CN115918523B

CN115918523B - Intelligent sterile line propagation method of millet independent of seed sorting machine

Info

Publication number: CN115918523B
Application number: CN202310095585.9A
Authority: CN
Inventors: 刁现民; 张伟; 智慧; 任玉双; 贾冠清; 张林林; 汤沙; 高远瞩
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2023-02-10
Filing date: 2023-02-10
Publication date: 2024-03-15
Anticipated expiration: 2043-02-10
Also published as: CN115918523A

Abstract

The invention discloses an intelligent millet sterile line propagation method independent of a seed sorting machine. Transferring fertility gene editing vectors and expression cassette vectors containing intelligent sterile linkage elements into wild millet to obtain an intelligent millet maintainer line; selfing the intelligent millet maintainer line to obtain selfed seeds of the intelligent millet maintainer line, wherein the selfed seeds with red color are the seeds of the intelligent millet maintainer line; yellow selfed seeds are intelligent sterile line seeds of millet and can be distinguished by naked eyes; planting self-bred seeds of the intelligent millet maintainer line and intelligent sterile line seeds of the millet, and completing screening of sterile lines and maintainer lines by manually distinguishing single spikes of the fertile and sterile plants in the field; meanwhile, the sterile line is fertilized with the maintainer line pollen to realize rapid mass propagation of pure sterile line seeds. The millet has the characteristics of small grains, high propagation coefficient, small seed quantity for sowing in unit area and the like, and the method can avoid large-scale seed sorting steps and the dependence on a high-precision seed sorting machine.

Description

Intelligent sterile line propagation method of millet independent of seed sorting machine

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an intelligent millet sterile line propagation method independent of a seed sorting machine.

Background

By crossing two parents with different traits to produce F ₁ The hybrid of the generation can exceed the parents in terms of vigor, viability, fertility, adaptability, yield and quality, so-called hybrid vigor. Therefore, the utilization of heterosis is also an important means for improving yield and quality in crops. For self-pollinated crops represented by rice, in order to facilitate the production of hybrid seeds, it is necessary to use a male sterile line as the female parent for hybrid seed production. Millet also has hybrid vigor, and at present, the production and application of hybrid vigor are realized by means of the millet of the high male sterile line. However, the method has the problems that the purity of the hybrid seeds is uneven, the millet restorer needs to be cultivated into a dominant herbicide-resistant type, and the seed propagation of the sterile line is difficult due to low fruiting rate of the high male sterile line.

The sterility of common nuclear male sterile line can reach 100%, the purity of the produced hybrid is high, however, the sterile line can not be self-matured and the maintainer line is difficult to find. By introducing the intelligent sterile linkage element expression cassette consisting of fertility restorer gene, transgenic pollen inactivating gene and transgenic seed color marker gene into a common nuclear male sterile line, the generated transgenic plant selfing can generate 50% of seeds containing color markers and 50% of seeds without color markers, wherein the seeds containing color markers are intelligent maintainer line seeds, and the seeds without color markers are intelligent sterile line seeds and do not contain transgenic components. The seeds can be further sorted by a high-precision fluorescent seed sorter so as to further reproduce sterile lines or be used for preparing hybrid seeds. At present, such intelligent sterility systems are also established on millet. However, the small grain size of millet causes sorting difficulty and lacks high-precision equipment for sorting seeds of the intelligent sterile system of millet, thus seriously affecting the application of the intelligent sterile system of millet.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a millet intelligent sterile line seed propagation method, which avoids large-scale seed sorting steps and dependence on a high-precision seed sorting machine.

In order to achieve the purpose, the invention provides a propagation method of intelligent sterile line seeds of millet.

The propagation method of the intelligent sterile line seeds of millet provided by the invention comprises the following steps:

1) Transferring a complete set of vectors for creating the intelligent millet maintainer line into wild millet to obtain transgenic millet, and screening the transgenic millet to obtain the intelligent millet maintainer line; selfing the intelligent millet maintainer line to obtain intelligent millet maintainer line selfing seeds, wherein the intelligent red-colored selfing seeds are intelligent millet maintainer line seeds, and the intelligent yellow-colored selfing seeds are intelligent sterile seeds;

2) Planting the intelligent maintenance line selfing seeds and the intelligent sterile line seeds of the millet, wherein the intelligent maintenance line selfing seeds of the millet are used as parent lines, and the intelligent sterile line seeds of the millet are used as parent lines;

3) During the flowering period, searching and marking a male-fertile plant with full anther and normal cracking and scattering powder in the parent line, wherein the male-fertile plant is the intelligent maintenance line of the millet, marking a male-sterile plant with shrunken anther or without cracking and scattering powder in the parent line, and the male-sterile plant is the intelligent sterile line of the millet;

