Application of polynucleotide, protein and biological material in regulation and control of plant nutrient substance storage organ development

Abstract

The invention relates to the technical field of gene engineering, and provides application of polynucleotide, protein and a biological material in regulation and control of plant nutrient substance storage organ development. The polynucleotide has a sequence as shown in SEQ ID NO: 1; or a complementary sequence, a degenerate sequence or a homologous sequence thereof; or polynucleotide hybridized with the nucleotide sequence as shown in SEQ ID NO: 1 under strict conditions or a complementary sequence of the polynucleotide. The polynucleotide is related to the development of plant nutrient substance storage organs, promotes the development of the plant nutrient substance storage organs through overexpression, and can be used for cultivating plant varieties.

Description

Technical Field

[0001] This invention relates to the technical field of genetic engineering, specifically to the application of polynucleotides, proteins, and biomaterials in regulating the development of plant nutrient storage organs. Background Technology

[0002] Potato (Solanum tuberosum L.) is an important economic and food crop, widely cultivated around the world. As the asexual reproduction and storage organ of potatoes, increasing tuber size or weight has always been a key goal in potato breeding. Tuber number and average tuber weight are important components of tuber weight. Potato tuber size or weight is a quantitative trait regulated by multiple genes. Although a number of genes affecting tuber size or weight have been reported in recent years, most of these genes are homologous genes whose functions have been verified in other crops such as Arabidopsis thaliana and rice.

[0003] APETALA2 / ETHYLENE RESPONSIVE FACTOR (AP2 / ERF) transcription factors are a class of plant-specific transcription factors that play a crucial role in regulating plant growth and development, as well as various biotic and abiotic resistances. Previous studies have shown that AP2 / ERF transcription factors play a key role in regulating grain size, quality, and grain shattering. In rice, a series of AP2 / ERF transcription factors have been identified that can participate in regulating grain size and increasing rice weight. However, there are no reports of AP2 / ERF transcription factors regulating tuber size or weight in potatoes. Summary of the Invention

[0004] This invention provides the application of polynucleotides, proteins, and biomaterials in regulating the development of plant nutrient storage organs, as well as related products and cultivation methods. The polynucleotides can participate in regulating the development of plant nutrient storage organs, improving tuber size and increasing yield.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of a polynucleotide in regulating the development of plant nutrient storage organs, wherein the polynucleotide comprises at least one of the following nucleotide sequences:

[0007] (a1) The nucleotide sequence shown in SEQ ID NO:1;

[0008] (a2) A complementary, degenerate, truncated or homologous sequence of the sequence shown in SEQ ID NO:1, wherein the homologous sequence is a polynucleotide having 80% or more identity with the nucleotide shown in SEQ ID NO:1;

[0009] (a3) A polynucleotide or its complementary sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO:1 under strict conditions.

[0010] In some embodiments, the present invention provides the application of the above-mentioned polynucleotides in regulating the development of potato nutrient storage organs.

[0011] Diploid cultivars *S. tuberosum* Group Stenotomum and *S. tuberosum* Group Phureja are rich in genetic variation and are excellent materials for studying potato tuber weight and quality. This invention, through genome-wide association analysis of diploid potato cultivars, located the QTL loci controlling tuber size or weight within the ~370kb (7.31-7.68Mb) region on chromosome 3, with the peak SNP (Chr03:43,644,193) located upstream of the Soltu.DM.03G005790 gene. Transcriptome data from 12 potato samples of *S. tuberosum* Group Stenotomum (STN) and *S. tuberosum* Group Phureja (PHU) during tuber enlargement and maturity stages revealed differential expression of the gene Soltu.DM.03G005790 during these stages, suggesting it as a candidate gene regulating tuber size by controlling the development of nutrient storage organs. Soltu.DM.03G005790 encodes a drought-response element-binding protein (DREB), named StTW3.1. Overexpression of StTW3.1 significantly increased potato tuber size and yield, effectively addressing yield increase issues in potato production. This research lays the foundation for studying the genetic basis and molecular regulatory mechanisms of potato tuber size, and is beneficial for molecular breeding.

[0012] The polynucleotide sequence of StTW3.1 is not necessarily completely identical in different potato materials. This invention provides the polynucleotide sequences of StTW3.1 from six other potato materials obtained during the research process. Their polynucleotide sequences have 80% to 100% similarity to the sequence shown in SEQ ID NO:1, and the sequences that are not completely identical are shown in SEQ ID NOs:2 to 7.

[0013] In some embodiments, the nucleotide sequence of the polynucleotide is the nucleotide sequence shown in SEQ ID NO:1.

[0014] In some embodiments, the nucleotide sequence of the polynucleotide is a complementary sequence to the sequence shown in SEQ ID NO:1. The complementary sequence is a complementary sequence formed according to the base pairing principle, and can be an incompletely complementary sequence or a completely complementary sequence having the same function as the nucleotide sequence shown in SEQ ID No.1.

