WO2023098481A1 - 甘薯IbSAP15基因在调控甘薯叶型与花型中的应用 - Google Patents

甘薯IbSAP15基因在调控甘薯叶型与花型中的应用 Download PDF

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WO2023098481A1
WO2023098481A1 PCT/CN2022/132557 CN2022132557W WO2023098481A1 WO 2023098481 A1 WO2023098481 A1 WO 2023098481A1 CN 2022132557 W CN2022132557 W CN 2022132557W WO 2023098481 A1 WO2023098481 A1 WO 2023098481A1
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sweet potato
ibsap15
gene
flower
type
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French (fr)
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刘亚菊
谢昊
杨强强
王笑笑
李染秋
闫会
后猛
唐维
王欣
张允刚
李强
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江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention belongs to the technical field of plant genetic engineering, and relates to an application of sweet potato IbSAP15 gene in regulating sweet potato leaf type and flower type.
  • the zinc finger domain contains multiple cysteine and histidine residues, which can bind to zinc ions through these residues and fold themselves into a "finger" shape, so they are called zinc fingers.
  • Proteins containing zinc finger domains are called zinc finger proteins (Kluget et al., 1987).
  • Zinc finger proteins can interact with DNA, RNA or other proteins to regulate gene transcription, translation and other processes.
  • Members of the plant stress-associated protein (SAP) family contain A20/AN1 zinc finger domains, which have ubiquitin ligase activity or transcription factor activity, or can sense changes in the redox state in cells through conformational changes, and regulate plant stress Biological and abiotic stress responses and growth and development.
  • Sweet potato is an important food crop, but also an important vegetable, feed and industrial raw material crop. Since the 21st century, sweet potatoes have gradually been used as ornamental plants. According to the different parts used for viewing, ornamental sweet potatoes can be divided into four types: leaf-viewing type, vine-viewing type, flower-viewing type, and potato-viewing type (Ren Yun et al., 2005).
  • the leaves of sweet potato have various shapes, such as heart-shaped, palm-shaped, scherd-shaped, etc.
  • the leaves of sweet potato for foliage are mainly notched and chicken-claw-shaped, or the leaf color is purple, tender green, and mixed colors.
  • the corolla of the sweet potato is funnel-shaped, composed of 5 petals, which is consistent with the flower shape of the morning glory, and the flower colors are mainly white, lavender, and light pink.
  • Sweet potatoes for flower viewing require easy flowering, large flower volume, and long flowering period. Flowering can be induced by short days (Meng Yusha et al., 2019), and the flower volume of sweet potato can be increased by applying fertilizers (Qiu Caifei et al., 2010). Ornamental sweet potatoes can be grown indoors through hydroponics or substrate cultivation to decorate the indoor environment. Hydroponics is easy to operate, low in cost, and can form a vibrant ecosystem with aquatic organisms such as ornamental fish, turtles or other aquatic plants. Potted sweet potatoes can be used for decoration in living rooms, balconies, offices, conference halls, etc. Ornamental sweet potatoes planted outdoors can be used for three-dimensional decoration, slope greening, sidewalk greening, and rock greening.
  • the invention provides an application of the sweet potato IbSAP15 gene in regulating the leaf shape and flower shape of the sweet potato.
  • the IbSAP15 gene of the present invention is a gene encoding AN1 zinc finger protein cloned from sweet potato, and its encoded protein contains two conserved AN1 zinc finger domains, which is a DNA molecule with a specific sequence, and its CDS is 579bp , the nucleotide sequence is shown in SEQ ID NO.1.
  • the present invention also provides the protein IbSAP15 encoded by the above-mentioned IbSAP15 gene, which contains 192 amino acid residues, and the amino acid sequence is shown in SEQ ID NO.2.
  • the present invention also provides a method for regulating the leaf type and flower type of sweet potato, comprising the following steps: overexpressing the IbSAP15 gene described in the above technical scheme in sweet potato.
  • the present invention finds for the first time that the overexpression of the sweet potato IbSAP15 gene in sweet potato can deepen the notch of the leaves, crack the funnel-shaped corolla, change the leaf and flower shapes of sweet potatoes, and is suitable for cultivating sweet potato germplasm with different leaf and flower shapes, increasing the Flower-viewing/leaf-viewing sweet potato varieties.
