Metasequoia glyptostroboides ein3 protein, its coding gene and application in regulating plant root development

Abstract

The application provides a Taxodium ascendens EIN3 protein, a coding gene thereof and application in regulating plant root development, and belongs to the technical field of plant genetic engineering.The application provides a Taxodium ascendens EIN3 protein, and the amino acid sequence is shown as SEQ ID NO:1.The Taxodium ascendens EIN3 protein has a negative regulation effect in the adventitious root regeneration of woody plants.Embodiments show that with the development of the adventitious roots of Taxodium ascendens, the expression level of ThEIN3 is gradually reduced, the expression amount in mature Taxodium ascendens cuttings is significantly higher than that in rejuvenation cuttings;compared with WT, the development of adventitious roots of the ThEIN3 overexpression plant is inhibited, meanwhile, the gibberellin content is significantly increased, the auxin and trans-zeatin riboside contents are reduced, and it is speculated that the Taxodium ascendens EIN3 protein may inhibit the development of adventitious roots by regulating the hormone level.The application provides a high-quality molecular resource for Taxodium ascendens molecular breeding, and has important application value in the field of forest tree genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a Metasequoia glyptostroboides EIN3 protein and its encoding gene (ThEIN3) and its application in regulating plant root development. Background Technology

[0002] Zhongshanshan (Taxodium hybrid 'Zhongshanshan') is a collective term for superior clones with certain super-parental traits, bred by the Zhongshanshan research team at the Jiangsu Institute of Botany, Chinese Academy of Sciences, through interspecific hybridization of the genus *Taxodium*. Years of indoor comparative trials, intermediate demonstration trials, and regional trials have shown that Zhongshanshan has advantages such as rapid growth, salt tolerance, flood resistance, wind resistance, disease resistance, and superior timber quality. It has broad application prospects and huge market demand in afforestation of lakes, wetlands, water networks, tidal flats, and plains in the Yangtze River Basin, southeastern coastal areas, and inland regions of China.

[0003] The propagation of Metasequoia glyptostroboides seedlings mainly uses softwood cuttings. However, during the propagation process, it was found that the root regeneration capacity declines significantly with the physiological age of the clonal lines, limiting the breeding and promotion of superior Metasequoia glyptostroboides varieties. Gene editing methods can be used to regulate the regeneration capacity of plant roots, but there are currently few reports on proteins that affect root development and growth. Summary of the Invention

[0004] In view of this, the present invention provides a Metasequoia EIN3 protein that can negatively regulate the development of adventitious roots in plants.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a Metasequoia glyptostroboides EIN3 protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] This invention provides a gene encoding the EIN3 protein of *Taxus chinensis*, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0008] The present invention provides a gene derivative comprising the said gene, including at least one of the following products: expression cassette, recombinant vector and recombinant bacteria.

[0009] This invention provides the application of the EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative in at least one of the following: regulating plant root development, regulating plant hormone levels, and cultivating transgenic plant varieties;

[0010] The plants include woody plants and / or herbaceous plants.

[0011] Preferably, the method for regulating plant root development is to negatively regulate plant root development using the Metasequoia glyptostroboides EIN3 protein, the gene, or the gene derivative.

[0012] The root system includes adventitious roots.

[0013] Preferably, the plant hormone includes at least one of auxin, trans-zeatin nucleoside, and gibberellin;

[0014] The method for regulating plant hormone levels includes at least one of the following:

[0015] The EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative positively regulates the gibberellin level in plants;

[0016] The EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative negatively regulates the auxin level and / or trans-zeatin nucleotide level in plants.

[0017] This invention provides a method for regulating the development of adventitious roots in plants, thereby regulating the expression level of the EIN3 protein or the gene of Metasequoia glyptostroboides in plants.

[0018] This invention provides a method for assessing the adventitious root development capacity of plants by detecting the relative expression level of the EIN3 protein or gene of *Taxus chinensis* in plants, wherein the relative expression level is inversely proportional to the adventitious root regeneration capacity of plants.

[0019] This invention provides a method for plant propagation by cuttings, which involves detecting the relative expression level of the EIN3 protein or gene of *Taxus chinensis* in the plant, and selecting cuttings with a relatively low expression level for propagation.

