CN110423764A - Anther development control gene and its application in brassica plant male sterility - Google Patents

Abstract

The invention discloses a kind of anther development control gene and its application in brassica plant male sterility, which is in β -1,3- glucanase geneBrA01G6On the basis of eliminate X8 structural domain, early stage anther specific expression promoterAP3Start the expression of the gene, the degradation in advance of callose during anther development can be caused, so that influencing microspore further buds into mature pollen grain, therefore the gene can be used for preparing brassica plant male sterile line.

Description

Genes controlling anther development and their application in male sterility in Brassica plants

technical field

The invention belongs to the field of genetic engineering and plant biotechnology, and specifically relates to a gene regulating anther development and its application in preparing male sterile lines of Brassica plants.

Background technique

The hereditary phenomenon that plants cannot produce pollen normally during sexual reproduction is called male sterility. In angiosperms, anthers are important organs for pollen production and sexual reproduction. In 1999, Sanders et al. carried out cytological slices on the anthers of Arabidopsis thaliana, and divided the germination of stamen primordia to the final anther maturation into two stages, which were subdivided into 14 stages. The first stage is stage 1-8, this stage is from the differentiation of stamen primordia until the tetrad releases microspores. The second stage is stage 9-14, in which microspores develop into mature pollen grains and are finally released from the anther to the extracellular space. The tetrad formed by pollen mother cell after meiosis is surrounded by callose wall and pollen mother cell wall, and the subsequent process of tetrad separation and release of microspores involves two steps of degradation of callose wall and pollen mother cell wall . The callose wall is mainly composed of β-1,3-glucan, and at a specific time, the callose wall wrapping the tetrad is secreted by the callose enzyme (β-1,3-glucan enzyme), the degraded haploid microspores are released into the anther cavity and further develop into mature pollen grains.

Non-heading cabbage (Brassica campestrisssp. Chinensis Makino), also known as Chinese cabbage, green vegetables, and feather vegetables, is widely cultivated in East Asia and is an important leafy vegetable. Non-heading Chinese cabbage belongs to Brassicaceae Brassica subspecies, and is a typical cross-pollination crop with obvious heterosis. At present, the use of male sterile lines for seed production is an effective way to utilize plant heterosis. Therefore, the research on the selection and application of male sterile lines in Chinese cabbage has received great attention from breeders.

There is diversity in β-1,3-glucanase genes, and there are different β-1,3-glucanase genes among different species, although the same species also contains different β-1,3- Glucanase genes, for example, 50 different β-1,3-glucanase genes have been found in Arabidopsis thaliana, and current research on this gene mainly focuses on disease resistance and fertility. Tohru Tsuchiya et al. used the β-1,3-glucanase gene isolated from soybean (without CTTP domain) to be fused to the promoter of the Osg6B gene specifically expressed in rice tapetum to transform tobacco and found that the transgenic plants were sterile. Cytology Early degradation of callose was observed at the level (Tsuchiya T, Toriyama K, Yoshikawa M, Ejiri S, Hinata K. Tapetum-specific expression of the gene for an endo-P-1,3-glucanase causes malesterility in transgenic tobacco. Plant and Cell Physiology, 1995, 3:487-494). In rice, the Osg1 gene encodes β-1,3-glucanase, which is expressed to varying degrees in different tissues. After suppressing the expression of this gene through interference experiments, it was found that RNAi inhibited the male sterile phenotype of the plants, and the anther development process was analyzed, and it was found that the degradation of the callose wall was delayed, and pollen abortion eventually occurred (Wan L L, Zha W J, Cheng X Y,Liu C,Lv L,Liu C X,Wang Z Q,Du B,Chen R Z,Zhu L L,He G C.A rice P-1,3-glucanasegene Osgl is required for callose degradation in pollen development.Planta,2011,2 :309-323). The expression of β-1,3-glucanase gene has time-space specificity, and early or delayed expression will cause abnormal callose wall degradation and male sterility phenotype.

