Application of populus tomentosa pttch4 gene in regulating plant growth and development

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to the application of the Populus tomentosa PtTCH4 gene in regulating plant growth and development. The nucleotide sequence of the Populus tomentosa PtTCH4 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. This invention, by overexpressing the PtTCH4 gene, results in transgenic plants with lighter inflorescence color and larger inflorescences compared to the wild type; this regulation of plant inflorescence development is of great significance for promoting plant genetic improvement and breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of the PtTCH4 gene of Populus tomentosa in regulating plant growth and development. Background Technology

[0002] The morphology and color characteristics of plant inflorescences play a crucial role in natural reproduction. Plants with brightly colored inflorescences are more likely to attract pollinating insects such as bees and butterflies, thus significantly improving pollination efficiency. Inflorescences with lighter colors or variations in hue may, to some extent, expand the variety and activity range of pollinating insects, facilitating gene exchange between different regions or species. It is noteworthy that variations in inflorescence color not only differ between different populations but may also serve as an evolutionary strategy, reducing interspecific pollen interference, increasing reproductive success rates, and enhancing plant survival under adverse conditions such as drought, high temperatures, and salinity. Therefore, the optimization and regulation of inflorescence traits are not only important results of natural selection but also a key trait that urgently needs attention in the breeding of economic plants such as forest trees and crops.

[0003] Populus tomentosa ( Populus tomentosa Belonging to the Populus family, it is most concentrated in the plains of the middle and lower reaches of the Yellow River, with a suitable altitude below 1500 meters. Due to its upright shape and beautiful crown, this species is an excellent choice for shelterbelt construction and roadside greening, as well as a high-quality source of industrial timber. However, in current forestry production practices, many woody plants, including the Populus tomentosa, suffer from unstable inflorescence traits or low pollination efficiency to varying degrees, which to some extent limits their natural reproduction rate and artificial breeding efficiency.

[0004] Currently, there is a certain amount of research on the molecular mechanisms affecting inflorescence development and color formation both domestically and internationally. However, for specific tree species such as Populus tomentosa, especially regarding the association between the TCH4 gene and inflorescence traits, there is a lack of systematic research reports. The TCH4 gene may have important biological significance in plant cell wall metabolism, morphogenesis, and environmental response, but its potential inflorescence regulatory function remains unknown. Therefore, in-depth exploration of unique forest resources such as Populus tomentosa, identification of key functional genes related to inflorescence traits, and application of these genes to novel molecular breeding techniques will not only help optimize inflorescence characteristics, improve pollination efficiency and seed yield, but also significantly enhance the survival rate and economic value of trees under adverse conditions. Summary of the Invention

[0005] Based on the deficiencies in the prior art, the purpose of this invention is to provide the application of the Populus tomentosa PtTCH4 gene in regulating plant growth and development.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the Populus tomentosa PtTCH4 gene in regulating plant growth and development, characterized in that the nucleotide sequence of the Populus tomentosa PtTCH4 gene is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the protein encoded by the Populus tomentosa PtTCH4 gene is shown in SEQ ID NO.2.

[0008] Preferably, the application in plant growth and development is one or more of the following: (1) Regulating the inflorescence area of ​​plants; (2) Regulating the color of plant inflorescences; (3) Regulating plant leaf length; (4) Regulate the width of plant leaves.

[0009] Preferably, the application involves overexpressing the PtTCH4 gene in Populus tomentosa to reduce the color of the inflorescence, decrease the inflorescence area, increase the leaf length, and decrease the leaf width.

[0010] Preferably, the method for overexpressing the Populus tomentosa PtTCH4 gene includes the following steps: An overexpression vector for the PtTCH4 gene of Populus tomentosa was constructed. The plants were genetically transformed using Agrobacterium infection, and transgenic lines overexpressing the PtTCH4 gene of Populus tomentosa were screened to achieve overexpression of the PtTCH4 gene of Populus tomentosa.

[0011] Preferably, the plant is tobacco or poplar.

