A specific promoter sequence pAkCSE3 in konjac tuber and application thereof

Abstract

This invention discloses a specific promoter sequence pAkCSE3 for konjac tubers and its applications, relating to the field of genetic engineering technology. The specific promoter sequence pAkCSE3 provided by this invention has the nucleotide sequence shown in SEQ ID NO.6. Verification has shown that overexpression of this promoter in tubers promotes the expression of specific genes in tubers and can be used to regulate the synthesis of specific genes in tubers. Furthermore, this experiment also clarified that the specific promoter sequence pAkCSE3 driving the overexpression of AkCSLA9 in konjac and potato tubers can promote glucomannan synthesis, providing a gene resource for improving the nutritional quality of tuber plants and enhancing glucomannan production using systems biology methods, showing promising application prospects in improving tuber-specific traits. The effect is particularly significant in plants such as potatoes and konjac.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a konjac tuber-specific promoter pAkCSE3 and its application in enhancing the expression of specific genes in tubers. Background Technology

[0002] Promoters are core components of gene regulation. Differences in their function lead to variations in gene expression efficiency and spatiotemporal specificity. Research on their structure and function is crucial for understanding plant growth and development, responses to environmental stress, and evolution. Tissue-specific promoters drive gene expression only in specific tissues or organs, such as roots, stems, leaves, flowers, or seeds. This effectively avoids silencing the target gene during transformation, improves the expression efficiency of exogenous genes, and avoids the metabolic burden and energy waste caused by non-specific continuous gene expression in plant cells. Based on these advantages, tissue-specific promoters can replace constitutive promoters such as CaMV 35S to avoid the side effects of 35S, such as reduced biomass and gene silencing. However, currently, tissue-specific promoters are predominantly derived from rice, maize, and wheat; their discovery from other species is still in its early stages. Previously reported tuber-specific promoters are mostly derived from potatoes, such as the promoter of the potato tuber-specific gene patatin. Konjac (Amorphophallus konjac) is a perennial herbaceous plant belonging to the Araceae family. Its tubers are rich in glucomannan, making it the only plant in nature from which glucomannan can be extracted on a large scale, thus possessing significant economic and medicinal value. Compared to other plants, konjac exhibits a high tuber enlargement coefficient and yield; however, promoters specifically expressed in konjac tubers have not yet been reported. Researching and utilizing konjac tuber-specific promoters is of great significance for improving the tuber enlargement coefficient, yield, and quality of konjac and other tuberous plants. Summary of the Invention

[0003] The purpose of this invention is to provide a specific promoter sequence pAkCSE3 for konjac tubers, which can be used to improve the expression of specific genes in tubers.

[0004] To achieve the above objectives, the present invention provides a specific promoter sequence pAkCSE3 for konjac tubers, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0005] The promoter sequence pAkCSE3 provided by this invention can be used to promote gene expression and synthesis in tubers.

[0006] The present invention also provides an expression box comprising the above-mentioned promoter sequence pAkCSE3, the expression box further comprising a coding sequence connected after the promoter sequence pAkCSE3.

[0007] Preferably, the coding sequence includes the coding sequence of the GUS gene or the coding sequence of the AkCSLA9 gene.

[0008] The present invention also provides a recombinant vector comprising the above-mentioned promoter sequence pAkCSE3.

[0009] Preferably, the recombinant vector is selected from pBI121-GUS.

[0010] The present invention also provides a recombinant bacterium containing the above-described promoter sequence pAkCSE3; or the above-described expression cassette; or the above-described recombinant vector.

[0011] Preferably, the recombinant bacteria are selected from Agrobacterium, and more preferably from GV3101.

[0012] The promoter sequence pAkCSE3 or expression cassette provided by this invention can be used for glucomannan synthesis, especially for improving glucomannan synthesis in the tubers of plants such as konjac and potato.

[0013] The present invention has the following advantages: This invention discloses the tuber-specific expression gene AkCSE3 cloned from konjac, the promoter sequence pAkCSE3, and the key enzyme gene AkCSL9 for konjac glucomannan synthesis. pAkCSE3 has been shown to be highly expressed in both konjac and potato tubers. A pAkCSE3-AkCSLA9 plant expression vector was constructed, and this vector was used to increase the glucomannan content in konjac and potato tubers. This invention reveals the function of the key gene AkCSLA9 in the biosynthesis of konjac glucomannan, providing a foundation for targeted breeding of konjac. Furthermore, the pAkCSE3-AkCSLA9 plant expression vector provides an effective approach for targeted breeding of plant germplasm resources containing high levels of glucomannan to meet the needs of the food nutrition industry. Attached Figure Description

[0014] Figure 1 This is a heatmap of the expression of a gene specifically expressed in konjac tubers screened in Example 1 of this invention.

