Use of expression promoter of ptpta1 gene in regulating branching development of plant vegetative organs

Abstract

The application discloses an application of an expression promoter of a PtPTA1 gene in regulating development of plant vegetative organs, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the PtPTA1 gene is shown as SEQ ID NO.1.It is found that the PtPTA1 gene can positively regulate development of plant vegetative organs, so as to significantly improve plant yield.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of PtPTA1 gene expression promoters in regulating the branching and development of plant vegetative organs. Background Technology

[0002] Branching development of plant vegetative organs is a crucial trait in plant growth and development, directly affecting plant structure, photosynthetic efficiency, nutrient accumulation, and ultimately, yield. The rational regulation of branching number is one of the core objectives of crop genetic improvement. For food crops and medicinal plants, appropriate branching can increase the number of effective photosynthetic organs, improve overall photosynthetic efficiency, and promote nutrient accumulation, thereby increasing yield. For ornamental plants, branching determines the fullness of the plant structure, directly impacting its ornamental value.

[0003] Pinellia ternata ( Pinellia ternat *Pinellia ternata* (Thunb.) Breit. is a perennial herbaceous medicinal plant belonging to the genus *Pinellia* in the family Araceae. Its tuber is a traditional Chinese medicine with effects such as drying dampness and resolving phlegm, relieving nausea and vomiting, and dissipating lumps and nodules. It is widely used in the field of traditional Chinese medicine. The typical form of *Pinellia ternata* has only three leaves. It prefers a cool, moist environment and is sensitive to high temperatures. Under high-temperature stress, leaves easily wither, petioles die, and the plant collapses, resulting in a shortened growth period and insufficient accumulation of photosynthetic products. Simultaneously, its leaves have weak photosynthetic capacity, making it impossible to compensate for the photosynthetic deficit by increasing the number of effective photosynthetic organs, nor is it easy to delay the collapse by increasing the number of petioles. Ultimately, this leads to low tuber yields, making it difficult to meet market demand. In recent years, with global warming and frequent high-temperature stress, the problem of seedling collapse in Pinellia ternata has become increasingly prominent. Therefore, by using genetic engineering technology to discover key genes that regulate the branching of Pinellia ternata petioles, and to achieve targeted improvement of its branching traits, it is of great significance to increase the yield of Pinellia ternata and promote the development of the medicinal plant industry. This is because increasing the number of petiole branches can compensate for insufficient photosynthesis in the leaves, expand the photosynthetic area, and improve the assimilation capacity of products. It can also delay seedling collapse caused by high temperature and prolong the growth and development time by increasing the number of petioles.

[0004] Plant lectins are a class of proteins with at least one non-catalytic domain that can reversibly bind to specific monosaccharides or oligosaccharides. They are widely distributed in various plant tissues and are mainly involved in plant disease and pest responses as well as responses to stresses such as high salinity. However, there are currently no reports on the regulation of branching in vegetative organs by plant lectin genes. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of a PtPTA1 gene expression promoter in regulating the branching and development of plant vegetative organs.

[0006] The application of PtPTA1 gene expression promoters in regulating the branching development of plant vegetative organs, wherein the nucleotide sequence of the PtPTA1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2, and the regulation of plant vegetative organ branching development is manifested in promoting the formation and increase of plant vegetative organ branches.

[0007] SEQ ID NO.1 is as follows: ATGCTAACAAGCCAGCATCGCGGCGAAGAAGAAGAGTTAGCTAGGGTTTTGCACTCTCAGTACGTAGCCAGCCAGCTAGCAGCAACCCCGTTCCTGATCATGGCCTCCAAGCTCCTCCTCTTCCTCCTCCCGGCCATTCTCGGCCTCGGGTCTGCCGCTGCAGTGGGCACCAACTATATGCTGTCCGGCGAAACCCTGGACACGAATGGCCATCTCAGGAACGGCGACTTCGACTTGGTCATGCAGGAAGACTGCAACGCCGTCCTGTACAACGGCAACTGGCAGTCCAACACGGCCAACAAAGGACGGGACTGCAAGCTCACCCTCACCGACCGCGGCGAGCTCCTCATCAACAACGGCAACGGAGCCACCGTCTGGAGGAGCGGCTCCCAGCAGTCCGTGAAGGGCAACTACGCCGCCGCCGTCCATCCGGAGGGGAGGCTGGTCATCTACGGCCCATCCGTCTTCAATATCAACCCATGGGTCCCCGGCCTCAACAGCCTGCAGCTCGGCAACGTTCCTTTCGCGGGCAACATGATCTTCTCCGGCCAAGCCCTCCATGAAGAAGGCAGGCTCACGGCCAGGAACCACAGGCTCGTGATGCAGGGCGACTGCAACCTGGTCCTCTACGGCGGCAAGTTCGGCTGGCAGTCCAACACCCACGGCAACGGCGAGCACTGTTTCGTGAGGCTGAACCACAAGGGCGAGCTCATCATCAAAGACGACGACTTCAAGAGCATCTGGAGCAGCCGATCCAGCTCCAAGCAGCAGGGTGAGTACGTCTTCATCCTCCAGGACAACGGCTTCGGCGTCATCTACGGCCCTGCCATCTGGGACACTCGCTCGAAGAGCCCGGTGGAGAAGATGATCGGTATGGTGACTGAGAAGTGA。

