A method for constructing a millet intelligent maintenance line with convenient identification

Abstract

The application discloses a millet intelligent maintenance line construction method which is convenient to identify. The application prepares a millet intelligent maintenance line by transferring a complete set of vectors for creating the millet intelligent maintenance line into wild type millet. The complete set of vectors for creating the millet intelligent maintenance line comprises a fertility gene knockout vector and an intelligent sterility expression cassette vector. The fertility gene knockout vector comprises an sgRNA expression cassette and a Cas9 nuclease expression cassette. The intelligent sterility expression cassette vector comprises an aminoglycoside phosphotransferase expression cassette, a fertility restoration gene expression cassette, a corn alpha amylase expression cassette and a betalain expression cassette. The selfed offspring of the millet intelligent maintenance line prepared by the application can generate 50% of the maintenance line seeds and the sterile line seeds respectively. The red seeds have betalain synthesis and are intelligent maintenance line seeds. The yellow seeds have no betalain synthesis and are intelligent sterile line seeds, and naked eyes can distinguish them.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a millet intelligent maintainer line construction method. BACKGROUND

[0002] By crossing two parents with different traits, the F1 hybrid can exceed the parents in growth potential, viability, fecundity, adaptability, yield and quality, which is called hybrid vigor. Therefore, in crops, using hybrid vigor is an important means to improve yield and quality. For self-pollinating crops represented by rice, in order to facilitate the production of hybrid seeds, male sterile lines are used as the female parent for hybrid seed production. Millet also has hybrid vigor, and at present, hybrid vigor production and application have been achieved by using highly male sterile lines. However, this method has problems such as uneven hybrid purity, the need to cultivate millet restorer lines into dominant herbicide-resistant types, and the difficulty in seed multiplication of sterile lines due to low seed setting rate of highly male sterile lines.

[0003] Ordinary cytoplasmic male sterile lines have a sterility rate of up to 100%, and the hybrid seeds produced by them have high purity. However, such sterile lines cannot be self-pollinated and set seeds, and it is difficult to find a maintainer line. By introducing an intelligent sterile linkage element expression cassette composed of fertility restoration genes, transgenic pollen inactivation genes and transgenic seed color marker genes into ordinary cytoplasmic male sterile lines, the transgenic plants produced by self-pollination can produce 50% of seeds containing color markers and 50% of seeds without color markers. 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. Further, the sterile line seeds can be sorted by high-precision seed color sorting machines and used to prepare hybrid seeds. SUMMARY

[0004] The purpose of the present application is to provide a millet intelligent maintainer line seed production system. Using the millet intelligent maintainer line seed production system, millet intelligent maintainer lines can be prepared, and further, millet intelligent maintainer line seeds and millet intelligent sterile line seeds can be produced using the millet intelligent maintainer lines.

[0005] To achieve the above-mentioned purpose, the present application first provides a complete set of vectors for creating millet intelligent maintainer lines.

[0006] The complete set of vectors for creating millet intelligent maintainer lines provided by the present application includes a fertility gene knockout vector and an intelligent sterile expression cassette vector;

[0007] The fertility gene knockout vector includes an sgRNA expression cassette and a Cas9 nuclease expression cassette;

[0008] The intelligent sterility expression cassette vector comprises an aminoglycoside phosphotransferase expression cassette, a fertility restoration gene expression cassette, a corn alpha amylase expression cassette, and a betalain expression cassette.

[0009] The betalain expression cassette expresses protein A, protein B, and protein C.

[0010] The protein A is A1) or A2):

[0011] A1) is a protein with an amino acid sequence as shown in SEQ ID NO: 7 in the sequence listing;

[0012] A2) is a protein with an amino acid sequence as shown in SEQ ID NO: 7 in the sequence listing, which has been substituted and / or deleted and / or added by one or more amino acid residues and has the same function;

[0013] The protein B is B1) or B2):

[0014] B1) is a protein with an amino acid sequence as shown in SEQ ID NO: 8 in the sequence listing;

[0015] B2) is a protein with an amino acid sequence as shown in SEQ ID NO: 8 in the sequence listing, which has been substituted and / or deleted and / or added by one or more amino acid residues and has the same function;

[0016] The protein C is C1) or C2):

[0017] C1) is a protein with an amino acid sequence as shown in SEQ ID NO: 9 in the sequence listing;

[0018] C2) is a protein with an amino acid sequence as shown in SEQ ID NO: 9 in the sequence listing, which has been substituted and / or deleted and / or added by one or more amino acid residues and has the same function.