[0015] Figure 2 This shows the GUS staining of transgenic konjac tubers transformed with the pAkCSE3-GUS recombinant vector in this invention; wherein, Figure 2 In the diagram, A represents the GUS staining of the entire transgenic konjac plant, while B and C represent the GUS staining of the tuber (indicated by arrows).

[0016] Figure 3The image shows GUS staining in the microbulbs of transgenic potatoes obtained by genetic transformation of the pAkCSE3-GUS recombinant vector in this invention; WT represents the untransformed wild type; OE1 and OE2 are two different transgenic potato lines. Three different tubers from each line were stained with GUS (1, 2, and 3 in the image).

[0017] Figure 4 The following are the transcriptome analysis results of konjac corms at different developmental stages in this invention. A shows the WGCNA analysis of transcriptome data from different tissues, in which a co-expressed Brown module highly correlated with glucomannan content was identified (r=0.91, P=2e-05); B shows the gene expression trend within the Brown module; C shows the Go enrichment results of the Brown module; and D shows the KEGG enrichment results of genes in the Brown module.

[0018] Figure 5 These are the results of the analysis of key genes (hub genes) in the co-expression network of konjac corms in this invention.

[0019] Figure 6 The results of qRT-PCR analysis of AkCSLA9 expression levels in six different tissues of konjac in this invention are shown.

[0020] Figure 7 The figures show the glucomannan content in transgenic konjac expressing the pAkCSE3-AkCSLA9 recombinant vector and its tubers in this invention. A represents wild-type (WT) and transgenic konjac plants (OECSL-1); B represents harvested wild-type (WT) and transgenic (OECSL-1, OECSL-2, and OECSL-4) konjac tubers; C represents the relative expression level of the AkCSLA9 gene in transgenic konjac plants detected by qRT-PCR; and D represents the glucomannan (KGM) content in tubers of different transgenic konjac lines.

[0021] Figure 8 The results of the analysis of the phenotype, yield per plant, and tuber size of the transgenic T1 generation potato plants expressing the pAkCSE3-AkCSLA9 recombinant vector were shown in A and B, respectively. Figure 8 (A) and harvested tubers ( Figure 8 In the figures, B); C represents the AkCSLA9 expression level of the transgenic potato lines; and D represents the fresh weight of individual tubers of wild-type (WT) and transgenic OE1, OE3, and OE4 plants.

[0022] Figure 9 Results of the determination of monosaccharide composition of cell wall material (AIR) in transgenic potatoes. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Example 1: Cloning of the konjac tuber-specific expression gene AkCSE3 1. Screening of genes specifically expressed in konjac tubers (1) Transcriptome sequencing of different tissues of konjac Healthy Amorphophallus konjac first-generation seed (average weight 20 g / corm) from Fuyuan, Yunnan Province were used as material. The plants were grown in a greenhouse under natural light at 25℃ and 60% relative humidity. Leaves, petioles, roots, and corms of healthy, representative plants at 60 days of growth were collected. Total RNA was extracted using TRIzol reagent (Tiangen, Beijing), and the quality of the total RNA was checked using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, China). After passing the tests, the samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for transcriptome sequencing. Raw reads were filtered using FastP (https: / / github.com / OpenGene / fastp) and aligned to the Amorphophallus konjac reference genome (GCA_022559845.1) using hisat2 software. Gene expression levels were calculated using feature counts software and expressed as FPKM values. Transcriptome data were uploaded to the Araceae Functional Genomics Database InternalDB-001 of the Araceae Genomics Database (http: / / www.araceaedb.com / ).

[0026] (2) Screening of tuber-specific expression genes Gene expression values ​​were extracted from leaves, petioles, roots, and tubers. After standardizing the results, gene expression heatmaps were generated using the HeatMap function of TBtools software (see [link to relevant documentation]). Figure 1Ten genes highly expressed in tubers, including evm.model.HIC_ASM_3.189_Akon, were initially screened. These genes showed extremely low expression in leaves, petioles, and roots, indicating that they are tuber-specific genes. Among them, evm.model.HIC_ASM_3.189_Akon, named AkCSE3, exhibited the most significant high expression.