[0015] In some embodiments, the nucleotide sequence of the polynucleotide is a degenerate sequence of the sequence shown in SEQ ID NO:1. A degenerate sequence means that changing one or more nucleotides of the SEQ ID NO:1 does not change the types of amino acids encoded by the positions of the changed nucleotides, and will not affect the function and expression level of the gene.

[0016] In some embodiments, the polynucleotide sequence is a truncated sequence of the nucleotide sequence shown in SEQ ID NO.1. A truncated sequence refers to a shorter nucleotide sequence extracted or cut from the nucleotide sequence in SEQ ID NO.1, and the cDNA sequence or the type of amino acid encoded by the corresponding position of the nucleotide sequence remains unchanged, so as not to affect the function and expression level of the gene.

[0017] In some embodiments, the nucleotide sequence of the polynucleotide is a homologous sequence to the sequence shown in SEQ ID NO:1. Homologous sequences include, but are not limited to, polynucleotides having about 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity with the nucleotide shown in SEQ ID NO:1.

[0018] Furthermore, in (a2), the homologous sequence having 80% or more identity with the nucleotide shown in SEQ ID NO:1 includes at least one of the nucleotide sequences shown in SEQ ID NOs:2 to 7.

[0019] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:2.

[0020] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:3.

[0021] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:4.

[0022] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:5.

[0023] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:6.

[0024] In some embodiments, the homologous sequence is the nucleotide sequence shown in SEQ ID NO:7.

[0025] In some embodiments, the nucleotide sequence of the polynucleotide is a polynucleotide or its complementary sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO:1 under stringent conditions.

[0026] For example, the "strict condition" refers to a condition under which the probe hybridizes with its target sequence to a level of detectability exceeding that with other sequences (e.g., at least twice the background). Strict conditions are sequence-dependent and vary depending on the environment. By controlling the strictness of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified. Alternatively, strict conditions can be adjusted to allow for some sequence mismatches, resulting in the detection of a lower degree of similarity.

[0027] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides having 80% or higher identity with the nucleotide sequence isolated according to this invention, as long as they encode the protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0028] Furthermore, the polynucleotide includes a heterologous regulatory element operatively linked to the nucleotide sequence. This heterologous regulatory element can be used to enhance the expression level of the polynucleotide, resulting in the production of more protein or RNA products in the host cell, i.e., overexpression of the polynucleotide. Different types of heterologous regulatory elements can precisely regulate the expression of the polynucleotide in cells, including temporal and spatial expression regulation. Introducing exogenous regulatory elements can also endow genes with new regulatory properties, such as promoters that respond to external stimuli, programmable expression regulation systems, etc., thereby creating plant strains that meet specific needs.

[0029] Preferably, the heterologous regulatory element comprises at least one of a promoter, enhancer, transposon, terminator, leader sequence, and marker gene.

[0030] This invention also provides the application of a protein in regulating the development of plant nutrient storage organs, wherein the protein is at least one of the following sequences:

[0031] (b1) The protein shown in SEQ ID NO:8;

[0032] (b2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:8;

[0033] (b3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:8.

[0034] (b4) A protein that has 80% or more of the same amino acid sequence as shown in SEQ ID NO:8 and has the same function.

[0035] In some embodiments, the protein is the protein shown in SEQ ID NO:8.

[0036] In some embodiments, the protein is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:8.

[0037] In some embodiments, the protein is a protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the amino acid sequence shown in SEQ ID NO:8.

[0038] In some embodiments, the protein is a protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO:8 and has the same function. The protein includes, but is not limited to, proteins that have about 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, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity with the nucleotides shown in SEQ ID NO:8 and have the same function.

[0039] The present invention provides the amino acid sequences of StTW3.1 from six other potato materials obtained during the research process, which have 90% to 100% similarity to the sequence shown in SEQ ID NO:8. In (b4), the amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO:8 and has the same function includes at least one of the amino acid sequences shown in SEQ ID NOs:9 to 14.

[0040] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:9. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, with the addition of G(glycine) and R(arginine) sequentially between G(glycine) at position 16 and position 17 of the original sequence.

[0041] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:10. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, with the original sequence having V (valine) at position 13 changed to M (methionine), and G (glycine) and R (arginine) added sequentially between G (glycine) at positions 16 and 17 of the original sequence.

[0042] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:11. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, with the addition of G(glycine), G(glycine), and R(arginine) sequentially between G(glycine) at position 15 and G(glycine) at position 16 of the original sequence.

[0043] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:12. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, with an R (arginine) inserted between the G (glycine) at position 17 and position 18 of the original sequence.

[0044] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:13. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, with the G (glycine) at position 18 and position 21 both replaced by R (arginine), and the S (serine) at position 98 replaced by I (isoleucine).

[0045] In some embodiments, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:14. This amino acid sequence is based on the amino acid sequence shown in SEQ ID NO:8, except that the G (glycine) at position 15 of the original sequence is deleted, the G (glycine) at position 19 of the original sequence is replaced with R (arginine), and the S (serine) at position 98 of the original sequence is replaced with I (isoleucine).