4) After open pollination, seeds are harvested from the marked male sterile plants, namely the intelligent sterile line seeds of the millet (pure intelligent sterile line seeds of the millet);

the complete set of vectors for creating the intelligent millet maintainer line comprises a fertility gene knockout vector and an intelligent sterile expression cassette vector;

the fertility gene knockout vector comprises an sgRNA expression cassette and a Cas9 nuclease expression cassette;

the intelligent sterile expression cassette vector comprises an aminoglycoside phosphotransferase expression cassette, a fertility restorer gene expression cassette, a corn alpha amylase expression cassette and a betalain expression cassette;

the betalain expression cassette expresses protein a, protein B and protein C;

the protein a is A1) or A2):

a1 Amino acid sequence is protein shown as sequence 7 in a sequence table;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 7 in the sequence table and has the same function;

the protein B is B1) or B2):

b1 Amino acid sequence is protein shown as sequence 8 in a sequence table;

b2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 8 in the sequence table and has the same function;

the protein C is C1) or C2):

c1 Amino acid sequence is protein shown as a sequence 9 in a sequence table;

c2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 9 in the sequence table and has the same function.

In the method, the male parent row and the female parent row can be planted according to the proportion of 1:2 or other proportions.

Further, the planting mode can be drill seeding,

still further, the row spacing for planting may be 0.35 meters or other row spacing.

In the above method, the fertility gene may be any gene known in the art that can regulate fertility of millet (i.e., the fertility of millet containing the gene is normal, the knockout of the gene is a male sterile line), such as the Sinp1 gene, the Sinks 2 gene in "Zhang W, zhi H, tang S, zhang H S, sui Y, jia G Q, wu C Y, diao X M,2021.Identification of no pollen 1provides acandidate gene for heterosis utilization in foxtail millet[J ]. Crop J.,2021,9 (6): 1309-1319", and the like.

The sgrnas target the fertility gene target sequences.

In a specific embodiment of the invention, the fertility gene is a SiPKS2 gene, and the nucleotide sequence of the SiPKS2 gene is shown as a sequence 1 in a sequence table.

The target sequence of the sgRNA is shown as a sequence 2 in a sequence table.

Further, the Cas9 nuclease is Csn1 endonuclease; the Csn1 endonuclease is D1) or D2):

d1 Amino acid sequence is protein shown as sequence 4 in a sequence table;

d2 Amino acid sequence shown in sequence 4 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and has the same function.

Still further, the Cas9 nuclease expression cassette comprises, in order, a promoter sequence, a simian virus 40 nuclear localization signal sequence, a Csn1 endonuclease encoding gene sequence, a dual-component nuclear localization signal sequence of nucleolytic enzyme, and a terminator sequence.

Further, the promoter is a maize ubiquitin promoter, and the nucleotide sequence of the promoter is shown in the 2539-4525 positions of the sequence 1 in table 1.

The simian virus 40 nuclear localization signal sequence is shown in Table 1 at positions 4533-4553 of sequence 1.

The bi-component nuclear localization signal sequence of the plasmin is shown in the 8679-8726 of the sequence 1 in table 1.

The terminator is nopaline synthase terminator, and the nucleotide sequence of the terminator is shown in the 8739-8991 positions of the sequence 1 in the table 1.

In the above method, the fertility restorer gene may be any gene known in the art that can regulate fertility of millet (i.e., the fertility of millet containing the gene is normal, the knockout of the gene is a male sterile line), such as the Sinp1 gene, the Sinks 2 gene in "Zhang W, zhi H, tang S, zhang H S, sui Y, jia G Q, wu C Y, diao X M,2021.Identification of no pollen 1provides acandidate gene for heterosis utilization in foxtail millet[J ]. Crop J.,2021,9 (6): 1309-1319", and the like.

In a specific embodiment of the invention, the fertility restorer gene is a SiPKS2 gene.

Further, the fertility restorer gene expression cassette comprises, in order, a promoter sequence, a SiPKS2 genomic DNA sequence, and a terminator sequence.

Further, the promoter is a SiPKS2 promoter, and the nucleotide sequence of the promoter is shown in the 2232-4374 positions of the sequence 2 in the table 1.

The SiPKS2 genomic DNA sequence is shown at positions 4375-5692 of sequence 2 in Table 1.

The terminator is the nopaline synthase terminator.

In the above method, the aminoglycoside phosphotransferase is E1) or E2):

e1 Amino acid sequence is protein shown as sequence 5 in a sequence table;

e2 Amino acid sequence shown in sequence 5 in the sequence table is substituted and/or deleted and/or added by one or more amino acid residues and has the same function.

Further, the aminoglycoside phosphotransferase expression cassette sequentially comprises a promoter sequence, an aminoglycoside phosphotransferase coding gene sequence and a terminator sequence.

Further, the promoter is the tobacco mosaic virus 35S promoter.

The coding gene sequence of the aminoglycoside phosphotransferase is shown in 485-1282 positions of a sequence 2 in table 1.

The terminator is a tobacco mosaic virus 35S polyadenylation signal sequence, and the nucleotide sequence is shown in the 303 th to 477 th positions of the sequence 2 in the table 1.

In the above method, the corn alpha amylase is F1) or F2):

f1 Amino acid sequence is protein shown as a sequence 6 in a sequence table;

f2 Amino acid sequence shown in sequence 6 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and has the same function.