[0019] In the above complete vector for creating a smart maintainer line of foxtail millet, the fertility gene can be any gene known in the art that can regulate the fertility of foxtail millet (i.e., foxtail millet containing the gene has normal fertility, and foxtail millet with the gene knocked out is a male sterile line), such as the SiNP1 gene, the SiPKS2 gene, etc. 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 1 provides a candidate gene for heterosis utilization in foxtail millet [J]. Crop J., 2021, 9(6): 1309-1319”.

[0020] The sgRNA targets the fertility gene target sequence.

[0021] In one specific embodiment of the present application, the fertility gene is SiPKS2 gene, the nucleotide sequence of SiPKS2 gene is shown as sequence 1 in the sequence listing.

[0022] The target sequence of the sgRNA is shown as sequence 2 in the sequence listing.

[0023] Further, the Cas9 nuclease is Csn1 endonuclease; the Csn1 endonuclease is D1) or D2):

[0024] D1) the protein with the amino acid sequence shown as sequence 4 in the sequence listing;

[0025] D2) a protein with the amino acid sequence shown as sequence 4 in the sequence listing after substitution and / or deletion and / or addition of one or several amino acid residues and having the same function.

[0026] Still further, the Cas9 nuclease expression cassette comprises, in order, a promoter sequence, a simian virus 40 nuclear localization signal sequence, a Csn1 endonuclease coding gene sequence, a two-component nuclear localization signal sequence of nucleolysin, and a terminator sequence.

[0027] Still further, the promoter is a maize ubiquitin promoter, the nucleotide sequence of which is shown as sequence 1, positions 2539-4525 in Table 1.

[0028] The simian virus 40 nuclear localization signal sequence is shown as sequence 1, positions 4533-4553 in Table 1.

[0029] The two-component nuclear localization signal sequence of nucleolysin is shown as sequence 1, positions 8679-8726 in Table 1.

[0030] The terminator is a nopaline synthase terminator, the nucleotide sequence of which is shown as sequence 1, positions 8739-8991 in Table 1.

[0031] The fertility restorer gene can be any gene known in the technical field that can regulate the fertility of foxtail millet (i.e., foxtail millet with the gene has normal fertility, and foxtail millet with the gene knocked out is a male sterile line), such as the SiNP1 gene and the SiPKS2 gene in "Zhang W, Zhi H, Tang S, Zhang H S, Sui Y, Jia G Q, Wu C Y, Diao XM, 2021. Identification of no pollen 1 provides a candidate gene for heterosis utilization in foxtail millet [J]. Crop J., 2021, 9(6): 1309-1319".

[0032] In one specific embodiment of the present application, the fertility restorer gene is the SiPKS2 gene.

[0033] Further, the fertility restorer gene expression cassette comprises, in sequence, a promoter sequence, a SiPKS2 genomic DNA sequence, and a terminator sequence.

[0034] Still further, the promoter is the SiPKS2 promoter, and the nucleotide sequence is shown in Table 1, Sequence 2, positions 2232-4374.

[0035] The SiPKS2 genomic DNA sequence is shown in Table 1, Sequence 2, positions 4375-5692.

[0036] The terminator is the above-mentioned nopaline synthase terminator.

[0037] In the above-mentioned complete set of vectors for creating intelligent maintainer lines of foxtail millet, the aminoglycoside phosphotransferase is E1) or E2):

[0038] E1) is a protein with the amino acid sequence shown in Sequence 5 in the Sequence Listing;

[0039] E2) is a protein with the amino acid sequence shown in Sequence 5 in the Sequence Listing, with one or more amino acid residues substituted, deleted, and / or added and having the same function.

[0040] Further, the aminoglycoside phosphotransferase expression cassette comprises, in sequence, a promoter sequence, an aminoglycoside phosphotransferase-encoding gene sequence, and a terminator sequence.

[0041] Still further, the promoter is the above-mentioned tobacco mosaic virus 35S promoter.

[0042] The aminoglycoside phosphotransferase-encoding gene sequence is shown in Table 1, Sequence 2, positions 485-1282.

[0043] The terminator is tobacco mosaic virus 35S polyadenylation signal sequence, the nucleotide sequence is shown as 303-477 of sequence 2 in Table 1.

[0044] In the complete set of vectors for creating the intelligent maintainer line of millet, the corn alpha amylase is F1) or F2):

[0045] F1) the protein with the amino acid sequence shown in sequence 6 in the sequence listing;

[0046] F2) a protein with the amino acid sequence shown in sequence 6 in the sequence listing after substitution and / or deletion and / or addition of one or more amino acid residues and having the same function.