[0046] In another aspect, the present invention provides a specific primer for amplifying the polynucleotide, the specific primer comprising or consisting of the following sequences:

[0047] Upstream primer: The nucleotide sequence shown in SEQ ID NO:15;

[0048] Downstream primer: The nucleotide sequence shown in SEQ ID NO:16.

[0049] It should be noted that other primers capable of amplifying the polynucleotide that are deemed feasible by those skilled in the art are within the scope of protection of this invention.

[0050] The present invention also provides a biomaterial, said biomaterial being any one of the following (c1) to (c5):

[0051] (c1) An expression cassette containing the polynucleotide of claim 1 or 2;

[0052] (c2) A recombinant vector containing the polynucleotide of claim 1 or 2;

[0053] (c3) Recombinant microorganisms containing the polynucleotides of claim 1 or 2;

[0054] (c4) A plant cell line containing the polynucleotide of claim 1 or 2;

[0055] (c5) Introduce any of the plant protoplasts, cells or callus tissues from (c1) to (c4).

[0056] In some embodiments, the expression cassette (c1) further includes a promoter operatively linked to the aforementioned nucleic acid molecule. Optionally, the expression cassette (c1) also includes other regulatory elements, such as enhancers, leader sequences, transposons, terminators, marker genes, etc.

[0057] In some embodiments, the recombinant vector described in (c2) is not specifically limited in type, and a suitable vector can be selected as needed. For example, the vector includes, but is not limited to, pCAMBIA1305, pCAMBIA1300, pSuper1300, pMAL-c5x, pET28b, and pGEX-4T-1, with pCAMBIA1305 being preferred.

[0058] In some embodiments, the recombinant microorganisms described in (c3) have a microbial chassis including, but not limited to, at least one of Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli.

[0059] In this invention, the plant cell line may or may not include propagation material.

[0060] In another aspect, the present invention provides the application of the aforementioned biomaterial in regulating the development of plant nutrient storage organs.

[0061] In some embodiments, the plant is selected from tuberous plants, fruit plants, and leafy plants.

[0062] In some embodiments, the plant is selected from potato, sweet potato, cassava, rice, yam, taro, Jerusalem artichoke, pinellia or senna, with potato being preferred.

[0063] In some embodiments, the plant nutrient storage organs include tubers, fruits, leaves, and seeds.

[0064] The above applications are not specifically limited, and any form of using the biological material to regulate the development of plant nutrient storage organs is within the scope of protection of this invention.

[0065] Furthermore, in the above-mentioned applications of the present invention, the regulation of the development of plant nutrient storage organs includes regulating one or more of the following: whether tubers are formed, the time of tuber formation, the number of tubers, the size of tubers, whether fruits are formed, the time of fruit formation, the number of fruits, the size of fruits, whether leaves are formed, the time of leaf formation, the number of leaves, the size of leaves, whether seeds are formed, the time of seed formation, the number of seeds, and the size of seeds.

[0066] In some preferred embodiments, the regulation of plant nutrient storage organ development includes regulating whether tubers form, the time of tuber formation, the number of tubers, and the size of tubers.

[0067] In some implementations, the application includes regulating whether potato tubers form, the time of tuber formation, the number of tubers, and the size of the tubers.

[0068] In some implementations, the application includes regulating whether tomatoes develop fruit, the time of fruit formation, the number of fruits, and the size of the fruits.

[0069] In some implementations, the application includes regulating whether tobacco leaves form, the time of leaf formation, the number of leaves, and the size of the leaves.

[0070] In some implementations, the application includes regulating rice seed formation time, seed number, and seed size.

[0071] The present invention also provides a method for identifying the development of plant nutrient storage organs, which uses the presence of molecular markers to predict whether the polynucleotide is overexpressed or whether the protein is expressed.

[0072] In some implementations, the StTW3.1 gene itself is used as a molecular marker, for example, by designing primers to amplify the StTW3.1 gene and identifying whether the StTW3.1 gene is overexpressed in potato samples by the results of PCR amplification.

[0073] In one aspect, the present invention also provides a method for cultivating plants, comprising overexpressing the polynucleotides or proteins described in the present invention in a recipient plant to obtain a target plant; compared with the recipient plant, the target plant has the following characteristics: earlier tuber formation time, increased number of tubers, larger tuber size, increased tuber yield, earlier fruit formation time, increased number of fruits, larger fruits, increased fruit yield, earlier leaf formation time, increased number of leaves, larger leaves, increased number of leaves per plant, whether seeds are formed, seed formation time, number of seeds, and seed size.

[0074] In some preferred embodiments, a method for cultivating a plant includes overexpressing the polynucleotide or protein described in this invention in a recipient plant to obtain a target plant; compared with the recipient plant, the target plant has an earlier tuber formation time, an increased number of tubers, larger tubers, and a higher tuber yield.

[0075] In some embodiments, the tuber formation time of the target plant is at least 30% earlier than that of the recipient plant. In some embodiments, the tuber formation time of the target plant is 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% earlier than that of the recipient plant. In some embodiments, the tuber formation time of the target plant is at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 30 days, or at least 40 days, at least 50 days, or at least 60 days earlier than that of the recipient plant.

