A peony PoAGL15 gene and its amino acid sequence and application

Abstract

The present invention discloses a peony PoAGL15 gene and its amino acid sequence and application. The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the gene is shown in SEQ ID No. 2. The peony PoAGL15 gene provided by the present invention is related to somatic embryogenesis, will be accumulated and expressed in the early embryo, and has an important regulatory role in the late stage of peony somatic embryo maturation, and can accelerate the efficiency of peony in vitro breeding. Constructing a eukaryotic expression vector and transforming Arabidopsis thaliana can enhance the induction of Arabidopsis leaf callus and make the leaves larger. The peony PoAGL15 gene and its amino acid sequence provided by the present invention can not only provide a certain theoretical basis for establishing a complete plant regeneration and genetic transformation system for peony, but also can be applied to the breeding of other plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a peony PoAGL15 gene and an amino acid sequence and application thereof. Background Art

[0002] AGL15 (AGAMOUS-like 15) belongs to the MIKC subfamily of the MADS-box gene family. It is the only gene member in the family that appears in all embryonic cell tissues and is preferentially expressed in plant embryos (immature embryos, endosperm, suspensor cells, etc.) and somatic embryo development. It plays an important role in floral organ formation, flowering period regulation, vegetative organ development, seeds, and somatic embryogenesis. It can activate somatic embryogenesis transcription factors. AGL15 also participates in multiple hormone regulation pathways such as auxin, gibberellin, and ethylene to enhance embryogenesis ability and promote the formation of somatic embryos. It is a functional gene with research value in the process of plant somatic embryogenesis.

[0003] Somatic embryogenesis is one of the important ways of plant in vitro regeneration, an effective way of rapid propagation, and the focus of future peony tissue culture. In particular, the direct generation of somatic embryos into seedlings without going through the callus stage can greatly accelerate the efficiency of peony in vitro propagation and provide good experimental material receptors for research such as plant genetic engineering.

[0004] At present, most of the important genes related to flower shape, flower color and flowering period regulation in peony have been discovered, but there are few reports on functional genes related to somatic embryogenesis. Therefore, exploring the functional characteristics of the embryogenic gene AGL15 and the characteristics of its expression during the entire somatic embryogenesis process is of great significance for establishing a complete plant regeneration and genetic transformation system for peony. For the genes related to peony somatic embryogenesis that have been reported so far, it has not been found that they have the effect of enhancing the induction of plant leaf callus and increasing leaf size. Summary of the invention

[0005] The object of the present invention is to provide a peony PoAGL15 gene and its amino acid sequence and application to solve the problems existing in the above-mentioned prior art. The peony PoAGL15 gene provided by the present invention is related to somatic embryogenesis, will be accumulated and expressed in the early embryo, and has an important regulatory role in the late maturation of peony somatic embryos. A eukaryotic expression vector is constructed and transformed into Arabidopsis, which can enhance the induction of Arabidopsis leaf callus and increase the size of the leaves.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Solution 1: The present invention provides a peony PoAGL15 gene, which is derived from the peony variety 'Fengdan' (P.ostii), and the sequence table of the gene is shown in SEQ No.1.

[0008] Solution 2: The present invention provides a protein encoded by the above-mentioned peony PoAGL15 gene, and its amino acid sequence is shown in SEQ No.2.

[0009] Solution 3: The present invention provides an expression cassette, a recombinant expression vector or a recombinant microorganism containing the above-mentioned peony PoAGL15 gene.

[0010] Solution 4: The present invention provides an application of the above-mentioned peony PoAGL15 gene or the protein encoded by it in the process of plant somatic embryogenesis.

[0011] Scheme 5: The present invention provides a method for preparing a transgenic plant, comprising the following steps:

[0012] The peony PoAGL15 gene is transformed into plant cells or tissues through a recombinant expression vector containing a PoAGL15 gene expression cassette by using an Agrobacterium-mediated method, and the transformed plant tissues are cultivated into plants to obtain transgenic plants.

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] The present invention provides a peony PoAGL15 gene and its amino acid sequence and application. The gene is related to peony somatic embryogenesis, accumulates and is expressed in early embryos, and has an important regulatory effect in the late maturation of peony somatic embryos. It can accelerate the efficiency of in vitro breeding of peony and provide a certain theoretical basis for establishing a complete plant regeneration and genetic transformation system for peony.