  • the present invention also provides a recombinant plant expression vector, which includes a starting vector and the IbSAP15 gene described in the above technical solution.
  • the starting vector is a plant expression vector pGWB12.
  • Figure 1 is the linearized entry vector Schematic
  • Figure 2 is the recombination entry vector Schematic
  • Fig. 3 is a schematic diagram of the structure of the plant overexpression vector pGWB12;
  • Fig. 4 is the structural representation of recombinant plant expression vector pGWB12-IbSAP15;
  • Fig. 5 is the detection result figure of IbSAP15-OE strain, and Zi8, -CK, +CK are the detection result of cultivated species, negative control and positive control respectively, and IbSAP15-OE is the detection result of different IbSAP15-OE strains;
  • Fig. 6 is a graph showing the relative expression level of IbSAP15 in the IbSAP15-OE strain and the control strain;
  • Fig. 7 is a comparison diagram of the leaf type of the IbSAP15-OE strain and the cultivar strain, the arrow points to the position where the notch becomes deeper, and the scale bar is 2cm;
  • Figures 8 to 9 are comparison diagrams of the flower types of the IbSAP15-OE strain and the cultivar strain, wherein Fig. 8 is a front view of the flower of the IbSAP15-OE strain and the cultivar strain, and Fig. 9 is a view of the flower of the IbSAP15-OE strain and the cultivar strain The top view of the flower of the cultivar line, the scale bar is 2 cm.
  • Embodiment 1 Cloning of IbSAP15 gene and construction of overexpression vector
  • the sweet potato variety used in the examples is Xu Zishu No. 8 (Zi8). Through cross-breeding, the parents are Xu Zishu No. 3 and Wanzi 56. It is a fresh potato with high yield, high dry rate and high anthocyanin content. food variety. Xu Zishu No. 8 has a scherd-shaped leaf shape and is easy to flower. It is also a high-quality ornamental sweet potato.
  • the CDS sequence of IbSAP15 was obtained from the transcriptome sequencing data of sweet potato (see GenBank accession number MW661075, https://www.ncbi.nlm.nih.gov/nuccore/2026807425).
  • Design specific primers tIbSAP15-F (SEQ ID No.3) and tIbSAP15-R (SEQ ID No.4) according to the CDS sequence of IbSAP15 amplify the CDS full-length of IbSAP15 from each tissue mixed sample cDNA of sweet potato by PCR, and connect to the starter vector above, the recombinant entry vector was constructed Through the LR reaction, the sequenced correct The IbSAP15 fragment above was substituted into the plant expression vector pGWB12 to obtain the recombinant plant expression vector pGWB12-IbSAP15.
  • the specific steps are:
  • RNA extraction and integrity detection of sweet potato mixed samples Collect samples of hydroponic sweet potato leaves, adventitious roots, and flowers, grind them into powder in liquid nitrogen, and mix them in equal proportions. Take about 100mg of sample powder, and use Huayueyang Universal Rapid Plant RNA Extraction Kit to extract total RNA from the mixed sample according to the instructions. Use a Nano drop 1000 UV spectrophotometer to detect the concentration of RNA, and take 1 ⁇ g of total RNA to detect the integrity by 1% agarose gel electrophoresis.
  • First-strand cDNA synthesis Use the ReverTra qPCR RT Master Mix with gDNARemover (Toyobo) cDNA synthesis kit to synthesize the first-strand cDNA. Using 500ng RNA as a template, the total system is 10 ⁇ L, and the specific synthesis steps refer to the kit instructions.
  • Amplification of IbSAP15 and construction of an entry vector use the primer pair tIbSAP15-F (SEQ ID NO.3) and tIbSAP15-R (SEQ ID NO.4) to perform PCR amplification using the sweet potato mixed sample cDNA as a template.
  • the DNA polymerase used was Taq TM Version 2.0plus dye (TaKaRa).
  • the total reaction system was 50 ⁇ L, including 25 ⁇ L of TaqVersion 2.0plus dye (2 ⁇ ), 1 ⁇ L of cDNA template, 2 ⁇ L of 10 mmol ⁇ L -1 forward and reverse primers, and 20 ⁇ L of ddH 2 O.
