New application of heat shock factor StHSFA6b gene in potato tuberization breeding

Abstract

The application provides a new application of a heat shock transcription factor StHSFA6b gene in potato tuber breeding, takes the StHSFA6b gene as an object, clones the ORF full length of the gene, and analyzes the sequence structure, evolutionary relationship, tissue expression and the like of the gene. The qRT-PCR result shows that the StHSFA6b gene expression has tissue specificity, and the expression is higher in the root and tuber maturation stages. Then, a silencing vector Gate8-StHSFA6b is constructed, and the agrobacterium is infected into potato plants, and the phenotype is observed after being cultured with wild type plants. The result shows that after the StHSFA6b gene is silenced, the tuber size and weight are significantly reduced, and the number of stolon is increased, so the StHSFA6b gene can be used as a new variety breeding candidate gene of high-yield potato, and provides a strong biological basis for the breeding of the potato tuber size.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a novel application of the heat shock transcription factor StHSFA6b gene in potato tuber breeding. Background Technology

[0002] Potato (Solanum tuberosum L.) is the world's fourth largest food crop, after rice, wheat, and corn. Potato tubers provide essential nutrients and carbohydrates, playing a vital role in ensuring my country's food security. In-depth research into candidate genes involved in regulating potato tuber development is of great significance for accelerating the breeding of high-yielding potato varieties.

[0003] Heat shock transcription factors (HSFs) are an important class of transcription factors in plant stress responses, playing a crucial role in both biotic and abiotic stresses (Yu et al., 2022). HSFs are classified into three classes based on their structure: HSFA, HSFB, and HSFC. All HSFs share a common core structure consisting of an N-terminal DNA-binding domain (DBD) and an adjacent oligomerization domain (OD). The hydrophobic core of the DBD forms a helical-turn-helical repeat (HTH). This HTH repeat sequence is responsible for the specific recognition of HSEs, thereby activating the expression of downstream genes (Sakurai and Enoki, 2020). The OD domain enables HSFs to form trimers, thus binding efficiently to DNA and activating the expression of related genes. Class A HSFs also contain a C-terminal transcription activation domain (CTAD). The short peptide motifs (AHA motifs) within the CTAD domain are rich in aromatic and macrohydrophobic amino acid residues embedded in an acidic environment, which are essential for the transcriptional activity of HSFs. Another well-defined domain of HSFs is the nuclear localization signal (NLS). In addition, some HSFs contain a leucine-rich nuclear export signal (NES).

[0004] Heat shock transcription factors were initially defined as transcriptional regulators of heat shock proteins, acting as molecular chaperones in protein folding and assembly, and protecting cells from proteotoxic damage under heat stress (Hu et al., 2009). Members of the A1 class of heat shock transcription factors in Arabidopsis play a major role in activating the transcription of heat-induced genes; when plants are subjected to heat shock, they can trigger a transcriptional cascade composed of many transcription factors. In rice, OsHSFA3 not only participates in the regulation of plant heat tolerance but also enhances drought tolerance by regulating polyamine biosynthesis dependent on ABA and reactive oxygen species levels (Zhu et al., 2020). In tomato and Arabidopsis, HSFA4 has been shown to be a potent activator of HSF gene expression, while HSFA5 inhibits HSFA4 activity (Baniwal et al., 2007). A study in Arabidopsis showed that HSFA6b is a positive regulator involved in ABA-mediated salt and drought resistance (Huang et al., 2016).

[0005] HSFs not only participate in plant responses to various abiotic stresses but also regulate multiple plant growth and development processes. In pollen, HSFA2 is an important coactivator of HSFA1a during HSR. Inhibition of HSFA2 reduces the viability and germination rate of pollen under stress during microsporogenesis and meiosis, but has no effect on the viability and germination rate of pollen under stress later. Under non-stress conditions, the developmental regulation of HSFA2a and several HS response genes mediated by HSFA1a partially alleviates this sensitivity. Therefore, HSFA2 is an important factor in initiating this process, which maintains pollen heat tolerance during microsporogenesis (Fragkostefanakis et al., 2016). Furthermore, heterologous overexpression of the pepper heat shock transcription factor gene CaHsfA1d in Arabidopsis significantly delayed senescence and significantly improved seed viability (Gai et al., 2023). Heterologous overexpression of two apple heat shock transcription factor genes, MdHsfA1d and MdHsfA9b, in Arabidopsis thaliana resulted in transgenic Arabidopsis exhibiting an earlier flowering phenotype (Zhang et al., 2022). Under heat stress, StHSFA5 and StHSFA8 were strongly upregulated in leaves and stolons (Dutta et al., 2022).

