Application of a peony PoAIL6 gene and its encoded protein

Abstract

The invention discloses an application of a peony PoAIL6 gene and its encoded protein, and belongs to the field of biotechnology. The invention clones the peony PoAIL6 gene from the peony cultivar "Fengdan" and performs bioinformatics analysis. The invention transfers the peony PoAIL6 gene into Arabidopsis thaliana, and finds that the number of lateral roots of the transgenic Arabidopsis thaliana increases significantly through phenotypic comparison, indicating that overexpression of the peony PoAIL6 gene can promote the development of the Arabidopsis root system. Arabidopsis thaliana root is used to induce callus tissue and perform callus differentiation, and it is found that overexpression of the peony PoAIL6 gene inhibits callus formation and promotes callus differentiation into buds, indicating that the peony PoAIL6 gene can regulate Arabidopsis somatic embryo formation. The invention provides a certain theoretical basis for establishing a complete plant regeneration and genetic transformation system for peony.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of a peony PoAIL6 gene and a protein encoded thereby. Background Art

[0002] AINTEGUMENTA-LIKE (AIL) belongs to the AP2 subfamily of the AP2 / ERF transcription factor family, and is characterized by containing two AP2 / ERF domains (AP2-R1 and AP2-R2) and a linker region. This type of regulatory protein plays an important role in various growth and development stages of plants. The AP2 subfamily is divided into the ANT group and the euAP2 group. Depending on whether the first AP2 / ERF domain and the N-terminal region contain conservative amino acid insertions, the ANT group is divided into euANT and basalANT. There are eight euANT genes in Arabidopsis: ANT, AIL1, AIL2 / BBM / PLT4 and PLETHORAs (PLTs: AIL3 / PLT1, AIL4 / PLT2, AIL6 / PLT3, AIL5 / PLT5, AIL7 / PTL7). The eight members of euANT all contain several classic motifs, and the structural similarity makes their functions redundant in plants. Among them, BBM has one more unique and highly conserved bbm-1 motif than the other seven members. This bbm-1 motif plays an important role in transcriptional regulation of plant growth and development.

[0003] AILs genes are widely involved in the regulation of developmental processes, such as embryogenesis, meristem maintenance, and induction of adventitious growth in plants. AIL2 / BBM, AIL3 / PLT1, AIL4 / PLT2, AIL5 / PLT5, and AIL6 / PLT3 regulate cell proliferation during embryogenesis and maintenance of root apical meristems, and promote cell division of stem cell progeny in roots. Some AILs genes are also involved in rooting and adventitious bud formation. Kareem et al. revealed a dual mechanism of action of PLTs proteins in adventitious bud regeneration: first, PLT3 / 5 / 7 activates root stem cell regulators PLT1 and PLT2 to form a pluripotent state; then, they promote the formation of adventitious buds by activating the expression of the CUP-SHAPED-COTYLEDON2 (CUC) gene. ANT and AIL5 / 6 / 7 have partially redundant effects on flower development, and their direct targets include genes such as LEAFY involved in polarity establishment, meristem and flower development, as well as the auxin signaling pathway. In addition, AIL5, AIL6 and AIL7 play important roles in the stability of leaf arrangement and lateral root emergence. ANT, AIL6 and AIL7 have the function of regulating the shoot apical meristem (SAM), and the SAM of the ant-ail6-ail7 triple mutant stops after only a few leaves are produced.

[0004] Although AILs genes play a key role in plant growth and development and have been used in many species, there are few studies on AILs genes in peony. Many AILs genes are considered to be candidate marker genes in the process of somatic embryo regeneration and are involved in a variety of physiological responses and signal transduction pathways. However, it is still unclear how they specifically regulate signal transduction and their specific roles in somatic embryo formation, cell differentiation and plant growth conversion. Therefore, exploring the expression and regulatory mechanism of AILs genes in peony during somatic embryo development is of great value for enhancing the ability of somatic embryo formation and improving the peony regeneration and genetic transformation system. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a peony PoAIL6 gene and its encoded protein to solve the problems existing in the above-mentioned prior art. Overexpression of the peony PoAIL6 gene can promote the root development of Arabidopsis thaliana and regulate the somatic embryo formation of Arabidopsis thaliana.