  • the PCR amplification program was: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 60°C for 15s, extension at 72°C for 15s, 35 cycles; full extension at 72°C for 2min; incubation at 4°C.
  • PCR products were detected by electrophoresis and gel recovery, and the gel recovery products were connected to a linearized (Invitrogen), construct the entry vector Escherichia coli DH5 ⁇ was transformed. Pick positive clones and send them to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and select single clones with correct sequences and directions to extract Plasmids were used in subsequent experiments.
  • tIbSAP15-F (SEQ ID NO.3): 5'-ATGGGAGGAGGAACAGAAGCT-3',
  • tIbSAP15-R (SEQ ID NO. 4): 5'-TCAAAAAGCTTTAACAGAAGGTATG GTAGTTGG-3'.
  • IbSAP15 expression vector construction take 100ng Mix well with 100ng pGWB12 plasmid, add 2 ⁇ L Gateway LR Clonase II Enzyme Mix (Invitrogen), rehydrate to 10 ⁇ L, and incubate at 25°C for 1h. Add 1 ⁇ L of proteinase K and incubate at 37°C for 10 min to terminate the reaction. Take 1 ⁇ L of the reaction solution and transform it into Escherichia coli DH5 ⁇ , pick positive clones to extract the plasmid, and obtain the plant expression vector pGWB12-IbSAP15.
  • Example 2 Overexpression of the IbSAP15 gene in sweet potato
  • the IbSAP15 gene was overexpressed in sweet potato, and the function of the IbSAP15 gene was analyzed by comparing the phenotypes of the overexpressed lines and cultivars.
  • Agrobacterium-mediated sweet potato genetic transformation methods include infection, co-cultivation, screening and identification, induction of seedlings, etc., followed by rapid propagation through tissue culture and cuttings after transplanting for propagation.
  • the antibiotics and hormones used include 2,4-dichlorophenoxyacetic acid (2,4-D), abscisic acid (ABA), kanamycin (Ka namycin, Kan), tide Homomycin B (Hyg), Rifampin (Rif), Cefotaxime sodium (Cef).
  • the medium used is: YEB (yeast extract 1g ⁇ L -1 , beef extract 5g ⁇ L -1 , sucrose 5g ⁇ L -1 , peptone 5g ⁇ L -1 , MgSO 4 7H 2 O 0.5g ⁇ L -1 1 ), MS (purchased from Beijing Kulaibo Technology Co., Ltd.), MSD (MS+2mg ⁇ L -1 2,4-D).
  • Other reagents are: Acetosyringone (AS). Specific steps are as follows:
  • the plant expression vector pGWB12-IbSAP15 was transformed into the competent Agrobacterium strain GV3101 by liquid nitrogen freeze-thaw method, and positive clones were picked and inoculated into 1 mL of YEB liquid medium containing 50 mg ⁇ L -1 Kan and 20 mg ⁇ mL -1 Rif , overnight at 28°C. Aspirate 500 ⁇ L of the overnight cultured bacterial solution, transfer it to a new 50 mL YEB liquid medium containing 50 mg ⁇ L -1 Kan and 20 mg ⁇ mL -1 Rif, and cultivate until the OD 600 reaches about 0.6.
  • tissue-cultured seedlings After the number of tissue-cultured seedlings was large, some of the seedlings with better growth were selected for transplanting. Clean the medium on the tissue culture seedlings with water, transplant them into nutrient soil, keep them moist with plastic wrap, and place them in a culture room at 28°C for light cultivation. After 1 day, remove the plastic wrap and culture normally.
  • Genomic DNA of positive lines and cultivar sweet potato was extracted using the improved CTAB method, and PCR amplification was performed using IbSAP15-OE detection primers FLAG-F (pGWB12) (SEQ ID NO.5) and ATTB2-R (SEQ ID NO.6).
  • IbSAP15-OE detection primers FLAG-F FLAG-F
  • ATTB2-R SEQ ID NO.6
  • the pGWB12-IbSAP15 plasmid was used as a positive control
  • the genomic DNA of cultivar Xuzishu No. 8 was used as a negative control
  • ddH 2 O was used as a blank control.
  • the DNA polymerase used was 2 ⁇ PCR Master Mix (CWBIO).
