StBSB, a transcription factor that regulates the accumulation of steroidal glycoside alkaloids in potatoes, and its applications.

Abstract

This application belongs to the field of agricultural biotechnology, specifically relating to the transcription factor StBSB, which regulates the accumulation of steroidal glycoside alkaloids in potatoes, and its applications. The transcription factor StBSB is encoded by the potato gene Soltu.DM.05G001620, the nucleotide sequence of which is shown in SEQ ID NO.1. This application provides new insights into the transcriptional regulatory mechanisms of SGAs in Solanaceae plants and offers crucial theoretical basis and genetic resources for the targeted improvement of the content of these anti-nutritional compounds through genetic means.

Description

Technical Field

[0001] This application belongs to the field of agricultural biotechnology, specifically relating to the transcription factor StBSB, which regulates the accumulation of steroidal glycoalkaloids in potatoes, and its applications. Background Technology

[0002] Steroidal glycoalkaloids (SGAs) are naturally occurring secondary metabolites in Solanaceae plants. They are a class of toxic natural anti-nutritional substances, widely distributed in Solanaceae plants such as potatoes, tomatoes, and eggplants. While they serve a defensive function in plants, effectively resisting pests, diseases, and environmental stresses, their toxicity to humans and animals cannot be ignored. In potatoes, α-solanine and α-carboxine account for over 90% of the total solanine alkaloids (SGAs); in tomatoes, α-tomatine and tomatidenol are the main SGAs in green tissues. Excessive intake of these compounds by humans or animals can cause neurotoxicity, easily leading to symptoms such as indigestion and vomiting, and in severe cases, even death.

[0003] Currently, transcription factors regulating potato SGA synthesis have been identified as light signal transduction factors (HY5, PIF3, and StMYB113) and AP2 / ERF family transcription factor GAME9 (also known as JRE4). Among them, GAME9 has been proven to be a key regulator in SGA biosynthesis. GAME9 activates the transcription of downstream genes such as DWF5, C5-SD, GAME4, and SSR2 by binding to elements such as the GCC-box and G-box; overexpression or silencing of GAME9 can significantly affect the level of SGAs in tomatoes and potatoes. However, many other transcription factors involved in regulating SGA synthesis and their interactions with existing functional networks still require further in-depth research.

[0004] Therefore, it is necessary to continue to explore the relevant factors involved in regulating the synthesis of SGAs in potatoes, and to investigate the specific mechanisms by which they regulate the synthesis of SGAs in potatoes. Summary of the Invention

[0005] The problem this application aims to solve is to provide a transcription factor StBSB that regulates the accumulation of steroidal glycoalkaloids in potatoes and its applications, providing new insights into the transcriptional regulation mechanism of SGAs in Solanaceae plants, and providing key theoretical basis and genetic resources for the targeted improvement of the content of these anti-nutritional compounds through genetic means.

[0006] To address the above problems, this application provides the following solution:

[0007] On the one hand, this application provides a potato transcription factor StBSB, which is encoded by the potato gene Soltu.DM.05G001620, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] On the other hand, this application provides the application of the above-mentioned potato transcription factor StBSB in reducing the accumulation of steroidal glycoside alkaloids in potatoes. The application reduces the synthesis of steroidal glycoside alkaloids in potatoes by inhibiting the expression of genes related to the transcription factor StBSB.

[0009] Furthermore, the inhibition of StBSB-related gene expression includes knocking out the StBSB-related genes.

[0010] Furthermore, the reduction of steroidal glycoalkaloid synthesis in potatoes includes reducing the expression level of GAME9, a core regulatory factor related to steroidal glycoalkaloid synthesis. GAME9 is encoded by the potato gene Soltu.DM.01G031000, the sequence of which is shown in SEQ ID NO.2.

[0011] On the other hand, this application provides the application of the above-mentioned potato transcription factor StBSB in reducing the accumulation of steroidal glycoalkaloids in other Solanaceae plants. The application includes constructing homologous transcription factors BSB of other Solanaceae plants based on the StBSB, and then inhibiting the expression of related genes of the homologous transcription factor BSB in other Solanaceae plants, thereby reducing the synthesis of steroidal glycoalkaloids in other Solanaceae plants.