[0015] The gene was used to construct a eukaryotic expression vector and transformed into Arabidopsis, which can enhance the induction of Arabidopsis leaf callus and increase leaf size. The verification of this function enables the characteristics of the peony PoAGL15 gene and its amino acid sequence to be applied to the breeding of other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is an RNA agarose gel electrophoresis detection diagram, in which M is the DL2000 maker band; 1-10 are the different developmental stages of somatic embryos and 0d; 5d; 10d; 15d; 20d; cotyledon growth period; somatic embryo formation period; secondary embryo germination; secondary embryo growth; somatic embryo maturity RNA bands;

[0018] Figure 2 Electrophoresis diagram of target gene PCR amplification M.DL2000 maker; L1. Target band;

[0019] Figure 3 The CDS sequence and the deduced amino acid sequence of the PoAGL15 gene, the solid line indicates the MADS domain, and the dotted line indicates the K-box domain;

[0020] Figure 4 Prediction of the secondary structure of PoAGL15 protein;

[0021] Figure 5 Prediction of the tertiary structure of PoAGL15 protein;

[0022] Figure 6 Comparative analysis of amino acid sequences of PoAGL15 and other plant homologs;

[0023] Figure 7 For phylogenetic analysis of PoAGL15 and other plants, RcAGL15-2 represents the AGL15 protein in Rosa chinensis ;

[0024] Figure 8 is the expression pattern of PoAGL15 gene during somatic embryogenesis;

[0025] Fig. 9 This is the growth condition of Arabidopsis thaliana transformed with the PoAGL15 gene. The horizontal axis represents the wild-type Arabidopsis thaliana, and the different transgenic Arabidopsis thaliana lines AGL15-1, AGL15-2, AGL15-5, and AGL15-6 from left to right. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The materials used in the embodiment are seeds of peony 'Fengdan' (P.ostii) collected from the fields in Heze, Shandong in August 2018, and stored in a laboratory -4°C refrigerator for standby use. After tissue culture of Fengdan embryos as explants through direct somatic embryogenesis, plant tissue materials (numbered from 1 to 10, respectively) in 10 consecutive culture stages (0d; 5d; 10d; 15d; 20d; cotyledon growth period; somatic embryo formation period; secondary embryo germination period; secondary embryo growth period; maturity period) during Fengdan somatic embryogenesis were obtained for subsequent gene cloning and the study of its expression characteristics during somatic embryo development.

[0032] The Arabidopsis used was from the wild type Arabidopsis thaliana (Col) from Columbia, which was preserved and used in the laboratory for a long time.

[0033] The LB liquid medium used was 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0

[0034] The LB solid medium used was prepared by adding 15 g of agar per liter of LB liquid medium.

[0035] Example 1 Total RNA extraction and cDNA synthesis

[0036] RNA was extracted from the materials of somatic embryos of sterile peony tissue culture seedlings at various developmental stages using the Quick RNA Isolation Kit (Huayueyang, China). It should be noted that most of the plant tissues at various stages of peony are rich in polysaccharides, polyphenols and other substances, and it is not easy to obtain high-quality RNA with little pollution and no degradation. Therefore, during extraction, it is necessary to pay attention to fully lyse the cells and operate quickly and accurately to prevent RNA from being unstable and degraded. The RNA concentration was determined by NanoDrop 2000 Nucleic Acid Quantifier Spectrophotometer, and 1% agarose gel electrophoresis was used to determine whether the RNA bands were intact. The RNA was stored at -80°C, and an equal amount of the total RNA extracted from each stage was mixed as a reverse transcription template.

[0037] The first strand of cDNA was synthesized by reverse transcription using a reverse transcription system (A3500, Promega). The specific steps are as follows:

[0038] (1) Add the components according to the reverse transcription system in Table 3-1;

[0039] (2) Mix gently and incubate at 42°C for 30 min;

[0040] (3) Place in a constant temperature water bath at 85°C for 5 seconds;

[0041] (4) Immediately return the sample to the ice box and place it in the refrigerator for 5 min. The resulting cDNA template was stored in a -20°C refrigerator until use.

[0042] The reverse transcription reaction system is shown in Table 1.

[0043] Table 1 Reverse transcription reaction system

[0044]

[0045] The OD260 / 280 ratio of total RNA extracted from 10 stages of direct somatic embryogenesis was determined by NanoDrop 2000 nucleic acid quantification instrument, and the OD260 / 230 ratio was between 2.0-2.1, and the OD260 / 230 ratio was between 1.8-2.0. After 1% agarose gel electrophoresis, the RNA bands were clear and bright, and the brightness of the 28S RNA band was about twice that of the 18S RNA band, with no tailing phenomenon ( Figure 1 ), indicating that the total RNA at each stage has no protein contamination, high purity, no degradation and good integrity.