[0008] SEQ ID NO.2 is as follows: MLTSQHRGEEEELARVLHSQYVASQLAATPFLIMASKLLLFLLPAILGLGSAAAVGTNYMLSGETLDTNGHLRNGDFDLVMQEDCNAVLYNGNWQSNTANKGRDCKLTLTDRGELLINNGNGATVWRSGSQQSVKGNYAAAVHPE GRLVIYGPSVFNINPWVPGLNSLQLGNVPFAGNMIFSGQALHEEGRLTARNHRLVMQGDCNLVLYGGKFGWQSNTHGNGEHCFVRLNHKGELIIKDDDFKSIWSSRSSSKQQGEYVFILQDNGFGVIYGPAIWDTRSKSPVEKMIGMVTEK.

[0009] This invention cloned the lectin gene PtPTA1 from Pinellia ternata, clarified its nucleotide and amino acid sequences, and for the first time discovered the function of lectin genes in regulating plant branching, confirming that the PtPTA1 gene can positively regulate the branching and development of plant vegetative organs.

[0010] Preferably, the plant is Arabidopsis thaliana or Pinellia ternata.

[0011] Preferably, the plant's vegetative organs include leaves and stems.

[0012] Preferably, the expression promoter of the PtPTA1 gene is used to increase plant yield.

[0013] Preferably, the expression promoter of the PtPTA1 gene is used to improve the high-temperature response of plants.

[0014] Preferably, the expression promoter of the PtPTA1 gene includes a recombinant plasmid carrying the PtPTA1 gene or a recombinant bacterium carrying the PtPTA1 gene.

[0015] Preferably, the recombinant plasmid is obtained by ligating the PtPTA1 gene into a plant expression vector.

[0016] Preferably, the recombinant bacteria are obtained by introducing the recombinant plasmid into Agrobacterium.

[0017] Preferably, promoting the formation and increase of branching of plant vegetative organs includes: introducing the PtPTA1 gene into plant cells or plants, thereby promoting the development of branching of plant vegetative organs.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention cloned the lectin gene PtPTA1 from Pinellia ternata, clarified its nucleotide and amino acid sequences, and for the first time discovered the function of the lectin gene in regulating plant branching and increasing yield.

[0019] This invention, through verification of the function of Pinellia ternata itself and verification of heterologous expression in Arabidopsis thaliana, confirms that the PtPTA1 gene can positively regulate the branching and development of plant vegetative organs and can significantly increase plant yield.

[0020] The PtPTA1 gene described in this invention has a wide range of applications and can be used for the genetic improvement of various plants such as Pinellia ternata, grain crops, and ornamental plants. It can not only solve the problem of low yield of Pinellia ternata, but also provide technical support for the optimization of plant type and yield improvement of other plants, and has broad application prospects.

[0021] The technical solution of this invention is simple and feasible. The constructed plant expression vector has high transformation efficiency, and the transgenic plants are easy to screen and readily applied on a large scale. It has significant theoretical research value and practical application value. It provides a novel gene target and technical pathway for optimizing the branching of vegetative organs and increasing yield in Pinellia ternata and other plants (such as ornamental plants and food crops) through genetic improvement, and has significant theoretical research value and promising prospects for agricultural applications. Attached Figure Description

[0022] Figure 1 This is an electrophoresis image of the PCR amplification of the Pinellia ternata lectin gene PtPTA1. In the image, M is the DNA molecular weight standard DL2000 DNA Marker, N is the negative control using water as a template, and I is the PCR amplification product of the PtPTA1 gene using Pinellia ternata cDNA as a template.