[0012] The other Solanaceae plants mentioned include tomatoes.

[0013] Furthermore, the inhibition of BSB-related gene expression includes knocking out the BSB-related genes.

[0014] Furthermore, the nucleotide sequence of the homologous transcription factor BSB is shown in SEQ ID NO.3.

[0015] Furthermore, the reduction of steroidal glycoalkaloid synthesis in other Solanaceae plants includes reducing the expression level of GAME9, a core regulatory factor related to steroidal glycoalkaloid synthesis.

[0016] This application has the following beneficial effects:

[0017] This application successfully screened a light-induced transcription factor, StBSB, that can directly regulate the synthesis of steroidal glycosides (SGAs) in potatoes, and elucidated the molecular mechanism by which StBSB regulates SGAs. StBSB can form a heterodimer with GAME9 and synergistically activate the transcription of multiple SGA synthases, promoting SGA biosynthesis. Based on the functional and structural conservation of StBSB in SGA biosynthesis, a transcription factor BSB capable of regulating other Solanaceae plants was successfully constructed. In the future, it is hoped that by combining molecular marker-assisted selection technology, superior allelic variants of this gene can be rapidly screened and aggregated to cultivate new germplasm that does not easily accumulate steroidal glycoside alkaloids. This could lead to the creation of new, safer Solanaceae plant varieties without introducing exogenous genes, and could provide support for the quality improvement of Solanaceae crops such as potatoes and tomatoes. Toxic SGAs are substances that can enhance plant resistance to diseases and pests. Rational design of potato SGAs based on the transcription factor StBSB and optimization of SGA metabolism to improve edibility, safety and resistance to diseases and pests is a key direction in potato breeding. Attached Figure Description

[0018] Figure 1 The expression characteristics analysis of StBSB includes: (a. Co-expression network of genes in SGA metabolic modules; b. Tissue expression profile analysis of BBSB and other SGA biosynthetic genes; c. Manhattan plot of GWAS analysis based on the total SGA content of 132 genotypes in tubers, with red arrows indicating variant sites in the chromosomal regions where StBSB is located; d. Co-localization results of StBSB and nuclear marker proteins in tobacco leaves, with green fluorescence indicating GFP fusion protein, red fluorescence indicating fusion markers of nuclear localization signals and mKate, Bright indicating bright-field images, and Merged indicating merged images; e. In situ RNA hybridization detection of BBSB in shoots and tubers, with tuber sections at the top and shoot sections at the bottom, and arrows indicating regions of high gene expression).

[0019] Figure 2 The effects of changes in BSB expression on the content of major SGAs in potatoes were investigated (a. expression level of StBSB in leaves of potato overexpression lines; b. content of α-carboxine and α-solanine in potato overexpression lines; c. content of α-carboxine and α-solanine in potato knockout lines, P<0.05, P<0.01, P<0.001, ****P<0.0001, ns indicates P>0.05).

[0020] Figure 3The effects of changes in BSB expression on the content of major SGAs in tomato were investigated (a. expression level of SlBSB in leaves of tomato overexpression lines; b. content of α-tomatine in tomato overexpression lines; c. content of lycopene and lycopene glycosides in leaves of tomato knockout lines, P<0.05, P<0.01, P<0.001, ****P<0.0001, ns indicates P>0.05).

[0021] Figure 4 The expression changes of genes involved in the SGA biosynthesis pathway in potatoes and tomatoes caused by BSB functional variations are shown in the figures (a. overlap of differentially expressed genes in four potato BSB knockout lines; b. overlap of differentially expressed genes in two tomato BSB knockout lines; c. expression changes of genes involved in SGA biosynthesis in leaves of potato knockout lines, overexpression lines, and tomato knockout lines; the numerical values ​​represent the percentage of gene expression in transgenic lines relative to wild-type controls, purple indicates upregulation, green indicates downregulation, and * indicates no expression of the gene).