Contents of the invention

The object of the present invention is to provide a kind of anther development control gene, this gene removes the X8 structural domain on the basis of β-1,3-glucanase gene BrA01G6, and the anther-specific expression promoter AP3 activates the gene in the early stage of anther development The expression can cause early degradation of callose in the process of anther development, thereby affecting the further development of microspores into mature pollen grains, so the gene can be used to prepare male sterile lines of plants (Brassica).

In order to achieve the above object, the present invention takes the following technical measures:

The gene regulating anther development, its nucleotide sequence is shown in SEQ ID NO.1, the gene is obtained by removing the X8 domain on the basis of the β-1,3-glucanase gene BrA01G6, and expresses in the early stage of anther development The expression of the gene driven by the AP3 promoter can be used to prepare male sterile lines of Brassica plants, and the specific method is as follows:

Construct a recombinant expression vector containing the AP3 promoter and the sequence described in SEQ ID NO.1, transform Brassica plants (preferably Arabidopsis thaliana and non-heading Chinese cabbage) to obtain male sterile target plant T0 generation, through the method of continuous hybridization Obtain sterile plants capable of stable inheritance.

When the gene of the present invention is used to prepare male sterile lines, in order to facilitate the screening of transgenic plants, the plant expression vectors used are processed, and the GUS genes that can be expressed in plants and can produce color changes and resistant genes are added. Antibiotic markers such as kanamycin or hygromycin.

The expression vector described in the present invention refers to any vector known in the prior art that can express in plants, for example, it is suitable for constructing the expression vector described in the present invention, including but not limited to, such as: PbI101, pCAMBIA2300, etc. .

Compared with the prior art, the present invention has the following advantages:

In Arabidopsis, the β-1,3-glucanase gene family contains 50 members, and these 50 β-1,3-glucanase genes have a certain degree of similarity in structure, but at the same time There are differences. What they have in common is that the N-terminal has a glycosyl hydrolase family GH-17 functional domain, but some of the genes have a sugar-binding module X8 that recognizes β-1,3-glucan at the C-terminal. In order to verify whether the X8 domain is directly related to the function of the β-1,3-glucanase gene, we used a specific promoter expressed in early anthers, AP3, to ensure that the gene is involved in β-1,3-glucan Enzymatic degradation of callose. Our results confirmed that the early expression of the deletion of the X8 domain of the BrA01G6 gene will lead to the early degradation of callose in the anthers of the transformed single plant, and the early degradation of callose directly affects the formation of the outer wall of the microspores, which in turn leads to the failure of the microspores to develop into mature pollen. grain. However, the transformation of the full-gene ORF expression vector with X8 domain in advance had no effect on the degradation of callose of the transformed single plant.

Description of drawings

Figure 1: GUS staining results of flower buds of Arabidopsis thaliana T ₀ generation transformed plants, in the T ₀ generation transformed plants transformed with the promoter analysis vector pBI-GUS, only the larger flower buds and flowers that are about to bloom can see obvious GUS Coloring signals. (A) in Figure 1 shows the GUS staining of the inflorescence of a transformed single plant, (B) shows the ninth stage of anther development, (C) shows the tenth stage of anther development, and (D) shows the eleventh stage of anther development.

Fig. 2: Morphological observation of transformed plants obtained by transfecting Arabidopsis thaliana with BrA01G6 gene and X8 domain. (A) in Fig. 2 is a side branch of the transformed plant of Arabidopsis thaliana without the X8 domain, (B) is a side branch of the transformed plant of Arabidopsis thaliana with the complete BrA01G6 gene, (C) The picture shows a lateral branch of a transformed plant of wild-type Arabidopsis thaliana.

Figure 3: Scanning electron microscope observation of pollen of wild-type and transformed sterile single plants. (A) in Figure 3 is the anther of the open flower of wild-type Arabidopsis, (B) is the pollen grain of the open flower of wild-type Arabidopsis, (C) and (D) are wild-type The pollen exine structure of Arabidopsis, (a) is the anther of the open flower of the transformed sterile plant, (b) is the pollen grain of the open flower of the transformed sterile plant, (c) and (d) are Transformation of pollen exine structures of sterile plants.

