CN110714022B - A gene STK1 for pollen competitiveness; 2 and application thereof in improving efficiency of propagating plant nuclear male sterile line - Google Patents

Abstract

The invention discloses a pollen competitiveness gene STK1;2 and the application thereof in improving the efficiency of propagating the nuclear male sterile line of the plant. The invention reduces the expression level of pollen fertilization competitiveness regulatory genes STK1 and STK2 (serine threonine kinase1; 2) on the RNA level, reduces the competitiveness of corn pollen participating in fertilization, combines the corn pollen with a transgenic technology plant male sterile line propagation technical system, and improves the production efficiency of the sterile line.

Description

Expression regulation of pollen competitiveness gene STK1; 2 and its role in increasing nuclear male Application of SCM Efficiency

technical field

The invention belongs to the field of plant genetic breeding and seed production, and specifically relates to regulating the expression regulation of pollen fertilization competitive genes STK1 and STK2 and its application in improving the efficiency of plant nuclear male sterile lines.

Background technique

Due to the existence of heterosis, the biomass, resistance to diseases and insect pests, and stress tolerance (drought, high temperature, low temperature, saline-alkali, etc.) of hybrids are considerably improved compared with their parents, such as the yield of hybrid corn and hybrid rice. higher than that of the homozygous parents. The method usually used to produce hybrids is: plant the female parent and the male parent together, remove the tassels of the female parent, and keep the tassels of the male parent, and the seeds harvested by the female parent are hybrids.

There are three types of plants in nature: self-pollination, cross-pollination and normal cross-pollination. Self-pollination refers to the phenomenon that the pollen of a plant pollinates the pistil of the same individual. In plants with bisexual flowers, it can be divided into the same flower pollination between the stamen and pistil of the same flower (Phaseolus), adjacent flower pollination between different flowers in one inflorescence (individual), and different flowers of the same plant. Cross-pollination of the same plant for pollination between flowers. In some plants, stamens and pistils do not grow in the same flower, or even on the same plant, and cannot self-pollinate. Their pistils can only get pollen from other flowers, which is called cross-pollination. A class of crops with a natural hybridization rate higher than 50% and selfing decline is classified as abnormal cross-pollinated crops, such as corn.

Corn is monoecious, and the male and female flowers are located in different parts of the plant. Corn can reproduce through self-pollination and cross-pollination. Under natural conditions, when the wind blows pollen from the tassel to the filament of the ear That is, natural pollination is complete.

In corn breeding, a homozygous maize inbred line should be developed first, and then the two inbred lines should be crossed, and the yield and stress resistance of the hybrid offspring should be evaluated to determine whether it has commercial potential. Each of these inbred lines may have one or more good traits that the other inbred line lacks, or complement one or more bad traits that the other inbred line lacks. The first-generation seeds of two inbred lines crossed are F1 generation seeds, and the F1 generation plants are obtained after the F1 generation seeds germinate. Both have strong advantages and have more biomass.

Hybrids can be produced by manually detasseling the female parent, that is, removing the tassels of the female parent that has not loosened powder (it can be sown with the male parent at intervals in the field, such as sowing 5 rows of female parents and one row of male parents), and retaining the tassels of the male parent . Subsequently, as long as the foreign corn pollen is isolated, the ear of the female parent can only accept the pollen of the male parent, and the resulting seed is a hybrid (F1), which can be used for agricultural production.

In the process of producing hybrids, due to changes in the environment, the plants may become tasseled after emasculation, or the emasculation is incomplete. Both of the above two situations can lead to self-pollination of the female parent, resulting in mixed production of hybrids. For the seeds of the female parent inbred line, the yield of the female parent inbred line is much lower than that of the hybrid. Such seeds are unqualified products, which will not only affect the income of farmers but also affect the reputation of the seed production company. The company bears the corresponding liability for compensation.

Female stock can also be detasseled by machine, which is about as reliable as manual detasseling, but is faster and less expensive. However, most detasseling machines cause more damage to the plant than manual detasseling, so there is currently no completely satisfactory detasseling method and people are still looking for less costly and more complete detasseling alternatives .

The stable male sterile system provides a simple and efficient means, and by using inbred lines with nuclear-cytoplasmic male sterile (CMS), onerous detasseling work can be avoided in some genotypes. The method includes three main materials, namely the sterile line: male sterile material, the maintainer line: can provide pollen for the sterile line, so that the offspring of the sterile line are still sterile lines, and the restorer line: can restore the sterile line Fertility. The CMS line is crossed with the restorer line to produce F1, the hybrid used for agricultural production. More specifically, the nuclear-cytoplasmic interaction sterility type shows nuclear-cytoplasmic interaction inheritance. Not only the sterility gene S in the cytoplasm is required, but also the homozygous sterility gene (rfrf) in the nucleus is required, and the two exist at the same time to make the plant show male sterility. If the cytoplasmic gene is fertile N, the male is fertile regardless of whether the nuclear gene is fertile (RfRf) or sterile (rfrf). Similarly, if there is a fertile gene (RfRf) or (Rfrf) in the nucleus, no matter whether the cytoplasmic gene is fertile N or sterile S, the male is also fertile. This male sterile line formed by nuclear-cytoplasmic interaction has a genetic composition of S(rfrf), which cannot produce normal pollen, but can be used as a hybrid female parent. Since the maintainer line N(rfrf) can be found [use it to cross with the sterile line, the F1 produced can still maintain male sterility, that is: S(rfrf)(♀)×N(rfrf)→S(rfrf)(not Fertility)] and can accept the restorer line S(RfRf) or N(RfRf) [using them to cross with the sterile line, the F1 produced are all fertile, that is: S(rfrf)(♀)×S(RfRf) →S(Rfrf)(F1)(fertile), or pollen of S(rfrf)(♀)×N(RfRf)→S(Rfrf)(F1)(fertile)] to restore F1 to male fertility, F1 plants are self-crossed to produce F2, so they can be widely used in agricultural production. The male sterile line can avoid artificial detasseling, save manpower, reduce seed cost, and ensure the purity of the seed. At present, crops such as rice, corn, sorghum, onion, castor, sugar beet and rapeseed have used nuclear-cytoplasmic male sterile to produce hybrid seeds; nuclear-cytoplasmic male sterile lines of other crops are also being widely used Research.

CMS also has its defects. First, it is observed that some CMS materials are susceptible to disease, and second, it is difficult to find restorer lines. These problems hinder the wide application of CMS system in seed production.

One type of genetic sterility is disclosed in US Patents 4,654,465 and 4,727,219 to Brar et al. However, this type of genetic sterility requires the maintenance of corresponding genotypes at multiple distinct loci within the genome, and molecular marker tracking of these loci is required in each generation. Patterson also described a potentially useful but more complex system of chromosomal translocations (see US Patent Nos. 3,861,709 and 3,710,511).

Attempts have been made to optimize male sterility systems, for example, Fabijanski, et al. developed methods for making plants male sterile (EPO 89/3010153.8 publication number 329308 and PCT application PCT/CA90/ 00037). Mainly through the following two ways to suppress the male flower fertility of plants, one method is to connect the promoter specifically expressed by the male tissue with the cytotoxic gene and transfer it into the plant, so that the male flowers cannot normally loose pollen and do not affect other traits; the other It is to interfere with the gene that has been cloned to control the male flower fertility of the plant through transgenic means by means of gene interference, so that it cannot function normally. There is also a means of inhibiting gene expression through some gene regulatory elements, thereby affecting plant fertility (WO90/08829).

In most cases, only plants that are homozygous for the recessive nuclear gene that controls male sterility (msms) will be male sterile, and since male sterile plants cannot self-fertilize, they can only be crossed with heterozygous plants (Msms) , to obtain male sterile plants (msms). And on the same ear, male sterile grains (msms) and fertile heterozygous grains (Msms) exist at the same time. It is impossible to distinguish which are sterile grains and which are fertile grains through the grains. can be distinguished.

In recent years, there are also methods of using transgenes to maintain the sterility of male sterile plants (US6743968). In this method, the pollen lethal gene and the male fertility restoration gene are constructed in a vector, and introduced into the male sterile plant, and the transgenic offspring are fertile, but can only produce pollen without the restoration gene. When such plants are crossed with male sterile plants, the homozygous recessive state of the recessive sterile plants is maintained. It first constructs a transgenic vector, which contains a pollen cell lethal gene, and meanwhile, the vector also contains a dominant gene for restoring plant fertility. The vector is transformed into a male sterile plant, and the vector exists in a heterozygous state in the transgenic plant, and the plant is fertile due to the presence of the restorer gene, and when it is crossed with the male sterile plant, due to the pollen containing the restorer gene (Msms) contains a lethal gene at the same time, making the pollen sterile, so only the pollen (ms) without the restorer gene can be crossed with the male sterile plant female gametes (ms), and the offspring are all recessive homozygous individuals (msms).

As mentioned above, one of the important issues in many works using the male sterile system for seed production is how to use the male sterile gene and how to distinguish male sterile seeds and plants from fertile seeds and plants. Individual sterility is maintained.

A variety of male sterile mutants have been identified in maize (Skibbe et al. 2005), see the table below:

Table 1 Male sterile mutants caused by nuclear genes

The above genes have been cloned successively, such as ms45 (Albertsen et al.1993) and ms26 (PTC/US2006/024273). At the same time, some male sterility genes in rice have been cloned successively, such as dpw (Jing Shi et al.2011) And some male sterility genes identified in Arabidopsis, such as (Aarts, et al.1993).

Contents of the invention

The purpose of the invention is to improve the production efficiency of the sterile line.

The method provided by the invention is used to maintain the homozygous recessive state of male sterile plants, the method comprising:

(a) providing a first plant comprising a homozygous recessive allele that makes the plant male sterile (the homozygous recessive allele is specifically ms45ms45 in the embodiment);

(b) providing a second plant comprising the same homozygous recessive allele that renders the plant male sterile as said first plant and comprising a construct (in an embodiment a transgenic element Ms45-RNAi) exists in a heterozygous state in the second plant (the hybrid Ms45-RNAi/- insert exists in only one chromatid, and its sister chromatid does not contain the transgenic element), the construct Include:

i. a first nucleotide sequence which, when expressed in said first plant, restores male fertility in said first plant;

ii. a second nucleotide sequence which, when present in a heterozygous state, affects pollen fertilization competitiveness;

The first nucleotide sequence (the Ms45 gene expression element shown in sequence 1 in the embodiment) is closely linked to the second nucleotide sequence (interfering fragments of the STK1 and STK2 genes in the embodiment), and the two A nucleotide sequence exists simultaneously in the plant (referred to as the transgenic element Ms45-RNAi in the embodiments of the present invention);

(c) fertilizing the female gametes of the first plant with the male gametes of the second plant to produce offspring that retain the homozygous recessive state of the first plant.

