A PheANT gene in moso bamboo, its expressed protein, and its applications

Abstract

This invention discloses a type of moso bamboo. Paint This invention relates to genes, their expressed proteins, and applications, and pertains to the field of plant genetic engineering technology. The first disclosed gene is a moso bamboo. Paint The gene, whose nucleotide sequence is shown in SEQ ID NO. 1, and whose corresponding amino acid sequence is shown in SEQ ID NO. 2, is constructed using the gene from moso bamboo. Paint Gene overexpression vector; constructing moso bamboo Paint The gene overexpression vector was transformed into Arabidopsis thaliana; transgenic plants with significantly altered phenotypes were obtained through screening and identification. The results of this invention's embodiments indicate that overexpression of *Phyllostachys edulis*... Paint The gene can significantly promote leaf growth and development. Transgenic Arabidopsis plants show obvious leaf phenotypes such as a significant increase in the number of rosette leaves and changes in leaf morphology.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a PheANT gene of moso bamboo, its expressed protein, and its applications. Background Technology

[0002] Moso bamboo (Phyllostachys edulis), an important bamboo species in the Phyllostachys genus used for both shoots and timber, boasts advantages such as rapid growth, abundant species, and wide distribution. It also plays a crucial role in maintaining forest ecosystem functions and promoting biodiversity. As the primary organ for photosynthesis and energy accumulation, the normal development of leaves is vital for bamboo growth. The three-dimensional morphogenesis of plant organs is a highly complex biological process involving cell proliferation, differentiation, and their precise spatiotemporal coordination. However, compared to other organs in bamboo plants, research on the cellular mechanisms by which leaf primordia gradually differentiate and develop into mature leaves remains relatively scarce. Moso bamboo seedlings, with their large leaves and distinctive morphological characteristics, are ideal model materials for in-depth research into the developmental patterns of leaves in bamboo species.

[0003] The AP2 / EREBP (APETALA2 / Ethylene-Responsive Element Binding Protein) transcription factor family plays a multi-layered regulatory role in plant leaf development. Members of this family participate in the regulation of key processes such as cell division, proliferation, differentiation, and polarity establishment by binding to GCC-boxes or DRE / CRT cis-acting elements in the promoters of downstream target genes. Some members of the ERF subfamily influence leaf size, thickness, and stomatal distribution by integrating auxin, cytokinin, and environmental signals. Furthermore, this family mediates the balance between abiotic stress and leaf development, coordinating resource allocation under adverse conditions to maintain basic photosynthetic structure. The function of PheANT in bamboo development has not yet been reported. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the technical problem to be solved by this invention is to provide a *PheANT* gene from *Phyllostachys pubescens*. Another technical problem to be solved by this invention is to disclose a protein that regulates plant leaf development, which is the expression protein of the *PheANT* gene from *Phyllostachys pubescens*. A further technical problem to be solved by this invention is to provide applications of the *PheANT* gene or protein from *Phyllostachys pubescens* for regulating plant leaf development and leaf morphogenesis. Finally, the technical problem to be solved by this invention is to disclose an *Arabidopsis thaliana* species with increased rosette leaf number and / or altered leaf morphology.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A first aspect of the present invention is to provide a PheANT gene from moso bamboo, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] A second aspect of this invention discloses a protein that regulates plant leaf development, the amino acid sequence of which is shown in SEQ ID NO. 2. It is the expression protein of the PheANT gene in moso bamboo.

[0008] A third aspect of the invention is to provide an expression vector or host cell containing the PheANT gene or protein from bamboo.

[0009] A fourth aspect of this invention discloses the application of the aforementioned PheANT gene or protein from bamboo in regulating the growth and development of rosette leaves in Arabidopsis thaliana. This regulation of rosette leaf development in Arabidopsis thaliana results in a significant increase in the number of rosette leaves and changes in leaf morphology.

[0010] The application of the PheANT gene or protein from bamboo in regulating the growth and development of rosette leaves in Arabidopsis thaliana includes the following specific steps:

[0011] 1) Construct an overexpression vector for the PheANT gene in moso bamboo;

[0012] 2) The constructed overexpression vector of the PheANT gene from moso bamboo was transformed into Arabidopsis thaliana;

[0013] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with increased rosette leaves and altered leaf morphology.

