca tgcggccgcc 120

gcgagcttca tgtaaagttt ttttctctct aattcctcca cctggccttt tgctgttttc 180

tttgattcag ctggcctttt tgtgctcttt ctgtttgctg ttgttcttga attcttaatc 240

gatcgatcca tccacgcccg tccatcggtt tctttggcct ggatgggttt gatttggttg 300

gtcgtggact ccctgatcat ctctctttct ttcactgatc gacttcggtt cttgtgagct 360

cgctttcctc tctcctctat ctagatagga aggtagggtg ccgacctgat gatatggcac 420

cgaattaatt caccgtgcct ttcctctttc cagttccaca gatttttttc atttaattgt 480

tgcctttaat ttgcactgat cttgtttgtt tccccccttt cactctattt atgaaaaaaa 540

attatgtggt tcttgattct gcaacagaac cataaatcag ctgacttgct acacattaaa 600

aaaatatatt tgagtgcgat tgctaacttc gatatggcac accggatttg attcttcgat 660

atcttgttct tccttcctac ttccaagagg acaaaagtgt ggtagatggt gaccaaacca 720

actatatcga tttgaaaaat ctggtgcttt aatttgattt gttattggtg ccagatagtt 780

cagttgcagt gaattaattg attaacgaat cactagctat agtctttctt ttaaatcctg 840

gatccattca aggggcagta attgttttca gaaagaggac aaaaaacaat actgatttat 900

attaattagt acatgcagca gctgatatat tgctacctct atctttattt tgcatctctt 960

tcaacgattc tatagcttgc ctgcagtcac cacacatgaa atttcgagac tatagatgcg 1020

tagcgtaggt agccatattt ttatgggctg ccagatataa atgctacaag gctagagaag 1080

attccatatt attctcctgt tcccatcctg cagaggttgg accagagata aatggccatc 1140

atactacgca gtcacacaca tacataggcg ccaagcttag agcagcatga tttcatgatg 1200

atatatgcat ggcagcacag cgccggcctt tcagagtttt cagagcgtct atatcagtag 1260

tgtcacacag tcacatcagt gttgttacag atgtatccat gtgtgtattt ctgtttcaca 1320

agcaggagcg gcaaggaggc gtcaggggcg tatgaccatc tgggcgagct ggaccaggcg 1380

ctgttcatgt acctggatca cggcagcagc catggcggcg gcgccacgca gcaagagcga 1440

aggcgtaagt gtgttagtac tactacacgc tagctagctt gcaacatgca cttctctctg 1500

acgacgaccg gaattcaact gcggctgctg ccggccgtgt gtgtgttttg cttttgcatt 1560

cctgtgttgt gttctgttct gttctgcagg aaagtcaaat gcaactgacc agcagtagta 1620

acgtacagta cgtatactcg tagactagtg cagtactagc atgtttaatt tttggctgca 1680

tttacattta gtggccagtg gtaatactga tggcataggc ccagaaacat atgtgtcgat 1740

cgtagtggac ggggatagca gtgtagctgc agggattcct ccgatcccat atcgtcctct 1800

cgatcgatgc agtcagatag atgcatgcat gcagtagagt gcagaaacgc gcgcatcctt 1860

ttccgtggtc agtcatgtga ggatcaagag agcgcggcga tacgctggcc tgcatgcagc 1920

tgtgcagatt ctcaggtagt actcccattt gaccaggatg ctgtgctgca aatcgatctg 1980

cagctgctag agcggtttgc aggacccagc agaccagcag gaaatcggca acaacataca 2040

tactgcaagc tcaaagtcca tgcacgaagc caacaggccg gcctctccct atagcttttt 2100

ggccttttgt ttcccttgct tcttgtccgt tctcttctct ctctctctct ctctctctct 2160

ctctctctct ctctctctct ctctctctct ctctctctct ctctctctcc ccatgcatct 2220

tctcttctcc atctctcccc ctacctccgg ccggtcggac tgtgacatgt aacggactgc 2280

tcaatgattt ccccgcgatc gagctgcatg tgcccgcggc agagcagtac acgcagacgg 2340

gatcatctcc atcggccagt actacgccag tggctacact agtctcgctt tctctctcat 2400

gcatgcatgt atatgccctc ctcttcctct ctctctcctg cacgctgtgc agcaagtgca 2460