[0047] Further, the corn alpha amylase expression cassette comprises, in order, a promoter sequence, a corn alpha amylase coding gene sequence, and a terminator sequence.

[0048] Further, the promoter is a pollen-specific promoter, the nucleotide sequence is shown as 5957-8732 of sequence 2 in Table 1.

[0049] The corn alpha amylase coding gene sequence is shown as 8733-10220 of sequence 2 in Table 1.

[0050] The terminator is the above-mentioned nopaline synthase terminator.

[0051] In the complete set of vectors for creating the intelligent maintainer line of millet, the betalain expression cassette comprises, in order, 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.

[0052] Further, the self-cleaving oligopeptide is P2A.

[0053] Further, the promoter is a barley aleurone-specific promoter, the nucleotide sequence is shown as 10500-11300 of sequence 2 in Table 1.

[0054] The protein A coding gene sequence is shown as 11307-12797 of sequence 2 in Table 1.

[0055] The protein B coding gene sequence is shown as 12864-13688 of sequence 2 in Table 1.

[0056] The protein C coding gene sequence is shown as 13755-15263 of sequence 2 in Table 1.

[0057] The self-cleaving oligopeptide sequence is shown as 12798-12863 of sequence 2 in Table 1.

[0058] The fertility gene knockout vector in the set of vectors for creating the smart maintainer line of foxtail millet can be specifically a SiPKS2 knockout vector. The SiPKS2 knockout vector is a vector obtained by replacing the DNA fragment between positions 8997-9737 of a pYLCRISPR / Cas9Pubi-H vector with the DNA molecule shown as SEQ ID NO: 3 in the 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 SEQ ID NO: 1 in Table 1.

[0059] The smart sterile expression cassette vector can be specifically a pSiPKS2-RUBY vector. The nucleotide sequence of the pSiPKS2-RUBY vector is shown as SEQ ID NO: 2 in Table 1.

[0060] To achieve the above-mentioned purposes, the application further provides a new use of the set of vectors for creating the smart maintainer line of foxtail millet.

[0061] The application provides an application of the set of vectors for creating the smart maintainer line of foxtail millet in the preparation of a smart maintainer line of foxtail millet.

[0062] The application further provides an application of the set of vectors for creating the smart maintainer line of foxtail millet in the preparation of a smart maintainer line of foxtail millet seed and a smart sterile line of foxtail millet seed.

[0063] To achieve the above-mentioned purposes, the application finally provides a method as described in 1) or 2) below:

[0064] 1) A method for preparing a smart maintainer line of foxtail millet, comprising the following steps: introducing the set of vectors for creating the smart maintainer line of foxtail millet into a wild-type foxtail millet to obtain a transgenic foxtail millet; and screening a smart maintainer line of foxtail millet from the transgenic foxtail millet.

[0065] 2) A method for preparing a smart maintainer line of foxtail millet seed and a smart sterile line of foxtail millet seed, comprising the following steps: obtaining a smart maintainer line of foxtail millet according to the method of 1), and selfing the smart maintainer line of foxtail millet to obtain selfing seeds, wherein red selfing seeds are smart maintainer line seeds of foxtail millet, and yellow selfing seeds are smart sterile line seeds of foxtail millet.

[0066] In the method 1) above, the screening method is to select a foxtail millet homozygous mutant containing a smart sterile expression cassette and a fertility gene knockout from the transgenic foxtail millet, and the foxtail millet homozygous mutant is the smart maintainer line of foxtail millet.

[0067] Further, the fertility gene is SiPKS2 gene. The smart sterile expression cassette is composed of the above-mentioned aminoglycoside phosphotransferase expression cassette, the above-mentioned fertility restoration gene expression cassette, the above-mentioned corn alpha amylase expression cassette and the above-mentioned betalain expression cassette.

[0068] Further, the foxtail millet homozygous mutant containing the smart sterile expression cassette and the fertility gene knockout is a foxtail millet homozygous mutant containing the smart sterile expression cassette and the deletion of the 405th nucleotide of the SiPKS2 gene (sequence 1 in the sequence table) (i.e. the deletion of the 405th nucleotide of the SiPKS2 gene in the homologous chromosome).

[0069] In any of the above-mentioned methods, the wild-type foxtail millet is specifically CY464.