[0076] In some embodiments, the number of tubers, tuber size, or tuber yield of the target plant is at least 30% higher than that of the recipient plant. In some embodiments, the number of tubers, tuber size, tuber yield, or average tuber yield per plant of the target plant is 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% higher than that of the recipient plant. In some embodiments, the number of tubers of the target plant is at least 1, at least 2, at least 3, at least 5, at least 10, or at least 20 higher than that of the recipient plant. In some embodiments, the average tuber yield per plant of the target plant is at least 10g, at least 20g, at least 30g, at least 40g, at least 50g, at least 100g, at least 150g, or at least 200g higher than that of the recipient plant.

[0077] Furthermore, according to the above-described plant cultivation method, the overexpression of the polynucleotide or protein in the recipient plant includes one or more of the following techniques: promoter editing, codon optimization, using strong promoters, inserting introns, using viral vectors, and fusion protein technology.

[0078] It should be noted that other methods of overexpressing the polynucleotides or proteins in the recipient plants that are deemed feasible by those skilled in the art are within the scope of protection of this invention.

[0079] In some embodiments, the overexpression method includes the following optional methods:

[0080] A. Achieving overexpression using various promoter editing techniques. Promoter editing techniques include the following options: a) Small-scale alterations to the endogenous promoter (base deletion or omission, but all operations are performed on the original endogenous promoter) (Rodriguez-Leal et al., 2017): Using CRISPR / Cas9 technology, sequences covering the entire promoter region are designed. The resulting altered promoter sequence affects the binding ability of transcription factors, thus affecting the expression of downstream genes. Some promoters contain uORFs encoding small peptides. During gene transcription, the transcription of uORFs affects the transcription of downstream genes, thus affecting their expression. Altering these uORFs using CRISPR / Cas9 technology can also alter the expression of downstream genes. b) Using CRISPR / Cas technology to insert enhancers or other gene-enhancing elements into the original endogenous promoter to enhance gene expression. c. Directly replace the endogenous promoter corresponding to the gene: First, use knock-in to replace the endogenous promoter with a strong promoter (such as 35S, or a constitutive strong expression promoter); Second, use CRISPR / Cas9 technology to induce chromosome inversion, thereby exchanging promoters of different genes and increasing the expression of the target gene (Schwartz et al., 2020).

[0081] B. Introducing the DNA sequence encoding the target gene into cells via transgenic methods. The following methods can be used individually or in combination: First, altering the DNA sequence corresponding to the endogenous gene through codon optimization; Second, utilizing strong promoters (constitutive strong promoters achieve overexpression in all tissues, while tissue-specific promoters achieve overexpression in specific tissues); Third, certain introns can enhance gene expression, and inserting these introns into the target gene can enhance its expression (Gallegos and Rose, 2019); Fourth, fusing certain solubilizing or anti-degradation tags or protein sequences at the end of the target gene to enhance the expression level of the target protein or prevent its degradation.

[0082] C. Overexpression of the target gene using viral vectors (DNA virus and RAN virus) (Torti et al., 2021). The virus replicates extensively in plant cells, thereby greatly increasing the copy number of the target gene and achieving the purpose of overexpression.

[0083] D. Other ways to achieve the purpose of overexpression.

[0084] The present invention also provides a method for cultivating potatoes, wherein the polynucleotide or protein described in the present invention is overexpressed in a recipient potato plant to obtain a target potato plant; compared with the recipient potato plant, the target potato plant has an earlier tuber formation time, an increased number of tubers, larger tubers, and a higher tuber yield.

[0085] The present invention also provides a method for cultivating tomatoes, wherein the polynucleotide or protein described in the present invention is overexpressed in a recipient tomato plant to obtain a target tomato plant; compared with the recipient tomato plant, the target tomato plant has an earlier fruit formation time, an increased number of fruits, larger fruits, and a higher fruit yield.

[0086] The present invention also provides a method for cultivating tobacco, wherein the polynucleotide or protein described in the present invention is overexpressed in a recipient tobacco plant to obtain a target tobacco plant; compared with the recipient tobacco plant, the target tobacco plant has an earlier leaf formation time, an increased number of leaves, larger leaves, and more leaves per plant.

[0087] Another aspect of the present invention provides a plant, the plant part, tuber or tuber portion, or plant cells, pollen or seeds thereof, which is one of the following:

[0088] (d1) The plant formed by the growth of plant cell lines, plant protoplasts, cells or callus tissues in the above-mentioned biological materials of the present invention, the plant part, tuber or tuber part, or plant cells, pollen or seeds thereof; or the plant obtained by the cultivation method described in any one of the above-mentioned inventions, the plant part, tuber or tuber part, or plant cells, pollen or seeds thereof.

[0089] (d2) The offspring formed by the self-pollination of the plant in (d1), and the plant formed by the growth of the offspring, including its plant parts, tubers or tuber portions, or its plant cells, pollen or seeds;

[0090] (d3) The offspring formed by the hybridization of the plant in (d1) with other varieties, and the plant formed by the growth of the offspring, including its plant parts, tubers or tuber portions, or its plant cells, pollen or seeds.