Further, the corn alpha amylase expression cassette sequentially comprises a promoter sequence, a corn alpha amylase coding gene sequence and a terminator sequence.

Further, the promoter is a pollen specific promoter, and the nucleotide sequence of the promoter is shown in the 5957-8732 th position of the sequence 2 in the table 1.

The coding gene sequence of the corn alpha amylase is shown in 8733-10220 of sequence 2 in table 1.

The terminator is the nopaline synthase terminator.

In the method, the betalain expression cassette sequentially comprises a promoter sequence, a protein A coding gene sequence, a self-cleaving oligopeptide sequence, a protein B coding gene sequence, a self-cleaving oligopeptide coding sequence, a protein C coding gene sequence and a terminator sequence.

Further, the self-cleaving oligopeptide is P2A.

Further, the promoter is a barley aleurone layer specific promoter, and the nucleotide sequence of the promoter is shown in the 10500-11300 position of the sequence 2 in the table 1.

The sequence of the protein A coding gene is shown in 11307-12797 of sequence 2 in table 1.

The coding gene sequence of the protein B is shown in 12864-13688 of the sequence 2 in the table 1.

The coding gene sequence of the protein C is shown in 13755-15263 positions of sequence 2 in table 1.

The self-cleaving oligopeptide sequence is shown at positions 12798-12863 of sequence 2 in table 1.

In the above method, the fertility gene knockout vector may specifically be a SiPKS2 knockout vector. The SiPKS2 knockout vector is obtained by replacing a DNA fragment between 8997-9737 th sites of the pYLCRISPR/Cas9Pubi-H vector with a DNA molecule shown in a sequence 3 in a sequence table, and keeping other sequences of the pYLCRISPR/Cas9Pubi-H vector unchanged. The nucleotide sequence of the pYLCRISPR/Cas9Pubi-H vector is shown as sequence 1 in Table 1.

The intelligent sterile expression cassette vector can be specifically a pSiPKS2-RUBY vector. The nucleotide sequence of the pSiPSS 2-RUBY vector is shown as sequence 2 in Table 1.

In the method, the screening method is to select Gu Zichun synthetic mutant which contains intelligent sterile expression cassette and has fertility gene knocked out from the transgenic millet, and the Gu Zichun synthetic mutant is the intelligent millet maintainer line.

Further, the fertility gene is a SiPKS2 gene. The intelligent sterile expression cassette consists of the aminoglycoside phosphotransferase expression cassette, the fertility restoration gene expression cassette, the corn alpha amylase expression cassette and the betalain expression cassette.

Furthermore, the Gu Zichun synthetic mutant containing the intelligent sterile expression cassette and having the fertility gene knocked out is a Gu Zichun synthetic mutant containing the intelligent sterile expression cassette and having the SiPKS2 gene (sequence 1 in the sequence table) deleted at the 405 th nucleotide (namely, deletion of all the 405 th nucleotides of the SiPKS2 gene in the homologous chromosome).

In the above method, the wild millet is specifically CY464.

The application of the method in the hybrid seed production of millet also belongs to the protection scope of the invention.

In order to achieve the aim, the invention finally provides a method for producing seeds by hybridization of millet.

The method for hybrid seed production of millet provided by the invention comprises the steps of carrying out hybrid seed production by taking the intelligent sterile line of millet obtained by the method as a female parent (sterile line) and taking a wild millet variety as a male parent (restorer line).

Compared with the prior art, the invention has the following beneficial effects:

1. the seeds of the pure millet sterile line can be rapidly propagated in large quantity without a large-scale seed sorting step.

2. The sterile line has sterility rate and sterility degree up to 100%, and the sterile line can be used as female parent to produce pure millet hybrid, so that it has important application in agricultural production.

The invention provides a seed propagation method of intelligent sterile line of millet, which comprises the steps of transferring fertility gene editing vectors and expression cassette vectors containing intelligent sterile linkage elements into wild millet by means of transgenic means to obtain intelligent maintenance line of millet. Selfing the intelligent millet maintainer line, wherein the selfing offspring can generate 50% of maintainer line seeds and sterile line seeds respectively, and the red seeds are synthesized by betalains and contain transgenic components; yellow seeds are not synthesized by betalain, are intelligent sterile line seeds, do not contain transgenic components, and can be distinguished by naked eyes. The sterile line plants are in male sterility in the field due to lack of fertility gene expression cassettes; and the maintainer line plants are male-fertile in the field due to the fertility restorer gene expression cassette. Based on the method, the efficient screening of the sterile line and the maintainer line can be completed by manually distinguishing the single spike of the fertile plant from the single spike of the sterile plant in the field. Meanwhile, the sterile line is further fertilized with the maintainer line pollen to realize rapid mass propagation of pure sterile line seeds. The propagation method provided by the invention is very suitable for propagating intelligent sterile line seeds of the millet, and avoids large-scale seed sorting steps and dependence on a high-precision seed sorting machine because the millet has the characteristics of small seeds, high propagation coefficient, small seed quantity for sowing in unit area and the like.