[0070] The application provides a set of vectors for creating a smart maintenance line of foxtail millet, which comprises a fertility gene editing vector and a smart sterile linkage element expression cassette vector. The set of vectors for creating a smart maintenance line of foxtail millet is introduced into a wild-type foxtail millet by transgenic means to obtain a smart maintenance line of foxtail millet. The selfed offspring of the smart maintenance line of foxtail millet can produce each 50% of the maintenance line seed and the sterile line seed (the sterility degree of the sterile line plant reaches 100%), wherein the red seed has betalain synthesis, which is a smart maintenance line seed containing a transgenic component; the yellow seed has no betalain synthesis, which is a smart sterile line seed not containing a transgenic component, and can be distinguished by naked eye. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 For the genetic background of Ci846, the structure of SiPKS2 gene, target design and gene editing type at the target site.

[0072] Figure 2 For the smart maintenance line sipks2#5 selfed seed diagram based on fluorescence sorting.

[0073] Figure 3 For the wild-type Ci846, the smart maintenance line sipks2#5-8B, the smart sterile line sipks2#5-A floret and pollen I2-KI diagram.

[0074] Figure 4 For the genetic background of CY464, the structure of SiPKS2 gene, target design and gene editing type at the target site.

[0075] Figure 5 For the smart maintenance line sipks2#26 selfed seed diagram based on betalain synthesis.

[0076] Figure 6The schematic diagram of wild type CY464, smart maintainer line sipks2#26-B, smart sterile line sipks2#26-A small flower and pollen I2-KI. DETAILED DESCRIPTION

[0077] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the application.

[0078] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0079] In the following examples, the millet DNA is extracted using the improved CTAB method. The specific steps are as follows: take 0.1-0.2 grams of leaf blades into a small mortar, add an appropriate amount of liquid nitrogen, grind immediately to powder, load into a 2 ml centrifuge tube, add 800 μl of 65℃ preheated CTAB solution into the centrifuge tube, mix carefully and then put into a 65℃ water bath, take out the centrifuge tube after 20 minutes, add 800 μl of chloroform / isopentanol solution (chloroform:isopentanol = 24:1), mix vigorously, centrifuge at 12000 rpm for 10 minutes, take the supernatant, add 800 μl of chloroform / isopentanol solution (chloroform:isopentanol = 24:1) again, centrifuge at 12000 rpm for 10 minutes, take the supernatant into a new centrifuge tube, add 600 μl of isopropyl alcohol, mix, and then put in a -20℃ refrigerator for more than half an hour. Centrifuge the precipitated DNA at 12000 rpm for 10 minutes. Remove the supernatant, wash the precipitate with 500 μl of 70% ethanol twice, centrifuge to dryness, dissolve in 100 μl of deionized water, and store in a -20℃ refrigerator.

[0080] The genomic sequence of SiPKS2 gene in the following examples is shown in sequence 1 in the sequence listing.

[0081] The breeding method of foxtail millet CY464 in the following examples is as follows: foxtail millet Ci846 is used as the female parent, foxtail millet Yugu 1 is used as the male parent to carry out hybridization, and hybrid offspring is obtained, and then the hybrid offspring is continuously selfed for 5 generations (to make the foxtail millet genome tend to be homozygous), and foxtail millet CY464 is obtained. Among them, foxtail millet Ci846 is recorded in the literature “Zhao M C, Tang S, Zhang H S et al. DROOPY LEAF1 controls leaf architecture by orchestrating early brassinosteroid signaling[J]. PNAS, 2020, 117(35): 21766-21774.”. Foxtail millet Yugu 1 is recorded 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 1 provides a candidate gene for heterosis utilization in foxtail millet[J]. Crop J., 2021, 9(6): 1309-1319”.

[0082] The partial sequences involved in the following examples are shown in Table 1.

[0083] Table 1

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Note: No. 1 is the nucleotide sequence of pYLCRISPR / Cas9Pubi-H vector; No. 2 is the nucleotide sequence of pSiPKS2-RUBY vector; No. 3 is the nucleotide sequence of pSiPKS2-DsRed vector.

[0106] Example 1, obtaining of millet intelligent maintenance line

[0107] I. Knockout target design

[0108] According to the SiPKS2 gene sequence, the SiPKS2 gene knockout target sequence was designed by using the online target design website CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) of plant CRISPR / Cas9, and the finally screened SiPKS2 gene knockout target sequence was as follows: 5'-TCTGGACATCTCCAACAAGG-3' (sequence 2 in sequence listing).

