Transcription factor StBSB with regulation effect on steroid glycoalkaloid accumulation of potatoes and application of transcription factor StBSB

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a transcription factor StBSB with a regulation effect on steroid glycoalkaloid accumulation of potatoes and application of the transcription factor StBSB, the transcription factor StBSB is encoded by a potato gene Soltu.DM.05G001620, and the nucleotide sequence of the potato gene Soltu.DM.05G001620 is shown as SEQ ID NO.1. The invention further discloses a preparation method of the transcription factor StBSB. According to the application, a new insight is provided for understanding a transcriptional regulation mechanism of SGAs in solanaceae plants, and a key theoretical basis and genetic resources are provided for directionally improving the content of anti-nutritional compounds through genetic means.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, and particularly relates to a transcription factor StBSB having a regulatory effect on the accumulation of steroidal glycoalkaloids in potatoes and an application thereof. BACKGROUND

[0002] Steroidal glycoalkaloids (SGAs) are secondary metabolites naturally produced in Solanaceae plants, are a class of toxic natural anti-nutritional substances, and are widely distributed in Solanaceae plants such as potatoes, tomatoes, eggplants, etc. They have a defense function in plants and can effectively resist pests and environmental stress, but their toxicity to humans and animals cannot be ignored. In potatoes, α-solanine and α-chaconine account for more than 90% of the total SGA content; and in tomatoes, α-tomatine and tomatidenol are the main SGAs in green tissues. These compounds can cause neurotoxicity if taken in excess by humans or animals, and can easily cause symptoms such as indigestion and vomiting, and in severe cases, can even cause life-threatening.

[0003] At present, transcription factors regulating the synthesis of potato SGAs have been identified, including 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 regulatory factor in SGA biosynthesis. GAME9 activates the transcription of downstream genes such as DWF5, C5-SD, GAME4 and SSR2 by binding to GCC-box, G-box and other elements; overexpression or silencing of GAME9 can significantly affect the level of SGAs in tomatoes and potatoes. However, further research is needed on other transcription factors involved in the regulation of SGA synthesis and their interactions with existing functional networks.

[0004] Therefore, it is necessary to continue to explore the factors involved in the regulation of SGA synthesis in potatoes and to explore the specific mechanism of StBSB in regulating the synthesis of SGAs in potatoes. SUMMARY

[0005] The problem to be solved by the present application is to provide a transcription factor StBSB having a regulatory effect on the accumulation of steroidal glycoalkaloids in potatoes and an application thereof, to provide new insights into the transcriptional regulation mechanism of SGAs in Solanaceae plants, and to provide a key theoretical basis and genetic resources for directional improvement of the content of such anti-nutritional compounds through genetic means.

[0006] In order to solve the above problems, the present application provides the following solutions: In one aspect, the application provides a transcription factor StBSB of potato, which is encoded by potato gene Soltu.DM.05G001620, the nucleotide sequence of which is shown as SEQ ID NO. 1.

[0007] In another aspect, the application provides an application of the above-mentioned transcription factor StBSB of potato in reducing the accumulation of steroidal glycoalkaloids in potato, which reduces the synthesis of steroidal glycoalkaloids in potato by inhibiting the expression of the related gene of the transcription factor StBSB.

[0008] Further, the inhibition of the expression of the related gene of StBSB includes knocking out the related gene of StBSB.

[0009] Further, the reduction of the synthesis of steroidal glycoalkaloids in potato includes reducing the expression level of the core regulatory factor GAME9 related to the synthesis of steroidal glycoalkaloids, which is encoded by potato gene Soltu.DM.01G031000, the sequence of which is shown as SEQ ID NO. 2.

[0010] In another aspect, the application provides an application of the above-mentioned transcription factor StBSB of potato in reducing the accumulation of steroidal glycoalkaloids in other solanaceous plants, which includes constructing a homologous transcription factor BSB of other solanaceous plants based on the StBSB, and then reducing the synthesis of steroidal glycoalkaloids in other solanaceous plants by inhibiting the expression of the related gene of the homologous transcription factor BSB of other solanaceous plants; In the application, the other solanaceous plants include tomatoes.

[0011] Further, the inhibition of the expression of the related gene of BSB includes knocking out the related gene of BSB.

[0012] Further, the nucleotide sequence of the homologous transcription factor BSB is shown as SEQ ID NO. 3.