The above method is a method for multiplying male sterile lines of plants;

And/or, the plant, the first plant and the second plant are all dicotyledonous plants or monocotyledonous plants;

And/or, the plant, the first plant and the second plant can be not only corn (Zea mays), but also rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticum aestivum), soybean (Glycine max), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) and other crops.

In the above method, the first nucleotide sequence includes a gene that controls male fertility, such as the wild-type allele Ms45 of ms45 in Table 1, and the gene that controls male fertility is not limited to the genes listed in Table 1 , genes controlling male fertility in maize or in other species can also achieve the purpose of the present invention, and therefore are also within the protection scope of the present invention.

In the above method, in an embodiment of the present invention, the first plant is the maize male sterile mutant ms45, specifically ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)), which is derived from ms45 Homozygous recessive mutant (MaizeGenetics Cooperation Stock Center, 905I; mutation site is recorded in Table 1 ms45) and Zheng 58 (zheng58) backcross offspring.

In the embodiment of the present invention, the first nucleotide sequence is a Ms45 gene expression element, and the Ms45 gene expression element expresses the protein Ms45 in the first plant.

The protein Ms45 is as follows a) or b):

a) a protein composed of amino acid residues shown in Sequence 2;

b) A protein having the same function as Sequence 2 through substitution and/or deletion and/or addition of one or several amino acid residues.

In the above method, the Ms45 gene expression elements include Ms45 gene promoter, Ms45 gene 5'UTR, Ms45 gene exon, Ms45 gene intron, Ms45 gene 3'UTR and Ms45 gene terminator;

The Ms45 gene expression element is a DNA molecule as shown in 1) or 2) or 3) as follows:

1) The coding region comprises a DNA molecule of sequence 1 or the coding region comprises a DNA molecule of the reverse complementary sequence of sequence 3 from the 5' end position 4762-8265;

2) The coding region is the DNA molecule of sequence 1 or the DNA molecule whose coding region is the reverse complementary sequence of sequence 3 from the 5' end position 4762-8265;

3) 1) or 2) undergoes one or several nucleotide substitutions and/or deletions and/or additions and has the same function as 1) or 2).

In the above method, when the second nucleotide sequence exists in a heterozygous state in the second plant, it reduces pollen fertilization competitiveness, but does not affect pollen fertility.

In the above method, the second nucleotide sequence includes an interference fragment of a specific gene; the specific gene is a gene capable of regulating pollen fertilization competitiveness. The reduced expression of this gene does not affect the fertility of the corresponding pollen (different from pollen lethality), and the pollen can develop normally, but the pollen that changes the expression of the transgene and the wild-type pollen have obvious differences in fertilization competitiveness, which is different from the wild-type pollen. Under mixed conditions, the competitiveness of transgenic pollen to participate in fertilization decreased greatly. However, the transgenic pollen can complete normal pollination and fertilization, pollinate under the condition that all of the transgenic pollen (without competition) can bear fruit normally, and achieve a high fruit setting rate.

In one embodiment of the present invention, the second nucleotide sequence includes interference fragments of STK1 and STK2 genes; the reverse complementary sequence of the interference fragments is as in sequence 3 in the sequence listing from 11282-11875 at the 5' end shown. The second nucleotide sequence, that is, the reverse complementary sequence of the complete expression element of the interference fragment is shown in the 10982-13578 position from the 5' end in the sequence 3 of the sequence listing.

The invention relates to nucleotide sequences for controlling male fertility and pollen competitiveness of plants, and a method for amplifying male sterility of plants developed by using these nucleotide sequences and transgenic technology.

Another object of the present invention is to provide a DNA construct.

The construct provided by the present invention is the construct described in the above method (the circular plasmid shown in the sequence 3 of the sequence listing in the embodiment).

The above-mentioned DNA construct can restore the fertility of the male sterile mutant and at the same time change the pollen competitiveness of the plant.

A third object of the present invention is to provide any of the following substances.

The following materials provided by the invention:

1) a plant, the plant is the second plant described in the above method;

2) a tissue culture of regenerative cells produced by the plant of 1);

3) the protoplast produced by the tissue culture described in 2);

4) A plant which is a homozygous recessive male sterile plant produced by the above method.

The above-mentioned second plant can maintain the sterility of the male sterile plant.

The use of the homozygous recessive male sterile plants produced by the above method in the production of hybrids is also within the protection scope of the present invention.

The key of the present invention is to construct the male restorer gene Ms45 and the elements controlling the pollen competitiveness of the plants in a vector, the vector can restore the fertility of the male sterile mutant ms45, and at the same time, the pollen competitiveness of the plants containing the transgenic elements decreases, Therefore, during the breeding process of sterile lines, the proportion of plants (fertile individuals) containing transgenic elements decreases, and the proportion of sterile individuals (sterile lines) increases, thereby realizing the expansion of sterile individuals (sterile lines).

In one embodiment of the present invention, the inventor first constructed a plant transformation vector, which contained expression elements for restoring male fertility genes and interference fragments of corn pollen fertilization competitiveness regulation genes STK1 and STK2 genes, and transformed the vector into into HiIIA×HiIIB corn hybrids, and then use male sterile plants to backcross the obtained transgenic plants, so as to control the plant male fertility restoration gene Ms45 and the interference fragments of the corn pollen fertilization competitiveness regulation genes STK1 and STK2 genes. Male sterile plant ms45. Due to the presence of the restorer gene Ms45, the transgenic plants appeared fertile. When the transgenic heterozygous plant (Msmsms) is crossed with the male sterile plant (msms), the following two offspring will be produced, one is a male sterile individual (sterile line, genotype is msms) that does not contain the transgenic sequence, and the The sterile line can be restored by any wild-type plant, and can be used as a sterile line in the seed production process; the other is a fertile grain containing a transgenic element (maintainer, genotype Msmsms), the maintainer line It is recessive and homozygous at the locus controlling male fertility. Because it contains a complementary transgene sequence, the plant is fertile. Because it also contains interference fragments of the STK1 and STK2 genes that regulate the fertilization competitiveness of corn pollen, the plant pollen competition The force decreases, the ratio of maintainer lines to sterile lines decreases, and the proportion of sterile lines increases, realizing the expansion of sterile lines.

Other objects of the invention will be apparent from the following description and claims.

The present invention and the existing method for multiplying plant nuclear male sterile lines (such as the patent "

Morphological marker method to propagate the plant nuclear male sterile line "), has an outstanding advantage, that is, the ratio of the obtained sterile line is significantly improved (the ratio of the sterile rate obtained by the present invention is increased to about 80%, and the ratio obtained by the existing method The infertility rate is only about 50%.

The present invention reduces the competitiveness of corn pollen to participate in fertilization by reducing the expression level of pollen fertilization competitiveness regulation genes STK1 and STK2 (serinethreonine kinase1; 2) at the RNA level, and carries out this method with the male sterile line technology system of transgenic technology to expand plants Combined to improve the production efficiency of the sterile line.

Description of drawings

Figure 1 shows the male flower phenotypes of the male fertility mutant ms45 (A) and the wild type Ms45 (B).

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

All technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Unless otherwise specified, the technologies used or mentioned herein are standard technologies recognized by those of ordinary skill in the art. Materials , methods and examples are for illustration only, not limitation.

Nuclear male sterility is the result of mutations, suppression or other effects of key genes in the process of microspore formation, and these genes are collectively referred to as male sterility genes. The pollen development pathway is controlled by many genes, so the mutation of many genes will eventually lead to male sterility. At present, a large number of male sterile mutants have been identified in maize (as shown in Table 1), and each male sterile gene has With its specific restorer gene, each male sterile mutant can only be restored by its wild-type allele.

The present invention is example with a male sterile mutant in table 1, as ms45, mutant male flower can not loose powder normally (shown in Fig. 1, A is male sterile mutant, B is wild type), and this mutant male flower can be Restored by the Ms45 gene in wild-type plants. In the present invention, the wild-type restorer gene Ms45 is derived from the inbred line B73, and its sequence is shown in sequence 1, which contains the complete expression elements of the Ms45 gene, including promoter, 5'UTR, exon, intron, 3'UTR and terminators. After the nucleotide shown in sequence 1 is transferred into the ms45 male sterile mutant, the mutant plants become fertile. The Ms45 gene expression element shown in sequence 1 encodes the Ms45 protein shown in sequence 2.

The present invention introduces functional elements in the process of propagating plant nuclear male sterile lines, and the functional elements include promoters, interference fragments of corn pollen fertilization competitiveness regulation genes STK1 and STK2 genes and terminators. Expression of functional elements can lead to decreased competitiveness of pollen pollination.

The accession number of STK1 gene in MaizeGDB is Zm00001d045056.

The accession number of STK2 gene in MaizeGDB is Zm00001d003377.

The following more detailed description is provided by illustration and illustration, which are not intended to limit the scope of the invention.

Embodiment 1, construction comprises the plant transformation vector of male fertility gene Ms45 and functional element and selectable marker gene

The present invention takes the ms45 male sterile mutant in Table 1 as an example to specifically illustrate the implementation.

The complete expression elements and functional elements of the above-mentioned Ms45 gene were constructed into the vector to obtain the transformation vector (the circular plasmid shown in sequence 3 of the sequence listing) constructed by the method. The transformation vector comprises male fertility gene Ms45, functional elements and selection marker gene bar.

In the sequence 3 of the sequence list, the reverse complementary sequence of the complete expression element of the Ms45 gene from the 5' end 4762-8265 (when constructing the vector amplification, a mutation occurs from the 5' 5664th position in the sequence 3, which is the same as the sequence 1 A difference of 1 base is produced, but it does not lead to a change in the encoded amino acid sequence, does not affect the function of the protein, and the rest of the bases do not change), the 10982-13578th position is the reverse complementary sequence of the functional element (including the corn pollen fertilization competition The complete expression elements of the interference fragments of the force regulation genes STK1 and STK2 genes; wherein, the reverse complementary sequence of the interference fragment is shown in position 11282-11875, the reverse complementary sequence of the promoter is shown in position 11876-13578, and the termination 10986-11238), the 13899-15353 is the complete expression element of the selectable marker gene bar (wherein, the 13899-14576 encodes the promoter, and the 14621-15172 encodes the selectable marker gene bar, positions 15179-15353 encode terminators). The male fertility gene Ms45, functional elements and selection marker gene bar are located on the same T-DNA.