[0022] Figure 5 The analysis of the interaction between StBSB and GAME9 includes: (a) changes in GAME9 expression in potato lines overexpressing and knocking out BSB; (b) the three-dimensional structure of the BSB–GAME9 heterodimer predicted by AlphaFold2-multimer-v2, with blue representing GAME9 amino acid residues and green representing BSB amino acid residues; (c) bimolecular fluorescence complementation experiment of the StBSB–GAME9 heterodimer in tobacco leaves, with yellow YFP fluorescence indicating the interaction signal of the fusion protein, red representing the autofluorescence of chloroplasts, bright representing bright field, and merged representing the superimposed image; (d) the Co-IP experiment using tobacco leaves to verify the interaction between BSB and GAME9; and (e) the Pull-down experiment to further verify the interaction between BSB and GAME9. Detailed Implementation

[0023] The technical solutions in some embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments provided in this application, all other embodiments obtained by those skilled in the art are within the scope of protection of this application.

[0024] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open and encompassing, that is, "including, but not limited to".

[0025] In describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0026] Example 1: Gene Screening

[0027] Transcriptome data from various potato tissues and under various conditions published in the PGSC database were used. This dataset covers different tissues and developmental stages, as well as transcriptome sequencing data under various hormone treatments, totaling 98 RNA-seq libraries. Genes with a mean TPM <1 in each tissue were then removed to improve the specificity of the co-expression network. The remaining genes were used to construct a weighted gene co-expression network using the R package WGCNA. Then, under default parameters, candidate genes were predicted and transcription factors were identified using the online database PlantTFDB. Potato whole genome GO terms annotated with InterProScan were downloaded from PGSC as the background set for enrichment analysis. Module genes related to SGA synthesis were extracted, and GO enrichment analysis was performed using the online platform OmicShareTools with thresholds set at p < 0.01 and q < 0.05.

[0028] Weighted gene co-expression network analysis identified an Ivory module associated with solanine synthesis. This module's genes were significantly enriched in GO entries related to phytosterol metabolism, such as sterol metabolic process (GO:0016125, P=0.0027), sterol biosynthetic process (GO:0016126, P=0.0027), and steroid metabolic process (GO:0008202, P=0.0047). The Ivory module contains 16 identified genes involved in solanine biosynthesis (e.g., ...). Figure 1 (as shown in a), including the recently identified reductase-encoding genes RPG1 / 2 and cholesterol glucuronide transferase-encoding gene GAME15 involved in potato SGA synthesis. These genes are expressed at high levels in tissues such as stems, immature fruits, and carpels (e.g., ...). Figure 1(as shown in b). Among the 1209 genes within the module, there are 69 transcription factors, including the identified core regulator of SGA synthesis, GAME9, and a 520-amino acid Basic helix-loop-helix leucine zipper-type transcription factor encoded by the gene Soltu.DM.05G001620 (SEQ ID NO.1), named StBSB in this study. Amino acids 329-379 of the StBSB protein constitute the bHLH conserved domain. Phylogenetic analysis showed that StBSB has one homolog in each of tomato, eggplant, and pepper, and two homologs in tobacco. StBSB is located near a previously reported variation site associated with total SGA content in potato tubers. GWAS analysis of total SGA content in tubers from 132 potato lines revealed a LOD value of 4.6, a phenotypic variance explained by 12.4%, and an additive effect of 0.29 (e.g., ...). Figure 1 (as shown in c).