[0013] Further, the reduction of the synthesis of steroidal glycoalkaloids in other solanaceous plants includes reducing the expression level of the core regulatory factor GAME9 related to the synthesis of steroidal glycoalkaloids.

[0014] The application has the following beneficial effects: The application successfully screens a transcription factor StBSB which is induced by light and can directly regulate the synthesis of SGAs in potatoes, and proves the molecular mechanism of StBSB regulating SGAs. StBSB can form a heterodimer with GAME9, and cooperatively activate the transcription of a plurality of SGAs synthetase to promote the biosynthesis of SGAs. Based on the function and structural conservation of StBSB in the biosynthesis of SGAs, a transcription factor BSB which can regulate other solanaceous plants is successfully constructed, and in the future, it is expected to combine molecular marker assisted selection technology, quickly screen and aggregate excellent allelic variations of the gene, and cultivate new germplasm which is not easy to accumulate steroidal glycoside alkaloids, so as to create new varieties of solanaceous plants which are safer for eating without introducing exogenous genes, and can provide support for the quality improvement of solanaceous crops such as potatoes and tomatoes. Toxic SGAs are substances that can improve the resistance of plants to diseases and pests, and based on the transcription factor StBSB, the rational design of potato SGAs is carried out, and the SGAs metabolism is optimized to improve the edibility, safety and resistance to diseases and pests, which is a key direction of potato breeding. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 4 is the expression characteristics analysis of StBSB (a. Gene co-expression network of SGAs metabolism related modules; b. Tissue expression profile analysis of BSB and other SGAs biosynthesis genes; c. Manhattan plot of GWAS analysis based on the total SGAs content of tubers of 132 genotypes, the red arrow indicates the variation site in the chromosome region of StBSB; d. Colocalization results of StBSB and nuclear marker protein in tobacco leaves, green fluorescence shows the GFP fusion protein, red fluorescence is the fusion marker of nuclear localization signal and mKate, Bright is the bright field image, and Merged is the merged image; e. RNA in situ hybridization detection of BSB in sprouts and tubers, the upper part is the tuber section, and the lower part is the sprout section, the arrow indicates the high expression region of the gene); Figure 2 Figure 5 is the influence of BSB expression change on the content of main SGAs in potatoes (a. Expression level of StBSB in leaves of potato overexpression lines; b. Content of α-solanine and α-chaconine in potato overexpression lines; c. Content of α-solanine and α-chaconine in potato knockout lines, P<0.05, P<0.01, P<0.001, ****P<0.0001, and ns represents P>0.05); Figure 3Effects of BSB expression changes on the contents of major SGAs in tomato (a. Expression levels of SlBSB in tomato overexpression lines; b. Contents of a-tomato base in tomato overexpression lines; c. Contents of tomatidine and tomatinol glycosides in tomato knockout lines, P<0.05, P<0.01, P<0.001, ****P<0.0001, ns represents P>0.05). Figure 4 Synchronous expression changes of genes in SGA synthesis pathway caused by BSB functional variation in potato and tomato (a. Overlapping of differentially expressed genes in four potato BSB knockout lines; b. Overlapping of differentially expressed genes in two tomato BSB knockout lines; c. Expression changes of genes involved in SGA biosynthesis in potato knockout lines, overexpression lines and tomato knockout lines; numerical values represent the percentage of gene expression in transgenic lines relative to wild type control, purple represents up-regulation, green represents down-regulation, * represents that the expression of the gene is not detected). Figure 5 Interaction analysis of StBSB and GAME9 (a. Expression changes of GAME9 in potato lines overexpressing and knocking out BSB; b. Three-dimensional structure of BSB-GAME9 heterodimer predicted based on AlphaFold2-multimer-v2, blue represents GAME9 amino acid residues, green represents BSB amino acid residues; c. Bimolecular fluorescence complementation experiment of StBSB and GAME9 heterodimer in tobacco leaves, YFP yellow fluorescence shows the interaction signal of fusion proteins, red is chloroplast autofluorescence, Bright is bright field, Merged is the superimposed image; d. Co-IP experiment using tobacco leaves to verify the interaction of BSB and GAME9; e. Pull-down experiment to further verify the interaction of BSB and GAME9). DETAILED DESCRIPTION

[0016] The technical solutions in some embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments provided in the present application, all other embodiments obtained by those of ordinary skill in the art fall within the scope of protection of the present application.

[0017] Unless otherwise required by the context, throughout the specification and claims, the term "comprising" is interpreted to mean "including, but not limited to".