[0012] The present invention has the following technical effects and advantages: This invention proposes that overexpression of the Populus tomentosa PtTCH4 gene can regulate tobacco inflorescence development, which is of great significance for promoting plant genetic improvement and breeding. Attached Figure Description

[0013] Figure 1 The backbone map of the expression vector pCAMBIA1300-35S-GFP containing a strong 35S promoter, which is provided for embodiments of the present invention, shows how the coding sequence of the PtTCH4 gene was cloned into the vector using homologous recombination. Figure 2 The image shows an electrophoresis diagram of RNA extraction from Populus tomentosa provided in an embodiment of the present invention. Each RNA has three bands, namely 5S, 18S, and 28S. Figure 3The image shows a 1% agarose gel electrophoresis result of the PtTCH4 gene provided in this embodiment of the invention. The DNA marker is DL2000. Lane 1 is a negative test result for ddH2O, and lanes 2, 3, 4, and 5 are positive test results. Figure 4 The image shows a 1% agarose gel electrophoresis image of a single colony of Escherichia coli PtTCH4 gene provided in an embodiment of the present invention. The DNA marker is DL2000. Lane 1 shows the Agrobacterium plasmid result, and lanes 2, 3, and 4 show the transgenic tobacco detection result. Figure 5 The image shows Agrobacterium colonies on 1% agarose gel electrophoresis provided in this embodiment of the invention. The DNA marker is DL2000. Lane 10 is the negative control, and lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent the Agrobacterium detection results. Figure 6 The image shows an RT-PCR result of PtTCH4 identification in transgenic tobacco provided in this embodiment of the invention. The DNA marker is DL2000, lane 1 is the positive control, lanes 2, 3, and 4 are the detection results of transgenic tobacco, and the primers are quantitative fluorescent primers. Figure 7 The results of detecting the transcriptional expression level of 35Spro::PtTCH4-GFP transgenic tobacco provided in the embodiments of the present invention; Figure 8 The growth status of transgenic tobacco PtTCH4 and wild-type tobacco WT in adventitious root differentiation medium provided in this embodiment of the invention. WT is wild-type tobacco and TCH4 is transgenic tobacco. Figure 9 A width comparison diagram of overexpressed tobacco PtTCH4 and wild-type tobacco WT provided in an embodiment of the present invention; Figure 10 A statistical diagram of leaf length of overexpressed tobacco PtTCH4 and wild-type tobacco WT provided in an embodiment of the present invention; Figure 11 A comparison diagram of inflorescence size and color between overexpressed tobacco PtTCH4 and wild-type tobacco WT provided in an embodiment of the present invention; Figure 12 A statistical diagram of inflorescence size of overexpressed tobacco PtTCH4 and wild-type tobacco WT provided in an embodiment of the present invention. Detailed Implementation

[0014] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Example 1

[0016] Cloning of the PtTCH4 gene in Populus tomentosa The protein sequence of the Arabidopsis thaliana AtTCH4 gene was found on the Tair website, and the sequence was obtained from Populus tomentosa (Populus tomentosa). Populus tomentosa The gene most homologous to it in the genome is PtTCH4; amplification primers were designed using SnapGene software, and the protein coding sequence of PtTCH4 (abbreviated as "TCH4") was obtained by PCR amplification using Populus tomentosa genomic cDNA as a template. The specific process is as follows: 1. Extraction of total RNA from Populus tomentosa leaves: Prepare 0.2g of Populus tomentosa leaves, grind them into powder under liquid nitrogen, and extract RNA using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit from Tiangen Biotech Co., Ltd. The results are as follows: Figure 2 As shown, there are bands at 28S, 18S and 5S respectively; 2. Reverse transcription of RNA into cDNA: cDNA of Populus tomentosa was obtained using the All-Gold EasyScript One-Step gDNA Removal and cDNA Synthesis Kit. 3. PCR amplification of the target gene PtTCH4: 3.1 Analysis and design of homologous amplification primers for the PtTCH4 coding region: PtTCH4-F (shown in SEQ ID NO.3): GAGCTCGGTACCATGGCTGCTGCTTATCCGTG; PtTCH4-R (shown as SEQ ID NO.4): GACTCTAGAGGATCCTATGTCTCTGTCTCTCTTGCATTCT; 3.2 The PCR reaction system is shown in Table 1: Table 1