[0020] Preferably, the reagent for detecting the relative expression level includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention provides a Metasequoia glyptostroboides EIN3 protein, the amino acid sequence of which is shown in SEQ ID NO: 1. Metasequoia glyptostroboides is mainly propagated through softwood cuttings, but its adventitious root regeneration ability significantly declines with physiological age, leading to low propagation efficiency and difficulties in promoting superior varieties. This invention aims to study the function of the core transcription factor EIN3 / EIL in the ethylene signal transduction pathway of Metasequoia glyptostroboides during adventitious root development. The ThEIN3 gene was cloned, and through expression analysis, subcellular localization, and genetic transformation, the molecular characteristics of the Metasequoia glyptostroboides EIN3 protein and its regulatory function in adventitious root development and plant hormone levels were demonstrated, providing a high-quality molecular resource for molecular breeding of Metasequoia glyptostroboides.

[0023] This invention provides the application of the *Taxus chinensis* EIN3 protein, the gene encoding the *Taxus chinensis* EIN3 protein, or a gene derivative thereof in at least one of the following: regulating plant root development, regulating plant hormone levels, and breeding transgenic plant varieties; the plants include woody plants and / or herbaceous plants. Using 'Taxus chinensis 302' as research material, this invention cloned the ThEIN3 gene, detected its spatiotemporal expression pattern using qRT-PCR, and identified its function by overexpressing ThEIN3 in tobacco using an Agrobacterium-mediated expression system. The results showed that as adventitious roots developed in *Taxus chinensis*, the expression level of ThEIN3 gradually decreased, and the expression level in mature *Taxus chinensis* cuttings was significantly higher than that in juvenile cuttings; compared with WT, the adventitious root development of ThEIN3-overexpressing plants was inhibited, while the GA3 content significantly increased and the IAA and TZR contents decreased, indicating that ThEIN3 may inhibit adventitious root development by regulating hormone levels. This invention lays the foundation for in-depth research on the regulatory mechanism of adventitious root development in Metasequoia glyptostroboides and provides gene reserves and theoretical guidance for molecular breeding of Metasequoia glyptostroboides. Attached Figure Description

[0024] Figure 1 Image of the amplified product of the ThEIN3 gene of Metasequoia glyptostroboides;

[0025] Figure 2 This is a comparison diagram of the amino acid sequence of EIN3 in Metasequoia glyptostroboides and other species;

[0026] Figure 3 Phylogenetic analysis of EIN3 in Metasequoia glyptostroboides and other species;

[0027] Figure 4 Subcellular localization map of ThEIN3, scale bar at 20 μM;

[0028] Figure 5 This is a graph showing the gene expression changes of ThEIN3 during key developmental stages of adventitious roots of Metasequoia glyptostroboides, based on transcriptome data from adventitious root development.

[0029] Figure 6 A graph showing the gene expression changes of ThEIN3 in mature and rejuvenated cuttings;

[0030] Figure 7 The image shows a comparison of the overall phenotypes of transgenic tobacco overexpressing ThEIN3 and non-transgenic tobacco, with the top image representing non-transgenic tobacco and the bottom image representing transgenic tobacco.

[0031] Figure 8 Image showing the detection of basal hormones in transgenic tobacco plants overexpressing ThEIN3 and non-transgenic tobacco plants. Detailed Implementation

[0032] The present invention provides a Taxodium 'zhongshanshan' EIN3 protein, the amino acid sequence of which is as shown in SEQ ID NO: 1 (MGFYEEMGYSENFDYLTLPPEGDSMCENVCENACDNEGEVMHDEDLSED EIDVDELEKRMWKDRIRLRRIKEQHKGKEQTDTAKQRQSQEQARRKKMSRAQDGILKYMLKMMEVCKAQGFVYGIIPEKGKPVSGASDNLRAWWKEKVRFDRNGPAAIAKYQAEHALPGSNEDSMIVAPTPHTLQELQDTTLGSLLSALMQHCDPPQRRFPLEKGISPPWWPTSDEDWWPQVGLPKGQGPPPYKKPHDLKKAWKVGVLTAVIKHMSPDIAKIRKLVRQSKCLQDKMTAKESATWLAVVNQEEALARQQNPNACPPTSMSLSANAGALTFSSSSEYDVEGFEEDPNTIFSNDDVQDRKPHDFDLFNSEVQECKPLDFELFNAGISKERVPSSLQIVDNVDMIRKRKLPEESSMEDQKIYTCPYEQCPHHPRQFGFLDQNSRNSHQAICAYRTDFQSIGYQRSEPQANNNLFCMRMGQPNQLQVQGKGNNFPARMDGQAAPNATVSLGQPNQLQAQGKGGAFATRMDGQATPNATVNLGPGSTACLAAPANSQQPINELLALYDSGLHQNKTSTLGSLPIMNNPSQREVNMHRAPQGRELLRMTTDENFFGQGVLPCNSGTDTSLNMQQLMKEGLNLDQSRVFDQGFVHQAQEMNEDFKFGSPYNIAIDYGDSFPRVPEAVPKYENPIWYFGA).

