Application of polynucleotide, protein and biological material in regulation and control of plant seed traits and improvement method

Abstract

The invention relates to the technical field of biology, in particular to application of polynucleotide, protein and biological materials in regulation of plant seed traits and an improvement method. The polynucleotide comprises a CDPK27 gene sequence as shown in SEQ ID NO: 3 or a related sequence of the CDPK27 gene sequence; a CDPK26 gene sequence or a related sequence thereof is shown as SEQ ID NO: 11. The CDPK gene cloned by the invention can indirectly influence seed development by adjusting energy distribution in tomato fruits, can reduce the number and weight of seeds, provides gene resources and theoretical basis for creating seedless, less-seed and / or small-seed tomatoes, and has wide application prospects. Meanwhile, a new solution is provided for controlling the number or size of the seeds and improving the sugar content of the fruits.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the application and improvement methods of polynucleotides, proteins and biomaterials in regulating plant seed traits. Background Technology

[0002] The propagation and evolution of flowering plants largely depend on the production of robust seeds and effective seed dispersal. However, with the continuous improvement of people's living standards, higher demands are being placed on the quality of fruits and vegetables. Some fruit and vegetable crops have too many seeds, which seriously affects their taste. The cultivation of seedless fruits, such as seedless watermelons and seedless grapes, has greatly satisfied people's needs.

[0003] Tomatoes are perennial plants in their natural environment. Ripe tomatoes have soft flesh, rich in sugar and other nutrients, attracting small rodents and birds, which facilitates seed dispersal. Simultaneously, the ripening of tomato fruit is a highly coordinated developmental process, providing a suitable environment for seed maturation. Seeds of fleshy tomato varieties fill in situ during the later stages of development, and seed dry weight reaches a stable level after the fruit reaches the green ripening stage (MG stage). However, tomatoes contain a relatively large number of hard seeds, which can affect the taste to some extent. Therefore, high-quality tomatoes such as seedless or low-seed tomatoes have considerable commercial value.

[0004] Currently, there is no research or cultivation of seedless or low-seed tomatoes. Summary of the Invention

[0005] In view of this, the present invention provides the application and improvement methods of polynucleotides, proteins, and biological materials in regulating plant seed traits. The CDPK gene cloned in this invention can reduce seed number and weight, providing genetic resources and a theoretical basis for creating seedless, low-seeded, and / or small-seeded, as well as high-sugar tomatoes.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides the application of a polynucleotide in regulating plant seed traits, the polynucleotide comprising at least one of the following nucleotide sequences:

[0008] (11) The CDPK27 gene sequence shown in SEQ ID NO:3;

[0009] (12) A complementary, degenerate, or homologous sequence of the CDPK27 gene sequence shown in SEQ ID NO:3, wherein the homologous sequence is a sequence that has 90% or more identity with the CDPK27 gene sequence shown in SEQ ID NO:3;

[0010] (13) A sequence or its complementary sequence that hybridizes with the CDPK27 gene sequence shown in SEQ ID NO:3 under strict conditions;

[0011] (14) The cDNA sequence of any one of the sequences (11)-(13);

[0012] (15) The CDPK26 gene sequence shown in SEQ ID NO:11;

[0013] (16) A complementary, degenerate, or homologous sequence of the CDPK26 gene sequence shown in SEQ ID NO:11, wherein the homologous sequence is a sequence that has 90% or more identity with the CDPK26 gene sequence shown in SEQ ID NO:11.

[0014] (17) A sequence or its complementary sequence that hybridizes with the CDPK26 gene sequence shown in SEQ ID NO:11 under strict conditions;

[0015] (18) cDNA sequence of any one of the sequences (15)-(17).

[0016] In a specific embodiment provided by the present invention, the CDS sequence of CDPK27 includes at least one of the following sequences:

[0017] (1) The sequence shown in SEQ ID NO:1;

[0018] (2) A complementary, degenerate, or homologous sequence of the sequence shown in SEQ ID NO:1, wherein the homologous sequence is a sequence that has 75% or more of the same identity as the sequence shown in SEQ ID NO:1;

[0019] (3) A sequence or its complementary sequence that hybridizes with the sequence shown in SEQ ID NO:1 under strict conditions;

[0020] (4) cDNA sequence of any one of sequences (1)-(3).

[0021] In a specific embodiment provided by the present invention, the CDS sequence of CDPK26 includes at least one of the following sequences:

[0022] (1) The sequence shown in SEQ ID NO:9;

[0023] (2) A complementary, degenerate, or homologous sequence of the sequence shown in SEQ ID NO:9, wherein the homologous sequence is a sequence that has 75% or more of the same identity as the sequence shown in SEQ ID NO:9;

[0024] (3) A sequence or its complementary sequence that hybridizes with the sequence shown in SEQ ID NO:9 under strict conditions;

[0025] (4) cDNA sequence of any one of sequences (1)-(3).

[0026] Furthermore, a homologous sequence is a nucleotide sequence that is approximately 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the original nucleotide sequence, or its corresponding cDNA molecule.

[0027] In this invention, homologous sequences that have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the original nucleotide sequence and have the same function are all within the scope of protection of this invention.

[0028] In some embodiments, homologous sequences that have 75%-85%, 76%-86%, 77%-87%, 78%-88%, 79%-89%, 80%-90%, 81%-91%, 82%-92%, 83%-93%, 84%-94%, 85%-95%, 86%-96%, 87%-97%, 88%-98%, 89%-99%, 90%-95%, 91%-96%, 92%-97%, 93%-98%, 94%-99%, 95%-100%, 85%-90%, 86%-91%, 87%-92%, 88%-93%, or 89%-94% identity with the original nucleotide sequence and have the same function are all within the scope of protection of this invention.

[0029] Furthermore, homologous sequences encode protein conserved domains that are identical to the original nucleotide sequence. Specifically, protein conserved domains include... Figure 1 As shown.

[0030] Furthermore, the homologous sequences and the original nucleotide sequences originate from the same species and the same gene. Specifically, the homologous sequences to SEQ ID NO: 3, originating from the same species and the same gene, are shown in SEQ ID NO: 26-39; the homologous sequences to SEQ ID NO: 1, originating from the same species and the same gene, are shown in SEQ ID NO: 40-68; the homologous sequences to SEQ ID NO: 11, originating from the same species and the same gene, are shown in SEQ ID NO: 98-111; and the homologous sequences to SEQ ID NO: 9, originating from the same species and the same gene, are shown in SEQ ID NO: 112-155.

[0031] The nucleotide sequence molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; it can also be RNA, such as mRNA or hnRNA.

[0032] The nucleotide sequences of CDPK27 and / or CDPK26 are not necessarily identical in different tomato materials. This invention provides the nucleotide sequences of CDPK27 and / or CDPK26 in other tomato materials obtained during the research process. Among them, the genomic identity of CDPK27 among different varieties is 98.734% to 100%, and the CDS identity is 92.83% to 100% (see Table 1 for details). The sequences that are not completely identical to SEQ ID NO: 3 are as shown in SEQ ID NO: 23-36 in the patent publication number CN118207227A (referred to as SEQ ID NO: 26-39 in this application), and the sequences that are not completely identical to SEQ ID NO: 1 are as shown in SEQ ID NO: 37-65 in the patent publication number CN118207227A (referred to as SEQ ID NO: 37-65 in this application). The genomic identity of CDPK26 among different varieties is 98.734%–100%, and the CDS identity is 90.59%–100% (see Table 2 for details). Among them, the sequences that are not completely identical to SEQ ID NO: 11 are the sequences shown in SEQ ID NO: 95-108 in the patent with publication number CN118207227A (referred to as the sequences shown in SEQ ID NO: 98-111 in this application), and the sequences that are not completely identical to SEQ ID NO: 9 are the sequences shown in SEQ ID NO: 109-152 in the patent with publication number CN118207227A (referred to as the sequences shown in SEQ ID NO: 112-155 in this application).

[0033] In embodiments of the present invention, seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

[0034] In some embodiments, regulating plant seed traits includes: reducing the average number of seeds per fruit and reducing the average weight of a thousand seeds, laying the foundation for creating plants with seedless, few-seeded, or small-seeded fruits and improving fruit taste; or increasing the average number of seeds per fruit and increasing the average weight of a thousand seeds, laying the foundation for seed production.

[0035] In some embodiments, the recipient plant is modified by reducing the average number of seeds per fruit and reducing the average weight of a thousand seeds to obtain the target plant.

[0036] Preferably, the average number of seeds per fruit or the average weight of 1,000 seeds in the fruit of the target plant is reduced by at least 5% compared to the recipient plant. In some embodiments, the average number of seeds per fruit or the average weight of 1,000 seeds in the fruit of the target plant is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the recipient plant.

[0037] In some embodiments, the seed germination rate of the target plant is substantially similar to that of the recipient plant. In some embodiments, the difference between the seed germination rate of the target plant and the recipient plant is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%.

[0038] In the embodiments provided by the present invention, the plant is a plant with edible parts such as fruit, tuber, and rhizome.

[0039] In some embodiments, the plant is one of the following: berry-bearing plants, plants of the Solanaceae family, plants of the Rosaceae family, plants of the Rutaceae family, plants of the *Ipomoea* genus of the Convolvulaceae family, plants of the Dioscoreaceae family, and plants of the Cucurbitaceae family. However, it is not limited to these plant species; any plant species recognized by those skilled in the art are within the scope of protection of this invention.

[0040] In the embodiments provided by this invention, berry plants include, but are not limited to, tomatoes, grapes, kiwifruit, papaya, pomegranate, ginseng fruit, blueberries, persimmons, passion fruit, dragon fruit, wax apples, etc.

[0041] In the embodiments provided by this invention, the solanaceae plants include, but are not limited to, potatoes, eggplants, tomatoes, peppers, goji berries, etc.

[0042] In the embodiments provided by this invention, Rosaceae plants include, but are not limited to, pear, apple, peach, apricot, cherry, plum, prune, crabapple, loquat, hawthorn, strawberry, raspberry, etc.

[0043] In the embodiments provided by this invention, Rutaceae plants include, but are not limited to, grapefruit, phellodendron, lemon, orange, mandarin orange, citrus, etc.

[0044] In a specific embodiment provided by the present invention, the plant is a tomato.

