Stsp5g-b gene for regulating starch synthesis in potato tubers and application thereof

Abstract

The application provides a StSP5G-B gene for regulating starch synthesis in potato tubers and an application thereof. Taking StSP5G-B as an object, the full length of the ORF of the gene is cloned, and the sequence structure, evolutionary relationship and tissue expression of the gene are analyzed. qRT-PCR analysis shows that the gene exhibits differential expression at different development stages of potato tubers. The StSP5G-B gene is cloned, and the sequence structure, evolutionary relationship and tissue expression of the gene are analyzed. Meanwhile, an overexpression vector is constructed and transformed into E3 potato for functional analysis. The results show that the StSP5G-B gene positively regulates the starch content of tubers and is closely related to the tuber morphology. The results not only lay an important foundation for the development of the research on the regulation theory of potato starch synthesis and tuber shape, but also provide important gene resources and molecular markers for the subsequent use of molecular assisted breeding to develop new varieties with high starch and different tuber shapes.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a StSP5G-B gene that regulates starch synthesis in potato tubers and its applications. Background Technology

[0002] Starch and tuber morphology are the main nutrients and commercial characteristics of potatoes. Currently, my country's potato starch production is severely insufficient, with an annual output of only 400,000 tons, accounting for less than 7% of the global annual output (Dong Haoran, 2023). Tuber morphology also severely restricts the development of the potato deep-processing industry. In-depth exploration of candidate genes involved in the regulation of potato starch and tuber morphology is of great significance for improving the quality and efficiency of my country's potato industry. The SP5G gene is a member of the FT subfamily of the phosphatidyl ethanolamine-binding protein (PEBP) gene family (Shannon S, 1991). In the photoperiod-sensitive Andigena potato variety, SP5G is considered an inhibitor of tuber formation, mainly by inhibiting the transcription of the tuber formation promoter SP6A, thereby inhibiting the tuberization process of potatoes under long-day conditions (Abelenda et al, 2016). SP5G is highly expressed in potato leaves under long-day conditions (Abelenda et al., 2016). Transgenic silenced lines of this gene can produce tubers under non-tuber-inducing long-day conditions, leading to the activation of the transcription of the tuber-promoting factor SP6A in leaves (Abelenda et al., 2016). Previous studies have also shown that inhibiting SP5G transcription can promote CK biosynthesis and disrupt hormone balance in potato leaves and tubers, resulting in tubers becoming insensitive to auxin and indirectly inhibiting JA signaling in both tissues (Eduard Cruz Oról, 2017). Furthermore, SP5G has been shown to play an important role in the morphogenesis of new shoots during tuber sprouting (Eduard Cruz Oról, 2017). The potato tuber starch synthesis pathway has been reported (Tong et al., 2023). Sucrose synthesized in potato leaves is transported long distances through the phloem into the parenchyma cells of the underground tuber. Subsequently, sucrose in the parenchyma cells is broken down into fructose and uridine diphosphate glucose (UDPG) under the catalysis of sucrose synthase (SUS). Next, UDPG is converted to glucose-1-phosphate (G-1-P) by uridine diphosphate glucose pyrophosphorylase (UDPase). G-1-P enters the amyloid body via plasma membrane transport proteins and is converted to adenosine diphosphate glucose (ADPG) by adenosine diphosphate glucose pyrophosphorylase (AGPase). Finally, ADPG in the amyloid body is synthesized into amylose or amylopectin through the combined action of granule-bound starch synthase (GBSS), starch synthase (SSⅠ / II / III), starch branching enzyme (SBE I / II), and debranching enzyme (DBE) (Farhad and Visser, 2017).Currently, research on potato tuber shape is relatively underdeveloped, although several QTLs related to tuber shape have been identified. However, research on SP5G mainly focuses on the regulation of the photoperiodic tuber formation pathway in leaves, and there are few reports on the relationship between SP5G and potato starch content and tuber morphology. Summary of the Invention

[0003] This invention provides a StSP5G-B gene that regulates starch synthesis in potato tubers and its application, and can also regulate potato tuber morphology, providing a powerful technical means for the study of potato starch synthesis pathways and tuber morphology.

[0004] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0005] This invention provides a StSP5G-B gene that regulates starch synthesis in potato tubers, the sequence number of which is Soltu.DM.05G024040.1 in the potato genome database.

[0006] The present invention also provides a vector containing the above-mentioned StSP5G-B gene.

[0007] This invention further provides an application of the StSP5G-B gene that regulates starch synthesis in potato tubers.

