Polypeptide gene stucg5p for regulating potato tubers and use of polypeptide gene stucg5p

Abstract

A polypeptide gene StUCG5P for regulating potato tubers and a use of the polypeptide gene StUCG5P. The CDS sequence of the polypeptide gene StUCG5P is as shown in SEQ ID No. 1, 79 amino acids are encoded, and the sequence is as shown in SEQ ID No. 2. A protein encoded by the gene is localized in a nucleus and cytoplasm. The number of tubers can be increased by increasing the number and length of stolons, and the time of microtuber formation can be promoted to be advanced under short-day conditions.

Description

StUCG5P, a polypeptide gene regulating potato tubers, and its application. Technical Field

[0001] This invention relates to the fields of plant genetic engineering and potato breeding technology, specifically to a polypeptide gene StUCG5P that regulates potato tubers and its application. Background Technology

[0002] The potato (Solanum tuberosum L.) is my country's fourth largest food crop, after rice, wheat, and corn. More than two-thirds of the world's countries cultivate potatoes, with a yield of approximately 320 million tons. According to statistics from the Food and Agriculture Organization of the United Nations in 2021, my country's potato planting area reached 4,606,000 hectares, with an average annual yield of 80 million tons, ranking among the world's top producers. As a dual-purpose crop (food and vegetable), the potato plays a vital role in ensuring my country's food security, improving the dietary structure of urban and rural residents, and contributing to poverty alleviation.

[0003] As the main harvested organ of potatoes, tubers significantly impact economic value through their yield and quality. The formation and development mechanisms of potato tubers have long been a focus of scientific attention. Potato tubers form from the swelling of the subapical portion of underground stolons; the occurrence, elongation, and swelling of the subapical portion of the stolons all influence tuber formation. Generally, the tuber formation rate from stolons is 50%–70%, and the number of tubers increases accordingly. The transition from stolons to tubers is a crucial developmental stage in potatoes, influenced not only by genetics but also by numerous external conditions (light, photoperiod, water, temperature, etc.) and internal factors (endogenous hormones, carbohydrates, etc.). Studies have shown that short-day conditions, cool temperatures, high light intensity, high sucrose levels, and low nitrogen application promote potato tuber formation; while long-day conditions, high temperatures, low light intensity, and high nitrogen application delay tuber formation. Changes in plant hormones play a crucial regulatory role in stolon and tuber formation and development. Generally, gibberellin (GA) is believed to stimulate stolon elongation and inhibit tuber formation, while abscisic acid (ABA) inhibits stolon elongation and stimulates tuber formation. Cytokinin (CK) can initiate new tuber formation, with high concentrations exhibiting an inhibitory effect and low concentrations a promoting effect. Auxin (IAA) promotes both longitudinal elongation and lateral expansion of tubers. Compared to other hormones, GA and ABA play a vital role in stolon and tuber formation and development. Under long-day (LD) conditions, GA levels in stolons remain high while ABA levels are low, which is conducive to elongation. Conversely, under short-day (SD) conditions, GA levels decrease at the beginning of the transition from stolon to tuber, while ABA levels increase, inducing tuber formation. Other studies have also reported that tuber formation is hindered by GA3, while the application of GA biosynthesis inhibitors promotes tuber formation.

[0004] In recent years, researchers have elucidated the preliminary regulatory network of tuber formation based on various influencing factors. However, there are few reports on the role of small molecules, especially peptides, in potato growth and development. Therefore, exploring the functions and molecular mechanisms of potato peptides in stolon development, tuber initiation, and formation can provide new insights into the regulatory network of potato tuber formation and offer theoretical support for potato production and breeding. Technical issues

[0005] The purpose of this invention is to provide a polypeptide gene StUCG5P that regulates potato tubers and its application. Technical solutions

[0006] This invention provides a polypeptide gene StUCG5P that regulates potato tubers. Its CDS sequence is 240 bp long, and its nucleotide sequence is shown in SEQ ID No. 1, encoding 79 amino acids. The amino acid sequence is shown in SEQ ID No. 2.

[0007] The present invention also provides an application of the polypeptide gene StUCG5P that regulates potato tubers in potato tuber improvement breeding. The CDS sequence of the polypeptide gene StUCG5P is 240 bp long, and the nucleotide sequence is shown in SEQ ID No.1, encoding 79 amino acids, and the amino acid sequence is shown in SEQ ID No.2.

[0008] Furthermore, the application involves overexpressing the peptide gene StUCG5P in potatoes to increase the number of potato tubers.