[0045] In the embodiments provided by this invention, the tomato varieties include, but are not limited to, SV7845TH, SVTH4018, Antres, SV4224TH, SV8795TG, SV3026TG, Meishuai, SV0313TG, Hongsui, Diliya, Deruis, Fengege, Oushuo, Huifu, SVTH1366, Ouke, Meibei, BOLZANO, DRC 564, DRK 936, and Tomimaru. MUCHOO), DELISHER, Strabena, DR0607TC, Anbes, Jiuli, Camry, Dongnuan, Sibeide, Kevin, Jiuli 200, Tennessee No. 3, Ruifei No. 5, Ruifei No. 2, Ameizi No. 5, PF207008, Julie, Beiying, Zidali, Ruifei, Kaisa, Ono, Pink Beiying, Labi, 72-CH0353, Harvest 74-560, Zulfia (73-610), Florentino (72-163), Fulltom (72-729), Forticia (72-719) Glorioso (74-112), Cappricia (72-466), Brioso (72-130), Kawaguchi (72-541), Kivu (72-629), Tatami (72-175), Tarffy (76-IM6917), Genery (72-192), Moscatel (74-104), Siberite (73-915), Siberite (73-916) 916, Plumola (72-001), Messina (73-47), Paulanca (72-534), Logure (73-571), Santiana (72-763), Operano (72-187), Ternetto (72-190), Tomary (72-191), TY (73-882)12. Beril (73-14), Abellus (73-583), Fortesa (72-152), Lidertom (74-254), Solarino (72-150), Vacetto (72-164), Reddery (72-008), Endeavour (72-487), Suncree (74-132), Gourami (72-021), Pareso (72-126), Hibachi (72-241), and others. Aruru (72-193), Lauster, Maggino (72-195), Yingla, Gutian, Solana, Andara, Andolina, Keren, Summer Sunshine, Honglilai, Diruisen, Qiulian, Nicola, Saint Laurent, Fendi, Dali, Hanyu 522, Anila, Burberry, Russell, Jinlong, Walter, Kailong, Dorothy, Fleissing, Raphael, Haiyue, Lola, Annecy, Ruila, Sidi, Fora 3661, Shilochi, Davidson, Golden Shed No. 8, Golden Shed No. 101, Golden Shed No. 950, Golden Shed No. 2095, Golden Shed Saint-Bia 45, Golden Shed Saint-Bia 50, Golden Shed No. 8 Type B, Golden Shed No. 9, Golden Shed No. 10, Golden Shed No. 148, Golden Shed No. 152. Jinpeng 218, Jinpeng 261, Jinpeng 322, Jinpeng 945, Jinpeng 951, Jinpeng 1186, Jinpeng 1198, Jinpeng Qiusheng, Jinpeng Rongwei, Jinpeng Tianyi No. 2, Jinpeng 102, Jinpeng 1828, Jinpeng M708, Jinpeng M5038, Jinpeng No. 11, Jinpeng 12-2, Jinpeng 18-98, Jinpeng 236, Jinpeng 1729, Jinpeng Heiyuanshuai, Jinpeng Shengbia 04, Jinpeng 11-21, Jinpeng 1521, Jinpeng 1605, Jinpeng M6, Jinpeng M7, Jinpeng M158, Jinpeng No. 5, Jinpeng Xia Nai, Jinpeng No. 1, Jinpeng 128, Jinpeng M215, Jinpeng 513, Jinpeng 515, Jinpeng No. 6, Jinpeng No. 3, Jinpeng Hongchuan 132, Golden Shed Pink Girl No. 2, Golden Shed Little Yellow Crown, Golden Shed Pink Girl No. 1, Golden Shed Little Yellow Crown 58, Golden Shed No. 19, Golden Shed No. 44, Golden Shed No. 53, Golden Shed No. 75, Golden Shed No. 151, Golden Shed Showgirl, Holy Banquet 1523, Xilaid No. 1, Darwin, Bora, Ferrari, Osina, Orina, Newnem 1618, Newnem 1718, Praise No. 2, Holy Banquet 3767, Blooming Red 6415, Jin Xiaoling, Pink Girl, Pink Girl No. 2, Lifei, Pink Taro No. 3, Delicious 925, Purple Taro, Athena, Xiu Taro, Qing Taro, Pink Lide, Pink Lide No. 2, Red Rise, Red Rise No. 2, Beautiful 601, Yellow Crown 16, Yellow Crown No. 2, Yellow Crown No. 3, Golden Pear, Yellow Little Y, Black Pearl No. 2 F1 Number Two No. 3 F1 No. 6 Number 7 Number 8 Number Nine Number 10 No. 11 227. Number One Number Two No. 3 No. 5, Guyu Tianci No. 1, Guyu Tianci No. 2, Guyu Tianci No. 3, Guyu Tianci No. 4, Guyu Tianci No. 5, Guyu Tianci No. 6, Guyu Tianci 595, Guyu Tianci 585, Guyu Tianci 575, Guyu Tianci 565F1, Guyu Tianqi No. 1, Guyu Tianqi No. 2, Guyu Tianqi No. 3 Number One Number Two No. 3 No. 4, Red Kerry No. 1, Guyutianmi No. 1, Guyutianmi No. 2, Guyutianmi No. 3, Hehong No. 1 Tomato, Hehong No. 2 Tomato, Hehong No. 3 Tomato, Yingjiahe No. 3, Yingjiahe No. 2, Yingjiahe No. 5, Yingjiahe No. 4, Yingjiahe No. 1, Pengbo No. 3, Pengbo No. 2, Pengbo No. 1, Herunsheng No. 4, Herunsheng No. 3, Herunsheng No. 2, Herunsheng No. 1, Baoliyuan No. 6, Baoliyuan No. 5, Baoliyuan No. 4, Baoliyuan No. 3, Baoliyuan No. 2, Baoliyuan No. 1, Millennium, Fengzhu, Xiaoxia, Chuntao, Guanghui 101, Money Maker, M82, Ailsa Craig, Provence, Ruixing Dabao, Fenbeibei, Zhongshu No. 4, Zhongshu No. 5, Busan 88, Fentaro, etc.

[0046] In some embodiments, tomatoes are selected from the group consisting of: Money Maker, M82, Ailsa Craig, Millennium, Provence, Ruixing Dabao, Zidali, Pink Baby, Summer Sunshine, Lola, Zhongshu No. 4, Zhongshu No. 5, Busan 88, Pink Taro, Antlers, Rabi, Lauster, Jinpeng No. 8, Jinpeng Xiaohuangguan, Guyu Tianci No. 2, and Chuntao.

[0047] Furthermore, this invention also provides the application of any one of the aforementioned polynucleotides, proteins, and biological materials in reducing the average number of seeds per fruit, reducing the average weight of a thousand seeds, and improving the quality of plant fruits. That is, the application of this invention can improve the quality of plant fruits while simultaneously reducing the average number of seeds per fruit and the average weight of a thousand seeds.

[0048] In specific embodiments provided by the present invention, improving the quality of plant fruits includes increasing the content of SSC, sugar, and citric acid in plant fruits, reducing the content of malic acid, and maintaining fruit weight.

[0049] In a specific embodiment of the present invention, the plant is tomato, and the contents of SSC, sugar, citric acid, and malic acid in the fruit, as well as the fruit weight, are measured during the red ripening period of the tomato fruit.

[0050] In the embodiments provided by the present invention, sugar includes one or more of glucose, fructose, and sucrose.

[0051] In specific embodiments provided by the present invention, sugar includes glucose and / or fructose.

[0052] In some embodiments, the recipient plant is modified by reducing the average number of seeds per fruit, reducing the average weight of a thousand seeds, and increasing the content of SSCs, sugars, or citric acid in the fruit, while decreasing the content of malic acid in the fruit, to obtain the target plant. This modification method lays the foundation for creating plants with seedless, few-seeded, or small-seeded fruits with excellent flavor.

[0053] In some embodiments, based on the regulation of seed traits, the content of SSC, sugar, or citric acid in the fruit of the target plant is increased by at least 10% compared to the recipient plant. In some embodiments, the content of SSC, sugar, or citric acid in the fruit of the target plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the recipient plant.

[0054] In some embodiments, based on the regulation of seed traits, the malic acid content in the fruit of the target plant is reduced by at least 10% compared to that of the recipient plant. In some embodiments, the malic acid content in the fruit of the target plant is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to that of the recipient plant.

[0055] Secondly, this invention provides the application of a protein in regulating plant seed traits, wherein the protein is at least one of the following sequences:

[0056] (21) The CDPK27 protein sequence shown in SEQ ID NO:2;

[0057] (22) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the CDPK27 protein sequence shown in SEQ ID NO:2;

[0058] (23) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the CDPK27 protein sequence shown in SEQ ID NO:2.

[0059] (24) A protein that has 90% or more identity with the CDPK27 protein sequence shown in SEQ ID NO:2 and has the same function;

[0060] (25) The CDPK26 protein sequence shown in SEQ ID NO:10;

[0061] (26) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the CDPK26 protein sequence shown in SEQ ID NO:10;

[0062] (27) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the CDPK26 protein sequence shown in SEQ ID NO:10.

[0063] (28) A protein that has 90% or more identity with the CDPK26 protein sequence shown in SEQ ID NO:10 and has the same function.

[0064] Specifically, proteins with the same function obtained through the substitution and / or deletion and / or addition of one or more amino acid residues refer to proteins with completely identical conserved protein domains. More specifically, conserved protein domains include... Figure 1 As shown.

[0065] Furthermore, the homologous sequence can also be a protein sequence that is approximately 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the original protein sequence.

[0066] In this invention, the homologous sequence may also be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the original protein sequence and have the same function, all of which are within the scope of protection of this invention.

[0067] In some embodiments, the homologous sequence may also be 75%-85%, 76%-86%, 77%-87%, 78%-88%, 79%-89%, 80%-90%, 81%-91%, 82%-92%, 83%-93%, 84%-94%, 85%-95%, 86%-96%, 87%-97%, 88%-98%, 89%-99%, 90%-95%, 91%-96%, 92%-97%, 93%-98%, 94%-99%, 95%-100%, 85%-90%, 86%-91%, 87%-92%, 88%-93%, or 89%-94% identical to the original protein sequence and have the same function, all of which are within the scope of protection of this invention.

[0068] Furthermore, the homologous sequence shares identical conserved protein domains with the original protein sequence. Specifically, the conserved protein domains include... Figure 1 As shown.

[0069] Furthermore, the homologous sequences and the original protein sequences originate from the same gene in the same species. Specifically, the homologous sequences to SEQ ID NO: 2 from the same gene in the same species are shown in SEQ ID NO: 69-97, and the homologous sequences to SEQ ID NO: 10 from the same gene in the same species are shown in SEQ ID NO: 156-199.

[0070] The protein sequences of CDPK27 and / or CDPK26 are not necessarily identical in different tomato materials. This invention provides the protein sequences of CDPK27 and / or CDPK26 in other tomato materials obtained during the research process. Among them, the identity of CDPK27 protein sequences among different varieties is 93.007% to 100% (see Table 1 for details). The sequences that are not completely identical to SEQ ID NO: 2 are shown in the patent with publication number CN118207227A, such as the sequences shown in SEQ ID NO: 66-94 (referred to as the sequences shown in SEQ ID NO: 69-97 in this application). The identity of CDPK26 protein sequences among different varieties is 90.037% to 100% (see Table 2 for details). The sequences that are not completely identical to SEQ ID NO: 10 are shown in the patent with publication number CN118207227A, such as the sequences shown in SEQ ID NO: 153-196 (referred to as the sequences shown in SEQ ID NO: 156-199 in this application).

[0071] In embodiments of the present invention, seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

[0072] Thirdly, the present invention provides the application of a biomaterial in regulating plant seed traits, wherein the biomaterial is any one of the following (31) to (35):

[0073] (31) Knockout cassette of the polynucleotide of claim 1;

[0074] (32) Knockout vector of the polynucleotide of claim 1;

[0075] (33) Recombinant microorganisms that knock out the polynucleotide of claim 1.

[0076] (34) Plant cell lines with the above polynucleotides knocked out;

[0077] (35) Import any of the plant protoplasts, cells or callus tissues from (31)-(33).

[0078] In embodiments of the present invention, seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

[0079] Fourthly, the present invention provides a method for improving plants or seeds, comprising: mutating the CDPK gene to inactivate the CDPK protein kinase, thereby obtaining improved plants or seeds, wherein the CDPK gene is the aforementioned polynucleotide and the CDPK protein is the aforementioned protein.

