Sweet potato conditional inducible promoter and application thereof in target gene coupling expression system

Abstract

The present application belongs to the field of plant genetic engineering and molecular breeding technology, and particularly relates to a sweet potato conditional inducible promoter and application thereof in a target gene coupled expression system. The present application mines a kind of endogenous conditional inducible promoter of sweet potato, a cis-acting element modification body thereof, a recombinant expression vector containing the promoter, and a method for spatiotemporal specific regulation of a target gene using the promoter. The promoter has both tissue specificity (specifically activated in anthocyanin accumulation tissues) and hormone inducibility (activity significantly enhanced after treatment with abscisic acid or calcium nitrate), and can be widely used as a precision regulation tool in plant molecular breeding, functional genomics research and biotechnological improvement. As a preferred application, the present application also provides a method for creating a conditionally induced flowering sweet potato germplasm by using the inducible promoter to drive flowering integrated gene IbFT2.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically relating to a conditionally inducible promoter for sweet potato and its application in a target gene-coupled expression system. Background Technology

[0002] I. Technological Requirements for the Development of Conditionally Inducible Promoters in the Field of Plant Genetic Engineering

[0003] Promoters are cis-regulatory DNA sequences located upstream of the coding region of a gene. Through interactions with trans-acting factors such as transcription factors and RNA polymerases, they precisely control the initiation time, spatial location, and expression intensity of gene transcription. In the field of plant genetic engineering, promoter selection directly determines the spatiotemporal pattern and efficiency level of exogenous gene expression, and is a key technical factor in the success or failure of transgenic trait improvement.

[0004] Currently, promoters commonly used in plant genetic engineering can be divided into three categories: first, constitutive promoters, such as CaMV35S, maize Ubiquitin-1, and rice Actin-1, which are continuously highly expressed in almost all tissues and at all developmental stages; second, tissue-specific promoters, such as the Arabidopsis oocyte-specific AtDD45 promoter, the potato tuber-specific Patatin promoter, and the wheat endosperm-specific HMW-GS promoter; and third, inducible promoters, such as Arabidopsis RD29A (drought-induced) and maize In2-2 (chemically induced). Developing promoters that combine tissue specificity and condition inducibility has always been a technical challenge in plant genetic engineering to meet the needs of precise spatiotemporal regulation.

[0005] Conditional inducible expression systems are important tools for achieving precise gene regulation. Common induction systems include chemical induction systems such as tetracycline, ethanol, and dexamethasone induction systems, which control gene switching through exogenous chemical reagents; hormone induction systems utilize response elements in the signaling pathways of endogenous plant hormones (such as abscisic acid ABA, gibberellin GA, and jasmonic acid JA) to construct hormone-inducible promoters, achieving physiologically relevant regulation of target genes. The advantages of hormone induction systems are that the inducer is a signaling molecule already present in the plant, resulting in fewer side effects on plant growth and development; and the induction intensity and duration can be precisely controlled by adjusting the spray concentration and treatment frequency, achieving precise regulation of gene expression.

[0006] II. The urgent need for specialized promoters in sweet potato genetic engineering

[0007] Sweet potato (Ipomoea batatas [L.] Lam., 2n=6x=90) is an important food, feed, and industrial raw material crop in my country, with an annual planting area exceeding 60 million mu and an annual output accounting for more than 70% of the world's total output. As a typical asexually propagated polyploid crop, sweet potato is characterized by self-incompatibility, complex genetic background, and a large number of chromosomes. Conventional hybridization breeding is inefficient, making molecular breeding technology a key approach to overcome the bottleneck of variety improvement.

[0008] However, current sweet potato genetic engineering research relies almost entirely on exogenous constitutive promoters. Due to differences in transcriptional regulatory mechanisms among species, the expression activity of exogenous promoters in sweet potato is often unstable, and precise regulation of target genes cannot be achieved. More importantly, as a hexaploid crop, sweet potato has a large number of homologous sequences in its genome, and exogenous promoters may interact non-specifically with endogenous transcription factors, leading to deviations from expected expression patterns. Many important agronomic traits of sweet potato (such as tuber development, starch accumulation, and flowering induction) have strict spatiotemporal specificity, requiring precise expression of target genes at specific developmental stages and in specific organs. Current technologies lack effective spatiotemporal control tools, severely restricting the breeding application of sweet potato functional genes.

[0009] III. The unique value of bHLH2 transcription factor and its promoter in the regulation of anthocyanin synthesis

[0010] The basic helix-loop-helix (bHLH) transcription factor family is a core regulator of plant secondary metabolism. In the anthocyanin biosynthesis pathway, the bHLH2 protein forms the MBW ternary complex (MYB-bHLH-WD40) with the R2R3-MYB transcription factor and the WD40 repeat protein, synergistically activating the expression of downstream structural genes (CHS, CHI, F3H, DFR, ANS, UFGT), and is the "core switch" for the regulation of anthocyanin biosynthesis.

[0011] Researchers have identified more than 200 bHLH genes in sweet potato, among which IbbHLH2 has been confirmed as a key positive regulator of anthocyanin accumulation in purple sweet potato (Guo F, Meng X, Hong H, Liu S, Yu J, Huang C, DongT, Geng H, Li Z, Zhu M. Systematic identification and expression analysis of bHLH gene family reveal their relevance to abiotic stress response and anthocyanin biosynthesis in sweetpotato. BMC Plant Biol. 2024 Mar 1;24(1):156.). IbbHLH2 is highly expressed in purple tissues such as sweet potato shoot tips, young leaves, petioles, and purple tubers, while maintaining low expression levels in green tissues (mature leaves and white tubers). Furthermore, the expression level of the IbbHLH2 gene is significantly induced upon exposure to jasmonic acid and mechanical damage signals, suggesting that the promoter pIbbHLH2 of this gene may possess typical conditionally inducible characteristics.