[0046] Example 2 Cloning of the PoAGL15 gene of 'Fengdan'

[0047] The specific primers were designed as shown in Table 2. The first strand of cDNA obtained by reverse transcription was used as a template to amplify the PoAGL15 gene. The PCR reaction program was 94°C pre-denaturation for 1 min; 35 cycles of denaturation at 98°C for 10 s; annealing at 56°C for 30 s; extension at 72°C for 1 min; and finally extension at 72°C for 10 min.

[0048] Table 2 Primer sequences

[0049]

[0050] The PCR amplification product was detected by agarose gel electrophoresis and showed obvious target bands such as Figure 2 As shown, the length of the obtained nucleotide sequence is consistent with the expected length. The single target band was recovered with reference to the TIANGEN Universal DNA Purification Kit. The recovered product was connected to the pMD19-T Vector vector at room temperature overnight for 12 hours and then transformed into E. coli DH5α competent cells. Then, it was evenly plated on LB solid agar medium containing AMP and cultured for 12-16 hours to select single clones for colony PCR. The positive single clone was shaken and the plasmid was extracted using the TIANGEN prep Mini Plasmid Kit and plasmid PCR was performed to verify the reliability. After verification, it was sent to Invitrogen TM The complete sequence of the coding region of the PoAGL15 gene was obtained by sequencing with Invitrogen.

[0051] ATGGGTCGAGGTAAGATTGAGATCAAGAGGATCGAGAATGCAAATAGCAGACAAGTCACTTTCTCTAAGAGACGTGCTGGATTGCTGAAGAAGGCGAAGGAACTTGCGATTCTTTGTGATGCTGAGGTTGCGGTTATAATTTTCTCTAATACCGGAAAGCTTTTTGAGTTCTCGAGTTCTGGCATGAAGCGAACACTTACAAGATATACGAAGTGTCAGAATTCTTCAGAGACGGCTATTGTGGAATACAAAGCAGAGAAACAAGATTCAATGGAGGCGGAAATTCTTAAAGATGAAATCACAAACCTACAAATGAAACAATTGCGGTTGTTGGGTAAGGAACTTACAGGCTTGAGCTTAAAAGATTTGCAGCACCTAGAACAACAGCTGAATGAAGGGTTATTATCCGTTAAGGAGAGGAAGGAGCAATTATTGATGGAACAATTAGAGCAGTCAAGAGTACAGGAAAAGCGAGCTATGCTGGAGAATGAAACTTTGCGCAGACAGGTTGAGGAGCTTCGAGGATTCTTTCCATCAACGGATAATTCACTGCCGTCTTATCTTGAGTACTATCCAGTAGAAAGGAAACATTCCATGCCAAAAAATGGTGTAATTGGTCCTGACGTGCTCTGCAACTGTGCAATGGAGAAAGAAGATTCAGACACCACCTTACAATTGGGGCTTCCATCTGATGTTTATCGGAAGAGGAAGGTACCCGAGAGAGAGAGCTGTTCCAACAATTCGGGAAGTCAAATTGGCATGGTCTGA

[0052] The gene sequence was deduced into the amino acid sequence using DNAMAN 7.0 software as follows:

[0053] MGRGKIEIKRIENANSRQVTFSKRAGLLKKAKELAILCDAEVAVIIFSNTGKLFEFSSSSGMKRTLTRYTKCQNSSETAIVEYKAEKQDSMEAEILKDEITNLQMKQLRLLGKELTGLSLKDLQHLE QQLNEGLLSVKERKEQLLMEQLEQSRVQEKRAMLENETLRRQVEELRGFFPSTDNSLPSYLEYYPVERKHSMPKNGVIGPDVLCNCAMEKEDSDTTLQLGLPSDVYRKRKVPERESCSNNSGSQIGMV

[0054] The coding sequence of the PoAGL15 gene is 768 bp long and encodes 255 amino acids. The amino acids 1-61 and 91-168 at the N-terminus contain two conserved domains of the MADS-box gene family, namely the MADS domain and the K-box domain ( Figure 3 ).