[0014] In a preferred embodiment, the leaf morphology changes include: a heart-shaped leaf with a forked midrib, a pair of dorsoventrally symmetrical leaves, or a pair of asymmetrical leaves at the petiole apex.

[0015] A fifth aspect of the present invention is to provide an Arabidopsis thaliana with increased rosette leaf number and / or altered leaf morphology, which contains the aforementioned PheANT gene from bamboo or expresses the aforementioned protein. The expression is described as overexpression.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention discloses for the first time the PheANT gene from moso bamboo, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of which is shown in SEQ ID NO. 2. This invention constructs an overexpression vector for the PheANT gene from moso bamboo; transforms the constructed overexpression vector into Arabidopsis thaliana; and cultivates, screens, and obtains transgenic Arabidopsis plants with increased rosette leaves and altered leaf morphology. The results of the embodiments of this invention show that overexpression of the PheANT gene from moso bamboo can significantly promote leaf growth and development, and the transgenic Arabidopsis plants exhibit obvious leaf phenotypes such as increased rosette leaves and altered leaf morphology.

[0018] The increase in the number of rosette leaves not only expands the photosynthetic area, providing a sufficient material basis for the later growth of crops, thus becoming a key breeding trait for increasing biomass and final yield; at the same time, this trait also provides ideal genetic material for in-depth analysis of the mechanisms of plant meristematic tissue activity, organ number determination, and the regulatory network of source-sink relationships.

[0019] Three patterns of leaf morphological changes—forked midrib heart-shaped leaves, dorsoventrally symmetrical double leaves, and asymmetrical double leaves at the petiole apex—can significantly improve light energy utilization and plant environmental adaptability in breeding by optimizing canopy structure and reducing mutual shading of leaves. At the same time, these morphological variations are valuable models for studying the establishment of developmental polarity in plant organs, the formation of leaf vein networks, and the plant's response mechanism to environmental signals. Attached Figure Description

[0020] Figure 1 Electrophoresis diagram of PCR amplification of the PheANT gene in bamboo (lane 1 is DNA Marker, lanes 2-4 are PCR amplification products of PheANT).

[0021] Figure 2 The structure diagram of the expression vector pCAMBIA1302;

[0022] Figure 3 Subcellular localization map of PheANT in moso bamboo (scale bar is 20 μm);

[0023] Figure 4 Phenotypic observations of transgenic Arabidopsis thaliana after three and four weeks of planting (Control is the control plant, #1 is the overexpression line pCAMBIA1302-PheANT-1, #2 is the overexpression line pCAMBIA1302-PheANT-2 and #3 is the overexpression line pCAMBIA1302-PheANT-3);

[0024] Figure 5 A graph showing the number of rosette leaves in transgenic Arabidopsis thaliana four weeks after planting (Col represents control plants, PheANT represents overexpression lines);

[0025] Figure 6 Phenotypic observation of leaf development changes in transgenic Arabidopsis thaliana four weeks after planting;

[0026] Figure 7 This is a graph showing the quantitative analysis of PheANT in the rosette leaves of transgenic Arabidopsis thaliana plants and control plants. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0028] The plant material used in this invention is bamboo leaves, collected from the Beida Mountain Laboratory of Nanjing Forestry University. The Arabidopsis thaliana (L.) Heynh. used in the genetic transformation experiment is of the Columbia variety (Col-0).

[0029] Example 1

[0030] 1. Total RNA extraction and cDNA synthesis

[0031] Total RNA was extracted from bamboo leaf tissue using the Novizan RNA Extraction Kit (RC 411). The procedure was strictly followed according to the product instructions to ensure the quality and integrity of the extracted RNA. Using the extracted total RNA as a template, reverse transcription was performed using the Total Gold Long Chain Reversal Kit (AT 311). The resulting cDNA product was then used for subsequent analysis.

[0032] 2. Cloning of the PheANT gene

[0033] Based on the *Phyllostachys pubescens* transcriptome database (https: / / gigadb.org / dataset / 100498#), full-length primers with restriction enzyme sites were designed using Primer 5 software to amplify the PheANT gene. Using cDNA from *Phyllostachys pubescens* leaves as a template, the PheANT fragment was amplified using the Novizan High Fidelity Polymerase Kit (P526) and corresponding specific primers. The specific PCR conditions and primer sequences are shown below:

[0034] PheANT-F:

[0035] 5'-ATGAGAGCAATGGCTAGTAGCG-3'(SEQ ID NO.3),

[0036] PheANT-R:

[0037] 5'-TTATGCATCCGTCCAAGCAG-3' (SEQ ID NO. 4).