[0006] A total of 27 heat shock transcription factors (HSFs) have been identified in potatoes, including 16 from subfamily A, 8 from subfamily B, and 1 from subfamily C (Tang Ruimin, 2018). Currently, functional studies of HSFs in potatoes mainly focus on resistance to abiotic stress. For example, the StHSFA3 gene in potatoes plays a positive regulatory role in the response to heat stress in overexpressed transgenic lines and induces the expression of StHsp26-CP and StHsp70 (Tang Ruimin et al., 2021). However, the function of heat shock transcription factors in regulating potato tuber formation and development has not yet been reported. Summary of the Invention

[0007] This invention provides a gene that regulates the heat-shock transcription factor StHSFA6b in potato tuber formation.

[0008] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0009] This invention provides a heat shock transcription factor StHSFA6b gene that regulates tuber formation in potatoes. The sequence number of the gene in the potato database is: Soltu.DM.06G015000.1.

[0010] The present invention also provides a vector of the above-mentioned StHSFA6b gene.

[0011] This invention further provides a novel application of the heat shock transcription factor StHSFA6b gene in potato tuber breeding.

[0012] The beneficial effects of this invention are as follows:

[0013] A total of 27 heat shock transcription factors (HSFs) have been identified in potatoes, including 16 from subfamily A, 8 from subfamily B, and 1 from subfamily C. Currently, functional studies of HSFs in potatoes mainly focus on resistance to abiotic stress. This invention uses the StHSFA6b gene as the target, cloning its full-length ORF and analyzing its sequence structure, evolutionary relationships, and tissue expression. qRT-PCR results show that StHSFA6b gene expression is tissue-specific, with higher expression in the root and tuber maturation stages. The silencing vector Gate8-StHSFA6b was then constructed, and Agrobacterium-mediated transformation into this vector was used to infect potato plants. Phenotypic analysis was conducted after co-culturing with wild-type plants. Results showed that silencing the StHSFA6b gene significantly reduced tuber size and weight while increasing the number of runners. This gene can serve as a candidate gene for breeding high-yielding potato varieties, providing a strong biological basis for breeding in terms of tuber size. Attached Figure Description

[0014] Figure 1 Phylogenetic analysis of the StHSFA6b gene.

[0015] Figure 2 : StHSFA6b protein sequence alignment.

[0016] Figure 3 : StHSFA6b promoter sequence analysis.

[0017] Figure 4 Expression analysis of StHSFA6b in 'E3' tissues.

[0018] Figure 5 Subcellular localization of StHSFA6b.

[0019] Figure 6 StHSFA6b-RNAi transgenic potato tubers harvested on day 90.

[0020] Figure 7 The weight and number of potatoes per plant in the StHSFA6b-RNAi transgenic line. Specific Implementation

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0022] Example 1: Cloning and Bioinformatics Analysis of the StHSFA6b Gene

[0023] (1) Plant materials

[0024] Intact and uncontaminated 'Emashan No. 3' (E3) tissue culture seedlings were selected. After washing the culture medium from the roots, they were transplanted into nutrient soil and planted in a growth chamber. Five weeks later, tissue samples were collected from runners (S1), slightly enlarged runners (S2), tuber formation (S3), small tubers (S4), medium tubers (S5), large tubers (S6), roots, stems, leaves, petioles, and flowers for tissue expression pattern analysis. Samples from different stages of tuber development were collected in equal proportions and mixed as gene cloning template materials. All experiments were performed in triplicate. After sampling, the samples were immediately flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0025] (2) RNA extraction from various potato tissues

[0026] RNA was extracted from the above tissues using a plant tissue RNA extraction kit, and cDNA was obtained by reverse transcription using a Yisheng reverse transcription kit.