[0055] Example 3 Bioinformatics Analysis

[0056] 1. Physicochemical properties and structural function analysis of PoAGL15 protein

[0057] Protparam was used to analyze the basic physical and chemical properties of proteins, WOLF PSORT was used to predict subcellular localization, SOPMA was used to predict the composition and characteristics of protein secondary structures, and SWISS-MODEL and PyMol were used to obtain and visualize protein three-dimensional structures. The results showed:

[0058] The molecular formula of the protein encoded by PoAGL15 is C 1256 H 2076 N 362 O 401 S 14 , relative molecular mass is 29113.32, protein isoelectronic value is 7.57, instability index is 54.36, hydrophilicity index is -0.654 (less than 0 indicates hydrophilicity), lipophilicity index is 83.33, it is speculated that the protein is an unstable hydrophilic protein with strong fluidity. The subcellular localization prediction results are nucl: 12.5, cyto_nucl: 7, plas: 1, indicating that the protein is likely to be located in the cell nucleus. Most of the secondary structure of the protein is composed of α-helix and random coil, and these two structures account for 86.67% of the total structure ( Figure 4 ). Modeling and prediction are performed through SWISS-MODEL, such as Figure 5As shown, the three-dimensional structure of the protein in two conserved domains from amino acid 2 to amino acid 78 and from amino acid 61 to amino acid 140 was obtained, and it was found that its β-folded structure only appeared in the MADS domain.

[0059] 2. Homology analysis of PoAGL15 gene and construction of phylogenetic tree

[0060] Homology analysis was performed using Blast and ClustalX in NCBI; the conserved domains were predicted using the CDD Tool in NCBI. The results showed that:

[0061] The amino acid sequence encoded by PoAGL15 was aligned with the known AGL15 amino acid sequences of 17 other plants with high sequence identity in NCBI by ClustalX, and the phylogenetic tree was constructed by MEGA6.0: Jatropha curcas (JcAGL15, XP_012065634.1), Ricinus communis (RcAGL15, XP_002534454.2), Rubber (HbAGL15, XP_002534454.2), Juglans regia (JrAGL15, XP_018817259.1), Quercus variabilis (QsAGL15, XP_023925149.1), Jujube (ZjAGL15, XP_002534454.2), and Polymorphic tree (ZhAGL15, XP_002534454.3). 015881556.1), peach (PpAGL15, XP_007219474.2), plum (PmAGL15, XP_008231548.1), rose (RcAGL15#, XP_024186607.1), sweet cherry (PaAGLK15, XP_021821687.1), grape (VvAGL15, XP_010659213.1), Tokyo evening cherry (PyAGL15, PQQ19640.1), orange (CsAGL15, KDO83296.177), Clementine (CcAGL15, XP_006438919.1), acacia (ApAGL15 XP_027356129.1), cassava (MeAGL15, XP_021625470.1), cocoa (TcAGL15, XP_017975249.1), the average sequence similarity is as high as 72.18%, among which the amino acid sequence encoded by JcAGL15 of Jatropha curcas has the highest similarity, reaching 80.00%, and the lowest similarity with the rose (Rosa chinensis) of the Rosaceae family is 69.00%. The results are shown in Figure 6, where superscript "*" indicates completely conserved (40.00%). Superscript ":" indicates highly conserved (18.43%), and superscript "." indicates relatively highly conserved (7.06%). An unrooted phylogenetic tree was constructed using the Neighbor-joining method of MEGA 6.0, with the Bootstrap value set to 1000 and the Partial deletion value set to 30, to analyze and explore its phylogenetic relationship. The results are shown in Figure 7 As shown, PoAGL15 and grape VvAGL15 are clustered on the same branch, indicating that they are most closely related to grape.

[0062] Example 4 Expression pattern of PoAGL15 gene during somatic embryogenesis

[0063] Ubiquitin ligase-3 (E3) was selected as the internal reference gene. According to the CDS sequence of PoAGL15 gene and the design principle of qRT-PCR primers, the fluorescent real-time quantitative primers were designed as shown in Table 1. The fluorescent quantitative PCR was performed using a fluorescent quantitative kit. Premix ExTaqTM (Perfect Real Time), the reaction was carried out on a QTOWER real-time fluorescence quantitative PCR instrument (analytikjena), using a two-step method: 95℃ pre-denaturation, 3min; 40 cycles: 95℃ denaturation, 5s; 62℃ annealing, 20s. The melting curve was 60 to 95℃, and the temperature was increased by 1℃ every 15s. Each reaction was set up with 3 biological replicates and 3 technical replicates. The gene expression level on day 0 was used as the control, and the 2-ΔΔCT method was used to calculate the transcriptional expression characteristics of the gene in 10 periods during direct somatic embryogenesis. The results are shown in Figure 8 As shown in the figure, S1-S10 represent: 0d; 5d; 10d; 15d; 20d; cotyledon growth period; somatic embryo formation period; secondary embryo germination period; secondary embryo growth period; maturity period. It was found that the relative expression levels of PoAGL15 at the 5th day, 10th day, and somatic embryo maturity period were 568.480, 530.054, and 526.347, respectively, showing consistent high-level expression characteristics, while the relative expression level was the lowest at 6.513 when the two cotyledons of the embryo were fully opened and turned green on the 20th day. Subsequently, its relative expression level increased to 178.115 in the somatic embryo formation (S6) stage and then decreased to form a "small peak". The expression level of PoAGL15 changed dramatically in two periods of time: first, the expression level decreased rapidly during the culture from the 10th day to the 15th day, and second, the gene expression level increased rapidly during the growth of secondary embryos to somatic embryo maturity. It can be speculated that the PoAGL15 gene in peony will accumulate and express in the early embryo, and also play an important regulatory role in the late maturation of peony somatic embryos.