Detailed ways

Below in conjunction with specific embodiment, further define the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow conventional conditions such as the conditions described in "Molecular Cloning: A Laboratory Guide" (New York: Cold Spring Harbor Laboratory, 1989), or according to the operation provided by the manufacturer method suggested in the manual. Experimental Materials:

The material "He B'ai" used in this experiment was discovered in the northern park of Wuhan Academy of Agricultural Sciences and preserved in the northern park of Wuhan Academy of Agricultural Sciences. The applicant distributed the biological material to the public within 20 years from the date of application for verification tests.

Example 1: Preparation of non-heading Chinese cabbage β-1,3-glucanase gene BrA01G6

(a) Extraction of RNA

The whole flower bud of non-heading Chinese cabbage "He Bai" was taken as the material, and the RNA was extracted according to the requirements of the Trizol extraction kit, and the absorbance values of the RNA at 260nm and 280nm were respectively detected with a protein detector (DU 650BECKMAN, USA). Combined with 1% (mass volume ratio) agarose gel electrophoresis to identify the purity and concentration of RNA.

(b) Synthesis of cDNA

Using the RNA obtained above as a template, carry out reverse transcription according to the following scheme: add 1 μl of DNAase to 2 μg of RNA, 1 μl of DNAase Buffer+MgCl ₂ , make DEPC H ₂ O to 10 μl, and keep at 37°C for 30 minutes; immediately put it on ice, and add 1 μl of EDTA, 65°C for 10 minutes; add 1 μl Oligo(dT), 65°C, 5 minutes, then immediately place on ice for 5 minutes, briefly centrifuge, add 5×M-MLV Buffer 4 μl, dNTP (10mmol/L) 2 μl, RNase Inhibitor 1μl, RTase 1μl, 42°C, 60min; 70°C, 5min, the obtained cDNA was aliquoted and stored in a -80°C refrigerator for later use.

(c) Amplification of BrA01G6 gene

The complete CDS sequence (SEQ ID NO.2) of the BrA01G6 gene and the CDS sequence (SEQ ID NO.1) without the X8 domain both contain the complete ORF reading frame and the initiation codon ATG. Primers were designed according to the CDS sequence obtained from the above analysis, using the cDNA of non-heading Chinese cabbage "He Bai" as a template, the complete CDS primers were named (F1 and R1), and the primers without the X8 domain were named (F2 and R2) , F1 is forward primer 5'-ATGGCTTTTTTTGCTCTGTTCAT-3', R1 is reverse primer 5'-TTACAGGGTAACGCT AGGGAACT-3', F2 is forward primer 5'-ATGGCTTTTTTTGCTCTGTTCATTTT-3', R2 is reverse primer 5'-TTACAGGGTAACGCTAGGCCACACGT -3', using hot-start high-fidelity long fragment polymerase PhusionHigh-Fidelity DNA Polymerase (Thermo Scientific, http://www.thermoscientificbio.com) for PCR amplification. The PCR amplification system is as follows:

PCR amplification program: total denaturation at 98°C for 1 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 4 min, 34 cycles; total extension at 72°C for 10 min. After the amplified product was detected by agarose gel electrophoresis, the product was excavated and recovered using the agarose gel recovery kit of Tiangen (Beijing) Biotechnology Co., Ltd. (http://www.tiangen.com/) (according to the kit instructions), the product was purified and treated with A: 10×buffer, 1μl; 25mmol/LMgCl ₂ , 0.8μl; 10mmol/L dNTP, 0.2μl; Taq enzyme, 0.1μl; recovered product, 7.9μl, in PCR In the instrument at 72°C for 30 min, 2 μl of it was connected to the pMD18-T vector (purchased from Dalian Bao Biological Engineering Co., Ltd.) for sequencing. The analysis results showed that the CDS sequence of BrA01G6 was obtained, and its sequence was shown in SEQ ID NO.2 in the sequence table, while the CDS sequence without X8 domain was shown in SEQ ID NO.1.