  • the total reaction system is 20 ⁇ L, including 10 ⁇ L of 2 ⁇ PCR Master Mix, 1 ⁇ L of template, 0.5 ⁇ L of 10 mmol ⁇ L -1 forward and reverse primers, and 8 ⁇ L of ddH 2 O.
  • the PCR amplification program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, and 35 cycles; full extension at 72°C for 2 min; incubation at 4°C.
  • FLAG-F (pGWB12) (SEQ ID NO.5): 5'-ATGAGCGACTACAAGGAT GACGAT-3', ATTB2-R (SEQ ID NO.6): 5'-ACCACTTTGTACAAGAAAGC TGGG-3'.
  • the RNA of the leaves of the IbSAP15-OE transgenic line was extracted and reverse-transcribed into cDNA.
  • the expression of IbSAP15 was detected by RT-qPCR.
  • Use SYBR Green Realtime PCR MasterMix (Toyobo) kit for RT-qPCR detection the reaction system is 10 ⁇ L, including 50 ⁇ L of 2 ⁇ SYBR Green Realtime PCR MasterMix (Toyobo), 2 ⁇ L of 20-fold diluted cDNA template, 10 mmol L -1 positive and negative 0.5 ⁇ L each of primers qIbSAP15-F (SEQ ID NO. 7) and qIbSAP15-R (SEQ ID NO.
  • the RT-qPCR program used was: first stage: pre-denaturation at 95°C for 10 min; second stage: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 20 s, 40 cycles; third stage: 65°C-95°C °C to detect the melting curve.
  • the sweet potato ADP-ribosylation factor (ADP-ribosylation factor, IbARF) gene (Park et al., 2012) was used as an internal reference, and the primer sequences were qIbARF-F (SEQ ID NO.9) and qIbARF-R (SEQ ID NO.10).
  • IbSAP15 relative to cultivar Xuzishu 8 in each line was calculated by 2- ⁇ Ct method.
  • the expression levels of the three IbSAP15-OE strains are shown in Figure 6, which are 123.56, 30.78, and 16.16 times that of the cultivar Xu Zishu No. 8, and are named OE1, OE2, and OE3, respectively.
  • qIbSAP15-F (SEQ ID NO.7): 5'-GATCACGCTTGCAAAGGCAG-3',
  • qIbSAP15-R (SEQ ID NO.8): 5'-CGTAGAATCCCTGCTCTTGTTTCC-3',
  • qIbARF-F (SEQ ID NO.9): 5'-CTTTGCCAAGAAGGAGATGC-3',
  • qIbARF-R (SEQ ID NO. 10): 5'-TCTTGTCCTGACCACCAACA-3'.
  • Embodiment 4 Comparison of IbSAP15-OE strain and No. 8 leaf type and flower type of cultivar Xu Zishu
  • the leaves of the overexpression lines changed in leaf shape.
  • the leaves of the OE1 and OE2 strains are smaller than those of the cultivars, and the notches are deeper, especially for the OE1 strain, the leaves are similar to chicken feet and have more ornamental value (Figure 7).
  • the IbSAP15-OE strain and the cultivar Xu Zishu No. 8 were planted in flowerpots, cultured under normal sunlight, and the flower types were compared after flowering. During this period, flowering can be accelerated by short-day treatment, that is, 8h light/16h dark cultivation.
  • the corolla of the cultivar is funnel-shaped, and the 5 petals of each corolla are united together, while the corolla of the IbSAP15-OE strain has different degrees of dehiscence.
  • the flower tube of the OE1 strain is completely cracked, and the flower tube of the OE2 strain is partially cracked, which is extremely rare in sweet potato and its related species, and has a very high ornamental value (Fig. 8 and Fig. 9).