[0008] The present invention has the following beneficial effects:

[0009] This invention focuses on the StSP5G-B gene, cloning its full-length ORF and analyzing its sequence structure, evolutionary relationship, and tissue expression. qRT-PCR analysis showed differential expression of this gene at different developmental stages of potato tubers. The invention also cloned the StSP5G-B gene and analyzed its sequence structure, evolutionary relationship, and tissue expression. Simultaneously, an overexpression vector was constructed and transformed into E3 potatoes for functional analysis. Results showed that the StSP5G-B gene contains a conserved PEBP domain of FT proteins, indicating that StSP5G-B is a typical PEBP protein with tissue-specific expression. Expression levels were lowest in leaves, petioles, flowers, and roots under short-day conditions, and higher in tubers and stems. Expression was significantly upregulated during potato tuber development. Silent transgenic lines showed significantly reduced starch content in tubers, and tuber shape changed from round to oblong. Overexpression transgenic lines showed significantly increased potato starch content. These results indicate that the StSP5G-B gene positively regulates tuber starch content and is closely related to tuber morphology. This result will not only lay an important foundation for the theoretical research on potato starch synthesis and tuber shape regulation, but also provide important gene resources and molecular markers for the subsequent use of molecular-assisted breeding to select new varieties with high starch content and different tuber morphologies. Attached Figure Description

[0010] Figure 1 : PCR amplification diagram of the StSP5G-B gene.

[0011] Figure 2 Phylogenetic analysis of the potato PEBP family genes and analysis of conserved protein domains.

[0012] Figure 3 : Relative expression levels of StSP5G-B in different potato tissues and tuber development. Different letter targets indicate significant differences.

[0013] Figure 4 Phenotypic diagram of StSP5G-B transgenic potato tubers.

[0014] Figure 5 : StSP5G-B transgenic potato tuber starch content chart. Detailed Implementation

[0015] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0016] Example 1: Cloning of the StSP5G-B gene

[0017] (1) Plant materials

[0018] Three groups of 'E' potato variety 3 (E3) with uniform growth at 8 weeks were selected as sampling seedlings. Flowers, petioles, leaves, stems, and roots of E3 potatoes were used as materials for tissue expression analysis. Three groups of E3 potatoes with uniform growth at 8 weeks were also selected as sampling seedlings, and runners at different developmental stages were collected according to morphological characteristics for fruit development analysis. Tubers of E3 potatoes were collected as template materials for gene cloning. All experiments were performed in triplicate. After sampling, samples were immediately flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0019] (2) Cloning and bioinformatics analysis of the StSP5G-B gene sequence

[0020] The base and amino acid sequences of the StSP5G-B gene (Soltu.DM.05G024040.1) were obtained from the Potato Genome Database (http: / / spuddb.uga.edu / index.shtml). Primers SP5G-F and SP5G-R (Table 1) were designed based on the ORF sequence of the StSP5G-B gene using Primer Premier 5.0 and synthesized by Sangon Biotech Co., Ltd. (Shanghai). RNA was extracted from E3 potato tubers using the plant RNA extraction kit from Sangon Biotech Co., Ltd. The StSP5G-B gene was cloned using the PrimeScript RT-PCR kit from Takara Bio Inc., following the manufacturer's instructions. cDNA was reverse transcribed as a template for PCR amplification. The amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles (denaturation-extension); 72℃ extension for 10 min; and storage at 4℃. The amplified products were gel-cleaved, purified, and ligated into the pMD18-T vector, transformed into DH5α competent cells, and positive clones were screened by PCR. Positive single clones were selected and sent to Sangon Biotech Co., Ltd. (Shanghai) for sequencing.

[0021] Protein domains were predicted using the online software SMART (http: / / smart.emblheidelberg.de / ), and the isoelectric point and molecular weight of the protein were analyzed using ExPASy (http: / / expasy.org / tools / ). Based on the cloned cDNA sequence, amino acid homology was compared using BLASTp, and amino acid sequence homology and phylogenetic analysis were performed using MEGA5 software. A Neighbor-Joining phylogenetic tree was constructed with 1000 replicates; all other settings were default.

[0022] Example 2: StSP5G-B gene expression analysis

[0023] Based on the cloned StSP5G-B gene sequence, qRT-PCR primers SP5G-QF and SP5G-QR were designed (Table 1), and the specificity of the primers was ensured using the BLASTn test in NCBI. The potato Ef1α gene (Soltu.DM.06G005580.1) was used as an internal reference gene, and the specific primer sequences are shown in Table 1.

[0024] Table 1 Primer information used

[0025]

[0026] The qRT-PCR reaction was performed using a Roche LightCycler 480 instrument, and the PCR enzyme was Takara's SYBR Green Master Mix. The reaction volume was 20 μL, containing 40 ng of template cDNA, 250 nM each of forward and reverse primers, 10 μL of SYBR Green Master Mix, and the remainder was added with ddH2O. The reaction program was: 94℃ pre-activation for 5 min; 94℃ for 10 s, 59℃ for 20 s, 72℃ for 30 s, for 40 cycles, followed by melting curve plotting (95→65℃, 0.1℃ / s). Using 2... -ΔΔCt The relative expression level of the StSP5G-B gene was calculated. All samples were tested in triplicate, with negative controls included. Mean values ​​were analyzed using Excel, and one-way ANOVA was performed using SPSS to determine the significance of differences in gene expression across different tissues and materials (P < 0.05). Graphs were plotted using SigmaPlot 12.5.