aagcagccaa gcacagggca cagcatctgt acatgcatgc attgtttgac tcgatcctat 2520

ccgcttatct ttcggtttca gtacaggcac gccttagctg tggccgctgt gcgaatccct 2580

gctgtcccgt atctcccagc tttctcgtgc tactgactgt tgtactagtc ctactacact 2640

gcacagagcc cagctagtat cgccatgatt gaatactgca actcctgctg cgtgctgcat 2700

gccacaccag agagcagcgt ttcaagctct tccccatagt tcagtacttc agtatatatg 2760

tgtgtgttgt ctgatgagaa cgtacatggc cctctagcta gctccggctc cgcccactct 2820

ctatccagta gctagccaca ggctctcatc cgcctcttgt tccttgtctt gttcatggat 2880

gcagagacac tcaacatctt cccttcccag cccatgcatg tggagccgtc gccgaaggta 2940

cgccaaacat gtatctcaga tttgttcaag cactcgatgt cttttcagac gcgcacaact 3000

gctgccacgg atgagattgc agcttatttg gttttggttg tttaactgat cggttttaat 3060

ttctggtgtg attaattggt agggggagat tagtttagtc ctgtccccgg cgccggtggg 3120

atccaagcag cctaggtcac cggaccatca ccatcaccag caggcggcca tggaggagtt 3180

ggcggggagc aggaggcagc aggagcacct ccacctgcag caccaccaac cctttgctgc 3240

cgctgctgag ccagcagcac ccgggatgat caaagatgtc aagccactgg ccaaggtgag 3300

tggttaacta gtgctccact acttgaatac caatctagaa caaggctact aaattgatta 3360

aatgtgttct ctctcgattt gaccattgct gttttgtgta tatatgatta ttttaatttc 3420

catggtgtta gtaatattgt tttttttctc gattttttgc tgatgtatag aaggatcaca 3480

ggagaggcac atcgaccgct gaacgcgacc caaaagtatg gccggattgt gatactgctc 3540

ttaattaagt agcaattatt gatgaattat ttaaccgctc agtctaccta cttgaaaaga 3600

aaacactact tgtcatcatt tttgctgcac taactttgtt tcttgtttct gcaccatgct 3660

gtaatgaaca cgcctgcaga cgctgagaag gctggcccag aacagggagg ctgcaaggaa 3720

aagcaggcta aggaagaagg tgagcgagtc tcgatactga tgacctacat caagattgga 3780

acaaaagaac gagaaaaaat ataatggcaa agagttaaca tcgttttctt tgtttttttt 3840

gaagtttgac atggttcaaa ttcttgtctc caccatccaa tgacagtgta gatttatatt 3900

gttttacttc tcttttgctg catgcaacag gcttacatcc agcaactcga gtccagcagg 3960

atcagactag ctcagctcga acaagaactg cacactgcaa gagctcaggt atatgattag 4020

tatgcaacag atttaataat aaccacaaag gaaaaatgaa aaaaaaagaa acgtagtgct 4080

tgtatatctt gcctctcgac ccttgttggc ttcgtaaagg ttgaattttt ttaactgatc 4140

tctagataaa tatgtattct ttctgcaggg agttttcttc cccaacagtg gcatcctcgc 4200

tgaccaaggt gtcgccggca aaggcgtccc catcggcggc atcgacggcc tcagctcagg 4260

tacaaactaa accacctctc ctcgcattaa ttccattcgg ttgataagtt cgtccagcaa 4320

atatttcctt ttctgattaa gagctatata tatactcgta cgtcggccat atatacgggg 4380

attccagagg cggcgatgtt cgacgtggag tacgggcggt ggcaggagga gcactaccgg 4440

ctgatgtacg agcttcgggc ggcgctgcag cagcacctgc cggaggggga gctgcagatg 4500

tacgtggaga gctgcctggc tcaccacgac gagatggtgg gcatcaaaga gggcgccatc 4560

aagggcgacg tcttccacct catctccggc gtctggcgga gccccgccga gcgctgcttc 4620

ctctggctcg gcggcttccg cccctccgaa gtcatcaagg tacgtgcaca cgtgtccctc 4680

cctcccttac tagcgcagta gcgctccata taattgtcat cgggagaaac gaacccgcaa 4740

cttaacccta aacctagaca gtcaatggtt gccgtcatga ttgatttgcg ttagaacgtc 4800