Embodiment 2, plant transformation vector transformation corn obtains transgenic corn

(1) Transformation of maize with plant transformation vector

The invention obtains the transgenic plant through the method of agrobacterium infecting the immature corn embryo. The plant transformation vector of Example 1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium, and then the recombinant Agrobacterium was used to infect corn immature embryos, as follows:

The plant transformation vector of Example 1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

The recipients used in the transgenic process in the laboratory were the hybrid F1 generation of the inbred lines HiIIIA and HiIIIB. Maize inbred lines HiIIA and HiIIB (Armstrong C L, Green C E and Phillips R L. Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation News Letter, 1991, 65:92-93).

First plant corn inbred lines HiIIA and HiIIB in the field, and bag them separately when the inbred lines are loosened; then prepare for pollination, there are two pollination methods: HiIIA as the female parent, HiIIIB as the male parent; HiIIA as the male parent, HiIIB as the female parent In this case, 9-11 days after pollination, the immature hybrid immature embryos on the grains of pollinated ears were taken; then the Agrobacterium tumefaciens EHA105 infection was carried out indoors, and the immature embryos invaded by Agrobacterium tumefaciens EHA105 were placed on the selection medium Multiple screenings were carried out to obtain resistant callus, and the resistant callus was regenerated into seedlings to obtain transgenic T0 generation plants. After the transgenic T0 generation was obtained, the pollen of the T0 transgenic plants was used to cross some seed-producing female parents and Ms45 male sterile materials, and observe the phenotype.

The plant transformation vector of Example 1 was introduced into the immature embryos of recipient plants by the Agrobacterium tumefaciens EHA105 infection method, and transgenic plants were obtained after selection by the herbicide bialaphos. The specific method is:

The specific method is:

1. Peel off the immature embryos

1. Remove the bract leaves. Cut off the top of the F1 ear of the hybrid F1 generation of HiIIA and HiIIB about 1cm, insert the ear from the top with tweezers, so that the tweezers can be used as a handle, which is convenient for operation, and then put the ear into a beaker containing disinfectant. According to actual needs, you can Put 4-6 ears in the same beaker.

2. Add about 700ml of disinfectant solution (50% bleach or 5.25% sodium hypochlorite, and add a drop of Tween20) to the beaker to soak the fruit ears. During the 20 minutes of disinfection, rotate the fruit ears from time to time and pat the beaker gently to remove Bubbles on the surface of the grains, so as to achieve the best disinfection effect. After disinfection, take out the ear and put it into a beaker filled with sterilized water, wash it in the water for 3 times, and then prepare to peel the embryo.

3. Put one end of the sterilized ear on a large Petri dish, and use a large scalpel to cut off the top of the grain (1.5-1.8mm). During this process, frequently disinfect the tools used, such as: scalpel blade , petri dish, embryo peeling knife, etc.

4. Insert the tip of the embryo stripping knife between the embryo and the endosperm, then gently pry out the young embryo, and gently lift the young embryo with the tip of a small scalpel to ensure that the young embryo will not be damaged. The hypocotyl surface of the embryo is close to the N6E medium with filter paper, and the density of the embryo is about 2×2cm (30 embryos/dish).

5. Seal the culture dish with a parafilm, and culture in the dark at 28 degrees for 2-3 days.

2. Agrobacterium infection

1. Recombinant Agrobacterium should be cultured on YEP (containing Kana33mg/L and Str100mg/L antibiotics) medium one week in advance, and stored in a 4-degree refrigerator for about one month, and long-term storage should be stored in -80-degree glycerol

2. Recombinant Agrobacterium should be cultured on YEP medium at 19°C for 3 days, and Kana (33mg/L) and Str (50mg/L) should be added at the same time.

3. After 3 days, pick the recombinant Agrobacterium into a 50ml centrifuge tube containing 5mL dipping medium, add 100uM AS (inf+AS) at the same time, and shake the bacteria at room temperature (25 degrees) at 75rpm for 2-4 hours.

4. Dipping immature embryos, put the freshly stripped immature embryos into a centrifuge tube containing inf+AS liquid medium (2ml), about 20-100 immature embryos in each tube, wash 2 times with such medium, and then add 1-1.5ml of Agrobacterium with a specific concentration (OD550=0.3-0.4), gently invert the centrifuge tube 20 times, and then place it upright in a dark box for 5 minutes to ensure that the immature embryos are completely immersed in the Agrobacterium liquid, and avoid vortex oscillation during the whole process .

3. Co-cultivation

1. After dipping, transfer the dipped immature embryos to the co-cultivation medium (the solute is as shown in Table 2, and the solvent is water), so that the hypocotyls of the immature embryos contact the surface of the medium, and simultaneously remove excess Agrobacterium on the surface of the medium;

2. Seal the culture dish with a parafilm, and culture it in the dark at 20 degrees for 3 days.

4. Resting cultivation

1. After 3 days of co-cultivation, transfer the immature embryos to the resting medium (the solute is shown in Table 2, and the solvent is water), and at the same time seal the culture dish with a parafilm, and culture it in the dark at 28 degrees for 7 days.

Five, choose

1. After 7 days, transfer all the immature embryos to the selection medium (the solute is as shown in Table 2, and the solvent is water) (35 immature embryos/dish), cultivate for two weeks, and the selection medium contains 1.5 mg/dish of bialaphos L, after two weeks of subculture, the concentration of bialaphos can rise to 3mg/L.

2. After immersion for about 5 weeks, the cells containing the transformants will grow into visible type II callus.

6. Regeneration of transgenic plants

1. Grow for 3 weeks on the regeneration medium I (the solute is shown in Table 2, and the solvent is water), and then germinate on the regeneration medium II (the solute is shown in Table 2, and the solvent is water) (in the light culture room) to obtain the T0 generation GMO corn.

2. When the regenerated seedlings grow 3-4 leaves, transfer them to the greenhouse, and pollinate them when they grow to the stage of spinning and loosening powder.

Solute and content of the medium in Table 2

In Table 2, the MS salt was purchased from Phyto Technology Laboratories, the product number is M524.

(2), analysis of the transgenic plants obtained

The T0 transgenic maize obtained in (1) was crossed with the ms45 homozygous recessive mutant (ms45 male sterile material, MaizeGenetics Cooperation Stock Center, 905I) to obtain hybrid offspring.

1. Detect the genotype of hybrid offspring

Determine whether the transgenic element Ms45-RNAi is contained by the detection of the bar gene; the bar gene detection method: use the following primers Bar669F and Bar669R: carry out PCR amplification to the hybrid offspring, if it contains a 669bp target fragment, the hybrid offspring contains the transgenic element Ms45- If the hybrid progeny of RNAi does not contain the target fragment with a size of 669bp, the hybrid progeny is a hybrid progeny that does not contain the transgenic element Ms45-RNAi.

The sequences of the above primers Bar669F and Bar669R are as follows:

Bar669F: 5' TCTCGGTGACGGGCAGGAC 3'

Bar669R: 5'TGACGCACAATCCCACTATCCTT 3'

Table 3 shows the statistics of the proportion of grains containing expression elements on the ears of some hybrid progenies.

table 3

The results showed that the proportion of grains containing expression elements on the ears of three transgenic lines was less than 20%, which was due to the decreased competitiveness of pollen pollination of plants containing the transgenic elements, and the transgenic plants were crossed with the male sterile mutant ms45 In the offspring, the proportion of grains containing transgenic elements was low.

STK-68, STK-111 and STK-125 were used in subsequent experiments.

Embodiment 3, using the male sterile maintainer line to carry out large-scale propagation of ms45 male sterile inbred line

The transgenic plants prepared in the examples can be used to multiply the ms45 male sterile inbred line, as follows:

(1) Ms45Ms45 wild-type inbred line transformed into the first plant ms45ms45 homozygous recessive inbred line

The ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 905I, as the female parent) was crossed with the inbred line (such as Zheng 58 (zheng58)), and the F1 obtained was continued to be used with the maize inbred line Zheng 58 (Henan Province Agricultural Institute of Grain Research, Academy of Sciences) for backcrossing, and genotype analysis of the obtained BC1 population, screening for heterozygous plants at the Ms45 site to continue backcrossing with Zheng 58, and after 5-6 generations of backcrossing, use molecular markers to screen for Ms45 The point is heterozygous, and the agronomic traits and phenotypes are similar to those of Zheng 58, so as to obtain the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)), which can be used as a sterile line , which will be called the first plant;

The method for screening the genotype of the Ms45 locus is as follows: use the following primers Ms45 F and Ms45 R for PCR amplification, the size of the target fragment of Ms45: 859bp, and the size of the target fragment of ms45: 811bp. If the 859bp and 811bp target fragments are obtained at the same time, the Ms45 site is heterozygous Ms45/ms45, if there is no 811bp fragment, the Ms45 site is Ms45/Ms45 dominant homozygous; if there is no 859bp target fragment, it is ms45/ms45 Recessive homozygosity.

Ms45 F:5'-CTTGAGCGACAGCGGGAACT-3',

Ms45R: 5'-TGTTGTTTCTTGGCAAAGGTCAG-3'.

(2), prepare the second plant

Preparation of the second plant Ms45-RNAi heterozygous and ms45 homozygous

The ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (first plant, female parent) obtained in (1) was crossed with the T0 generation Ms45-RNAi corn (male parent) obtained in Example 2, Then take the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (the first plant) as the recurrent parent to carry out multi-generation backcrossing, and the T0 generation transfer Ms45-RNAi corn from Example 2 is transformed into containing An inbred line that is heterozygous for Ms45-RNAi and homozygous recessive at the ms45 locus is the second plant that is heterozygous for Ms45-RNAi and homozygous for ms45, also known as Zheng 58 (Ms45-Lc heterozygous and ms45 homozygous recessive).

The specific method is as follows:

The inbred line Zheng 58 (Zheng 58 (ms45ms45)) was hybridized, and the grain containing the transgenic element was selected from the hybrid offspring (determined whether the transgenic element was determined by detecting the bar gene, the same below) and then the T0 generation in Example 2 was sown to the field. Ms45-RNAi corn (male parent) and the ms45 homozygous obtained in (1) were sprayed with 200mM bialaphos recessively, and the surviving plants were continued to be backcrossed with Zheng 58 (ms45ms45), and after 5-6 generations of such backcrossing, Since the grain or plant containing the transgenic element is always selected for crossing with the first plant during the backcross process, the grain or plant containing the transgenic element contains the transgenic site (Ms45-RNAi), so the grain or plant containing the transgenic element in the obtained offspring is transgenic loci (Ms45-RNAi) were all heterozygous. If the first plant is pollinated with the grain or plant pollen containing the transgenic element, if the obtained grain or normal plant without the transgenic element is sterile, the transgenic site Ms45-RNAi of the plant providing the pollen is heterozygous and ms45 The point is homozygous for stealth, which is the second plant.