[0012] Our previous research data showed that the IbMYB1 transcription factor can specifically bind to the MRE motif of the IbbHLH2 gene promoter to activate gene expression, while the IbbHLH2 transcription factor encoded by the IbbHLH2 gene can bind to the G-box cis-acting element in the IbMYB1 gene promoter region to enhance IbMYB1 gene expression, thus forming a signal amplification regulatory loop (Dong F, Huang Y, Dong W, Zhang Y, Kang S, Xiang W, Yao J, Gong Y, Zhang C, Li Q, Zhang D. IbMYB1-4 positively regulates purple stem and influences leaf color via dosage effect of gene expression in sweetpotato (Ipomoeabatatas (L.) Lam.). Theor Appl Genet. 2026 Jan 19;139(1):40.). At the protein level, transcription factors IbbHLH2 and IbMYB1 interact to form the MBW complex, which binds to and activates the promoters of downstream anthocyanin synthesis genes such as IbDFR, IbANS, and IbUFGT (Deng et al., 2020, Hou et al., 2023). This demonstrates that the regulation of the promoter pIbbHLH2 by the purple gene IbMYB1 in sweet potato is highly precise. Therefore, pIbbHLH2 should be an ideal endogenous promoter backbone for constructing a conditionally inducible expression system in sweet potato.

[0013] IV. Breeding Bottlenecks in Sweet Potato Flowering Regulation and the Solution Strategies of This Invention

[0014] Sweet potato is a short-day plant, and its flowering induction is strictly regulated by the photoperiod. Most cultivated varieties are the result of long-term asexual reproduction and selection, and their flowering ability under long-day conditions has gradually degenerated during domestication. Under the widespread natural cultivation conditions in my country, sweet potato hybridization breeding faces the breeding dilemma of "narrow flowering window, uncontrollable flowering period, and low hybridization efficiency." To solve the flowering problem of sweet potato, breeders have developed various artificial flowering induction techniques. These include strategies such as simulating short-day treatment through shading, grafting induction, induction by spraying exogenous plant growth regulators, and winter breeding in southern regions to induce flowering. However, these strategies all have limitations such as high cost, complex operation, and the ability to complete only one hybridization cycle per year. In traditional breeding work, a complete breeding cycle for a sweet potato variety usually takes 8-10 years.

[0015] In recent years, with the deepening of molecular biology research on plant flowering, the core role of the FLOWERING LOCUS T (FT) gene and its homologs as "florigen" has been widely confirmed. After synthesis in leaves, the FT protein is transported to the shoot apical meristem via the phloem, where it interacts with the bZIP transcription factor FD to activate the expression of floral meristem characteristic genes such as AP1 and SOC1, thereby initiating the flowering program. In model plants (Arabidopsis thaliana) and various crops (tomato, rice, citrus, etc.), overexpression of FT homologs has been shown to effectively promote flowering and shorten the growth period.

[0016] Therefore, how to overcome the photoperiodic flowering limitation of sweet potato using the florigen gene while achieving precise control over flowering time and establishing an efficient transgenic plant screening system has become a key technical problem urgently needing to be solved in the field of sweet potato molecular breeding. This invention aims to provide a method for creating sweet potato germplasm resources that can conditionally induce the expression of target genes, for example, by combining the modified pIbbHLH2 promoter with the target gene to achieve precise control over the crop and the corresponding traits of the target gene. Summary of the Invention

[0017] In order to discover more specific promoters for sweet potato genetic engineering and to obtain a breeding method that can promote flowering and shorten the growth period of sweet potatoes, this application discloses the following technical solutions:

[0018] This invention discloses a sweet potato conditionally inducible promoter, the nucleotide sequence of which is shown in SEQ ID NO:7.

[0019] A modified cis-acting element of a sweet potato conditionally inducible promoter is disclosed. The modified cis-acting element retains the sequences in the -1067 bp to -877 bp, -780 bp to -668 bp, -594 bp to -516 bp, and -511 bp to -367 bp regions of the sweet potato conditionally inducible promoter shown in SEQ ID NO:7. This process deletes all cis-acting elements involved in light signal response or binding to transcription factors such as MYB and MYC within these regions, including B-box, I-box, G-box, and MRE. Simultaneously, cis-acting elements for specific plant hormone signal responses are added at the partially deleted cis-acting element positions, and a TMVΩ sequence is added downstream to stabilize the modified promoter activity.

[0020] Preferably, the cis-acting element of the plant hormone signal response is an abscisic acid or gibberellin signal response cis-acting element; the nucleotide sequence of the modified cis-acting element of the sweet potato conditionally inducible promoter is shown in SEQ ID NO: 8.

[0021] Application of a sweet potato conditionally inducible promoter or its cis-acting element modified form in the construction of a target gene coupled expression system.

[0022] Preferably, the target gene is the sweet potato florigen gene IbFT2, whose nucleotide sequence is shown in SEQ ID NO:9, whose CDS nucleotide sequence is shown in SEQ ID NO:10, and whose amino acid sequence is shown in SEQ ID NO:11.

[0023] A method for breeding sweet potatoes that continuously flower and have purple stems and leaves is disclosed. The specific method involves constructing an expression vector by combining a conditionally inducible promoter of sweet potato with the IbFT2 gene, and then transferring the vector into sweet potato plants with purple stems and leaves for expression. This yields sweet potatoes that continuously flower and have purple stems and leaves. The method for preparing the sweet potato plants with purple stems and leaves involves highly expressing the IbMYB1 gene in the stems and leaves of sweet potatoes.

[0024] A method for conditionally inducing flowering in sweet potato breeding involves coupling a modified cis-acting element of a conditionally inducible promoter of sweet potato with the target gene IbFT2, constructing an expression vector, and then transferring it into the cultivar Xushu 48, which is not prone to flowering. The leaves are then treated with an appropriate concentration of abscisic acid or calcium nitrate solution to induce flowering as needed.