[0091] In another aspect, the present invention provides a food or feed made from the above-mentioned potato plant, its plant parts, tubers or tuber portions, or its plant cells, pollen or seeds.

[0092] Furthermore, in the aforementioned food or feed, the food includes: fresh potatoes, dried potatoes, frozen potatoes, French fries, potato chips, potato flour, or potato starch; the feed includes: liquid feed, solid feed, semi-solid feed, or feed ingredients.

[0093] In one aspect, the present invention also provides a method for manufacturing a commercial plant product, comprising obtaining a potato plant, plant part, tuber or tuber part, as described above, and manufacturing the commercial plant product.

[0094] The plant products mentioned therein are selected from the group consisting of: fresh tuber material, frozen tuber material, dehydrated tuber material, tuber liquid, tuber strips, tuber flakes, tuber granules, tuber powder, fresh fruit, frozen fruit, dehydrated fruit material, fresh leaves, frozen leaves, and dehydrated leaves.

[0095] The present invention, by adopting the above technical solution, has the following beneficial effects:

[0096] (1) The present invention provides an application of polynucleotides and proteins in regulating the development of plant nutrient storage organs. The polynucleotides and / or proteins can be used in tuber plant breeding, fruit plant breeding or leaf plant breeding, especially in regulating the development of potato nutrient storage organs and improving nutrient storage organ development-related traits, such as shortening tuber formation time, improving tuber size and increasing yield.

[0097] (2) The polynucleotides and proteins provided by this invention can also provide new gene resources for increasing plant yield and can be used to modify the chassis of plant genetic engineering strategies. In particular, they can provide new gene resources for increasing potato yield and provide a solid foundation for the modification of potato genetic engineering strategies. The application of these gene resources can play an important role in the field of plant breeding and promote plant growth and development and increase yield.

[0098] (3) This invention provides a plant breeding method that utilizes the overexpression of the polynucleotides and / or proteins to improve the phenotypic traits of plant development, especially the phenotypic traits of potato tubers, effectively solving problems such as potato yield increase, and providing a method reference for potato variety selection.

[0099] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and should be understood to include values ​​close to those ranges or values. For numerical ranges, endpoint values ​​of various ranges, endpoint values ​​of various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In this document, unless otherwise specified, data ranges include endpoints. Attached Figure Description

[0100] Figure 1 The image shows the QTL sites that regulate tuber size traits, located using genome-wide association analysis (GWAS). Figure 1In this context, 'a' represents the GWAS localization related to tuber size traits in potatoes; Figure 1 In this context, 'b' represents the Linkage Disequilibrium (LD) map between SNPs within the location area. Figure 1 In this context, 'c' represents the peak SNP. ATW The average tuber weight corresponding to samples with genotypes A, G, and R.

[0101] Figure 2 The figure shows the phenotypic evaluation of mean tuber weight and yield per plant for overexpression and knockout lines. Figure 2 In this context, 'a' represents the overall situation of tubers from overexpressing lines OE-13, OE-23, wild-type WT, and knockout line KO-90 at harvest time in 2024. Figure 2 In this context, 'b' represents the relative expression levels of the overexpression lines OE-13 and OE-23. Figure 2 In this context, 'c' represents the average tuber weight of overexpression and knockout lines. Figure 2 In this context, 'c' represents the tuber yield per plant in overexpression and knockout lines.

[0102] Figure 3 The image shows the editing status of the knockout lines and the phenotypic evaluation of the average tuber weight and yield per plant in the overexpression lines. Among them, Figure 3 In the table, 'a' indicates the editing status of the knockout line KO-90, red text indicates the sequences of the two target sites, and blue text indicates the insertion or deletion of sequences; 'b' indicates the average tuber weight of the overexpression lines OE-13 and OE-23 in Dingxi in 2023; 'c' indicates the yield per plant of the overexpression lines OE-13 and OE-23 in Dingxi in 2023; 'd' indicates the average tuber weight of the overexpression lines OE-13 and OE-23 in Hohhot in 2023; and 'e' indicates the yield per plant of the overexpression lines OE-13 and OE-23 in Dingxi in 2023. Detailed Implementation

[0103] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0104] Terminology Explanation:

[0105] In this invention, the term "StTW3.1" can refer to the StTW3.1 gene or the protein encoded by the StTW3.1 gene, and its specific meaning can be determined in conjunction with the context.

[0106] In this invention, the term "polynucleotide" can refer to a polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers described above. Nucleotide can refer to ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is generally at least 10 bases in length unless otherwise stated. The term can refer to RNA or DNA molecules of indeterminate length. This term includes both single-stranded and double-stranded DNA. Nucleic acid molecules may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotides.

[0107] In this invention, the term "identity" refers to sequence similarity to a natural nucleic acid sequence or amino acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0108] In this invention, the term "homology" is sometimes used to refer to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acid or amino acid sequences, expressed as a percentage of positional identity. Homology also refers to the concept of evolutionary relevance, typically demonstrated by similar functional properties between different nucleic acids or proteins sharing similar sequences.