[0027] 2. RNA extraction from konjac tubers and cloning of the AkCSE3 gene (1) RNA was extracted from konjac tubers using RNAiso PLUS (Takara) reagent; the extracted RNA was reverse transcribed into cDNA (Vayme) using cDNA first strand kit (TIANGEN).

[0028] (2) AKCSE3 gene amplification: Gene-specific primers CSE3-F / R (sequence details below) were designed. Using the above cDNA as a template, the target gene fragment was amplified using TaKaRaLA-MIX. The PCR reaction program was: 95℃ for 3 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1.5 min, 30 cycles, 72℃ for 10 min. After the program was completed, the sample was stored at 4℃.

[0029] CSE3-F (SEQ ID NO.1):atgaagctcttcttcctcctcc; CSE3-R (SEQ ID NO. 2): ctaggccacagcatgcatgttg.

[0030] (3) Product recovery and sequencing: After the PCR product electrophoresis was completed, the target band was cut off, and the target band was recovered using the TIANGEN purification and recovery kit. The band was then subcloned into the pMD19-T vector for sequencing. The coding sequence of the obtained AkCSE3 gene is shown in SEQ ID NO.3.

[0031] AkCSE3 gene (SEQ ID NO.3): Experiment Example 2: Cloning of the AkCSE3 gene promoter sequence pAkCSE3 and construction of the GUS reporter gene fusion expression vector 1. pAkCSE3 amplification (1) Extraction of konjac genomic DNA: 1.0 g of mature konjac leaves were ground in liquid nitrogen and DNA was extracted using the conventional CTAB method (2XCTAB solution). Using the extracted DNA as a template, PCR amplification was performed using promoter-specific primers pAkCSE3-F / pAkCSE3-R (specific sequences below) as primers, and TaKaRa LA-MIX was used for amplification. The PCR amplification reaction program was: 95℃ for 3 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1.5 min, 30 cycles, 72℃ for 10 min, and the product was stored at 4℃ after the program was completed.

[0032] pAkCSE3-F (SEQ ID NO.4): CAGCTATGACCATGATTACGGGTTATCGTGCAGGGGCTCTCTC; pAkCSE3-R (SEQ ID NO.5): ACAGGACGTAACATGGATCCGGTTGGCTTAGCTAGCTAGGTAAC.

[0033] (2) PCR product recovery and sequencing: After the PCR products were electrophoresed, the target band was cut off and recovered using the TIANGEN purification and recovery kit. The target band was then subcloned into the pMD19-T vector and sequenced. The obtained promoter sequence pAkCSE3 has the DNA sequence shown in SEQ ID NO.6.

[0034] AkCSE3 gene promoter sequence pAkCSE3 (SEQ ID NO.6):

[0035] 2. Construction of pAkCSE3-GUS fusion expression vector Using Snapgene bioinformatics software to find available restriction enzyme sites, the plasmid MD19-T-pAkCSE3 was amplified using PCR to introduce HindIII and XbaI restriction sites. The PCR products were digested separately with the pBI121-GUS empty vector, and the two digested products were ligated at 22℃ for 2 hours. After ligation, the ligated products were transformed into DH5α E. coli competent cells. Single colonies were selected, and the GUS sequence was directionally amplified by colony PCR using GUS-specific primers GUS-F / R (specific sequence shown below). Amplification was performed using TaKaRaLA-MIX. The PCR amplification program was: 95℃ for 3 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1.0 min, 30 cycles, 72℃ for 10 min, and then stored at 4℃ after completion. The successfully validated plasmid was genetically transformed into Agrobacterium GV3101 to obtain the recombinant strain pAkCSE3-GUS-GV3101.