[0018] In describing some embodiments, it will be understood that the terms "at least one" and "one or more of A, B, and C" are used interchangeably, and that each is to be taken to include one of A, B, or C alone, a combination of A, B, and 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.

[0019] Example 1 Gene screening Using the published transcriptome data of potato under multiple tissues and conditions in PGSC database, which covers transcriptome sequencing data under different tissues and developmental stages, as well as under multiple hormone treatment conditions, a total of 98 RNA-seq libraries, and then genes with TPM mean <1 in each tissue were excluded to improve the specificity of co-expression network; the remaining genes were used to construct a weighted gene co-expression network using R package WGCNA; then under the default parameters, the candidate genes were predicted and identified transcription factors using online database PlantTFDB; the GO terms of the whole genome of potato annotated by InterProScan were downloaded from PGSC as the background set for enrichment analysis; the module genes related to SGA synthesis were extracted, and GO enrichment analysis was performed using online platform OmicShareTools, with threshold set as p<0.01, q<0.05.

[0020] An Ivory module related to solanidane synthesis was identified through weighted gene co-expression network analysis, and the genes in this module were significantly enriched in 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) and other GO entries related to plant sterol metabolism. The Ivory module contains 16 identified genes involved in solanidane biosynthesis (as shown in a of Figure 1 , including the recently identified reductase encoding gene RPG1 / 2 and cholesterol glucuronosyltransferase encoding gene GAME15 involved in potato SGA synthesis. These genes have high expression levels in tissues such as stems, immature fruits, and carpels (as shown in Figure 1of the 1209 genes within the module, there are 69 transcription factors, including the 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 whose nucleotide sequence is shown as SEQ ID NO. 1, which is named StBSB in this study. The 329~379 amino acids of StBSB protein constitute the bHLH conserved domain. Phylogenetic analysis shows that StBSB has one homologous gene in each of tomato, eggplant and pepper, and two homologous genes in tobacco. StBSB is located near a reported variation site associated with total SGA content in potato tubers. Through GWAS analysis of the total SGA content of 132 potato lines, the LOD value of this site is 4.6, the phenotypic variation explanation rate is 12.4%, and the additive effect is 0.29 (as shown in Fig. 1c). Figure 1

[0021] ​After the recombinant vector 35S:StBSB CDS-eGFP is constructed, the tetraploid cultivated potato (Qingshu No. 9, Solanum tuberosum) is based on the recombinant vector 35S:StBSB CDS-eGFP, and the overexpression strain is constructed by Agrobacterium GV3101 infection of explants (potato stem segments); the vector pNK2-cas9-S-Ng is constructed, and the diploid potato (M6, Solanum chacoense) is constructed by CRISPR / Cas9 technology, using the vector pNK2-cas9-S-Ng, and the knockout strain is constructed by Agrobacterium GV3101 infection of explants (potato stem segments). Then the constructed tetraploid cultivated potato overexpression strain and diploid potato knockout strain are planted in advance in an artificial climate chamber, and the growth conditions are: a light period of 16 hours of light (24°C) and 8 hours of darkness (18°C), a light intensity of 18,000 Lux, and Sanger or second-generation sequencing is used to identify variations, and two potato StBSB overexpression strains (OE2, OE7) and four potato StBSB knockout strains (KO32, KO64, KO158, KO205) are successfully obtained. Then according to the annotation information of the public database PGSC, primers are designed to clone the full-length coding region sequence of the target gene from the tetraploid cultivated potato "Qingshu No. 9", and the upstream and downstream primers shown in SEQ ID NO. 4 and SEQ ID NO. 5 are designed; then the expression vector 35S:CDS-GFP is constructed by homologous recombination, wherein CDS is the coding region of the target gene; the recombinant plasmid is transformed into Agrobacterium GV3101 competent cells, which are cultured at 30°C for 2 days, resuspended, mixed with Marker bacterial liquid at a volume ratio of 1:1, and injected into Nicotiana benthamiana leaves. After the transformed plants are cultured in weak light for 2 days, tobacco leaves injected with Agrobacterium are prepared into sections, and fluorescence observation is performed using a laser confocal microscope Nikon C2-ER. Among them, the excitation light of the green fluorescent protein GFP is 488 nm, and the emission light is 510 nm; the far-red fluorescent protein mKate fused with the nuclear localization signal (MDPKKKRKV) has an excitation light of 561 nm and an emission light of 580 nm; and the excitation light of the chloroplast is 640 nm, and the emission light is 675 nm. Finally, from the observation of the confocal microscope, it can be seen that StBSB is co-localized with the nuclear marker (as shown in d of FIG. 8). Figure 1