(3) Acquisition of maintenance system

The second plant of Zheng 58 (Ms45-RNAi heterozygous and ms45 homozygous) obtained in (2) was used as the male parent, and the ms45 homozygous recessive inbred line Zheng 58 obtained in (1) (Zheng 58 (ms45ms45)) (female This) cross, the offspring not only has Zheng 58 (ms45ms45), but also has the male sterile Zheng 58 maintainer line Zheng 58 (Ms45-RNAi heterozygous ms45ms45). The grains of Zheng 58 (ms45ms45) did not contain transgenic elements, while the grains of its maintainer Zheng 58 (Ms45-RNAi heterozygous ms45ms45) contained transgenic elements.

(4), using the male sterile maintainer line to carry out large-scale propagation of the ms45 male sterile inbred line

Taking the Zheng 58 inbred line as an example, the first male sterile line Zheng 58 (ms45ms45) obtained in (1) and the male sterile maintainer Zheng 58 (Ms45-RNAi heterozygous ms45ms45) obtained in (3) were inoculated into In the field, the two materials are sown at intervals, and each row of the maintainer line is planted with 5 rows of the CMS line to ensure that no other corn is sown within 300 meters around the seeding, allowing the CMS line and the maintainer line to be naturally pollinated in the field. The maintainer line can only accept its own pollen, and in the offspring produced, since the grains containing homozygous transgenic elements cannot be distinguished from the heterozygous grains, these grains are discarded, and the grains that do not contain transgenic elements can be used as sterile lines. The male sterile line material received the pollen of the maintainer line, and the progeny that did not contain the transgenic element were the sterile line, while those that contained the transgenic element were the maintainer line. All the maintainer lines will be used to propagate the CMS and maintainer lines in the next year, most of the sterile lines will be used to produce commercial varieties, and the remaining small part will be used to propagate the CMS and maintainer lines in the next year. Since the competitiveness of the pollen pollination of plants containing the transgenic element Ms45-RNAi decreases, the proportion of the maintainer line is much smaller than that of the sterile line (the maintainer line accounts for about 20%, and the sterile line accounts for about 80%), thereby achieving sterility The improvement of the multiplication coefficient and the reduction of cost.

Embodiment 4, utilize the male sterile line among the embodiment 3 large-scale production hybrids

The sterile line produced in Example 3 is a recessive homozygous sterile line controlled by the nucleus, and the sterile line can be restored to fertility by any wild-type plant (Ms45Ms45).

Therefore, as long as one chooses a male sterile (ms45ms45) inbred line, such as a male sterile Zheng 58 (ms45ms45) inbred line with high combining ability, such as Chang 7-2, hybrids with excellent agronomic properties can be produced. . In order to achieve the above purpose, the male sterile inbred line and the wild type inbred line are sown alternately in the field to ensure that there is no other corn sowing within 300 meters around the breeding line, and the ear of the sterile line can only accept the pollen of the wild type inbred line. Wild-type inbred lines can only self-bred. The seeds produced on the ears of the sterile lines are hybrids.

sequence listing

<110> China Agricultural University

<120> Expression Regulation of Pollen Competitiveness Gene STK1;2 and Its Application in Improving the Efficiency of Propagation of Plant Genic Male Sterile Lines