[0109] In this invention, the term "gene" is defined as a genetic unit (usually represented by a DNA sequence) that occupies a specific location on a chromosome and contains genetic instructions that contribute to a plant's potential phenotypic characteristics or traits. In some contexts, the term "gene" refers to a nucleic acid molecule that expresses a specific protein. A "gene" includes the DNA region encoding the gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to coding and / or transcriptional sequences. Therefore, a gene includes, but is not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, introns, and locus control regions.

[0110] In this invention, the term "expression cassette" refers to a DNA fragment into which nucleic acids or polynucleotides can be inserted at specific restriction sites or through homologous recombination. As used herein, the DNA fragment contains a polynucleotide encoding a target polypeptide, and the expression cassette and restriction sites are designed to ensure that the expression cassette is inserted into the appropriate reading frame for transcription and translation. In one embodiment, the expression cassette may include a polynucleotide encoding a target polypeptide and, in addition to the polynucleotide, elements that promote the transformation of a specific host cell. In one embodiment, the expression cassette may also include elements that allow enhanced expression of the polynucleotide encoding the target polypeptide in host cells. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, responsive elements, terminator sequences, polyadenylated sequences, etc.

[0111] In this invention, the term "vector" is used interchangeably with "construct," "cloning vector," and "expression vector," and means a vector that can introduce a DNA or RNA sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. "Non-viral vector" refers to any vector that does not contain a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence containing at least one DNA replication origin and at least one selective marker gene. Examples include, but are not limited to, plasmids, phages, bacterial artificial chromosomes (BACs), or viruses that carry foreign DNA into the cell. Vectors may also include one or more genes, antisense molecules, and / or selective marker genes, and other genetic elements known in the art. The vector can transduce, transform, or infect cells, resulting in the expression of nucleic acid molecules and / or proteins encoded by the vector.

[0112] In this invention, the term "expression" refers to the biosynthesis of a gene product, including transcription and / or translation of the gene product. "Expressing" or "producing" a protein or polypeptide from a DNA molecule means transcribing and translating the coding sequence to produce the protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule means translating the RNA coding sequence to produce the protein or polypeptide.

[0113] Expression vectors containing all the necessary expression elements are commercially available and well-known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, 1989. Cellular genetic engineering is performed by introducing exogenous DNA / RNA into cells. This exogenous DNA / RNA is placed under the efficient control of transcriptional elements to allow the exogenous DNA to be expressed in the host cell.

[0114] In this invention, the "strict conditions" can be any of low-strict, medium-strict, or high-strict conditions. "Low-strict conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. "Medium-strict conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C. "High-strict conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, the higher the temperature, the more efficiently high-homogeneity DNA can be obtained. Factors affecting hybridization strictness include temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc. Those skilled in the art can achieve the same strict conditions by appropriately selecting these factors.

[0115] In this invention, the term "overexpression" means that the expression level and amount of StTW3.1 in the target potato material are higher than those in the wild type (e.g., recipient potato material).

[0116] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0117] The biological materials, sequences, reagents, or instruments used in the embodiments of this invention are all commercially available.

[0118] Note: The S. tuberosum Group Phureja (PHU) potato strain originated from the S. tuberosum Group Stenotomum (STN) strain.

[0119] The present invention will be further illustrated below with reference to the embodiments:

[0120] Example 1: Genome-wide association analysis and candidate gene mining of potato tuber weight

[0121] The tuber weight phenotype of the diploid potato cultivars *S. tuberosum* Group Stenotomum (STN) and *S. tuberosum* Group Phureja (PHU) was evaluated, revealing rich phenotypic variation in tuber weight. To further locate tuber weight-related QTLs, genome-wide association analysis (GWAS) was performed on the tuber weight phenotype in this population. Significant SNPs associated with tuber weight were detected on nine chromosomes, such as… Figure 1 As shown in Figure a, the peak SNP (SNP) ATW Located on chromosome 3 (Chr03:43,644,193), such as Figure 1The LD block diagram shows that SNPs exceeding the threshold in the ~370kb (7.31-7.68Mb) range are highly linked. Simultaneously, SNPs in the native cultivated populations of *S. tuberosum* Group Stenotomum (STN) and *S. tuberosum* Group Phureja (PHU) were analyzed. ATW Genotype of the locus ( Figure 1 c) SNP was discovered. ATW The tuber size of the sample with genotype "A" was significantly smaller than that of the samples with genotypes "R" and "G", indicating that one or more genes in this region may be involved in regulating potato tuber weight.

[0122] Based on the annotations of the potato reference genome DM1-3 (v6.1), genes within the Chr03:7.31-7.68 Mb region were analyzed. Among the five candidate genes, the peak SNP located upstream of Soltu.DM.03G005790 was identified. ATW (Chr03:43,644,193); In addition, using transcriptome data from 12 STN and PHU samples measured previously, analysis revealed that only the gene Soltu.DM.03G005790 showed differential expression during the tuber enlargement and maturity stages. The expression level during the enlargement stage was about 8 times that during the maturity stage, indicating that Soltu.DM.03G005790 may be involved in the development of nutrient storage organs, thereby regulating tuber size. Soltu.DM.03G005790 was named StTW3.1.