[0022] ​The tubers, stems and stem bud tissues of tetraploid cultivated potato were rinsed with PBS and immediately placed in a plant-specific in situ hybridization fixing solution at 4°C for not less than 12 hours. After fixation, the target blocks of about 3 mm in thickness were cut in a fume hood, and then dehydrated with gradient alcohol and transparentized with xylene. The paraffin-embedded sections were prepared with a sectioning machine, and then baked in an oven at 62°C for 2 hours. The sections were deparaffinized with deparaffinization transparent liquid and gradient alcohol, and finally soaked with DEPC water. According to the type of the tissue and the experimental requirements, an appropriate repair solution was selected for antigen repair. Then, 20 μg / ml proteinase K was added for digestion at 37°C, and then the sections were washed with PBS. Then, pre-hybridization solution was added dropwise and incubated at 37°C for 1 hour, and then hybridization solution containing the probe was added for overnight hybridization in a constant temperature box. The sequence of the BSB gene probe is shown in SEQ ID NO. 6. After hybridization, the sections were washed with 2×SSC, 1×SSC and 0.5×SSC, respectively. According to the non-specific hybridization, formamide washing can be added. Then, the sections were blocked with normal rabbit serum at room temperature for 30 minutes, and then AP-labeled anti-DIG-AP antibody was added and incubated at 37°C for 40 minutes, and then washed with TBS. The color developing step used BCIP / NBT color developing liquid, and the positive results were observed under a microscope. Glycerin gelatin was used for mounting, and finally, microscopic examination and image acquisition analysis were performed. Finally, it can be seen that the outer layer of potato tuber is the main site of SGA synthesis and cholesterol accumulation. RNA in situ hybridization analysis shows that StBSB is mainly expressed in the tuber bud and tuber skin, but not in the tuber flesh (as shown in e of Figure 1 , which is basically consistent with the expression profile shown by the transcriptome data.

[0023] Example 3 Function of StBSB in regulating the synthesis of potato SGAs After the mature potato StBSB overexpression lines OE2 and OE17 and the knockout lines KO32, KO64, KO158 and KO205 were subjected to continuous light for 12 hours, they were immediately frozen in liquid nitrogen and vacuum freeze-dried using a freeze dryer (Scientz-100F). In order to evaluate the precision of the method and the stability of the instrument, all sample extracts were mixed in equal amounts to prepare a quality control sample. Then, after grinding, dissolving, vortexing and centrifugation, UPLC-MS / MS analysis of a- solanine and a-solanine was performed. The instrument system used for data acquisition mainly includes ultra-high performance liquid chromatography and tandem mass spectrometry.

[0024] It was found after the experiment that the expression level of StBSB in the overexpression lines OE2 and OE17 was significantly increased (as shown in Figure 2(As shown in a); 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 knockout lines, the levels of both decreased by 9%–15% and 21%–33% respectively (as shown in b). Figure 2 (as shown in c in the diagram).

[0025] Example 4: Functional conservation of StBSB in SGAs biosynthesis Design upstream and downstream primers as shown in SEQ ID NO.7 and SEQ ID NO.8, 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 has an amino acid sequence identity of 94.04% with StBSB. After constructing the recombinant vector 35S::SlBSBCDS-eGFP, diploid tomato (MicroTom, Solanum lycopersicum) was used to construct overexpression lines by infecting explants (tomato cotyledons) with Agrobacterium GV3101 based on the recombinant vector 35S::SlBSBCDS-eGFP. The vector pHSbdcas9i-tRNA-ccdb(K5) was constructed, and diploid tomato (MicroTom, Solanum lycopersicum) was used to construct knockout lines by infecting explants (tomato cotyledons) with Agrobacterium GV3101 using CRISPR / Cas9 technology with the vector pHSbdcas9i-tRNA-ccdb(K5). The constructed tomato overexpression and knockout lines were then planted in an artificial climate chamber under the following growing conditions: 16 hours of light (24°C) and 8 hours of darkness (18°C), with a light intensity of 18,000 Lux. The mutations were identified using Sanger or next-generation sequencing, and one tomato SlBSB overexpression line (OE14) and four tomato SlBSB knockout lines (KO1, KO5, KO7, and KO9) were successfully obtained.