[0080] Improved plants or seeds include:

[0081] (i) a reduction of at least 5% in the average number of seeds per fruit compared to the control plant or seed; and / or,

[0082] (ii) The average weight of 1,000 seeds is reduced by at least 5% compared to the control plant or seeds;

[0083] The control plants or seeds and the improved plants or seeds contain essentially the same genetic background.

[0084] In the embodiments provided by the present invention, the plant is a plant with edible parts such as fruit, tuber, and rhizome.

[0085] In some embodiments, the plant is one of the following: berry-bearing plants, plants of the Solanaceae family, plants of the Rosaceae family, plants of the Rutaceae family, plants of the *Ipomoea* genus of the Convolvulaceae family, plants of the Dioscoreaceae family, and plants of the Cucurbitaceae family. However, it is not limited to these plant species; any plant species recognized by those skilled in the art are within the scope of protection of this invention.

[0086] In the embodiments provided by this invention, berry plants include, but are not limited to, tomatoes, grapes, kiwifruit, papaya, pomegranate, ginseng fruit, blueberries, persimmons, passion fruit, dragon fruit, wax apples, etc.

[0087] In the embodiments provided by this invention, the solanaceae plants include, but are not limited to, potatoes, eggplants, tomatoes, peppers, goji berries, etc.

[0088] In the embodiments provided by this invention, Rosaceae plants include, but are not limited to, pear, apple, peach, apricot, cherry, plum, prune, crabapple, loquat, hawthorn, strawberry, raspberry, etc.

[0089] In the embodiments provided by this invention, Rutaceae plants include, but are not limited to, grapefruit, phellodendron, lemon, orange, mandarin orange, citrus, etc.

[0090] In a specific embodiment provided by the present invention, the plant is a tomato.

[0091] In a specific embodiment of the present invention, a method for mutating the CDPK gene to inactivate CDPK protein kinase includes:

[0092] (a) Knockout of a multiple of 3 bases at the active site of the CDPK gene, resulting in a deletion mutation at at least one amino acid site with kinase activity in the CDPK protein, thus losing kinase activity and obtaining a deletion mutant; and / or,

[0093] (b) Inserting bases in multiples of 3 at the active site of the CDPK gene, resulting in an insertion mutation at at least one kinase-active amino acid site in the CDPK protein, thus losing kinase activity and obtaining an insertion mutant; and / or,

[0094] (c) Knockout of bases not in multiples of 3 in the coding region of the CDPK gene, resulting in a frameshift mutation at at least one amino acid site in the CDPK protein, leading to premature termination of protein translation, thus obtaining a prematurely terminated translation mutant; and / or,

[0095] (d) Insertion of bases that are not integer multiples of 3 into the coding region of the CDPK gene causes a frameshift mutation at at least one amino acid site in the CDPK protein, resulting in premature termination of protein translation and obtaining a prematurely terminated translation mutant.

[0096] In the specific embodiments provided by the present invention, after deletion or insertion mutations are made at the active site of the CDPK gene, the amino acid site with kinase activity (i.e., the ATP-binding domain) will be changed, resulting in loss of kinase activity and alteration of protein function; frameshift mutations are made in the coding region of the CDPK gene, which can cause premature termination of protein translation, protein truncation, and kinase inactivation.

[0097] In specific embodiments provided by the present invention, for example, the knockout of bases that are multiples of 3 is the knockout of 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 bases. In this case, only the amino acid at the CDPK knockout site is missing, causing CDPK to lose its kinase activity, but it does not affect the translation of the entire CDPK protein.

[0098] In specific embodiments provided by the present invention, for example, the insertion of bases in multiples of 3 is the insertion of 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 bases. In this case, only the amino acid type of the kinase active site at the CDPK insertion site is changed, so that CDPK loses kinase activity, but does not affect the translation of the entire CDPK protein.

[0099] In specific embodiments provided by the present invention, for example, the knockout of bases that are not integer multiples of 3 is the knockout of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or 31 bases. In this case, a frameshift mutation occurs in CDPK translation, causing premature termination of translation and only a truncated sequence that loses kinase activity is obtained.

[0100] In specific embodiments provided by the present invention, for example, the insertion of bases that are not integer multiples of 3 is an insertion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or 31 bases. In this case, a frameshift mutation occurs in CDPK translation, causing premature termination of translation and only a truncated sequence that loses kinase activity is obtained.

[0101] In specific embodiments provided by this invention, the amino acid sites with kinase activity include at least one of the following: L62, G63, R64, G65, T70, A83, K85, V118, M134, E135, L136, C137, E141, E184, N185, L187, I200, and D201. In these specific embodiments, only the L62 and G63 sites are mutated, but other sites or combinations of sites can also achieve the same result.

[0102] In specific embodiments provided by this invention, the methods for mutating the CDPK gene include at least one of CRISPR / Cas9 gene editing technology, guided editing technology, promoter editing technology, TALEN technology, T-DNA insertion, EMS mutagenesis, and ZFN technology. Specific mutations in CDPK can be repeatedly achieved using the above methods.

[0103] Fifthly, the present invention provides a method for improving tomato plants or seeds, which involves knocking out an integer multiple of 3 bases at the active site of the CDPK27 gene, resulting in a deletion mutation at at least one amino acid site with kinase activity in the CDPK27 protein, thereby losing kinase activity and obtaining a deletion mutant, further yielding improved tomato plants or seeds. The improved tomato plants or seeds comprise:

[0104] (i) The average number of seeds per fruit is reduced by at least 5% compared to the control tomato plant or seed;

[0105] The control tomato plants or seeds and the improved tomato plants or seeds contained essentially the same genetic background.

[0106] In a specific embodiment provided by the present invention, the deletion mutant is MM-SlCDPK27-CR1.

[0107] Sixthly, the present invention provides a method for improving tomato plants or seeds, wherein the coding regions of both the CDPK27 and CDPK26 genes are knocked out in multiples of 3 (not integer multiples of 3), resulting in a frameshift mutation at at least one amino acid site in the CDPK27 protein and a frameshift mutation at at least one amino acid site in the CDPK26 protein, leading to premature termination of protein translation, thereby obtaining a double-gene premature translation termination mutant, and further obtaining an improved tomato plant or seed, the improved tomato plant or seed comprising:

[0108] (ii) The average weight of 1,000 seeds was reduced by at least 5% compared to the control tomato plants or seeds;

[0109] The control tomato plants or seeds and the improved tomato plants or seeds contained essentially the same genetic background.

[0110] In a specific embodiment provided by the present invention, the dual-gene translation premature termination mutant is MM-CDPK27-CR2 / MM-CDPK26-CR1.

[0111] In a seventh aspect, the present invention provides a mutant protein, wherein the mutant protein is at least one of the following sequences:

[0112] (41) The CDPK27 mutant protein sequence shown in SEQ ID NO:18;

[0113] (42) The CDPK27 mutant protein sequence shown in SEQ ID NO:21;

[0114] (43) The CDPK26 mutant protein sequence shown in SEQ ID NO:24;

[0115] (44) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the sequences (41)-(43).

[0116] Eighthly, the present invention provides a polynucleotide, wherein the polynucleotide is at least one of the following sequences:

[0117] (51) The nucleotide sequence of the CDPK27 mutant gene shown in SEQ ID NO:19;

[0118] (52) The nucleotide sequence of the CDPK27 mutant gene shown in SEQ ID NO:22;

[0119] (53) The nucleotide sequence of the CDPK26 mutant gene shown in SEQ ID NO:25;

[0120] (54) The nucleotide sequence of the mutant protein shown in any of the coding sequences (41)-(44) other than (51)-(53).

[0121] In a ninth aspect, the present invention provides the application of the above-mentioned mutant proteins and / or polynucleotides in regulating plant seed traits.

[0122] The seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

[0123] In a specific embodiment of the present invention, the present invention provides a method for improving a plant or its seeds by transferring the following mutant polynucleotides and / or mutant proteins into the plant to be improved, thereby obtaining the improved plant or its seeds, wherein the plant to be improved is a plant other than tomato.

[0124] The CDPK27 mutant gene shown in SEQ ID NO:19 is a deletion mutant gene obtained by knocking out 6 bases at the kinase active site of the CDPK27 gene. This CDPK27 mutant gene causes deletion mutations at L62 and G63 in the CDPK protein, resulting in the CDPK27 deletion mutant protein shown in SEQ ID NO:18. Both the CDPK27 deletion mutant gene and the CDPK27 deletion mutant protein can be synthesized artificially and then transferred into the plant to be improved to obtain seedless or low-seed improved plants.

[0125] The CDPK27 mutant gene shown in SEQ ID NO:22 is a deletion mutant gene obtained by knocking out 5 bases at the kinase active site of the CDPK27 gene. This CDPK27 deletion mutant gene causes a frameshift mutation in the CDPK protein starting from amino acid position 62, leading to premature termination of translation, resulting in the truncated CDPK27 premature translation mutant protein shown in SEQ ID NO:21. The CDPK26 mutant gene shown in SEQ ID NO:25 is a deletion mutant gene obtained by knocking out 5 bases at the kinase active site of the CDPK26 gene. This CDPK26 deletion mutant gene causes a frameshift mutation in the CDPK protein starting from amino acid position 64, leading to premature termination of translation, resulting in the truncated CDPK26 premature translation mutant protein shown in SEQ ID NO:24. The aforementioned CDPK27 deletion mutant gene, CDPK27 translation premature termination mutant protein, CDPK26 deletion mutant gene, and CDPK26 translation premature termination mutant protein can all be synthesized artificially and then transferred into the plant to be improved to obtain seedless or small-seeded improved plants.

[0126] In some embodiments, the plant to be improved is one of the following: berry plants, solanaceae plants, Rosaceae plants, Rutaceae plants, Convolvulaceae plants (Ipomoea spp.), Dioscorea cirrhosa plants, and Cucurbitaceae plants. For example, the plant to be improved includes grapes, kiwifruit, papaya, pomegranate, ginseng fruit, blueberries, persimmons, passion fruit, dragon fruit, wax apples, eggplants, chili peppers, goji berries, pears, apples, peaches, apricots, cherries, plums, apricots, crabapples, loquats, hawthorns, strawberries, raspberries, grapefruits, phellodendron bark, lemons, oranges, tangerines, and other citrus fruits.

[0127] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0128] In previous studies, it was found that a lack of SlCDPKs increased the sugar content of the fruit (see patent application number 202211616622.8, publication number CN118207227A). In this invention, research revealed that specific SlCDPKs mutations not only increase the sugar content of the fruit but also reduce the number or weight of seeds, while germination remains normal, indicating that photosynthetic substances can be redistributed from seeds to the fruit during ripening. Excessive seed quantity or oversized seeds can lead to excessive nutrient accumulation, thus affecting sugar accumulation in the fruit. These results suggest that the SlCDPKs gene cloned in this invention can indirectly affect seed development by regulating energy distribution in tomato fruits, reducing seed number and weight, maintaining seed germination rate, and providing genetic resources and a theoretical basis for creating seedless, low-seeded, and / or small-seeded, as well as high-sugar tomatoes. It also provides a new solution for manipulating seed number or size and increasing fruit sugar content. Attached Figure Description

[0129] Figure 1 This shows the protein sequence information of the tomato CDPK27 gene; among which, Figure 1 A shows the predicted conserved domains of the tomato CDPK27 protein sequence. Figure 1 B shows detailed information on the location of conserved domains in the tomato CDPK27 protein sequence;

[0130] Figure 2 The tomato CDPK27 gene homozygous mutant lines MM-SlCDPK27-CR1 and MM-SlCDPK27-CR2 are shown.