Example 2: Promoter Analysis of BrA01G6

(a) Promoter sequence amplification: In this example, the upstream non-coding segment 1160 bp of BrA01G6 was amplified by designing primers as the promoter segment of the gene (promoter for short), and the forward primer was 5'-TAATTGAGAGAAGGAGACAGCCACA-3 ', the reverse primer is 5'-CAAAATGTTGTTGATAGGAGA GGGA-3', with HindIII and BamHI as the restriction sites of the forward and reverse primers, the leaf DNA of "He Bai" was extracted as a template, and the hot start high-fidelity long Fragment polymerase Phusion High-Fidelity DNA Polymerase (Thermo Scientific, http://www.thermoscientificbio.com) was used for PCR amplification. Its PCR amplification system and product recovery are consistent with the above steps. The recovered product was directly double-digested with the fast restriction endonuclease of fermentas (Thermo Scientific, http://www.thermoscientificbio.com), and the double-digestion system was as follows:

The enzyme digestion reaction was carried out in a water bath at 37°C for 2 hours. The digested product was recovered with a DNA purification kit from Beijing Tiangen (Beijing) Biotechnology Co., Ltd. (http://www.tiangen.com/). The transformation vector was ligated with the enzyme-digested recovery product of the amplified fragment of the candidate gene and then transformed into Escherichia coli DH5ɑ (purchased from Takara Bioengineering Dalian Co., Ltd. TAKARA Co., Ltd.). sample sequencing. After the sequencing was correct, the plasmid was extracted and transformed into Agrobacterium strain GV1301 (Chen Wei et al., 2006), and positive clones were selected and stored at -70°C at ultra-low temperature.

(b) Construction of a promoter analysis vector: the wild-type plants of Arabidopsis thaliana were transformed by conventional Agrobacterium staining method. In 1/2MS medium containing 25mg/L kanamycin (1/2MS medium: Murashige&Skoog basic medium (MS for short, MS medium or MS basic medium for short, the three are synonymous) 2.2g/L; Sucrose (Sucrose) 10g/L; agar powder (Agarose) 6g/L; use ultrapure water (ddH ₂ O) to make the volume to 1L; adjust the pH to 5.8-5.9) to screen the seeds of transformed Arabidopsis plants, resistance The morphological characteristics of the plants are that the roots are elongated, the plants are larger, and the color of the cotyledon and true leaves is bright green; the morphological characteristics of the non-resistant plants are that the roots cannot be elongated, the plants are short, and the cotyledons are yellow. After the screened resistant plants grew to 3-5 true leaves, they were transplanted into nutrient soil, and a total of 20 resistant plants were obtained. Extract the total DNA of positive plant leaves of Arabidopsis resistant plants (Stewart et al., 1993), use the sequence on the pbI101 (Jefferson et al., 1987) vector to design primers as NP-F and NP-R, and pass PCR Transformed plants were further identified.

The DNA sequences of primers NP-F and NP-R are as follows:

NP-F (forward primer): 5'-GCCACAGTCGATGAATCCAG-3';

NP-R (reverse primer): 5'-ATGGTGGAGCACGACACTCT-3'.

(c) GUS staining: the flower buds of the obtained T0 generation positive transgenic plants at different stages were subjected to GUS staining. Reagents to be prepared:

(1) 20mM X-gluc: Weigh 100mg X-gluc, add 9.58ml DMSO;

(2) 50mM phosphate buffer (PH=7.0):

Solution A: Weigh 3.12g NaH ₂ PO ₄ 2H ₂ O, dissolve in 100ml sterilized ddH ₂ O;

Solution B: Weigh 7.17g Na ₂ HPO ₄ ·12H ₂ O, dissolve in 100ml sterilized ddH ₂ O;

(3) Reagent I:

(4) Reagent II:

Take 19ml of reagent I, add 1ml of 20mM X-gluc, and mix to form reagent II.

Dyeing steps:

Sampling and fixation: Remove the tissue material with tweezers, put it into a 50ml centrifuge tube filled with 90% acetone, and place it on ice for 20-30min;

Rinse: Pour out the acetone in the test tube, then rinse with reagent Ⅰ, 4°C, 30min, repeat once;

Staining: Pour off the reagent Ⅰ, put the sample into the reagent Ⅱ, evacuate it with a vacuum pump for 10 minutes, then place it in a 37°C incubator, and dye it overnight;

Decolorization: pour out the reagent II, add 70% ethanol to the test tube for decolorization. The 70% ethanol was replaced at least three times in the middle. In order to speed up decolorization, the test tube can be placed in a 37°C incubator.