[0025] The beneficial effects of this invention are:

[0026] This invention identifies a conditionally inducible promoter endogenous in sweet potato, a modified cis-acting element thereof, a recombinant expression vector containing the promoter, and a method for spatiotemporally specific regulation of target genes using the promoter. The promoter possesses both tissue specificity (specific activation in anthocyanin-accumulating tissues) and inducibility (significantly enhanced activity after treatment with abscisic acid, gibberellin, or calcium nitrate), making it a widely applicable tool for precise regulation in plant molecular breeding, functional genomics research, and biotechnology improvement. As a preferred application, this invention also provides a method for creating conditionally induced flowering sweet potato germplasm by using the inducible promoter to drive the flowering integration gene IbFT2. Attached Figure Description

[0027] Figure 1 The pIbbHLH2 promoter sequence varies between different cultivars;

[0028] Figure 2 The FPKM values ​​of target gene expression between purple and green tissues in sweet potato cultivars were analyzed using transcriptome analysis (Xiang 2091 purple and Xiang 2091 green were leaf samples, and g39534.t1 and g39535.t1 were transcripts of the first and last segments of the IbbHLH2 gene after high-throughput sequencing sequence assembly).

[0029] Figure 3 To analyze the relative expression levels of target genes between purple and green tissues in sweet potato cultivars using RT-qPCR (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001);

[0030] Figure 4 This is a phylogenetic tree relationship based on the amino acid sequence homology of the PEBP gene family genes;

[0031] Figure 5 A schematic diagram for constructing the 35S::IbFT2 expression vector;

[0032] Figure 6 This is the phenotype of the 35S::IbFT2 transgenic positive line of Xushu 48; Figure 6 A represents the trait of a positive strain exhibiting flower buds during the seedling stage. Figure 6 B represents the flowering trait of a transgenic positive line. Figure 6 C represents a cross-sectional comparison of transgenic positive strains and wild-type cultivated varieties during the same period. Figure 6 D is a comparison of the aerial phenotypic traits of transgenic positive strains and wild-type cultivated strains during the same period;

[0033] Figure 7 To analyze the relative expression level of the target gene IbFT2 in different genetic materials using RT-qPCR (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001); Figure 7 The top left shows that the relative expression level of IbFT2 in the leaves of the 35S::IbFT2 transgenic positive line of Xushu 48 was significantly higher than that in the wild-type cultivated variety. Figure 7 The top right image shows the offspring of the cross between the ΔpIbbHLH2::IbFT2 transgenic line of Xushu 48 and the black leaf transgenic line. The relative expression level of IbFT2 in the leaves of the transgenic line with purple stems and leaves was significantly higher than that of the ΔpIbbHLH2::IbFT2 transgenic line of Xushu 48. Figure 7 The lower left shows the difference in relative expression of IbFT2 in the leaves of the ΔΔpIbbHLH2::IbFT2 transgenic line of Arabidopsis thaliana between water spray treatment and ABA treatment. Figure 7 The bottom right shows the difference in relative expression levels of IbFT2 in the leaves of the ΔΔpIbbHLH2::IbFT2 transgenic line of Xushu 48 after spraying with water, ABA, and calcium nitrate.

[0034] Figure 8 A schematic diagram for constructing the ΔpIbbHLH2::IbFT2 expression vector;

[0035] Figure 9 The phenotypic differences of the transgenic ΔpIbbHLH2::IbFT2 line in non-purple and purple transgenic sweet potato varieties under natural growth conditions; Figure 9 A represents the phenotype of the Xushu 48 transgenic line. Figure 9 B represents the phenotype of the transgenic line with purple stems and leaves. Figure 9 C represents the flowering status of the transgenic line with purple stems and leaves;

[0036] Figure 10 A schematic diagram for constructing the ΔΔpIbbHLH2::IbFT2 expression vector;

[0037] Figure 11 The ΔpIbbHLH2::IbFT2 transgenic Arabidopsis thaliana was conditionally induced to express the early flowering trait by ABA spraying; Figure 11 The transgenic lines on the left were treated with ABA. Figure 11 The right side shows the control material of the same strain that was not sprayed;

[0038] Figure 12 The difference in flowering traits of the ΔΔpIbbHLH2::IbFT2 transgenic line of Xushu 48 under conditions ( Figure 12 From left to right: water spraying treatment, ABA spraying treatment, and calcium nitrate spraying treatment. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0040] Cloning of the sweet potato pIbbHLH2 promoter sequence

[0041] Using the promoter sequence of the IbbHLH42 gene from Xu Zishu No. 8 (i.e., the IbbHLH2 gene, GenBank: OP558349.1, as shown below) as a reference, PCR amplification was performed on DNA samples from different cultivars Black_leaf, Purple_leaf, Purple_X2091, and Green_X2091 using primer combinations pbHLH2F:5'-gttggactaattaggcagtag-3' (SEQ ID NO:1) / pbHLH2R:5'-tcgcagaccacactgcagagg-3' (SEQ ID NO:2). The corresponding promoter sequences were obtained. Sequence alignment revealed certain SNP and InDel sequence differences in the promoter sequence of this gene among different cultivars. Figure 1 The prediction of cis-acting elements indicates that the differences in cis-regulatory elements caused by these SNP and InDel sequence differences do not necessarily correspond to the purple trait.