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

[0007] The present invention provides application of peony PoAIL6 gene in promoting plant root development. The nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides the use of a protein expressed by the peony PoAIL6 gene in promoting plant root development, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0009] Preferably, the plant is Arabidopsis thaliana; and the root system development comprises an increase in the number of lateral roots.

[0010] The present invention also provides a method for promoting plant root development, comprising introducing a peony PoAIL6 gene into a plant to obtain a transgenic plant stably expressing the peony PoAIL6 gene, and promoting the transgenic plant to produce more lateral roots; the nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1.

[0011] Preferably, the plant is Arabidopsis thaliana.

[0012] The present invention also provides an application of the peony PoAIL6 gene in regulating plant somatic embryogenesis. The nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1.

[0013] The present invention also provides application of a protein expressed by the peony PoAIL6 gene in regulating plant somatic embryogenesis, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0014] Preferably, the plant is Arabidopsis thaliana; and the somatic embryogenesis of the plant includes the formation of callus and differentiation of callus into shoots.

[0015] The present invention also provides a method for regulating plant somatic embryogenesis, comprising introducing a peony PoAIL6 gene into a plant to obtain a transgenic plant stably expressing the peony PoAIL6 gene, inhibiting the formation of the plant callus, and promoting the differentiation of the plant callus into buds; the nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1.

[0016] Preferably, the plant is Arabidopsis thaliana.

[0017] The present invention discloses the following technical effects:

[0018] The present invention clones the peony PoAIL6 gene from the cultivated peony "Fengdan", and constructs a phylogenetic tree with the AILs genes of other plants, and performs amino acid sequence comparison and conservative motif identification. At the same time, the peony PoAIL6 gene is transferred into Arabidopsis to construct a transgenic plant. By phenotypic comparison, it is found that the number of lateral roots of transgenic Arabidopsis increases significantly, indicating that overexpression of the peony PoAIL6 gene can promote the development of the Arabidopsis root system. Afterwards, Arabidopsis root-induced callus tissue is used, and it is found that the callus tissue induced by transgenic Arabidopsis is less, but the buds differentiated from the callus tissue are more, and the differentiation is more rapid, indicating that overexpression of the peony PoAIL6 gene can inhibit callus formation and promote callus differentiation into buds, that is, the peony PoAIL6 gene has the function of regulating the formation of Arabidopsis somatic embryos. The present invention uses Arabidopsis as a model plant, verifies that the peony PoAIL6 gene has the function of regulating growth and development and somatic embryo formation, and provides a certain theoretical basis for the establishment of a complete plant regeneration and genetic transformation system for peony. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 is a map of the expression vector pHG;

[0021] Figure 2 This is the PCR electrophoresis diagram of the tree peony PoAIL6 gene; M is the DL2000 maker, and 1 is the target band;

[0022] Figure 3 This is the phylogenetic tree of peony PoAIL6 and AIL genes of other species; the red triangle arrow indicates peony PoAIL6;

[0023] Figure 4 The figure shows the comparison analysis of the protein motifs of PoAIL6 and the identification of 10 conserved motifs; a is the phylogenetic tree and conserved motif analysis, peony PoAIL6 is marked with a red triangle arrow, and the 10 conserved motifs are represented by square blocks of different colors; b is the 10 conserved motifs;

[0024] Figure 5 The phenotypic records and statistical graphs of wild-type and PoAIL6 transgenic Arabidopsis; a is the phenotype of PoAIL6 overexpressing plants after 34 days of cultivation; b, d and e are the phenotypic records and plant height statistics after 44 days of cultivation, ns indicates no significant difference; c is the root phenotype of PoAIL6 transgenic Arabidopsis; f is the statistics of the number of lateral roots of PoAIL6 transgenic Arabidopsis, **** indicates P < 0.0001;

[0025] Figure 6 The phenotypes of root induction and differentiation of PoAIL6 transgenic Arabidopsis thaliana are recorded and statistically shown; a is a photo taken when the roots were grown on SIM medium for 30 days; b is a photo taken when the roots were grown on SIM medium for 35 days; c is the statistical data of callus area when the roots were grown on SIM medium for 30 days, **** indicates P<0.0001;

[0026] Figure 7 The expression pattern of PoAIL6 gene in transgenic Arabidopsis thaliana; *** indicates P<0.001, **** indicates P<0.0001. DETAILED DESCRIPTION

[0027] 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.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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. The intermediate value in any stated value or stated range, and each smaller range between 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.