[0029] Constructing a recombinant vector 35S:: StBSB After CDS-eGFP, tetraploid cultivated potatoes (Qingshu No. 9) were used. Solanum tuberosum Based on the recombinant vector 35S:: StBSB CDS-eGFP was used to construct overexpression lines by infecting explants (potato stem segments) with Agrobacterium GV3101; the vector pNK2-cas9-S-Ng was constructed to express diploid potatoes (M6, Solanum chacoenseUsing CRISPR / Cas9 technology and the vector pNK2-cas9-S-Ng, knockout lines were constructed by infecting explants (potato stem segments) with Agrobacterium GV3101. The constructed tetraploid potato overexpression lines and diploid potato knockout lines were then pre-planted in an artificial climate chamber under the following conditions: a 16-hour photoperiod (24℃) and an 8-hour dark period (18℃), with a light intensity of 18,000 Lux. Sanger sequencing or next-generation sequencing was used to identify the mutations, successfully obtaining two potato StBSB overexpression lines (OE2, OE7) and four potato StBSB knockout lines (KO32, KO64, KO158, KO205). Then, based on the annotation information in the public database PGSC, primers were designed to clone the full-length coding region sequence of the target gene from the tetraploid cultivated potato variety "Qingshu No.9", and upstream and downstream primers as shown in SEQ ID NO.4 and SEQ ID NO.5 were designed. Then, the expression vector 35S::CDS-GFP was constructed using homologous recombination, where CDS is the coding region of the target gene. The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells, cultured at 30℃ for 2 days, resuspended, and mixed with nuclear marker (Marker) bacterial solution at a 1:1 volume ratio. The mixture was then injected into leaves of Tobacco Benzovia. After culturing the transformed plants under low light conditions for 2 days, sections of the Tobacco leaves injected with Agrobacterium were prepared and fluorescence was observed using a Nikon C2-ER laser confocal microscope. Among them, the excitation light of green fluorescent protein GFP was 488 nm, and the emission light was 510 nm; the far-red fluorescent protein mKate, fused with the nuclear localization signal (MDPKKKRKV), was excited at 561 nm and emitted at 580 nm; the excitation light of chloroplasts was 640 nm, and the emission light was 675 nm. Finally, observation under a confocal microscope showed that StBSB co-localized with nuclear markers (such as... Figure 1 (as shown in d).

[0030] The tubers, stem peels, and shoot buds of tetraploid cultivated potatoes were rinsed with PBS and immediately placed in plant-specific in situ hybridization fixation medium. Fixation was performed at 4°C for at least 12 hours. After fixation, target blocks approximately 3 mm thick were cut from the tissues in a fume hood. These blocks were dehydrated with a gradient of alcohols and cleared with xylene, then embedded in paraffin. Paraffin sections were 6 μm thick, cut using a microtome, spread, and baked in a 62°C oven for 2 hours. Dewaxing was performed sequentially using dewaxing and clearing solutions followed by a gradient of alcohols, and finally soaked in DEPC water. An appropriate retrieval solution was selected based on the tissue type and experimental requirements for antigen retrieval. Subsequently, 20 μg / ml proteinase K was added, and digestion was performed at 37°C, followed by rinsing with PBS. Subsequently, pre-hybridization solution was added at 37°C and incubated for 1 hour, followed by hybridization solution containing the probe, and hybridization was performed overnight in an incubator. The BSB gene probe sequence is shown in SEQ ID NO. 6. After hybridization, the sections were washed sequentially with 2×SSC, 1×SSC, and 0.5×SSC. Formamide washing could be added depending on the non-specific hybridization results. The sections were then blocked with normal rabbit serum at room temperature for 30 minutes, followed by the addition of AP-labeled anti-DIG-AP antibody, incubated at 37°C for 40 minutes, and washed with TBS. The chromogenic step used BCIP / NBT chromogenic solution, and positive results were observed under a microscope. The sections were mounted with glycerol gelatin, and finally, microscopic examination and image acquisition and analysis were performed. Ultimately, it was observed that the outer layer of the potato tuber is the main site of SGA synthesis and cholesterol accumulation. RNA in situ hybridization analysis showed that StBSB is mainly expressed in the tuber buds and tuber skin rather than the tuber flesh (e.g., ...). Figure 1 (as shown in e), which is basically consistent with the expression profile shown by the transcriptome data.

