Use of agpase small subunit in increasing yield of root crops

Abstract

The present application provides the role of AGPase small subunit in improving yield traits or plant type traits of root crops. The present application discloses a novel regulation mechanism of AGPase small subunit for root crops, and prepares root crops with changed traits, which has important significance for genetic improvement of root crop traits.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to the application of the AGPase small subunit in increasing the yield of root crops. Background Technology

[0002] Root crops are a class of terrestrial plants (crops) with enlarged root systems. Their tubers are formed from the localized enlargement of lateral or adventitious roots. A single root crop plant can produce multiple tubers. These tubers consist entirely of roots, excluding the hypocotyl and stem. Sweet potato and fleeceflower root tubers belong to this category. Sweet potato tubers are relatively irregular in shape. Approximately 20-30 days after planting, some adventitious roots begin to enlarge and form tubers. The main components of tubers are starch and sugars, along with a large amount of water and a small amount of protein; tubers are a type of storage root.

[0003] Sweet potato, a common root crop, possesses high efficiency in utilizing light, heat, and water resources, high biomass per unit area, high starch content, and suitability for planting in arid and barren land without competing with food crops for land. It is currently one of the most promising energy crops for development. Sweet potato is an important food, feed, and industrial raw material crop worldwide, playing a vital role in ensuring biomass energy and food security. Belonging to the Convolvulaceae family, Ipomoea genus, and Ipomoea species, it is a vining herbaceous plant. The sweet potato plant can be divided into roots, stems, leaves, flowers, fruits, and seeds. Sweet potato is not only an important food crop but also a crucial raw material for the food processing industry, boasting rich nutritional value. Therefore, research on sweet potato variety optimization and cultivation / regeneration technologies is of great significance. Other common plants with tuberous root structures similar to sweet potato include cassava and Polygonum multiflorum (He Shou Wu).

[0004] Storage roots in root crops are important energy storage organs, and their development is a complex biological process, including the initiation of swelling and subsequent thickening. These processes are regulated not only by endogenous hormones and genes but also by the external environment. However, the molecular mechanisms regulating the swelling and development of storage roots are still poorly understood.

[0005] Meanwhile, the starch processing industry using plant tubers as raw materials is still in its developmental stage, but the processing of various products places diverse demands on the quality of the raw starch (such as the ratio of amylose to amylopectin). Relying on traditional breeding to modify starch quality is time-consuming and requires significant human and material resources, making it difficult to meet the urgent needs of the starch processing industry. Researchers in this field have attempted to regulate the composition and content of starch in plants by modulating the main genes involved in starch synthesis, and the exploration of proteins related to starch production has industrial application significance.

[0006] In summary, it is necessary to study superior genes in root crops that can regulate potato storage roots, thereby providing new avenues for plant improvement. Summary of the Invention

[0007] The purpose of this invention is to provide the application of AGPase small subunit in increasing the yield of root crops.

[0008] In a first aspect of the invention, a method is provided for improving (optimizing) the yield or plant type traits of a root crop or for preparing a root crop with improved yield or plant type traits, comprising: upregulating the expression or activity of the small subunit of AGPase in the root crop.

[0009] In one or more embodiments, the improved root crop yield traits are: increasing root biomass or yield, increasing starch content in root tubers, and / or increasing aboveground biomass.

[0010] In one or more embodiments, the improved plant type traits of the root crop are: promoting robust plant type (increased stem diameter), reducing internode spacing of stems, and / or increasing leaf area.

[0011] In one or more embodiments, the upregulation of the expression or activity of the AGPase small subunit includes: transferring the gene encoding the AGPase small subunit or an expression construct or vector containing the gene into a root crop;

[0012] Gain-of-function mutations were performed on the AGPase small subunit; AGPase small subunit expression was promoted by expressing an enhancing promoter or a tissue-specific promoter; or AGPase small subunit expression was promoted by an enhancer.

[0013] In another aspect of the invention, there is a use of the AGPase small subunit or an upregulator thereof for: improving (optimizing) the yield or plant type traits of root crops or preparing root crops with improved yield or plant type traits.

[0014] In one or more embodiments, the improved root crop yield traits are: increasing root biomass or yield, increasing starch content in root tubers, and / or increasing aboveground biomass.

[0015] In one or more embodiments, the improved plant type traits of the root crop are: promoting robust plant type (increased stem diameter), reducing internode spacing of stems, and / or increasing leaf area.

[0016] In one or more embodiments, the AGPase small subunit upregulator includes: an exogenous AGPase small subunit encoding gene or an expression construct or vector containing the encoding gene.

[0017] In one or more embodiments, the expression construct includes an enhancing promoter, a tissue-specific promoter, or an enhancer; or, a reagent for gain-of-function point mutation of the AGPase small subunit; preferably, a reagent for reverting the mutant to the wild type of the AGPase small subunit in root crops with AGPase small subunit mutations.

[0018] In one or more embodiments, the terms up, promote, improve, increase, enlarge or enhance indicate a significant up, promote, improve, increase, enlarge or enhance, such as up, promote, improve, increase or enhance by 20%, 40%, 60%, 80%, 90% or higher.