Drawings

FIG. 1 shows the SiPKS2 gene structure, target design and target gene editing type in the Ci846 genetic background.

FIG. 2 is a schematic representation of a fluorescent sorting based inbred seed of the intelligent maintainer line sipks2#5.

FIG. 3 shows wild Ci846, intelligent maintainer line sipks2#5-8B, intelligent sterile line sipks2#5-A floret and pollen I ₂ -KI schematic.

FIG. 4 shows the SiPKS2 gene structure, target design and target gene editing type in CY464 genetic background.

FIG. 5 is a schematic representation of the selfed seed of the intelligent maintainer line sipks2#26 based on betalain synthesis.

FIG. 6 shows wild CY464, intelligent maintainer line sipks2#26-B, intelligent sterile line sipks2#26-A floret and pollen I ₂ -KI schematic.

Fig. 7 is a schematic diagram of a seed propagation strategy for intelligent sterile line of millet independent of a seed sorter.

FIG. 8 is a schematic diagram showing the setting of the intelligent maintainer line sipks2#26-B and the sterile line sipks2#26-A in the strict bagging and open pollination states.

FIG. 9 is a graph showing the seed setting rate statistics and the spike number statistics of the intelligent millet maintainer line sipks2#26-B and the sterile line sipks2#26-A under the conditions of strict bagging and open pollination.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The extraction of millet DNA in the examples below was performed using a modified CTAB method. The method comprises the following specific steps: 0.1-0.2 g of blade is put into a small mortar, a proper amount of liquid nitrogen is added, the powder is immediately ground into a powder, a 2ml centrifuge tube is filled, 800 mu l of CTAB solution preheated at 65 ℃ is added into the centrifuge tube, water bath at 65 ℃ is put after the mixture is carefully mixed for 20 minutes, the centrifuge tube is taken out, 800 mu l of chloroform/isoamyl alcohol solution (chloroform: isoamyl alcohol=24:1) is added after 20 minutes, the mixture is vigorously mixed and centrifuged at 12000rpm for 10 minutes, the supernatant is taken, 800 mu l of chloroform/isoamyl alcohol solution (chloroform: isoamyl alcohol=24:1) is added again, the supernatant is taken out and centrifuged at 12000rpm for 10 minutes, 600 mu l of isopropanol is added into a new centrifuge tube for mixing, and the mixture is put at-20 ℃ for more than half an hour. The precipitated DNA was centrifuged at 12000rpm for 10 minutes. The supernatant was removed and the pellet was washed twice with 500. Mu.l of 70% ethanol, dried by centrifugation, dissolved in 100. Mu.l of deionized water and stored in a refrigerator at-20 ℃.

The genomic sequence of the SiPKS2 gene in the following examples is shown as sequence 1 in the sequence table.

The cultivation method of millet CY464 in the following example is as follows: the millet Ci846 is used as a female parent and the millet Yugu 1 is used as a male parent for hybridization to obtain filial generation, and then the filial generation is continuously selfed for 5 generations (the genome of the millet is made to be homozygous) to obtain the millet CY464. Millet Ci846 is described in the literature "Zhao M C, tang S, zhang H S et al DROOPY LEAF1 controls LEAF architecture byorchestrating early brassinosteroid signaling [ J ]. PNAS 2020,117 (35): 21766-21774. Millet yucu 1 is described in the literature "Zhang W, zhi H, tang S, zhang H S, sui Y, jia G Q, wu C Y, diao X M,2021.Identification of no pollen 1provides a candidate gene for heterosis utilization in foxtail millet[J ]. Crop j.,2021,9 (6): 1309-1319".

The partial sequences involved in the examples below are shown in Table 1.

TABLE 1

/>

Note that: the nucleotide sequence of the pYLCRISPR/Cas9Pubi-H vector is numbered 1; the nucleotide sequence of the pSiPKS2-RUBY vector is numbered 2; the nucleotide sequence of the pSiPKS2-DsRed vector is numbered 3.

Example 1 obtaining Intelligent maintenance System of millet

1. Design of knockout target point

According to the SiPKS2 gene sequence, a plant CRISPR/Cas9 online target point is utilized to design website CRISPR-P2.0 (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /) to design a SiPKS2 gene knockout target sequence, and finally the SiPKS2 gene knockout target sequence obtained by screening is as follows: 5'-TCTGGACATCTCCAACAAGG-3' (SEQ ID NO: 2 of the sequence Listing).

2. Fragment acquisition for fusion ligation and containing target sequences

1. First round PCR

Using pYLsgRNA-OsU aLacZ plasmid as template, 1 μl of each of primers U-F (5'-CTCCGTTTTACCTGTGGAATCG-3') and gR-R (5'-CGGAGGAAAATTCCATCCAC-3') and 0.5 μl of each of primers LAP5-gRT (5'-TCTGGACATCTCCAACAAGGGTTTTAGAGCTAGAAAT-3') and LAP5-OsU aT (5'-CCTTGTTGGAGATGTCCAGACGGCAGCCAAGCCAGCA-3') were added to 50 μl of the system. PCR reaction conditions: 94℃for 10s,58℃for 15s,68℃for 20s,25-28 cycles.