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3504

<212>DNA

<213> Artificial Sequence

<400> 1

tgctgagttc tccttgggtt atccatggtg tctctatgaa aaagatgagt acaatgtgtc 60

tatatccgtt ttcttagggt cccttcttct gccttattac tgactgaatc ggggttacaa 120

aaaaacttcc acgggtgcat gatctccatg ttccacttct cccacctcgc gttgcacatt 180

tcttggatgt cggtggttcc catctgaccg aggcccatca gacacctttc gggacaccca 240

tcaagggcct ttcggatggc ccacgagacg tatcgggtcg tggtgatcca ggggatatat 300

gtcccccaca atcgtcacct atattattat tctttagata ttattaatt tttggaaaaa 360

taacaaactt atacttttgt gtagggcctc agcatagatt ttcgcttagg gcccagaaat 420

gcgaggacca gccatgtcta gtgtccacta ttggcactac ccagaacaag atttaaaaaa 480

ataaccaaag taactaatcc actcgaaagc tatcatgtaa tgtttaaaga aacatctatt 540

aaaaccacga tcctcttaaa aaacaagcat atttcgaaag agacaaatta tgttacagtt 600

tacaaacatc taagagcgac aaattatatc gaaaggtaag ctatgacgtt cagatttttc 660

tttttcattc ttgttatttt gttattgttt ttatatacat tttcttctct tacaatagag 720

tgattttctt ccgattttat aaaatgacta taaagtcatt tttatataag agcacgcatg 780

tcgtagattc tcgttcaaaa atctttctga tttttttaag agctagtttg gcaaccctgt 840

ttctttcaaa gaattttgat tttttcaaaa aaaattagtt tattttctct ttataaaata 900

gaaaacactt agaaaaatag agttgccaga ctagccctag aatgttttcc caataaatta 960

caatcactgt gtataattat ttggccagcc ccataaatta tttaaaccga aactgaaatc 1020

gagcgaaacc aaatctgagc tatttctcta gattagtaaa aagggagaga gagaggaaga 1080

aatcagtttt aagtcattgt ccctgagatg tgcggtttgg caacgatagc caccgtaatc 1140

atagctcata ggtgcctacg tcaggttcgg cagctctcgt gtcatctcac atggcatact 1200

acatgcttgt tcaaccgttc gtcttgttcc atcgtccaag ccttgcctat tctgaaccaa 1260

gaggatacct actcccaaac aatccatctt actcatgcaa cttccatgca aacacgcaca 1320

tatgtttcct gaaccaatcc attaaagatc acaacagcta gcgttctccc gctagcttcc 1380

ctctctcctc tgccgatctt tttcgtccac cagcatggag aagaggaacc tgcagtggcg 1440

gcgagggcgt gatggcatcg tgcagtaccc tcacctcttc ttcgcggccc tggcgctggc 1500

cctcctagtc gcggacccgt tcggcctcag tccgctggcc gaggtcgact accggccggt 1560

gaagcacgag ctcgcgccgt acggggaggt catgggcagc tggcccagag acaatgccag 1620

ccggctcagg cgcgggaggc tggagttcgt cggcgaggtg ttcgggccgg agtctatcga 1680

gttcgatctc cagggccgcg ggccgtacgc cggcctcgcc gacggccgcg tcgtgcggtg 1740

gatgggcgag gaggccgggt gggagacgtt cgccgtcatg aatcctgact ggtaagtgct 1800

cgatatcgct ccggcgtcca ctcgttacat gctataatat agtagtacta agatattttg 1860

atctgatttt ttgcattctt gggagaaacg tcatgcaaaa tttgttgttt cttggcaaag 1920

gtcagaagaa gtctgtgcca atggagtgaa ctcaacgacg aggaagcagc acgagaagga 1980

ggagttctgc ggccggccgc tcggcctgag gttccacggg gagaccggcg agctctacgt 2040

cgccgacgcg tactacggtc tcatggtcgt tggccagagc ggcggcgtgg cgtcctccgt 2100

cgcgagggaa gccgacgggg acccccatccg gttcgcgaac gacctcgatg tgcacaggaa 2160

tggatccgta ttcttcactg acacgagcat gagatacagc agaaagtgag caaagcgacg 2220

taacaatccg gcttctcatt ttcaaacgcc tctgtattct ctgctgaaag agtagctcac 2280

cagacaagag ctgaatttgc agggaccatc tgaacatcct gttagaagga gaaggcaccg 2340

ggaggctgct caggtatgat ccagaaacaa gcggtgtcca tgtcgtgctc aaggggctgg 2400

tgttcccaaa cggcgtgcag atctcagagg accatcagtt tcttctcttc tccgagacaa 2460

caaactgcag gtaacaaaaa tactatctga cgatgctcat gattctaccg tatccatagt 2520

catgaacaca aaccacga atctggcctt gaccaggata atgaggtact ggctggaagg 2580

cccaagagcg ggcgaggtag aggtgttcgc gaacctgccg ggcttccccg acaacgtgcg 2640

ctccaacggc aggggccagt tctgggtggc gatcgactgc tgccggacgc cggcgcagga 2700

ggtgttcgcc aagaggccgt ggctccggac cctgtacttc aagttcccgc tgtcgctcaa 2760

ggtgctcact tggaaggccg ccaggaggat gcacacggtg ctcgcgctcc tcgacggcga 2820

agggcgcgtc gtggaggtgc tcgaggaccg gggccacgag gtgatgaagc tggtgagcga 2880

ggtgcggggag gtgggccgca agctgtggat cggaaccgtg gcgcacaacc acatcgccac 2940

catcccctac cctttagagg actaaccatg atctatgctg tttcaatgcc tcctaatctg 3000

tgtacgtcta taaatgtcta atgcagtcac tggttgtaat cttgtttgtg tttggcaaat 3060

tggcataata atggacagat tcaatgggca ttggtgctgt agtcgcatca cactaattga 3120

atgggatcat gttgagctct cactttgcta caatttgctc cagcttgtac ggttgtaccc 3180

tcttgctcgt ctatagtaag ggccatctaa aaaaaactca aattagatct gcaatacaag 3240

tatgattggg ccgaatttgg attgtcacgg gtccgcgacc gcgaattggg ctcggtttga 3300

tttagccgac atagtagtga ccgacccgag ccggcggcga gccaaaccga gcggacgccg 3360

ccatggatcg cgagtggggc tccaagcccg gcagcggcgg cgccgcctcc gcgcagaatg 3420

aggccatcga ccggcgggag cgcctccgcc gcctggccct cgagaccatc gacctcgcca 3480

aggacccccta tttcatgcgc aacc 3504

<210> 2

<211> 412

<212> PRT

<213> Artificial Sequence

<400> 2

Met Glu Lys Arg Asn Leu Gln Trp Arg Arg Gly Arg Asp Gly Ile Val

1 5 10 15

Gln Tyr Pro His Leu Phe Phe Ala Ala Leu Ala Leu Ala Leu Leu Val

20 25 30

Ala Asp Pro Phe Gly Leu Ser Pro Leu Ala Glu Val Asp Tyr Arg Pro

35 40 45

Val Lys His Glu Leu Ala Pro Tyr Gly Glu Val Met Gly Ser Trp Pro

50 55 60

Arg Asp Asn Ala Ser Arg Leu Arg Arg Gly Arg Leu Glu Phe Val Gly

65 70 75 80

Glu Val Phe Gly Pro Glu Ser Ile Glu Phe Asp Leu Gln Gly Arg Gly

85 90 95

Pro Tyr Ala Gly Leu Ala Asp Gly Arg Val Val Arg Trp Met Gly Glu

100 105 110

Glu Ala Gly Trp Glu Thr Phe Ala Val Met Asn Pro Asp Trp Ser Glu

115 120 125

Glu Val Cys Ala Asn Gly Val Asn Ser Thr Thr Arg Lys Gln His Glu

130 135 140

Lys Glu Glu Phe Cys Gly Arg Pro Leu Gly Leu Arg Phe His Gly Glu

145 150 155 160

Thr Gly Glu Leu Tyr Val Ala Asp Ala Tyr Tyr Gly Leu Met Val Val

165 170 175

Gly Gln Ser Gly Gly Val Ala Ser Ser Ser Val Ala Arg Glu Ala Asp Gly

180 185 190

Asp Pro Ile Arg Phe Ala Asn Asp Leu Asp Val His Arg Asn Gly Ser

195 200 205

Val Phe Phe Thr Asp Thr Ser Met Arg Tyr Ser Arg Lys Asp His Leu

210 215 220

Asn Ile Leu Leu Glu Gly Glu Gly Thr Gly Arg Leu Leu Arg Tyr Asp

225 230 235 240

Pro Glu Thr Ser Gly Val His Val Val Leu Lys Gly Leu Val Phe Pro

245 250 255

Asn Gly Val Gln Ile Ser Glu Asp His Gln Phe Leu Leu Phe Ser Glu

260 265 270

Thr Thr Asn Cys Arg Ile Met Arg Tyr Trp Leu Glu Gly Pro Arg Ala

275 280 285

Gly Glu Val Glu Val Phe Ala Asn Leu Pro Gly Phe Pro Asp Asn Val

290 295 300

Arg Ser Asn Gly Arg Gly Gln Phe Trp Val Ala Ile Asp Cys Cys Arg

305 310 315 320

Thr Pro Ala Gln Glu Val Phe Ala Lys Arg Pro Trp Leu Arg Thr Leu

325 330 335

Tyr Phe Lys Phe Pro Leu Ser Leu Lys Val Leu Thr Trp Lys Ala Ala

340 345 350

Arg Arg Met His Thr Val Leu Ala Leu Leu Asp Gly Glu Gly Arg Val

355 360 365

Val Glu Val Leu Glu Asp Arg Gly His Glu Val Met Lys Leu Val Ser

370 375 380

Glu Val Arg Glu Val Gly Arg Lys Leu Trp Ile Gly Thr Val Ala His

385 390 395 400

Asn His Ile Ala Thr Ile Pro Tyr Pro Leu Glu Asp

405 410

<210> 3

<211> 21708

<212>DNA

<213> Artificial Sequence

<400> 3

gtttacccgc caatatatcc tgtcaaacac tgatagtta attcccgatc tagtaacata 60

gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt ttctatcgcg 120

tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa taacgtcatg 180

cattacatgt taattattac atgcttaacg taattcaaca gaaattatat gataatcatc 240

gcaagaccgg caacaggatt caatcttaag aaactttat gccaaatgtt tgaacgatcg 300

gggaaattcg agctggtcac caagtttcgc ttcggcgtgc ttgctgctgc tgttgcagct 360

cgcaggagcg tcgcatgtcg tctacaacta caaggacctc gaagccgagg ctgctgcggc 420

gacggaccag gtgccgccgt ccatcgtcaa ccccctgctc aggacggggt accacttcca 480

gccccccaag aactggatca atgatcccaa cgcgcccatg tactacaagg ggtggtacca 540

tttcttctac caatacaatc ccaagggcgc cgtatggggc aacatcgtgt attggtccct 600

ctcccgtccc agagaaaccc tccctccctc ctccattgga ctgcttgctc cctgttgacc 660

attgggtatt ctgaaccatc gagccatggc tgccaagctg actcgcctcc acagtcttcg 720

cgaacgcctt ggtgccacct tctcctccca tcccaatgaa ctgatagcac tcttttccag 780

gtatgttcac cagggcaagg gaatgcttca ggagggtagc tggacccccga ctgccgagct 840

acctctgcga caggacccgg cggtggacga cgacctgccg caggtggtgg tccaggagaa 900

gcagcatggc ggcggtgctg ccgctcggac tgcgatggcg acggcgacgg cgacgcggga 960

cgggtcggtg ggttggagcg gcaagccgtg ccgcagagac aggtccgggg gatggttcgc 1020

cggcttcctc atgctcgcag tggtggatcg gtcgtgagga gttccttaac atttcttgcg 1080

acatggagta caaaaagagg gtggatgcgc tagtgttttt ctctttactt ctcctaggat 1140

actttgctgc tcatgcgcat gggaacggtc atgtcacgga tgatgtcaat gtttctgctc 1200

cagctgaaga aggaattttg cgagaaaaaa gagcacaatg cgctcaaggg tttcttccat 1260

gaatcctaac aattgattac gaggttaaat cattgtctaa gaaaagtgga ataaaaaggt 1320

caaaataata gtaaaatgtt tagttttgga taagttttgc ttaattaccc tctcatggaa 1380

gaaacccttg agcgcattgt gctctttttt ctcgcaaaat tccttcttca gctggagcag 1440

aaacattgac atcatccgtg acatgaccgt tcccatgcgc atgagcagca aagtatccta 1500