(d) GUS stained section analysis

Take GUS-stained flower buds and wild plants at different development stages, fix them in FAA fixative solution (formaldehyde: glacial acetic acid: 50% ethanol = 5:5:90) for 16 hours, dehydrate them with graded alcohol, and then put them in the resin pre-infiltration solution 1-2h, then stand still in the resin permeate solution for 1-2h, then polymerize in the embedding plate, and bake in a constant temperature oven at 37°C for about 4 days. The anthers were cross-cut on an ultramicrotome with a thickness of 1 μm, stained with toluidine blue slices, washed with water and dried for observation. Resin-embedded semi-thin sectioning techniques are as follows:

(1) Fixation: take mutant and wild-type tassels of different lengths (0.5cm to 24cm), fix them in 50% FAA fixative solution (formaldehyde: glacial acetic acid: 50% ethanol = 5:5:90), and slowly fix them with a vacuum pump. Pump air (the pumping process lasts for 10 minutes), place it in a near-vacuum state for 15 minutes, and then slowly deflate (the deflation process lasts for 10 minutes), and it can be observed that there are air bubbles coming out, and the sample sinks to the bottom of the bottle; replace it with a new FAA to fix it. solution, placed at room temperature for 16-24h; after settling, transfer to 70% ethanol for storage (cut the spikelets from the spikelets with scissors, and store them respectively according to the size of the spikelets);

(2) Gradient alcohol dehydration: gradients are: 50% ethanol (the plant tissue should sink at the bottom of the bottle, indicating that the pumping is sufficient), 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 95% ethanol , 100% ethanol, 100% ethanol, 60min for each level at room temperature;

(3) Pre-infiltration (1h-2h);

(4) Penetration (1h-2h);

(5) Polymerization: This step requires quick action. First, use a 1.5mL centrifuge tube or other embedding container to pack the sample, then calculate the dosage to prepare the polymerization drug and then polymerize immediately (note that the polymerization solution must be filled up to completely cover the sample to prevent volatilization. ), after 2 hours at room temperature, wait for a little solidification, and then put it into a 37°C incubator for 3d-4d polymerization. A good polymerization solution should generally be completed within 5min;

(6) Drop a drop of distilled water on the glass slide, and after slicing, take the sliced material and spread it on the glass slide; after the slice, it can be observed under a microscope and photographed;

Reagents and formulations:

Resin: Hereaus Kulzer Technovit 7100Resin, including base liquid Technovit7100, hardener I (powder), hardener II (40mL, will solidify at 4°C, store at room temperature).

Pre-permeation solution: mix equal volume of 95% or absolute alcohol with base liquid Technovit 7100, store at 4°C.

Penetration solution: 1g induritore I (hardening agent I, ie one package) was dissolved in 100mL base liquid Technovit7100, stirred and mixed, and stored at 4°C.

Polymerization solution: Ready to use, add 1mL Hardener II to 15mL penetrating solution and mix well to embed the sample. The prepared polymer solution should generally be completed within 5 minutes.

The results of promoter analysis showed that GUS coloring signals could be observed in flower buds, and the color of GUS gradually became lighter from the inside to the outside, and the larger flower buds did not express (Figure 1A). Semi-thin sections of the flower buds after GUS staining and photography showed that GUS was clearly expressed in the microspores and tapetum of the anthers, and the staining was darker at the 9th stage (as shown in Figure 1B) and the 10th stage (as shown in Figure 1C) , and to the 11th stage (as shown in Figure 1D), the staining is very light. Therefore, BrA01G6 is expressed in the middle and late anthers.