[0038] The PCR reaction system consisted of: 12.5 μL 2×Phanta UniFi Master Mix [Dye Plus], 1 μL PheANT-F, 1 μL PheANT-R, 1 μL cDNA, and ddH2O to 25 μL.

[0039] The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 56℃ annealing for 10 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ for 5 min.

[0040] PCR amplification products were detected by 1% agarose gel electrophoresis. The electrophoresis results showed that the target gene band was single and clear. Figure 1 This indicates that the PCR amplification has good specificity and amplification efficiency. Under ultraviolet light irradiation, the electrophoretic band corresponding to the target gene was accurately located and cut using a UV transilluminator. Subsequently, the cut gel was processed using the Novizan gel extraction kit (DC 301) according to the manufacturer's instructions to purify and recover the PCR product of the target gene.

[0041] 3. Constructing an overexpression vector for the PheANT gene

[0042] The pCAMBIA1302 plant expression vector was double-digested with restriction enzymes NcoI-HF and BstEII-HF (purchased from New England Biotechnology Co., Ltd.). (Vector information is as follows) Figure 2 (As shown).

[0043] The double enzyme digestion reaction system was as follows: 10 μL pCAMBIA1302 plasmid, 5 μL 10×rcut smart, 1 μL NcoI-HF, 1 μL BstEII-HF, ddH2O to 50 μL.

[0044] The double enzyme digestion reaction procedure is as follows: digestion at 37℃ for 2 hours, followed by inactivation at 80℃ for 20 minutes.

[0045] The target gene PCR product was cloned into the linearized pCAMBIA1302 expression vector using the Novinogene Recombinant Gene Recombinant Kit (C115) in a one-step direct cloning process.

[0046] The ligation reaction system consisted of 2.5 μL of 2×CE Mix V3, 1.5 μL of gel-recovered product, and 1 μL of linearized vector. After thoroughly mixing the above ligation system, the mixture was reacted at 50°C for 5 min to allow the target gene and the linearized vector to ligate effectively via homologous recombination. After the reaction was complete, the ligation product was immediately cooled on ice to prepare for subsequent transformation of competent E. coli cells. The specific steps are as follows:

[0047] 1) Take 5 µL of ligation product and add it to 50 µL of slightly soluble E. coli competent cells DH5α. Gently mix and let stand on ice for 30 min.

[0048] 2) Heat shock in a water bath at 42℃ for 30 seconds, then quickly place on ice for 3 minutes;

[0049] 3) Add 900 μL of antibiotic-free LB liquid medium, mix well, and place in a shaker at 37°C and 200 rpm for 1 h; centrifuge at 5000 rpm for 5 min, discard 700 µL of supernatant on a clean bench, gently pipette the bacterial cells to fully suspend the bacterial solution, and take 100 µL of the bacterial solution to spread on LB agar plates containing Kan (50 μg / mL);

[0050] 5) Invert the container and incubate overnight in a 37°C incubator. The incubation time should not exceed 16 hours.

[0051] Inside a clean bench, select a single colony with a regular morphology (white, with smooth and regular edges), and suspend it in 10 μL of ddH2O, mixing thoroughly. Use a portion of the bacterial suspension as a PCR template and perform rapid PCR amplification using Novizan 2×Rapid TaqMaster Mix (P222).

[0052] The PCR reaction system consisted of the following components: 10 μL of 2×Rapid Taq Master Mix, 2 μL of bacterial culture, 1 μL of upstream primer, 1 μL of downstream primer, and 20 μL of ddH2O.

[0053] The PCR amplification program was set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃ annealing for 15 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ for 5 min.

[0054] PCR products were detected by 1% agarose gel electrophoresis, and positive clones whose target band length matched the expected length were selected for further processing.

[0055] The PCR-identified single-clone colonies were transferred to 350 μL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 3 h at 200 rpm. The cultured bacterial solution was then sent to Qingke Biotechnology Co., Ltd. for DNA sequencing verification. The sequencing results confirmed the correctness of the target gene sequence in the recombinant plasmid. The bacterial solution with correct sequencing results was used for long-term preservation. Specifically, an equal volume of 50% glycerol was added to the bacterial solution, mixed thoroughly, and stored at -80°C. The extracted recombinant plasmid was stored at -20°C for later use.