2. Second round PCR

The first round PCR product was diluted 10-fold as template, and primers U-GAL (5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GAR were added to 50. Mu.l of the system

(5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGCTCTT G-3') 1.5. Mu.l each. PCR reaction conditions: 98℃10s,58℃15s,68℃1min,28-30 cycles.

The second round PCR product is the DNA fragment which is used for the ligation of the fusion and contains the target sequence, and the nucleotide sequence of the DNA fragment is shown as a sequence 3 in a sequence table.

3. Construction of SiPKS2 knockout vectors

1. The pYLCRISPR/Cas9Pubi-H vector was digested for 5H at 37℃using restriction enzyme Bsa I (Cat No. R3535L, new England Biolabs, united States) to obtain a pYLCRISPR/Cas9Pubi-H linear vector.

The nucleotide sequence of the pYLCRISPR/Cas9Pubi-H vector is shown as a sequence 1 in a table 1, wherein, the 103 th-277 th positions of the sequence 1 are tobacco mosaic virus 35S polyadenylation signal sequences, the 317 th-1342 th positions are coding gene sequences of hygromycin B phosphotransferase, and the 1410 th-2087 th positions are tobacco mosaic virus 35S promoter sequences; the 2539-4525 th site is corn ubiquitin promoter sequence, the 4533-4553 th site is simian virus 40 nuclear localization signal sequence, the 4578-8678 th site is coding gene sequence of Cas9 (Csn 1) endonuclease of streptococcus pyogenes II type CRISPR/Cas system, the coding sequence is Csn1 endonuclease shown in sequence 4 in the sequence table, the 8679-8726 th site is bi-component nuclear localization signal sequence of nucleolytic enzyme, and the 8739-8991 th site is nopaline synthase terminator.

2. The DNA fragment for ligation of the ligation obtained In the second step and containing the target sequence was ligated into the pYLCRISPR/Cas9Pubi-H linear vector using In-Fusion HD Cloning Kits (Cat No.639648, takara, japan) to obtain a ligation product.

3. The ligation products were transformed into E.coli competent cells Trans-T1 (Cat no.CD501, transGen Biotech, china) and plated on kanamycin-containing solid LB medium, and the single clones were picked up and sequenced using primers 5'-GTGGTGATAAGCGTCCTG-3' and 5'-AAGGCGATTAAGTTGGGT-3' (target product sequence size 1235 bp) to obtain the SiPKS2 knockout vector.

Sequencing results showed that: the SiPKS2 knockout vector is obtained by replacing a DNA fragment between 8997-9737 th positions of the pYLCRISPR/Cas9Pubi-H vector with a DNA molecule shown in a sequence 3 in a sequence table, and keeping other sequences of the pYLCRISPR/Cas9Pubi-H vector unchanged.

4. Construction of intelligent sterile vector for millet

1. Construction of intelligent sterile vector pSiPSS 2-RUBY of millet based on betalain synthesis

The DNA fragment containing the transgenic pollen inactivating element and the betalain synthesizing element is connected into the pCAMBIA-1305 vector through EcoRI and Pml I enzyme cutting sites to obtain an intelligent sterile intermediate vector, and then a SiPKS2 expression cassette (the SiPKS2 expression cassette sequentially comprises a SiPKS2 promoter sequence and a SiPKS2 genome sequence) is connected into the intelligent sterile intermediate vector through HindIII enzyme cutting sites to obtain a millet intelligent sterile vector pSiPKS2-RUBY based on betalain synthesis.

The nucleotide sequence of the pSiPSS 2-RUBY vector is shown as a sequence 2 in a table 1, wherein the 303 th to 477 th positions of the sequence 2 are a tobacco mosaic virus 35S polyadenylation signal sequence, the 485 th to 1282 th positions are coding gene sequences of aminoglycoside phosphotransferase, the coding sequence is the aminoglycoside phosphotransferase shown as a sequence 5 in a sequence table, and the 1345 th to 2022 nd positions are a tobacco mosaic virus 35S promoter sequence; 2232-4374 are SiPKS2 promoter sequences, 4375-5692 are SiPKS2 genomic sequences, 5699-5951 are nopaline synthase terminators; 5957-8732 is pollen specific promoter sequence, 8733-10220 is corn alpha amylase coding gene sequence, coding corn alpha amylase shown in sequence 6 in the sequence table, 10221-10473 is nopaline synthase terminator; the 10500-11300 th site is a barley aleurone layer specific promoter sequence, the 11307-15263 th site is a betalain synthesis element sequence (11307-12797 th site is a coding gene sequence of protein A, the 12798-12863 th site is a self-cleaving oligopeptide sequence, the 12864-13688 th site is a coding gene sequence of protein B, the 13689-13754 th site is a self-cleaving oligopeptide sequence, the 13755-15263 th site is a coding gene sequence of protein C, the 15304-15556 th site is a nopaline synthase terminator).