ggagaagtaa agagaaaaac actagcgcat ccaccctctt tttgtactcc atgtcgcaag 1560

aaatgttaag gaactcctca cgaccgatcc accactgcga gcatgaggaa gccggcgaac 1620

catcccccgg acctgtctct gcggcacggc ttgccgctcc aacccaccga cccgtcccgc 1680

gtcgccgtcg ccgtcgccat cgcagtccga gcggcagcac cgccgccatg ctgcttctcc 1740

tggaccacca cctgcggcag gtcgtcgtcc accgccgggt cctgtcgcag aggtagctcg 1800

gcagtcgggg tccagctacc ctcctgaagc attcccttgc cctggtgaac atacctggaa 1860

aagagtgcta tcagttcatt gggatggggag gagaaggtgg caccaaggcg ttcgcgaaga 1920

ctgtggaggc gagtcagctt ggcagccatg gctcgatggt tcagaatacc caatggtcaa 1980

caggggagcaa gcagtccaat ggaggaggga gggagggttt ctctgggacg ggagagggac 2040

caatacacga tgttgcccca tacggcgccc ttgggattgt attggtagaa gaaatggtac 2100

caccccttgt agtacatggg cgcgttggga tcattgatcc agttcttggg gggctggaag 2160

tggtaccccg tcctgagcag ggggttgacg atggacggcg gcacctggtc cgtcgccgca 2220

gcagcctcgg cttcgaggtc cttgtagttg tagacgacat gcgacgctcc tgcgagctgc 2280

aacagcagca gcaagcacgc cgaagcgaaa cttaagcttg ggggtgctat ttgtactgtg 2340

caggcgcagg ccggccggcc ggccggagcg ccggctgacc ggctgcttcg tcgtcccaca 2400

aggggagatat accactacta ctaaaacata ctctctacca caaatctgtg gatctttttg 2460

acgaatatac aaaaatctgt gggttgactc tgaggtctgg agttataagc atatattgct 2520

atatctctgg attaaagttt ttttttgtac gaaggtcagt tgaccatttt agctacgtgt 2580

ctgtctatct agctatacta ttatagaaac aaacttgttg aaaactagct cttagctgcc 2640

atatcacatt ctctatctca cctcttattt caaaattcac tctctaaaca acataaatat 2700

tgctatctac agtgtaaaat agtgttttgc atagctatat atatgatctg ctgaacacaa 2760

ctttagtcgt atgaatctat atatatctac tccatcccaa cctgtctatt ccatatgtcg 2820

ccttaaatat atcctatcat ttctaaaata aatgcacatc gtctatcttt ctgaggagcc 2880

aaatctttta aagttcgatc aaatatataa aagtaattat tgttagatta agcattaaat 2940

gtgtcgacgt ttcgagaccg gggggtccct gggccgacga gtgagtgtcg ccgcgtgccc 3000

cagccccagat gggtcgagcg cgagggcgag cgcgaagggg ggagagcgag gcggccggag 3060

accggcgtga gagaggtggg aatcccgcgg ccttcatgtt cgtcccgcgc cccggtcggg 3120

tgcgcttgca gtagggggtt acaagcgtcc acgcgggaga gggagcgagc ggctccaggc 3180

gagcgcctgt ctcgtcctcg tccccgcgcg gccaaccctc tccaagaggg ccctggtcct 3240

tccttttata ggcgtaagga gaggatccag gtgtacaatg gggggtgtag cagagtgcta 3300

cgtgtctagc ggaggagagc tagcgcccta agtacatgcc gttgtggcag acggagagac 3360

tttggcaccc agctggtgtg atgtcgtggc cgtcggagga gcgatggagc ctggcggagg 3420

gacagctgtc ggagcggttg ggtccttgct gacgtcctct tgcttccgta agggggctga 3480

gagccgccgt cgttacagag tatgcggggc gccatcattg cctatctggc ggagctagcc 3540

agatgggacg ccggtcttgt tccctgcggc ccgagtcagc tcggggtagg gtgatgatgg 3600

cgcctcctgttgacgtggct ggcctgcgcc ctaggttggg cgatgtggag gctcctccga 3660

agccgaggtc gagtctgtct tccgtggccg aggccgagtc cgagcccctg ggtcgggcga 3720

ggcggaggcc gtcggctgag gccagggcgg agtccgagcc ctgggtcggg cgaagcggag 3780

ttcgtcgtct tctggggctg agcccgagtc cgagccctgg gtcgggcgga gcggagttcg 3840

ccgtcttccg ggacttagcc cgagtccgag ccctgggtcg ggcggagcgg agttcgccgt 3900

cttccgggac ttagcccgag tccgagccct gggtcgggcg gagcggagtt cgccgtcttc 3960

cgggacttag cccgagtccg agccctgggt cgggcgaagc ggagcttcct atggtgcctt 4020

cggcagggcc tgactgcctg tcagtctcac tctgtcaagt ggcactgcag tcagagtggc 4080

gcaggcgacg ctgtccttct gtcaggccgg tcagtggagc ggcgaagtga cggcggtcac 4140

ttcggctctg ccggggggcg cgcgtcagga taaaggtgtc aggccacctt tgcattaaat 4200

gctcctgcga tttggtcggt cggtgcggtg atttagtcag ggttgcttct tagcgaaggc 4260

agggctcggg cgagccggaa atatgttcgc cgttggaggg gggcctcggg cgagacggaa 4320

atccttcggg gtcggctgcc cttgtccgag gctaggctcg ggcgaggcgt gatcgagtcg 4380

ctcgaacgga ctaatccctg acttaatcgc acccatcagg ccttagcagc tttatgctga 4440

tggggggttac cagctgagaa ttaggagtct tgagggtacc cctaattatg gtccccgaca 4500

gtagcccccg agcctcgaag ggagtgttag cactcgcttg gaggctttcg tcgcactttt 4560

ttgcaagggg accagccttt ctcggttgca ttttgttccg gtgggtgcgc gcgagcgcac 4620

ccgccgggtg tagcccccga ggcctcggag gagtggtttc actcctttga ggtcttaatg 4680

cctcgcgtaa tgcttcggct ggtctggttg ttctctcatg cgagcccggg ctagggttta 4740

gggcgggtta cctgccgagg tggttgcgca tgaaataggg gtccttggcg aggtcgatgg 4800

tctcgagggc caggcggcgg aggcgctccc gccggtcgat ggcctcattc tgcgcggagg 4860

cggcgccgcc gctgccgggc ttggagcccc actcgcgatc catggcggcg tccgctcggt 4920

ttggctcgcc gccggctcgg gtcggtcact actatgtcgg ctaaatcaaa ccgagcccaa 4980

ttcgcggtcg cggacccgtg acaatccaaa ttcggcccaa tcatacttgt attgcagatc 5040

taatttgagt tttttttaga tggcccttac tatagacgag caagagggta caaccgtaca 5100

agctggagca aattgtagca aagtgagagc tcaacatgat cccattcaat tagtgtgatg 5160

cgactacagc accaatgccc attgaatctg tccattatta tgccaatttg ccaaacacaa 5220

acaagattac aaccagtgac tgcattagac atttatagac gtacacagat taggaggcat 5280

tgaaacagca tagatcatgg ttagtcctct aaagggtagg ggatggtggc gatgtggttg 5340

tgcgccacgg ttccgatcca cagcttgcgg cccacctccc gcacctcgct caccagcttc 5400

atcacctcgt ggccccggtc ctcgagcacc tccacgacgc gcccttcgcc gtcgaggagc 5460

gcgagcaccg tgtgcatcct cctggcggcc ttccaagtga gcaccttgag cgacagcggg 5520

aacttgaagt acagggtccg gagccacggc ctcttggcga aacacctcctg cgccggcgtc 5580

cggcagcagt cgatcgccac ccagaactgg cccctgccgt tggagcgcac gttgtcgggg 5640

aagcccggca ggttcgcgaa cacttctacc tcgcccgctc ttgggccttc cagccagtac 5700

ctcattatcc tggtcaaggc cagattcgtg tggtttgtgt tcatgactat ggatacggta 5760

gaatcatgag catcgtcaga tagtattttt gttacctgca gtttgttgtc tcggagaaga 5820

gaagaaactg atggtcctct gagatctgca cgccgtttgg gaacaccagc cccttgagca 5880

cgacatggac acctgcttgtt tctggatcat acctgagcag cctcccggtg ccttctcctt 5940

ctaacaggat gttcagatgg tccctgcaaa ttcagctctt gtctggtgag ctactctttc 6000

agcagagaat acagaggcgt ttgaaaatga gaagccggat tgttacgtcg ctttgctcac 6060

tttctgctgt atctcatgct cgtgtcagtg aagaatacgg atccattcct gtgcacatcg 6120

aggtcgttcg cgaaccggat ggggtccccg tcggcttccc tcgcgacgga ggacgccacg 6180

ccgccgctct ggccaacgac catgagaccg tagtacgcgt cggcgacgta gagctcgccg 6240

gtctccccgt ggaacctcag gccgagcggc cggccgcaga actcctcctt ctcgtgctgc 6300

ttcctcgtcg ttgagttcac tccatggca cagacttctt ctgacctttg ccaagaaaca 6360

acaaattttg catgacgttt ctcccaagaa tgcaaaaaat cagatcaaaa tatcttagta 6420

ctactatatt atagcatgta acgagtggac gccggagcga tatcgagcac ttaccagtca 6480

ggattcatga cggcgaacgt ctcccacccg gcctcctcgc ccatccaccg cacgacgcgg 6540

ccgtcggcga ggccggcgta cggcccgcgg ccctggagat cgaactcgat agactccggc 6600

ccgaacacct cgccgacgaa ctccagcctc ccgcgcctga gccggctggc attgtctctg 6660

ggccagctgc ccatgacctc cccgtacggc gcgagctcgt gcttcaccgg ccggtagtcg 6720

acctcggcca gcggactgag gccgaacggg tccgcgacta ggagggccag cgccagggcc 6780

gcgaagaagaga ggtgagggta ctgcacgatg ccatcacgcc ctcgccgcca ctgcaggttc 6840

ctcttctcca tgctggtgga cgaaaaagat cggcagagga gagagggaag ctagcggggag 6900

aacgctagct gttgtgatct ttaatggatt ggttcaggaa acatatgtgc gtgtttgcat 6960

ggaagttgca tgagtaagat ggattgtttg ggagtaggta tcctcttggt tcagaatagg 7020

caaggcttgg acgatggaac aagacgaacg gttgaacaag catgtagtat gccatgtgag 7080

atgacacgag agctgccgaa cctgacgtag gcacctatga gctatgatta cggtggctat 7140

cgttgccaaa ccgcacatct cagggacaat gacttaaaac tgatttcttc ctctctctct 7200

ccctttttac taatctagag aaatagctca gatttggttt cgctcgattt cagtttcggt 7260

ttaaataatt tatggggctg gccaaataat tatacacagt gattgtaatt tatgggaaa 7320

acattctagg gctagtctgg caactctatt tttctaagtg ttttctattt tataaagaga 7380

aaataaacta attttttttg aaaaaatcaa aattctttga aagaaacagg gttgccaaac 7440

tagctcttaa aaaaatcaga aagatttttg aacgagaatc tacgacatgc gtgctcttat 7500

ataaaaatga ctttatagtc attttataaa atcggaagaa aatcactcta ttgtaagaga 7560

agaaaatgta tataaaaaca ataacaaaat aacaagaatg aaaaagaaaa atctgaacgt 7620

catagcttac ctttcgatat aatttgtcgc tcttagatgt ttgtaaactg taacataatt 7680

tgtctctttc gaaatatgct tgttttttaa gaggatcgtg gttttaatag atgtttcttt 7740

aaacattaca tgatagcttt cgagtggatt agttactttg gttatttttt taaatcttgt 7800

tctgggtagt gccaatagtg gacactagac atggctggtc ctcgcatttc tgggccctaa 7860

gcgaaaatct atgctgaggc cttacacaaa agtataagtt tgttattttt ccaaaaatta 7920

aataatatct aaagaataat aatataggtg acgattgtgg gggacatata tcccctggat 7980

caccacgacc cgatacgtct cgtgggccat ccgaaaggcc cttgatgggt gtcccgaaag 8040

gtgtctgatg ggcctcggtc agatgggaac caccgacatc caagaaatgt gcaacgcgag 8100

gtgggagaag tggaacatgg agatcatgca cccgtggaag tttttttgta accccgattc 8160

agtcagtaat aaggcagaag aagggaccct aagaaaacgg atatagacac attgtactca 8220

tctttttcat agagacacca tggataaccc aaggagaact cagcatcaca ggcccgatct 8280

agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta tattttgttt 8340

tctatcgcgt attaaatgta taattgcggg actctaatca taaaaaccca tctcataaat 8400

