Application of potato StHSF1 gene in alkaloid biosynthesis regulation

Abstract

The invention discloses application of a potato StHSF1 gene in alkaloid biosynthesis regulation and control, and belongs to the technical field of biology. According to the invention, potatoes with different pulp colors are studied, 48 alkaloids are identified and the accumulation dynamics of the alkaloids are determined, and the colored pulp potato clone is found to have richer alkaloid types and higher alkaloid content than a yellow pulp clone. A key structural gene is screened by systematically analyzing synthesis pathways of various alkaloids, transcription factors possibly participating in regulation are mined based on a co-expression network, and the StHSF1 gene is identified as a potential core hub regulation factor. Functional verification experiments show that transient overexpression of StHSF1 can significantly increase the total alkaloid content and specifically up-regulate the expression of 15 alkaloids. The invention provides a key genetic resource and a theoretical basis for cultivating a new potato variety with optimized alkaloid components, and is beneficial for increasing the nutritional value or reducing toxic alkaloid to guarantee the edible safety and the like by enriching functional alkaloid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving an integrated analysis of the accumulation characteristics and metabolic pathways of potato alkaloids. StHSF1 Application of genes in the regulation of alkaloid biosynthesis. Background Technology

[0002] Potatoes, a globally important food crop, are rich in starch, protein, vitamins, and other nutrients, as well as secondary metabolites such as flavonoids, alkaloids, and phenolic acids. Among these, alkaloids, the core components of potato secondary metabolites, not only provide a natural defense barrier for plant growth and development through their unique chemical structure, protecting against fungi, pests, and herbivores, but also hold significant potential for applications in human health, potentially leading to the development of fortified functional foods.

[0003] Alkaloids are a class of nitrogen-containing compounds with potent biological activity, widely distributed in plants, and constitute one of the largest categories of plant secondary metabolites. Potato alkaloids are primarily glycoside alkaloids, but also include isoquinolines, indoles, tropanes, and other types, with solanine and chalcone accounting for over 95% of the total alkaloid content. These alkaloids possess various pharmacological activities, including antibacterial, antiviral, anti-inflammatory, analgesic, and immunomodulatory effects. Some indole alkaloids also exhibit cerebral vasodilatory, anticancer, hypotensive, and antiarrhythmic effects. However, most alkaloids are also significantly toxic. When the content of glycoside alkaloids in potato tubers exceeds the safe threshold, it can damage the human digestive and nervous systems, causing poisoning symptoms. Therefore, strictly controlling the alkaloid content in crops is crucial.

[0004] Currently, some research has been conducted on the regulatory mechanisms of plant alkaloid biosynthesis, and several transcription factor families, such as AP2 / ERF, bHLH, MYB, and WRKY, have been confirmed to participate in the regulation of alkaloid synthesis. However, the developmental dynamics of potato alkaloids and their transcriptional regulatory mechanisms still require further investigation, especially the accumulation patterns of alkaloids and key regulatory genes at different developmental stages in potatoes with different flesh colors. Therefore, identifying key regulatory genes in potato alkaloid biosynthesis and elucidating their regulatory mechanisms is of great significance for breeding potato varieties with optimized alkaloid content, while ensuring both nutritional value and food safety. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides the accumulation characteristics of alkaloids in potatoes of different flesh colors at different developmental stages and clarifies multiple alkaloid metabolic pathways. StHSF1The application of this gene in the regulation of alkaloid biosynthesis has clarified the sequence characteristics of the gene and its regulatory function in alkaloid synthesis, providing new gene resources and theoretical basis for molecular breeding of potato alkaloid metabolism and development of functional foods.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide StHSF1 The application of genes in the regulation of alkaloid biosynthesis, the StHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The regulation includes the regulation of the biosynthesis of various alkaloids such as steroids, phenols, isoquinolines, and pyridines.

[0008] Furthermore, the aforementioned StHSF1 Genes have a positive regulatory effect on alkaloid biosynthesis, and their overexpression can significantly increase the types and / or contents of alkaloids.

[0009] The second objective of this invention is to provide a primer combination for amplifying the aforementioned... StHSF1 The gene, whose nucleotide sequence is shown in SEQ ID NO.2~SEQ ID NO.3.

[0010] A third objective of this invention is to provide a reagent kit containing the aforementioned primer combination.