[0042] >Xuzishu8

[0043] gttggactaattaggcagtagaatggtagatataagaagaatgagaagggtaggaaagtgtcgagatcagagcagtcaccatctatatttataattcaacttttataaagttggttaacattattgaatgaaacttatatttaaaaattcattgaaaatcaaataattattattttctttggaataaaaattggccgggatttgacccggaaaaaaaattgatactcaaatactacaaatgacctttgcatacaaggaggccgaaaaattattattaaataatatatttaaaaaaagaaagacaaacaaaaggattttattttacctcaaaatattaaaatgatgactagtgaaatgaaac ccagaaaaaaaaaagtatcttttaaatttagggattaataaataaaaatatagtggaagttgagaattttaatgtataaaaagaataattatttgtgataaactactgatcgcaattgagtaattacgattccacctagacctaatgcatcaatcacatgttatatatcccattgtgcgtcgcaattacgtccttgcatcaaaatcataaacttttttgtgaacaaaaacaagtacatacaagtatacaacgtaaccaccgtaacgttgttgtctgcagattcaatctttaggcttcgacactgacaaatataatttttgagaataagatgctttttaagatactttttgggtgaagggtaagaca agaagttgtagccacaca taacttataatctttaagtatgatgatcatattttattcattaagctactctttttggagtaaccatatcatgttt gtctaatttttcatttaatagctagagagaatatttaaagatcataaatttgatttttgtcaaaattttcttagtc gatcttgctgcacaaaattttcttagtgtagtttacctctgcagtgtggtctgcgagttgttacagattaataaaa tttattcaaataaactctcagataataaaagggggtttcttataaaaaaaaataaaaattaaaaataaactatagg aacagaaaatagtcatactcttgataaataagagatgatgaagaattgaagacctcttgtgcgcagagtgcacagg taagcgatgaaaacttgaagccagataaggcaacttacaatttacctctactagaaattcttaggtagtatgtggt tctaaacgtttagaattaattagagttgtcgcatggcagcttgatcaactggtcacatgtgtgaagtttagaagaa gaaatcagggatcgagtctcatcagtggtaatgtaggagcaaccccttaaagtgagggggtccttgtgtccggttt agtccactgaggctcaaatccacccccatatgagggtgaaatcgggtgccactaaatcacaagtctttgacagaag aattaaatagagctgaaaaacctaactaaatttataataaacaaaataaaaaaaataaaaagtgacactaatatag taatatgatacacgtgggacacataaaaggcaaggacaaaaacctaatcttgaattctcctattttgtcgtctctt tccccagtccctatacccgaccggttgacaccaaccagtcaaaatccaactccccgacaaacaaataaattcaacc ttaaccccttcaactcttaatttcatatgtaaaccactctgtctcggatctttccatcttcaattcatcatcgtca cgcaaatctgtgttgcagatcagagattccacccccgccatgcatgcatcgccgccttaattagcttctctgtaat ctatat (SEQ ID NO:3)

[0044] Black leaf

[0045]

[0046] >Purple_X2091

[0047]

[0048] >Green_X2091

[0049]

[0050] Tissue-specific expression activity analysis of pIbbHLH2 gene in sweet potato

[0051] Three groups of experimental materials from sweet potato cultivars with mutations in the purple trait were selected respectively:

[0052] The first group is the purple sweet potato cultivar Zhezishu No. 1. After bud mutation, the IbMYB1-2 gene in this material was deleted, resulting in the loss of expression of the purple gene IbMYB1. The tubers lost the purple trait and became yellow-skinned and light orange-red flesh. The material after bud mutation was named Xiangshu 99. The color trait of the bud mutation line is stable (Zhang D, Tan Y, Dong F, Zhang Y, Huang Y, Zhou Y, Zhao Z, Yin Q, Xie X, Gao X, Zhang C, Tu N. The Expression of IbMYB1 Is Essential to Maintain the Purple Color of Leaf and Storage Root in Sweet Potato [Ipomoea batatas (L.) Lam]. Front Plant Sci. 2021 Sep 23;12:688707.).

[0053] The second group consists of Purple_X2091, a material whose stems, leaves, and other above-ground parts are purple. This material has a deletion mutation in the IbMYB1-3 gene, causing the encoded purple gene IbMYB1 to lose its expression ability. The stems, leaves, and other above-ground parts change from purple to green. The bud mutation material is named Green_X2091, and the green trait is stable in the bud mutant (Zhang D, Tan Y, Dong F, Zhang Y, Huang Y, Zhou Y, Zhao Z, Yin Q, Xie X, Gao X, Zhang C, Tu N. The Expression of IbMYB1 Is Essential to Maintain the Purple Color of Leaf and Storage Root in Sweet Potato [Ipomoea batatas (L.) Lam]. Front Plant Sci. 2021 Sep 23;12:688707.).

[0054] The third group of materials came from the cultivated variety Xiangshu 20. After bud mutation, the IbMYB1-4 gene was obtained. The purple gene IbMYB1 encoded by this gene is mainly expressed in the outer periphery of the stem. The vines changed from green to purple. The material before bud mutation was named Xiangshu 20 Green, and the material after bud mutation was named Xiangshu 20 Purple. The purple trait of the bud mutation line was stable (Dong F, Huang Y, Dong W, Zhang Y, Kang S, Xiang W, Yao J, Gong Y, Zhang C, Li Q, Zhang D. IbMYB1-4 positively regulates purple stem and influences leaf color via dosage effect of gene expression in sweetpotato (Ipomoea batatas (L.) Lam.). Theor ApplGenet. 2026 Jan 19;139(1):40.).

[0055] Transcriptome data Figure 2 Analysis showed that under natural growth conditions, the expression of the IbbHLH2 gene in tissues such as roots, stems, and leaves was highly correlated with the expression level of the IbMYB1 gene. When the IbMYB1 gene was highly expressed in the tubers of Zhezishu No. 1, or in the purple stems and leaves of Purple_X2091, or in the purple stems of X20, the expression level of the IbbHLH2 gene was significantly upregulated. In other non-purple parts, the expression level of the IbbHLH2 gene remained low.

[0056] Figure 2 FPKM values ​​of target gene expression between purple and green tissues in sweet potato cultivars were analyzed using transcriptome analysis (Xiang 2091 purple and Xiang 2091 green were leaf samples).