[0029] 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.

[0030] 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 invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] 1.1 Materials

[0035] The experimental material is the cultivar "Fengdan" which has been cultivated and evolved for a long time and has ornamental, medicinal, oil-use characteristics and stable genetic traits. The mature "Fengdan" seeds of the year were selected, and the seeds came from Heze, Shandong. The peony tissue culture seedlings are developed from the embryo as explants. After 7 days of dark treatment, the explants are transferred to the tissue culture room for normal culture. The normal culture conditions in the tissue culture room are alternating 16 hours of light and 8 hours of darkness, and the light intensity is 40μmol·m -2 ·s -1 , temperature 24±1℃. Subculture once every 20 days. RNA was extracted from the roots, stems, leaves and callus of peony seeds and tissue culture seedlings for real-time fluorescence quantitative analysis.

[0036] The Arabidopsis thaliana used in the present invention were all Columbia-0 (Col-0) wild type, planted in an artificial climate room with a light intensity of 40 μmol·m -2 ·s -1 , humidity 60~70%, temperature 24±1℃.

[0037] 1.2 Methods

[0038] 1.2.1 Total RNA extraction and cDNA synthesis

[0039] RNA was extracted from sterile peony tissue culture seedlings using a Quick RNA Isolation Kit (Huayueyang, China). The RNA concentration was determined by spectrophotometer. The integrity of the RNA band was confirmed by 1% agarose gel electrophoresis and the RNA was stored in a -80°C ultra-low temperature refrigerator for later use. The extracted RNA was used as a template and PrimeScript TM The first-strand cDNA was synthesized by reverse transcription according to the instructions of RTreagent Kit (Perfect Real Time) (RR047A, TAKARA).

[0040] 1.2.2 Cloning of the Paeonia suffruticosa PoAIL6 gene

[0041] According to the primer design principle, the primers were designed using SnapGene (V2.3.2) software (as shown in Table 1). The target sequence was amplified by a high-fidelity enzyme (Novozyme, Nanjing) of Max Master Mix (P515), and the target band was detected by 1% agarose gel electrophoresis. When the length of the target band was consistent with that of the marker sequence, it indicated that the target sequence was obtained, and the CDS sequence of the peony PoAIL6 gene was cloned. The gel was cut on a UV gel cutting table, and the product was recovered according to the steps of the DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd., Beijing). The gel recovery product was connected to the cloning vector using the pCE2TA / Blunt-Zero vector (C601) (Novozyme, Nanjing) kit for transformation. Take the competent cells of Escherichia coli DH5α, transform according to the steps, and then evenly plate on LB solid agar medium containing AMP, invert and culture at 37°C for 12-16h to select single clones for colony PCR, shake the positive single clones, use the plasmid mini-extraction kit (TIAN prep Mini Plasmid Kit, DP103-03, Beijing) of Tiangen Company to extract the plasmid and take 10 μL of the plasmid solution to send to Anshengda (Anshengda, Beijing) Company for sequencing to verify that the target sequence is obtained correctly. The nucleotide sequence of the PoAIL6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the PoAIL6 protein is shown in SEQ ID NO.2.

[0042] SEQ ID NO.1:

[0043]

[0044] SEQ ID NO.2:

[0045] MEMLSSPDHSQMMSYDGSSAASPQYFIDNFYANGWGNPKSHVNFTQGDDTRAKLEVEQAQIMSSSSIFTSFVDHHTQHQTVVPKLEDFLGGDSSSIVPYSDSQTETQDSSLTHIYDQSSAYFGDQQDLKAIAEFQAFSTNSGSEVEDSGSINRTQLTSIEFVQGQSIESGAEMAFSNGATGGALSLGVTQNTERAVSVDLDSSKKIADTFGQRTSIYRGVTRHRWTGRYEAHLWDNSCRREGQARKGRQVYLGGYDKEEKAARAYDLAALKYWGPTATTNFPLSDYAKEVEEMKNVTKQEFIASLRRKSSGFSRGASMYRGVTRHHQQGRWQARIGRVAGNKDLYLGTFATEQEAAEAYDIAAIKFRGVNAVTNFEMNRYDVEAIASSSLPIGGAAKRLKLSLETEKKPLVNHSQQPPCSSTNGNNISFASVQPVSAIPCGIPLDTMYQHNLYHHFQSNNIAGTSDSTSSIGTPLTFLPSPADQFFIWPHQSY。