[0009] The present invention also provides the protein encoded by the above-mentioned polypeptide gene StUCG5P that regulates potato tubers, the amino acid sequence of which is shown in SEQ ID No. 2.

[0010] This invention also provides the application of the protein encoded by the above-mentioned polypeptide gene StUCG5P, which regulates potato tubers, in increasing the number of potato tubers.

[0011] The present invention also provides a recombinant expression vector for the above-mentioned polypeptide gene StUCG5P that regulates potato tubers.

[0012] Furthermore, the recombinant expression vector is a StUCG5P overexpression vector.

[0013] Furthermore, the recombinant expression vector uses the pCAMBIA1300-eGFP vector as its backbone.

[0014] This invention also provides the application of the recombinant expression vector of the above-mentioned polypeptide gene StUCG5P that regulates potato tubers in potato tuber improvement breeding.

[0015] Furthermore, the application involves genetically transforming the overexpression vector of the polypeptide gene StUCG5P that regulates potato tubers into potatoes using Agrobacterium-mediated transformation to obtain StUCG5P overexpression transgenic lines. The obtained StUCG5P overexpression transgenic lines show an increase in the number and length of stolons and the number of tubers.

[0016] The present invention also provides a method for increasing the number of potato tubers, wherein the method is to increase the number of tubers by overexpressing StUCG5P in potatoes, wherein the CDS sequence of StUCG5P is 240 bp long, the nucleotide sequence is shown in SEQ ID No.1, encoding 79 amino acids, and the amino acid sequence is shown in SEQ ID No.2.

[0017] Furthermore, the method involves constructing a StUCG5P overexpression vector using pCAMBIA1300-eGFP as the backbone, and then genetically transforming it into potatoes using Agrobacterium-mediated transformation to obtain transgenic lines with StUCG5P overexpression. The obtained StUCG5P overexpression transgenic lines show an increase in the number and length of stolons and the number of tubers.

[0018] The process of this invention is as follows: The inventors screened a polypeptide gene with high expression levels in flowers, mature fruits, and tuber buds from transcriptome data of different tissues of potatoes. This gene, numbered (Soltu.DM.04G033200.1) and named StUCG5P, was identified. In Spud DB, this polypeptide gene is annotated as an uncharacterized protein (2023 annotation) or a G5 domain-containing protein (2022 annotation). Search results in Spud DB show that the StUCG5P gene is 398 bp in length, containing 2 exons and 1 intron, with a CDS sequence of 240 bp. The nucleotide sequence is shown in SEQ ID No. 1, encoding 79 amino acids, and the amino acid sequence is shown in SEQ ID No. 2. The protein sequence of the StUCG5P gene was predicted using the SignalP-5.0 and TMHMM-2.0 online databases, respectively. The results showed that the protein encoded by this gene lacks a signal peptide and a transmembrane domain. Subcellular localization revealed that the target polypeptide gene was located in the nucleus and cytoplasm. To further investigate its function, potato genetic transformation was performed using Agrobacterium infection to obtain overexpressing transgenic lines. Phenotypic analysis showed that StUCG5P promoted the number and length of stolons, the number of tubers, and the timing of tuber development in vitro. Compared to the wild type, the overexpressing lines showed increased stolons and length, increased tuber development, and earlier tuber development under short-day conditions. Beneficial effects

[0019] This invention provides a polypeptide gene, StUCG5P, that regulates potato tuber formation and its applications. The CDS sequence of the polypeptide gene StUCG5P is shown in SEQ ID No. 1, encoding 79 amino acids, and its sequence is shown in SEQ ID No. 2. The protein encoded by this gene is located in the cell nucleus and cytoplasm, increasing the number and length of stolons to increase tuber formation, and can promote earlier tuber development in vitro under short-day conditions. This provides new insights into the regulatory network of potato tuber formation, is of great significance for elucidating the molecular mechanism of potato tuber formation, and also provides theoretical support and new materials for potato production and breeding. Attached Figure Description

[0020] Figure 1 shows the structure of the StUCG5P gene.

[0021] Figure 2 shows the evolutionary analysis of StUCG5P and its homologous genes; where A is the protein sequence alignment and B is the phylogenetic tree.

[0022] Figure 3 shows the expression pattern analysis of StUCG5P; where A and B are RNA-seq data of StUCG5P in different tissues of DM and RH varieties; and C is qRT-PCR data of StUCG5P in different tissues of RH variety.

[0023] Figure 4 shows the subcellular localization of StUCG5P.