[0031] Example 3: Function of StBSB in regulating potato SGA synthesis

[0032] Potatoes matured after 12 hours of continuous light exposure and StBSB overexpression lines OE2 and OE7, as well as knockout lines KO32, KO64, KO158, and KO205, were immediately frozen in liquid nitrogen and then freeze-dried under vacuum using a Scientz-100F freeze dryer. To assess the precision of the method and the stability of the instrument, all sample extracts were mixed in equal volumes to prepare quality control samples. Subsequently, all samples were processed by grinding, dissolving, vortexing, and centrifugation before UPLC-MS / MS analysis of α-carboxane and α-solanine. The instrument system used for data acquisition mainly included ultra-high performance liquid chromatography and tandem mass spectrometry.

[0033] Experiments revealed that the expression level of StBSB was significantly increased in the overexpression lines OE2 and OE17 (e.g., Figure 2As shown in a); liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis showed that, compared with the wild type, the levels of α-carboxine and α-solanine in the leaves of the overexpression lines increased by 15%–27% and 16%–26%, respectively (e.g., as shown in a). Figure 2 (as shown in b), while in the leaves of the knockout strains, the levels of both decreased by 9%–15% and 21%–33%, respectively (as shown in b). Figure 2 (as shown in c).

[0034] Example 4: Functional conservation of StBSB in SGAs biosynthesis

[0035] The upstream and downstream primers shown in SEQ ID NO. 7 and SEQ ID NO. 8 were designed to obtain the homologous tomato transcription factor BSB based on StBSB, named SlBSB. SlBSB is encoded by the gene Solyc05g010610.3 as shown in SEQ ID NO. 3, and its amino acid sequence identity with StBSB is 94.04%. The recombinant vector 35S:: was constructed. SlBSB After CDS-eGFP, diploid tomatoes (MicroTom, Solanum lycopersicum Based on the recombinant vector 35S:: SlBSB CDS-eGFP was used to construct an overexpression line by infecting explants (tomato cotyledons) with Agrobacterium GV3101; the vector pHSbdcas9i-tRNA-ccdb(K5) was constructed to express diploid tomato (MicroTom) cotyledons. Solanum lycopersicum Using CRISPR / Cas9 technology and the vector pHSbdcas9i-tRNA-ccdb(K5), knockout lines were constructed by infecting explants (tomato cotyledons) with Agrobacterium GV3101. The constructed tomato overexpression and knockout lines were then grown in an artificial climate chamber under the following conditions: a 16-hour photoperiod (24°C) and an 8-hour dark period (18°C), with a light intensity of 18,000 Lux. Sanger sequencing or next-generation sequencing was used to identify the mutations, successfully obtaining one tomato SlBSB overexpression line (OE14) and four tomato SlBSB knockout lines (KO1, KO5, KO7, and KO9).

[0036] Tomato SlBSB overexpression line OE14 and knockout lines KO1, KO5, KO7, and KO9, which matured after 12 hours of continuous light exposure, were immediately frozen in liquid nitrogen and then freeze-dried in vacuum using a Scientz-100F freeze dryer. To evaluate the precision of the method and the stability of the instrument, all sample extracts were mixed in equal volumes to prepare quality control samples. Subsequently, all samples were processed by grinding, dissolving, vortexing, and centrifugation. α-Tomatine in the tomato samples was quantitatively analyzed using an Agilent 6460 triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system, referring to standard NY / T 3951-2021 and publicly reported (Zhang, et al., 2019).

[0037] The experiment revealed that the expression level of SlBSB increased 39-fold in the overexpression lines (e.g., Figure 3 As shown in a); liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis showed that, compared with the wild type, the level of α-tomatine in the leaves of the overexpressing tomato lines increased by approximately 38% (e.g., as shown in a). Figure 3 (As shown in b), while in the leaves of the knockout lines, the levels of lycopene and lycopene glycoside, important intermediates in α-lycopene synthesis, decreased by 6%–24% and 48%–92%, respectively (e.g., ...). Figure 3 (as shown in c). These results indicate that BSB plays a conserved role in promoting the synthesis of major SGAs in potatoes and tomatoes.