[0036] GUS-F (SEQ ID NO.7): GCTTCGCGTCGGCATCCGG GUS-R (SEQ ID NO.8): CCGTATTCGGTGATGATAATCGG Experimental Example 3: Transformation of konjac callus tissue with pAkCSE3-GUS recombinant vector The obtained recombinant vector was transferred into konjac, as follows: (1) Preparation of Agrobacterium infection solution of pAkCSE3-GUS-GV3101: In a clean bench, take 1 mL of positive strain and add 5 mL of LB (100 mg / L Rif Rifampicin + 100 mg / L Kanamycin) liquid culture medium. Shake at 180 rpm for 12 h at 28℃. (2) Take out the bacterial culture that has been shaken for 12 hours, add 100 mL of LB (100 mg / L Rif + 100 mg / L Kana) liquid medium, and incubate at 28°C and 180 rpm for 1 day, waiting for OD. 600 =0.5, let stand at room temperature for 3 hours; (3) Centrifuge at 5000 rpm for 10 min, discard the supernatant, collect the bacterial cells, resuspend the bacterial cells in sterile MS liquid medium (containing 100 μmol / L AS acetylsuccinone), and incubate at room temperature in the dark for 2 h to adjust OD. 600 =0.5, to obtain the inoculum; (4) Agrobacterium infection of konjac petioles: Petioles of konjac tissue culture seedlings (after leaves have fully expanded) were cut into 1 cm segments and cultured in induction medium for approximately 15-30 days. Once the ends began to swell, Agrobacterium infection was performed. The swollen petiole segments were then immersed in the infection solution with shaking for 15 minutes. Residual infection solution was blotted dry with sterile filter paper, and the segments were placed in co-culture medium and incubated in the dark at room temperature for 2 days. After 2 days, they were transferred to selection medium and cultured under 25°C, 16 hours of light, and 8 hours of darkness. Subculture was performed approximately every 30 days until plantlets emerged from the callus. DNA was extracted from the leaves of the regenerated plantlets, the GUS gene fragment was amplified, and positive transgenic plantlets were screened.

[0037] (5) GUS tissue viability staining of transgenic plants: Fix plant material with 90% acetone for 20 min; After discarding the acetone, rinse the plant material once with X-Glu buffer; Add staining solution (4 mL X-Glu buffer + 100 μL X-Glu stock solution), vacuum treatment for 20 min; Incubate in water bath at 37℃ for 24 h; Decolorize with anhydrous ethanol until colorless, observe the experimental results and take pictures.

[0038] Figure 2 In the diagram, A represents the GUS staining of the entire transgenic konjac plant, while B and C represent the GUS staining of the tuber (indicated by arrows). It can be seen that the tuber of the transgenic konjac seedling exhibits a distinct GUS blue color (…). Figure 2 (as indicated by arrows B and 2C), while the leaf blade and petiole showed no obvious GUS staining ( Figure 2 A) indicates that pAkCSE3 can drive high expression of the GUS gene in tubers.

[0039] The konjac induction medium consisted of: 4.74 g of sugar-free MS powder (Solarbio), 2 mg of 6-BA, 0.8 mg of NAA, 30 g of sucrose, diluted to 1 L with distilled water, pH=5.8, and 8 g of agar. The medium was sterilized at 121 °C for 20 min.

[0040] Konjac co-culture medium: 4.74 g of sucrose-free MS powder (Solarbio), 2 mg of 6-BA, 0.8 mg of NAA, 30 g of sucrose, distilled water to a final volume of 1 L, pH=5.8, 8 g of agar, sterilized at 121 °C for 20 min; after cooling, add 100 μmol / L AS (acetylsyleugenol).

[0041] Screening medium: sucrose-free MS powder (Solarbio), 6-BA 2mg, NAA 0.5mg, sucrose 30g, distilled water to a final volume of 1L, pH=5.8, agar 8g, autoclaved at 121℃ for 20min, after sterilization, add 400mg / L Cef (cephalosporin) and 200mg / L Kanamycin (kanamycin).

[0042] X-Glu buffer: 80 mL phosphate buffer (pH=7.00), 0.744 g EDTA, 0.1 mL Tritium X, 16.4 mg potassium ferricyanide, 21.1 mg potassium ferrocyanide, and RNase-free ddH2O to 100 mL; X-Glu stock solution: Weigh 10 mg of X-Glu powder and dissolve it in 1 mL of methanol. Store at -20℃ protected from light. GUS staining solution: X-Gluc 50µL, GUS buffer 1mL.

[0043] Experimental Example 4: Genetic transformation of potato tubers using the pAkCSE3-GUS recombinant vector 1. Take tubers of *Potato 3* var. *evodiae* with a diameter of approximately 0.5 cm that have grown for 84–140 days and are grown in vitro, cut them into 1 mm–2 mm slices, and soak them in activated *Agrobacterium tumefaciens* pAkCSE3-GUS infection solution (see Experiment 3) with shaking for 10 min. After soaking, remove the slices and blot dry the surface bacterial solution with sterile filter paper, then transfer them to co-culture medium and incubate in the dark at 26°C for 2 days. Transfer them to selection medium supplemented with kanamycin and termethin, and incubate them under conditions of 2000 lx light intensity, 16 h light / 8 h dark photoperiod, and 23 ± 1°C. Replace the medium with fresh medium every 7–10 days, and remove and discard any young shoots at the edges of the tuber slices. After 21–28 days, resistant buds grow from the center of the test tube potato slices. When the resistant buds reach 0.5 cm–1 cm in length, they are cut off and transferred to a selection medium to induce rooting. After the potato seedlings grow strong, they are transferred to a potato induction medium and cultured in the dark for about 30 days to induce tuber formation in the test tube potato seedlings.