[0057] RT-qPCR analysis confirmed this conclusion, showing that the IbbHLH2 gene maintained low expression levels under non-IbMYB1 gene activation conditions, but its expression was significantly upregulated after IbMYB1 gene activation, meeting the basic requirements for conditional expression induction. Figure 3 (as described). Figure 3 To analyze the relative expression levels of target genes between purple and green tissues in sweet potato cultivars using RT-qPCR (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001).

[0058] Bioinformatics analysis identified the core sequence of the pIbbHLH2 gene promoter region as ΔpIbbHLH2 (underlined below). The sequences in the -1067 bp to -877 bp, -780 bp to -668 bp, -594 bp to -516 bp, and -511 bp to -367 bp regions of the sweet potato conditionally inducible promoter shown in SEQ ID NO:6 were retained. All cis-acting elements (such as B-box, I-box, G-box, and MRE) involved in light signal response or binding to transcription factors like MYB and MYC in these regions were deleted. Cis-acting elements for specific plant hormone signal responses were added at the partially deleted cis-acting element positions, and a TMVΩ sequence was added downstream to stabilize the modified promoter activity, resulting in the modified abscisic acid and gibberellin-specific inducible epiprogenitor sequence.

[0059] pIbbHLH2 Startup Core Sequence

[0060] The boxes indicate the main functional cis-acting elements in the core sequence range of the pIbbHLH2 start-up region, while the underlined segments are replaceable or deleteable sequences.

[0061] agaagttgtagccacacataacttataatctttaagtatgatgatcatattttattcattaagctactctttttggagtaaccatatcatgtttgtctaatttttcatttaatagctagagagaatatttaaagatcataaatttgatttttgtcaaaattttcttagtcgatcttgctgcacaaaattttcttagtgtagtttacctct GCAGTGtggt ctgcgagttgttacagATTAAT aaaatttattcaaataaactctcagataataaaaagggggtttcttataaaaaaaaataaaaattaaaaataaactataggaacagaaaatagtcatactcttgataaataagagatgatgaagaattgaagacctcttgtgcgcagagtgcacaggtaagcgatgaaaacttgaagcc AGATAAGGcaacttacaatttacctcta ctagaaattcttaggtagtatgtggttctaaACGTTTAGAATTAATtagagttgtcgcatggcagcttgatcaactggtcacatgtgtgaagtttagaagaagaaatcagggatcgagtctCATCAGTGgtaatgtaggagcaaccccttaaagtgagg gggtccttgtgtccggtttagtccactgaggctcaaatccacccccatatgagggtgaaatcgggtgccactaaatcacaagtctttgacagaagaattaaatagagctgaaaa ACCTAActaaatttataataaacaaaataaa aaaaataaaaagtgacactaatatagtaatatgatacacgtgggacacataaaaggcaaggacaaaACCTAAtctt gaattctcctattttgtcgtctctttccccagtccctatacccgaccggttgacaccaaccagtcaaaatccaact ccccgacaaacaaataaattcaaccttaaccccttcaactcttaatttcatatgtaaaccactctgtctcggatct ttccatcttcaattcatcatcgtcacgcaaatctgtgttgcagatcagagattccacccccgccatgcatgcatcg ccgccttaattagcttctctgtaatctatat (SEQ ID NO:7)

[0062] >The modified inducible promoter ΔΔpIbbHLH2 (SEQ ID NO:7) First underscore The gibberellic acid (GA) responsive cis-acting element replaces the original SEQ ID NO:6. First underscore sequence ; Delete the original SEQ ID NO:6 second Underscore sequence; second underscore The abscisic acid ABA-responsive cis-acting element replaces the original SEQ ID NO:6 CATCAGTG motif; Third underscore The gibberellic acid (GA) responsive cis-acting element replaces the original SEQ ID NO:6. Third time Underline sequence; fourth underline (The sequence is a TMVΩ sequence, used to maintain the functional stability of the promoter sequence). After the sequence modification design is completed, the company directly synthesizes the target sequence fragment.

[0063] AGAATTTGTAGCCACGCATAACTTATAATCTTAAGTATGATGATCATATTTTATTCATTAAGCTACTCTTTTTGGAGTAACCATATCATGTTTGTCTAATTTTTCATTTAATAGCTAGAGAATATTTAAAGATCATAAATTTGATTTTTGTCAAAATTTTCTTAGTCGATAGCTGCACAAAATTTTCTTAGTGTAGTTTACCTCTG TACGTAAAACAG AACCTTTCTTACTCTGTTGTAACAAGATATCCTTAACAAAATCTGTTGACCTTTCTTACAAAATTTAATAAACTATAGGAACAGAAAATAGGCATACTCTTGATAAATAAGAGATGATGAAGAATTGAAGACCTCTTGTGCGCAGAGTGCACAAGTAAGCGATGAAAACTTGAAGACTAGAGTTGTCGCATGGTAGCTTGATCAATTGGTCACATGTGTGAAGTTTGGAAGAAGAAATCAAGGATCGAGTCTCAT GTCAGAAAGCTTGCCACGTGGCTGCAGTGCCATTGCCACCGGATCAGCC ACGTGGCTCCAGTGCCATTGCCACCGGATGACGCACAATCCCAC GCAATGTAGGAGCAACCCCTTAAAGTGAGGGGGTCCTTGTGCCTGGTTTAGTCCACTGAGGCTCAAATCCACCCCATCCACCCATATGAAGGTGAAACCGGGTGTCACTAAATCACAAGTCTTTGACAGAAGAATTAAATAGAGCTGAAAA TACGTAAAACAGAACCTTTCTTACTCTGTTG TAACAAGATATCCTTAACAAAATCTGTTGACCTTTCTTAC ATATCATACCTGCAG ACAATTACCAACAACAACAAA CAACAAACAACATTACAATTACTATTTACAATTAC (SEQ ID NO:8)