[0064] Example 5 Construction of PoAGL15 gene expression vector and transformation of Arabidopsis thaliana for functional verification

[0065] 1. Construction of gene expression vector

[0066] The gene expression vector is pHG-AGL15, and the primers and restriction sites used are shown in Table 3.

[0067] Table 3 Primer list

[0068]

[0069] 1) Construction of plasmid using high-fidelity enzyme PCR reaction

[0070] The 20 μl PCR reaction system was 2 μl 10× buffer, 2 μl 2 mM dNTPs, 2 μl DNA template, 1 μl DMSO, 0.8 μl 10 pmol / μl upstream and downstream primers, 0.5 U kod DNA polymerase (KOD-401, TOYOBO), and water was added to make up to 20 μl.

[0071] The PCR amplification program was pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 50-64°C for 30 s, extension at 68°C for 1 min / kb, 40 cycles, and insulation at 68°C for 8 min.

[0072] 2) DNA electrophoresis, recovery and enzyme digestion:

[0073] Electrophoresis: Add an appropriate amount of 10× loading buffer to each reaction tube and perform electrophoresis on a 1%-3% agarose gel (10ul EB, 1-3g agarose / 100ml 0.5×TBE buffer). Perform electrophoresis at 5-10V / cm in 0.5×TBE buffer, and take photos in a gel imaging system after the electrophoresis is completed.

[0074] Agarose gel DNA recovery (GK2042, Jierui Biotechnology): Carefully cut off the DNA target band and put it into a 1.5ml EP tube. Add 400ul of banding B to the tube and put it in a 70℃ water bath until the gel is completely dissolved. Add 100ul of isopropanol to the tube, let it stand at room temperature for 1 minute, centrifuge at 5000rpm for 1 minute to pass through the column, repeat step 3, add 500ul wash buffer, wash twice at 12000rpm, centrifuge at 10000rpm for 1 minute, add 40ul of double distilled water to the column, let it stand at 37℃ for 2 minutes, and centrifuge at 12000rpm for 1 minute to collect.

[0075] Enzyme digestion: 40 μl of the enzyme digestion system is plasmid (30 ul), 4 μl of 10× enzyme digestion buffer, 4 μl of 10× BSA (with or without reference to the instructions), 6U of restriction endonuclease (NEB), add water to make up to 40 μl, and treat in a 37 degree water bath for about 1 hour.

[0076] 3) Plasmid construction steps:

[0077] After the PCR fragments were recovered by agarose gel, they were mixed with the empty vectors recovered by enzyme digestion and connected by EasyGenoDNA recombination system (#VI201-02, Tiangen Bio). The 10ul recombination system was: 5ul 2×EasyGeno Assembly Mix, 2.5ul enzyme-cut vector DNA, and 2.5ul fragment DNA. The reaction system was added to a 250ul EP tube, placed in a 50℃ water bath for 30 minutes, and then transformed into E. coli plates. After 16 hours in a 37℃ incubator, the bacteria were picked and sent for sequencing (the sequencing results were in the "Plasmid Construction Sequencing Results" folder). The PHG-AGL15 plasmid was sequenced correctly, and the extracted plasmid was stored at -20℃ for a long time.