[0033] In this invention, the ThEIN3 gene was cloned for the first time, and its molecular characteristics and regulatory function in adventitious root development of *Taxodium spp.* were verified through expression analysis, subcellular localization, and genetic transformation, providing high-quality gene resources for molecular breeding of *Taxodium spp.*. The *Taxodium spp.* EIN3 protein contains 690 amino acids, possesses a typical conserved domain of the EIN3 superfamily, and is located in the cell nucleus. As adventitious roots develop, the expression level of ThEIN3 gradually decreases, with significantly higher expression levels in mature *Taxodium spp.* cuttings than in juvenile cuttings. Compared to wild-type tobacco (WT), adventitious root development was inhibited in ThEIN3-overexpressing plants, with significantly increased gibberellin content and decreased indoleacetic acid and trans-zeatin nucleotide content. This indicates that the *Taxodium spp.* EIN3 protein and ThEIN3 gene can regulate adventitious root development and plant hormone levels, and that they may inhibit adventitious root development by regulating hormone levels.

[0034]

[0035] In this invention, the gene encoding the EIN3 protein of Metasequoia glyptostroboides is 2073 bp in length. As the adventitious roots of Metasequoia glyptostroboides develop, the expression level of the ThEIN3 gene gradually decreases, and the expression level in mature Metasequoia glyptostroboides cuttings is significantly higher than that in juvenile cuttings. Compared with wild-type tobacco (WT), the adventitious root development of ThEIN3-overexpressing plants is inhibited, the gibberellin content is significantly increased, and the content of indoleacetic acid and trans-zeatin nucleoside is decreased. This invention reveals for the first time the negative regulatory role of the ThEIN3 gene in the regeneration of adventitious roots in woody plants, providing a molecular mechanism basis for elucidating the reproductive obstacles of Metasequoia glyptostroboides. It also provides gene reserves and theoretical guidance for molecular breeding of Metasequoia glyptostroboides, and has important application value in the field of forest genetic engineering.

[0036] The present invention provides a gene derivative comprising the said gene, including at least one of the following products: expression cassette, recombinant vector and recombinant bacteria.

[0037] In this invention, the backbone vector of the recombinant vector preferably includes pCAMBIA1305. The recombinant expression vector is preferably obtained by inserting the ThEIN3 gene into the multiple cloning sites BstEII and BglII of the backbone vector. The host bacterium of the recombinant bacteria preferably includes Agrobacterium strain GV3101. The recombinant bacteria are preferably obtained by introducing the recombinant expression vector into Agrobacterium strain GV3101. The introduction method preferably includes the freeze-thaw method.

[0038] This invention provides the application of the EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative in at least one of the following: regulating plant root development, regulating plant hormone levels, and cultivating transgenic plant varieties;

[0039] The plants include woody plants and / or herbaceous plants.

[0040] In this invention, the woody plants preferably include plants of the genus *Taxodium*. The *Taxodium* plants preferably include *Taxodium hybrid 'zhongshanshan'*, more preferably *Taxodium hybrid 'Zhongshanshan 302'*. 'Zhongshanshan 302' is an excellent forest tree variety bred in my country, possessing high ornamental value, excellent wood quality, and tolerance to waterlogging and salinity. It can grow normally in soil conditions with a pH < 8.5 and a salt content < 0.3%. In 2002, it passed the national forestry and grassland administration's approval for improved forest tree varieties (National S-SC-TDM-004-2002), becoming one of the first batch of nationally approved improved varieties. The herbaceous plants preferably include dicotyledonous plants, more preferably plants of the Solanaceae family, further preferably plants of the genus *Nicotiana*, and most preferably *Nicotiana*. Since tobacco is a model organism, this invention has verified in tobacco that the EIN3 protein of Metasequoia glyptostroboides, the gene or the gene derivative has a negative regulatory effect on the development of adventitious roots in plants, and this effect can be found in dicotyledonous plants.