[0064] Cloning of sweet potato apolloniferin gene IbFT2

[0065] Homologous sequence alignment from the sweet potato reference genome database (https: / / sweetpotato.uga.edu / ) yielded PEBP gene family genes homologous to the FT (FLOWERING LOCUS T) of Arabidopsis thaliana or morning glory. Among them, IbFT2 is most closely related to the FT of Arabidopsis thaliana or morning glory (e.g., Figure 4 As shown in the reference (Huang Zherui, Xin Shuli, Zhao Tian, ​​Liu Yonghua, Zhu Guopeng. Identification of PEBP Gene Family in Sweet Potato and Screening of Key PEBP Genes Regulating Storage Root Development, Journal of Tropical Crops, 2024, 45(3): 459-472.), and IbFT2 is mainly expressed in the stem and leaf parts. The CDS sequence of IbFT2 can be cloned from the cDNA of sweet potato cultivars such as Xiangshu 99 under short-day induction treatment using the primer combination IbFT2F: 5'-ATGAGAAGGGGAATAGTAGAC-3' / IbFT2R: 5'-TTAACGGGAAGGACCAGGGTAATC-3'. The gene sequence, CDS and amino acid sequence of this gene are shown below.

[0066] The gene sequence of IbFT2 has the ID Ibat.Brg.11B_G021460 in the reference genome. In the following sequences, the underlined parts are exons, and the remaining parts are introns.

[0067] ATGAGAAGGGGAATAGTAGACCCTCTGGTGGTGGGGCGCGTGATCGGAGACGTTGTGGATCCGTTCAC TCGGTCCGTTGATCTTAGGGTGATTTACAACAACGAGGTGGATATCAGGAATGGGTGTGAGATGAGGCCTTCTCAG CTCATCAACCCACCTAGGGTTGAAATCGGCGGACACGATCTCCGTACTTTCTACACTCTG GTACCTTCCACCACCCAAACTACCTAGCTAGGGTTTTTAATTTCTCCCTTATGCCAAGGGCTTTTGTGGTCCAGTGGCATCAAACCCTTCCTGTATACGGGAGGTGGTAGGTTTGAGCCTGGTTGAAAGTGTTGCTGTAATAGAGCCAGCAGCATTTTCAAAAAGTTCTCCCTCTTATTATTCTATATGTATATATATATATATATATATATATATATATATATGACTTTGTGTTTTTTAAG GTTATGGTGGATCCTGATGCTCCAAGCCCAACCTCTCCAACCCTCAGGGAATACCTCCACTGGTCAGTCCCCAACTATATATTTCTTCCCCAATATAATAACACTACCGCCCTCCGTTTGGTAAATAATCAGCCTATCAGCCAATTTTGGCTTATTTCACCACTATTAGTTGTTTGGTTAATAAGCTTTTTGTAACTCTAAAATGCTAAAATTCAAAAGGCTACTCAAACAAGCCTTTTCAATTAGCTTTTTGAGAAA AACGAAATTATACTAAACACCTATCAACTAACAGCTAATCTATAAACAACTTTCTACATTATCAACAAATCATACCTTTTAACCCAAACAGTCAACCCAATCAGCTAACAGCCATTACCAAAAGGGCCATTTTTTATTGAATCAAGTTATTTTTTATTCAATTTTTCATAATTTTAAGTGTAGTATTATATATATAT ATATATATATATATATATATATATATATATATATATATATATATATATATAAAATTAAAGTATTATTAAACACAAAAAAATAAATTTAAAAATAATATAGAAAATAAACAAAGAAAAGGAATTAGTTTAATGAATAAATAGTAAACCTAAAACGGGACATGGTGTGAGTATTAGTGAATACAATCTTTATATAGTT TTTATTTTTATTTTAAATATTAGTTAAACACACACACAAACAAAACAAAAAAAAAAAAAATTCAAATCAATAAGAAGTTAAACGGTTAAAGTAAATAAGCAAATCGATATGATGAAGCATATATAATAGTGTAGATGAGATTAGATAGCAGAGGTTTCTTCTCTCTTAACCTAATTAATGATCGATTTGGTACTACAG GTTGGTGACTGATATACCAGGAACTACGGGAGCAAGTTTCGGCAATGAAGCGATATTCTACGAGCCTCCGAGGCCGTCAATGGGAATCCACCGTTTTGTGTTTGTG CTTTTCCGGCAACTTGGCCGGCAAACCGTTTATGCACCGCATTGGCGCCAAAATTTCAACACTCGAAACTTTGCTG AGCTTTACAATCTTGGTTTGCCAGTGGCCGTCACTTACTTTAACGGCCAAAGGGAGGGTGGCACCGGCGGTCGATC TCCGGCGGAGCCCTGGGCAGCCGATTACCCTGGTCCTTCCCGTTAA (SEQ ID NO: 9)

[0068] >CDS of IbFT2

[0069] ATGAGAAGGGGAATAGTAGACCCTCTGGTGGTGGGGCGCGTGATCGGAGACGTTGTGGATCCGTTCACTCGGTCCGTTGATCTTAGGGTGATTTACAACAACGAGGTGGATATCAGGAATGGGTGTGAGATGAGGCCTTCTCAGCTCATCAACCCACCTAGGGTTGAAATCGGCGGACACGATCTCCGTACTTTCTACACTCTGGTTATGGTGGATCCTGATGCTCCAAGCCCAACCTCTCCAACCCTCAGGGAATACCTCCACTGGTTGGTGACTGATATACCAGGAACTACGGGAGCAAGTTTCGGCAATGAAGCGATATTCTACGAGCCTCCGAGGCCGTCAATGGGAATCCACCGTTTTGTGTTTGTGCTTTTCCGGCAACTTGGCCGGCAAACCGTTTATGCACCGCATTGGCGCCAAAATTTCAACACTCGAAACTTTGCTGAGCTTTACAATCTTGGTTTGCCAGTGGCCGTCACTTACTTTAACGGCCAAAGGGAGGGTGGCACCGGCGGTCGATCTCCGGCGGAGCCCTGGGCAGCCGATTACCCTGGTCCTTCCCGTTAA(SEQ ID NO: 10)