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Abstract

本发明公开一种甘薯IbSAP15基因在调控甘薯叶型与花型中的应用。与徐紫薯8号相比，IbSAP15过表达株系叶片的缺刻更深、花冠开裂，具有较高的观赏价值。本发明将甘薯IbSAP15基因在甘薯中过表达，能够加深叶片缺刻，并使得漏斗状花冠开裂，改变甘薯的叶型和花型，适用于培育不同叶型、花型的甘薯种质，增加观花型/观叶型甘薯品种。

Description

甘薯IbSAP15基因在调控甘薯叶型与花型中的应用
本申请要求于2021年12月03日提交中国专利局、申请号为CN202111465395.9、发明名称为“甘薯IbSAP15基因在调控甘薯叶型与花型中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于植物基因工程技术领域，涉及一种甘薯IbSAP15基因在调控甘薯叶型和花型中的应用。
背景技术
锌指结构域中包含多个半胱氨酸和组氨酸残基，能通过这些残基与锌离子结合，自我折叠为“手指”状，故称锌指。包含锌指结构域的蛋白被称为锌指蛋白(Kluget al.,1987)。锌指蛋白可以与DNA、RNA或其他蛋白相互作用，调控基因的转录、翻译等过程。植物胁迫相关蛋白(Stress-associatedprotein，SAP)家族成员包含A20/AN1锌指结构域，具有泛素连接酶活性或转录因子活性，或可通过构象改变感受细胞内氧化还原状态的变化，调控植物的生物、非生物胁迫响应与生长发育。
甘薯是重要的粮食作物，同时也是重要的蔬菜、饲料和工业原料作物。21世纪以来，甘薯也逐渐被人们作为观赏植物。按照用于观赏的部位不同，观赏甘薯可分为观叶型、观藤型、观花型、观薯型4种(任韵等，2005)。甘薯的叶片形状多样，有心形、掌状、戟形等，观叶甘薯叶形以复缺刻、鸡爪形为主，或叶色为紫色、嫩绿色、混色。甘薯的花冠呈漏斗状，由5个花瓣联合组成，与牵牛花花型一致，花色主要为白色、淡紫、淡粉。
观花甘薯要求易开花、花量大、花期长，可通过短日照诱导开花(孟羽莎等，2019)，并通过施用肥料提高甘薯的花量(邱才飞等，2010)。观赏甘薯可在室内通过水培或基质栽培，装点室内环境。水培操作简单、成本低，且可以与观赏鱼、乌龟或其他水草等水生生物构成生机盎然的生态圈。盆栽甘薯则可用于客厅、阳台、办公室、会议厅等的装饰。室外种植观赏甘薯则可用于立体装饰、坡体绿化、行道绿化、岩石绿化。与室内种植不同，室外种植观赏甘薯的面积更大，可以通过不同叶色、株型的观赏甘薯搭配，或观赏甘薯与其他 植被搭配，形成更具美感的景观(孟羽莎等，2019)。目前，对于观花型甘薯花型的研究较少，而且市场观花型甘薯品种较少。
发明内容
本发明提供一种甘薯IbSAP15基因在调控甘薯叶型与花型中的应用。
本发明所述的IbSAP15基因是从甘薯中克隆出的一种AN1锌指蛋白编码基因，其编码的蛋白包含两个保守的AN1锌指结构域，为具有特定序列的DNA分子，其CDS为579bp，核苷酸序列如SEQ ID NO.1所示。
本发明还提供上述IbSAP15基因编码的蛋白IbSAP15，其包含192个氨基酸残基，氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种调控甘薯叶型与花型的方法，包括如下步骤：在甘薯中过表达上述技术方案所述的IbSAP15基因。
本发明首次发现将甘薯IbSAP15基因在甘薯中过表达，能够加深叶片缺刻，并使得漏斗状花冠开裂，改变甘薯的叶型和花型，适用于培育不同叶型、花型的甘薯种质，增加观花型/观叶型甘薯品种。
本发明还提供了一种重组植物表达载体，所述重组植物表达载体包括出发载体和上述技术方案所述的IbSAP15基因。
优选的，所述出发载体为植物表达载体pGWB12。
附图说明
图1是线性化的入门载体
Figure PCTCN2022132557-appb-000001
结构示意图；
图2是重组入门载体
Figure PCTCN2022132557-appb-000002
结构示意图；
图3是植物过表达载体pGWB12结构示意图；
图4是重组植物表达载体pGWB12-IbSAP15的结构示意图；
图5是IbSAP15-OE株系的检测结果图，Zi8、-CK、+CK分别为栽培种、阴性对照和阳性对照的检测结果，IbSAP15-OE为不同IbSAP15-OE株系的检测结果；