[0017] 3.3 PCR reaction conditions are shown in Table 2: Table 2

[0018] 3.4 Identification by 1% agarose gel electrophoresis: Weigh 0.5 g of agarose, add 50 mL of 1×TAE, heat to melt, cool to room temperature, add 5 μL of nucleic acid dye, and after the agarose gel cools, add the PCR product to the sample wells and perform electrophoresis for 15 min; the electrophoresis results are as follows. Figure 3 As shown, the DNA Marker is DL2000, and lane 1 is... Negative test, target bands in lanes 2, 3, 4, and 5 were positive; the target band was cut off with a scalpel, placed in a 1.5 mL centrifuge tube, and weighed; the 873 bp target gene fragment was recovered according to the instructions of the DNA gel recovery kit from Beijing TransGen Biotech Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO.1, specifically: Atggctgctgcttatccgtggactttgtttcttggcatgctggttatggtatctggaacaatgggagctgccctgaggaagccagtggatgtggcgttcggtaggaactatgttccgacatgggcttttgaccacattaagtacttcaatggaggcaatgagattcagctacacttggataaatacacaggtactggtttccaatcaaaaggttcatacttatttggacatttcagtatgcaaatgaagttggttcctggtgactcagctggaacagttactgctttctatctatcctcacaaaactcggagcatgacgagatagactttgagttcttaggaaacaggactggccagccctacattttgcagactaatgttttcacaggaggcaagggggatagagaacagaggatttacctctggtttgacccaaccaaggaattccaccactattctgtcctctggaacatgttcctcgtggatgatgtgccaatcagagtgttcaagaactgcaaagatttgggagtgaagtttccattcaaccagccaatgaagatctactcaagcctatggaatgccgatgattgggctaccaggggtggactcgagaagacagactggtccaaggcacccttcattgcctcctacaggagtttccacatagatgggtgcgaggcctccgtggaagccaagttctgcgccacacagggtgctagatggtgggaccagaaggagttccaagatctggatgccttccagtacaggaggctcagctgggtccgccagaaatataccatctacaactactgcactgatagatcaagatacccttcaatgcccccagaatgcaagagagacagagacatataa. SED ID No.2: Amino acid sequence of Populus tomentosa xyloglucosyltransferase TCH4 protein MASLKTVLVPLVALLVTVASASNFYNDFDITWGDGRAKILSNGELLTLNLDKASGSGFQSRNEYLFGKIDMQLKLVPGNSAGTVTAYYLSSKGSAWDEIDFEFLGNLSGDPYILHTNVFSQGKGNREQQFYLWFDPTADFHTY SILWNPQRIVFSVDGTPIREFKNLESMGVPFPKNQPMRIYSSLWNADDWATRGGLVKTDWALAPFTASYRNFNAEACVLSNGASSCGTTTSPPASTSNAWFSEELDSTRQERLKWVRENYMVYNYCKDVNRFPQGLPPECSMS. Example 2 Construction and genetic transformation of plant overexpression vectors for the PtTCH4 gene I. Construction of PtTCH4 gene plant expression vector The coding sequence of the PtTCH4 gene obtained in Example 1 was cloned into an expression vector containing a 35S strong promoter using homologous recombination. ,like Figure 1 As shown, the specific process is as follows: 1. Vector double digestion: KpnⅠ and BamHI were selected as digestion sites, and the reaction mixture was added according to Table 3. Digestion was carried out at 37℃ for 20 min. Table 3

[0019] 2. Using a homologous recombination kit purchased from Tiangen Biotech (Beijing) Co., Ltd., the target gene and vector were ligated; 3. The ligation product was transferred into competent *E. coli* cells DH5α, incubated on ice for 30 min, heat-shocked for 45 s, and then incubated on ice again for 2 min. 600 μL of LB liquid culture medium was added, and the cells were placed in a constant-temperature shaker at 37°C and 220 rpm for 1 h to recover. 100 μL of activated *E. coli* was evenly spread onto LB solid medium containing Kana antibiotic. The cells were incubated upside down at 37°C for 12-16 h. After colony growth, single colonies were picked and placed in 700 μL of LB liquid medium containing Kana antibiotic. The colonies were grown at 37°C for 6 h. Single-colony PCR was performed according to the reaction system described in Table 4, and identification was performed by agarose gel electrophoresis. The 1% agarose gel electrophoresis image of a single *E. coli* colony is shown below. Figure 4 As shown, the DNA marker is DL2000, lane 1 represents the Agrobacterium plasmid results, and lanes 2, 3, and 4 represent the transgenic tobacco detection results. Bacterial cultures showing the observed target band were sent to the company for sequencing, positive clones were screened, and the vector was named [vector name missing]. .

[0020] The primers for single colony PCR are: PtTCH4-F (shown in SEQ ID NO.3): GAGCTCGGTACCATGGCTGCTGCTTATCCGTG; PtTCH4-R (shown as SEQ ID NO.4): GACTCTAGAGGATCCTATGTCTCTGTCTCTCTTGCATTCT; Table 4

[0021] Colonies with correct sequencing results were incubated at 37°C for 12 h. Plasmids were extracted using the TIANprep plasmid kit, and Agrobacterium was transformed using GV3101. Single colonies were picked for PCR detection, and the results are as follows: Figure 5 As shown, the DNA marker is DL2000, lane 10 is the negative control, and lanes 1, 2, 3, 4, 5, 6, 7, 8, and 9 represent the Agrobacterium detection results.