[0056] The nucleotide sequence determined based on the sequencing results is shown in SEQ ID NO. 1, and the gene is named the PheANT gene. The amino acid sequence of the protein it encodes is shown in SEQ ID NO. 2.

[0057] SEQ ID NO. 1:

[0058]

[0059] SEQ ID NO. 2:

[0060] MRAMASSGNWLGFSLSPHMAMEVPSSSSEPAPAHPPPPASAISSSSNNATTCNFLFSPPAQMAAPYPGYYYVGGAYGDGTSTAGVYYSHLPVMPVKSDGSVCMMEGMMPSSSPKLEDFLGGGGNGSGHDTATYYSHHQGQEEEASRGYQHHHQ LVPYNFQPLTEAEMLQEAAAPMEEAMAAAKNFLVTSYGACYSNGEMQPLSLSMSPGSQSSSCVSAAPQQHQMAVAAAQGGSNGGGEQCVGKKRGNGKGGQKQPVHRKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSCKKDGQTRKGRQV YLGGYDTEDKAARSYDLAALKYWGPSTHINFPLENYRDELEEMKGMTRQEYVAHLRRRSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGLNAVTNFDITRYDVDKIMESSLLPGEAARKVKA IEAGNGVSVMQNSGRELIPAEEASSTGTHWRMVLHGSPQQAVPCAEVTDPHSSLHGIVGLDVDSAAHDIDVSGKIGCINFSNSSSLVSSLSNSREGSPERLGLAMLYAKHPNAVSLASMSPWMSMPAPTATHALRPPNAVAHLPVFAAWTDA

[0061] 4. PheANT subcellular localization

[0062] Agrobacterium-containing recombinant plasmid pCAMBIA1302-PheANT was cultured in LB liquid medium (containing 50 μg / mL rifampin and 50 μg / mL kanamycin) at 28°C in a shaker until the OD600 reached 0.8. The cells were then collected by centrifugation at 4500 rpm for 13 minutes. The cells were resuspended in buffer until OD600 = 0.6 and incubated in the dark for 4 hours. Following the transient expression technique for tobacco, the bacterial culture was injected into tobacco leaves for subcellular localization observation. Results are as follows: Figure 3 As shown, consistent with typical transcription factors, PheANT is located in the cell nucleus.

[0063] Example 2

[0064] 1. Constructing overexpression vectors

[0065] The pCAMBIA1302-PheANT plant overexpression vector was constructed using homologous recombination. Homologous recombination primers containing specific homologous arms were used, and their sequences are shown in the table below:

[0066] PC1302-PheANT-F:

[0067] 5'- aacacgggggactcttgaccATGAGAGCAATGGCTAGTAGCG-3' (SEQ ID NO.5),

[0068] PC1302-PheANT-R:

[0069] 5'-ggaaattcgagctggtcaccTTATGCATCCGTCCAAGCAG-3' (SEQ ID NO. 6).

[0070] DNA fragments containing the target gene PheANT were obtained by PCR amplification. The amplification product was mixed with the linearized pCAMBIA1302 plant expression vector, which had been digested with enzymes, and homologous recombination was performed in one step using the ClonExpress Ultra One Step Cloning Kit V3. The successfully constructed recombinant vector was transformed into Agrobacterium GV3101 competent cells, and positive single colonies were selected for subsequent transformation experiments.

[0071] 2. Genetic transformation in Arabidopsis thaliana

[0072] 1) Transformation of Agrobacterium

[0073] Add 5 μL of recombinant plasmid DNA to 50 μL of GV3101 competent cells and gently tap the bottom of the tube to mix. Perform the following treatments sequentially: incubation on ice for 5 min, cold shock in liquid nitrogen for 5 min, water bath at 42°C for 5 min, and ice bath for 5 min each. Then add 900 μL of antibiotic-free LB broth and incubate at 28°C on a shaker for 3 h at 200 rpm. After centrifugation at 5000 rpm for 1 min, enrich the bacterial cells and discard 700 μL of supernatant on a clean bench. Resuspend the bacterial cells by pipetting, and spread 100 μL of the bacterial solution onto LB agar plates containing kanamycin (Kan 50 μg / mL) and rifampin (Rif 20 μg / mL). Incubate upside down at 28°C for 2 days to obtain positive Agrobacterium colonies containing the recombinant plasmid.