[0109] II. Obtaining of fragment for infusion connection and containing target sequence

[0110] 1. First round of PCR

[0111] Using pYLsgRNA-OsU6aLacZ plasmid as a template, 1 μl each of primers UF (5'-CTCCGTTTTACCTGTGGAATCG-3') and gR-R (5'-CGGAGGAAAATTCCATCCAC-3') and 0.5 μl each of primers LAP5-gRT#+ (5'-TCTGGACATCTCCAACAAGGGTTTTAGAGCTAGAAAT-3') and LAP5-OsU6aT#- (5'-CCTTGTTGGAGATGTCCAGACGGCAGCCAAGCCAGCA-3') were added to a 50 μl system. PCR reaction conditions: 94℃ for 10 s, 58℃ for 15 s, 68℃ for 20 s, for 25-28 cycles.

[0112] 2. Second round of PCR

[0113] The first-round PCR product was diluted 10-fold as a template. Primers U-GAL (5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3') and Pgs-GAR were added to a 50 μl volume.

[0114] 1.5 μl each of (5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGCTCTTG-3'). PCR reaction conditions: 98℃ for 10 s, 58℃ for 15 s, 68℃ for 1 min, 28-30 cycles.

[0115] The second-round PCR product is a DNA fragment containing the target sequence used for infusion ligation, and its nucleotide sequence is shown in Sequence 3 of the sequence listing.

[0116] III. Construction of SiPKS2 knockout vector

[0117] 1. The pYLCRISPR / Cas9Pubi-H vector was digested with restriction endonuclease BsaⅠ (Cat no. R3535L, New England Biolabs, United States) at 37℃ for 5 h to obtain the pYLCRISPR / Cas9Pubi-H linear vector.

[0118] The nucleotide sequence of the pYLCRISPR / Cas9Pubi-H vector is shown in Table 1, Sequence 1, wherein the sequence 1 is from 103 to 277, which is a tobacco mosaic virus 35S polyadenylation signal sequence; from 317 to 1342, which is a hygromycin B phosphotransferase coding gene sequence; from 1410 to 2087, which is a tobacco mosaic virus 35S promoter sequence; from 2539 to 4525, which is a maize ubiquitin promoter sequence; from 4533 to 4553, which is a simian virus 40 nuclear localization signal sequence; from 4578 to 8678, which is a coding gene sequence of Cas9 (Csn1) endonuclease of Streptococcus pyogenes type II CRISPR / Cas system; Csn1 endonuclease shown in Sequence 4 in the coding sequence table; from 8679 to 8726, which is a two-component nuclear localization signal sequence of nucleolysin; from 8739 to 8991, which is a nopaline synthase terminator.

[0119] 2. The DNA fragment obtained in step two for infusion connection and containing the target sequence is connected to the pYLCRISPR / Cas9Pubi-H linear vector by using In-Fusion HD Cloning Kits (Cat no. 639648, Takara, Japan) to obtain a ligation product.

[0120] 3. The ligation product is transformed into E. coli competent cells Trans-T1 (Cat no. CD501, TransGen Biotech, China) and plated on solid LB medium containing kanamycin, and single colonies are picked and identified by sequencing using primers 5'-GTGGTGATAAGCGTCCTG-3' and 5'-AAGGCGATTAAGTTGGGT-3' (the target product sequence is 1235 bp in size) to obtain the SiPKS2 knockout vector.

[0121] The sequencing results show that the SiPKS2 knockout vector is a vector obtained by replacing the DNA fragment between 8997 and 9737 of the pYLCRISPR / Cas9Pubi-H vector with the DNA molecule shown in Sequence 3 in the sequence table, and keeping other sequences of the pYLCRISPR / Cas9Pubi-H vector unchanged.

[0122] Four, construction of a millet intelligent sterile vector

[0123] 1. Construction of a millet intelligent sterile vector pSiPKS2-RUBY based on betalain synthesis

[0124] The DNA fragment containing the transgenic pollen inactivation element and the betalain synthesis element is connected into the pCAMBIA-1305 vector through EcoR I and Pml I enzyme cutting sites to obtain an intelligent sterility intermediate vector, and then the SiPKS2 expression cassette (the SiPKS2 expression cassette includes a SiPKS2 promoter sequence and a SiPKS2 genomic sequence in sequence) is connected into the intelligent sterility intermediate vector through a Hind III enzyme cutting site to obtain a millet intelligent sterility vector based on betalain synthesis, namely pSiPKS2-RUBY.