[0044] 2. The mini-tubers of the GUS-positive transgenic potatoes were subjected to GUS staining (method as in Experiment 3). The staining results are as follows: Figure 3 As shown, the tubers of the two transgenic plants (denoted as OE1 and OE2, respectively) exhibited different degrees of blue coloration, while the untransformed WT (wild-type) mini-tubers showed no GUS signal, indicating that pAkCSE3 can drive high expression of the GUS gene in potato tubers.

[0045] The potato co-culture medium consisted of: 4.74 g of sugar-free MS powder (Solarbio), 0.02 mg of NAA, 2 mg of Zentin, 0.02 mg of GA3, 30 g of sucrose, diluted to 1 L with distilled water, pH 5.8, and 8 g of agar. The medium was sterilized at 121 °C for 20 min. After cooling, 100 μmol / L AS (acetylsyleugenol) was added. Potato selection medium: 4.74 g sucrose-free MS powder (Solarbio), 0.02 mg NAA, 2 mg Zentin, 0.02 mg GA3, 30 g sucrose, diluted to 1 L with distilled water, pH=5.8, 8 g agar, sterilized at 121℃ for 20 min; after cooling, add 200 mg / L kanamycin and 200 mg / L timentin. Miniature potato induction medium: 4.74 g of sucrose-free MS powder (Solarbio), 5 mg of 6-BA, 8% sucrose, distilled water to a final volume of 1 L, pH=5.8, 8 g of agar, sterilized at 121℃ for 20 min.

[0046] Example 5: Cloning of the key enzyme gene AkCSLA9 for konjac glucomannan synthesis and construction of the pAkCSE3-AkCSLA9 recombinant vector 1. Transcriptome analysis of konjac corms at different developmental stages The genome of *Amorphophallus konjac* and transcriptome data for different corm development stages (Stages 1-4 represent different stages of tuber formation: formation, budding, swelling, and maturity) were downloaded from the Aracea Genome Database (http: / / www.araceaedb.com / ). A co-expression network for *Amorphophallus konjac* corm development was constructed. The transcriptome analysis results for different developmental stages of *Amorphophallus konjac* corms are as follows: Figure 4 As shown. A Brown co-expression module highly correlated with glucomannan content was also identified (r=0.91, P=2e-05) (see details). Figure 4 (As shown in A in the diagram). The gene in this module is highly expressed during the expansion phase (Stage 3) (see Figure 1). Figure 4 (As shown in B in the figure). Go enrichment analysis showed that the Brown module genes were significantly enriched in biological processes such as cytokinin response, polysaccharide metabolism, nucleotide synthesis, glucose metabolism, and starch metabolism (see Figure B). Figure 4 (As shown in C in the figure), KEGG enrichment results also showed that a large number of genes were significantly enriched in metabolic pathways such as autophagy, TCA cycle, oxidative phosphorylation, fructose and mannose metabolism, and starch and sucrose metabolism (see C in the figure). Figure 4 (As shown in D in the diagram). The top 100 key genes (Hubgenes) of this module were calculated, revealing AkCSLA9 as the core Hub gene. The results of the co-expression network Hub gene analysis are shown in [the diagram]. Figure 5 As shown.

[0047] 2. Quantitative analysis of AkCSLA9 expression levels in konjac tissues Six tissue samples were extracted from fully leaf-spreading Amorphophallus plants: mature leaves, young leaves, petioles, petiole bases, tubers, and roots. Total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing, China). DNA was removed using a DNA remover (TIANDZ), and cDNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novizan). Quantitative primers AkCSLA9-qRT-F / R (sequence shown below) were designed. Eukaryotic initiation factor 4A (EIF4A) of Amorphophallus was used as an internal reference gene (primer sequence EIF4A-F / R, sequence shown below) for normalization. qRT-PCR reactions were performed using the Taq Pro Universal SYBR qPCR Master Mix kit (Novizan), and the relative expression levels of the genes were calculated using the 2-ΔΔCT method. Results are as follows. Figure 6 As shown, the results indicate that AkCSLA9 was expressed at the highest level in the tuber, about 13 times higher than in other parts, while its expression level was lower in other parts, and lowest in young leaves and petioles. This demonstrates that AkCSLA9 has a very obvious tuber expression characteristic.