[0078] 4) Plasmid extraction

[0079] Inoculate a single colony in 3 ml LB liquid medium containing appropriate antibiotics, and culture overnight at 37°C and 200 rpm; take 1.5 ml of the culture (3 ml for low-copy plasmids), centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant, suspend the bacteria in 100 μl solution Ⅰ (Glucose 50 mmol / L, EDTA 10 mmol / L, Tris-HCl 25 mmol / L, pH 8.0); add 200 μl of freshly prepared solution Ⅱ (NaOH 0.2 mol / L, SDS 1%), and immediately mix gently up and down; add 150 μl solution Ⅲ (KAc5 mol / L, pH 4.8), quickly mix up and down, let stand at room temperature for 5 minutes; centrifuge at 12,000rpm for 10 minutes; transfer the supernatant to another centrifuge tube, add 2 volumes of ethanol, mix; centrifuge at 12,000rpm for 10 minutes; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000rpm for 1 minute, remove the supernatant; vacuum dry the precipitate; dissolve in 60μl double distilled water containing 10μg / ml RNase A.

[0080] 2. Transformation of Agrobacterium to obtain positive clones and sequencing verification

[0081] (I) Transformation of Agrobacterium tumefaciens and identification

[0082] 1) Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while until it partially melts. When it is in an ice-water mixture, insert it into ice; add 0.1μg (volume not more than 10μl) plasmid DNA for every 100μl competent state, stir the bottom of the tube by hand to mix, let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, and ice bath for 5 minutes; add 700μl LB liquid culture medium without antibiotics, and culture at 28℃, 200rpm shaking for 2-3 hours; centrifuge at 6000rpm for one minute to collect the bacteria, retain about 100μl of supernatant, gently blow and resuspend the bacteria block, apply it on the LB plate containing 50μg / ml kanamycin, and invert it in a 28℃ incubator for 2-3 days. Randomly select a single colony, perform colony PCR, identify the correct Agrobacterium monoclonal clone, and mark it for use;

[0083] 2) Use a sterile pipette tip to pick up the marked Agrobacterium monoclone and inoculate it into 1.5 ml LB liquid medium containing the corresponding antibiotics (use a 50 ml blue-capped centrifuge tube) and culture it at 30 degrees and 200 rpm for 24 hours;

[0084] 3) Inoculate the Agrobacterium culture at a ratio of 1% into 100 ml of LB liquid medium containing antibiotics, and culture at 30°C with shaking until the OD 600 = about 1.0;

[0085] 4) Centrifuge at 20°C, 4,000 rpm for 15 min to collect the cells;

[0086] 5) Use transformation buffer (Table 4) to evenly disperse the cells and resuspend them to OD 600 =about 1.0.

[0087] Table 4 Bacterial transformation Buffer

[0088]

[0089] 3. Screening of transgenic Arabidopsis and transgenic Arabidopsis

[0090] Preparation of culture medium: Arabidopsis thaliana culture medium is 1 / 2MS (0.8% agar powder, without sucrose, pH 5.8).

[0091] Seed disinfection: 70% ethanol for 1 minute, add 1 ml of 7% sodium hypochlorite solution (containing 1 drop of Tween) for 10 minutes, mix by inversion for 5 minutes, and rinse with sterile water 5 times;

[0092] The sterilized seeds were resuspended in 100 μl sterile water, and were aspirated with a 1 ml pipette tip, and spotted on a 1 / 2 MS medium (selective antibiotics were added to the medium: 50 μg / ml KAN or 30 μg / ml HYG or 50 μM Glufosinate-ammonium) plate;

[0093] The plate was sealed and vernalized in a refrigerator at 4 degrees for 48 hours, and then placed in an artificial climate chamber to start germination and growth. The plant growth environment is 60% relative humidity; constant temperature 20-22 degrees; photoperiod is 16 hours of light and 8 hours of darkness; light intensity is 80-200μmol / M2 / S;

[0094] Observe after 8-15 days, identify the positive ones and transplant them into planting soil.

[0095] Stable 35S::PoAGL15 overexpressing transgenic Arabidopsis plants were obtained by Agrobacterium-mediated inflorescence infection. T2 overexpressing transgenic Arabidopsis and wild-type controls were planted and cultured under the same culture conditions. The results showed that the gene significantly enhanced the induction of Arabidopsis leaf callus and increased leaf size ( Fig. 9 ), the leaf area of ​​the wild type is 69.55 mm 2 The leaf areas of transgenic Arabidopsis lines AGL15-1, AGL15-2, AGL15-5, and AGL15-6 were 183.89 mm 2 、120.13mm 2 、109.28mm 2 、164.77mm 2 , increased by 164.40%, 72.72%, 57.13% and 136.91% respectively compared with the control, all reaching extremely significant levels.