[0131] Figure 3 The MM-CDPK26-CR1 genotype of the tomato CDPK27 and CDPK26 homozygous double mutant lines MM-CDPK27-CR2 / MM-CDPK26-CR1 is shown.

[0132] Figure 4 The specific protein sequences and active sites of the tomato CDPK27 gene homozygous mutant lines MM-SlCDPK27-CR1 and MM-SlCDPK27-CR2 are shown.

[0133] Figure 5 The tomato MM-SlCDPK27-CR1 protein can be encoded normally, but it has lost its kinase activity;

[0134] Figure 6 The specific protein sequence of the MM-CDPK26-CR1 genotype of the tomato CDPK27 and CDPK26 homozygous double mutant lines MM-CDPK27-CR2 / MM-CDPK26-CR1 is shown.

[0135] Figure 7 The results of tomato seed phenotypic identification are shown, where a is the average number of seeds per fruit, b is the average weight of 1,000 seeds, c is the seed germination rate, and *** indicates P-Value < 0.001. Detailed Implementation

[0136] This invention discloses the application and improvement methods of polynucleotides, proteins, and biomaterials in regulating plant seed traits. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0137] This application compared the genomic, CDS, and protein identities of 46 tomato varieties CDPK27 and CDPK26:

[0138] Table 1. Identification of CDPK27 genome, CDS, and proteins.

[0139]

[0140]

[0141] Table 2. Identification of CDPK26 genome, CDS, and proteins.

[0142]

[0143]

[0144] Among different varieties, the genomic identity of CDPK27 was 98.734%–100%, the CDS identity was 92.83%–100%, and the protein sequence identity was 93.007%–100%; the genomic identity of CDPK26 was 98.734%–100%, the CDS identity was 90.59%–100%, and the protein sequence identity was 90.037%–100%.

[0145] Note: In this application, CDPK27 gene is the abbreviation of Solyc11g065660 gene (recorded as CDPK8 in patent application number 202211616622.8 and publication number CN118207227A), and CDPK26 gene is the abbreviation of Solyc01g008440 gene (recorded as CDPK9 in patent application number 202211616622.8 and publication number CN118207227A).

[0146] CDPK27, CDPK26, or CDPK27, CDPK26 indicate that the gene or protein can be identified based on the context.

[0147] The reagents, instruments, strains or biological materials used in this invention can all be obtained through commercial channels.

[0148] The present invention will be further illustrated below with reference to the embodiments:

[0149] Example 1: Knocking out the CDPK27 gene using the CRISPR / Cas9 system

[0150] The tomato CDPK27 gene protein sequence information is as follows:

[0151] Figure 1 According to the conserved domain prediction of the tomato CDPK27 protein sequence, CDPK27 contains 533 amino acid residues. At the N-terminus, it contains a low-complexity region (LCR); a serine / threonine protein kinase domain (S_TKc) with protein kinase activity, which can catalyze the phosphorylation of serine and threonine hydroxyl groups in the protein; and at the C-terminus, it has four EF-Hand domains that can bind to calcium ions, causing conformational changes in the protein. Figure 1 B shows detailed information on the location of conserved domains in the tomato CDPK27 protein sequence. Data source: SMART website (http: / / smart.embl-heidelberg.de / ).

[0152] The CDPK27 gene in tomato was knocked out using the CRISPR / Cas9 system to obtain a CDPK27 gene knockout mutant. The specific steps are as follows:

[0153] Step 1: Selection of sgRNA sequence

[0154] A target site sequence with a length of 19 bp was designed on the CDPK27 gene.

[0155] Sequence 1 (CDPK27-CDS):

[0156] ATGGGGAATTGCTGTGGGACACCTGGTAATTCTTCTGAGAATAAGAAGAAGAAGAACAAA

[0157] CCAAACCCTTTTGCTCTTGATTATGGTGCAACTCAAGCATCTGGAGGTGATG GAAACAAG C

[0158] TTGTTGTGTTG AAAGATCCAACAGGACACAATATTCAAGAAAAATATGATC TTGGTTGTG

[0159] AGCTAGGAAG AGGAGAATTTGGGGTTACATATTTATGTACTGATGTTGATACAGGGGAC

[0160] AAATATGCTTGCAAATCGATATCAAAGAAGAAACTTAGGACTGCTGTAGATATAGATGAT

[0161] GTTAGGAGAGAAGTTGAGATCATGAAGCATTTGCCTAAAACATCCTAATATTGTGACCTTG

[0162] AAGGACACTTATGAGGATGATAATGCAGTGCATATTGTGATGGAACTTTGTGAGGGGGG

[0163] GAGTTGTTTGATAGGATTGTTGCCAGGGGACATTATACAGAGAGAGCAGCTGCGGTTATT

[0164] ATGAAGACTATAGTAGAAGTTGTTCAGATGTGTCATATGCATGGAGTAATGCATCGTGAT

[0165] CTCAAACCTGAGAACTTTCTGTTTGGTAACAAGAAAGAAACTGCTCCTTTGAAGGCTATTG

[0166] ACTTTGGATTATCAGTGTTCTTTAAAACCCGGGAACGCTTTAATGAGATTGTGGGAAGTCC

[0167] TTATTACATGGCTCCAGAGGTCCTAAAACGCAATTATGGCCCGGAGGTTGATGTCTGGAGT

[0168] GCTGGAGTTATCCTTTATATTCTTCTTTTGTGGTGTTCCACCTTTCTGGGCAGAGACTGAACA

[0169] AGGAGTAGCCCAAGCAATTATTCGGTCTGTGGTTGATTTCAAGAGAGATCCATGGCCTAA

[0170] GGTTTCTGATAATGCAAAGGATCTTGTAAAGAAGATGCTTGACCCGGATCCAACTCGACG

[0171] ACTCACAGCTCAGCAGGTTCTTGAGCACACCTGGTTACAAAATATAAAGAAAGCACCAAA

[0172] TGTCTCATTGGGTGAGACTGTGAAAGCAAGGCTTAAACAATTTTCAGTAATGAACAAGCT

[0173] CAAGAAAAGAGCTCTAACGATTATGGCCGAGTTTTTATCAGCGGAAGAAGTGGCTGGAAT

[0174] GAAGGACGCATTTGATATGATGGATACAGGAAAGAAAGGCAAGATTAACCTTGGAGAAC

[0175] TTAAAAATGGTCTGCAAAAGCTTGGCCATCAGATCCCTGATGTTGATCTTCAGATTCTTAT

[0176] GGAAGCTGCGGATGTTGATGGAGATGGAAGCTTAAATTATGGGGAGTTTGTTGCTGTATC

[0177] TGTTCATCTCAGAAAAATGGCAAATGATGAGCACCTGCACAAAGCATTTTCAGTTTTTGAC

[0178] AGAGATCAGAGTGGTTACATAGAAATCGAGGAGCTGCGCAGTGCCTTGAGTGATGAGGAT

[0179] GGTGGCAACAGTGAGGAAGTCATCAATGCCATTATGCATGATGTTGACACTGACAAGGAT

[0180] GGCCGCATTAGTTACGAGGAATTTGCTGCAATGATGAAGGCTGGCACAGATTGGAGAAAA

[0181] GCATCGAGACAGTATTCTCGTGAAAGATTCAACAGTCTCAGCTTAAAATTGATGAGAGAT

[0182] GGCTCGATACAAGTCGGAAAGGAAGAAGGTAGATGA

[0183] Sequence 2 (CDPK27 - protein):

[0184] MGNCCGTPGNSSENKKKKNKPNPFALDYGATQASGGDGNKLVVLKDPTGHNIQEKYDLGCE

[0185] LG RGEFGVTYLCTDVDTGDKYACKSISKKKLRTAVDIDDVRREVEIMKHLPKHPNIVTLKDTY

[0186] EDDNAVHIVMELCEGGELFDRIVARGHYTERAAAVIMKTIVEVVQMCHMHGVMHRDLKPEN

[0187] FLFGNKKETAPLKAIDFGLSVFFKPGERFNEIVGSPYYMAPEVLKRNYGPEVDVWSAGVILYIL

[0188] LCGVPPFWAETEQGVAQAIIRSVVDFKRDPWPKVSDNAKDLVKKMLDPDPTRRLTAQQVLEH

[0189] TWLQNIKKAPNVSLGETVKARLKQFSVMNKLKKRALTIMAEFLSAEEVAGMKDAFDMMDTG

[0190] KKGKINLGELKNGLQKLGHQIPDVDLQILMEAADVDGDGSLNYGEFVAVSVHLRKMANDEH

[0191] LHKAFSVFDRDQSGYIEIEELRSALSDEDGGNSEEVINAIMHDVDTDKDGRISYEEFAAMMKA

[0192] GTDWRKASRQYSRERFNSLSLKLMRDGSIQVGKEEGR

[0193] Sequence 3 (CDPK27 - genome):