[0023] Figure 2 This is a schematic diagram of the structure of the plant expression vector pCAMBIA1305-PtPTA1 for the PtPTA1 gene.

[0024] Figure 3To investigate the effects of PtPTA1 transformation on branching and yield in Arabidopsis thaliana, (A) shows the PCR detection of the hygromycin gene in transformed plants, and M represents the DNA molecular weight standard DL2000 DNA. Marker: P represents the plasmid containing PtPTA1; N represents the negative control using water as a template; WT represents the control using wild-type Arabidopsis DNA as a template; 1–4 represent Arabidopsis lines transgenic with PtPTA1 selected by hygromycin; (B) shows the expression level of the PtPTA1 gene in transgenic plants analyzed by RT-PCR; WT represents the control using wild-type Arabidopsis cDNA as a template; OE-1–OE-3 represent independent transgenic lines; AtAct represents the Arabidopsis Actin gene used as an internal control; (C) compares the phenotypes of transgenic and wild-type Arabidopsis; (D) compares the yields of transgenic and wild-type Arabidopsis; (E) shows the branch number statistics of transgenic and wild-type Arabidopsis; (F) shows the pod number statistics of transgenic and wild-type Arabidopsis; (G) shows the seed weight per plant of transgenic and wild-type Arabidopsis. Different letters represent… P <0.05.

[0025] Figure 4 To investigate the effects of PtPTA1 transgenic plants on petiole branching and yield of Pinellia ternata, the following data were analyzed: (A) PCR detection of hygromycin gene in transgenic plants, M is the DNA molecular weight standard DL2000 DNA Marker, P is a plasmid containing PtPTA1, N is a negative control using water as a template, and 1–4 are PtPTA1 transgenic Pinellia ternata lines selected by hygromycin screening; (B) RT-qPCR analysis of PtPTA1 gene expression in transgenic plants, WT is a control using wild-type Pinellia ternata cDNA as a template, and OE-2, OE-4, and OE-5 are independent transgenic lines; (C) comparison of tuber yield between transgenic and wild-type Pinellia ternata; (D) comparison of phenotype between potted transgenic and wild-type Pinellia ternata; (E) comparison of plant phenotype between transgenic and wild-type Pinellia ternata; and (F) comparison of tuber size between transgenic and wild-type Pinellia ternata. Different letters in (B) and (C) represent… P <0.05.

[0026] Figure 5 This study compares the heat tolerance of PtPTA1-transgenic and wild-type Pinellia ternata under high temperatures. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0028] Example 1: Cloning and sequence analysis of the Pinellia ternata lectin gene PtPTA1 Experimental materials: The Pinellia ternata used in the experiment was the artificially cultivated Pinellia ternata variety "Su Banxia". Healthy Pinellia ternata plants free from diseases and pests were selected, fresh leaves were collected, washed, and the surface moisture was absorbed with filter paper. They were then stored in a refrigerator at -80℃ for later use.

[0029] Reagents: Trizol reagent (Invitrogen), reverse transcription kit (TaKaRa), PCR amplification kit (TaKaRa), DNA gel recovery kit (Omega), vector ligation kit (TaKaRa), Escherichia coli DH5α competent cells (Tiangen Biotech Co., Ltd.), etc.

[0030] Instruments: High-speed refrigerated centrifuge, PCR instrument, gel imaging system, clean bench, constant temperature incubator, etc.

[0031] 1. Extraction and reverse transcription of total RNA from Pinellia ternata According to the Trizol reagent instructions, total RNA was extracted from Pinellia ternata leaves: 0.1g of Pinellia ternata leaves were ground into powder with liquid nitrogen, transferred to a centrifuge tube, 1mL of Trizol reagent was added, vigorous shaking was performed for 15s, and the mixture was allowed to stand at room temperature for 5min; 0.2mL of chloroform was added, vigorous shaking was performed for 15s, and the mixture was allowed to stand at room temperature for 2min, then centrifuged at 12000g for 15min at 4℃; the upper aqueous phase was aspirated and transferred to a new centrifuge tube, 0.5mL of isopropanol was added, and the mixture was gently inverted to mix, allowed to stand at room temperature for 10min, and then centrifuged at 12000g for 10min at 4℃; the supernatant was discarded, 1mL of 75% ethanol (prepared with DEPC water) was added, the precipitate was gently washed, and the mixture was centrifuged at 7500g for 5min at 4℃; the supernatant was discarded, the precipitate was air-dried at room temperature, 20μL of DEPC water was added to dissolve the RNA, and the mixture was stored at -80℃ for later use.