[0125] The nucleotide sequence of the pSiPKS2-RUBY vector is shown in sequence 2 in Table 1, wherein sequence 2 is 303-477, which is a tobacco mosaic virus 35S polyadenylation signal sequence, 485-1282, which is an amino sugar phosphotransferase coding gene sequence, the amino sugar phosphotransferase shown in sequence 5 in the coding sequence table, 1345-2022, which is a tobacco mosaic virus 35S promoter sequence; 2232-4374, which is a SiPKS2 promoter sequence, 4375-5692, which is a SiPKS2 genomic sequence, 5699-5951, which is a nopaline synthase terminator; 5957-8732, which is a pollen-specific promoter sequence, 8733-10220, which is a maize alpha amylase coding gene sequence, the maize alpha amylase shown in sequence 6 in the coding sequence table, 10221-10473, which is a nopaline synthase terminator; 10500-11300, which is a barley aleurone-specific promoter sequence, 11307-15263, which is a betalain synthesis element sequence (11307-12797, which is a protein A coding gene sequence, the protein A shown in sequence 7 in the coding sequence table, 12798-12863, which is a self-cleavage oligopeptide sequence, 12864-13688, which is a protein B coding gene sequence, the protein B shown in sequence 8 in the coding sequence table, 13689-13754, which is a self-cleavage oligopeptide sequence, 13755-15263, which is a protein C coding gene sequence, the protein C shown in sequence 9 in the coding sequence table), 15304-15556, which is a nopaline synthase terminator.

[0126] 2. Construction of a millet intelligent sterility vector based on fluorescent labeling, namely pSiPKS2-DsRed

[0127] The DNA fragment containing the transgenic pollen inactivation element and the fluorescent marker element is connected into the pCAMBIA-1305 vector through EcoR I and Pml I enzyme cutting sites to obtain an intelligent sterility intermediate vector, and then the SiPKS2 expression cassette (the SiPKS2 expression cassette includes a SiPKS2 promoter sequence and a SiPKS2 genomic sequence in sequence) is connected into the intelligent sterility intermediate vector through a Hind III enzyme cutting site to obtain a foxtail millet intelligent sterility vector pSiPKS2-DsRed based on a fluorescent marker.

[0128] The nucleotide sequence of the pSiPKS2-DsRed vector is shown as sequence 3 in Table 1, wherein the sequence 3 is 303-477 for a tobacco mosaic virus 35S polyadenylation signal sequence, 485-1282 for an aminoglycoside phosphotransferase coding gene sequence, 1345-2022 for a tobacco mosaic virus 35S promoter sequence; 2238-4380 for a SiPKS2 promoter sequence, 4381-5698 for a SiPKS2 genomic sequence, 5705-5957, 10227-10479 and 11998-12250 for nopaline synthase terminator sequences, 5963-8738 for a pollen-specific promoter sequence, 8739-10226 for a maize alpha amylase coding gene sequence, 10485-11285 for a barley aleurone-specific promoter sequence, and 11286-11963 for a red fluorescent protein coding gene sequence.

[0129] V. Obtaining and identifying of the foxtail millet intelligent maintainer line

[0130] 1. Obtaining of the sipks2#5 and sipks2#7

[0131] The SiPKS2 knockout vector and the millet smart sterile vector pSiPKS2-DsRed based on fluorescent labeling were introduced into Agrobacterium EHA105 to obtain a recombinant bacterium; the SiPKS2 knockout vector and the smart sterile vector pSiPKS2-DsRed based on fluorescent labeling were co-transformed into Ci846 immature embryo callus of millet by using Agrobacterium-mediated genetic transformation, to obtain T0 generation transgenic plants. Then, the T0 generation transgenic plants were subjected to DNA detection by using primers 5'-AACACCAACTGCGACGACC-3' and 5'-TTGACGAGACGAACAGAGGC-3' (the sequence size of the target product is 1227 bp), to determine the editing mode and the homozygous editing site of the transgenic plants. At the same time, the T0 generation transgenic plants were subjected to DNA detection by using primers 5'-CTCGAATTTCGGAATCAT-3' and 5'-ACGCCGCTATCAGGGACT-3' (the sequence size of the target product is 1287 bp), to determine the smart sterile expression cassette transgenic positive plants. Sequencing found that the target point was a homozygous editing type and contained a single plant of the smart sterile expression cassette, which were named as sipks2#5 and sipks2#7, respectively.

[0132] As shown in Table 1, compared with the wild type millet Ci846 genomic DNA, the difference of sipks2#5 is only that one base A is deleted in the gene sequence encoding SiPKS2 protein shown in sequence 1 in the sequence table, and the deletion position of the base A is located at the 405th position of sequence 1, thereby causing a frame shift and premature termination, and the SiPKS2 protein is functionally deleted. Figure 1 As shown in Table 1, compared with the wild type millet Ci846 genomic DNA, the difference of sipks2#7 is only that 14 bp of base deletion occurs in the gene sequence encoding SiPKS2 protein shown in sequence 1 in the sequence table, and the deletion base is located at the 393-408th position of sequence 1 (CATCTCCAACAAGG), thereby causing a frame shift and premature termination, and the SiPKS2 protein is functionally deleted.