gctcgatggc cattgacagc gacgtcggta gacgtgtgag gaaaagagct gttgtttctc 4860

tacaaactgg tgcatgaaaa catgcatgta aaaccctatt ttttttttgc tatagcaatt 4920

tcaggcaata catgactaac tacaggcaag ggttttaatt tcaatattcg aacttttttc 4980

agtcacctcc aaaatttgct aaatttcata aaaaatgtta gagatatata tgatgatttt 5040

ctaagaccta aacaaaacat gactacacac ctgagtagaa aaatataacc ctttttaaac 5100

aacaaagaca atatgagcct agagtacgtt agatattcat gataaagcac atccattagt 5160

gtaatacaaa aacttgaata ttatttattt cagcctctct cgaattcacg aaatttcacc 5220

gaaatttcgg aacttgatac atgcatgtac tagatctaat atataatcca ccttgttact 5280

aggtcatata tggggattga tccggtcact ttggactaac tacgaggttg gccattccag 5340

ataggcagcg tttacaatcc ctagataccc aaataaaaga atgcattgga cccgcctgaa 5400

atgtagcagt aaacgtgtgt gatgtgtacg cagtccgata tttagaatat tagtgtaaaa 5460

tataaaatat tagatttaaa ggaaagaaaa actaaagtac tatttagaag caacccagtt 5520

tttaataaat tgatttttat cttttatgta ggagagacca gcttcatggt tttttaagaa 5580

actgataatt caatttctat aaattgaagc ataaattggt atgtttggag ccacctcaat 5640

ttttataaac cagcttttta gaaactgggt gcttccaaat aggccctaaa ttgtcttgct 5700

agaagtatgg tcttagctcc cttttgcggg gctcttttaa aggatcctct aacaactctt 5760

tcaaaaaaaa atctaccaaa cagaaagctc ctttggtgga ttctttgtaa aactaggaag 5820

gagctatccc tagaaatcga actaagtgag gagccaaaaa aaggcttctt cgactctcta 5880

ctcctttcct gtcccacgaa aatagaagga gctatatttt caaccaaata gagtgacgat 5940

agtgctccta tagaggctct tcatgaaaaa aaattaatga aggaactgtt tcacataaaa 6000

ggcaaagccg gagctagagt tgttaataca agtgccttat caccctgtgg tagggctctt 6060

ttaaaggctc ctccaaccgc gtcttcaaaa aatctgtaaa tgagatgctc ccttactgag 6120

ttctccgtaa aaataaggag ttgtccctaa aaaccgatca aaggcgagag caccaaaagg 6180

aggcttctgt cgttctttct tgttctcccc tcctccatga aaatagaagg aactatgttt 6240

ccaatcaaat aaagtgctcc tttataaact cttcctgaaa ataaagagag aagagtgcaa 6300

gatctctaca taaaagggcc taaatcacat tagatcagta gccgtttcac aatatttgtt 6360

ttacaatatt tttagctgac taactattag ttctagtaca ttcaaacacc cttaaaagta 6420

gccatctaga acctgatcca ccagtagcat tgatgacgaa gccgtgagag gagttgatat 6480

gtgtatgccc cggtgtgtgc agcggttgaa agtagcagca atgatgggcg ttcgttcccg 6540

cagcgccgtg ggtgacggca gccgaaatgt tccttcggta ggtacggctg ccgataatgg 6600

aagtggcagg ccttgcccgg cagcgctctt gaaggcttcc cgccttcccc agcggcgaac 6660

cggggcgcgc ggcattgaag cgccggggcc ctcgtgaaat ctctgtcgat ggagcgccgt 6720

acaggcatgc gcgccccgtc atatacgcag cgcatgcatg cacacatgct gctgtgcagc 6780

cccgacatgg acgatcatag tatatatagt aagacgtaag tactaagtag taggtgtgaa 6840

gtctggaaac agtaggtttg caataaagtt ttgtatacga tcgtcgtata atcaaattag 6900

caaaaattac gaaaatttct taaaagttat tgtttactat actaacatat tagattttaa 6960

attataattc attctatatt acagcatgta aaaatagtaa aattattgtt tttcatcctt 7020

cgtattgaat tgcatattcc ctttttatat cccttaacat atagtgaatt agaaacttaa 7080

aatttgatat attagagggt gtttggtttg tagggactaa tttttagtct ctctatttta 7140