2. Construction of fluorescent-labeled-based intelligent millet sterile vector pSiPKS2-DsRed

The DNA fragment containing the transgenic pollen inactivating element and the fluorescent marker element is connected into the pCAMBIA-1305 vector through EcoRI and Pml I enzyme cutting sites to obtain an intelligent sterile intermediate vector, and then a SiPKS2 expression cassette (the SiPKS2 expression cassette sequentially comprises a SiPKS2 promoter sequence and a SiPKS2 genome sequence) is connected into the intelligent sterile intermediate vector through HindIII enzyme cutting sites to obtain the fluorescent marker-based millet intelligent sterile vector pSiPKS2-DsRed.

The nucleotide sequence of the pSiPSS 2-DsRed vector is shown as a sequence 3 in a table 1, wherein, the 303 th to 477 th positions of the sequence 3 are tobacco mosaic virus 35S polyadenylation signal sequences, the 485 th to 1282 th positions are coding gene sequences of aminoglycoside phosphotransferase, and the 1345 th to 2022 nd positions are tobacco mosaic virus 35S promoter sequences; the 2238-4380 site is SiPKS2 promoter sequence, the 4381-5698 site is SiPKS2 genome sequence, the 5705-5957 site, the 10227-10479 site and the 11998-12250 site are nopaline synthase terminator sequence, the 5963-8738 site is pollen specific promoter sequence, the 8739-10226 site is corn alpha amylase coding gene sequence, the 10485-11285 site is barley aleurone layer specific promoter sequence, and the 11286-11963 site is red fluorescent protein coding gene sequence.

5. Obtaining and identifying millet intelligent maintainer line

1. Acquisition of sipks2#5 and sipks2#7

Introducing a SiPKS2 knockout vector and a fluorescence-marker-based millet intelligent sterile vector pSiPKS2-DsRed into agrobacterium EHA105 to obtain recombinant bacteria; the SiPKS2 knockout vector and the intelligent sterile vector pSiPKS2-DsRed based on fluorescent markers are used for co-transforming the Ci846 immature embryo callus of the millet by using an agrobacterium-mediated genetic transformation means to obtain a T0 generation transgenic plant. Then, DNA detection is carried out on the T0 generation transgenic plant by using primers 5'-AACACCAACTGCGACGACC-3' and 5'-TTGACGAGACGAACAGAGGC-3' (the size of a target product sequence is 1227 bp), and the editing mode and the heterozygosity of the editing site of the transgenic plant are determined. Meanwhile, primers 5'-CTCGAATTTCGGAATCAT-3' and 5'-ACGCCGCTATCAGGGACT-3' (the sequence size of a target product is 1287 bp) are utilized to carry out DNA detection on the T0 generation transgenic plant, and the intelligent sterile expression cassette transgenic positive plant is determined. The sequencing finds that the target spot is a single plant which is homozygous and edited and contains the intelligent sterile expression cassette, and the single plant is named as sipks2#5 and sipks2#7 respectively.

As shown in FIG. 1, the difference of sipks2#5 compared with the genomic DNA of wild-type millet Ci846 is that only one deletion of base A, which is located at 405 st position of sequence 1, occurs in the gene sequence encoding SiPKS2 protein shown in sequence 1 in the sequence table, thereby causing frame shift and premature termination, and the function of SiPKS2 protein is deleted.

As shown in FIG. 1, the difference of sipks2#7 compared with the genomic DNA of wild-type millet Ci846 is that only in the gene sequence encoding SiPKS2 protein shown in sequence 1 in the sequence table, a base deletion of 14bp occurs, and the deleted base is located at 393-408 (CATCTCCAACAAGG) of the sequence 1, thereby causing frame shift and premature termination, and the function of SiPKS2 protein is deleted.

2. Acquisition of sipks2#26

Introducing a SiPKS2 knockout vector and an intelligent sterile vector pSiPKS2-RUBY synthesized based on betalain into agrobacterium EHA105 to obtain recombinant bacteria; and (3) co-transforming young embryo callus of the millet CY464 with a SiPKS2 knockout vector and an intelligent sterile vector pSiPKS2-RUBY based on betalain synthesis by using an agrobacterium-mediated genetic transformation means to obtain a T0 generation transgenic plant. Then, DNA detection is carried out on the T0 generation transgenic plant by using primers 5'-AACACCAACTGCGACGACC-3' and 5'-TTGACGAGACGAACAGAGGC-3' (the size of a target product sequence is 1227 bp), and the editing mode and the heterozygosity of the editing site of the transgenic plant are determined. Meanwhile, primers 5'-CTCGAATTTCGGAATCAT-3' and 5'-ACGCCGCTATCAGGGACT-3' (the sequence size of a target product is 1287 bp) are utilized to carry out DNA detection on the T0 generation transgenic plant, and the intelligent sterile expression cassette transgenic positive plant is determined. The target was found to be a homozygous editing type individual strain containing the intelligent sterile expression cassette by sequencing, and was designated sipks2#26.