[0133] Figure 1 2. Obtaining of sipks2#26

[0134] 2. Obtaining of sipks2#26

[0135] ​The SiPKS2 knockout vector and the smart sterile vector based on betalain synthesis pSiPKS2-RUBY are introduced into Agrobacterium EHA105 to obtain a recombinant bacterium; the SiPKS2 knockout vector and the smart sterile vector based on betalain synthesis pSiPKS2-RUBY are co-transformed into foxtail millet CY464 young embryo callus using Agrobacterium-mediated genetic transformation to obtain T0 generation transgenic plants. Then, the T0 generation transgenic plants are subjected to DNA detection using primers 5'-AACACCAACTGCGACGACC-3' and 5'-TTGACGAGACGAACAGAGGC-3' (the target product sequence size is 1227 bp) to determine the transgenic plant editing mode and the pure heterozygous editing site. At the same time, the T0 generation transgenic plants are subjected to DNA detection using primers 5'-CTCGAATTTCGGAATCAT-3' and 5'-ACGCCGCTATCAGGGACT-3' (the target product sequence size is 1287 bp) to determine the smart sterile expression cassette transgenic positive plant. Sequencing finds that the target point is a homozygous editing type and contains a smart sterile expression cassette, and the single plant is named sipks2#26.

[0136] As shown in Figure 4 , compared with the wild type foxtail millet CY464 genomic DNA, the difference of sipks2#26 is only that in the gene sequence encoding SiPKS2 protein shown in sequence 1, a base A deletion occurs at position 405 of sequence 1, which causes a frameshift and premature termination, and the SiPKS2 protein function is lost.

[0137] Six, fertility analysis of foxtail millet smart maintainer line

[0138] 1. Smart maintainer line sipks2#5 self-crossed seeds based on fluorescence sorting

[0139] Mature smart maintainer line sipks2#5 self-crossed seeds are taken for observation.

[0140] The results are shown in Figure 2 . Without shelling, the sipks2#5 self-crossed seeds cannot be sorted under the fluorescence field of view Figure 2 (left); with shelling, the sipks2#5 self-crossed seeds can be sorted under the fluorescence field of view Figure 2 (right), in which the red fluorescent seeds are smart maintainer line (sipks2#5-8B) seeds containing transgenic components, and the non-red fluorescent seeds are smart sterile line (sipks2#5-A) seeds not containing transgenic components.

[0141] 2. Smart maintainer line sipks2#26 self-crossed seeds based on betalain synthesis

[0142] The mature self-pollinated seeds of the intelligent maintainer line sipks2#26 were taken for observation.

[0143] The results are shown in Table 1. Figure 5 As shown in Table 1, the self-pollinated seeds of the intelligent maintainer line sipks2#26 can be sorted, whether or not the hulls are removed, with the red seeds being the seeds of the intelligent maintainer line (sipks2#26-B) and containing the transgenic components, and the yellow seeds being the seeds of the intelligent sterile line (sipks2#26-A) and not containing the transgenic components.

[0144] 3. Pollen I2-KI staining

[0145] 1) The anthers of the wild type millet variety Ci846, the intelligent maintainer line sipks2#5-8B and the intelligent sterile line sipks2#5-A at the flowering stage were taken and placed on a slide, 1 drop of distilled water was added, the anthers were thoroughly crushed with a pair of tweezers to release the pollen grains, 1-2 drops of I2-KI solution was added, a cover glass was placed thereon, and the observation was made under a low-power microscope. The pollen grains that were stained black were the ones with starch and stronger vitality, and the pollen grains that were stained yellowish brown were the ones with poor development.

[0146] The results are shown in Table 2. Figure 3 As shown in Table 2, when the pollen of the wild type millet variety Ci846, the intelligent maintainer line sipks2#5-8B and the intelligent sterile line sipks2#5-A was subjected to I2-KI staining, it was found that the pollen of the wild type millet variety Ci846 could be normally colored (left), half of the pollen of the intelligent maintainer line sipks2#5-8B could be normally colored (middle), and the pollen of the intelligent sterile line sipks2#5-A could not be normally colored (right). Figure 3 Figure 3 Figure 3

[0147] 2) The anthers of the wild type millet variety CY464, the intelligent maintainer line sipks2#26-B and the intelligent sterile line sipks2#26-A at the flowering stage were taken and placed on a slide, 1 drop of distilled water was added, the anthers were thoroughly crushed with a pair of tweezers to release the pollen grains, 1-2 drops of I2-KI solution was added, a cover glass was placed thereon, and the observation was made under a low-power microscope. The pollen grains that were stained black were the ones with starch and stronger vitality, and the pollen grains that were stained yellowish brown were the ones with poor development.