图6是IbSAP15-OE株系及对照株系中IbSAP15的相对表达量结果图；
图7是IbSAP15-OE株系与栽培种株系叶型的比较图，箭头所指为缺刻变深的部位，比例尺为2cm；
图8～9是IbSAP15-OE株系与栽培种株系花型的比较图，其中，图8为IbSAP15-OE株系与栽培种株系花的主视图，图9为IbSAP15-OE株系与栽培种 株系花的俯视图，比例尺为2cm。
具体实施方式
为了使本领域技术人员更加清楚地明白本发明的目的、技术方案及优点，下面结合附图和具体实施例，对本发明做进一步详细描述。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
下述实施例中所用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。
实施例1：IbSAP15基因的克隆及过表达载体的构建
实施例中使用的甘薯品种为徐紫薯8号(Zi8)，通过杂交育种选育，亲本为徐紫薯3号和万紫56，是产量高、干率高、花青素含量高的鲜食品种。徐紫薯8号叶型为戟形，易开花，也是优质的观赏甘薯。
通过在甘薯的转录组测序数据中得到IbSAP15的CDS序列(见GenBank登录号MW661075，https://www.ncbi.nlm.nih.gov/nuccore/2026807425)。根据I bSAP15的CDS序列设计特异引物tIbSAP15-F(SEQ ID No.3)和tIbSAP15-R(SEQ ID No.4)，通过PCR从甘薯各组织混合样cDNA中扩增到IbSAP15的CDS全长，并连接到入门载体
Figure PCTCN2022132557-appb-000003
上，构建得到重组入门载体
Figure PCTCN2022132557-appb-000004
通过LR反应，将测序正确的
Figure PCTCN2022132557-appb-000005
Figure PCTCN2022132557-appb-000006
上的IbSAP15片段置换到植物表达载体pGWB12上，得到重组植物表达载体pGWB12-IbSAP15。具体步骤为：
1、甘薯混合样RNA提取及完整性检测：收取水培甘薯叶片、不定根、花的样品，分别在液氮中研磨成粉末，等比例混合。取约100mg样品粉末，使用华越洋通用快速植物RNA提取试剂盒，参照说明书提取混合样总RNA。使用Nano drop 1000紫外分光光度计检测RNA的浓度，并取1μg总RNA通过1％琼脂糖凝胶电泳检测完整性。
2、第一链cDNA合成：使用ReverTra qPCR RT Master Mix with gDNARemover(Toyobo)cDNA合成试剂盒进行第一链cDNA的合成。以500ng RNA为模板，总体系10μL，具体合成步骤参照试剂盒说明书。
3、IbSAP15的扩增和入门载体构建：使用引物对tIbSAP15-F(SEQ ID NO.3)和tIbSAP15-R(SEQ ID NO.4)，以甘薯混合样cDNA为模板进行PCR扩增，所使用的DNA聚合酶为Taq TM Version 2.0plus dye(TaKaRa)。反应总体系50μL， 包括TaqVersion 2.0plus dye(2×)25μL，cDNA模板1μL，10mmol·L -1正反向引物各2μL，ddH 2O 20μL。PCR扩增程序为：98℃预变性30s；98℃变性10s，60℃退火15s，72℃延伸15s，35个循环；72℃充分延伸2min；4℃保温。PCR产物进行电泳检测和胶回收，通过TA克隆将胶回收产物连接在线性化的
Figure PCTCN2022132557-appb-000007
(Invitrogen)上，构建入门载体
Figure PCTCN2022132557-appb-000008
转化大肠杆菌DH5α。挑取阳性克隆送至生工生物工程(上海)股份有限公司测序，选取序列及方向均正确的单克隆提取
Figure PCTCN2022132557-appb-000009
质粒用于后续实验。
tIbSAP15-F(SEQ ID NO.3)：5’-ATGGGAGGAGGAACAGAAGCT-3’，
tIbSAP15-R(SEQ ID NO.4)：5’-TCAAAAAGCTTTAACAGAAGGTATG GTAGTTGG-3’。