[0194] ATGGGGAATTGCTGTGGGACACCTGGTAATTCTTCTGAGAATAAGAAGAAAGAACAAA

[0195] CCAAACCCTTTTGCTCTTGATTATGGTGCAACTCAAGCATCTGGAGGTGATG GAAACAAG C

[0196] TTGTTGTGTTG AAAGATCCAACAGGACAATATTCAAGAAAAATATGATC TTGGTTGTG

[0197] AGCTAGGAAG AGGAGAATTTGGGGTTACATATTTATGTACTGATGTTGATACAGGGAC

[0198] AAATATGCTTGCAAATCGATATCAAAGAAGAAACTTAGGACTGCTGTAGATATAGATGAT

[0199] GTTAGGAGAAGTTGAGATCATGAAGCATTTGCCTAAACATCCTAATATTGTGACCTTG

[0200] AAGGACACTTATGAGGATGATAATGCAGTGCATATTGTGATGGAACTTTGTGAGGGGGGT

[0201] GAGTTGTTTGATAGGATTGTTGCCAGGGACATTATACAGAGAGAGCAGCTGCGGTTATT

[0202] ATGAAGACTATAGTAGAAGTTGTTCAGGTACAGTTTTACTAATTAATGCTTGTCTTTGCTTT

[0203] TCATCTATAATTGTTACTTGTTTGTTTATTTCATGTAATTTAGGGTTGCTAATTTAGGATT

[0204] ATGTTAATGAAGTTTGAATTGACAATGCCAACTGAAAGTGTCAATTTAGACTAGAAAGGA

[0205] ATGTTGAGATTGAAGTTTCATGTTTGTAAGCAGAATGAATGTTCTTGTCGAAGAATCTCTT

[0206] TTAGAATATCTTTCCCACTTCTTCAGTAAATTTTCCTACTTTCTTATGTCGATAACTGCCA

[0207] CTCCTTTTCTGAGTTTGCCTGCACTCTCTTGGTAGATAGGACACTCCCTTTCTTAGCTCAGG

[0208] ATCTACTTTGCTTGTGTAAATGAGTGCTTAATGTTTTTATTTGATTCATTTTACACGTTGAG

[0209] ATCCTATAGCATTTAATTAGACTAGATTTTATCTTCCAATATCACTAAACCTAGATGTCAA

[0210] ATATAGTAAAAAGTTATGTTGCTCGAACTCTTCAAAGATATCGACATGTGTGTGTCAGATC

[0211] CTCCAAGGTAGTGCATTTTTGGAGGATCCGACGGGGGTGGGGCAACAATTTTGGAGAGT

[0212] TCGAGCAACTTAGGTAAAAAGTGTATTCATGCTCTAAATGTTGTTAGGTTTTCTATCTTTCT

[0213] TATCTTGTCTTGTGAATTTTGCTTTCTTGGCAACTGTTTCATTGTCCTATATTTTTCTCATGC

[0214] AAATACCTTTAAAATTATATACTTTCGTGCCAACCATTTAAATGAAACTTAAGGAAGCTAA

[0215] TCACACAGGATTTCACCATCCAAATGTCGAAAATGAATCGCAATTTGTGATAGTTTTAGAC

[0216] AGAGAGCTTATTTTTATAGATGCAATTAGAATAATAGAAATGTAAAATAACTATCAGTAA

[0217] CCAGTAAGATGCCGGTCTTTATGGTCTATTTTCAGAGGGGGAGATCTATTGTAGCTTGCTT

[0218] TCAGCATCAGTTAGTATAGCATAGGACTGTTGCTAACAGGGTTCAATTTTAAACTGTATTT

[0219] GGTGAACATAATGACATGAGAGCTCAACTTACATAACTACAGATTAGAGAGAGAGACATTCA

[0220] TTTTATGGAATGGATTTAAGAAGTTCATGTTTCTACATTTAGTTGTTATAAGAACAGGGTG

[0221] GTAACTGGTAACCGACGCAAACATGATGGCTAATTATCCCAAGTTTATACAATTTAATCTA

[0222] TGTGGTTGCTCATACATCTTTGGTCAATGTTCACTTTACAGTTGCATTATGTTTCCTCAAGA

[0223] AATGGCTGTTCTTCTTTCAATTTTCAACTTACGGAGTTTCAATTGACATTGCAGATGTGTCA

[0224] TATGCATGGAGTAATGCATCGTGATCTCAAACCTGAGAACTTTCTGTTTGGTAACAAGAAA

[0225] GAAAACTGCTCCTTTGAAGGCTATTGACTTTGGATTATCAGTGTTCTTTAAACCCGGTAATG

[0226] CAATAAGTAATGATAGTGTGTTGTAACTTTTTTTCATGCTAAAAAAGATTAATGTTGGAAT

[0227] CATCTTTTAATATGTTTAACAGGGGAACGCTTTAATGAGATTGTGGGAAGTCCTTATTACA

[0228] TGGCTCCAGAGGTCCTAAAACGCAATTATGGCCCGGAGGTTGATGTCTGGAGTGCTGGAG

[0229] TTATTCCTTTATATTCTTCTTTGTGGTGTTCCACCTTTCTGGGCAGGTCCGTTTCTTTTATTTG

[0230] CCTTCTTTAGGTAACAAGTAATACCTCGGTTCACCGACAAAATAACAAAGTAAATCAACCTT

[0231] GGTGCATACTATTCATTTCTTAGAACTTGGTAATTTATTAATGAGTGTCAAAATGAAATGG

[0232] AATTTGAAACATATTGATGCGTTGTTAACATCGTCGATGACTAGGTGAGGGTGGCTGCAA

[0233] GAGTGAATCCCACCTCTTCCTGGACCAATTCAAAATCAAAGTTCCTTTTTTTCCTTTTGGCC

[0234] TGTGATAGTCAGTACTTTGTGCTTCCATTAATTTACTCACAGCTTTCTCAGTAATTGCCTAA

[0235] AATTCTACTGAATATGTGCAGAGACTGAACAAGGAGTAGCCCAAGCAATTATTCGGTCTG

[0236] TGGTTGATTTCAAGAGAGATCCATGGCCTAAGGTTTCTGATAATGCAAAGGATCTTGTAAA

[0237] GAAGATGCTTGACCCGGATCCAACTCGACGACTCACAGCTCAGCAGGTTCTTGGTAATGTT

[0238] TGTTAATCCTTGAGATGCTATACTTTCATTTGCTTGCTGTTTGTTCTCTCTTAGTTTCATAAG

[0239] TTCAATTTTGTGACTTTTTGGGGCTTTCGTGCATATACATGAATGCAGAGCACACCTGGTT

[0240] ACAAAATATAAAGAAAGCACCAAATGTCTCATTGGGTGAGACTTGGAAAGCAAGGCTTAA

[0241] ACAATTTTCAGTAATGAACAAGCTCAAGAAAGAGCTCTAACGGTAAAATATTCCCTTTTC

[0242] ATTTGCAGTTACATATCATTAGCGTTCTGCACCTTCTCAGAAGGATATCTTCATATGCCTA

[0243] ATTCTTAAAGCCCAAAATAAGGACAAAGATTTTCTGCCTTCCACAAAGTTTCGGGTGT

[0244] TATATTCTTTCTTCATTGCTTGCACAGATTATGGCCGAGTTTTTATCAGCGGAAGAAGTGG

[0245] CTGGAATGAAGGACGCATTTGATATGATGGATACAGGAAAGAAAGGCAAGATTAACCTTG

[0246] GAGAACTTAAAAATGGTCTGCAAAGCTTGGCCATCAGATCCCCTGATGTTGATCTTCAGAT

[0247] TCTTATGGAAGCTGTAAGCATTCTCATGCTCTTCATTTCACTTGATTGGATTTTCTTTCAAC

[0248] TTTACGAAGACTAAATATATATTATTATAAGTCAAGCGTCTAATATTATTATAATTGTTCC

[0249] CTTTTTTATCTTCTGCTTCACTTTTATAGGCGGATGTTGATGGAGATGGAAGCTTAAATTAT

[0250] GGGGAGTTTGTTGCTGTATCTGTTCATCTCAGAAAAATGGCAAATGATGAGCACCTGCAC

[0251] AAAGCATTTTCAGTTTTTGACAGAGATCAGAGGTGGTTACATAGAAATCGAGGAGCTGCGC

[0252] AGTGCCTTGAGTGATGAGGATGGTGGCAACAGTGAGGAAGTCATCAATGCCATTATGCAT

[0253] GATGTTGACACTGACAAGGTTAGAATTTTTTTTGTGTCCATTCATGTATATCTACTGGATTT

[0254] CATACTAGTGCATATACTTCTTTTATAAATGTCTCTTTAATTGCTTGTGGAACCATAAATAC

[0255] CCCCCTTATCTTGTTCCGCAGCTCCAACTACACACGAAACTATGTTGAAGACGTATTACCC

[0256] CCTTGTACTTGTAAAATGTGTATCTTCCGTCATCCTGAGAGGCATATATGATGTAACCAAA

[0257] TGTGAACATTTAAGTATATGCATGTGCTGCCTCATGTCATTTACCCCTTTTTCCCTTTTCAC

[0258] TTCTCTTCCATCTCCGCAACATGTTAATGTCCTCTAGCAAATCTGAAAAAAGATGGAGTGA

[0259] GCTTCTCCAACGGAAAAAATCGGATTCACCCTAGTTTTAGTTTTCTGGCAACATAAACAGCT

[0260] CAAGTCGATACAAAAAAACAAATGTTCCTATCAAAAAACTCAAATTAGTAGAAAGGAAA

[0261] AGAAGGGTCATAGGTAAGGGAAGAACAAGAAAGGAATAGAAAGACCCTTCGGGGTGGCCC

[0262] AGTTATTTGGGCTTGGGGCTTGGGGCTTGGGACTTCCATGTTGGAGGTCTCAAGTTCAAAA

[0263] CCCCTTGCCAATGAAAGCAACGGGTTTTCCTTCTACGTTGACTGTCGAGCTCGTCGCACCG

[0264] GGCTTACCTAGTGCGGTTTACCTCTCCTTTGCGTGCTATAGGAATAGGGGTTTTACCCTGT

[0265] GTGCACTCAAAGGGTAGCGACTGCGGATTTCCCAAGAAAAGGAAGAGAAAGGTAAA

[0266] GAAAACATTTTAAAAAAGAGTAGGATGGTTATAATAGAAGAAATGCTACAATATTTTTTC

[0267] ACCTCAGGCGCTTTAAGAGAGTAAATACTCTACGTTACATGGCGTCCCAATGTTGTCGTAG

[0268] TTACATTTTGGACAAGAACAGGGAGTAATAGGCCTCCCGCAAAGTTAAAATGTAGTGGGG

[0269] AATGGGGGAATAATTGAGGAAGGTATTTATATATTTTCTCCTTTCTAAATCATATTTTCTTT

[0270] TCCATTCACTGCTCTTCTGTTCAACCTTATCATTCATTTGGACTAAATGTTGAATTGATTTT

[0271] GAGCTTTAGTTTGTTACCACAGCTTCAAAGTTCATCAGTTACATTGTCTATAACCATGCA

[0272] GGATGGCCGCATTAGTTACGAGGAATTTGCTGCAATGATGAAGGCTGGCACAGATTGGAG

[0273] AAAAGCATCGAGACAGTATTCTCGTGAAAGATTCAACAGTCTCAGCTTAAAATTGATGAG

[0274] AGATGGCTCGATACAAGTCGGAAAGGAAGAAGGTAGATGA

[0275] The target site is located at positions 173-191 of sequence 1 (CDS sequence) (as shown in the box in sequence 1, where the bolded GCTAGG is...). Figure 2 The 6 bases missing in the MM-CDPK27-CR1 mutant shown are located at positions 173-191 of sequence 3 (genomic sequence), and the target site 1 sequence is TTGGTTGTGAGCTAGGAAG (Sequence 4, as shown in the boxed sequence in sequence 1 and sequence 3).

[0276] The target site design sgRNA sequence is as follows:

[0277] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (sequence 5)

[0278] The DNA molecule encoded by this sgRNA is:

[0279] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (Sequence 6).

[0280] Step 2: Construction of CRISPR / Cas9 vector

[0281] The original vector contains an sgRNA sequence. The target site 1 sequence from step 1 is then inserted into the vector to obtain a CRISPR / Cas9 vector.

[0282] Step 3: Obtaining transgenic plants

[0283] The CRISPR / Cas9 vector obtained in step 2 was transformed into Agrobacterium competent cells EHA105 (Agrobacterium EHA105 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., and are available to the public through purchase) via heat shock transformation to obtain recombinant bacteria EHA105 / CRISPR / Cas9.

[0284] The recombinant bacteria EHA105 / CRISPR / Cas9 were then transformed into tomatoes using the Agrobacterium tumefaciens infection method (the recombinant Agrobacterium tumefaciens was propagated at 28°C, and the propagated bacterial solution was used to infect tomatoes). After kanamycin resistance screening, T0 generation transgenic tomato plants were obtained.

[0285] Step 4: Identification of transgenic plants with CDPK27 gene mutations

[0286] Leaves of T0 generation transgenic tomato plants obtained in step 3 of Example 1 were collected, and genomic DNA was extracted as a template. PCR amplification was performed using the following primer pairs to obtain PCR amplification products of different strains.

[0287] The sequences of the primers for detecting the CDPK27 mutation are as follows:

[0288] CDPK27-test-F:ATGGGGAATTGCTGTGGGAC(Sequence 7)

[0289] CDPK27-test-R:TTCATAATAACCGCAGCTGCTCTCTCT(Sequence 8)

[0290] PCR amplification products from different strains were subjected to Sanger sequencing, and the sequencing results were compared with the wild-type (MoneyMaker) CDPK27 gene. The CDPK27 genotype was identified according to the following principles.