[0046] Table 1 Primer Information Table

[0047]

[0048] 1.2.3 Bioinformatics Analysis

[0049] The basic physicochemical properties of the amino acid sequence of PoAIL6 were predicted and analyzed online using the ExPASy-ProtParam website. The secondary structure of the PoAIL6 protein was analyzed using SOPMA. TMHMM-v.2.0 and SignalP-v.6.0 were used to predict the presence or absence of transmembrane domains and signal peptides of PoAIL6, respectively. Plant mPLoc was used to predict the subcellular localization of PoAIL6 protein. The PlantTFDB plant transcription factor database (https: / / planttfdb.gao-lab.org / ) and the NCBI public database (https: / / www.ncbi.nlm.nih.gov / ) were used to search for homology to the AILs protein sequences of other species. ClustalWv.2.0.11 software was used to perform multiple sequence alignment of the peony AIL6 amino acid sequence with the AILs amino acid sequences of 12 species including Arabidopsis thaliana, rapeseed, soybean, Medicago truncatula, Rosa canina, grape, corn, tobacco, Populus trichocarpa, cocoa, rice, and lotus. The NJ (Neighbor-Joining) neighbor-joining method of MEGA11 was used to construct a phylogenetic tree by combining the AILs protein sequences of other species and the PoAIL6 protein sequences. 1000 bootstrap replicates were set, and other parameters were set to default values. iTOL (https: / / itol.embl.de / ) was used for online beautification.

[0050] DNAMAN 9.0 software was used to perform multiple sequence alignment of AILs protein sequences of peony, Arabidopsis, rapeseed, soybean, Medicago truncatula, grape, cocoa, tobacco, lotus, poplar, corn and Rosa canina. MEME online software was used to perform motif analysis of PoAIL6 protein sequences with AIL6 of other species, and the number of motifs was set to 10. The saved XML file and the Newick file obtained in the phylogenetic tree construction were input into the Gene Structure View (Advanced) window of TBtools v.2.4.0.119028 software for visualization and analysis.

[0051] 1.2.4 Functional verification of peony PoAIL6 gene transfection into Arabidopsis thaliana

[0052] The PoAIL6 gene was cloned into the plant expression vector pHG / PBI121 (pHG map as shown in Figure 1 ), a stable inherited 35S::PoAIL6 overexpressing transgenic Arabidopsis plants were obtained by Agrobacterium-mediated inflorescence infection. T3 generation overexpressing transgenic Arabidopsis and wild type controls were planted and cultured under the same culture conditions, and observed and recorded.

[0053] 1.2.4.1 Observation of transgenic plant growth phenotype

[0054] The wild-type and transgenic Arabidopsis T3 positive strains were sterilized and sown in culture dishes containing MS medium. After vernalization for 7 days, they were transplanted into planting soil and cultured in an artificial climate chamber. The number of leaves of Arabidopsis thaliana was counted at the same time after germination. The biological and technical replicates were repeated three times, and their growth was observed and photographed.

[0055] 1.2.4.2 Observation of callus induction and differentiation phenotype of transgenic plants

[0056] The wild-type and transgenic Arabidopsis T3 seeds were sterilized and sown in culture dishes containing MS medium and cultured for about 15 days after vernalization. 1-2 cm Arabidopsis roots were cut in a clean bench and placed in CIM medium and cultured in the dark in the tissue culture room for 7-10 days for callus induction. They were then transferred to SIM medium and cultured normally in the tissue culture room for callus differentiation. The differentiation of the control and experimental groups was observed and photographed for record.

[0057] 1.2.4.3 Phenotypic observation of transgenic plant roots

[0058] The wild-type and transgenic Arabidopsis T3 positive lines were sterilized and sown neatly side by side in a culture dish containing MS medium and cultured for 30 days after vernalization. The root development of Arabidopsis was observed and photographed.