[0123] Example 2: Cloning of the StTW3.1 gene and obtaining overexpression and knockout lines.

[0124] Using cDNA from DM1-3 tuber tissue as a template, PCR amplification was performed using specific primers (see Table 1). The target fragment was purified and ligated into the pSuper1300 vector. Positive single clones were selected for sequencing to obtain the cDNA sequence of StTW3.1. The full-length cDNA sequence of StTW3.1 is 525 bp, encoding 174 amino acids. An AP2 functional domain, belonging to the APETALA2 / ethylene-responsive element binding factor (AP2 / ERF) transcription factor family CBF / DREB subset, is located between residues 35-94 of the amino acid sequence.

[0125] The candidate gene was overexpressed using the binary expression vector pCAMBIA-1305.4, and the target fragment was inserted between BamHI and PmlI using homologous recombination. The successfully constructed recombinant plasmid was transformed into Agrobacterium GV301. Using stem segments of diploid potato S. tuberosum Group Phureja S15-65 (hereinafter referred to as CIP65, with the International Potato Center Germplasm Bank number CIP 703541) as explants, genetic transformation was carried out using Agrobacterium-mediated transformation. After positive identification, the transgenic lines OE-13 and OE-23, which successfully overexpressed StTW3.1, were screened based on the expression levels of StTW3.1 in the leaves and tubers of the transgenic lines for phenotypic evaluation.

[0126] Table 1. Primers for StTW3.1 gene-specific amplification

[0127] Upstream primer (SEQ ID No:15) 5'-ATGGAGGTTGACGGCGTCA-3' Downstream primer (SEQ ID No:16) 5'-TCACCAATAATCAAATTCCGGG-3'

[0128] Using the gDNA sequence of the StTW3.1 gene from the receptor material CIP65 as a reference, CRISPR / Cas9 target sites were designed. After considering factors such as location information, GC content, and off-target efficiency, two highly specific target sites were screened: Target Site 1: GCGTCAGAAGCGGCGATGC (SEQ ID NO:17); Target Site 2: AGCTGTTAGTCGGTGATA (SEQ ID NO:18). The designed target sites were introduced into primers and ligated into the BsaI-digested linearized pKSE402 vector. Positive single clones were screened by colony PCR, and after Sager sequencing and alignment analysis, the pKSE402-StTW3.1 dual-target CRISPR / Cas9 knockout vector was successfully constructed. The successfully constructed recombinant plasmid was transferred into Agrobacterium GV301, and genetic transformation was performed using Agrobacterium-mediated transformation with CIP65 as the recipient. After positive identification, the StTW3.1 gene sequence was amplified and sequenced to obtain the transgenic line KO-90 with both strands edited, which was then used for phenotypic evaluation.

[0129] Example 3: Phenotypic evaluation of transgenic plants

[0130] The transgenic lines OE-13, OE-23, and KO-90, which overexpressed StTW3.1, were evaluated for tuber phenotype in two locations: the South Campus of Inner Mongolia University in Hohhot, Inner Mongolia (40°45′N, 111°40′E, 1040m.asl) and Dingxi, Gansu (35°44′N, 104°50′E, 2094m.asl). Wild-type WT (CIP65) and transgenic lines were planted in greenhouses with at least 30 plants per line. Normal water and fertilizer management was implemented, and tuber yield and average tuber weight were evaluated at harvest time.

[0131] The results showed that, compared with wild-type CIP65, the overexpression lines OE-13 and OE-23 had significantly higher average tuber weight and total tuber yield (e.g., Figure 2 and Figure 3 As shown in the figure, the overall yield of tubers per plant increased by 29.15%–54.27.5%, and the average tuber weight increased by 22.72%–25.88%. However, the average tuber weight and total tuber yield of the knockout line KO-90 decreased significantly, by 23.3% and 19.8%, respectively.

[0132] In summary, this invention demonstrates that the StTW3.1 gene discovered during screening can participate in the regulation of potato tuber size and yield.

[0133] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a polynucleotide for modulating the development of a plant nutrient storage organ, characterized in that, The polynucleotide comprises at least one of the following nucleotide sequences: (a1) the nucleotide sequence shown in SEQ ID NO: 1; (a2) a complementary sequence, a degenerate sequence, a truncated sequence or a homologous sequence of the sequence shown in SEQ ID NO: 1, wherein the homologous sequence is a polynucleotide having 80% or more identity with the nucleotide shown in SEQ ID NO: 1; (a3) a polynucleotide or its complementary sequence which hybridizes to the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions.

2. Use according to claim 1, characterized in that, In the (a2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 2-7.

3. Use according to claim 1, characterized in that, The polynucleotide further comprises a heterologous regulatory element operably linked to the nucleotide sequence; Preferably, the heterologous regulatory element comprises at least one of a promoter, an enhancer, a transposon, a terminator, a leader sequence, a marker gene.