[0011] The fourth objective of this invention is to provide a recombinant expression vector containing the aforementioned... StHSF1 Gene.

[0012] The fifth objective of this invention is to provide a host bacterium containing the aforementioned... StHSF1 The gene may contain the recombinant expression vector.

[0013] The sixth objective of this invention is to provide a method for regulating the types and / or contents of alkaloids in crops, which involves introducing the recombinant vector or the host bacteria into recipient crop material.

[0014] The seventh objective of this invention is to provide the application of the primer combination, the kit, the recombinant expression vector, or the host bacterium in the breeding of crop alkaloid biosynthesis regulation.

[0015] Furthermore, the crop includes potatoes.

[0016] Furthermore, the breeding includes developing nutritionally fortified potato varieties with enriched alkaloid content or developing food-safe potato varieties with reduced alkaloid content.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention analyzes the accumulation characteristics of alkaloids and the metabolic pathways of glycosidic alkaloids and amino acid-derived alkaloids in potatoes of different flesh colors (yellow, red and purple potatoes) at different developmental stages (tuber formation period, tuber enlargement period and tuber maturity period), including the expression characteristics of related alkaloids and the expression level of key structural genes.

[0018] (2) This invention preliminarily clarifies the potato StHSF1 The regulatory function of genes in alkaloid biosynthesis was confirmed through transient overexpression experiments. StHSF1 It can significantly increase the total alkaloid content by 28.56% and specifically upregulate the accumulation of 15 different types of alkaloids, including steroidal, phenolic, isoquinoline, and pyridine alkaloids, indicating that it has a wide range of regulatory effects.

[0019] (3) This invention discovers through co-expression network analysis that, StHSF1 Key structural genes in the alkaloid biosynthesis pathway PGA1 , SGT1 There is a strong correlation, and it may participate in the regulation of alkaloid synthesis by regulating the expression of genes such as glycosyltransferases, thus enriching the transcriptional regulatory network of potato alkaloid biosynthesis.

[0020] (4) The present invention provides StHSF1 Genes and their related primers, vectors, and host bacteria can be directly applied to potato molecular breeding. By regulating the expression level of these genes, the alkaloid content of potatoes can be precisely optimized. This can provide nutritionally fortified varieties for the development of functional foods, as well as cultivate low-toxicity and high-safety potato varieties, with broad application prospects.

[0021] In summary, this invention provides characteristics of potato alkaloid accumulation and multiple alkaloid biosynthesis pathways, and discovers the application of another gene—the potato StHSF1 gene—in the regulation of alkaloid biosynthesis, providing new gene resources and theoretical basis for molecular breeding of potato alkaloid metabolism and the development of functional foods. Attached Figure Description

[0022] Figure 1 The K-means of the morphological characteristics, total alkaloid content, differentially expressed genes, and differentially expressed metabolites of the potato tubers tested in Example 1 of this invention; Figure 2 Analysis of the differences in alkaloid accumulation at different developmental stages of the three types of flesh-colored potatoes in Example 1 of this invention; Figure 3 This is an integrated analysis of differentially expressed genes and differentially accumulated alkaloids in Example 1 of the present invention; Figure 4Analysis of the biosynthetic pathway of glycoside alkaloids (steroidal alkaloids) in Example 1 of this invention; Figure 5 Analysis of the biosynthetic pathways of tyramine (phenolic amine) and indole (plumeria alkaloid) alkaloids in Example 1 of this invention; Figure 6 Analysis of the biosynthetic pathways of other amino acid-derived alkaloids in Example 1 of this invention; Figure 7 For the expression analysis of alkaloid-related transcription factors in Example 1 of this invention and StHSF1 Functional verification; Figure 8 In Embodiment 1 of the present invention StHSF1 Analysis of differentially expressed genes and alkaloid metabolites in tobacco transiently overexpressed; Figure 9 This is a schematic diagram illustrating the dynamic changes and biological significance of alkaloids during the developmental stage of potato tubers in Example 1 of the present invention. Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0025] Example 1 1. Plant materials and sample preparation Three homologous potato clones with different flesh colors (Genesys: https: / / www.genesys-pgr.org / ): yellow-skinned, yellow-fleshed potato “BJ47” (CIP 302281.17), red-skinned, red-fleshed potato “BJ46” (CIP302281.25), and purple-skinned, purple-fleshed potato “BJ48” (CIP 302281.15), were selected and planted in the experimental field of Dingxi Academy of Agricultural Sciences using a ridge-furrow mulching and drip irrigation method. Tuber samples were collected at three key developmental stages: tuber formation (S1), tuber enlargement (S2), and tuber maturity (S3) for metabolomics and transcriptomics analysis.