[0048] AkCSLA9-qRT-F (SEQ ID NO.9): AGGAAGATGGCAATAGAA; AkCSLA9-qRT-R (SEQ ID NO. 10): ACACGACACAATAGAAGA; EIF4A-F (SEQ ID NO. 11): AACAGATGAGGAGCAGGG; EIF4A-R (SEQ ID NO. 12): GGTGATAAGGACACGAGA.

[0049] 3. Construction of pAkCSE3-AkCSLA9 recombinant vector Based on the full-length AKCSLA9 gene sequence (evm.model.HIC_ASM_10.2728_Akon), specific cloning primers AkCSLA9-F / R (sequence details below) were designed. Using konjac tuber cDNA as a template, PCR amplification was performed using TaKaRa LA. The PCR reaction program was: 95℃ for 3 min, 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1.5 min, 30 cycles, 72℃ for 10 min. After completion, the product was stored at 4℃. After electrophoresis of the PCR products, the target band was excised, purified using a TIANGEN purification kit, and subcloned into the pMD19-T vector for sequencing.

[0050] AkCSLA9-F (SEQ ID NO. 13): ATGGAGGGCCTAATGAAG; AkCSLA9-R (SEQ ID NO. 14): CTACTTTTCACTAGGAACAA.

[0051] The AKCSLA9 gene fragment and the pAkCSE3-GUS fusion expression vector were digested with restriction endonucleases EcoRI and BamHI, respectively. The digestion products were verified by gel electrophoresis and recovered using a universal DNA purification and recovery kit (Tiangen), and stored at -20℃ for later use. The full-length AKCSLA9 gene fragment was ligated into a linearized vector using T4 ligase (T4 DNA Ligase, Nanjing Novizan). The recombinant pAkCSE3-AkCSLA9 product was transformed into DH5α and verified by colony PCR.

[0052] Experiment Example 6: Application and Verification of pAkCSE3-AkCSLA9 Recombinant Vector in Increasing Glucomannan Content in Konjac Tubers 1. Agrobacterium GV3101 was transformed with the pAkCSE3-AkCSLA9 recombinant vector. Take 5 μL of pAkCSE3-AkCSLA9 plasmid and gently add it to GV3101 Agrobacterium competent cells in an ice-water mixture. Mix gently and let stand on ice for 30 min. Quickly transfer to liquid nitrogen and freeze for 5 min. Quickly transfer to a 37°C water bath and incubate for 5 min. After that, quickly place on ice and let stand for 5 min. Add 600 μL of LB medium containing rifampicin in a clean bench and shake at 150 rpm for 2 h at 28°C. After that, centrifuge at 4000 rpm and discard the excess supernatant in a clean bench, leaving 60-100 μL in a tube. Mix gently and spread on LB solid medium containing kanamycin and rifampicin. Incubate upside down at 28°C for 2 days. Then, use AkCSLA-9F / R primers (same as in Experiment 5) to amplify the AkCSLA9 gene target fragment by PCR to verify positive colonies.

[0053] Incubate positive colonies overnight at 28°C and 200-220 rpm. Take 1 mL of the overnight culture and add 50 mL of LB liquid medium containing Rif and Kana. Incubate until OD600 = 0.5-0.6. Transfer the culture to a 50 mL sterile centrifuge tube and centrifuge at 4°C and 5000 rpm for 5 min. Discard the supernatant and add an equal volume of MS staining solution to suspend the cells for later use.

[0054] 2. Obtaining transgenic konjac and determining the glucomannan content in its tubers The petioles of konjac tissue culture seedlings were cut into 1 cm segments and cultured in induction medium for about 15-30 days. After the ends began to swell, Agrobacterium infection was performed. The infection method and the induction method for transgenic konjac were the same as in Experiment 3.

[0055] Rooted, untransformed control plants (WT) and transgenic konjac plants were transferred into the soil (see...). Figure 7 (A) The potting mix is ​​a 3:2 (v / v) mixture of peat moss and garden soil. Cultivate in a greenhouse for 4-5 months. After the above-ground parts wither, harvest the underground konjac tubers (see section A). Figure 7 (B in the middle).