[0291] If a sequence has a bimodal characteristic starting from the target site sequence, then the genotype of the strain is heterozygous (the CDPK27 gene on one of the two homologous chromosomes is mutated, while the CDPK27 gene on the other chromosome is not mutated), and the strain is a T0 generation transgenic tomato heterozygous mutant strain.

[0292] If a line has a bimodal sequence starting from the target site and the CDPK27 gene is mutated in both homologous chromosomes, then the line is a T0 generation transgenic tomato biallelic mutant line.

[0293] If a sequence with a specific single-peak characteristic starting from the target site sequence is identical to the CDPK27 gene sequence of wild-type tomato, then the genotype of the strain is wild-type, meaning that the CDPK27 gene sequence has not been mutated; if it is different from the CDPK27 gene sequence of wild-type tomato, then the genotype of the strain is homozygous (the CDPK27 gene on both homologous chromosomes has been mutated), and the strain is a T0 generation transgenic tomato homozygous mutant strain.

[0294] This case identified a T0 generation homozygous mutant line of the CDPK27 gene (e.g., Figure 2 (As shown), it is used to identify the following tomato seed quantity, thousand-seed weight, and germination rate phenotypes.

[0295] Figure 2 The genotypes MM-SlCDPK27-CR1 and MM-SlCDPK27-CR2 represent homozygous mutant lines of the tomato CDPK27 gene. Compared to the wild type (WT), the MM-SlCDPK27-CR1 mutant line has a 6-base deletion mutation, resulting in the loss of two amino acids, L (leucine) and G (glycine). Figure 4 The missing two amino acids are essential for the CDPK27 protein to maintain its kinase activity. After their deletion, the SlCDPK27 protein can still encode the kinase, but it loses its kinase activity (e.g., ...). Figure 5 As shown in SlCDPK27-CR1, the MM-SlCDPK27-CR2 mutant line has a 5-base deletion mutation, which causes a frameshift mutation in the translation of the SlCDPK27 protein, leading to premature termination of translation and encoding only the 72 amino acids at the N-terminus. Figure 4 Therefore, both MM-SlCDPK27-CR1 and MM-SlCDPK27-CR2 are loss-of-function mutants of the SlCDPK27 gene. Among them, MM-SlCDPK27-CR1 is a CDPK27 kinase activity-deficient mutant, and MM-SlCDPK27-CR2 is a CDPK27 translation premature termination mutant.

[0296] Example 2: Knocking out the CDPK26 gene using the CRISPR / Cas9 system

[0297] Based on the MM-SlCDPK27-CR2 mutant line obtained in Example 1, the CDPK26 gene in tomato was knocked out using the CRISPR / Cas9 system to obtain a CDPK26 gene knockout mutant. The specific steps are as follows:

[0298] Step 1: Selection of sgRNA sequence

[0299] A target site sequence with a length of 19 bp was designed on the CDPK26 gene.

[0300] Sequence 9 (CDPK26 - CDS):

[0301] ATGGGTAATTGCTGTGTGAAACCGGGTAAATCTGCCGAAAAAAAGAATAAAAAGAACAA

[0302] CAGCAAACCTAACCCTTTTTCAATTGATTATGGGGGGACTAAACATGCCTCTGGGAGTGG

[0303] AAACAAGCTAGTTGTATTA AAAGAACCAACAGGGCAAAATATACATGATAAGTATGATT T

[0304] GGGGCATGAGCTCGGAAG AGGAGAATTTGGGGTTACTTATCTATGTACTGACTTGGAAG

[0305] GAGGAGAAAAATATGCTTGCAAATCGATATCTAAAAAGAAGCTAAGGACTGCAGTGGAT

[0306] ATTGATGATGTTAGGAGAGAGGTTGAGATCATGAAACATTTGCCTGTGCATCCGAATATT

[0307] GTGACCTTGAAGGATACTTATGAGGATGATAATGCAGTGCACATTGTGATGGAATTGTGT

[0308] GAGGGTGGGGAGTTGTTTGATCGAATCGTTGCTAGAGGCCACTATACAGAGAGAGCAGCT

[0309] GCTGGAATTTTGAAGACTGTCGTGGAAGTCGTTCAGATGTGTCACAGGCAAGGGGTAATG

[0310] CATCGTGATCTCAAACCTGAGAATTTTCTTTTTGGTAACAAGAAAGAAACAGCTCCACTGA

[0311] AGGCTATTGACTTTGGGTTGTCTGTTTTCTTTAAACCTGGGGAACGCTTTAATGAGATAGT

[0312] GGGAAGTCCTTATTACATGGCTCCTGAGGTCCTAAAGCGCAATTATGGACCAGAGGTCGA

[0313] TGTCTGGAGTGCTGGAGTTATACTTTACATTCTTCTATGCGGTGTTCCACCTTTCTGGGCAG

[0314] AGACTGAACAAGGAGTAGCCCAAGCGATTATTCGTTCTGTGATTGATTTCAAGAGGGATC

[0315] CATGGCCTAAAGTTTCTGATAATGCAAAGGATCTTGTAAAGAAAATGCTTGATCCAGATC

[0316] CAACTCGACGGCTCACAGCTCATCAAGTTCTTGAGCATCCCTGGTTACATAATATAAAGAA

[0317] AGCACCAAACGTCTCATTGGGTGAGACTGTTAAAGCAAGACTCAAGCAGTTTTCAGTAAT

[0318] GAACAAGCTCAAGAAAAAAGCTCTGACGGTTATAGCTGAGTTTTTGTCTGCGGAGGAAGT

[0319] CGCTGGAATGAAGGAAGCATTTGAAATGATGGATACCGGAAAGAAGGGCAAGATAAACC

[0320] TGAATGAACTTAAAGATGGCTTGCAGAAGCTTGGCCATCAAATCCCTGATGCTGATCTTCA

[0321] TATTCTCATGGAAGCGGCTGACGTTGATGGAGATGGAAGTTTAAATTATCCAGAGTTTGTT

[0322] GCTGTATCTATTCATCTTAGAAAGATGGCCAATGATGAACACCTGCACAAAGCATTTTCAT

[0323] TTTTCGACAAAAATCAGAGTGGTTTCATAGAAATCGAAGAGCTTCGTAGTGCTTTGAGGG

[0324] ATGAAGACGACAGCAACAGCGAGGAAGTCACCAATGCCATTATGCATGACGTTGATACAG

[0325] ACAAGGATGGTCGGATTAGTTATGAGGAATTTGCTGCGATGATGAAGGCTGGTACGGACT

[0326] GGAGAAAAGCATCGAGACAGTATTCTCGTGAACGTTTTAACAGTCTAAGCTTAAAGTTGA

[0327] TGAGGGAAGGCTCATTACAAGTTGAAAACAAAGTCTAG

[0328] Sequence 10 (CDPK26 - Protein):

[0329] MGNCCVKPGKSAEKKNKKNNSKPNPFSIDYGGTKHASGSGNKLVVLKEPTGQNIHDKYDLG

[0330] HE LG RGEFGVTYLCTDLEGGEKYACKSISKKKLRTAVDIDDVRREVEIMKHLPVHPNIVTLKD

[0331] TYEDDNAVHIVMELCEGGELFDRIVARGHYTERAAAGILKTVVEVVQMCHRQGVMHRDLKP

[0332] ENFLFGNKKETAPLKAIDFGLSVFFKPGERFNEIVGSPYYMAPEVLKRNYGPEVDVWSAGVIL

[0333] YILLCGVPPFWAETEQGVAQAIIRSVIDFKRDPWPKVSDNAKDLVKKMLDPDPTRRLTAHQVL

[0334] EHPWLHNIKKAPNVSLGETVKARLKQFSVMNKLKKKALTVIAEFLSAEEVAGMKEAFEMMD

[0335] TGKKGKINLNELKDGLQKLGHQIPDADLHILMEAADVDGDGSLNYPEFVAVSIHLRKMANDE

[0336] HLHKAFSFFDKNQSGFIEIEELRSALRDEDDSNSEEVTNAIMHDVDTDKDGRISYEEFAAMMK

[0337] AGTDWRKASRQYSRERFNSLSLKLMREGSLQVENKV

[0338] Sequence 11 (CDPK26 - Genome):