[0019] In one or more embodiments, the term "reduction" indicates a significant reduction, such as a reduction of 20%, 40%, 60%, 80%, 90%, or lower.

[0020] In one or more embodiments, the AGPase small subunit includes its homologs (e.g., genes / proteins that are sequence homologous to and functionally identical to the AGPase small subunit and found in root crops other than sweet potato).

[0021] In one or more embodiments, the root crop is a plant containing the AGPase small subunit or its homologs.

[0022] In one or more embodiments, the root crops include (but are not limited to): sweet potato, cassava, potato, yam, taro, kudzu root, konjac, Jerusalem artichoke, snow lotus fruit, Panax notoginseng, and Polygonum multiflorum.

[0023] In one or more embodiments, the amino acid sequence of the AGPase small subunit is selected from the group consisting of:

[0024] (i) A polypeptide with the amino acid sequence shown in SEQ ID NO:2;

[0025] (ii) A polypeptide derived from (i) having the regulatory trait function formed by substituting, deleting or adding one or more (e.g., 1-30, 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:2.

[0026] (iii) A polypeptide having the regulatory trait function, with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95% or ≥98%) to the amino acid sequence shown in SEQ ID NO:2;

[0027] (iv) The active fragment of the polypeptide with the amino acid sequence shown in SEQ ID NO:2; or

[0028] (v) A polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

[0029] In another aspect of the invention, there is provided the use of a small subunit of AGPase (including a gene or protein) as a molecular marker for identifying traits of root crops; said traits include yield or plant type traits.

[0030] In one or more embodiments, the yield trait is: root biomass or yield, starch content in root, and aboveground biomass; or, the plant type trait is: stem diameter, internode spacing and / or leaf area.

[0031] In one or more embodiments, plant traits are identified by analyzing the expression level of AGPase small subunits in plants; if the expression level of AGPase small subunits in the tested plant is equal to or higher than the average expression level of AGPase small subunits in that type of plant, then the biomass or yield of its tuberous roots is increased, the starch content in the tuberous roots is increased, and / or the biomass of the aboveground parts is increased; or, its plant type is robust (stem diameter increases), the internode spacing of the stem is decreased, and / or the leaf area is increased.

[0032] In one or more embodiments, if the expression level of the small AGPase subunit in the tested plant is equal to or lower than the average expression level of the small AGPase subunit in that type of plant, then its trait is not ideal.

[0033] In another aspect of the present invention, a method for targeted selection or identification of root crops is provided, the method comprising: identifying the expression or sequence characteristics of AGPase small subunits in a test root crop; if the test root crop has high expression of AGPase small subunits, it is a high-yielding plant; if the test root crop has low expression or no expression of AGPase small subunits, it is a low-yielding plant.

[0034] In one or more embodiments, the high expression or high activity includes a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical root crops.

[0035] In one or more embodiments, the low expression or low activity includes a statistically significant reduction in expression or activity compared to the average expression or activity of similar or identical root crops.

[0036] In another aspect of the present invention, a method is provided for screening substances (potential substances) that can improve the yield trait of root crops, comprising: (1) adding the candidate substance to a system expressing an AGPase small subunit; (2) detecting the system and observing the expression or activity of the AGPase small subunit therein, and if its expression or activity is increased, it indicates that the candidate substance is a substance that can be used to improve the yield trait of root crops.

[0037] In one or more embodiments, the method further includes setting up a control group to clearly distinguish the difference in AGPase small subunit expression or activity between the test group and the control group.

[0038] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulators, small molecule compound gene editing constructs, etc.) designed to target the small subunit of AGPase or its encoding gene or their upstream or downstream proteins or genes.

[0039] In one or more embodiments, the system is selected from, but not limited to, plant cell systems (or cell culture systems), subcellular systems (or subcellular culture systems), solution systems, tissue systems, organ systems, etc.

[0040] In another aspect of the invention, a plant cell, tissue, or organ is provided that expresses an exogenous AGPase small subunit or a homolog thereof; preferably, the expression cassette includes: a promoter, a gene encoding the AGPase small subunit or a homolog thereof, and a terminator; preferably, the expression cassette is contained in a construct or expression vector.

[0041] In one or more embodiments, the plant cells, tissues, or organs are non-plant propagation materials, or they will not regenerate into living plants.

[0042] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0043] Figure 1 Manhattan diagram of AGPase small subunit (IbAGPS).

[0044] Figure 2 A binary gene expression cassette was constructed and integrated into the pCAMBIA1301 vector, with the overexpression vector on top and the RNAi vector on the bottom.

[0045] Figure 3 Gene expression in transgenic plants (top) and AGPase enzyme activity assay (bottom).

[0046] Figure 4Changes in the above-ground parts of the transgenic plants after one month of growth in the greenhouse.

[0047] Figure 5 Changes in leaf growth of transgenic plants after one month of growth in a greenhouse.

[0048] Figure 6 Changes in the tuber growth of transgenic plants after two months of greenhouse growth.