[0148] The results are shown in Table 3. Figure 6 As shown in Table 3, when the pollen of the wild type millet variety CY464, the intelligent maintainer line sipks2#26-B and the intelligent sterile line sipks2#26-A was subjected to I2-KI staining, it was found that the pollen of the wild type millet variety CY464 could be normally colored (left), half of the pollen of the intelligent maintainer line sipks2#26-B could be normally colored (middle), and the pollen of the intelligent sterile line sipks2#26-A could not be normally colored (right). Figure 6 ​​​(Left), the intelligent retention system SIPKS2#26-B showed that half of the pollen could color normally, while the other half could not. Figure 6 (in the middle), while the pollen of the intelligent sterile line sipks2#26-A cannot be properly colored (in the middle), Figure 6 right).

Claims

1. A set of vectors for creating a smart maintainer line of foxtail millet, the set of vectors comprising a fertility gene knockout vector and a smart sterile expression cassette vector; the fertility gene knockout vector comprises an sgRNA expression cassette and a Cas9 nuclease expression cassette; the target sequence of the sgRNA is shown in SEQ ID NO: 2 in the sequence listing; the Cas9 nuclease is a Csn1 endonuclease; the Csn1 endonuclease is a protein with an amino acid sequence shown in SEQ ID NO: 4 in the sequence listing; the smart sterile expression cassette vector comprises an aminoglycoside phosphotransferase expression cassette, a fertility restorer gene expression cassette, a maize alpha amylase expression cassette and a betalain expression cassette; the fertility restorer gene is a SiPKS2 gene, and the nucleotide sequence of the SiPKS2 gene is shown in SEQ ID NO: 1 in the sequence listing; the betalain expression cassette expresses a protein A, a protein B and a protein C; the protein A is a protein with an amino acid sequence shown in SEQ ID NO: 7 in the sequence listing; the protein B is a protein with an amino acid sequence shown in SEQ ID NO: 8 in the sequence listing; the protein C is a protein with an amino acid sequence shown in SEQ ID NO: 9 in the sequence listing.

2. The set of vectors for creating intelligent maintainer lines of foxtail millet as claimed in claim 1 wherein: the fertility gene is a SiPKS2 gene.

3. The set of vectors for creating intelligent maintainer lines of foxtail millet according to claim 1 or 2, wherein: 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 two-component nuclear localization signal sequence of a nucleic acid endonuclease and a terminator sequence.

4. The set of vectors for creating intelligent maintainer lines of foxtail millet according to claim 1 or 2, wherein: the aminoglycoside phosphotransferase is a protein with an amino acid sequence shown in SEQ ID NO: 5 in the sequence listing; or, the maize alpha amylase is a protein with an amino acid sequence shown in SEQ ID NO: 6 in the sequence listing.

5. The set of vectors for creating intelligent maintainer lines of foxtail millet according to claim 1 or 2, wherein: the betalain expression cassette comprises, in order, a promoter sequence, a protein A-encoding gene sequence, a self-cleaving oligopeptide sequence, a protein B-encoding gene sequence, a self-cleaving oligopeptide sequence, a protein C-encoding gene sequence and a terminator sequence. 6.Use of the set of vectors for creating a smart maintainer line of foxtail millet according to any one of claims 1-5 in the preparation of a smart maintainer line of foxtail millet. 7.Use of the set of vectors for creating a smart maintainer line of foxtail millet according to any one of claims 1-5 in the preparation of a smart maintainer line of foxtail millet seed and a smart sterile line of foxtail millet seed. 8.A method for preparing a smart maintainer line of foxtail millet, comprising the following steps: introducing the set of vectors for creating a smart maintainer line of foxtail millet according to any one of claims 1-5 into a wild-type foxtail millet to obtain a transgenic foxtail millet; and screening a smart maintainer line of foxtail millet from the transgenic foxtail millet.

9. A method for preparing a seed of a millet intelligent maintainer line and a seed of a millet intelligent sterile line, comprising the steps of: obtaining a millet intelligent maintainer line according to the method of claim 8, selfing the millet intelligent maintainer line to obtain selfed seeds, wherein, The self-pollinated seeds with red color are smart maintainer line of foxtail millet seeds, and the self-pollinated seeds with yellow color are smart sterile line of foxtail millet seeds.

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