[0041] In this embodiment of the invention, after recombinant bacteria infect tobacco explants, they are co-cultured in a co-medium and then sequentially inoculated into callus induction medium and selection medium. The resulting callus is inoculated into differentiation medium for differentiation culture, and the resulting differentiation material is inoculated into seedling strengthening medium for seedling strengthening culture to obtain transgenic tobacco plants overexpressing the ThEIN3 gene. The tobacco explants preferably include leaves. The co-medium is preferably MS medium containing 0.1 mg / L NAA and 1.5 mg / L 6-BA. The co-culture conditions are preferably dark culture at 23℃ for 48 h. The callus induction medium is preferably MS medium containing 0.1 mg / L NAA, 0.5 mg / L 6-BA, and 0.3 mg / L 6-KT. The culture time for callus induction medium is preferably 10 days. After 10 days of culture, preferably vigorous callus is selected and inoculated into selection medium. The selection medium is preferably callus induction medium containing 400 mg / L cephalosporin. The selection culture time for selection medium is preferably 15 days. The selection culture is repeated twice. The differentiation medium is preferably MS medium containing 0.6 mg / L LBA, 0.6 mg / L 6-BA, 0.5 mg / L 6-KT, and 400 mg / L cephalosporin. Callus tissue on the differentiation medium forms seedlings, which are then subcultured in a seedling strengthening medium. The seedling strengthening medium is preferably MS medium containing 0.01 mg / L NAA, 0.1 mg / L 6-BA, and 400 mg / L cephalosporin. After seedling culture, genetic identification is preferably performed to obtain transgenic tobacco plants. The primers used for genetic identification preferably include a forward primer with the nucleotide sequence shown in SEQ ID NO: 5 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 6. The reaction system is preferably 20 μL, containing the following components in the following proportions: 10 μL 2×TaqPCR Mix, 1 μL transgenic tobacco plant DNA, 1 μL 10 μmol / L upstream primer, 1 μL 10 μmol / L downstream primer, and 7 μL ddH2O. The preferred procedure for detecting the relative expression level is: 95℃ for 3 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 2 min, for a total of 35 cycles; 72℃ for 10 min; 4℃ forever. Compared with wild-type tobacco (WT), transgenic tobacco plants overexpressing ThEIN3 showed inhibited adventitious root development, significantly increased gibberellin content, and decreased indoleacetic acid and trans-zeatin nucleotide content.

[0042] In this invention, the method for regulating plant root development is preferably the negative regulation of plant root development by the EIN3 protein of *Taxus chinensis*, the gene, or a gene derivative; the root system preferably includes adventitious roots. The method for negatively regulating plant root development preferably includes overexpressing the gene to inhibit plant root development.

[0043] In this invention, the plant hormone preferably includes at least one of auxin, trans-zeatin nucleoside, and gibberellin;

[0044] The method for regulating plant hormone levels preferably includes at least one of the following: the Metasequoia glyptostroboides EIN3 protein, the gene, or the gene derivative positively regulates the gibberellin level of the plant; the Metasequoia glyptostroboides EIN3 protein, the gene, or the gene derivative negatively regulates the auxin level and / or trans-zeatin nucleotide level of the plant.