[0074] 2) Preparation of infection solution

[0075] Pipette 5 μL of bacterial culture into 300 μL of double-antibiotic LB liquid medium (Kan 50 μg / mL, Rif 20 μg / mL) for expansion culture, and shake at 200 rpm at 28°C. Once the culture becomes turbid, transfer it to a shake flask containing 30 mL of double-antibiotic LB liquid medium and continue incubation at 28°C with shaking for 1 day until the culture turns orange-yellow. Measure the OD of the bacterial culture using a UV spectrophotometer. 600 A value of 0.8 indicates that the bacterial concentration has reached the target value. After centrifugation at 5000 rpm for 10 min, discard the supernatant, collect the bacterial cells, add an equal volume of conversion osmosis buffer (5% sucrose (w / v, g / 100 mL), 0.02% surfactant (v / v)), shake well to mix, and set aside for later use.

[0076] 3) Arabidopsis thaliana culture

[0077] The Arabidopsis thaliana growing substrate was prepared using a ratio of peat moss:vermiculite:perlite = 9:3:1 (by weight). At the initial bolting stage, the main stem was removed to ensure even bolting throughout the entire tray. Before Agrobacterium infection, the pods on the inflorescence were removed, leaving only the flower buds to improve conversion efficiency.

[0078] 4) Agrobacterium infection and transfection of Arabidopsis thaliana

[0079] The flower buds of Arabidopsis thaliana were placed in Agrobacterium infection solution for 30 seconds, the liquid on the plant surface was wiped dry, and then the plants were infected again for 30 seconds. After infection, the plants were placed in the dark for 24 hours and then transferred to the plant growth chamber for normal culture. A second infection was carried out one week after the first infection during the peak flowering period of the plants to improve the transformation efficiency. After the Arabidopsis thaliana matured, T0 generation seeds were collected by mixing single genes, dried at 28°C, and then placed in silica gel to keep dry for further identification.

[0080] 3. Screening and identification of transgenic Arabidopsis thaliana positive seedlings

[0081] On a clean bench, 1.5 mL of 75% ethanol was added to centrifuge tubes containing transgenic and wild-type Arabidopsis thaliana T0 seeds, and the tubes were shaken at a constant speed for 5 min to sterilize. The ethanol was discarded, and 1 mL of anhydrous ethanol was added. The seeds were pipetted out and spread evenly on sterilized filter paper until they were completely dry. The sterilized wild-type seeds were evenly scattered on 1 / 2 MS solid medium containing antibiotics (25 mg / L) for control and resistance testing. The transgenic seeds were evenly scattered on 1 / 2 MS solid medium containing hygromycin (25 mg / L) after sterilization. Vernalization was carried out at 4℃ for 2 days, and then the seeds were transferred to a light incubator for cultivation (culturing conditions: 24℃, 16 h light, 8 h dark). About 10 days after sowing, healthy seedlings with 4 true leaves were selected and transplanted into a mixed substrate for normal light cultivation.

[0082] The obtained T1 generation seeds were sown on 1 / 2 MS solid medium containing hygromycin for screening. Leaves from positive seedlings were collected and extracted using the Novizan RNA Extraction Kit (RC 411). Based on the nucleotide sequence of the PheANT gene, quantitative primers for the PheANT gene were designed using Primer 5 software (forward primer qPheANT-F: GCTACAGCAACGGGGAGATG (SEQ ID NO. 7), reverse primer qPheANT-R: TCTTCCCCACACACTGCTCC (SEQ ID NO. 8)). PheANT expression was detected by PCR using quantitative primers. The selected transgenic lines were routinely cultivated to the T2 generation, during which phenotypic observation and data measurement were performed.

[0083] 4. Statistical and quantitative analysis of transgenic plant phenotypes

[0084] T2 generation Arabidopsis thaliana plants from lines #1, #2, and #3 were planted in a culture room (culture conditions: 24℃, 16h light, 8h darkness). The number of rosette leaves for each line system was counted from 20 plants.