4、IbSAP15表达载体构建：取100ng
Figure PCTCN2022132557-appb-000010
与100ng pGWB12质粒，混合均匀，加入2μL Gateway LR Clonase II Enzyme Mix(Invitrogen)，补水至10μL，25℃孵育1h。加入1μL蛋白酶K，37℃保温10min后终止反应。取1μL反应液转化至大肠杆菌DH5α中，挑取阳性克隆提取质粒，获得植物表达载体pGWB12-IbSAP15。
实施例2：在甘薯中过表达IbSAP15基因
为研究IbSAP15基因的功能，将IbSAP15基因在甘薯中过表达，通过对比过表达株系和栽培种的表型来分析IbSAP15基因的功能。农杆菌介导的甘薯遗传转化方法包括侵染、共培养、筛选鉴定、诱导成苗等，后续通过组培快繁和移栽后扦插来进行扩繁。所用抗生素和激素有2,4-二氯苯氧乙酸(2,4-Dichloro phenoxyacetic acid，2,4-D)、脱落酸(AbscisicAcid，ABA)、卡那霉素(Ka namycin，Kan)、潮霉素(homomycin B，Hyg)、利福平(Rifampin，Rif)、头孢(Cefotaxime sodium，Cef)。所用培养基有：YEB(酵母提取物1g·L -1，牛肉浸膏5g·L -1，蔗糖5g·L -1，蛋白胨5g·L -1，MgSO 4·7H 2O 0.5g·L -1)、MS(购买自北京酷来搏科技有限公司)、MSD(MS+2mg·L -12,4-D)。其他试剂有：乙酰丁香酮(Acetosyringone，AS)。具体步骤如下：
1、农杆菌介导的甘薯遗传转化：
通过液氮冻融法将植物表达载体pGWB12-IbSAP15转入农杆菌菌株GV3101感受态中，挑取阳性克隆接种到1mL含有50mg·L -1Kan和20mg·mL -1 Rif的YEB液体培养基中，28℃过夜培养。吸取500μL过夜培养的菌液，转接到新的50mL含有50mg·L -1Kan和20mg·mL -1Rif的YEB液体培养基中，培养至OD 600达到0.6左右。4000rpm离心5min，收集菌体，以MSD液体培养基重悬，调整OD 600在0.1-0.2之间，并加入AS至终浓度30mg·L -1。将甘薯愈伤压碎成1～2mm的颗粒，使用MSD清洗3次，放入10mL重悬的农杆菌菌液中，40rpm、25℃避光培养30min，超声波处理15s。将愈伤取出，放置于垫有滤纸的MSD+30mg·L -1AS培养基中，避光共培养。3d后，使用MSD清洗至上清清澈，转移至MSD+300mg·L -1Cef液体培养基中，室温避光慢摇1h后，把愈伤接种到MSD+200mg·L -1Cef固体培养基上共培养2周。
2、抗性愈伤的筛选、诱导与移栽
将共培养2周后的愈伤转移至MSD+10mg·L -1Hyg+200mg·L -1Cef固体培养基上，2周后挑选状态良好的愈伤继代到新的培养基上再培养2周。将愈伤转移到MS+10mg·L -1Hyg+200mg·L -1Cef+1mg·L -1ABA固体培养基上诱导体细胞胚，每2周继代一次。体胚形成芽之后转移到固体培养基MS+10mg·L -1Hyg+200mg·L -1Cef上成苗。试管苗长至6cm以上后切段转移至MS固体培养基上扩繁，在组培苗数量较多后，选取一部分长势较好的进行移栽。用水清洗干净组培苗上的培养基，将其移栽至营养土中，用保鲜膜保湿，置于28℃培养室中光照培养。1d后去除保鲜膜，正常培养。
实施例3：IbSAP15-OE株系的鉴定与IbSAP15表达量的检测
1、IbSAP15-OE转基因株系的鉴定
使用改良的CTAB法提取阳性株系及栽培种甘薯的基因组DNA，使用IbSAP15-OE检测引物FLAG-F(pGWB12)(SEQ ID NO.5)和ATTB2-R(SEQ ID NO.6)进行PCR扩增，检测是否为IbSAP15-OE转基因，并以pGWB12-IbSAP15质粒为阳性对照，以栽培种徐紫薯8号基因组DNA为阴性对照，以ddH 2O为空白对照。所使用的DNA聚合酶为2×PCR Master Mix(CWBIO)。反应总体系20μL，包括2×PCR Master Mix 10μL，模板1μL，10mmol·L -1正反向引物各0.5μL，ddH 2O 8μL。PCR扩增程序为：94℃预变性2min；94℃变性30s，60℃退火30s，72℃延伸30s，35个循环；72℃充分延伸2min；4℃保温。反应结束后使用琼脂糖凝胶电泳分析，栽培种(Zi8)和阴性对照(-CK)未扩增出条带，阳性对照(+CK)扩增出950bp大小的条带，8个 拟转基因株系中有7个扩增出了与阳性对照一致的条带，确定这7个株系为IbSAP15-OE转基因株系(图5)。