aacgtcatgc attacatgtt aattattaca tgcttaacgt aattcaacag aaattatatg 8460

ataatcatcg caagaccggc aacaggattc aatcttaaga aactttatg ccaaatgttt 8520

gaacgatcgg ggaaattcga gctggtcacc tgtaattcac acgtgtcacc gcttccctat 8580

agctttgcga agagcctcgc tgatcatcca gggcacgacg gcaccggagc cagcaaactg 8640

agctcgtatc ttaagcccca tgaagccatc tggcgctgaa gcctgaaccg agaggacgtc 8700

caaatggagg ctcttgatgg cgtcgaacac tcgcgtcatc aggagctcct cccaccggca 8760

ctgcacctcc aggagcacgt ccttgtccga gacggtgacg gtgacgttgc tggtgccggc 8820

gtccatggtg aggaccgggg ggcgctccac gtcgtctctg ccgagctctg ggctcttcct 8880

cttggagccc gcgcagacct ccttcctcac actctcatta ttgccacggg agggccttgt 8940

tattagcctt gtcgtcgttt cggatgggcg cgacgcaggt tccctactgg actccagctc 9000

ttgcaccctt ctctgaagct ccttgaggta ggctatcgtt tcggcgagga tcgacgcttt 9060

gttcaccctg tgaatggacg gaagcagtga cttgaggacg aggaacatct cgttgagctt 9120

ctctcgtcgc tttcgctccg acatgacgtg gttcttggtg ccagtgccac tcatttcctg 9180

tgctgctccc gtcgcgccgc cacagctctc ccaagcaccg ccgccggcca ccactttctt 9240

cagcaatctc tgcggctcct cgatggccgg cactactgct gcgggcgccg cgtcgtcgga 9300

gcacgacgac tgctgcgagg acctcgtcca agccatgaaa ctcgtcgcgc gagagccgta 9360

gacgggtgcc cttgaggcgt cggcggcgac gttagcggta gccctgtcca ccggaggcgg 9420

cgctggctgc ggggaagagc aggggacctc gaaattggac gcgtccacgg tccagccgtc 9480

ctctagcggt agtggtagcg cctgcaggtc catttcgtcg cagaggctgt agaactcctc 9540

gatctccttg gtgatgtgct ccaggcttgc gtcgtctgac agggcctcgg cttctcttag 9600

tcttagctcc tcctcctgcc cgtgtccccc ggcggcggtc atcgcctcta tgtcgtccat 9660

gccattattg tggtcgagtt cctcgaacgc aaacgtgccg tcgtctgctg cggcctcgcc 9720

ggtctcgttt gctcgtcctg acgggctgga gctcgggcac tgcggctccc agaaagccgc 9780

ggttgctcgg ctgaccaagt ccggggcctc cggcaccgtg tcagttgtac caagctcaag 9840

cacgccgccc ataaccggga tgcagaggat tgactgaatg gacgcgctct tggccaggag 9900

cgcgcggggg aaggctttgc tgccggcgag gtgcgcgttg cacagccaga catgctcgtc 9960

gctcgcgaaa ctcctgccgg gcaacccttg gcctggccgg aaggcgtagg tcatggagac 10020

cacgtagtac cactcggtgt cgccgaggtc ctccggcgac agagagccgg ccggccgcgc 10080

aggcgcagcg cggcggtcgc cctcgcccga caggagggcc tcgtagagct cccggagctg 10140

gtcgctcctc tgcatgacga gctggtcgga tgtcagctcc acggagttgg agatcttccg 10200

cgtcttcacc tcgccgttgt agaacccgtc cgtccacgtc agcaccctg gttgagtgtc 10260

tgaaatggac cagaagaggg cgtagctcca gttgatgctc ctggcggctg cagcaagctg 10320

gctcctcatc agctgcctct cagcaggtcg ttgcagcagt tcttccgcct gctgaactcg 10380

ggaagctgaa agcgccatct cgatccgctc accacgaac ccgaaccccg gagaacctca 10440

cgaacccgaa cccgaaccgg aggggggagga gaagccaacc cacctctaag aaagcacgga 10500

gagggaatgc gtcccccgca gcgtctttat agccgagcgc ggggccgccg cggccgtcct 10560

gatcccatcc gacggcgcgg acgcccagtg gaatatgcgg ggcatcgggg tcgccaccgc 10620

gctacgagca cacgagcacc tcccggggac ggctttgttg ggacccggag caggacggga 10680

gcgtgaggcg gggcccagcg tgactgacct gacctcgttt tggccgctgc actgccggcc 10740

tgtgcaggcc accgaaaaat atctcgcgtg gcgaaatgcc atccttgccc tcgggagcac 10800

acggatcgga cggcaagcgg tcgtcgggtc gggtggtgga tatggacact tgctgatgac 10860

gtgggtccac gatggcagtg ggcgaagcca agtccacgcg tcacagtgac cgcacctagt 10920

gctcaaaaac gacggatcgg ataggaacga atacatgtca caattcacta ccttgtaatc 10980

tgcccgatct agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta 11040

tattttgttt tctatcgcgt attaaatgta taattgcggg actctaatca taaaaaccca 11100

tctcataaat aacgtcatgc attacatgtt aattattaca tgcttaacgt aattcaacag 11160

aaattatatg ataatcatcg caagaccggc aacaggattc aatcttaaga aactttatg 11220

ccaaatgttt gaacgatcgg ggaaattcga gctggtcacc tgtaattcac acgtgtcaca 11280

gcgcaaggtg ggcgagggcg ggtacggccc cgtgtacaag ggcttcctcg accacacgca 11340

ggtggccatc aaggtgctcc ggcccgacgc ggcgcagggc cggtcgcagt tccagcagga 11400

ggtggaggtg ctgagctgca tccggcaccc caacatggtg ctcctcctcg gcgcgtgccc 11460

ggagtacggc tgcctcgtgt acgagtacat ggccagcggc agcctggacg actgcctgtt 11520

ccaatcctaa caattgatta cgaggttaaa tcattgtcta agaaaagtgg aataaaaagg 11580

tcaaaataat agtaaaatgt ttagttttgg ataagttttg cttaattacc ctctggaaca 11640

ggcagtcgtc caggctgccg ctggccatgt actcgtacac gaggcagccg tactccgggc 11700

acgcgccgag gaggagcacc atgttggggt gccggatgca gctcagcacc tccacctcct 11760

gctggaactg cgaccggccc tgcgccgcgt cgggccggag caccttgatg gccacctgcg 11820

tgtggtcgag gaagcccttg tacacggggc cgtacccgcc ctcgcccacc ttgcgcttcg 11880

cagctcctgc tccaagattg acgtctctta ttaaagcacg ccattttagg gatgtggtgg 11940

ccgtttgggc cgtacgcgcc ggccaaaaat agagctgttt atcgggggct tcaacctcgt 12000

tcaacgggtg gccactggac aacggattca tgttcctgtg gaacacgttt ggttactcga 12060

ctttttattt acactttgga aacaaacagt caaacaccat cacaaaacta ccatggctca 12120

cgagcgcaca cgattgtaac ctatatactt attcgctaag gggtgtttga gagtgaagtt 12180

ttttcacagt tttggaagaa taaaatacta cggtattata tacagaattg tgtttggcaa 12240

gttggctaaa atctctgttt ttaaaactga agtattgcaa atactatagt tgttttaagt 12300

tattctaaac ttaggtctgg acctcagttt tcaaaattgt ggtattgtaa actgcagtat 12360

tgtcataact aaagtatact gtagtatttc aaaaactgtg gttttcaaaa actttgttcc 12420

caaacagggc ctaaatgggt aggcactaag cacagtacga cccgcgatgc tttgacacga 12480

cacggacacg atttatataa tgtcatacgg cacaggttta gtcacatctt tggaccacga 12540

cctccgcaca atggactgac atgaacacga cagtttagac cagatcccgt gacaatccgg 12600

cacaatccag tgccccatcc cctggtccac agccttcaca gctctggccc ggccttgcca 12660

tatcgtgcac gggggtgtgt ccagaatgta cctactggag cgcgctgcac gctggcgagc 12720

gatccactca ccgttgatcc caacaaactc cggtccccaa actccacagc accaacagct 12780

aagcccgatg cgctgcgtgc gcggcgatcc aaccgccggc tcacctaaaa atttcggcac 12840

gtctaactgc gactggcagg tgcgcacgcg tggtcgcgcg gaataaagcg gacacgttgc 12900

gcccccagcg aagcccgcac gcatcgcatt cgcatcgcat cgcaggtcgc atccgacgct 12960

agcggctagc ctagccgaac agcctgagcg cgcgaagatg gcgcccgccg acggcgagtc 13020

ctccccgccg ccgcacgtgg ccgtggtcgc cttcccgttc agctcccacg cggcggtgct 13080

gctctccatc gcgcgcgccc tggctgccgc cgcggcgccg tccggggcca cgctctcgtt 13140

cctctccacc gcgtcctccc tcgcgcagct ccgcaaggcc agcagcgcct ccgccgggca 13200

cgggctcccg gggaacctgc gcttcgtcga ggtaccggac ggcgcgcccg cggccgagga 13260

gaccgtgccg gtgccgcggc agatgcagct gttcatggag gccgcggagg ccggaggggt 13320

gaaggcctgg ctggaggcgg cccgcgccgc ggcgggcggc gccagggtga cctgcgtggt 13380

gggcgacgcg ttcgtgtggc ctgcggcgga cgcggccgcc tccgcggggg cgccgtgggt 13440

gccggtgtgg acggccgcgt cgtgcgcgct cctggcgcac gtccgcaccg acgcgctccg 13500

ggaggacgtc ggcgaccagg gtgcgttgga ttctactact acagtactac ttctctccct 13560

tccttgtccc ttcattgcga attcgtaatc atggtcatag ctgtttcctg tgtgaaattg 13620

ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 13680

tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 13740

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 13800

gcgtattggc tagagcagct tgccaacatg gtggagcacg acactctcgt ctactccaag 13860

aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 13920

atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat caaaaggaca 13980

gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa ggctatcgtt 14040

caagatgcct ctgccgacag tggtcccaaa gatggaccccc cacccacgag gagcatcgtg 14100

gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga taacatggtg 14160

gagcacgaca ctctcgtcta ctccaagaat atcaaagata cagtctcaga agaccaaagg 14220

gctattgaga cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca 14280

gctatctgtc acttcatcaa aaggacagta gaaaaggaag gtggcaccta caaatgccat 14340

cattgcgata aaggaaaggc tatcgttcaa gatgcctctg ccgacagtgg tcccaaagat 14400

ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag 14460

caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct 14520

tcgcaagacc ttcctctata taaggaagtt catttcattt ggagaggaca cgctgaaatc 14580

accagtctct ctctacaaat ctatctctct cgagtctacc atgagcccag aacgacgccc 14640

ggccgacatc cgccgtgcca ccgaggcgga catgccggcg gtctgcacca tcgtcaacca 14700

ctacatcgag acaagcacgg tcaacttccg taccgagccg caggaaccgc aggagtggac 14760

ggacgacctc gtccgtctgc gggagcgcta tccctggctc gtcgccgagg tggacggcga 14820

ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc aacgcctacg actggacggc 14880

cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg ggactgggct ccacgctcta 14940

cacccacctg ctgaagtccc tggaggcaca gggcttcaag agcgtggtcg ctgtcatcgg 15000

gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc ggatatgccc cccgcggcat 15060

gctgcgggcg gccggcttca agcacgggaa ctggcatgac gtgggtttct ggcagctgga 15120

cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc accgagattt gactcgagtt 15180