[0059] 1.2.5 Fluorescence quantitative analysis

[0060] The peony somatic embryos cultured in 1.1 were cultured at seven stages of development (0, 5, 10, 15, 20, 30 and 60 days, with day 0 being the day before dark treatment), five different peony tissues (seed, root, stem, leaf, callus), and the root, stem, leaf, flower, and fruit tissues of Arabidopsis thaliana transfected with PoAIL6 gene in the T3 generation. RNA was extracted and reverse transcribed for the first strand of cDNA. The cDNA was diluted 10 times with ddH2O and used as a template. The primers are shown in Table 1. The reaction was completed on a QTOWER real-time fluorescence quantitative PCR instrument (analytikjena, Germany) using the TB Green Premix ExTaqⅡ Fluorescence Quantification Kit (Tli RNaseH Plus, TaKaRa). The PCR reaction system was TB Green Premix Ex Taq 5μl; Template 1μl; Primer (F+R) 0.4μl; ddH2O was added to 10μl, and a total of 3 biological replicates were set. The reaction conditions were 95℃ pre-denaturation for 90s; 95℃ denaturation for 5s, 60℃ melting for 30s, 40 cycles; melting curve 60 to 95℃, 1℃ increase every 15s. Peony ubiquitin ligase and AtActin were used as internal reference genes, and the expression levels of peony somatic embryos on day 0, peony root tissues, and Arabidopsis root tissues were used as control groups, respectively. The relative expression level was measured using 2 -ΔΔCT The relative expression levels of PoAIL6 in various tissues were calculated and analyzed.

[0061] 2.1 Cloning and sequence analysis of tree peony PoAIL6 gene

[0062] Using the transcriptome data of peony somatic embryos at 10 different stages as a reference, cDNA from the stage with higher expression of the target gene was selected as a template for gene cloning. The electrophoresis diagram showed that an obvious specific fragment appeared at the position of about 1500 bp (such as Figure 2 As shown in the figure, the peony PoAIL6 gene is known to be 1482 bp, which is basically consistent with the expected target gene length. The target fragment is then connected to the cloning vector and sequenced to obtain 100% correct sequence information, and the desired target gene is cloned.

[0063] The basic physicochemical properties of the AIL6 gene were analyzed. The results showed that the AIL6 protein encodes 493 amino acids with a relative molecular weight of approximately 54.4 kDa. It is an unstable hydrophilic protein (instability coefficient > 40, and the hydrophilicity index is negative). The theoretical isoelectric point is 5.85, the instability coefficient II is 45.88, the overall average hydrophilicity is -0.582, and the fat coefficient is 61.81. Subcellular localization predicts that the AIL6 protein is located in the cell nucleus.

[0064] The predicted secondary structure of the protein showed that the peony AIL6 protein was composed of α-helix, β-pleated sheet, β-turn and random coil, among which α-helix accounted for 35.23%, β-pleated sheet accounted for 4.66%, β-turn accounted for 6.74%, and random coil accounted for 53.37%. There was no transmembrane domain and no signal peptide.

[0065] 2.2 Phylogenetic tree analysis of peony PoAIL6 protein

[0066] To further understand the evolution and relationship between peony PoAIL6 and other species, the phylogenetic tree was constructed by combining the obtained peony PoAIL6 with AILs from Arabidopsis, tobacco, rapeseed, cocoa, grape, corn, poplar, rice, soybean, lotus, and Rosa canina. Figure 3 As shown, the results showed that the phylogenetic tree was basically consistent with the biological evolutionary relationship. Peony AIL6 and AIL6 members in other species clustered in the same branch and were closely related to Populus trichocarpa, Grape and Theobroma cacao.

[0067] 2.3 Multiple sequence alignment and conserved domain analysis of peony PoAIL6 protein

[0068] To analyze the similarity between PoAIL6 and AIL6 of other species, the amino acid sequence of PoAIL6 obtained was compared with the AIL6 sequences of other closely related species. The results showed that PoAIL6 all had two AP2 domains (AP2-R1 and AP2-R2), all belonged to the AP2 family, and all contained typical conserved motifs in the euANT system: euANT1, euANT2, euANT3, euANT4, euANT5, euANT6 and an amino acid insertion (H) in the AP2-R2 domain. The difference was that PoBBM contained a bbm-1 motif (GLSMIKTWLRNQ) unique to all species BBM members (e.g. Figure 4 shown).