[0026] After the continuous light for 12 hours, the mature tomato SlBSB overexpression line OE14 and knockout lines KO1, KO5, KO7, KO9 were immediately frozen in liquid nitrogen and vacuum freeze-dried using a freeze dryer (Scientz-100F). In order to evaluate the precision of the method and the stability of the instrument, all sample extracts were mixed in equal amounts to prepare a quality control sample. Subsequently, after grinding, dissolving, vortexing and centrifugation, the alpha-tomatine in the tomato samples was quantitatively analyzed by Agilent 6460 triple quadrupole liquid chromatography-mass spectrometry (LC-MS) according to the standard NY / T 3951-2021 and the published report (Zhang, et al., 2019).

[0027] It was found after the experiment that the expression level of SlBSB in the overexpression line was increased by 39 times (as shown in a of Figure 3 The liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis showed that compared with the wild type, the level of alpha-tomatine in the leaves of the overexpression tomato line was increased by about 38% (as shown in b of Figure 3 The level of the important intermediate product of alpha-tomatine synthesis, tomatenol and tomatenol glycoside, in the leaves of the knockout lines was reduced by 6%-24% and 48%-92% (as shown in c of Figure 3 These results showed that BSB had a conservative role in promoting the synthesis of the main SGAs of potato and tomato.

[0028] Example 5 StBSB-regulated metabolic pathways Transcriptome sequencing was performed on the leaf tissues of potato knockout lines KO32, KO64, KO158, KO205, overexpression line OE2 and their corresponding wild types. The results showed that in potato, StBSB knockout caused 3164 genes to be down-regulated and 1774 genes to be up-regulated (as shown in a of Figure 4 StBSB overexpression caused 867 genes to be up-regulated and 507 genes to be down-regulated. By comparing the down-regulated and up-regulated genes in the knockout and overexpression lines of potato, a core gene set consisting of 162 genes was identified, including 10 known SGA biosynthesis genes and the core regulatory factor GAME9, which was encoded by the potato gene Soltu.DM.01G031000, the sequence of which was SEQ ID NO. 2. The core regulatory factor GAME9, GAME15, SGT3, GAME6, SGT1 constitute a metabolic gene cluster. At the same time, transcriptome analysis was performed on the leaf tissues of tomato knockout lines KO5 and KO9 and their wild types. The results showed that in tomato, SlBSB knockout caused 216 genes to be down-regulated and 926 genes to be up-regulated (as shown inFigure 4

[0029] After analyzing the expression changes of genes in the SGA biosynthesis pathway in potato and tomato (as shown in c of Figure 4 In potato, StBSB overexpression led to the up-regulation of genes SSR2, SMO1-L, SMO2-L, C5-SD2, DWF5-L in the pathway from cycloartenol to cholesterol, while in the knockout lines of potato and tomato, the expression levels of these genes were generally decreased; similarly, in the pathway from cholesterol to tomatidenol, all the GAME genes (GAME15 / 6 / 8 / 11 / 4 / 12) were down-regulated in the knockout lines of potato and tomato, while up-regulated in the overexpression lines; in the potato-specific pathway from tomatidenol to α-solanine and α-chaconine, the genes including SGT1 / 2 / 3, DPS, RPG1 / 2 were significantly down-regulated in the BSB knockout lines, while up-regulated in the overexpression lines; in the tomato-specific pathway from tomatidenol to α-tomatine, the genes including S5αR2, GAME1 / 17 / 18 were also down-regulated in the BSB knockout lines. The above results showed that the functional variation of BSB caused similar expression changes of SGA synthesis pathway genes in potato and tomato.

[0030] Example 6 Mechanism of the interaction between StBSB and GAME9 Transcriptome sequencing was performed on the leaf tissues of potato knockout lines KO32, KO64, KO158, KO205, overexpression line OE2 and their corresponding wild type, and tomato knockout line KO9 and its wild type. The transcriptome data showed that in the potato StBSB overexpression lines, the expression level of GAME9 increased by 90%, while in the potato StBSB knockout lines, it decreased by 87%~99%; in the tomato BSB knockout lines, the expression level of GAME9 decreased by 26% (as shown in a of Figure 5 Cardenas, et al., 2016). It can be inferred that StBSB may play a regulatory role upstream of GAME9.