[0085] like Figure 4 and Figure 5 As shown, overexpression of PheANT in Arabidopsis thaliana resulted in an increased number of leaves. Figure 6 As shown, after overexpression of PheANT in Arabidopsis thaliana, transgenic plants exhibited significant changes in leaf morphology, which can be summarized into three main patterns: The first pattern is a forked, heart-shaped leaf with disordered vein development, resulting in two main veins on the leaf that radiate from the petiole tip to the leaf margin in a forked manner. The overall leaf shape is a broad heart shape, which is significantly different from the single-main-veined lanceolate leaf of the wild type. Figure 6 (A) The second pattern is a dorsoventrally symmetrical bifoliate pattern, in which the blades differentiate symmetrically along the dorsoventrally axis (i.e., the axis between the front and back of the blade) at the tip of a single petiole, forming two blades arranged in the back direction. Figure 6 (B in the text); the third pattern is asymmetrical double lobes at the petiole apex, with a single petiole differentiating into two leaf blades ( Figure 6 The CD pattern was the most abundant of the three patterns. These phenotypic characteristics indicate that overexpression of the PheANT gene has a significant regulatory effect on leaf number and vein development in Arabidopsis thaliana.

[0086] 5. Expression analysis of PheANT in transgenic Arabidopsis thaliana

[0087] Leaves from T2 generation transgenic and wild-type Arabidopsis thaliana were collected before bolting for gene relative expression level analysis. Total RNA was extracted from leaf tissues of both transgenic and wild-type Arabidopsis thaliana using the Novizan RNA Extraction Kit (RC 411) following the product instructions, ensuring the quality and integrity of the extracted RNA. Using the extracted total RNA as a template, reverse transcription was performed using the Tiangen Long Chain Reverse Transcription Kit (KR 107).

[0088] Using cDNA obtained after reverse transcription as a template, real-time quantitative PCR analysis was performed using the AG11701 kit (SYBR Green fluorescent dye). Atα-tubulin was selected as an internal control gene to eliminate differences in RNA extraction amount and reverse transcription efficiency among samples. Primer sequences are shown below:

[0089] Atactin-F:

[0090] 5'-CTCTCCCGCTTTGAATTGTCTCGTTG-3'(SEQ ID NO.9),

[0091] Atactin-R:

[0092] 5'- GGTACCATTGTCACACACGATTGGT -3' (SEQ ID NO. 10);

[0093] AtANT-F:

[0094] 5'- GCTACAGCAACGGGGAGATG -3' (SEQ ID NO.11),

[0095] AtANT-R:

[0096] 5'-TCTCTCCCCACACACTGCTCC-3' (SEQ ID NO. 12).

[0097] qRT-PCR analysis showed that, compared with the wild-type control, the relative expression level of the PheANT gene in leaf tissues of the transgenic lines was significantly increased. Figure 7 The above results indicate that the PheANT gene effectively influences the morphogenesis of Arabidopsis leaves in the early stages of development by regulating leaf polarity and cell proliferation and expansion.

[0098] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A PheANT gene from moso bamboo, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. A protein that regulates plant leaf development, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. An expression vector containing the PheANT gene of bamboo as described in claim 1 or expressing the protein as described in claim 2.

4. A host cell, characterized in that, It contains the PheANT gene of bamboo as described in claim 1 or an expression vector expressing the protein as described in claim 2.

5. The application of the PheANT gene from *Phyllostachys pubescens* as described in claim 1 or the protein as described in claim 2 in regulating rosette leaf development in *Arabidopsis thaliana*, characterized in that... The regulation of rosette leaf development in Arabidopsis thaliana resulted in a significant increase in the number of rosette leaves and changes in leaf morphology.

6. The application according to claim 5, characterized in that, The specific steps are as follows: 1) Construct an overexpression vector for the PheANT gene in moso bamboo; 2) The constructed overexpression vector of the PheANT gene from moso bamboo was transformed into Arabidopsis thaliana; 3) Breed, screen and obtain transgenic Arabidopsis strains with significantly increased number of rosette leaves and altered leaf morphology.

7. The application according to claim 5, characterized in that, The leaf morphology changes mentioned include: heart-shaped leaves with forked midribs, dorsoventrally symmetrical double leaves, or asymmetrical double leaves at the petiole apex.

8. An Arabidopsis thaliana with an increased number of rosette leaves and / or altered leaf morphology, characterized in that, It contains the PheANT gene of moso bamboo as described in claim 1 or expresses the protein as described in claim 2.

9. The Arabidopsis thaliana according to claim 8, characterized in that, The expression described is an overexpression.

Images