[0070] >Amino acid sequence of IbFT2

[0071] MRRGIVDPLVVGRVIGDVVDPFTRSVDLRVIYNNEVDIRNGCEMRPSQLINPPRVEIGGHDLRTFYTLVMVDPDAPSPTSPTLREYLHWLVTDIPGTTGASFGNEAIFYEPPRPSMGIHRFVFVLFRQLGRQTVYAPHWRQNFNTRNFAELYNLGLPVAVTYFNGQREGGTGGRSPAEPWAADYPGPSR(SEQ ID NO: 11)

[0072] RT-qPCR analysis of target gene expression levels in transgenic materials

[0073] The primers used for gene expression analysis during the implementation of this technology are listed in Table 1 below:

[0074] Table 1. Primer list used for gene expression analysis during the implementation of this technology.

[0075]

[0076] Promoters and their application in target gene coupled expression systems

[0077] Application Case 1

[0078] Flower-promoting function of the IbFT2 gene

[0079] Building upon pCAIMBIA1300, such as Figure 5 The expression vector of 35S::IbFT2::NOS Terminal shown was transformed into sweet potato cultivar Xushu 48 using Agrobacterium-mediated transformation of embryogenic callus suspension cell lines (Zang N, Zhai H, Gao S, Chen W, He S, Liu Q. (2009). Efficient production of transgenic plants using the bargene for herbicide resistance in sweetpotato. Scientia Horticulturae, 122(3),404). It was found that flower buds differentiated first in the positive embryogenic callus seedling stage before seedling formation, and the flowering habit of the positive plants after differentiation was no longer limited by the length of daylight. More than 15 genes in the PEBP gene family have been predicted for sweet potato cultivars. Some of these may be flowering-promoting genes, some may be flowering-inhibiting genes, and some may not be directly related to flowering regulation. Currently, there is no publicly available research data to prove which gene is the flowering-promoting gene. Functional identification of genes in higher plants is not an easy task, especially when transferring the gene into a plant. Verifying the function of transgenic plants usually requires a lot of complicated trial and error and screening. However, this data shows that the IbFT2 gene has a direct effect on promoting sweet potato flowering and can be directly used for positive regulation of plant flowering. Figure 6 This is the phenotype of the 35S::IbFT2 transgenic positive line of Xushu 48; Figure 6 A represents the trait of a positive strain exhibiting flower buds during the seedling stage. Figure 6 B represents the flowering trait of a transgenic positive line. Figure 6 C represents a cross-sectional comparison of transgenic positive plants and wild-type cultivated varieties under the same growth conditions. Figure 6D represents a top-view comparison of the traits of transgenic positive plants and wild-type cultivated varieties under the same growth conditions. For example... Figure 7 As shown in the top left figure, the expression level of the IbFT2 gene in the leaves of the corresponding transgenic lines was significantly upregulated.

[0080] Application Case 2

[0081] Sweet potato ΔpIbbHLH2 promoter core sequence combined with IbFT2 gene

[0082] Build as Figure 8 The pCAIMBIA1300::ΔpIbbHLH2::IbFT2::NOS terminal expression vector was transformed into sweet potato cultivar Xushu 48 using Agrobacterium-mediated embryogenic callus suspension cell line transformation (Zang N, Zhai H, Gao S, Chen W, He S, Liu Q. (2009). Efficient production of transgenic plants using the bargene for herbicide resistance in sweetpotato. Scientia Horticulturae, 122(3),404). It was found that the positive transgenic plants under natural growth conditions did not have obvious flowering traits (IbMYB1 was not expressed in the stems and leaves), indicating that in plants where the IbMYB1 gene was not expressed, the promoter activity of ΔpIbbHLH2 was not sufficient to induce the expression of downstream IbFT2 and thus flowering. However, by performing directional hybridization of the ΔpIbbHLH2::IbFT2 transgenic line Xushu 48 with Black Leaf, and screening the offspring to introduce the ΔpIbbHLH2::IbFT2 transgenic element into sweet potato plants with purple stems and leaves (IbMYB1 is highly expressed in stems and leaves; the method described in invention patents CN 114134154 A or CN 116103310A can yield sweet potato plants with purple stems and leaves), due to the positive regulatory effect of the transcription factor IbMYB1 on the core sequence of the ΔpIbbHLH2 promoter, germplasm resources that continuously express the florigen gene, flower, and have purple stems and leaves can be obtained. The creation of such materials enriches the categories of ornamental sweet potato germplasm resources. Figure 9 The phenotypic differences of the transgenic ΔpIbbHLH2::IbFT2 line in non-purple and purple transgenic sweet potato varieties under natural growth conditions; Figure 9 A represents the phenotype of the Xushu 48 transgenic line. Figure 9 B represents the phenotype of the transgenic line with purple stems and leaves. Figure 9 C shows flowering details of the transgenic line with purple stems and leaves. (Example) Figure 7As shown in the upper right corner, the expression level of the IbFT2 gene in the leaves of the corresponding transgenic line was significantly upregulated, indicating the function of induced expression under the ΔpIbbHLH2 promoter condition.