[0049] Figure 7 Changes in root yield and lignin and starch content of transgenic plants after 3 months of greenhouse growth. Detailed Implementation

[0050] Through in-depth research, the inventors have revealed the role of the AGPase small subunit (AGPS) in regulating yield or plant type traits in root crops. This invention discloses a novel mechanism involving the AGPase small subunit, which is of great significance for the genetic improvement of root crop traits.

[0051] Targets for targeted regulation

[0052] As used in this invention, unless otherwise specified, the AGPase small subunit refers to a polypeptide having the sequence shown in SEQ ID NO:2 or its encoding gene, and also includes sequence variations having the same function as the AGPase small subunit. The encoding gene may be gDNA or cDNA, and may also contain a promoter.

[0053] Variations of the AGPase small subunit include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 30, preferably up to 20 or 10, for example, up to 5, 3, or 2) at the C-terminus and / or N-terminus. Any protein with high homology to the AGPase small subunit (e.g., 70% or higher homology with the polypeptide sequence shown in SEQ ID NO:2); preferably 80% or higher homology; more preferably 90% or higher homology (e.g., 95%, 98%, or 99% homology) and having the same function as the AGPase small subunit is also included in this invention. In this invention, polypeptides derived from species other than sweet potato that have high homology with the sequence shown in SEQ ID NO:2, or that play the same or similar role in the same or similar regulatory pathways, are also included.

[0054] In this invention, the term "AGPase small subunit" also includes its homologs. It should be understood that while this invention preferably studies AGPase small subunits obtained from a specific species, sweet potato, other polypeptides or genes obtained from other species that are highly homologous to the AGPase small subunit (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0055] The polynucleotide (gene) encoding the small subunit of AGPase can be a natural gene from the plant or a degenerate sequence thereof.

[0056] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0057] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0058] Methods for improving root crops

[0059] As used herein, "crop" or "plant" includes plants expressing the AGPase small subunit, as well as homologs of the AGPase small subunit. Based on knowledge in the art, plants containing the AGPase small subunit inherently possess the mechanism of action claimed in this invention, and can achieve the technical effects claimed in this invention. In some preferred embodiments, the plant is a root crop. Preferably, the root crop may include (but is not limited to): sweet potato, cassava, potato, yam, taro, kudzu root, konjac, Jerusalem artichoke, snow lotus fruit, Panax notoginseng, Polygonum multiflorum, etc.

[0060] In the inventors' research, GWAS analysis of sweet potato hybrid populations identified an association between the small subunit of AGPase (IbAGPS) and tuber yield.

[0061] Further research showed that transgenic plants with downregulated AGPase small subunit exhibited significantly increased stem length and internode length, significantly thinner diameter, resulting in slender plants with decreased dry matter content and increased lignin content. Conversely, plants overexpressing the AGPase small subunit showed significantly shorter stem length and internode length, significantly thicker diameter, resulting in robust plants with increased dry matter content.

[0062] Further research showed that transgenic plants with downregulated AGPase small subunit had smaller leaves, reduced leaf area, significantly lower starch content, increased sucrose content, and decreased fructose and glucose content. Conversely, plants overexpressing the AGPase small subunit had larger leaves, significantly increased leaf area, and significantly increased starch content.

[0063] Further research showed that transgenic plants with downregulated AGPase small subunits did not exhibit root enlargement, had significantly reduced tuber yield, significantly increased lignin content, and significantly reduced starch content. Conversely, transgenic plants with overexpressed AGPase small subunits showed significantly increased tuber yield (including increased volume and weight), no significant change in lignin content, and significantly increased starch content.

[0064] The above research results indicate that the AGPase small subunit is essential for the yield of root crops, and that the AGPase small subunit can increase the yield of root crops.

[0065] Based on the inventor's new discovery, the present invention provides a method for improving plants, the method comprising: increasing the expression or activity of AGPase small subunits in plants, thereby improving the yield trait of root crops and promoting increased yield of root crops.

[0066] As used in this invention, the “improvement (or enhancement) of root crop yield traits” includes: increasing root biomass or yield, increasing starch content in root and / or increasing aboveground biomass.

[0067] As used in this invention, the "improved plant type traits of root crops" include: promoting robust plant type (increased stem diameter), reducing internode spacing of stems, and / or increasing leaf area.

[0068] As used in this invention, the term "above-ground part" also refers to a portion of the plant tissue that is located above the ground or the surface of the culture solution when the plant is planted in the soil or cultured in a nutrient solution. In this invention, the height of the "above-ground part" has the same meaning as the plant height.

[0069] It should be understood that, once the function of the AGPase small subunit is known, various methods well-known to those skilled in the art can be used to regulate the expression or activity of the AGPase small subunit. For example, various methods well-known to those skilled in the art can be used to overexpress the AGPase small subunit.

[0070] In this invention, the upregulators of the AGPase small subunit or its encoding gene include promoters, agonists, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant "upregulation" and "promotion." Any substance that can increase the activity of the AGPase small subunit, improve the stability of the AGPase small subunit, upregulate the expression of the AGPase small subunit gene, increase the effective duration of action of the AGPase small subunit, or increase the phosphorylation / activation level of various proteins can be used in this invention as substances useful for upregulating the AGPase small subunit. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0071] The present invention also provides a method for upregulating the expression of the small AGPase subunit in plants, the method comprising: transferring the coding gene of the small AGPase subunit or an expression construct or vector containing the coding gene into plants.