Example 3: Application of the BrA01G6 gene in the preparation of Arabidopsis male sterile plants

1. Construction of BrA01G6 transformation vector

In the present invention, the anther-specific expression promoter AP3 (as shown in SEQ ID NO.3) is adopted, and the primer sequence used is: AP3-pro(F) forward primer 5'-CAGTAACTGTGGCCAACTTAG-3', AP3-pro(R ) The reverse primer is 5'-ATTCTTCTCTCTTTGTTTAATC-3', HindIII and PstI are used as the restriction endonuclease sites of the forward and reverse primers respectively, and the hot-start high-fidelity long-fragment polymerase Phusion High-Fidelity DNA Polymerase is used for PCR Amplify (the amplification system is as described in the present embodiment above), the amplified fragment is reclaimed with a gel recovery kit (purchased from Shanghai Sangon Bioengineering Co., Ltd.), and the fragment of 867bp is recovered by double enzyme digestion with HindIII and PstI, and connected to On the expression vector pCAMBIA2300 (gifted by Professor Tu Jinxing, State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University) digested by HindIII and PstI, the ligated product was transformed into Escherichia coli strain DH5ɑ, on LB medium containing 50 μg/ml hygromycin Screen transformants, select a single colony to extract the plasmid, and use sequencing to detect that the nucleotide sequence is completely correct; analyze the restriction site according to the sequence information of the target gene, design primers combined with the multiple cloning site of the vector, and use cDNA as a template to separate Perform PCR to amplify the BrA01G6 gene and remove part of the sequence of the X8 domain. Amplify BrA01G6 primer F1 is the forward primer 5'-ATGGCTTTTTTTGCTCTGTTCAT-3', R1 is the reverse primer 5'-TTACAGGGTAACGCTAGGGAACT-3', using hot start high-fidelity long fragment polymerase Phusion High-Fidelity DNA Polymerase for PCR amplification, The product was recovered with a gel recovery kit (Tiangen (Beijing) Biotechnology Co., Ltd.), and recovered with PstI and KpnI double enzyme digestion to obtain a 1431bp long fragment, and the primer without the X8 domain sequence was amplified, and F2 was the forward primer 5 '-ATGGCTTTTTTTGCTCTGTTCATTTT-3', R2 is the reverse primer 5'-TTACAGGGTAACGCTAGGCCACACGT-3', both of which use PstI and KpnI as restriction sites, and use hot-start high-fidelity long-fragment polymerase for PCR amplification, recovery, and PstI and KpnI double enzyme digestion recovered a 1179bp long fragment, which was connected to the expression vector pCAMBIA2300 containing the Ap3 promoter digested by PstI and KpnI, and then the ligated product was transformed into E. Transformants were screened on the LB medium, and a single colony was selected to extract the plasmid, and the nucleotide sequence was proved to be completely correct by sequencing, and the correctly constructed recombinant plasmid vector was introduced into the Agrobacterium strain GV1301, and the positive single clone was selected and stored in a -80°C refrigerator save.

2. Arabidopsis transformation

The Agrobacterium bacterial strain obtained in the previous step is used to infect wild-type Arabidopsis inflorescences (according to conventional methods), and the harvested seeds are screened on the 1/2MS medium containing 25mg/ml kanamycin. After 12 days, the anti- The resistant plants will gradually grow taller and their roots will elongate, while the non-resistant plants will be shorter and will gradually turn yellow in the later stage. The resistant plants will be transplanted into nutrient soil for growth, and their phenotypes will be observed (Figure 2). The wild-type siliques grew normally, but the siliques of the complete BrA01G6 gene transformants were short and some could elongate; the siliques of the transformants without the X8 domain were all very short. The flowers were observed under a microscope, and it was found that the stamens of the wild-type and complete BrA01G6 gene transformants had pollen overflowing and stained the stigma, while the stamens of the transformants without the X8 domain had no pollen, showing a completely different appearance. education.