[0339] ATGGGTAATTGCTGTGTGAAACCGGGTAAATCTGCCGAAAAAAAGAATAAAAAGAACAA

[0340] CAGCAAACCTAACCCTTTTTCAATTGATTATGGGGGGACTAAACATGCCTCTGGGAGTGG

[0341] AAACAAGCTAGTTGTATTA AAAGAACCAACAGGGCAAAATATACATGATAAGTATGATT T

[0342] GGGGCATGAGCTCGGAAG AGGAGAATTTGGGGTTACTTATCTATGTACTGACTTGGAAG

[0343] GAGGAGAAAAATATGCTTGCAAATCGATATCTAAAAAGAAGCTAAGGACTGCAGTGGAT

[0344] ATTGATGATGTTAGGAGAGAGGTTGAGATCATGAAACATTTGCCTGTGCATCCGAATATT

[0345] GTGACCTTGAAGGATACTTATGAGGATGATAATGCAGTGCACATTGTGATGGAATTGTGT

[0346] GAGGGTGGGGAGTTGTTTGATCGAATCGTTGCTAGAGGCCACTATACAGAGAGAGCAGCT

[0347] GCTGGAATTTTGAAGACTGTCGTGGAAGTCGTTCAGGTATAGTTTTTATCAACTTCTTAGT

[0348] TCTCAGTTACCTACTGCTTTTATTACCTTTTGCTTAGGTTATCTTATTATATCCATTATTTTC

[0349] AGCATAGCTTCTTCATTACTGTATTTCCTTTTCATATTGTTTTTATATATGTCTTACTTAGGC

[0350] TGAGGGTCCATTGGAAATAGCCTCTCTACCTTCACAAGGTAGGAGTAAGGCTGCGTACAC

[0351] ACTACCCTTCCCACACCCCACTTGTGGGACTATACTAAGTATGTTGTTGTTTTTAGTTCTCAA

[0352] TTGATGCTTTTTGGTTTTTATGTAGTAACTTTGGGTGTTTCTTTTACTGAGTTCTTCAAAGCT

[0353] TTTGTTATGGGTTTTATATCATGACGGATGTATCCTGTATTGTTGTTTCGGGTTTTTATTAAA

[0354] GAAGTCTGGCAATTTCAAGTTAAAAAGAAAATCATATCTTAAAAGAGAAGCCTAATCTTG

[0355] AGCAAATCTTGAGCAGATAGCAATTTCAGGGTGAAATGTGAATATTCTTGGTGAAGTTGC

[0356] ATATGAACTGGTAACCATTATTCAATTCATTTCTAGTGTTGATCTTTGCAATCAATGGTTTC

[0357] ATTCTAGTGTACGTTCATGCTGTAGCATTTACCTTGATATCCTCATGGAATTTCCTTTACTG

[0358] CCAAAATTATAATGAGAAGTGGTTATCTTTTTAATAATGGTAAAGTATGAGAAGAGGTTT

[0359] TCTTATGTTCTCAGTCTTTGCTAGTTCTATTTCTTTACTCGTTTTACTTAACTTTTTTAGTTTC

[0360] TTGTGAACTGTAGTATTGCTCTATATCTTCTCACTCAAAATTCATGTTCTAAATCTTAACCAT

[0361] TCTAGTGATAATAATGAAGTTAATTGGTGATATAGGATATCCCCCTCCAAAATCTAACAAT

[0362] CTTTCAACTCATCATAGTCTTGGGCAGATAGCTAGTTAGACCGTAACAAATATTTAGGAA

[0363] AGTAGATCATTCGAAATAGTTATCCTTGTGAAATATTATTCTTTATCTTCATTATTGACCAT

[0364] TGGTTTTCAAACTCTAGTACTCTTAAGTGGAAGACTCACTTCTTGTTTCTGTGGAGTATTAG

[0365] CTATAGCGGCCTACAACAGAATTTGATTGTTGCACTCAAAGTTAAACAGAGTTCTACAGTT

[0366] TAGGGAAATGATAATGGAATTTAAATTTAGTTTGTATACTGAAGTAAATATGGTTGACACT

[0367] ATCAGAAACAAGTGCTTTTACTTCCAAATATTGATCTTTATCCTCATTGCGAACCATTGAT

[0368] GTCCAAACTGTTTTAGATGGAAGAGCAATTTGTTGTTCATTTGAGTGGAATAGTCACAAC

[0369] AACAGCTAATTAACTACGTATAGCTTAGAGAGATGCGAACCAATGGTAAATTTAAGTTGT

[0370] CTCCTGAAGTAGTTACTGATTGTTCATGATAAAGGTCATTAAAGTTGAATTCTCTATTTAG

[0371] TCAGTGTCCTTTTCTGCATCGACTATATAGTGGCCTGAATGACATTACAAAATATTTATGA

[0372] GTTGGTAGGAATCTTTTTTTCATATGTGAATCATGAATGTGTTCACAAATGATCAATCAGA

[0373] GAGTGATTATTGTATTATGTGGTGTTGATAACTTTTGAACTTTTCTTCATTGGTTTGTGTTT

[0374] AACTGTTATCTGAAAGGCTTGGTTGGTGGTACTAAAGCTATTGGATTAGCAGAAAACTTTA

[0375] TACATGTTTATGGTCATGTTTCGCTCTCCAGTCCCCGTTTTTTATTTGTCAAAGGTCAGTTCA

[0376] TATATTTCCAATGGTTTTGCAGATGTGTCACAGGCAAGGGTAATGCATCGTGATCTCAAA

[0377] CCTGAGAATTTTCTTTTTGGTAACAAGAAAGAACAGCTCCACTGAAGGCTATTGACTTTG

[0378] GGTTGTCTGTTTTTCTTTAAACCTGGTAATGGAATTGGAAAAAAACAGTGAGTTTAAGACT

[0379] TTTATCATGCTAAACAAAGGTTTAACATGTTGATTATTTTTAACAGGGGAACGCTTTAATGA

[0380] GATAGTGGGAAGTCCTTATTACATGGCTCCTGAGGTCCTAAAGCGCAATTATGGACCAGA

[0381] GGTCGATGTCTGGAGTGCTGGAGTTATACTTTACATTCTTCTATGCGGTGTTCCACCTTTCT

[0382] GGGCAGGTCTGCTTATTTTATTTCTTCGTTTTTTATAACCATGGAACGAACCTCAATGCAG

[0383] ATATTTTCATGTGCAATTTTTGGAACTTGATAATTGATTAACTAAGATTGTGAATATGAAG

[0384] TAGAACTTGATTCATGTTAGAATATGTTGTTGAAATATTAGATGAGGCAAGTGAGGT

[0385] GAAAGACTGTCCCTGCTTTAATCAATGTAGATCATGTTCAATTATTTTGGTTTCTCTTAATG

[0386] GTTCTTATGGGTTGGACTTTGCTAAATAACTTGCATAAATATGTTAACATATTCTTGATACT

[0387] TCCCTTAAGTTCCTCTGAATATTTGCAGAGACTGAACAAGGAGTAGCCCAAGCGATTATTC

[0388] GTTCTGTGATTGATTTCAAGAGGGATCCATGGCCTAAAGTTTCTGATAATGCAAAGGATCT

[0389] TGTAAAGAAAATGCTTGATCCAGATCCAACTCGACGGCTCACAGCTCATCAAGTTCTTGGT

[0390] AATGTTTGTGAATATTGAGATATTGTACTTTATTCATTCGTTGTTTTACTCCTCTCTCTCT

[0391] CTCTCTCTCTCTCTATCTATCTCTGATAAGTATTTTTTTCCGGGGCTTTGACTCTTGTGGTGC

[0392] AGAGCATCCCTGGTTACATAATATAAAGAAAGCACCAAACGTCTCATTGGGTGAGACTGT

[0393] TAAAGCAAGACTCAAGCAGTTTTCAGTAATGAACAAGCTCAAGAAAAAAGCTCTGACGGT

[0394] AAGCACACACTTAGTCTGCAATCATATTTATAAGCTCTGGATTCTTCACTGTCCACTAAAG

[0395] ATGAATTCATATGCTTTTCCTCATAGATCGAAATGAAGGAGAGAAGGTTGTGTCTTAATTA

[0396] TTTACTGCACTGCCTTGTGCTATTTAATAAAAACTTAATTCTCATCCGTAAAAAATGAGAGG

[0397] CGGTTCTCAAAATTTTGATCAAGTTTCGTTTTTTTATCTTGATATATGTACTTTCTTGTATTC

[0398] CAAACAGGTTATAGCTGAGTTTTTGTCTGCGGAGGAAGTCGCTGGAATGAAGGAAGCATT

[0399] TGAAATGATGGATACCGGAAAGAAGGGCAAGATAAACCTGAATGAACTTAAAGATGGCT

[0400] TGCAGAAGCTTGGCCATCAAATCCCTGATGCTGATCTTCATATTCTCATGGAAGCGGTAAG

[0401] CGGTCTCATGCCCTGCTTCTTGGCTCGATCAGATTTCTTATAGCTCTACCGGGGGTAACAT

[0402] AATATTTGAAAGTTAGCTGCTAAACTTTTCTGCCTCAAAGAGAAAACAGGTGCCTGAGAT

[0403] GATATGGCCATGTCTTACGTAGAACTGTAAATTACATCAATGGACATTATTTTAAAATGAC

[0404] CGAAAACTACATGGAATCCATGTGAACTTAGTAAAGAAAATATTCAATGGAAGTAAAGGA

[0405] TACCAAGTAGTTGGATTAAGGCTAGATTGTTGGTGGTACACTGACATTAGTGTCTCAATGT

[0406] TTACTAAGAGTCTCAGTTTGATTAGAGATTTGCACGTAGATGCAGGGATAACCGTGAGATT

[0407] AAGAAAATTTTAAGAAATTCACGGAGATGAAAAAGATCTGTAATACTCATATAGCTTAGG

[0408] GCCAAGCCCAACTAGTTTGGGATCAAATCGTAGTTGTTGTTGATAAGAATTTAGCATTAGA

[0409] CTTTTGTCTGTCACCCTACTGTCAAAAAATTGATTGATTACAAAATATATTTTGTCAATATA

[0410] AGTTGTAACATTTTTCTACCATTTCAATGTAGAAAAAAGGAAAATCTGTAGCAAAATTAGC

[0411] TTGACATTTCGCTTGTCTTAGCATCCCTGGTTCAATGCTTATTATTATCCAGAAAGGAAAA

[0412] AAAAAACATTTCTCTTGATCCTTTTGCATCTTCGACCTGAAGGATGCTTATCTTTTTTATTA

[0413] TTTTATTTCCTTGAACTTTTGTTTTATGTTTTTCTTATTCTGTATCGGTTTTACAGGCTGACG

[0414] TTGATGGAGATGGAAGTTTAAATTATCCAGAGTTTGTTGCTGTATCTATTCATCTTAGAAA

[0415] GATGGCCAATGATGAACACCTGCACAAAGCATTTTCATTTTTCGACAAAATCAGAGTGG

[0416] TTTCATAGAAATCGAAGAGCTTCGTAGTGCTTTGAGGGATGAAGACGACAGCAACAGCGA

[0417] GGAAGTCACCAATGCCATTATGCATGACGTTGATACAGACAAGGTCAGTATGTCCATTTAT

[0418] GTGCACGTACACCATTTAGTAACTTTTCGTTCTTTGTTCATACTTCATAGTGCAAATATTA

[0419] TGCATTGTTGCTAATGTTTACATCTTAATGAGTTCTCATTTTGAAGTTCTCTTCTGAACCCT

[0420] CGTTTGAGTTTAGAGCTCAAATACTCCACCTAGTTGTGACTTATTGTATCTCTAAAGTCCGT

[0421] CATATTATATAAACGTATTATACTTTGATGGCATGCACACTTTCTAGCACATATGTGTTGCT

[0422] AACGAAACTCTAGTGCTGTGACAAATGAAATACATGTATACATTGATGGCATCTGTTCTTC

[0423] CCATCACATATGTACTACCGGTCATTACTCTCTAGTGCTGTGACATTATGCTCTCCCTGTTC

[0424] GCTTCATGAGTTACACGGTTGGTTAACCTGCTGCTCGTTACATGTCTGCTACAGAATTCAA

[0425] GTTTTGTTAGCTGTTTTCCATCTCATCTCTAACAACATCCCCACTCATTTGTATTAGCTTCTT

[0426] ATCTGGAGTTTTGTGAGCTCAGTCTTGTATCGTCTTGTGTCAAAACGTGTGCAGGATGGTC

[0427] GGATTAGTTATGAGGAATTTGCTGCGATGATGAAGGCTGGTACGGACTGGAGAAAAGCAT

[0428] CGAGACAGTATTCTCGTGAACGTTTTAACAGTCTAAGCTTAAAGTTGATGAGGGAAGGCT

[0429] CATTACAAGTTGAAAACAAAGTCTAG

[0430] The target site is located at positions 179-197 of sequence 9 (CDS sequence) (as shown in the boxed bases in sequence 9, where the bolded CTCGG is...). Figure 3 The five bases missing in the MM-CDPK26-CR1 mutant shown are located at positions 179-197 of sequence 11 (genomic sequence), and the target site 2 sequence is TGGGGCATGAGCTCGGAAG (sequence 12, as shown in the boxed sequence in sequence 9 and sequence 11).

[0431] The target site design sgRNA sequence is as follows:

[0432] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (sequence 13)

[0433] The DNA molecule encoded by this sgRNA is:

[0434] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (Sequence 14).

[0435] Step 2: Construction of CRISPR / Cas9 vector

[0436] The original vector contains an sgRNA sequence. The target site 2 sequence from step 1 is then inserted into the vector to obtain a CRISPR / Cas9 vector.

[0437] Step 3: Obtaining transgenic plants

[0438] The CRISPR / Cas9 vector obtained in step 2 was transformed into Agrobacterium competent cells EHA105 (Agrobacterium EHA105 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., and are available to the public through purchase) via heat shock transformation to obtain recombinant bacteria EHA105 / CRISPR / Cas9.

[0439] The recombinant bacteria EHA105 / CRISPR / Cas9 were then transformed into tomatoes using the Agrobacterium tumefaciens infection method (the recombinant Agrobacterium tumefaciens was propagated at 28°C, and the propagated bacterial solution was used to infect tomatoes). After kanamycin resistance screening, T0 generation transgenic tomato plants were obtained.