FLAG-F(pGWB12)(SEQ ID NO.5)：5’-ATGAGCGACTACAAGGAT GACGAT-3’，ATTB2-R(SEQ ID NO.6)：5’-ACCACTTTGTACAAGAAAGC TGGG-3’。
2、IbSAP15-OE转基因株系IbSAP15表达量的检测
参照实施例1提取IbSAP15-OE转基因株系叶片的RNA，并反转录为cDNA。使用RT-qPCR检测IbSAP15的表达量。使用SYBR Green Realtime PCR MasterMix(Toyobo)试剂盒进行RT-qPCR检测，反应体系为10μL，其中2×SYBR Green Realtime PCR MasterMix(Toyobo)50μL，20倍稀释的cDNA模板2μL，10mmol·L -1正反向引物qIbSAP15-F(SEQ ID NO.7)和qIbSAP15-R(SEQ ID NO.8)各0.5μL，ddH 2O 2μL。所采用的RT-qPCR程序为：第一阶段：95℃预变性10min；第二阶段：95℃变性15s，60℃退火15s，72℃延伸20s，40个循环；第三阶段：65℃-95℃检测溶解曲线。以甘薯ADP核糖基化因子(ADP-ribosylationfactor，IbARF)基因(Park et al.,2012)为内参，引物序列为qIbARF-F(SEQ ID NO.9)和qIbARF-R(SEQ IDNO.10)。通过2 -ΔΔCt法计算IbSAP15在各个株系中相对栽培种徐紫薯8号的表达量。其中3个IbSAP15-OE株系的表达量如图6所示，分别为栽培种徐紫薯8号的123.56、30.78、16.16倍，分别命名为OE1、OE2和OE3。
qIbSAP15-F(SEQ ID NO.7)：5’-GATCACGCTTGCAAAGGCAG-3’，
qIbSAP15-R(SEQ ID NO.8)：5’-CGTAGAATCCCTGCTCTTGTTTCC-3’，
qIbARF-F(SEQ ID NO.9)：5’-CTTTGCCAAGAAGGAGATGC-3’，
qIbARF-R(SEQ ID NO.10)：5’-TCTTGTCCTGACCACCAACA-3’。
实施例4：IbSAP15-OE株系与栽培种徐紫薯8号叶型、花型的比较
过表达株系的叶片相比栽培种徐紫薯8号的叶片，其叶型发生了变化。OE1、OE2两个株系的叶片相比于栽培种更小，且缺刻变深，特别是OE1株系，叶片近似鸡爪状，更具观赏价值(图7)。
将IbSAP15-OE株系和栽培种徐紫薯8号栽种于花盆中，正常日照培养，待开花后比较花型。期间可通过短日照处理加速开花，即8h光照/16h黑暗培养。栽培种的花冠为漏斗状，每个花冠的5个花瓣相互联合在一起，IbSAP15-OE株 系的花冠则出现了不同程度的开裂。OE1株系的花筒完全开裂，OE2株系花筒部分开裂，这在甘薯及其近缘种中都是极其少见的，具有极高的观赏价值(图8和图9)。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (5)

  1. 甘薯IbSAP15基因在调控甘薯叶型与花型中的应用，其特征在于，所述的甘薯IbSAP15基因核苷酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的应用，其特征在于，所述的IbSAP15基因编码的蛋白IbSAP15的氨基酸序列如SEQ ID NO.2所示。
  3. 一种调控甘薯叶型与花型的方法，其特征在于，包括如下步骤：在甘薯中过表达权利要求1所述应用中的IbSAP15基因。
  4. 一种重组植物表达载体，其特征在于，所述重组植物表达载体包括出发载体和权利要求1所述应用中的IbSAP15基因。
  5. 根据权利要求4所述的重组植物表达载体，其特征在于，所述出发载体为植物表达载体pGWB12。
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