[0032] The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit: According to the kit instructions, 2 μg of total RNA was taken, 1 μL of Oligo(dT) primer was added, and DEPC water was added to a final volume of 12 μL. The mixture was incubated at 65°C for 5 min, followed by an ice bath for 2 min. Then, 4 μL of 5× reverse transcription buffer, 1 μL of RNase inhibitor, 2 μL of dNTP mixture, and 1 μL of reverse transcriptase were added. The mixture was gently mixed, and the reaction was terminated by incubating at 42°C for 60 min, followed by an ice bath for 15 min. The cDNA was obtained and stored at -20°C for later use.

[0033] 2. PCR amplification of the PtPTA1 gene Based on the transcriptome sequencing data of Pinellia ternata, a suspected lectin gene sequence was obtained. Specific primers were designed (upstream primer: 5'-ATGCTAACAAGCCAGCATCGC-3', denoted as SEQ ID NO.3; downstream primer: 5'-TCACTTCTCAGTCACCATAC-3', denoted as SEQ ID NO.4). PCR amplification was performed using Pinellia ternata cDNA as a template. The PCR reaction system (25 μL) consisted of: 12.5 μL of 2×PCR Mix, 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L), 1 μL of cDNA template, and 9.5 μL of ddH2O. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min; and storage at 4℃. After PCR amplification, 5 μL of the amplification product was taken and subjected to 1% agarose gel electrophoresis to observe the size of the target band; the band that met the expected size was recovered using a DNA gel extraction kit to obtain the PCR product of the PtPTA1 gene. Figure 1 ).

[0034] 3. Cloning and sequence analysis of the PtPTA1 gene The recovered PCR product was ligated to the pMD18-T vector according to the instructions of the vector ligation kit: 4 μL of the recovered PCR product was added to 1 μL of pMD18-T vector and 5 μL of ligation buffer, and the mixture was gently mixed and incubated overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium containing ampicillin, and incubated at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, and cultured at 37°C with shaking at 200g for 8–10 h. The plasmid was extracted and verified by double enzyme digestion and sequencing (performed by Sangon Biotech (Shanghai) Co., Ltd.).

[0035] Sequencing results showed that the CDS sequence of the cloned PtPTA1 gene was 891 bp in length (the specific length was determined according to SEQ ID NO.1), and the nucleotide sequence was shown in SEQ ID NO.1; the protein encoded by this gene contains 296 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0036] Sequence alignment analysis showed that the protein encoded by the PtPTA1 gene has a typical plant lectin domain and is homologous to proteins encoded by other plant lectin genes, but it is unique in nucleotide and amino acid sequences, confirming that it is a novel Pinellia ternata lectin gene.

[0037] Example 2: Construction of a plant expression vector containing the PtPTA1 gene Vector backbone: pCAMBIA1305 plant expression vector (containing CaMV 35S promoter and hygromycin resistance gene); Reagents: Restriction endonucleases (BamHI and SacHI, TaKaRa), DNA ligase (TaKaRa), plasmid extraction kit (Omega), etc. Other materials: pMD18-T recombinant plasmid containing the PtPTA1 gene obtained in Example 1, and Escherichia coli DH5α competent cells.

[0038] 1. Double enzyme digestion reaction The pMD18-T recombinant plasmid containing the PtPTA1 gene and the pCAMBIA1305 vector were double-digested with enzymes respectively. Enzyme digestion system (50 μL): 20 μL plasmid, 2 μL BamHI, 2 μL SacHI, 5 μL 10× digestion buffer, 21 μL ddH2O; digestion in a water bath at 37℃ for 4 h.

[0039] After enzyme digestion, 10 μL of the digestion product was subjected to 1% agarose gel electrophoresis to confirm successful digestion. Then, the target fragment of the PtPTA1 gene and the pCAMBIA1305 vector fragment were recovered using a DNA gel recovery kit.