ttctctaaat tatcaaatac ggaaattaaa actctatcta ttttcgtttt catattttat 7200

aaattagggc taaaatagaa taaaattgag ggactaaaaa ttagtcccta aaaatcaaac 7260

accacccctt agcagagtaa ataataaaat atatgtatac ctttttttag aatttttata 7320

tttttagtaa tttagttaca tagaggtgat ttttagtaat ttagttatat aaaattagtc 7380

tctaaaaatc aaacaccacc ccttagtgtg ataatacggt gatggctacc caaaacccga 7440

atatccgacg ggctctacct gatatcgaag tgggtatgag atgatttctt tactcgcaag 7500

tatgaatggg gaagaacttt tacttatcag gtagacaggt acaggtgtgg atgggtacta 7560

cacatacccg tatacccata gataaaatat acatgcttcg taccacaatt accatctaat 7620

aaattaaagc acgtcttaac taaaataaat tctttcgttg gctatagtta gttatatgat 7680

attttatgga taccgatata cactccacga actctttact gagtgtaact ctcggtggac 7740

tgtacatcga caacggtttc tttgccgagt actttttgtc ggtgtcacct ggtactcagc 7800

aaggacaaag agtcggattc cagtagtgat agagtggtac tatccgatag gtatgtatat 7860

atatacacgt ttctattctt agatatagtg tatatctaag taacattagt atatttagga 7920

aagacaaaaa gtcttgtaat ttagaatgga gggagcacca ttaaattgtt atgcaatgtg 7980

tttttagtgt gtttgtttag tgataaatac tgacgttttt ttttataaaa ttggttacac 8040

tttagatgac tagtgaaata tagaacttga attgcatttt ttgagatgga ggcagtaaat 8100

tcgagcatct cattatcgtt ccatcacatt tatttgtaca atgagtgcgt aatacatatg 8160

agccgggtta cgaagtatga attgctgcgt atataccatg catatatgac gtgtagatgc 8220

ttaaattaat taattgcacg tactcccata tgtatacaga tgctgctgag ccacgtagag 8280

cccctgaccg aacagcagat cgtgggcgtg tacgggctgc agcagtcggc gctggagacg 8340

gaggaagcgc tgagccaggg cctggaggcg ctctaccagt cgctgtctga caccgtggtc 8400

tccgacgcgc tcagctgccc ctcgaacgtc tccaactaca tgggtcagat ggccgtcgcc 8460

atgaacaagc tctccacgct cgagggcttc gtcagacaag taagtaacca aggccgttgg 8520

ccgatgcatc cgatccattc agttcgttgg tgctaacttg cactgcttgc ttcagcatca 8580

ctactactct tcccagctaa ttcgctgtcg ctgtaatggg ttagttatag ctgcaactaa 8640

aacaaatatt gatcaaagat ctatatagta tagtatagta gcactccata atatatcgtc 8700

agttgaagca aggtacgttg gatagcttgg atggtttacc taattatttg agctcgacag 8760

tccagttctt tagtcatact aaacagtggt ttagatatat cgagcatgca cgccaggtgc 8820

aggccggcaa agcagacgca tggattaggt atcaataaaa aagcaccgaa ctgtcgccat 8880

cccgaaattg gagaacctgc agcattgttg cacaaactcc agggctcatt cagattttca 8940

tgttgggtag gtacccttga tgccgacatt gtttaggccg gcttcacctg cccagggccc 9000

ggccctcata cagtcatact agttacgtgc gtggactgtg ttcattaatt aggacactgc 9060

aaatagtgct gctggctgat attattgact gattcgtttg tgacgatggc aagacgccag 9120

acacagtact gacaatgata tatatgatat gtcgtccccc taatcatttt gatctgttct 9180

atcttgtata tatgctgcgt ggtgtttgtt taattttgtg ttaattatta actatatatc 9240

gatcagcttc attctgacga acatcatgga cgcatgcatg acgcacaggc tgagaacctt 9300

cggcagcaga cgctgcaccg gctgcaccag gtcctgacga cgcggcagat ggcgcggtcg 9360

ctgctggccg tgtcggacta cttccaccgc ctccgcacgc tgagctcgct ctgggtcaca 9420

cgccccaggg caccgcagga gcaataa 9447

<210> 2

<211> 486

<212> PRT

<213> corn (Zea mays)