[0056] RNA was extracted from transgenic konjac tubers and reverse transcribed into cDNA. The expression level of AkCSLA9 in the transgenic konjac tubers was analyzed by qRT-PCR (method as in Experiment 5). Figure 7 As shown in C, in transgenic konjac, the expression of OECSL-1, OECSL-2, OECSL-3 and OECSL-4 lines was 1.8 to 3.3 times that of the control.

[0057] Tubers from transgenic lines OECSL-1, OECSL-2, and OECSL-4 with high expression levels were selected, washed, sliced, dried, and ground into powder. The powder was sieved through a 100-mesh sieve, retaining particles smaller than 0.150 mm for glucomannan content determination. The determination method followed the NY / T494-2010 standard promulgated in 2010. The results are shown below. Figure 7 As shown in Figure D, the glucomannan content in the tubers of the three transgenic konjac lines OECSL-1, OECSL-2, and OECSL-4 was significantly higher than that of the wild-type control (WT) (LSD, P < 0.05); compared with the wild-type, it increased by 28.5%–78.5%. These results demonstrate the role of the pAkCSE3-AkCSLA9 recombinant vector in increasing the glucomannan content of konjac tubers, providing gene resources and recombinant vectors for the subsequent creation of konjac materials with high glucomannan content.

[0058] Experiment 7: Verification of the application of the pAkCSE3-AkCSLA9 recombinant vector in increasing glucomannan content in potato tubers 1. Transformation of potatoes using the pAkCSE3-AkCSLA9 recombinant vector Take tubers of *Potato 3* var. *evodiae* with a diameter of approximately 0.5 cm after 84–140 days of growth, cut them into 1–2 mm slices, and soak them in activated *Agrobacterium* pAkCSE3-AkCSLA9 infection solution (same as in Example 6) with shaking for 10 min. After removal, blot dry the surface bacterial solution with sterile filter paper, transfer to co-culture medium, and incubate in the dark at 26°C for 2 days. Transfer to selection medium supplemented with hygromycin and termethin, and incubate under conditions of 2000 lx light intensity, 16 h light / 8 h dark photoperiod, and 23 ± 1°C. Replace with fresh medium every 7–10 days, and remove and discard any young shoots at the edges of the tuber slices. After 21-28 days, resistant shoots emerged from the center of the test-tube potato slices. When the resistant shoots reached 0.5-1 cm in length, they were cut off and transferred to a selection medium to induce rooting. When the potato seedlings reached 6-7 cm in height, the culture medium attached to them was carefully washed off, and they were transplanted into a sterile substrate (peat soil: river sand = 2:1) and cultured until the plants matured. RNA was extracted from the tubers of untransformed wild-type (WT) and five transgenic potato plants (OE1-5) and reverse transcribed into cDNA. The expression level of AkCSLA9 in the transgenic potato tubers was analyzed by qRT-PCR (method as in Experiment 5). Figure 8 As shown in Figure C, in transgenic potatoes, the expression of AkCSLA9 in the OE1, OE2, and OE4 lines was 2.2-fold, 1.6-fold, and 1.2-fold higher than that in the wild-type control, respectively, indicating that the tuber-specific pAkCSE3 promoter can drive the expression of the konjac AkCSLA9 gene in potato tubers. Furthermore, the transgenic potato lines OE1, OE2, and OE4, overexpressing AkCSLA9, were cultured to the T1 generation. Figure 8 In the diagram, A~B represent the obtained transgenic potato T1 generation plants (e.g., ... Figure 8 (A) and harvested tubers (such as A) Figure 8 (B) in the text. Figure 8 As shown in B and D, it can be seen that the transgenic plants (OE1, OE3, OE4) had significantly higher tuber yield (fresh weight) compared to wild-type plants, consistent with the previously predicted phenotype of AKCSLA9 promoting cell enlargement. Compared to wild-type, the AkCSLA9-overexpressing potato lines OE1, OE2, and OE4 showed tuber fresh weight increases of 67%, 48%, and 41%, respectively (see Figure 1). Figure 8 (D in the middle).

[0059] The induction medium and co-culture medium were the same as in Experiment 4.

[0060] Screening medium: 4.74 g of sucrose-free MS powder (Solarbio), 0.02 mg of NAA, 2 mg of Zentin, 0.02 mg of GA3, 30 g of sucrose, diluted to 1 L with distilled water, pH=5.8, 8 g of agar, sterilized at 121 °C for 20 min; after cooling, add 20 mg / L hygromycin and 200 mg / L timentin.