[0040] 2. Carrier connection and transformation The recovered PtPTA1 gene target fragment was ligated with the pCAMBIA1305 vector fragment: ligation system (20 μL): 5 μL vector fragment, 10 μL target fragment, 1 μL T4 DNA ligase, 2 μL 10× ligation buffer, 2 μL ddH2O; ligation was carried out overnight at 16℃.

[0041] The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB solid medium containing hygromycin. The cells were incubated at 37°C for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing hygromycin, and cultured at 37°C with shaking at 200 g for 8-10 h. The plasmid was extracted and verified by double enzyme digestion and PCR. The successfully verified recombinant plasmid was the plant expression vector containing the PtPTA1 gene, named pCAMBIA1305-PtPTA1. Figure 2 ).

[0042] Example 3: Heterologous expression and functional verification of the PtPTA1 gene in Arabidopsis thaliana The tested Arabidopsis thaliana variety was the Columbia ecotype (Col-0). Agrobacterium strain: GV3101; Reagents: Agrobacterium culture medium (LB medium, YEB medium), hygromycin, sterile water, Tween-80, etc.; Instruments: constant temperature shaking incubator, ultra-clean workbench, artificial climate chamber, etc.

[0043] 1. Agrobacterium-mediated transformation The pCAMBIA1305-PtPTA1 plant expression vector constructed in Example 2 was transformed into Agrobacterium GV3101 competent cells: 10 μL of recombinant plasmid was added to 100 μL of Agrobacterium competent cells and incubated on ice for 30 min; then frozen in liquid nitrogen for 5 min, incubated in water at 37°C for 5 min, and incubated on ice for 2 min; 1 mL of YEB liquid medium was added and cultured at 28°C with shaking at 200 g for 2–3 h (2 h in this example); 100 μL of bacterial culture was spread on YEB solid medium containing hygromycin and rifampin and cultured at 28°C for 2–3 days (2 days in this example); single colonies were picked and inoculated into YEB liquid medium containing the corresponding antibiotics and cultured overnight at 28°C with shaking at 200 g to obtain Agrobacterium bacterial culture.

[0044] 2. Arabidopsis thaliana transformation (flower dipping method) Arabidopsis thaliana plants (Col-0) that have reached full bloom were used as transformation recipients. Agrobacterium tumefaciens culture was centrifuged (5000g, 5min), the supernatant was discarded, and the culture was resuspended in infection solution (1 / 2 MS medium, containing 5% sucrose and 0.02% Tween-80). The OD600 value was adjusted to 0.8-1.0, and in this example, it was adjusted to 0.9. Arabidopsis thaliana inflorescences were immersed in the culture solution for 30s, and gently shaken to ensure that the inflorescences were fully in contact with the culture solution. The infected Arabidopsis thaliana plants were covered with plastic wrap and cultured in the dark for 24h, and then transferred to an artificial climate chamber for normal culture (16h light / 8h dark, 22℃, 70% humidity).

[0045] Once the Arabidopsis seeds mature, harvest them, dry them, and store them for later use.

[0046] 3. Screening and identification of transgenic Arabidopsis thaliana The harvested Arabidopsis seeds were disinfected with 75% ethanol for 30 seconds, rinsed 3-4 times with sterile water, disinfected with 0.1% mercuric chloride for 10 minutes, and rinsed 5-6 times with sterile water. The disinfected seeds were evenly spread on MS solid medium containing hygromycin (concentration 10 μg / mL), vernalized at 4℃ for 3 days, and then transferred to an artificial climate chamber for 7-10 days of cultivation (8 days in this example). Seedlings with hygromycin resistance (positive seedlings) were selected, transplanted into nutrient soil, and cultured normally.

[0047] Genomic DNA and RNA were extracted from positive Arabidopsis thaliana plants, and the hygromycin gene was detected by PCR (upstream primer: 5'-ACTCACCGCGACGTCTGT-3', SEQ ID NO. 5; downstream primer: 5'-TTTCTTTGCCCTCGGACG-3', SEQ ID NO. 6). Positive plants were selected for RT-PCR verification of PtPTA1 gene integration and expression, using upstream primer: 5'-AACAAAGGACGGGACTGCAA-3', SEQ ID NO. 7; downstream primer: 5'-TGAGCCTGCCTTCTTCATGG-3', SEQ ID NO. 8. The Arabidopsis thaliana Actin gene was used as an internal reference gene, and the amplification primers were: upstream primer: 5'-GTCGTACAACCGGTATTGTG-3', SEQ ID NO. 9; downstream primer: 5'-GAGCTGGTCTTTGAGGTTTC-3', SEQ ID NO. 10.