[0031] ​The protein StBSB and GAME9 interaction was analyzed based on AlphaFold2-multimer-v2 through the online service ColabFold (Mirdita, et al., 2022). The two chains were connected with a colon and then submitted to the ColabFold online server for complex structure prediction; the msa_mode was set to MMseqs2 (UniRef+Environmental) and the pair_mode was set to unpaired+paired. Based on the best prediction model, the number of residues and the solvent accessible surface area of the complex interaction interface were analyzed using the online server PDBePISA, and the possibility of protein interaction was evaluated through the complex structure significance score; the PyMOL software was used for complex spatial structure visualization (as shown in Fig. 1b). Figure 5 As shown in Fig. 1b, it can be seen that there is a certain possibility of direct interaction between StBSB and GAME9.

[0032] The expression vectors 35S::CDS-YC GFP (GFP C-terminal) and 35S::CDS-YN GFP (GFP N-terminal) were constructed by homologous recombination, wherein CDS is the coding sequence of the target gene; the recombinant plasmid was transformed into Agrobacterium GV3101 competent cells, which were resuspended after being cultured at 30°C for 2 days, and used to infect Nicotiana benthamiana leaves; after the transformed plants were cultured under weak light for 2 days, the tobacco leaves injected with Agrobacterium were sectioned, and fluorescence observation was performed using a laser confocal microscope Nikon C2-ER (as shown in Fig. 2c). Figure 5

[0033] 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 tag, respectively, to construct BSB-Myc and Flag-GAME9 fusion proteins; each fusion protein was transiently expressed by Agrobacterium GV3101 to infect 1-month-old Nicotiana benthamiana leaves, and then total protein was extracted; the total protein was incubated with MYC trap beads at 4°C overnight; the beads were washed 3 times with PBS buffer, and the precipitated protein was eluted in 4×SDS buffer at 99°C metal bath for 5 minutes. The immunoprecipitated protein was separated on a 12% or 15% SDS-PAGE gel, and immunoblotting was performed using MYC antibody (Abclonal, AE070) or FLAG antibody (MBL, M185-3L) (as shown in Fig. 4d). Figure 5

[0034] ​​The coding sequence (CDS) of each gene was cloned into expression vectors pET28a and pGEX-6P, respectively, to construct recombinant proteins fused with 6xHis or GST tags, named BSB-His and GST-GAME9, respectively. Each fusion protein was expressed in E. coli BL21 and purified in vitro using His-Tag Protein Purification Kit and GST Protein Purification Kit (Sangon Biotech, Shanghai, China). The pull down method was 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 binding protein (His tag) was mixed with the bait protein bound to the magnetic beads in PBS solution at 4°C for 16 hours. After washing with PBS buffer for 3 times, the denatured protein was subjected to anti-GST (mouse, YEASEN, China) and anti-His (mouse, Proteintech, China) antibody detection (as shown in e of FIG. 1). Figure 5

[0035] From the results of BiFC, Co-IP and Pull Down, it can be seen that StBSB and GAME9 have a direct interaction relationship both in vivo and in vitro. The above description is only a preferred embodiment of the present application, and it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein by the above teaching or related art or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the appended claims of the present application.​

Claims

1. A transcription factor StBSB of potato, characterized in that, The transcription factor StBSB is encoded by the potato gene Soltu.DM.05G001620, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the synthesis of steroidal glycoalkaloids in potatoes is reduced by inhibiting the expression of the relevant gene of the transcription factor StBSB.

2. Use according to claim 1, characterized in that, The inhibition of the expression of the relevant gene of StBSB includes knocking out the relevant gene of StBSB.

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

2.

4. Use of the potato transcription factor StBSB according to claim 1 for reducing the accumulation of steroidal glycoalkaloids in other solanaceous plants, characterized in that, The application includes constructing homologous transcription factor BSB of other solanaceous plants based on the StBSB, and then inhibiting the expression of the relevant gene of the transcription factor BSB of other solanaceous plants to reduce the synthesis of steroidal glycoalkaloids in other solanaceous plants. The other solanaceous plants include tomatoes.

5. Use according to claim 4, characterized in that, The inhibition of the expression of the relevant gene of BSB includes knocking out the relevant gene of BSB.

6. Use according to claim 4, characterized in that, The nucleotide sequence of the transcription factor BSB is shown as SEQ ID NO.

3.

7. Use according to claim 4, characterized in that, The reduction of the synthesis of steroidal glycoalkaloids in other solanaceous plants includes reducing the expression level of the core regulatory factor GAME9 related to the synthesis of steroidal glycoalkaloids.

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