[0038] Example 5: StBSB-regulated metabolic pathways

[0039] Transcriptome sequencing was performed on leaf tissues of potato knockout lines KO32, KO64, KO158, KO205, overexpression line OE2, and their corresponding wild-type plants. The results showed that in potatoes, StBSB knockout led to downregulation of 3164 genes and upregulation of 1774 genes (e.g., ...). Figure 4(as shown in a) StBSB overexpression upregulated 867 genes and downregulated 507 genes. By comparing the downregulated and upregulated genes in potato knockout and overexpression lines, a core gene set of 162 genes was identified, including 10 known SGA biosynthetic genes and the core regulatory factor GAME9. The core regulatory factor GAME9 is encoded by the potato gene Soltu.DM.01G031000, the sequence of which is shown in SEQ ID NO.2, where GAME15, SGT3, GAME6, and SGT1 constitute a metabolic gene cluster. Simultaneously, transcriptome analysis of leaves from tomato knockout lines KO5 and KO9 and their wild-type plants revealed that in tomato, StBSB knockout caused downregulation of 216 genes and upregulation of 926 genes (e.g., ...). Figure 4 (as shown in b).

[0040] In analyzing gene expression changes in the SGA biosynthesis pathway in potatoes and tomatoes (e.g., Figure 4 As shown in c), it was found that in potatoes, StBSB overexpression led to increased expression of genes SSR2, SMO1-L, SMO2-L, C5-SD2, and DWF5-L in the cycloartenol-to-cholesterol synthesis pathway, while the expression levels of these genes were generally decreased in potato and tomato knockout lines. Similarly, in the cholesterol-to-lycopene synthesis pathway, all GAME genes (GAME15 / 6 / 8 / 11 / 4 / 12) were present in potato and tomato knockout lines. In potato-specific pathways, the expression of genes involved in the SGA synthesis pathway (including SGT1 / 2 / 3, DPS, RPG1 / 2) was significantly reduced in BSB knockout lines but increased in overexpression lines. Similarly, in tomato-specific pathways, all genes involved in the SGA synthesis pathway (including S5αR2, GAME1 / 17 / 18) were downregulated in BSB knockout lines. These results indicate that BSB functional variations induce similar expression changes in genes involved in the SGA synthesis pathway in both potato and tomato.

[0041] Example 6: Mechanism of Action Between StBSB and GAME9

[0042] Transcriptome sequencing was performed on leaf tissues of potato knockout lines KO32, KO64, KO158, KO205, overexpression line OE2 and their corresponding wild-type plants, as well as on tomato knockout line KO9 and its wild-type plants. Transcriptome data showed that GAME9 expression was increased by 90% in potato StBSB overexpression lines, but decreased by 87%–99% in potato StBSB knockout lines; in tomato BSB knockout lines, GAME9 expression was decreased by 26% (e.g., ...). Figure 5 (as shown in a). Analysis of previous transcriptome data (Cardenas, et al., 2016) revealed no significant changes in the expression of StBSB or SlBSB in GAME9 overexpression or RNAi transgenic lines of potatoes and tomatoes. This suggests that StBSB may play a regulatory role upstream of GAME9.

[0043] Based on AlphaFold2-multimer-v2, the interaction between proteins StBSB and GAME9 was analyzed using the online service ColabFold (Mirdita, et al., 2022). The two strands were joined with a colon and submitted to the ColabFold online server for complex structure prediction; msa_mode was set to MMseqs2 (UniRef+Environmental), and pair_mode was set to unpaired+paired. Based on the optimal prediction model, the number of residues and solvent-accessible surface area at the complex interaction interface were analyzed using the online server PDBePISA, and the probability of protein-protein interaction was assessed by the complex structure significance score; the spatial structure of the complex was visualized using PyMOL software (e.g., ...). Figure 5 As shown in b), it can be seen that there is a certain possibility of direct interaction between StBSB and GAME9.