[0096] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention. Sequence Listing <110> International Bamboo and Rattan Center <120> A peony PoAGL15 gene and its amino acid sequence and application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 768 <212> DNA <213> Artificial Sequence <400> 1 atgggtcgag gtaagattga gatcagagg atcgagaatg aaatagcag acagtcact 60 ttctctaaga gacgtgctgg attgctgaag aaggcgaagg aacttgcgat tctttgtgat 120 gctgaggttg cggttataat tttctctaat accggaagc ttttgagtt ctcgagttct 180 ggcatgaagc gaacacttac aagatatacg aagtgtcaga attctcaga gacggctatt 240 gtggaataca aagcagagaa acagattca atggaggcgg aaattcttaa agatgaatc 300 aaaacctac aaatgaaca attgcggttg tgggtagg aacttacagg cttgagctta 360 aaagattgc agcacctaga acacagctg atgaagggt tattaccgt taggagagg 420 aaggagcaat tattgatgga acattagag cagtcagag tacaggaaa gcgagctatg 480 ctggagaatg aacttgcg cagacaggtt gaggagcttc gaggattct tccatcacg 540 gataattcac tgccgtctta tcttgagtac tatccagtag aaggaaaca ttccatgcca 600 aaaaatggtg taattggtcc tgacgtgctc tgcactgtg caatggagaa agagattca 660 gabaccacct tacattggg gcttccactct gatgtttatc ggaagaggaa ggtacccgag 720 agagagagct gttccaacaa ttcgggaagt caaattggca tggtctga 768 <210> 2 <211> 768 <212> PRT <213> Artificial Sequence <400> 2 Ala Thr Gly Gly Gly Thr Cys Gly Ala Gly Gly Thr Ala Ala Gly Ala 1 5 10 15 Thr Thr Gly Ala Gly Ala Thr Cys Ala Ala Gly Ala Gly Gly Ala Thr 20 25 30 Cys Gly Ala Gly Ala Ala Thr Gly Cys Ala Ala Ala Thr Ala Gly Cys 35 40 45 Ala Gly Ala Cys Ala Ala Gly Thr Cys Ala Cys Thr Thr Thr Cys Thr 50 55 60 Cys Thr Ala Ala Gly Ala Gly Ala Cys Gly Thr Gly Cys Thr Gly Gly 65 70 75 80 Ala Thr Thr Gly Cys Thr Gly Ala Ala Gly Ala Ala Gly Gly Cys Gly 85 90 95 Ala Ala Gly Gly Ala Ala Cys Thr Thr Gly Cys Gly Ala Thr Thr Cys 100 105 110 Thr Thr Thr Gly Thr Gly Ala Thr Gly Cys Thr Gly Ala Gly Gly Thr 115 120 125 Thr Gly Cys Gly Gly Thr Thr Ala Thr Ala Ala Thr Thr Thr Thr Cys 130 135 140 Thr Cys Thr Ala Ala Thr Ala Cys Cys Gly Gly Ala Ala Ala Gly Cys 145 150 155 160 Thr Thr Thr Thr Thr Gly Ala Gly Thr Thr Cys Thr Cys Gly Ala Gly 165 170 175 Thr Thr Cys Thr Gly Gly Cys Ala Thr Gly Ala Ala Gly Cys Gly Ala 180 185 190 Ala Cys Ala Cys Thr Thr Ala Cys Ala Ala Gly Ala Thr Ala Thr Ala 195 200 205 Cys Gly Ala Ala Gly Thr Gly Thr Cys Ala Gly Ala Ala Thr Thr Cys 210 215 220 Thr Thr Cys Ala Gly Ala Gly Ala Cys Gly Gly Cys Thr Ala Thr Thr 225 230 235 240 Gly Thr Gly Gly Ala Ala Thr Ala Cys Ala Ala Ala Gly Cys Ala Gly 245 250 255 Ala Gly Ala Ala Ala Cys Ala Ala Gly Ala Thr Thr Cys Ala Ala Thr 260 265 270 Gly Gly Ala Gly Gly Cys Gly Gly Ala Ala Ala Thr Thr Cys Thr Thr 275 280 285 Ala Ala Ala Gly Ala Thr Gly Ala Ala Ala Thr Cys Ala Cys Ala Ala 290 295 300 Ala Cys Cys Thr Ala Cys Ala Ala Ala Thr Gly Ala Ala Ala Cys Ala 305 310 315 320 Ala Thr Thr Gly Cys Gly Gly Thr Thr Gly Thr Thr Gly Gly Gly Thr 325 330 335 Ala Ala Gly Gly Ala Ala Cys Thr Thr Ala Cys Ala Gly Gly Cys Thr 340 345 350 Thr Gly Ala Gly Cys Thr Thr Ala Ala Ala Ala Gly Ala Thr Thr Thr 355 360 365 Gly