[0072] In tuberous plants, a class of proteins may be found that interact with the small subunit of AGPase and inhibit its function. Downregulating such proteins or their encoding genes is beneficial for upregulating the expression or activity of the small subunit of AGPase. Methods for downregulating such proteins or their encoding genes in plants include, but are not limited to, targeted mutations, gene editing, or gene recombination. For example, methods for downregulating the expression of a specific protein or its encoding gene in plants include: (1) transferring an interfering molecule that interferes with expression into plant cells, tissues, organs, or seeds to obtain plant cells, tissues, organs, or seeds transformed with the interfering molecule; (2) regenerating plants from the plant cells, tissues, organs, or seeds transformed with the interfering molecule obtained in step (1). Preferably, the method further includes: (3) selecting plant cells, tissues, or organs transformed with the vector; and (4) regenerating plants from the plant cells, tissues, or organs in step (3).

[0073] The technical solution of this invention can be applied to molecular design breeding through various pathways.

[0074] This invention also provides genetically engineered host cells, tissues, or organs that express exogenous AGPase small subunits or their homologs in an expression cassette; preferably, the expression cassette includes: a promoter, a gene encoding the AGPase small subunit or its homolog, and a terminator; preferably, the expression cassette is contained in a construct or expression vector. Transformed plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation.

[0075] Molecular markers or targeted screening

[0076] Previous research in this field has not analyzed the function of the AGPase small subunit in regulating yield / plant type traits in tuberous plants. This invention is the first to discover this regulatory role of the AGPase small subunit. Based on this discovery, this invention also relates to the application of the AGPase small subunit as a molecular marker, which can serve as a tracking marker for the progeny of gene-transformed plants. This invention also includes a method for early determination of drought tolerance in plants, which is achieved by detecting the expression or activity of the AGPase small subunit in plants.

[0077] After understanding the function of the AGPase small subunit, it can be used as a molecular marker for targeted screening of root crops. This new discovery can also be used to screen for substances or potential substances that can target and regulate yield traits in root crops by modulating this mechanism.

[0078] Based on this, the present invention provides a method for targeted selection or identification of root crops, the method comprising: identifying the expression or sequence characteristics of AGPase small subunits in test plants; if the test plant has high expression of AGPase small subunits (compared with control plants), then it is a plant that can be used to improve the yield trait of root crops.

[0079] As used herein and as will be understood by those skilled in the art, selecting appropriate “control plants” is a routine part of experimental design and may include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties that are the same as or belong to the same class as the plant being evaluated. Control plants may also be individuals that have lost their transgenic components due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, such as tubers.

[0080] The present invention provides a method for regulating the yield trait of root crops, comprising: (1) adding a candidate substance to a system expressing an AGPase small subunit; (2) detecting the system and observing the expression or activity of the AGPase small subunit therein. If its expression or activity is increased, it indicates that the candidate substance can be used to increase the yield of root crops.

[0081] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0082] Through large-scale screening, a class of potential substances that specifically act on the small subunit of AGPase and have a regulatory effect on the yield traits of root crops can be obtained.

[0083] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0084] Materials and methods

[0085] I. Experimental Methods

[0086] 1. Sweet potato GWAS analysis

[0087] A large-scale investigation, genetic mapping, and GWAS analysis were conducted on various agronomic traits (leaf shape, leaf color, tuber quantity, tuber yield, tuber skin color, etc.) and gene expression data of sweet potato hybrid populations. Through research and analysis, the inventors identified genetic loci and candidate genes controlling these traits. Among them, the AGPase small subunit (IbAGPS) located on chromosome 13 is closely related to tuber yield, and further research on the regulation of agronomic traits was carried out on the AGPase small subunit gene.

[0088] 2. Carrier Construction

[0089] Using sweet potato leaf cDNA as a template, the target gene IbAGPS was amplified by PCR. The full-length gene was then inserted into the pCAMBIA1301 vector to construct a binary overexpression vector, and an RNAi binary expression vector was also constructed.

[0090] 3. Genetic transformation of sweet potato

[0091] Transgenic sweet potatoes were obtained via embryo regeneration using an Agrobacterium-mediated embryonic suspension cell genetic transformation system. Agrobacterium tumefaciens containing a binary vector was cultured in liquid YEB containing the appropriate antibiotics until OD (occurrence zone). 600 =0.5-1.0, centrifuge at 6000 rpm at 4℃ for 10 min to collect bacterial cells, resuspend in an equal volume of LCP-AS to wash the cells, centrifuge again, and resuspend the precipitate in 1 / 2 volume of LCP-AS to adjust OD. 600 Prepare version 1.0 for later use. Sweet potato embryogenic callus obtained from suspension culture was washed with LCP and the culture medium was drained. The tissue was then immersed in the prepared infection solution, vacuumed for 10-20 minutes, drained, and transferred to sterile filter paper (laid flat on an SBM-AS). The tissue was co-cultured in a light incubator for 4 days with the following parameters: temperature 25℃, relative humidity 50%, 16h light, 8h darkness, and light intensity approximately 50 μmol / m².-2 s -1 .