<400> 2

Met Val Ser Gly Gly Ser Thr Lys Glu Gln Gln His Glu Met Asn Ile

1 5 10 15

Ser Phe Gly Met Met Asn Gln His His Gln Pro Pro Ser Ser Ser Ser

20 25 30

Ser Ser Ser Met His Ala Ala Ala Ala Ser Phe Met Ser Gly Lys Glu

35 40 45

Ala Ser Gly Ala Tyr Asp His Leu Gly Glu Leu Asp Gln Ala Leu Phe

50 55 60

Met Tyr Leu Asp His Gly Ser Ser His Gly Gly Gly Ala Thr Gln Gln

65 70 75 80

Glu Arg Arg Gln Thr Leu Asn Ile Phe Pro Ser Gln Pro Met His Val

85 90 95

Glu Pro Ser Pro Lys Gly Glu Ile Ser Leu Val Leu Ser Pro Ala Pro

100 105 110

Val Gly Ser Lys Gln Pro Arg Ser Pro Asp His His His His Gln Gln

115 120 125

Ala Ala Met Glu Glu Leu Ala Gly Ser Arg Arg Gln Gln Glu His Leu

130 135 140

His Leu Gln His His Gln Pro Phe Ala Ala Ala Ala Glu Pro Ala Ala

145 150 155 160

Pro Gly Met Ile Lys Asp Val Lys Pro Leu Ala Lys Lys Asp His Arg

165 170 175

Arg Gly Thr Ser Thr Ala Glu Arg Asp Pro Lys Thr Leu Arg Arg Leu

180 185 190

Ala Gln Asn Arg Glu Ala Ala Arg Lys Ser Arg Leu Arg Lys Lys Ala

195 200 205

Tyr Ile Gln Gln Leu Glu Ser Ser Arg Ile Arg Leu Ala Gln Leu Glu

210 215 220

Gln Glu Leu His Thr Ala Arg Ala Gln Gly Val Phe Phe Pro Asn Ser

225 230 235 240

Gly Ile Leu Ala Asp Gln Gly Val Ala Gly Lys Gly Val Pro Ile Gly

245 250 255

Gly Ile Asp Gly Leu Ser Ser Glu Ala Ala Met Phe Asp Val Glu Tyr

260 265 270

Gly Arg Trp Gln Glu Glu His Tyr Arg Leu Met Tyr Glu Leu Arg Ala

275 280 285

Ala Leu Gln Gln His Leu Pro Glu Gly Glu Leu Gln Met Tyr Val Glu

290 295 300

Ser Cys Leu Ala His His Asp Glu Met Val Gly Ile Lys Glu Gly Ala

305 310 315 320

Ile Lys Gly Asp Val Phe His Leu Ile Ser Gly Val Trp Arg Ser Pro

325 330 335

Ala Glu Arg Cys Phe Leu Trp Leu Gly Gly Phe Arg Pro Ser Glu Val

340 345 350

Ile Lys Met Leu Leu Ser His Val Glu Pro Leu Thr Glu Gln Gln Ile

355 360 365

Val Gly Val Tyr Gly Leu Gln Gln Ser Ala Leu Glu Thr Glu Glu Ala

370 375 380

Leu Ser Gln Gly Leu Glu Ala Leu Tyr Gln Ser Leu Ser Asp Thr Val

385 390 395 400

Val Ser Asp Ala Leu Ser Cys Pro Ser Asn Val Ser Asn Tyr Met Gly

405 410 415

Gln Met Ala Val Ala Met Asn Lys Leu Ser Thr Leu Glu Gly Phe Val

420 425 430

Arg Gln Ala Glu Asn Leu Arg Gln Gln Thr Leu His Arg Leu His Gln

435 440 445

Val Leu Thr Thr Arg Gln Met Ala Arg Ser Leu Leu Ala Val Ser Asp

450 455 460

Tyr Phe His Arg Leu Arg Thr Leu Ser Ser Leu Trp Val Thr Arg Pro

465 470 475 480

Arg Ala Pro Gln Glu Gln

485

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtcctgtccc cggcgccgg 19

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctgctgagcc agcagcacc 19

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tacgtcggcc atatatacg 19

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctcaccacg acgagatgg 19

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cggcctcagc tcaggtacaa 20