[0083] Application Case 3

[0084] Sweet potato ΔΔpIbbHLH2 promoter core sequence coupled with IbFT2 gene for co-expression

[0085] Build as Figure 10 The expression vector pCAIMBIA1300::ΔΔpIbbHLH2::IbFT2::NOS terminal shown was transformed into wild-type Arabidopsis thaliana using the standard floral dip method. Homozygous F2 transgenic plants were obtained, but none showed obvious early flowering phenotype. Seven days after transplanting, Arabidopsis plants treated with 50 μM ABA showed significantly earlier bolting and flowering than wild-type and untreated control transgenic plants. Figure 7 As shown in the lower left, the expression level of the IbFT2 gene in the leaves of the corresponding transgenic line was significantly upregulated. This case demonstrates that the coupled expression of ΔΔpIbbHLH2 and IbFT2 in Arabidopsis thaliana can specifically induce flowering under appropriate ABA treatment conditions. Similarly, it can be anticipated that, in this application case, the coupled expression of the ΔΔpIbbHLH2 promoter core sequence with other target genes can also achieve the effect of specifically inducing the expression of target genes. Figure 11 The transgenic Arabidopsis thaliana with ΔpIbbHLH2::IbFT2 structure conditionally induced to express the early flowering trait; the left side shows the transgenic line treated with ABA, and the right side shows the control material of the same line without ABA treatment.

[0086] Application Case 4

[0087] Sweet potato ΔΔpIbbHLH2 promoter core sequence coupled with IbFT2 gene for co-expression

[0088] The already constructed Figure 10The expression vector pCAIMBIA1300::ΔΔpIbbHLH2::IbFT2::NOS terminal shown was transformed into Xushu 48 using Agrobacterium-mediated embryogenic callus suspension cell line transformation (Zang N, Zhai H, Gao S, Chen W, He S, Liu Q. (2009). Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato. Scientia Horticulturae, 122(3), 404). The transgenic positive plants showed no obvious flowering phenotype compared to the Xushu 48 cultivar. 30 days after transplanting, the transgenic plants were sprayed with 40 mg L... -1 ABA and 750 kg hm -2 The treatment involved spraying calcium nitrate twice, with a 7-day interval. Compared to the untreated transgenic lines, the treated group showed a large number of flower buds appearing in the axillary buds. Figure 7 As shown in the lower right corner, the expression level of the IbFT2 gene in the leaves of the corresponding transgenic line was significantly upregulated. This case illustrates that in sweet potato, the coupled expression of ΔΔpIbbHLH2 and IbFT2 can specifically induce flowering under appropriate concentrations of ABA or calcium nitrate treatment.

[0089] Similarly, it can be anticipated that in sweet potatoes, the ΔΔpIbbHLH2 promoter core sequence coupled with other target genes can also achieve the effect of specifically inducing the expression of target genes. Figure 12 The differences in flowering traits induced under the conditions of transgenic line Xushu 48ΔΔpIbbHLH2::IbFT2 (from left to right: water, ABA and calcium nitrate spraying treatments).

Claims

1. A conditionally induced promoter for sweet potatoes, characterized in that, The nucleotide sequence of the sweet potato conditionally inducible promoter is shown in SEQ ID NO:

7.

2. A modified cis-acting element of the sweet potato conditionally induced promoter as described in claim 1, characterized in that, The modified sweet potato conditionally inducible promoter cis-acting element is obtained by retaining the -1067 bp to -877 bp, -780 bp to -668 bp, -594 bp to -516 bp, and -511 bp to -367 bp regions of the sweet potato conditionally inducible promoter shown in SEQ ID NO:7, thereby deleting all cis-acting elements such as B-box, I-box, G-box, and MRE involved in light signal response or binding to transcription factors such as MYB and MYC in these regions. At the same time, cis-acting elements for specific plant hormone signal responses are added at the positions of the partially deleted cis-acting elements, and a TMVΩ sequence is added downstream to stabilize the activity of the modified promoter.

3. The modified cis-acting element of the sweet potato conditionally induced promoter according to claim 2, characterized in that, The cis-acting element of the plant hormone signal response is the cis-acting element of the abscisic acid or gibberellin signal response.

4. The modified cis-acting element of the sweet potato conditionally induced promoter according to claim 3, characterized in that, The nucleotide sequence of the modified cis-acting element of the sweet potato conditionally inducible promoter is shown in SEQ ID NO:

8.

5. A target gene coupled expression system, characterized in that, The target gene coupled expression system comprises the following: (1) the sweet potato conditionally inducible promoter of claim 1 or the cis-acting element modified body of any one of claims 2-4; and (2) the target gene.

6. The target gene coupled expression system according to claim 5, characterized in that, The target gene is the IbFT2 gene, which is homologous to the sweet potato florigen gene. Its nucleotide sequence is shown in SEQ ID NO:9, its CDS nucleotide sequence is shown in SEQ ID NO:10, and its amino acid sequence is shown in SEQ ID NO:

11.

7. A method for breeding sweet potatoes that continuously flower and have purple stems and leaves, characterized in that, The specific method of the sweet potato breeding method is as follows: after constructing an expression vector by combining the sweet potato conditionally inducible promoter described in claim 1 with the IbFT2 gene, the vector is transferred into sweet potato plants with purple stems and leaves for expression, thereby obtaining sweet potatoes that continuously flower and have purple stems and leaves.

8. The sweet potato breeding method according to claim 7, characterized in that, The method for preparing the purple-stemmed sweet potato plant is as follows: the IbMYB1 gene is highly expressed in the stems and leaves of sweet potato.

9. A method for conditionally inducing flowering in sweet potatoes, characterized in that, The specific method of the sweet potato breeding method is as follows: the cis-acting element of the sweet potato conditionally inducible promoter described in claims 2-4 is coupled with the target gene IbFT2 described in claim 6, and after constructing the expression vector, it is transferred into the cultivar Xushu 48, which is not easy to flower. The leaves are treated with an appropriate concentration of abscisic acid or calcium nitrate solution, and the plants can be induced to flower as needed.

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