[0024] Figure 5 shows the identification of StUCG5P over-transgenic lines; Figure A shows the PCR identification results of StUCG5P over-transgenic lines, with lane 1 as the positive control and lanes 2 and 3 as the negative controls for water and wild type, respectively; Figure B shows the qRT-PCR identification of wild type and StUCG5P over-transgenic lines; Figure C shows the Western blot results of wild type and StUCG5P over-transgenic lines.

[0025] Figure 6 shows the phenotype of 70-day-old stolons of the StUCG5P over-transgenic line; where A is the phenotype of underground stolons of potato plants; B is the statistics of the number of primary stolons; C is the statistics of secondary stolons; D is the statistics of the total length of primary stolons; and E is the statistics of the average length of primary stolons.

[0026] Figure 7 shows the tuber formation phenotype of the StUCG5P over-transgenic line at 70 days of age; where A is the tuber formation phenotype of 10 potato plants; B is the tuber number count per plant; C is the tuber weight count per plant; and D is the total yield of 10 plants.

[0027] Figure 8 shows the phenotypes of in vitro tubers from the StUCG5P over-transgenic lines under short-day conditions; where A represents the total in vitro tuber phenotype of 49 lines at 90 days; B represents the changes in total tubers from 49 in vitro plantlets during the period from 55 days to 80 days; and C represents the total yield of in vitro tubers for each line at 90 days. Embodiments of the present invention

[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that the experimental materials whose source is not specified in the embodiments of the present invention are all commercially available, and the experimental methods whose specific conditions are not specified in the embodiments of the present invention are generally carried out according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers. In addition, it should be noted that the potato E3 mentioned in the present invention is E'ma potato No. 3.

[0029] Example 1: Sequence characteristics and evolutionary analysis of StUCG5P

[0030] The inventors screened a polypeptide with high expression levels in flowers, mature fruits, and tuber buds from transcriptome data of different potato tissues. This polypeptide, numbered (Soltu.DM.04G033200.1) and named StUCG5P, was identified. A search on Spud DB (http: / / spuddb.uga.edu / ) showed that the full-length StUCG5P gene is 398 bp, containing 2 exons and 1 intron, with a CDS sequence of 240 bp encoding 79 amino acids (Figure 1). Prediction of the protein sequence of the StUCG5P gene using the SignalP-5.0 and TMHMM-2.0 online databases revealed that the protein encoded by this gene lacks both a signal peptide and a transmembrane domain.

[0031] Using the protein sequence encoded by this gene as the source sequence, BLAST was performed on NCBI and Phytozome websites, yielding a total of 18 sequences from 13 species. Homology comparison of these 18 sequences was performed, and a phylogenetic tree was constructed using the NJ method. The results showed that these sequences were relatively conserved at the C-terminus (Figure 2A). The 18 protein sequences were divided into two major branches. In the first branch, all sequences belonged to the Solanaceae family. StUCG5P showed the highest homology with the tomato sequence, followed by pepper, tobacco, and datura. This gene is relatively conserved within the Solanaceae family (Figure 2B).

[0032] Example 2: StUCG5P Expression Pattern Analysis

[0033] RNA-Seq data from different tissue sites in PGSC showed that the StUCG5P gene was highly expressed in flowers, mature fruits, and tuber buds (Figures 3A and 3B). qRT-PCR results also showed that the expression was highest in flowers, followed by leaves (Figure 3C).

[0034] To investigate the localization of StUCG5P protein in cells, a pCAMBIA1300-StUCG5P-eGFP vector initiated by CaMV35S was constructed. Subcellular localization was performed by injecting tobacco with Agrobacterium. The results showed that StUCG5P was localized in the nucleus and cytoplasm (Figure 4).

[0035] Example 3: Obtaining potato overexpression lines

[0036] To further investigate the function of StUCG5P in potato, an overtransformation vector for StUCG5P was constructed using pCAMBIA1300-eGFP as the backbone. E3 tube potatoes were used as recipients, and genetic transformation was performed via Agrobacterium-mediated transformation. After genomic PCR identification, 14 positive overtransformation lines were obtained (Figure 5A). The expression levels of these overtransformation lines were identified using qRT-PCR. The results showed that compared to the control (WT), the transcription levels of the overtransformation lines were all more than 100-fold, with the highest reaching over 4000-fold (Figure 5B). Western blot analysis of the expression level of the GFP-tagged StUCG5P protein transformed into the overtransformation lines showed that the transformed StUCG5P gene was successfully expressed in the overtransformation lines (Figure 5C).

[0037] Example 4: Phenotypic Identification

[0038] (1) Overexpression of StUCG5P promotes the occurrence and length of potato stolons.