As shown in FIG. 4, the difference of sipks2#26 compared with the genomic DNA of wild-type millet CY464 is that only one deletion of base A, which is at 405 st position of sequence 1, occurs in the gene sequence encoding SiPKS2 protein shown in sequence 1 in the sequence table, thereby causing frame shift and premature termination, and the function of SiPKS2 protein is deleted.

6. Fertility analysis of millet intelligent maintainer line

1. Intelligent maintainer line sipks2#5 selfed seed based on fluorescence sorting

The inbred seeds of the intelligent maintainer line sipks2#5 in the mature period are taken for observation.

As a result, as shown in fig. 2, sipks2#5 inbred seeds could not be sorted in the fluorescent field of view without dehulling (fig. 2 left); under the condition of threshing, the sipks2#5 selfed seeds can be sorted under the fluorescent view (right in fig. 2), wherein the seeds with red fluorescence are intelligent maintainer line (sipks2#5-8B) seeds, transgenic components are contained, and the seeds without red fluorescence are intelligent sterile line (sipks2#5-A) seeds, and transgenic components are not contained.

2. Intelligent maintainer line sipks2#26 selfing seed based on betalain synthesis

The inbred seeds of the intelligent maintainer line sipks2#26 in the mature period are taken for observation.

As shown in FIG. 5, the selfed seeds of Sipks2#26 can be sorted either without husking or with husking, wherein red colored seeds have betalain synthesis, are intelligent maintainer (Sipks2#26-B) seeds, and contain transgenic components; yellow seeds do not have betalain synthesis, are intelligent sterile line (sipks2#26-A) seeds, and do not contain transgenic components.

3. Pollen I ₂ Dyeing of KI

1) Placing wild millet variety Ci846, intelligent maintainer line sipks2#5-8B and intelligent sterile line sipks2#5-A anther in anthesis period on glass slide, adding 1 drop of distilled water, mashing with forceps to release pollen grains, and adding 1-2 drops of I ₂ KI solution, cover glass, and observe under low power microscope. The black colored pollen grains with strong activity containing starch are yellow brown pollen grains with dysplasia.

The results are shown in the graph3, I is carried out on wild millet variety Ci846, intelligent maintainer line sipks2#5-8B and intelligent sterile line sipks2#5-A pollen ₂ -KI staining found: the wild millet variety Ci846 pollen can be colored normally (left in FIG. 3), half of the pollen of the intelligent maintainer line sipks2#5-8B can be colored normally, half of the pollen can not be colored normally (right in FIG. 3), and the pollen of the intelligent sterile line sipks2#5-A can not be colored normally (right in FIG. 3).

2) Placing wild millet variety CY464, intelligent maintainer line sipks2#26-B and intelligent sterile line sipks2#26-A anther in anthesis period on a glass slide, adding 1 drop of distilled water, fully mashing the anther with forceps to release pollen grains, and adding 1-2 drops of I ₂ KI solution, cover glass, and observe under low power microscope. The black colored pollen grains with strong activity containing starch are yellow brown pollen grains with dysplasia.

As shown in FIG. 6, I was performed on wild millet variety CY464, intelligent maintainer line sipks2#26-B and intelligent sterile line sipks2#26-A pollen ₂ -KI staining found: the wild millet variety CY464 pollen can be colored normally (left in FIG. 6), half of the intelligent maintainer line sipks2#26-B pollen can be colored normally, half of the pollen can not be colored normally (right in FIG. 6), and the intelligent sterile line sipks2#26-A pollen can not be colored normally (right in FIG. 6).

Example 2 Intelligent seed propagation method for sterile line of millet

1. Intelligent sterile line seed propagation method for millet

Because the millet grain is small, and there is no high precision color selection equipment. The invention provides a method for efficiently propagating pure intelligent sterile line seeds and preparing hybrid seeds based on the preparation of an embodiment 1, which does not need a large-scale seed sorting step and does not depend on a seed sorting machine, and a specific flow chart is shown in fig. 7, wherein the specific implementation steps are as follows:

1. the intelligent maintainer line sipks2#26 selfed seeds (including the millet intelligent sterile line sipks2#26-A seed and the millet intelligent maintainer line sipks2#26-B seed) and the intelligent sterile line sipks2#26-A seed obtained in example 1 were planted in a greenhouse at the department of farm, china, 12 months 2021. The intelligent maintainer line sipks2#26 selfed seeds of the millet are used as parent lines, the intelligent sterile line sipks2#26-A seeds of the millet are used as parent lines, the parent lines and the parent lines are planted according to the proportion of 1:2, the area of a cell is 2.1 m multiplied by 3 m, the planting mode is drill seeding, and the planting line spacing is 0.35 m. The seedling and field management method in the later period are all conventional methods.