[0022] II. Transgenic Tobacco Conversion An overexpression vector containing the PtTCH4 gene coding sequence was transformed into tobacco using Agrobacterium-mediated transformation. After pre-culture, infection, dark culture, induction of adventitious shoots, induction of shoot rooting, propagation, hardening, and transplanting to a greenhouse, the process was detailed below: 1. Leaf pre-culture: Take leaves from wild-type tobacco tissue culture seedlings that are 4-5 weeks old and in good growth condition. In a clean bench, use sterile sterilization equipment to make cuts on the leaves along the direction perpendicular to the veins. Lay the leaves flat on the antibiotic-free pre-culture medium with the upper surface facing up and incubate at 25°C for 2 days. 2. Preparation of Agrobacterium infection solution: Pick a sample containing Agrobacterium from the plate. Single colonies of Agrobacterium (GV3101 competent cells) were inoculated into 100 mL of liquid LB medium containing Kana and Rif antibiotics and cultured at 28°C and 180 rpm until... ; 3. Infection and co-culture: In a clean bench, immerse the pre-cultured leaves in Agrobacterium bacterial solution for 15 minutes, gently shaking them 2-3 times during the process to ensure that the injured parts of the leaves are fully in contact with the bacterial solution. After infection, remove the leaves with sterile forceps and place them on pre-sterilized filter paper to absorb excess bacterial solution. Finally, inoculate the infected leaves onto a co-culture medium without antibiotics and incubate them in the dark at 25°C for 3 days. 4. Resistance culture: In a clean bench, leaves that have been dark-treated for 3 days are placed on pre-sterilized filter paper to remove excess bacteria, and then transferred to differentiation and selection medium. Under the conditions of 25℃, 16 h light / 8 h dark, adventitious buds resistant to hygromycin are induced and screened. 5. After 10 days of selective culture, in a clean bench, use sterile forceps to place the light green, dense callus tissue or leaves with adventitious buds onto a new selective culture medium so that the callus tissue or adventitious buds can have more sufficient nutrition to induce differentiation. 6. Continue to select and culture for about 2 weeks. When the adventitious buds grow to 3-4 cm or more, use a sterile scalpel in a clean bench to cut off the adventitious buds individually and place them on a rooting medium containing selection pressure (50 mg / L hygromycin) and sterilization (200 mg / L termethin) for rooting culture. Two weeks later, the adventitious buds will grow adventitious roots. After rooting, subculture for propagation (the first subculture medium contains termethin and hygromycin, and subsequent subcultures only contain termethin). 7. The culture medium formulations used for the above genetic transformations are shown in Table 5, 1L (adjusted to pH 5.8-6.0 with NaOH): Table 5

[0023] III. Identification of Transgenic Plants Using the above transformation method, a total of 18 resistant plants were obtained. Leaves from these resistant plants were harvested, and their genomic DNA was extracted. Identification primers were designed, and positive transgenic plants were identified by PCR, as detailed below: 1. Steps for rapid extraction of genomic DNA: Take 0.2 g of tobacco leaves and place them in a 2 mL centrifuge tube containing small steel balls. Quickly freeze in liquid nitrogen, then grind into powder using a grinder. Add 400 μL of Edwards extract, vortex for 15 s, and centrifuge at 12000 rpm for 3 min. Transfer the supernatant to a 1.5 mL centrifuge tube, add an equal volume of isopropanol (400 μL), and mix thoroughly by pipetting. After incubating on ice for 30 min, centrifuge at 12000 rpm for 5 min and discard the supernatant. Add 400 μL of 70% ethanol to the precipitate, centrifuge at 12000 rpm for 2 min, discard the supernatant, and dry overnight at 28°C. Add... , stored at 4℃; 2. PCR identification of transgenic tobacco: Forward primers were designed based on the PtTCH4 genome sequence, and vectors were used for this purpose. Reverse primers were designed for PCR amplification, followed by electrophoresis detection. Positive transgenic plants were verified using agarose gel electrophoresis. Transgenic tobacco PtTCH4 was identified. The results are as follows Figure 6As shown, the DNA marker is DL2000, lane 1 is the positive control, and lanes 2, 3, and 4 are the positive results of transgenic tobacco detection. 2.1 Identification primers: (shown in SEQ ID NO.5): ATGGCTGCTGCTTATCCGTG; (Shown in SEQ ID NO.6): CAGGGTCAGCTTGCCGTAGG; 2.2 The PCR reaction system is shown in Table 6: Table 6

[0024] 2.3 PCR reaction conditions are shown in Table 7: Table 7

[0025] 2.4. Take 10 mL of PCR product and perform 1% agarose gel electrophoresis to screen out 3 strains that were successfully transformed. Positive plants.