[0039] To investigate the effects of StUCG5P overexpression on potato tuber formation, harvested small potatoes were sprouted and transplanted into large pots for normal growth in a growing chamber (16 h light / 8 h dark, 22℃ / 18℃). Harvesting was conducted at 70 days, and the number and length of underground runners, as well as the number and weight of tubers, were recorded. The results showed that the number of primary runners (Fig. 6A and 6B) and secondary runners (Fig. 6A and 6C) in the overexpressing lines were significantly higher than those in the WT lines. Furthermore, the total length of primary runners in the overexpressing lines was also significantly higher than that in the WT lines (Fig. 6A and 6D), and the average length was significantly or extremely significantly higher than that in the WT lines (Fig. 6E). This indicates that StUCG5P promotes the number and length of potato runners, and that overexpression of StUCG5P will increase the number and length of runners.

[0040] (2) Overexpression of StUCG5P increases potato tuber formation rate

[0041] Statistical analysis of underground tubers harvested at 70 days in (1) showed that the number of tubers per plant in the StUCG5P overexpression line was higher or significantly higher than that in the WT line (Figs. 7A and 7B), but the tuber weight per plant was significantly lower than that in the WT line (Fig. 7C). Statistical analysis of the total tuber weight of 10 plants revealed that the total yield of the overexpression line was also lower than that of the WT line (Fig. 7D). This indicates that StUCG5P has a promoting effect on potato tuber development, and overexpression of StUCG5P will lead to an increase in the number of potato tubers.

[0042] (3) Overexpression of StUCG5P promotes the development of potato tubers in vitro.

[0043] To investigate the effect of StUCG5P overexpression on potato tuber formation, tuber formation was induced in vitro using MS solid medium with 8% sucrose under short-day conditions (8 h light / 16 h dark, 22℃ / 18℃). Seven bottles were used for each line, with seven stem segments per bottle, totaling 49 plantlets. Tuber number per bottle was counted starting at day 55. The two overexpressing lines started tuber formation earlier than the WT line. When 30 tubers were produced, the time required for WT (approximately 70 days) was significantly longer than for OE-6 (approximately 63 days) and OE-84 (approximately 57 days). At day 80, the number of tubers produced by OE-6 and OE-84 was significantly higher than that of the WT line (Figure 8B). At day 90, after harvesting the tubers, the total yield per bottle was counted. The results showed that the tuber weight per bottle in the overexpressing lines was also significantly and extremely significantly higher than that in the WT line (Figures 8A and 8C). This indicates that StUCG5P plays a promoting role in potato tuber formation under short-day conditions, and that overexpression of StUCG5P will advance the tuber formation time of potato tubers under short-day conditions.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the polypeptide gene StUCG5P, which regulates potato tubers, in potato tuber improvement breeding, characterized in that... The CDS sequence of the polypeptide gene StUCG5P is 240 bp long, and the nucleotide sequence is shown in SEQ ID No. 1, encoding 79 amino acids, the amino acid sequence of which is shown in SEQ ID No.

2. The application is to increase the number of potato tubers by overexpressing the polypeptide gene StUCG5P in potatoes.

2. The application of a recombinant expression vector of the polypeptide gene StUCG5P that regulates potato tubers in potato tuber improvement breeding, characterized in that, The CDS sequence of the polypeptide gene StUCG5P is 240 bp long, and the nucleotide sequence is shown in SEQ ID No. 1, encoding 79 amino acids, the amino acid sequence of which is shown in SEQ ID No.

2. The application involves genetically transforming the overexpression vector of the polypeptide gene StUCG5P that regulates potato tubers into potatoes using Agrobacterium-mediated transformation to obtain StUCG5P overexpression transgenic lines. The obtained StUCG5P overexpression transgenic lines show an increase in the number and length of stolons and the number of tubers.

3. A method for increasing the number of potato tubers, characterized in that, The method involves increasing the number of tubers by overexpressing StUCG5P in potatoes. The CDS sequence of StUCG5P is 240 bp long, and the nucleotide sequence is shown in SEQ ID No. 1, encoding 79 amino acids, the amino acid sequence of which is shown in SEQ ID No.

2.

4. The method according to claim 3, characterized in that, The method involves constructing a StUCG5P overexpression vector using pCAMBIA1300-eGFP as the backbone, and then genetically transforming it into potatoes using Agrobacterium-mediated transformation to obtain transgenic lines with overexpression of StUCG5P. The obtained StUCG5P overexpression transgenic lines show an increase in the number and length of stolons and the number of tubers.

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