[0044] Expression vectors 35S::CDS-YCGFP (GFP C-terminus) and 35S::CDS-YNGFP (GFP N-terminus) were constructed using homologous recombination, where CDS represents the coding sequence of the target gene. The recombinant plasmids were transformed into Agrobacterium GV3101 competent cells, cultured at 30°C for 2 days, and then resuspended for infection of Nicotiana benthamiana (…). Nicotiana benthamianais Leaves; after culturing the transformed plants under low light conditions for 2 days, tobacco leaves injected with Agrobacterium were used to prepare sections, and fluorescence observation was performed using a Nikon C2-ER laser confocal microscope (e.g., leaves). Figure 5 (as shown in c).

[0045] The coding sequences (CDS) of each gene were cloned into the expression vector pBWA(V)Hs-TMVΩ and fused with 4×Myc or 3×Flag tags, respectively, to construct BSB-Myc and Flag-GAME9 fusion proteins; 1-month-old Nicotiana benthamiana (Nickellea Benzyl) was infected with Agrobacterium GV3101. Nicotiana benthamianaTransient expression of each fusion protein was performed on leaves, followed by extraction of total protein. The total protein was incubated overnight at 4°C with MYC trap beads. The beads were washed three times with PBS buffer, and the precipitated protein was eluted in 4×SDS buffer by heating in a 99°C metal bath for 5 minutes. The immunoprecipitated protein was separated on a 12% or 15% SDS-PAGE gel and detected by Western blotting using MYC antibody (Abclonal, AE070) or FLAG antibody (MBL, M185-3L). Figure 5 (as shown in d).

[0046] The coding sequences (CDS) of each gene were cloned into expression vectors pET28a and pGEX-6P, respectively, to construct recombinant proteins fused with 6×His or GST tags, named BSB-His and GST-GAME9, respectively. The fusion proteins were expressed in *E. coli* BL21 and purified in vitro using a His-Tag protein purification kit and a GST protein purification kit (Sangon Biotech, Shanghai, China). The pull-down assay was performed as follows: 50 μL of equilibrated glutathione agarose magnetic beads (Solarbio, China) were mixed with 500 μg of bait protein (GST tag) in PBS buffer. Subsequently, 500 μg of the bait protein bound to the magnetic beads was placed in PBS solution and incubated at 4°C for 16 hours. After washing three times with PBS buffer and denaturing the protein in a boiling water bath, anti-GST (mouse, YEASEN, China) and anti-His (mouse, Proteintech, China) antibodies were detected (e.g., [example missing]). Figure 5 (as shown in e).

[0047] Results from bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and fusion protein deposition (Pull Down) showed that StBSB and GAME9 have a direct interaction relationship both in vivo and in vitro.

[0048] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. The application of the potato transcription factor StBSB in reducing the accumulation of steroidal glycoside alkaloids in potatoes, characterized by, The transcription factor StBSB is encoded by the potato gene Soltu.DM.05G001620, the nucleotide sequence of which is shown in SEQ ID NO.

1. By knocking out the gene related to the transcription factor StBSB, the synthesis of steroidal glycoside alkaloids in potatoes is reduced.

2. The application according to claim 1, characterized in that, The reduction of steroidal glycoalkaloid synthesis in potatoes includes reducing the expression level of GAME9, a core regulatory factor related to steroidal glycoalkaloid synthesis. GAME9 is encoded by the potato gene Soltu.DM.01G031000, the sequence of which is shown in SEQ ID NO.

2.

3. The application of the tomato transcription factor SlBSB in reducing the accumulation of steroidal glycoside alkaloids in tomatoes, characterized by, By knocking out the gene associated with the transcription factor SlBSB, the synthesis of steroidal glycoside alkaloids in tomatoes was reduced. The nucleotide sequence of the transcription factor SlBSB is shown in SEQ ID NO.

3.

4. The application according to claim 3, characterized in that, The reduction of steroidal glycoalkaloid synthesis in other Solanaceae plants includes reducing the expression level of GAME9, a core regulator of steroidal glycoalkaloid synthesis.

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