3. Scanning electron microscope observation

The mature anthers of transformed sterile plants and wild plants were selected at the flowering stage, fixed in 2.5% glutaraldehyde for 24 hours (4°C), and dehydrated through gradient alcohol (30% → 50% → 70% → 85% → 95% → 100% → 100%, 10 minutes per level), and then through the intermediate solution replacement (first soaked in a mixture of isoamyl acetate: ethanol = 1:1 for 10 minutes, then soaked in pure isoamyl acetate for 10 minutes, shake properly), and then the sample After critical point drying, sticking, and gold spraying, and then using a scanning electron microscope (S570, Hitachi, Tokyo, Japan) for microscopic observation and photography, the results are shown in Figure 3, and the surface of normal pollen grains has a regular network structure. However, the pollen grains of the transformed sterile plants had tumor-like protrusions on the surface, not a regular net-like structure.

summary

The X8 domain is mainly responsible for binding sugars. It is a conserved sequence containing one phenylalanine and six cysteines, which is essential for the activity of β-1,3-glucanase. An early expression vector was constructed for the entire ORF of the BrA01G6 gene and a part of the ORF missing the X8 domain. It was found that the early expression of BrA01G6 with the deletion of the X8 domain under the activation of the anther-specific expression promoter AP3 would lead to abortion phenotype. Cytological observation It was found that the callose was degraded in advance, which affected the formation of the outer wall of the microspores, resulting in the inability of the microspores to form normally mature pollen grains.

sequence listing

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ggaacagaga ggcattgggg aattttgaat cctgacggta cacagatcta cgatattgat 1080

ttgagtggga caagaccggt ttctagtctt ggttcgttgc ctaaaccaaa taacaatgtt 1140

ccgttcaagg gcaacgtgtg gtgtgtggcg gtagaaggag ctaacgagac ggagttaggg 1200

caggcgcttg actttgcttg tggaagaagc aatgccacat gtgcagcttt ggcaccggga 1260

agagagtgtt acgcaccagt ttcggttact tggcacgcaa gctatgcatt tagctcgtac 1320

tgggctcagt tccggaacca aagctcccag tgctacttca acggcttggc gcgtgagacc 1380

acgaccaacc caggaaatga acaatgcaag ttccctagcg ttaccctgta a 1431

<210> 3

<211> 867

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagtaactgt ggccaactta gttttgaaac aacactaact ggtcgaagca aaaagaaaaa 60

agagtttcat catatatctg atttgatgga ctgtttggag ttaggaccaa aattatcta 120

caaacaaaga cttttctcct aacttgtgat tccttcttaa accctagggg taatattcta 180

ttttccaagg atctttagtt aaaggcaaat ccgggaaatt attgtaatca tttggggaaa 240

catataaaag atttgagtta gatggaagtg acgattaatc caaacatata tatctctttc 300

ttcttatttc ccaaattaac agacaaaagt agaatattgg cttttaacac caatataaaa 360

acttgcttca cacctaaaca cttttgttta ctttagggta agtgcaaaaa gccaaccaaa 420

tccacctgca ctgatttgac gtttacaaac gccgttaagt ttgtcaccgt ctaaacaaaa 480

acaaagtaga agctaacgga gctccgttaa taaattgacg aaaagcaaac caagttttta 540

gctttggtcc ccctctttta ccaagtgaca attgatttaa gcagtgtctt gtaattatac 600

aaccatcgat gtccgttgat ttaaacagtg tcttgtaatt aaaaaaatca gtttacataa 660

atggaaaatt tatcacttag ttttcatcaa cttctgaact tacctttcat ggattaggca 720

atactttcca tttttagtaa ctcaagtgga ccctttactt cttcaactcc atctctctct 780

ttctatttca cttctttctt ctcattatat ctcttgtcct ctccaccaaa tctcttcaac 840

aaaaagatta aacaaagaga gaagaat 867

Claims (4)

1. A gene regulating anther development, characterized in that the nucleotide sequence of said gene is shown in SEQ ID NO.1. 2. The application of the gene according to claim 1 in the preparation of male sterile lines of Brassica plants, characterized in that the expression of the gene is driven by the AP3 promoter expressed in the early stages of anther development. 3. the method for the gene preparation of Brassica plant male sterile line according to claim 1, is characterized in that, constructs the recombinant expression vector that contains AP3 promotor and sequence described in SEQ ID NO.1, transforms Brassica plant and obtains In the T0 generation of the male sterile target plant, a sterile plant that can be stably inherited is obtained by continuous crossing. 4. The method according to claim 1, characterized in that, the Brassica plants are Arabidopsis thaliana and non-heading Chinese cabbage.