[0026] (5) Identification of expression levels in transgenic plants: Total RNA was extracted from transgenic poplar lines, and the expression level of the PtTCH4 gene was detected using real-time quantitative PCR. The results of real-time quantitative PCR are as follows: Figure 7 As shown, the highest expression level is Secondly, strain #2 was selected. As materials for subsequent experiments.

[0027] Example 3 Phenotypic analysis of PtTCH4 gene overexpression lines The overexpression line from Example 2 The specific steps for phenotypic analysis of experimental materials are as follows: 1. Total RNA extraction from tobacco was performed using a kit from Tiangen Biotech Co., Ltd. (RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit); 2. Tobacco RNA reverse transcription was performed using the EasyScript one-step gDNA removal and cDNA synthesis kit. 3. Detection of PtTCH4 expression level using real-time quantitative PCR: The cDNA obtained from reverse transcription was analyzed using Green qPCR SuperMix fluorescent quantitative enzyme from TransGen Biotech Co., Ltd. As specific primers, As an internal control primer, it was used for quantitative determination of gene expression, as detailed below: 3.1 Identification primers: qPtTCH4-F (shown in SEQ ID NO.7): TTCCTCGTGGATGATGTGCC; qPtTCH4-R (shown in SEQ ID NO.8): AGAACTTGGCTTCCACGGAG; Actin-F (shown in SEQ ID NO.9): AAGGGATGCGAGGATGGA; Actin-R (shown in SEQ ID NO. 10): CAAGGAAATCACCGCTTTGG.

[0028] 3.2 The Real-time PCR reaction system is shown in Table 8: Table 8

[0029] 3.3 The amplification conditions for real-time quantitative PCR are shown in Table 9: Table 9

[0030] Repeat the amplification step 40 times, and collect fluorescence signals after each cycle; at 65-95℃, increase the temperature by 0.5℃ every 5 seconds, and plot the melting curve.

[0031] Transgenic phenotypic analysis (see) Figure 8 Select transgenic lines with high expression levels. For wild-type tobacco, each line was propagated to more than 20 plants. Fifteen tissue culture seedlings with uniform growth were selected for soil culture at 24℃, with a 16-hour light / 8-hour dark cycle. Six-month-old wild-type tobacco and overexpression line seedlings were then collected from the soil culture (see...). Figure 9 The effects of PtTCH4 gene overexpression on tobacco growth and development were investigated by measuring and statistically analyzing inflorescence and leaf-related indicators, with 15 plants from each line being measured. Through the determination and analysis of growth phenotypes, transgenic tobacco The inflorescence color is significantly lighter compared to the wild type; the inflorescence of transgenic tobacco is pinkish-white, while that of wild-type tobacco is pink (see...). Figure 11 The inflorescences of the transgenic tobacco were significantly larger than those of the wild type, with the crown limb diameter being 1.27 times that of the wild type, and the length and width of the corolla tube increasing by 1.28 times and 1.34 times, respectively (see...). Figure 12 Compared with the wild type, the leaves of the overexpressing transgenic lines were significantly longer and significantly narrower, approximately 1.36 and 0.59 times that of the wild-type lines, respectively (see...). Figure 10Analysis of the results of various physiological indicators shows that PtTCH4 plays an important role in the growth of tobacco, participating in the development of tobacco inflorescences and leaves.

[0032] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the PtTCH4 gene in regulating plant growth and development in Populus tomentosa, characterized by, The nucleotide sequence of the Populus tomentosa PtTCH4 gene is shown in SEQ ID NO.1; The application involves overexpressing the PtTCH4 gene in Populus tomentosa to reduce the color of the inflorescence, increase the area of ​​the inflorescence, increase the length of the leaves, and decrease the width of the leaves. The plant in question is tobacco.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the Populus tomentosa PtTCH4 gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The method for overexpressing the PtTCH4 gene of Populus tomentosa includes the following steps: An overexpression vector for the PtTCH4 gene of Populus tomentosa was constructed. The plants were genetically transformed using Agrobacterium infection, and transgenic lines overexpressing the PtTCH4 gene of Populus tomentosa were screened to achieve overexpression of the PtTCH4 gene of Populus tomentosa.

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