[0092] After co-culture, the callus tissue was washed three times with sterile water and twice with 200 mg / L cephalosporin. It was then transferred to a medium containing hygromycin for screening and regeneration. After 1-2 months of culture, the obtained resistant embryoids were transferred to a medium without hygromycin and cultured for a longer period until they germinated to form sweet potato plantlets.

[0093] 4. Real-time PCR detection

[0094] 1 μg of total RNA was used to synthesize cDNA in a 20 μL reaction system using reverse transcriptase ReverTra Ace (catalog number TRT-101, TOYOBO, Shanghai), and then used for Real-time PCR detection.

[0095] PCR reactions were performed on a Bio-Rad CFX96 real-time PCR instrument. The 20 μL system included 10 μL of 2×SYBR Master Mix (catalog number QPK-201, TOYOBO, Shanghai), 50 ng of cDNA, and 400 nmol L-1 each of Forward Primer and Reverse Primer.

[0096] The reaction conditions were: denaturation at 95℃ for 1 min; denaturation at 95℃ for 15 sec; annealing and extension at 60℃ for 30 sec; 40-50 cycles; Actin gene was used as an internal control.

[0097] Each sample was tested in triplicate. The relative expression levels of the detected genes were calculated based on the relative Ct values. The formula for calculating the relative expression levels of each gene in a single sample is as follows:

[0098] ΔCt=Ct(Target gene)-Ct(Actin).

[0099] The formula for calculating the relative expression level of a specific gene in two samples is:

[0100] ΔΔCt=ΔCt(Sample 1)-ΔCt(Sample 2).

[0101] 5. Greenhouse phenotypic determination

[0102] Cut sweet potato vines obtained from greenhouse cultivation or seed potato seedlings into stem segments (about 20cm long) containing 2-3 lateral buds and use them as seedlings. Keep the seedlings from the same source and of similar quality in the same plot.

[0103] 6. Determination of major monosaccharides in sweet potato tissue

[0104] Freshly collected sweet potato tubers were completely dried at 80℃, ground thoroughly in a mortar, and 30 mg of sample was weighed into a 2.0 mL EP tube. 700 μL of 80% ethanol was quickly added, and the mixture was thoroughly shaken and mixed. The mixture was then centrifuged at 14000 g for 3 minutes. The supernatant was transferred to a clean centrifuge tube, 700 μL of pure water was added, and the tube was centrifuged at 1200 rpm for 5 minutes. 1 mL of the supernatant was transferred to a new centrifuge tube, 700 μL of chloroform was added, and the tube was centrifuged at 12000 rpm for 10 minutes. The extraction was repeated once to remove the pigment.

[0105] The sample was separated using an Agilent Technologies HPLC column (ZORBAX Carbohydrate column; 4.6 × 150 mm, 5 μM) on an HPLC1260-RI system. 10 μL of sample was injected, with 75% acetonitrile as the mobile phase, a flow rate of 0.8 mL / min, a separation temperature of 35 °C, and a differential refractive index detector. The blank control was 80% ethanol.

[0106] 7. Determination of starch content in sweet potato tissue

[0107] According to the Total Starch kit (K-TSTA, Megazyme), the dried plant tissue was first moistened with 50 μL of 75% ethanol, and the sample was repeatedly dispersed by shaking to prevent clumping during heating. 1.5 ml of Alpha-amylase was added, and the mixture was incubated in boiling water for 12 min to fully gelatinize the starch in the plant tissue. During this period, the mixture was shaken for 2 minutes each time to prevent the sample from spraying out of the EP tube due to alcohol evaporation. The EP tube was then transferred to a 50°C water bath, and 50 μL of amyloglucosidase was added. The mixture was incubated for 30 min, and all the liquid in the EP tube was transferred to a 15 mL test tube. The volume was adjusted to 10 mL, and approximately 100 μL of the sample was added to 3 mL of GOPOD. The mixture was incubated at 50°C for 20 min. The blank control was replaced with the same amount of water instead of the sample. The glucose control was 100 μL of glucose standard (1 mg / mL) + 3 mL of GOPOD reagent.

[0108] The absorbance of all samples was measured at 510 nm.

[0109]

[0110] Where: F = 100 (weight of glucose standard 100μg) / absorbance value of 100μg glucose standard;

[0111] FV: Final volume (10 mL);

[0112] ΔA: Absorbance of the sample;

[0113] W: Sample weight (mg).

[0114] 8. Lignin content determination

[0115] Fresh sweet potato stems and tubers were dried at 80℃ to constant weight, ground thoroughly in a mortar, and 1.0 mg of dried sample was accurately weighed for lignin content analysis according to the literature (Gui Jinshan et al., 2020, Fibre-specific regulation of ligninbiosynthesis improves biomass quality in Populus. New Phytologist, 226:1074-1087).