Cys Ala Gly Cys Ala Cys Cys Thr Ala Gly Ala Ala Cys Ala Ala 370 375 380 Cys Ala Gly Cys Thr Gly Ala Ala Thr Gly Ala Ala Gly Gly Gly Thr 385 390 395 400 Thr Ala Thr Thr Ala Thr Cys Cys Gly Thr Thr Ala Ala Gly Gly Ala 405 410 415 Gly Ala Gly Gly Ala Ala Gly Gly Ala Gly Cys Ala Ala Thr Thr Ala 420 425 430 Thr Thr Gly Ala Thr Gly Gly Ala Ala Cys Ala Ala Thr Thr Ala Gly 435 440 445 Ala Gly Cys Ala Gly Thr Cys Ala Ala Gly Ala Gly Thr Ala Cys Ala 450 455 460 Gly Gly Ala Ala Ala Ala Gly Cys Gly Ala Gly Cys Thr Ala Thr Gly 465 470 475 480 Cys Thr Gly Gly Ala Gly Ala Ala Thr Gly Ala Ala Ala Cys Thr Thr 485 490 495 Thr Gly Cys Gly Cys Ala Gly Ala Cys Ala Gly Gly Thr Thr Gly Ala 500 505 510 Gly Gly Ala Gly Cys Thr Thr Cys Gly Ala Gly Gly Ala Thr Thr Cys 515 520 525 Thr Thr Thr Cys Cys Ala Thr Cys Ala Ala Cys Gly Gly Ala Thr Ala 530 535 540 Ala Thr Thr Cys Ala Cys Thr Gly Cys Cys Gly Thr Cys Thr Thr Ala 545 550 555 560 Thr Cys Thr Thr Gly Ala Gly Thr Ala Cys Thr Ala Thr Cys Cys Ala 565 570 575 Gly Thr Ala Gly Ala Ala Ala Gly Gly Ala Ala Ala Cys Ala Thr Thr 580 585 590 Cys Cys Ala Thr Gly Cys Cys Ala Ala Ala Ala Ala Ala Thr Gly Gly 595 600 605 Thr Gly Thr Ala Ala Thr Thr Gly Gly Thr Cys Cys Thr Gly Ala Cys 610 615 620 Gly Thr Gly Cys Thr Cys Thr Gly Cys Ala Ala Cys Thr Gly Thr Gly 625 630 635 640 Cys Ala Ala Thr Gly Gly Ala Gly Ala Ala Ala Gly Ala Ala Gly Ala 645 650 655 Thr Thr Cys Ala Gly Ala Cys Ala Cys Cys Ala Cys Cys Thr Thr Ala 660 665 670 Cys Ala Ala Thr Thr Gly Gly Gly Gly Cys Thr Thr Cys Cys Ala Thr 675 680 685 Cys Thr Gly Ala Thr Gly Thr Thr Thr Ala Thr Cys Gly Gly Ala Ala 690 695 700 Gly Ala Gly Gly Ala Ala Gly Gly Thr Ala Cys Cys Cys Gly Ala Gly 705 710 715 720 Ala Gly Ala Gly Ala Gly Ala Gly Cys Thr Gly Thr Thr Cys Cys Ala 725 730 735 Ala Cys Ala Ala Thr Thr Cys Gly Gly Gly Ala Ala Gly Thr Cys Ala 740 745 750 Ala Ala Thr Thr Gly Gly Cys Ala Thr Gly Gly Thr Cys Thr Gly Ala 755 760 765

Claims

1. An application of the peony PoAGL15 gene in enlarging plant leaves, characterized in that: The nucleotide sequence of the peony PoAGL15 gene is shown in SEQ ID No. 1; and the plant is Arabidopsis thaliana.

2. A method for preparing a transgenic plant with enlarged leaves, characterized in that: The method comprises the following steps: introducing the peony PoAGL15 gene into a recipient plant to obtain a transgenic plant, wherein the nucleotide sequence of the peony PoAGL15 gene is shown as SEQ ID No. 1; and the plant is Arabidopsis thaliana.

3. The method according to claim 2, characterized in that The PoAGL15 gene is introduced into a recipient plant via a recombinant expression vector containing the PoAGL15 gene.

4. The method according to claim 3, characterized in that The recombinant expression vector containing the PoAGL15 gene is used to transform plant cells or tissues by using the Agrobacterium-mediated method, and the transformed plant tissues are cultivated into plants.

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