tctccataat aatgtgtgag tagttcccag ataagggaat tagggttcct atagggtttc 15240

gctcatgtgt tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct 15300

atcaataaaa tttctaattc ctaaaaccaa aatccagtac taaaatccag atcccccgaa 15360

ttaattcggc gttaattcag tacattaaaa acgtccgcaa tgtgtttatta agttgtctaa 15420

gcgtcaattt gtttacacca caatatatcc tgccaccagc cagccaacag ctccccgacc 15480

ggcagctcgg cacaaaatca ccactcgata caggcagccc atcagtccgg gacggcgtca 15540

gcgggagagc cgttgtaagg cggcagactt tgctcatgtt accgatgcta ttcggaagaa 15600

cggcaactaa gctgccgggt ttgaaacacg gatgatctcg cggagggtag catgttgatt 15660

gtaacgatga cagagcgttg ctgcctgtga tcaccgcggt ttcaaaatcg gctccgtcga 15720

tactatgtta tacgccaact ttgaaaacaa ctttgaaaaa gctgttttct ggtatttaag 15780

gttttagaat gcaaggaaca gtgaattgga gttcgtcttg ttataattag cttcttgggg 15840

tatctttaaa tactgtagaa aagaggaagg aaataataaa tggctaaaat gagaatatca 15900

ccggaattga aaaaactgat cgaaaaatac cgctgcgtaa aagatacgga aggaatgtct 15960

cctgctaagg tatataagct ggtgggagaa aatgaaaacc tatatttaaa aatgacggac 16020

agccggtata aagggaccac ctatgatgtg gaacgggaaa aggacatgat gctatggctg 16080

gaaggaaagc tgcctgttcc aaaggtcctg cactttgaac ggcatgatgg ctggagcaat 16140

ctgctcatga gtgaggccga tggcgtcctt tgctcggaag agtatgaaga tgaacaaagc 16200

cctgaaaaga ttatcgagct gtatgcggag tgcatcaggc tctttcactc catcgacata 16260

tcggattgtc cctatacgaa tagcttagac agccgcttag ccgaattgga ttacttactg 16320

aataacgatc tggccgatgt ggattgcgaa aactgggaag aagacactcc atttaaagat 16380

ccgcgcgagc tgtatgattt tttaaagacg gaaaagcccg aagaggaact tgtcttttcc 16440

cacggcgacc tgggagacag caacatcttt gtgaaagatg gcaaagtaag tggctttat 16500

gatcttggga gaagcggcag ggcggacaag tggtatgaca ttgccttctg cgtccggtcg 16560

atcagggagg atatcgggga agaacagtat gtcgagctat tttttgactt actggggatc 16620

aagcctgatt gggagaaaat aaaatattat atttactgg atgaattgtt ttagtaccta 16680

gaatgcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 16740

gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 16800

acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 16860

tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 16920

ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 16980

atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 17040

agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 17100

cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 17160

agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 17220

acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 17280

gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 17340

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 17400

gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 17460

gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 17520

gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 17580

cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 17640

ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 17700

acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 17760

ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagggtg 17820

ccttgatgtg ggcgccggcg gtcgagtggc gacggcgcgg cttgtccgcg ccctggtaga 17880

ttgcctggcc gtaggccagc catttttgag cggccagcgg ccgcgatagg ccgacgcgaa 17940

gcggcggggc gtagggagcg cagcgaccga agggtaggcg ctttttgcag ctcttcggct 18000

gtgcgctggc cagacagtta tgcacaggcc aggcgggttt taagagtttt aataagtttt 18060

aaagagtttt aggcggaaaa atcgcctttt ttctctttta tatcagtcac ttacatgtgt 18120

gaccggttcc caatgtacgg ctttgggttc ccaatgtacg ggttccggtt cccaatgtac 18180

ggctttgggt tcccaatgta cgtgctatcc acaggaaaga gaccttttcg acctttttcc 18240

cctgctaggg caatttgccc tagcatctgc tccgtacatt aggaaccggc ggatgcttcg 18300

ccctcgatca ggttgcggta gcgcatgact aggatcgggc cagcctgccc cgcctcctcc 18360

ttcaaatcgt actccggcag gtcatttgac ccgatcagct tgcgcacggt gaaacagaac 18420

ttcttgaact ctccggcgct gccactgcgt tcgtagatcg tcttgaacaa ccatctggct 18480

tctgccttgc ctgcggcgcg gcgtgccagg cggtagagaa aacggccgat gccgggatcg 18540

atcaaaaagt aatcggggtg aaccgtcagc acgtccgggt tcttgccttc tgtgatctcg 18600

cggtacatcc aatcagctag ctcgatctcg atgtactccg gccgcccggt ttcgctcttt 18660

acgatcttgt agcggctaat caaggcttca ccctcggata ccgtcaccag gcggccgttc 18720

ttggccttct tcgtacgctg catggcaacg tgcgtggtgt ttaaccgaat gcaggtttct 18780

accaggtcgt ctttctgctt tccgccatcg gctcgccggc agaacttgag tacgtccgca 18840

acgtgtggac ggaacacgcg gccgggcttg tctcccttcc cttcccggta tcggttcatg 18900

gattcggtta gatgggaaac cgccatcagt accaggtcgt aatcccacac actggccatg 18960

ccggccggcc ctgcggaaac ctctacgtgc ccgtctggaa gctcgtagcg gatcacctcg 19020

ccagctcgtc ggtcacgctt cgacagacgg aaaacggcca cgtccatgat gctgcgacta 19080

tcgcgggtgc ccacgtcata gagcatcgga acgaaaaaat ctggttgctc gtcgcccttg 19140

ggcggcttcc taatcgacgg cgcaccggct gccggcggtt gccggggattc tttgcggatt 19200

cgatcagcgg ccgcttgcca cgattcaccg gggcgtgctt ctgcctcgat gcgttgccgc 19260

tgggcggcct gcgcggcctt caacttctcc accaggtcat cacccagcgc cgcgccgatt 19320

tgtaccgggc cggatggttt gcgaccgtca cgccgattcc tcgggcttgg gggttccagt 19380

gccattgcag ggccggcaga caacccagcc gcttacgcct ggccaaccgc ccgttcctcc 19440

acacatgggg cattccacgg cgtcggtgcc tggttgttct tgattttcca tgccgcctcc 19500

tttagccgct aaaattcatc tactcattta ttcatttgct catttactct ggtagctgcg 19560

cgatgtattc agatagcagc tcggtaatgg tcttgccttg gcgtaccgcg tacatcttca 19620

gcttggtgtg atcctccgcc ggcaactgaa agttgacccg cttcatggct ggcgtgtctg 19680

ccaggctggc caacgttgca gccttgctgc tgcgtgcgct cggacggccg gcacttagcg 19740

tgtttgtgct tttgctcatt ttctctttac ctcattaact caaatgagtt ttgattaat 19800

ttcagcggcc agcgcctgga cctcgcgggc agcgtcgccc tcgggttctg attcaagaac 19860

ggttgtgccg gcggcggcag tgcctgggta gctcacgcgc tgcgtgatac gggactcaag 19920

aatgggcagc tcgtacccgg ccagcgcctc ggcaacctca ccgccgatgc gcgtgccttt 19980

gatcgcccgc gacacgacaa aggccgcttg tagccttcca tccgtgacct caatgcgctg 20040

cttaaccagc tccaccaggt cggcggtggc ccatatgtcg taagggcttg gctgcaccgg 20100

aatcagcacg aagtcggctg ccttgatcgc ggacacagcc aagtccgccg cctggggcgc 20160

tccgtcgatc actacgaagt cgcgccggcc gatggccttc acgtcgcggt caatcgtcgg 20220

gcggtcgatg ccgacaacgg ttagcggttg atcttcccgc acggccgccc aatcgcgggc 20280

actgccctgg ggatcggaat cgactaacag aacatcggcc ccggcgagtt gcagggcgcg 20340

ggctagatgg gttgcgatgg tcgtcttgcc tgacccgcct ttctggttaa gtacagcgat 20400

aaccttcatg cgttccccctt gcgtatttgt ttatttactc atcgcatcat atacgcagcg 20460

accgcatgac gcaagctgtt ttactcaaat acacatcacc tttttagacg gcggcgctcg 20520

gtttcttcag cggccaagct ggccggccag gccgccagct tggcatcaga caaaccggcc 20580

aggatttcat gcagccgcac ggttgagacg tgcgcgggcg gctcgaacac gtacccggcc 20640

gcgatcatct ccgcctcgat ctcttcggta atgaaaaacg gttcgtcctg gccgtcctgg 20700

tgcggtttca tgcttgttcc tcttggcgtt cattctcggc ggccgccagg gcgtcggcct 20760

cggtcaatgc gtcctcacgg aaggcaccgc gccgcctggc ctcggtgggc gtcacttcct 20820

cgctgcgctc aagtgcgcgg tacagggtcg agcgatgcac gccaagcagt gcagccgcct 20880

ctttcacggt gcggccttcc tggtcgatca gctcgcgggc gtgcgcgatc tgtgccgggg 20940

tgagggtagg gcgggggcca aacttcacgc ctcgggcctt ggcggcctcg cgcccgctcc 21000

gggtgcggtc gatgattagg gaacgctcga actcggcaat gccggcgaac acggtcaaca 21060

ccatgcggcc ggccggcgtg gtggtgtcgg cccacggctc tgccaggcta cgcaggcccg 21120

cgccggcctc ctggatgcgc tcggcaatgt ccagtaggtc gcgggtgctg cgggccaggc 21180

ggtctagcct ggtcactgtc acaacgtcgc cagggcgtag gtggtcaagc atcctggcca 21240

gctccgggcg gtcgcgcctg gtgccggtga tcttctcgga aaacagcttg gtgcagccgg 21300

ccgcgtgcag ttcggcccgt tggttggtca agtcctggtc gtcggtgctg acgcgggcat 21360

agcccagcag gccagcggcg gcgctcttgt tcatggcgta atgtctccgg ttctagtcgc 21420

aagtattcta ctttatgcga ctaaaacacg cgacaagaaa acgccaggaa aagggcaggg 21480

cggcagcctg tcgcgtaact taggacttgt gcgacatgtc gttttcagaa gacggctgca 21540

ctgaacgtca gaagccgact gcactatagc agcggagggg ttggatcaaa gtactttgat 21600

cccgagggga accctgtggt tggcatgcac atacaaatgg acgaacggat aaaccttttc 21660

acgccctttt aaatatccgt tattctaata aacgctcttt tctcttag 21708

Claims (5)

1. the method for keeping the homozygous recessive state of maize male sterile plant, described method comprises: (a) providing a first plant comprising a homozygous recessive allele that renders the plant male sterile; (b) providing a second plant comprising the same homozygous recessive allele that renders the plant male sterile as said first plant and comprising a construct, said construct being present in said second plant Existing in a heterozygous state, the construct comprises: i. a first nucleotide sequence which, when expressed in said first plant, restores male fertility in said first plant; ii. a second nucleotide sequence which, when present in a heterozygous state, affects pollen fertilization competitiveness; The first nucleotide sequence is closely linked to the second nucleotide sequence, and the two nucleotide sequences exist simultaneously in the second plant; The second nucleotide sequence includes an interference fragment of a specific gene; the specific gene is a gene capable of regulating pollen fertilization competitiveness; the reduction in the expression of the specific gene will lead to a decrease in pollen fertilization competitiveness, but does not affect pollen fertility sex; The second nucleotide sequence includes interference fragments of STK1 and STK2 genes; The reverse complementary sequence of the interference fragment is shown in the sequence 3 from the 5' end 11282-11875 in the sequence listing; (c) fertilizing the female gametes of the first plant with the male gametes of the second plant to produce offspring that retain the homozygous recessive state of the first plant. 2. The method according to claim 1, characterized in that: the method is a method for multiplying male sterile lines of plants. 3. The method according to claim 1 or 2, characterized in that: the first plant is maize male sterile mutant ms45; And/or, the first nucleotide sequence includes genes controlling male fertility. 4. The method according to claim 3, characterized in that: The gene controlling male fertility is a Ms45 gene expression element, and the Ms45 gene expression element expresses protein Ms45 in the first plant; The protein Ms45 is composed of the amino acid residues shown in Sequence 2. 5. Application of the method according to claim 1 in the production of hybrids.

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