[0116] 9. AGPase enzyme activity assay

[0117] Approximately 0.1 g of fresh sweet potato leaf tissue was weighed, and the AGPase activity was determined using an AGPase activity assay kit.

[0118] 10. Iodine staining of leaves

[0119] First, remove chlorophyll with 95% alcohol until the leaves are basically white. Then, soak them in 1% Lugol's solution (accurately weigh 3.75g KI and 1.25g I2 dissolved in 500mL distilled water) at room temperature for 30 minutes. Wash off the residual iodine solution with water and observe and take pictures.

[0120] Example 1: GWAS analysis of sweet potato hybrid populations

[0121] The inventors conducted research and analysis on the genes of a wide range of root crops, examining and genetically mapping various agronomic traits (leaf shape, leaf color, number of tubers, tuber yield, tuber skin color, etc.) and gene expression levels in root crop hybrid populations. Combined with experimental analysis, they identified genetic loci and candidate genes that effectively control root crop traits. Analysis revealed that the small subunit of AGPase (IbAGPS) located on chromosome 13 is associated with tuber yield. Figure 1 ).

[0122] The cDNA sequence of the small subunit of AGPase is as follows (SEQ ID NO: 1; 1563bp):

[0123]

[0124] The amino acid sequence of the small subunit of AGPase is as follows (SEQ ID NO:2; 521 amino acids):

[0125] MAAAIGAPKLAPYTCAAERNDGSARRAARFKSLSFASSNLSGDKLASLVSRRCSRSGGKSSERRNAPIIVSPKAVSDSQNSQTCLDPDASRSVLGIILGGGAGTRLYPLTKKRAKPAVPLGANYRLIDIP VSNCLNSNVSKIYVLTQFNSASLNRHLSRAYASNMGGYKNEGFVEVLAAQQSPENPNWFQGTADAVRQYLWLFEEHNVLEFLVLAGDHLYRMDYERFIQAHRETDADITVAALPMDEKRATAFGLMKIDE EGRIIEFAEKPKGEQLKAMKVDTTILGLDDQRAKELPFIASMGIYVISKNVMLNLLREKFPGANDFGSEVIPGATSIGMRVQAYLFDGYWEDIGTIEAFYNANLGITKKPVPDFSFYDRSAPIYTQPRYL PPSKMLDADVTDSVIGEVQNCKIHHSVVGLRSCISEGAIIEDSLLMGADYYETDADRRLLAAKGSIPIGIGRNSHIKRAIIDKNARIGDNVKIINSDDVQEAARETDGYFIKSGIVTVIKDALIPSGTVI

[0126] Example 2: Preparation of overexpression plants

[0127] 1. Constructing plant expression vectors

[0128] Based on the gene sequence of the AGPase small subunit, the target gene IbAGPS was obtained by PCR amplification using sweet potato leaf cDNA as a template. The full-length 1563 bp gene was then inserted into the pCAMBIA1301 vector to construct an overexpression vector. A schematic diagram of the main components of the overexpression vector is shown below. Figure 2 The image above.

[0129] A suitable sequence fragment for RNAi was selected. Using sweet potato leaf cDNA as a template, a 327 bp sequence from positions 145 to 471 of IbAGPS was amplified by PCR. After constructing a hairpin structure by inserting it into a PBS intermediate vector, the hairpin structure was inserted into the multiple cloning site of the pCAMBIA1301 vector to obtain the RNAi vector. A schematic diagram of the main components of the RNAi vector is shown below. Figure 2The image below.

[0130] The plant expression vector prepared above was used to transform the sweet potato variety Xushu 22.

[0131] 2. Preparation and Plant Regeneration of Transgenic Sweet Potatoes

[0132] Agrobacterium containing the aforementioned binary vector was used to transform sweet potato embryogenic callus, and transgenic plants were obtained through screening and regeneration.

[0133] 3. Detection of transgenic plants

[0134] Results of transgenic plant testing Figure 3 .

[0135] The expression of the IbAGPS gene in transgenic sweet potato was detected at the molecular level using qRT-PCR, showing that the gene expression level was significantly upregulated in overexpressing transgenic plants. Figure 3 (See the image above), AGPase activity was also significantly enhanced. Figure 3 (See image below).

[0136] In RNAi plants, the expression level of the IbAGPS gene was downregulated. Figure 3 (See the image above), AGPase activity also decreased significantly. Figure 3 (See image below).

[0137] Example 3: Phenotypic Changes in Transgenic Plants: Aboveground Phenotyps

[0138] After one month of growth in a greenhouse, the aboveground morphology of RNAi transgenic plants and overexpression transgenic plants was compared with that of wild-type Xushu 22 grown under the same conditions.

[0139] The results showed that RNAi plants exhibited significantly increased stem length and internode length, significantly thinner diameter, resulting in slender plants, decreased dry matter content, and increased lignin content. Overexpression plants showed significantly shorter stem length and internode length, significantly thicker diameter, resulting in robust plants and increased dry matter content. Figure 4 This also means that the aboveground biomass has increased significantly.