[0026] Experimental results are as follows Figure 1 As shown.

[0027] 2. Metabolomics and Transcriptomics Analysis Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used for broad-targeted metabolomics analysis to identify the types and contents of alkaloids in potato tubers. High-quality total RNA was extracted, and RNA libraries were constructed and high-throughput sequencing was performed to screen differentially expressed genes (DEGs). DEGs were functionally annotated and classified using databases such as KEGG, GO, and NR. A co-expression network module was constructed using Weighted Gene Co-expression Network Analysis (WGCNA) to analyze the correlation between genes and alkaloid metabolites.

[0028] Experimental results are as follows Figure 2 , Figure 3 As shown.

[0029] 3. Integrated analysis of alkaloid metabolic pathways Using functional annotation and classification from databases such as KEGG, and drawing on KEGG metabolic pathways and related research, we integrated and analyzed the metabolic pathways of glycosidic alkaloids and amino acid-derived alkaloids, including the dynamic accumulation characteristics of related alkaloids and the expression levels of key structural genes.

[0030] Experimental results are as follows Figure 4 , Figure 5 , Figure 6 As shown.

[0031] 4. Screening and expression analysis of transcription factors related to alkaloid biosynthesis Based on the combined analysis of metabolomics and transcriptomics, alkaloid-related transcription factors were screened, the expression levels of differentially expressed transcription factors were analyzed, and their potential core genes were analyzed through co-expression networks.

[0032] Experimental results are as follows Figure 7 As shown in AB.

[0033] 5. StHSF1 Gene cloning and recombination vector construction Based on combined metabolomics and transcriptomics analysis, an important transcription factor in alkaloid biosynthesis was identified. StHSF1 ( Soltu.DM.04G033520 This invention is based on... Soltu.DM.04G033520The target gene was cloned to analyze its role in alkaloid biosynthesis. The target gene sequence was located based on the potato genome sequence. Primers for amplification were used: 5'-ATGGCTTTAATGCTAGATAATTGTGAA-3' (SEQ ID NO. 2), and the reverse primer was 5'-AGATGAGAGAAGGAGGCATG-3' (SEQ ID NO. 3). The primer sequences were submitted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. High-fidelity polymerase (PrimeSTAR) from Takara was used. The cDNA of red potato variety was amplified using HS DNA Ploymerase. The specific PCR reaction system and amplification procedure are shown in Tables 1 and 2.

[0034] Table 1 PCR reaction system

[0035] Table 2 PCR amplification program

[0036] After the target fragment was amplified, the PCR products were detected by gel electrophoresis to verify their integrity. The verified intact PCR products were then recovered using a DNA gel extraction kit from JianShi Biotechnology; specific procedures were followed by referring to the instruction manual. The concentration of the obtained purified PCR products was determined using a micro spectrophotometer before subsequent vector ligation experiments.

[0037] Single-clone bacterial cultures with brighter bands were selected and cultured overnight, after which plasmids were extracted and sequenced. Sequencing analysis revealed that the cloned gene sequences shared 97.5% similarity with the reference genome.

[0038] StHSF1 The nucleotides (SEQ ID NO.1) are as follows: 6. Transient expression experiment and analysis in tobacco 6.1 Transient expression experiment in tobacco leaves Preliminary determination was made through transient expression experiments in tobacco. StHSF1 Its function in the biosynthesis of alkaloids. The specific operation is as follows: (1) Activation and culture of Agrobacterium: The bacterial culture stored at -80℃ was spread on LB solid medium containing Kan+Rif and cultured upside down in the dark at 28℃ for 2 days.

[0039] (2) Use an inoculation loop to scrape the cultured Agrobacterium, fill a loop, place it in the infection solution, and let it stand at 28°C for 1 h. Gently invert the loop to allow the bacterial solution at the bottom to float, and let it stand for another 1 h. The concentration of the bacterial solution OD600 should be between 0.8 and 1.0. Try to complete the bacterial solution injection within 4 h to avoid damage to the viability of the Agrobacterium strain.