[0069] 2.4 Phenotypic analysis of Arabidopsis thaliana overexpressing the PoAIL6 gene

[0070] 2.4.1 Growth and development phenotypes of PoAIL6 transgenic plants

[0071] The phenotypes of the PoAIL6 transgenic and wild-type Arabidopsis plants were observed. The leaf size and number of the two plants were similar, but the bolting time was slightly different: the wild-type plant began to bolt on the 28th day, while the PoAIL6 transgenic line bolted later than the wild-type plant, and began to bolt and flower on the 32nd day ( Figure 5In the late growth and development of Arabidopsis, the overall growth and development status of most PoAIL6 overexpressing plants was similar to that of the wild type, and the overall growth and development of some transgenic plants was slightly shorter than that of the wild type ( Figure 5 b, d and e). By comparing the root growth status of wild-type and PoAIL6 transgenic Arabidopsis in MS sterile medium, it was found that the roots of Arabidopsis overexpressing PoAIL6 produced more lateral roots than the wild-type, and the difference was extremely significant. The taproot of the wild-type was slightly longer than that of the transgenic, but the difference was not very obvious ( Figure 5 These results indicate that PoAIL6 plays an important role in the growth and development of Arabidopsis thaliana, especially in root development.

[0072] 2.4.2 Callus induction and differentiation phenotypes of PoAIL6 transgenic plants

[0073] To explore whether PoAIL6 plays a role in embryogenesis in overexpressed Arabidopsis, 1-2 cm roots of wild-type and PoAIL6 transgenic Arabidopsis were cultured in CIM induction medium in the dark for one week, and then cultured in SIM differentiation medium under normal light for observation. The results showed that after 30-35 days of culture on SIM medium, PoAIL6 overexpressed Arabidopsis induced less callus tissue than wild-type, but differentiated more buds, and the differentiation was faster. Figure 6 As shown, the PoAIL6 gene promotes the differentiation of Arabidopsis callus into buds, and it is speculated that it may also play a promoting role in the regeneration of peony somatic embryos.

[0074] 2.5 Expression pattern analysis

[0075] The RT-qPCR method was used to analyze the expression level of PoAIL6 gene in various tissues of Arabidopsis thaliana: root, stem, leaf, flower, and fruit (such as Figure 7 The results showed that the PoAIL6 gene was expressed in all transgenic lines, and the expression levels in the five parts of Arabidopsis thaliana (roots, stems, leaves, flowers, and fruits) were different. The expression level was highest in leaves, there was a certain expression in stems, the expression level in flowers and roots was lower, and there was almost no expression in fruits, which was consistent with that in peony.

[0076] When the T3 generation PoAIL6 transgenic Arabidopsis plants were cultured for about 45 days, the roots, stems, leaves, flowers and fruit pods were taken to extract RNA and reverse transcribed into cDNA, and the PoAIL6 gene was quantified in the roots, stems, leaves, flowers and fruit pods. Three biological replicates and three technical replicates were performed for each tissue. The results showed that the expression level of the PoAIL6 gene was the highest in the roots of transgenic Arabidopsis, much higher than other tissues. This is consistent with the above-mentioned speculation that the PoAIL6 gene plays an important role in the development of the Arabidopsis root system and that the callus tissue induced by the PoAIL6 transgenic Arabidopsis root system is easier to differentiate into buds than the wild type.

[0077] 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.

Claims

1. Application of the peony PoAIL6 gene in promoting root development of Arabidopsis thaliana, characterized in that: The nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1; The root system develops as an increase in the number of lateral roots.

2. Application of the protein expressed by the peony PoAIL6 gene in promoting root development of Arabidopsis thaliana, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.2; The root system develops as an increase in the number of lateral roots.

3. A method for promoting plant root development, characterized in that: The method comprises introducing the peony PoAIL6 gene into a plant to obtain a transgenic plant stably expressing the peony PoAIL6 gene, and promoting the transgenic plant to produce more lateral roots; The nucleotide sequence of the peony PoAIL6 gene is shown in SEQ ID NO.1; The plant is Arabidopsis thaliana.

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