[0048] The results showed that the PtPTA1 gene was normally transcribed and expressed in transgenic Arabidopsis plants, confirming that the PtPTA1 gene had been successfully integrated into the Arabidopsis genome and expressed, resulting in the acquisition of transgenic Arabidopsis plants. Figure 3 A, B), select independent homozygous lines by harvesting individual plants.

[0049] 4. The regulatory role of the PtPTA1 gene in Arabidopsis branching Transgenic Arabidopsis plants (T3 generation homozygous lines) were selected, with wild-type Arabidopsis (Col-0) as a control. They were planted in nutrient soil and cultured normally in artificial climate chambers. The growth status of the plants was observed and recorded regularly, with a focus on counting the number of branches of the stem (roselet stem).

[0050] After 40 days of cultivation, statistical results showed that the average number of stem branches in wild-type Arabidopsis thaliana was 1, while the average number of stem branches in transgenic Arabidopsis thaliana was 3-4, significantly higher than that in wild-type Arabidopsis thaliana (P<0.05), and seed yield was increased. Figure 3 C~G).

[0051] The above results indicate that heterologous expression of the PtPTA1 gene in Arabidopsis thaliana can significantly induce increased stem branching in Arabidopsis thaliana, and has a positive regulatory function on the branching development of vegetative organs in Arabidopsis thaliana.

[0052] Example 4: Functional verification of the PtPTA1 gene in Pinellia ternata Materials used for testing: tissue culture seedlings of "Staphyllum pinellia"; Agrobacterium strain: GV3101 (containing pCAMBIA1305-PtPTA1 plant expression vector); Reagents: MS medium, hygromycin, Agrobacterium infection solution, sterile water, etc. Instruments: Clean bench, constant temperature incubator, shaker, etc.

[0053] 1. Infection and transformation of Pinellia ternata tissue culture seedlings Take healthy Pinellia ternata tissue culture seedlings with a height of about 3-5 cm, remove the leaves, and retain the stem segments and petiole bases as transformation recipients; resuspend the Agrobacterium GV3101 bacterial suspension containing the pCAMBIA1305-PtPTA1 vector in infection medium (MS medium, containing 5% sucrose, 0.02% Tween-80), and adjust the OD600 value to 0.6-0.8, in this example, the OD600 value is adjusted to 0.7; immerse the Pinellia ternata tissue culture seedling stem segments in the bacterial suspension for 15-20 min, in this example, for 18 min, gently shaking during the process; after infection, blot the bacterial suspension on the surface of the stem segments with sterile filter paper, and inoculate them on co-culture medium (MS medium + 0.1 mg / L 6-BA + 0.05 mg / L NAA), and co-culture at 25℃ in the dark for 3 days.

[0054] 2. Screening and identification of genetically modified Pinellia ternata After co-culture, the Pinellia ternata tissue culture seedlings were transferred to the selection medium (MS medium + 0.1 mg / L 6-BA + 0.05 mg / L NAA + 50 μg / mL hygromycin) and cultured at 25℃ under 12h light / 12h dark conditions. The selection medium was changed every 2 weeks to screen for hygromycin-resistant callus tissue and regenerated seedlings. When the regenerated seedlings grew to 3-4 leaves, they were transplanted to the rooting medium (1 / 2 MS medium + 0.1 mg / L IAA) and cultured until rooting.

[0055] Genomic DNA and RNA were extracted from rooted transgenic Pinellia ternata plants, and the integration and expression of the PtPTA1 gene were verified by PCR and real-time quantitative PCR (RT-qPCR). Hygromycin gene was detected in DNA samples by PCR (primers and amplification conditions as above). Positive samples were further analyzed for expression levels using RT-qPCR. PtGAPDH The gene is an internal reference (upstream primer: 5'-ACTGTTGATGGACCTTCTG-3', denoted as SEQ ID NO.11, downstream primer: 5'-TTGGAACTCGGAATGACAT-3', denoted as SEQ ID NO.12).

[0056] The results showed that hygromycin-specific bands could be amplified in positive transgenic Pinellia ternata plants ( Figure 4 A), and RT-qPCR showed that the expression level of transgenic plants was more than 5 times that of wild-type plants (A). Figure 4B) indicates that the PtPTA1 gene has been successfully integrated into the Pinellia ternata genome and expressed, resulting in transgenic Pinellia ternata plants.