[0027] Example 3: Construction of overexpression vectors and interference vectors and functional verification of transgenic potatoes

[0028] Overexpression vectors were constructed using SP5G-OE-F / SP5G-OE-R (Table 1), with EcoRI restriction sites added to the 5' ends of the primers and Spe I restriction sites added to the 5' ends of the reverse primers. PCR amplification was performed using potato tuber cDNA as a template. The obtained PCR products were ligated into the pMD19-T vector and sequenced. Finally, plasmids with correct sequencing were extracted. The pRC26B vector and the correctly sequenced plasmid were double-digested with EcoRI and Spe I, respectively. A plant overexpression vector containing the StSP5G-B target gene was constructed using T4 DNA ligase and named pRC26B-StSP5G-B.

[0029] The StSP5G-B gene-specific sequence was analyzed using the Potato Genome Information Network (Spud DB). Forward primers SP5G-Ri-F1 and SP5G-Ri-F2, and reverse primers SP5G-Ri-R1 and SP5G-Ri-R2 (Table 1) were designed to amplify the StSP5G-B specific sequence. Primers F1 and R1 contain XhoI restriction sites at their 5′ ends, and primers F2 and R2 contain XbaI restriction sites at their 5′ ends. The obtained PCR positive and negative products of the StSP5G-B specific sequence were ligated into the pMD19-T vector and sequenced. Finally, plasmids with correct sequencing were extracted and digested with XhoI and XbaI enzymes respectively. The recovered sense sequence and the pHELLSGATE-8 vector digested with XhoI were used to construct a plant interference expression vector containing the sense StSP5G-B specific sequence using T4 DNA ligase. After correct sequencing, the plasmid was extracted and digested with XbaI again. The plant interference expression vector containing sense and antisense StSP5G-B specific sequences was constructed using T4 DNA ligase and named pHELLSGATE-8-StSP5G-B. The overexpression vector pRC26B-StSP5G-B and the interference expression vector pHELLSGATE-8-StSP5G-B were transformed into Agrobacterium strain GV3101 using the liquid nitrogen freeze-thaw method. Following the literature (Arshad W, Waheed MT, Mysore KS, et al. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens[J]. PLoS One, 2014, 9(5):e96979.), the overexpression vector and interference vector were transformed into E3 potatoes using the Agrobacterium infection method to obtain regenerated seedlings. Positive potatoes were screened on MS solid medium containing 30 μg / ml, and positive transgenic potato seedlings were detected using specific primers for pRC26B and pHELLSGATE-8 plasmids. The transgenic plants and wild-type plants were cultured in the same environment, and their tuber development phenotypes were compared.

[0030] Example 4, Results and Analysis

[0031] (1) Cloning and bioinformatics analysis of the StSP5G-B gene

[0032] Using E3 potato tuber cDNA as a template, a fragment of approximately 500 bp was amplified using SP5G-F / SP5G-R primers (Table 1). Figure 1 Sequencing results showed that the gene is 528 bp in size, encoding 176 amino acids, with a molecular weight of 19.7 kDa and a theoretical isoelectric point of 5.49. Amino acid sequence analysis indicated that StSP5G-B, like other PEBP family genes, contains a conserved PEBP domain, suggesting that StSP5G-B is a typical PEBP protein. Figure 2 ).

[0033] The amino acid sequence of StSP5G-B was homology-searched using BLASTp, and then a phylogenetic tree was constructed using MEGA 6.0 software. Figure 2 The results indicate that StSP5G-B is evolutionarily closely related to SP6A and SP3D, belonging to the FT subfamily.

[0034] (2) Analysis of tissue expression characteristics of StSP5G-B gene

[0035] qRT-PCR results showed that the StSP5G-B gene was expressed in all nine potato tissues tested, but the expression was tissue-specific, with the lowest expression in roots and the highest expression in large potatoes (approximately 960 times higher than in roots). Expression levels remained high throughout the entire stolon development stage. Figure 3 This result indicates that the StSP5G-B gene can specifically participate in potato tuber development.

[0036] (3) Phenotypic analysis of potatoes with overexpression of the StSP5G-B gene

[0037] Transgenic phenotypic results showed that potato plants overexpressing the StSP5G-B gene had a significantly higher tuber starch content of approximately 35.71% compared to the wild type. Figure 5 This result indicates that overexpression of the StSP5G-B gene significantly increases the starch content of potato tubers and can be used as a candidate gene for breeding new high-starch potato varieties.

[0038] (4) Phenotypic analysis of potato with StSP5G-B gene interference expression

[0039] Transgenic phenotypic results showed that the tuber starch content of potato plants with interference in StSP5G-B gene expression was significantly reduced by approximately 42.42% compared to the wild type. Figure 5 This result indicates that interfering with StSP5G-B gene expression significantly reduces the starch content of potato tubers, and the tuber shape of potatoes with interfered StSP5G-B gene expression changes from a prototypical shape to an elongated shape. Figure 4 This gene can be used as a candidate gene for breeding high-starch potato varieties and improving potato shape.

Claims

1. A method for regulating starch synthesis in potato tubers StSP5G-B The application of genes, the StSP5G-B The gene's sequence number in the potato genome database is Soltu.DM.05G024040.1; StSP5G-B Genes positively regulate the starch content of tubers.

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