2. Because the intelligent sterile line plant lacks a fertility gene expression cassette, the intelligent sterile line plant shows male sterility in the field; whereas intelligent maintainer plants contain a fertility restorer gene expression cassette and thus appear male-fertile in the field. Based on the method, the efficient screening of the sterile line and the maintainer line can be completed by manually distinguishing the single spike of the fertile plant from the single spike of the sterile plant in the field. The method comprises the following specific steps: and searching and marking 10 male-fertile plants with full anther and normal cracking and pollen scattering in the parent line of the district in the flowering period, wherein the male-fertile plants are the intelligent maintenance lines of the millet. Meanwhile, marking a male sterile plant with shrunken anther or without cracking and scattering powder in the cell female parent row, wherein the male sterile plant is the intelligent sterile line of millet.

To investigate the sterility of the intelligent sterile line, 10 marked millet intelligent sterile lines were randomly selected in the master line and subjected to strict bagging selfing. The remaining male sterile plants of the mother line are pollinated open.

3. And harvesting seeds from the male sterile single plant subjected to open pollination to obtain pure intelligent sterile line seeds of the millet, wherein the intelligent sterile line seeds of the millet can be further used as female parents for hybrid seed production.

2. Setting rate

Taking marked spikes of an intelligent maintainer line, an intelligent sterile line (strictly bagging) and an intelligent sterile line (open pollination) in the mature period, randomly counting the floret number and the real number of the 5 first-stage branches of each spike, calculating the setting rate according to the formula of setting rate= (setting number/floret number) multiplied by 100%, and respectively counting the setting rate of 5 strains of the intelligent maintainer line, the intelligent sterile line (strictly bagging) and the intelligent sterile line (open pollination).

As shown in fig. 8, the intelligent maintainer line can be normally set (left in fig. 8), the intelligent sterile line (strict bagging) cannot be set (in fig. 8), and the intelligent sterile line (open pollination) set is basically normal (right in fig. 8).

The statistical result of the setting rate is shown in fig. 9, the setting rate of the intelligent maintainer line is 81.39+ -3.00%, the setting rate of the intelligent sterile line (strict bagging) is 0, and the setting rate of the intelligent sterile line (open pollination) is 51.26+ -3.15%.

3. Spike and grain number

Taking the marked intelligent maintainer line, intelligent sterile line (strictly bagging) and intelligent sterile line (open pollination) single plant ears in the mature period, randomly counting the florets and the real numbers of 5 primary branches per ear, calculating the number of the ears according to the formula of 'ear number=ear weight/thousand weight multiplied by 1000', and respectively counting the number of the ears of 5 plants per intelligent maintainer line, intelligent sterile line (strictly bagging) and intelligent sterile line (open pollination).

The spike number statistics result is shown in fig. 9, the spike number of the intelligent maintainer line is 4400+/-448, the spike number of the intelligent sterile line (strictly bagging) is 0, and the spike number of the intelligent sterile line (open pollination) is 2168+/-215.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. A propagation method of intelligent sterile line seeds of millet comprises the following steps:

4) After open pollination, seeds are harvested from the marked male sterile plants, namely the intelligent sterile line seeds of the millet;

the complete set of vectors for creating the intelligent millet maintainer line comprises a fertility gene knockout vector and an intelligent sterile expression cassette vector; the fertility gene isSiPKS2A gene;

the target sequence of the sgRNA is shown as a sequence 2 in a sequence table;

the Cas9 nuclease is a protein with an amino acid sequence shown as a sequence 4 in a sequence table;

the amino acid sequence of the aminoglycoside phosphotransferase is a protein shown as a sequence 5 in a sequence table;

the corn alpha amylase is a protein with an amino acid sequence shown as a sequence 6 in a sequence table;

the betalain expression cassette expresses protein a, protein B and protein C;

the protein A is a protein with an amino acid sequence shown as a sequence 7 in a sequence table;

the protein B is a protein with an amino acid sequence shown as a sequence 8 in a sequence table;

the protein C is a protein with an amino acid sequence shown as a sequence 9 in a sequence table.

2. The method according to claim 1, characterized in that: and planting the male parent row and the female parent row according to a ratio of 1:2 or other ratios.

3. The method according to claim 1, characterized in that: the Cas9 nuclease expression cassette sequentially comprises a promoter sequence, a simian virus 40 nuclear localization signal sequence, a Csn1 endonuclease coding gene sequence, a dual-component nuclear localization signal sequence of nucleolytic enzyme and a terminator sequence.

4. The method according to claim 1, characterized in that: the fertility restorer gene isSiPKS2And (3) a gene.

5. The method according to claim 1, characterized in that: the betalain expression cassette sequentially comprises a promoter sequence, a protein A coding gene sequence, a self-cleaving oligopeptide sequence, a protein B coding gene sequence, a self-cleaving oligopeptide sequence, a protein C coding gene sequence and a terminator sequence.

6. Use of the method according to any one of claims 1-5 in the hybrid seed production of millet.

7. A method for hybrid seed production of millet, comprising the step of hybrid seed production by taking a intelligent sterile line of millet obtained by the method according to any one of claims 1 to 5 as a female parent and a wild type variety of millet as a male parent.