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtaagggagg gagggacacg t 21

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tggtaggggg agattagttt agt 23

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgatggtccg gtgacctag 19

Claims (12)

1.ZmTGA10The application of the gene in controlling the male reproductive development of the corn is characterized in that the amino acid sequence of the gene is shown as SEQ ID NO. 2.

2. The use of claim 1, wherein the nucleotide sequence of the gene is represented by SEQ ID No. 1.

3. A method for creating a male sterile line of maize, comprising inhibiting the expression and/or activity of the gene of claim 1 or 2 in maize and selecting a maize male sterile plant.

4. The method for creating a male sterile line of maize according to claim 3, wherein said method for suppressing gene expression and/or activity comprises any one of gene editing, RNA interference, T-DNA insertion.

5. The method for creating the male sterile line of maize according to claim 4, wherein the gene editing adopts CRISPR/Cas9 method.

6. The method for creating a maize male sterile line according to claim 5, wherein the CRISPR/Cas9 method comprises: designing CRISPR/Cas9 vector targets at a fourth exon, an eighth intron and a ninth exon of the gene, wherein the DNA sequence of the target is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

7. An obtainingtga10Method for male sterile lines, characterized in that obtained by the method according to any one of claims 3 to 6tga10Hybridizing and backcrossing the male sterile line with the target material to obtain the target materialtga10Traits of male sterility and genetic mutations.

8. Obtained by the method of any one of claims 3-6tga10The application of the male sterile line in cross breeding and seed production.

9. The use according to claim 8, wherein said cross breeding and seed production is to be performedtga10The male sterile line is used as a female parent to be hybridized with other male parents.

10. Use according to claim 8, comprising what is to be obtainedtga10Hybridizing and backcrossing the male sterile line with other target materials to obtain the target materialstga10Traits of male sterility and genetic mutations.

11. A kind oftga10The molecular marker primers ZmTGA10-F1 and ZmTGA10-R1 of the male sterile line are characterized in that the sequences of the primers ZmTGA10-F1 and ZmTGA10-R1 are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.

12. Corn male sterile linetga10The molecular marker primers ZmTGA10-F2 and ZmTGA10-R2 are characterized in that the sequences of the primers ZmTGA10-F2 and ZmTGA10-R2 are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10.

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