[0027] (3) Cloning and bioinformatics analysis of the StHSFA6b gene

[0028] The base and amino acid sequences of StHSFA6b (Soltu.DM.06G015000.1) were obtained from the potato database (http: / / spuddb.uga.edu / index.shtml). Primers StHSFA6b-F and StHSFA6b-R (Table 1) were designed based on the ORF sequence of the StHSFA6b gene using Premier 5.0 and synthesized by Qingke Biotechnology Co., Ltd. The StHSFA6b gene was amplified by PCR using cDNA from a mixed sample of tubers at different developmental stages in equal proportions as a template. The amplification conditions were: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 5 s, 72℃ extension for 6 s, 35 cycles (denaturation-extension); 72℃ extension for 5 min, and storage at 4℃. The amplified product was 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 Qingke Biotechnology Co., Ltd. for sequencing.

[0029] The corrected StHSFA6b base sequence was cloned, and the gene structure was constructed using the online software GSDS2.0. Amino acid sequences of Arabidopsis and heat shock transcription factor family genes were obtained from the Arabidopsis database TAIR (https: / / www.arabidopsis.org / ), and a phylogenetic tree was constructed using MEGA6.0. Multiple sequence alignment analysis was performed using DNAMAN software. The ~1.5kb promoter sequence upstream of the transcription start site of the StHSFA6b gene, extracted from the potato database, was submitted to the PlantCARE website for predictive analysis. Regulatory elements were screened, summarized, and visualized.

[0030] Example 2: Expression Pattern Analysis of StHSFA6b

[0031] (1) Analysis of tissue expression characteristics of StHSFA6b gene

[0032] Based on the StHSFA6b gene sequence obtained by cloning and sequencing in Example 1, qRT-PCR primers StHSFA6b-QF and StHSFA6b-QR (Table 1) were designed, and the specificity of the primers was ensured using the BLASTn test in NCBI. The potato StEF1a gene was used as an internal reference gene, and the specific primer sequences are shown in Table 1.

[0033] The potato tissues from Example 1 were amplified using qRT-PCR. The instrument used for the qRT-PCR reaction was a Bio-Rad CFX96 real-time quantitative PCR instrument, and the PCR enzyme was Takara's SYBR Green Master Mix. The reaction volume was 10 μL, containing 12.5 ng of template cDNA, 250 nM each of forward and reverse primers, 5 μL of SYBR Green Master Mix, and the remainder was made up 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 levels of the StHSFA6b gene in each tissue were calculated. All samples were tested in triplicate, with negative controls included. The mean values ​​were statistically analyzed using Excel software, and the significance of the differences in the target gene in different tissues and materials was analyzed using SPSS software (P<0.05). Graphs were plotted using Prism 12.5 software. (2) Subcellular localization of the StHsfA6b gene

[0034] Using reverse-transcribed cDNA from mature potato 'E3' tubers as a template, the CDS fragment of the StHSFA6b gene was amplified using the StHSFA6b-1300-F / R primers listed in Table 1. The pCAMBIA1300 vector was digested with XbaI and BamHI, and the amplified fragment was then homologously recombinated with the digested vector. After successful transformation and sequencing, plasmids from the correct bacterial culture were extracted to obtain the 35S::StHsfA6b-eGFP vector. This vector was then used to infect tobacco leaves using Agrobacterium-mediated transient transformation, and laser confocal microscopy was employed to determine the subcellular localization of StHsfA6b.