[0057] 3. The regulatory effect of the PtPTA1 gene on the branching and yield of Pinellia ternata petioles Transgenic Pinellia ternata plants were selected, and non-transgenic Pinellia ternata tissue culture seedlings were used as controls. They were transplanted into greenhouses and managed normally (temperature controlled at 20-25℃, humidity at 60-70%, and regular watering and fertilization). The growth status of the plants was observed and recorded regularly, with a focus on counting the number of petiole branches and the number of effective vegetative organs. After the Pinellia ternata tubers matured, the tuber yield was counted.

[0058] After 30 days of cultivation, statistical results showed that the control Pinellia ternata plants had an average of 1 petiole and 1-3 effective vegetative organs (leaves); while the transgenic Pinellia ternata plants had an average of 2-3 petiole branches and 4-9 effective vegetative organs, significantly higher than the control group. P <0.05); the yield of transgenic Pinellia tubers increased by more than 80% compared with the control group ( P <0.05)( Figure 4 (D~F). The results show that... PtPTA1 The gene promotes the branching of Pinellia ternata petioles, increases the number of effective vegetative organs, and improves photosynthetic efficiency and nutrient accumulation efficiency, thereby increasing the yield of Pinellia ternata.

[0059] Example 5: Functional verification of PtPTA1-transformed Pinellia ternata in high-temperature response Transgenic and control Pinellia tubers were planted in pots containing nutrient soil and cultured at 25℃ until they reached about 15cm in height. Then, they were subjected to high temperature stress at 40℃ for 4 days and then cultured at room temperature at 25℃ for 7 days before the plant phenotype was observed. Figure 5 It was observed that all wild-type Pinellia ternata died, but while some petioles of the transgenic Pinellia ternata withered, others still grew well. This indicates that increasing the number of petioles by transgenic Pinellia ternata with the PtPTA1 gene can effectively alleviate the seedling collapse caused by high temperatures.

[0060] In summary, the following objectives can be achieved using the content of this invention: Constructing plant expression vectors containing the Pinellia ternata lectin gene PtPTA1 (such as commonly used plant expression vectors like pCAMBIA1305 and pBI121), transforming them into target plants, and obtaining transgenic plants with increased branching of vegetative organs and improved yield. Editing homologous genes of the Pinellia ternata lectin gene PtPTA1 in target plants using gene editing technology to regulate the branching development of vegetative organs and increase the yield of target plants. Using the Pinellia ternata lectin gene PtPTA1 as a molecular marker to screen plant varieties with strong branching ability of vegetative organs and high yield. Utilizing the protein encoded by the Pinellia ternata lectin gene PtPTA1 to regulate the branching signaling pathway of plant vegetative organs, promoting plant branching and yield improvement.

[0061] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0062] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0063] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of PtPTA1 gene expression promoters in regulating branching development of plant vegetative organs, characterized in that, The nucleotide sequence of the PtPTA1 gene is shown in SEQ ID NO.1, and the regulation of branching development of plant vegetative organs is manifested in promoting the formation and increase of branches of plant vegetative organs.

2. The application according to claim 1, characterized in that, The plant in question is either Arabidopsis thaliana or Pinellia ternata.

3. The application according to claim 2, characterized in that, The plant's vegetative organs include leaves and stems.

4. The application according to claim 2, characterized in that, The expression promoter of the PtPTA1 gene is used to increase plant yield.

5. The application according to claim 2, characterized in that, The expression promoter of the PtPTA1 gene is used to improve the high-temperature response of plants.

6. The application according to claim 1, characterized in that, The expression promoter of the PtPTA1 gene includes a recombinant plasmid carrying the PtPTA1 gene or a recombinant bacterium carrying the PtPTA1 gene.

7. The application according to claim 6, characterized in that, The recombinant plasmid is obtained by ligating the PtPTA1 gene into a plant expression vector.

8. The application according to claim 6, characterized in that, The recombinant bacteria are obtained by introducing the recombinant plasmid into Agrobacterium.

9. The application according to claim 1, characterized in that, Promoting the formation and increase of branching in plant vegetative organs includes introducing the PtPTA1 gene into plant cells or plants, thereby promoting the development of branching in plant vegetative organs.

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