[0140] Example 4: Phenotypic changes in transgenic plants: leaves and their starch characteristics

[0141] After one month of growth in a greenhouse, RNAi transgenic plants and overexpression transgenic plants were compared with wild-type Xushu 22 grown under the same conditions in terms of leaf characteristics and starch traits.

[0142] After one month of growth in a greenhouse, compared with the wild type of Xushu 22, the RNAi plants showed smaller leaves, reduced leaf area, lighter iodine staining, significantly lower starch content, increased sucrose content, and decreased fructose and glucose content. Conversely, the overexpression plants showed larger leaves, significantly increased leaf area, darker iodine staining, and significantly increased starch content. Figure 5 ).

[0143] Example 5: Phenotypic Changes in Transgenic Plants: Tuberous Root Characteristics

[0144] After two months of growth in a greenhouse, RNAi transgenic plants and overexpression transgenic plants were compared with wild-type Xushu 22 grown under the same conditions for tuber traits.

[0145] Compared to the wild type of Xushu 22, the RNAi plants did not have enlarged roots, had significantly smaller root diameters, and significantly reduced root yield, while the overexpressing plants had larger tuber diameters and significantly higher tuber yields. Figure 6 ).

[0146] After three months of growth in a greenhouse, tuber harvesting revealed that, compared to the wild type of Xushu 22, RNAi plants showed no root enlargement, significantly reduced tuber yield, significantly increased lignin content, and significantly reduced starch content. In contrast, overexpression plants exhibited significantly increased tuber yield (including increased volume and weight), with no significant change in lignin content, but a significantly increased starch content. Figure 7 ).