[0061] 2. Analysis of cell wall components in transgenic potatoes (1) Extraction of alcohol-insoluble cell wall substances (AIR) Fresh tuber samples from wild-type (WT) and transgenic potatoes (OE1, OE3, and OE4) were homogenized with distilled water and passed through a 300-mesh sieve to remove starch, followed by freezing in a vacuum freeze dryer. The freeze-dried samples were stored at -20 °C. 2.0 g of the freeze-dried powder was taken, and 100 mL of 95% ethanol was added. The mixture was then incubated in a water bath at 85 °C for 20 min to remove soluble sugars. 100 mL of a methanol / chloroform solution (1:1, w / w) was added, and the mixture was shaken at 26 °C for 3 h before filtration. Finally, 20 mL of acetone was added, and the mixture was filtered again. The residue was dried at 45 °C to obtain the alcohol-insoluble cell wall material (AIR).

[0062] (2) Monosaccharide component analysis Sample pretreatment: Weigh an appropriate amount of AIR sample, add a small amount of anhydrous ethanol, shake to decolorize for 5 min, centrifuge and discard the supernatant. Add an appropriate amount of sterile water to the precipitate and extract at 70℃ for 2 hours. Centrifuge and collect an appropriate amount of supernatant, concentrate by rotation or blow dry under nitrogen. Add 1 ml of 2M TFA solution and heat at 121℃ for 2 hours. Blow dry under nitrogen, add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis. Monosaccharide component analysis was performed using a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) with a Dionex™ CarboPac™ PA20 (150*3.0mm, 10μm) liquid chromatography column.

[0063] like Figure 9As shown, there were no significant differences in the contents of arabinose (Ara) and rhamnose (Rha) in the cell wall material (AIR) of transgenic lines (OE1, OE3, and OE4) and wild-type (WT) potatoes. However, the galactose (Gal) content in the AIR of transgenic plants was significantly increased compared to wild-type potatoes (t-test, *, P < 0.05; **, P < 0.01), rising from a maximum of 67 ug / mg in the wild type to a maximum of 107 ug / mg. In particular, the mannose (Man) content in the AIR of transgenic potatoes increased from a maximum of 2.9 ug / mg in the wild type to a maximum of 45 ug / mg. Mannose is one of the main components of plant cell walls, and it is linked with monosaccharides such as glucose through glycosidic bonds to form complex polysaccharide chains. The significant increase in mannose can be inferred to be an increase in glucomannan content.

[0064] In summary, the tuber-specific promoter sequence pAkCSE3 provided by this invention drives the expression of the target gene in tubers and can be used to regulate tuber growth, development, and product synthesis. Furthermore, this experiment also clarified that overexpression of AkCSLA9 in konjac and potato tubers can promote glucomannan synthesis, providing a gene resource for improving the nutritional quality of tuber plants and enhancing glucomannan production using systems biology methods. This has promising applications in improving tuber-specific traits, especially in potatoes and konjac.

[0065] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A specific promoter sequence pAkCSE3 from konjac tubers, characterized in that, The nucleotide sequence of the promoter sequence pAkCSE3 is shown in SEQ ID NO.

6.

2. The application of the promoter sequence pAkCSE3 as described in claim 1 in promoting gene expression in tubers.

3. An expression box comprising the promoter sequence pAkCSE3 as described in claim 1, and a coding sequence connected to the promoter sequence pAkCSE3.

4. The expression box according to claim 3, characterized in that, The coding sequence includes the coding sequence of the GUS gene or the coding sequence of the AkCSLA9 gene.

5. A recombinant vector comprising the promoter sequence pAkCSE3 as described in claim 1.

6. The recombinant vector according to claim 5, characterized in that, The recombinant vector was selected from pBI121-GUS.

7. A recombinant bacterium comprising the promoter sequence pAkCSE3 as described in claim 1; or comprising the expression cassette as described in any one of claims 3-4; or comprising the recombinant vector as described in any one of claims 5-6.

8. The recombinant bacteria according to claim 7, characterized in that, The recombinant bacteria were selected from Agrobacterium.

9. The recombinant bacteria according to claim 8, characterized in that, The Agrobacterium was selected from GV3101.

10. The application of the promoter sequence pAkCSE3 as described in claim 1 or the expression cassette as described in claim 4 in glucomannan synthesis.

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