4. Use of a protein in the regulation of the development of a plant nutrient storage organ, characterized in that, The protein is at least one of the following sequences: (b1) the protein shown in SEQ ID NO: 8; (b2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 8; (b3) a protein having the same function as the protein shown in SEQ ID NO: 8, obtained by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence shown in SEQ ID NO: 8; (b4) a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 8 and having the same function.

5. Use according to claim 4, characterized in that, In the (b4), the amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 9 and having the same function includes at least one of the amino acid sequences shown in SEQ ID NOs: 9-14.

6. A biomaterial, characterized by, The biological material is any one of the following (c1) to (c5): (c1) an expression cassette comprising the polynucleotide of claim 1 or 2; (c2) a recombinant vector comprising the polynucleotide of claim 1 or 2; (c3) a recombinant microorganism comprising the polynucleotide of claim 1 or 2; (c4) a plant cell line comprising the polynucleotide of claim 1 or 2; (c5) a plant protoplast, a cell or a callus into which any one of (c1) to (c4) is introduced.

7. Use of the biological material of claim 6 in regulating development of a plant nutrient storage organ.

8. Use according to claim 7, characterized in that, The plant is selected from the group consisting of potato, sweet potato, cassava, rice, tomato, tobacco, yam, taro, Jerusalem artichoke, pinellia ternata and manroot, and more preferably is potato; Preferably, the plant nutrient storage organ includes a tuber, a fruit, a leaf, a seed.

9. Use according to claim 7, characterized in that, The regulation of development of the plant nutrient storage organ includes regulation of one or several of whether a tuber is formed, a tuber formation time, a tuber number, a tuber size, whether a fruit is formed, a fruit formation time, a fruit number, a fruit size, whether a leaf is formed, a leaf formation time, a leaf number, a leaf size, whether a seed is formed, a seed formation time, a seed number, a seed size. The plant is selected from the group consisting of potato, sweet potato, cassava, rice, tomato, tobacco, yam, taro, Jerusalem artichoke, pinellia ternata and manroot, and more preferably is potato; Preferably, the plant nutrient storage organ includes a tuber, a fruit, a leaf, a seed. The regulation of development of the plant nutrient storage organ includes regulation of one or several of whether a tuber is formed, a tuber formation time, a tuber number, a tuber size, whether a fruit is formed, a fruit formation time, a fruit number, a fruit size, whether a leaf is formed, a leaf formation time, a leaf number, a leaf size, whether a seed is formed, a seed formation time, a seed number, a seed size. Preferably, the modulating tuber organ development comprises modulating whether tubers form, the time of tuber formation, the number of tubers, the size of tubers.

10. A method for identifying the development of a plant nutrient storage organ, characterized in that, using the identification of the presence or absence of the molecular marker to predict whether the polynucleotide of any one of claims 1-3 is overexpressed, or to predict whether the protein of claim 4 or 5 is expressed; Preferably, the molecular marker comprises a KASP marker identifying the SNP at 43,644,193 on chromosome 3 of Solanum tuberosum.

11. A method for breeding a plant, characterized by, overexpressing the polynucleotide of claim 1 or 2, or the protein of claim 4 or 5 in a recipient plant, to obtain a plant of interest; wherein the plant of interest has an earlier time of tuber formation, an increased number of tubers, an increased size of tubers, an increased yield of tubers, an earlier time of fruit formation, an increased number of fruits, an increased size of fruits, an increased yield of fruits, an earlier time of leaf formation, an increased number of leaves, an increased size of leaves, an increased yield of leaves, an earlier time of seed formation, an increased number of seeds, an increased size of seeds, an increased yield of seeds, compared to the recipient plant.

12. The plant growing method according to claim 11, characterized in that, The manner of overexpressing the polynucleotide of claim 1 or 2, or the protein of claim 4 or 5 in a recipient plant comprises one or more of promoter editing techniques, codon optimization, use of strong promoters, insertion of introns, use of viral vectors, fusion protein techniques.

13. A plant, plant part, tuber or tuber part, or a plant cell, pollen or seed thereof, characterized in that, which is one of: (d1) a plant, plant part, tuber or tuber part, or plant cell, pollen or seed thereof, of the plant cell line, plant protoplast, cell or callus grown from the plant of claim 6, or obtained using the breeding method of any one of claims 11 or 12; (d2) progeny formed by selfing the plant of (d1), and plants, plant parts, tubers or tuber parts, or plant cells, pollen or seeds thereof, grown from the progeny; (d3) progeny formed by crossing the plant of (d1) with another variety, and plants, plant parts, tubers or tuber parts, or plant cells, pollen or seeds thereof, grown from the progeny.

14. A food or feed product made from the plant, plant part, tuber or tuber part, or plant cell, pollen or seed thereof of claim 13.

15. A method of making a commercial plant product, comprising obtaining the plant, plant part, tuber or tuber part, of claim 13, and making the commercial plant product, wherein the plant product is selected from the group consisting of fresh tuber material, frozen tuber material, dehydrated tuber material, tuber juice, tuber strips, tuber slices, tuber granules, tuber whole powder, fresh fruit, frozen fruit, dehydrated fruit material, fresh leaf, frozen leaf, dehydrated leaf.

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