[0440] Step 4: Identification of transgenic plants with CDPK26 gene mutations

[0441] Leaves of T0 generation transgenic tomato plants obtained in step 3 of Example 2 were collected, and genomic DNA was extracted as a template. PCR amplification was performed using the following primer pairs to obtain PCR amplification products of different strains.

[0442] The sequences of the primers for detecting the CDPK26 mutation are as follows:

[0443] CDPK26-test-F:AGAACAACAGCAAACCTAACCC(Sequence 15)

[0444] CDPK26-test-R:TCCAGCAGCTGCTCTCTCTG(Sequence 16)

[0445] PCR amplification products from different strains were subjected to Sanger sequencing, and the sequencing results were compared with those of the wild-type CDPK26 gene. The CDPK26 genotype was identified according to the following principles.

[0446] If a sequence has a bimodal characteristic starting from the target site sequence, then the genotype of the strain is heterozygous (the CDPK26 gene on one of the two homologous chromosomes is mutated, while the CDPK26 gene on the other chromosome is not mutated), and the strain is a T0 generation transgenic tomato heterozygous mutant strain.

[0447] If a line has a bimodal sequence starting from the target site and the CDPK26 gene is mutated in both homologous chromosomes, then the line is a T0 generation transgenic tomato biallelic mutant line.

[0448] If a sequence with a specific single-peak characteristic starting from the target site sequence is identical to the CDPK26 gene sequence of wild-type tomato, then the genotype of the strain is wild-type, meaning that the CDPK26 gene sequence has not been mutated; if it is different from the CDPK26 gene sequence of wild-type tomato, then the genotype of the strain is homozygous (the CDPK26 gene on both homologous chromosomes has been mutated), and the strain is a T0 generation transgenic tomato homozygous mutant strain.

[0449] This case identified a T0 generation homozygous mutant line of the CDPK26 gene (e.g., Figure 3 (As shown), it is used to identify the following tomato seed quantity, thousand-seed weight, and germination rate phenotypes.

[0450] Figure 3 The MM-CDPK26-CR1 genotype represents the homozygous double mutant tomato lines MM-CDPK27-CR2 / MM-CDPK26-CR1. Compared to the wild type, the mutant lines exhibit a 5-base deletion mutation, leading to a frameshift mutation in CDPK26 protein translation, causing premature translation termination and encoding only the N-terminal 74 amino acids. Figure 6 Therefore, MM-CDPK27-CR2 / MM-CDPK26-CR1 is a mutant with premature termination of translation of the CDPK27 and CDPK26 genes.

[0451] Example 3: Determination of seed number, thousand-seed weight, and seed germination in tomato mutants with CDPK27 and CDPK26 gene knockout.

[0452] Phenotypic investigations were conducted on homozygous T2 generation plants selected after two generations of self-pollination of the T0 generation in Examples 1 and 2. The obtained T2 generation CDPK27 homozygous mutant lines MM-CDPK27-CR1 and MM-CDPK27-CR2, and the CDPK27 and CDPK26 homozygous double-gene mutant line MM-CDPK27-CR2 / MM-CDPK26-CR1 were planted in the greenhouse of the Nankou Pilot Base of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences in the spring of 2022. The results of the average seed number per fruit, average 1000-seed weight, and seed germination rate of wild-type plants (Money Maker) and mutants are shown in [the table below]. Figure 7 And Table 3.

[0453] Table 3

[0454]

[0455] Note: *** indicates P<0.001.

[0456] Systematic identification and statistical analysis of seed number, thousand-seed weight, and seed germination rate of mutants revealed that MM-CDPK27-CR1 showed a significant decrease in seed number, while thousand-seed weight and seed germination rate remained unchanged; MM-CDPK27-CR2 showed no significant changes in seed number, thousand-seed weight, or seed germination rate; and the thousand-seed weight of homozygous double-gene mutant lines of CDPK27 and CDPK26 was significantly reduced, while seed number and germination rate remained unchanged. Figure 7 (a, b, c) indicates that the CDPK27 and CDPK26 genes can regulate the number of tomato seeds and the weight of a thousand seeds.

[0457] Example 4: Using gene editing or guided editing techniques, mutants of MM-CDPK27-CR1 or similar effects to MM-CDPK27-CR1 were obtained.

[0458] MM-CDPK27-CR1 is a CDPK27 kinase-deficient mutant. Amino acid sequence alignment revealed 14 additional amino acid sites in the CDPK27 kinase domain besides the missing L62 and G63 amino acids, which may affect the ATP-binding function of CDPK27 and thus its kinase activity. These 14 amino acid sites are R64, G65, T70, A83, K85, V118, M134, E135, L136, C137, E141, E184, N185, L187, I200, and D201. Deleting one or more of these sites, or site-directed mutations of one or more of these sites, using gene editing or guided editing techniques can create CDPK27 kinase-deficient mutants with similar effects to MM-CDPK27-CR1.

[0459] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a polynucleotide in regulating plant seed traits, characterized in that, The polynucleotide comprises at least one of the following nucleotide sequences: (11) The CDPK27 gene sequence shown in SEQ ID NO:3; (12) A complementary, degenerate, or homologous sequence of the CDPK27 gene sequence shown in SEQ ID NO:3, wherein the homologous sequence is a sequence that has 90% or more identity with the CDPK27 gene sequence shown in SEQ ID NO:

3. (13) A sequence or its complementary sequence that hybridizes with the CDPK27 gene sequence shown in SEQ ID NO:3 under strict conditions; (14) The cDNA sequence of any one of the sequences (11)-(13); (15) The CDPK26 gene sequence shown in SEQ ID NO:11; (16) A complementary, degenerate, or homologous sequence of the CDPK26 gene sequence shown in SEQ ID NO:11, wherein the homologous sequence is a sequence that has 90% or more identity with the CDPK26 gene sequence shown in SEQ ID NO:

11. (17) A sequence or its complementary sequence that hybridizes with the CDPK26 gene sequence shown in SEQ ID NO:11 under strict conditions; (18) The cDNA sequence of any one of the sequences (15)-(17); Preferably, the seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

2. The application of a protein in regulating plant seed traits, characterized in that, The protein is at least one of the following sequences: (21) The CDPK27 protein sequence shown in SEQ ID NO:2; (22) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the CDPK27 protein sequence shown in SEQ ID NO:2; (23) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the CDPK27 protein sequence shown in SEQ ID NO:

2. (24) A protein that has 90% or more identity with the CDPK27 protein sequence shown in SEQ ID NO:2 and has the same function; (25) The CDPK26 protein sequence shown in SEQ ID NO:10; (26) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the CDPK26 protein sequence shown in SEQ ID NO:10; (27) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the CDPK26 protein sequence shown in SEQ ID NO:

10. (28) A protein that has 90% or more identity with the CDPK26 protein sequence shown in SEQ ID NO:10 and has the same function; Preferably, the seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

3. The application of a biomaterial in regulating plant seed traits, characterized in that, The biomaterial is any one of the following (31) to (33): (31) Knockout cassette of the polynucleotide of claim 1; (32) Knockout of the polynucleotide knockout vector of claim 1; (33) Recombinant microorganisms that knock out the polynucleotides of claim 1; Preferably, the seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

4. A method for improving tomato plants or seeds, characterized in that, Mutating the CDPK gene to inactivate the CDPK protein kinase yields improved tomato plants or seeds, wherein the CDPK gene is the polynucleotide of claim 1 and the CDPK protein is the protein of claim 2. The modified tomato plant or seeds comprise: (i) at least a 5% reduction in the average number of seeds per fruit compared to control tomato plants or seeds; and / or, (ii) The average weight of 1,000 seeds was reduced by at least 5% compared to the control tomato plants or seeds; The control tomato plant or seed and the improved tomato plant or seed contain substantially the same genetic background.

5. The method according to claim 4, characterized in that, The method of mutating the CDPK gene to inactivate CDPK protein kinase includes: (a) Knockout of a multiple of 3 bases at the active site of the CDPK gene, resulting in a deletion mutation at at least one amino acid site with kinase activity in the CDPK protein, thus losing kinase activity and obtaining a deletion mutant; and / or, (b) Inserting bases in multiples of 3 at the active site of the CDPK gene, resulting in an insertion mutation at at least one kinase-active amino acid site in the CDPK protein, thus losing kinase activity and obtaining an insertion mutant; and / or, (c) Knockout of bases not in multiples of 3 in the coding region of the CDPK gene, resulting in a frameshift mutation at at least one amino acid site in the CDPK protein, leading to premature termination of protein translation, thus obtaining a prematurely terminated translation mutant; and / or, (d) Insertion of bases that are not integer multiples of 3 into the coding region of the CDPK gene causes a frameshift mutation at at least one amino acid site in the CDPK protein, resulting in premature termination of protein translation and obtaining a prematurely terminated translation mutant.

6. The method according to claim 5, characterized in that, The amino acid sites with kinase activity include at least one of the following: L62, G63, R64, G65, T70, A83, K85, V118, M134, E135, L136, C137, E141, E184, N185, L187, I200, D201.

7. A method for improving tomato plants or seeds, characterized in that, Knockout of the CDPK27 gene at its active site in claim 1 by an integer multiple of 3 bases results in a deletion mutation at at least one amino acid site with kinase activity in the CDPK27 protein, thereby losing kinase activity and obtaining a deletion mutant. This leads to an improved tomato plant or seed, wherein the improved tomato plant or seed comprises: (i) The average number of seeds per fruit is reduced by at least 5% compared to the control tomato plant or seed; The control tomato plant or seed and the improved tomato plant or seed contain substantially the same genetic background.

8. A method for improving tomato plants or seeds, characterized in that, Knockout of bases not in multiples of 3 from the coding regions of both the CDPK27 and CDPK26 genes as described in claim 1, resulting in a frameshift mutation at at least one amino acid site in the CDPK27 protein and a frameshift mutation at at least one amino acid site in the CDPK26 protein, leading to premature termination of protein translation, yielding a dual-gene premature translation termination mutant, further resulting in an improved tomato plant or seed, wherein the improved tomato plant or seed comprises: (ii) The average weight of 1,000 seeds was reduced by at least 5% compared to the control tomato plants or seeds; The control tomato plant or seed and the improved tomato plant or seed contain substantially the same genetic background.

9. A mutant protein, characterized in that, The mutant protein is at least one of the following sequences: (41) The CDPK27 mutant protein sequence shown in SEQ ID NO:18; (42) The CDPK27 mutant protein sequence shown in SEQ ID NO:21; (43) The CDPK26 mutant protein sequence shown in SEQ ID NO:24; (44) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the sequences (41)-(43).

10. A polynucleotide, characterized in that, The polynucleotide is at least one of the following sequences: (51) The nucleotide sequence of the CDPK27 mutant gene shown in SEQ ID NO:19; (52) The nucleotide sequence of the CDPK27 mutant gene shown in SEQ ID NO:22; (53) The nucleotide sequence of the CDPK26 mutant gene shown in SEQ ID NO:25; (54) The nucleotide sequence of the mutant protein shown in any of the coding sequences (41)-(44) other than (51)-(53).

11. The use of the mutant protein of claim 9 and / or the polynucleotide of claim 10 in regulating plant seed traits, characterized in that, The seed traits include at least one of the following: average number of seeds per fruit and average weight of 1,000 seeds.

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