[0040] (3) Select robust and appropriately timed tobacco plants for tobacco injection. EV was injected on one side of the tobacco leaf as a negative control, and the other side of the leaf was injected with... StHSF1 This was the experimental group. Using a 1 mL syringe, the needle was removed, and the bacterial solution was slowly injected onto the back of the tobacco. After a brief period of darkness, the injected tobacco was placed in an artificial climate chamber for cultivation. Samples were taken after 6 days to measure changes in alkaloids.

[0041] 6.2 StHSF1 Determination of expression level and total alkaloid content Total RNA was extracted from samples using the PureLink Plant RNA Extraction Kit (USA), and the RNA samples were reverse transcribed into cDNA using the PrimeScript Reverse Transcription Kit (Takara Bio Inc., Japan). Gene expression levels were verified using real-time quantitative polymerase chain reaction (qPCR). The reaction system used the TB Green Premix Ex Taq Real-Time PCR Kit (Takara Bio Inc., Japan), and amplification was performed on a Bio-Rad CFX96 real-time quantitative PCR detection system, with three technical replicates for each sample. Relative expression levels were measured using 2... - The ΔΔCt method was used for calculation, with the specific primer sequence being 5'-TAACCCGCGGCACAGTATTT-3' (SEQ ID NO.4) and the reverse primer being 5'-TACGTAACCGGTCGTTTGTCC-3' (SEQ ID NO.5). Furthermore, the total alkaloid content was determined spectrophotometrically. The total alkaloid content was determined using the G0150F kit (GraceBio, Suzhou, China), and the specific experimental method was referred to the instruction manual. Three biological replicates were set up for each group.

[0042] Experimental results are as follows Figure 7 CD shown.

[0043] 6.3 Detection of differentially expressed alkaloid metabolites and differentially expressed genes Transient overexpression StHSF1 After freeze-drying, the leaves of *Tobacco Bengal* and the negative control sample of EV were ground and extracted with 70% methanol (containing internal standard). After vortexing, standing, and centrifugation, the supernatant was filtered through a 0.22 μm PTFE membrane and targeted analysis of alkaloid metabolites was performed by UPLC-MS / MS to identify the types of alkaloids.

[0044] Using the PureLink Plant RNA Extraction Kit (USA), RNA was extracted from transiently overexpressed plants. StHSF1 Total RNA was extracted from leaves of *Nicotiana benthamiana* (EV control). The integrity, purity, and concentration of total RNA were detected and quantified by 1% agarose gel electrophoresis and spectrophotometry. High-quality total RNA was sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for library construction and sequencing. Gene expression profiles were visualized and analyzed using TBtools software.

[0045] Experimental results are as follows Figure 8 As shown.

[0046] The dynamic changes and biological significance of alkaloids during potato tuber development are as follows: Figure 9 As shown.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. StHSF1 The application of genes in the regulation of alkaloid biosynthesis is characterized by, The StHSF1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The StHSF1 Genes have a positive regulatory effect on alkaloid biosynthesis, and their overexpression can significantly increase the types and / or contents of alkaloids.

3. A primer combination, characterized in that, It is used to amplify the application described in claim 1. StHSF1 The gene, whose nucleotide sequence is shown in SEQ ID NO.2~SEQ ID NO.

3.

4. A reagent kit, characterized in that, It contains the primer combination described in claim 3.

5. A recombinant expression vector, characterized in that, It contains the application described in claim 1. StHSF1 Gene.

6. A host bacterium, characterized in that, It contains the application described in claim 1. StHSF1 The gene or contains the recombinant expression vector as described in claim 5.

7. A method for regulating the types and / or content of alkaloids in crops, characterized in that, It is carried out by introducing the recombinant vector of claim 5 or the host bacteria of claim 6 into the recipient crop material.

8. The application of the primer combination of claim 3, the kit of claim 4, the recombinant expression vector of claim 5, or the host bacterium of claim 6 in the breeding of crop alkaloid biosynthesis regulation.

9. The application according to claim 8, characterized in that, The crop includes potatoes.

10. The application according to claim 9, characterized in that, The breeding includes developing nutritionally fortified potato varieties with enriched alkaloid content or developing food-safe potato varieties with reduced alkaloid content.

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