[0147] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. sequence list <110> Shanghai Chenshan Botanical Garden <120> Application of AGPase small subunit in increasing yield of root crops <130> 222418 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1563 <212> DNA <213> *Ipomoea batatas* (L.) Lam.) <400> 1 atggcggcag caatcggagc tccgaagctc gccccgtata cctgcgcggc ggagaggaac 60 gacggctctg ctcggcgtgc tgcgaggttc aagagcctct cgttcgcgtc ttctaatctc 120 tccggcgaca aactcgcgtc gttagtttct cggcgctgca gccgttccgg agggaaatcg 180 tcggagaggc gtaatgctcc gattatcgtt tcccctaaag ctgtttccga ctcccagaat 240 tcgcagactt gcctcgatcc tgatgctagc cgaagtgtct tgggaattat tcttggaggt 300 ggagctggga cccggctcta ccctctaaca aagaagaggg caaagccagc tgttccactt 360 ggagcaaact atcgtctgat tgatatccct gttagcaatt gcttgaacag taatgtttcc 420 aagatctatg ttcttacaca gtttaactct gcatcactca atcgtcacct ctcacgggca 480 tatgcaagta acatgggcgg ttacaagaat gaaggtttcg ttgaagttct tgctgctcag 540 caaagtccag agaaccctaa ttggttccag ggtactgctg atgctgtgag gcagtatcta 600 tggctgtttg aggaacacaa tgttcttgaa ttcctagttc ttgctggaga tcatttgtac 660 cgaatggatt atgagagatt cattcaagcc cacagagaaa cagatgctga tattactgtt 720 gctgctctgc caatggatga gaagcgtgcc actgcatttg gtctcatgaa gattgatgaa 780 gaagggcgga ttattgaatt tgcagagaaa ccaaaaggag aacagttgaa agctatgaag 840 gttgatacta ctattttagg tcttgatgat cagagagcta aagagttgcc ttttattgca 900 agtatgggca tatatgtcat tagcaaaaat gtgatgttaa acctacttcg agaaaagttc 960 cctggagcca atgactttgg aagtgaagtc attcctggtg caacatccat tggaatgaga 1020 gttcaagctt atttgtttga cggctactgg gaagaatattg gtacaattga ggctttctat 1080 aatgccaact tggggatcac aaaaaagcca gtgccagatt tcagtttcta tgaccgatca 1140 gctccaatct acactcagcc tcgatattta cctccttcta aaatgcttga tgctgatgtc 1200 acggacagtg ttatggcga agtgcagaac tgtaaaattc accattctgt ggttgggctt 1260 agatcatgta tatcagaagg tgcaattat gaagattcac tactgatggg agctgattac 1320 tatgagactg atgctgacag gaggcttctg gcagcaaagg gcagcatccc aattggcatt 1380 ggcaggaact cccacattaa aagagcaatc attgacaaga atgcccgtat aggggacaat 1440 gtgaagatca ttaatagtga cgatgttcaa gaagcagcta gggaaactga tggatatttc 1500 atcaagagtg gaatcgtgac tgtcatcaag gatgcattga tccccagcgg aactgtaata 1560 taa 1563 <210> 2 <211> 520 <212> PRT <213> Sweet potato (Ipomoea batatas (L.) Lam.) <400> 2 Met Ala Ala Ala Ile Gly Ala Pro Lys Leu Ala Pro Tyr Thr Cys Ala 1 5 10 15 Ala Glu Arg Asn Asp Gly Ser Ala Arg Arg Ala Ala Arg Phe Lys Ser 20 25 30 Leu Ser Phe Ala Ser Ser Asn Leu Ser Gly Asp Lys Leu Ala Ser Leu 35 40 45 Val Ser Arg Arg Cys Ser Arg Ser Gly Gly Lys Ser Ser Glu Arg Arg 50 55 60 Asn Ala Pro Ile Ile Val Ser Pro Lys Ala Val Ser Asp Ser Gln Asn 65 70 75 80 Ser Gln Thr Cys Leu Asp Pro Asp Ala Ser Arg Ser Val Leu Gly Ile 85 90 95 Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Tyr Pro Leu Thr Lys Lys 100 105 110 Arg Ala Lys Pro Ala Val Pro Leu Gly Ala Asn Tyr Arg Leu Ile Asp 115 120 125 Ile Pro Val Ser Asn Cys Leu Asn Ser Asn Val Ser Lys Ile Tyr Val 130 135 140 Leu Thr Gln Phe Asn Ser Ala Ser Leu Asn Arg His Leu Ser Arg Ala 145 150 155 160 Tyr Ala Ser Asn Met Gly Gly Tyr Lys Asn Glu Gly Phe Val Glu Val 165 170 175 Leu Ala Ala Gln Gln Ser Pro Glu Asn Pro Asn Trp Phe Gln Gly Thr 180 185 190 Ala Asp Ala Val Arg Gln Tyr Leu Trp Leu Phe Glu Glu His Asn Val 195 200 205 Leu Glu Phe Leu Val Leu Ala Gly Asp His Leu Tyr Arg Met Asp Tyr 210 215 220 Glu Arg Phe Ile Gln Ala His Arg Glu Thr Asp Ala Asp Ile Thr Val 225 230 235 240 Ala Ala Leu Pro Met Asp Glu Lys Arg Ala Thr Ala Phe Gly Leu Met 245 250 255 Lys Ile Asp Glu Glu Gly Arg Ile Ile Glu Phe Ala Glu Lys Pro Lys 260 265 270 Gly Glu Gln Leu Lys Ala Met Lys Val Asp Thr Thr Ile Leu Gly Leu 275 280 285 Asp Asp Gln Arg Ala Lys Glu Leu Pro Phe Ile Ala Ser Met Gly Ile 290 295 300 Tyr Val Ile Ser Lys Asn Val Met Leu Asn Leu Leu Arg Glu Lys Phe 305 310 315 320 Pro Gly Ala Asn Asp Phe Gly Ser Glu Val Ile Pro Gly Ala Thr Ser 325 330 335 Ile Gly Met Arg Val Gln Ala Tyr Leu Phe Asp Gly Tyr Trp Glu Asp 340 345 350 Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn Leu Gly Ile Thr Lys 355 360 365 Lys Pro Val Pro Asp Phe Ser Phe Tyr Asp Arg Ser Ala Pro Ile Tyr 370 375 380 Thr Gln Pro Arg Tyr Leu Pro Pro Ser Lys Met Leu Asp Ala Asp Val 385 390 395 400 Thr Asp Ser Val Ile Gly Glu Val Gln Asn Cys Lys Ile His His Ser 405 410 415 Val Val Gly Leu Arg Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp 420 425 430 Ser Leu Leu Met Gly Ala Asp Tyr Tyr Glu Thr Asp Ala Asp Arg Arg 435 440 445 Leu Leu Ala Ala Lys Gly Ser Ile Pro Ile Gly Ile Gly Arg Asn Ser 450 455 460 His Ile Lys Arg Ala Ile Ile Asp Lys Asn Ala Arg Ile Gly Asp Asn 465 470 475 480 Val Lys Ile Ile Asn Ser Asp Asp Val Gln Glu Ala Ala Arg Glu Thr 485 490 495 Asp Gly Tyr Phe Ile Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala 500 505 510 Leu Ile Pro Ser Gly Thr Val Ile 515 520

Claims

1. A method for improving the plant type traits of root crops, comprising: Upregulates the expression or activity of the small subunit of AGPase in root crops; The improved plant type traits of the root crops are: promoting increased stem diameter, reducing internode spacing, and increasing leaf area; The amino acid sequence of the small subunit of AGPase is shown in SEQ ID NO: 2; the root crop is sweet potato.

2. The method as described in claim 1, characterized in that, Upregulating the expression or activity of the AGPase small subunit includes transferring the gene encoding the AGPase small subunit or an expression construct containing the gene into root crops.

3. The method as described in claim 2, characterized in that, Enhancers promote the expression of the small subunit of AGPase.

4. The use of an AGPase small subunit or its expression construct for: improving plant type traits of root crops; The improved root crop plant type traits are: promoting increased stem diameter, reducing internode spacing, and increasing leaf area; The amino acid sequence of the small subunit of AGPase is shown in SEQ ID NO: 2; the root crop is sweet potato.

5. The use of a reagent for detecting the small subunit of AGPase, the reagent being used to identify traits of root crops; the traits being plant type traits; the plant type traits being: increased stem diameter, decreased internode spacing, and increased leaf area; in, The amino acid sequence of the small subunit of AGPase is shown in SEQ ID NO: 2; the root crop is sweet potato.

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