[0035] Example 3: Construction of silencing vector and functional verification of transgenic potatoes

[0036] Using reverse-transcribed cDNA from mature potato 'E3' plants as a template, the StHSFA6b silencing fragment was amplified using the StHSFA6b-Ri-1F / 1R primers listed in Table 1. The pHELLSGATE-8 vector was digested with XhoI, and the amplified fragment was then homologously recombinated with the digested vector. After successful transformation and sequencing, the plasmid from the correct bacterial culture was extracted, and the correctly sequenced plasmid was digested with Xbal 1. A second vector was then constructed. Potato tuber cDNA was amplified using the StHSFA6b-Ri-2F / 2R primers listed in Table 1, amplifying another silencing fragment of the StHSFA6b gene. This fragment was then recombinated with the pHELLSGATE-8 vector containing the first silencing fragment. After successful transformation and sequencing, the plasmid from the correct bacterial culture was extracted, yielding the silencing vector Gate8-StHSFA6b containing both StHSFA6b silencing fragments. Gate8-StHSFA6b was transformed into Agrobacterium strain GV3101, and then transferred into potatoes via Agrobacterium infection to obtain regenerated seedlings. Positive transgenic potatoes were detected using pHELLSGATE-8 vector-specific primers. The obtained StHSFA6b-RNAi transgenic potato lines and wild-type potatoes were planted in a plant growth chamber to observe the tuber phenotypes of different lines.

[0037] Table 1 Primer information used

[0038]

[0039] Example 4, Results and Analysis

[0040] (1) Bioinformatics analysis of the StHSFA6b gene

[0041] The phylogenetic analysis of potato HSFs family proteins showed that the StHSFA6b protein belongs to the HSFA subfamily. Homology searches of the amino acid sequences of Arabidopsis HSFs were performed using BLASTp, and a phylogenetic tree was constructed using MEGA 6.0 software. The results indicated that potato StHSFA6b is most closely related to wild-type Arabidopsis AtHSFA6b in terms of evolutionary hierarchy. Figure 1 Homologous amino acid sequence analysis of HSFA6b revealed that StHSFA6b possesses a highly conserved DBD domain, oligomeric OD domain, nuclear localization signal NLS, and AHA motif. Figure 2To further investigate the physiological functions of the StHSFA6b gene, cis-regulatory element analysis was performed on the ~1.5kb sequence upstream of the transcription start site of the StHSFA6b gene obtained from NCBI using the online website Plant-CARE. The results showed that, in addition to basic promoter elements such as the TATA-box and CAAT-box, the StHSFA6b promoter contains various light-related elements, ABA-responsive elements (ABRE motif), and jasmonic acid-responsive elements (TGACG motif). Figure 3 ).

[0042] (2) Tissue expression characteristics analysis of the StHSFA6b gene

[0043] qRT-PCR results showed that the expression of the StHSFA6b gene was tissue-specific, with higher expression in the root and tuber maturation stages. Figure 3 This result suggests that the StHSFA6b gene may be specifically involved in potato tuber development.

[0044] (3) Subcellular localization of the StHSFA6b gene

[0045] To confirm and clarify the expression location of the StHSFA6b gene-encoded protein in plant cells, an expression vector was constructed by fusing the StHSFA6b gene with GFP (green fluorescent protein) using homologous recombination. The figure shows that the localization signal in tobacco leaf epidermal cells, using the p1300-GFP empty vector as a positive control, was nonspecific, while the StHSFA6b-GFP fusion protein exhibited green fluorescence in the cell nucleus, indicating that StHSFA6b is a nuclear localized protein.

[0046] (4) Analysis of tuber formation phenotype of transgenic lines

[0047] Wild-type and interventional lines were planted in a medium-day growth chamber and harvested after three months for phenotypic analysis. No significant differences in tuber morphology were observed between the transgenic and wild-type lines. Figure 6 Statistical analysis of the number and weight of tubers per plant revealed that the average tuber weight per plant was significantly lower in the three interference lines than in the wild type, while the number of tubers per plant did not show a significant difference. Furthermore, statistical analysis of primary and secondary runners showed that Ri-2 and Ri-18 had significantly higher numbers of primary and secondary runners than the wild type, and Ri-25 also had a slightly higher number, but the differences were not statistically significant. Figure 7 The results indicate that silencing StHSFA6b significantly reduces tuber size and weight while increasing the number of runners, making it a potential candidate gene for breeding high-yielding potato varieties.

Claims

1. A heat shock transcription factor StHSFA6b gene in potato tuber breeding, said StHSFA6b gene having the sequence number Soltu.DM.06G015000.1 in the potato database, said application specifically being to reduce tuber size and weight while increasing the number of stolons after silencing StHSFA6b the gene.

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