[0045] In this invention, *Taxus chinensis* faces a key bottleneck in large-scale propagation: as the physiological age of the mother plant increases, the regenerative capacity of adventitious roots in cuttings significantly declines, making it difficult to efficiently propagate high-quality germplasm at an advanced age. This invention addresses this by detecting the expression level of the ThEIN3 gene in *Taxus chinensis*. The results show that the expression level of ThEIN3 gradually decreases as adventitious roots develop. Grafting and juvenile grafting techniques can alter the endogenous hormone levels in cuttings, improving the regenerative capacity of adventitious roots in *Taxus chinensis*. This invention further examines the expression levels of ThEIN3 in mature and juvenile *Taxus chinensis* cuttings. The results show that the expression level of ThEIN3 in mature *Taxus chinensis* cuttings is significantly higher than that in juvenile cuttings. This invention also constructs transgenic tobacco plants overexpressing ThEIN3. The results show that, compared to wild-type tobacco (WT), the adventitious root development of ThEIN3-overexpressing plants is inhibited, gibberellin content is significantly increased, and indoleacetic acid and trans-zeatin nucleotide content are decreased. Therefore, the EIN3 protein and its encoding gene of Taxodium montana negatively regulate the development of adventitious roots, the levels of auxin and trans-zeatin nucleoside, and positively regulate the level of gibberellin. The EIN3 protein and its encoding gene of Taxodium montana may inhibit the development of adventitious roots by regulating hormone levels.

[0046] This invention provides a method for regulating the development of adventitious roots in plants, thereby regulating the expression level of the EIN3 protein or the gene of Metasequoia glyptostroboides in plants.

[0047] In this invention, increasing the expression level of the EIN3 protein or the gene of the Metasequoia glyptostroboides inhibits the development of adventitious roots in the plant; decreasing the expression level of the EIN3 protein or the gene of the Metasequoia glyptostroboides promotes the development of adventitious roots in the plant.

[0048] This invention provides a method for assessing the adventitious root development capacity of plants by detecting the relative expression level of the EIN3 protein or gene of *Taxus chinensis* in plants, wherein the relative expression level is inversely proportional to the adventitious root regeneration capacity of plants.

[0049] In this invention, the reagent for relative expression level detection preferably includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4. The system for relative expression level detection preferably contains the following components in the following amounts: 10 μL of 2×ChamQ Universal SYBR qPCR MasterMix, 2 μL of 100 ng / μL cDNA, 0.4 μL of 10 μmol / L forward primer, 0.4 μL of 10 μmol / L reverse primer, and 7.2 μL of ddH2O. The preferred procedure for relative expression level detection is: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 40 cycles; melting curve analysis uses the instrument's default acquisition program.

[0050] This invention provides a method for plant propagation by cuttings, which involves detecting the relative expression level of the EIN3 protein or gene of *Taxus chinensis* in the plant, and selecting cuttings with a relatively low expression level for propagation.

[0051] In this invention, the reagents, systems, and procedures for detecting the relative expression levels are preferably the same as those in the above-described method for comparing the regeneration capacity of adventitious roots in plants, and will not be repeated here.

[0052] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] In the following examples, operations not described in detail are all routine biological experimental procedures, which can be performed with reference to molecular biology experimental manuals and existing publicly available journal literature.

[0054] Example 1

[0055] Cloning and sequence analysis of ThEIN3

[0056] Total RNA was extracted from leaves of 'Zhongshanfeng 302' using the FastPure Universal PlantTotal RNAIsolation Kit (Nanjing Novizan Biotechnology Co., Ltd.), and simultaneously reverse transcribed into cDNA using the HiScript III 1st Strand cDNASynthesis Kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0057] Based on the genomic information of *Taxus chinensis*, upstream primer ThEIN3-F: 5'-ATGGGGTTTTATGAAGAAATGG-3' (SEQ ID NO: 5) and downstream primer ThEIN3-R: 5'-TGCTCCAAAATACCAGATTG-3' (SEQ ID NO: 6) were designed using Primer3 Plus software. PCR amplification was performed using cDNA as a template in a 50 μL reaction volume. The reaction program was: 95℃ for 3 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 2 min, for a total of 35 cycles; 72℃ for 10 min; 4℃ for forever. The PCR product was detected by 1% agarose gel electrophoresis, showing a clear band at 2073 bp. Figure 1 The DNA was extracted using the FastPure Gel DNA Extraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.). The target fragment was then ligated into pMD19-TVector (TaKaRa, Japan), transformed into *E. coli* DH5α competent cells, and subjected to colony-linked PCR verification. The PCR amplification procedure was the same as above. After 1% agarose gel electrophoresis, positive clones were screened and sent to Anhui General Biotechnology Co., Ltd. for sequencing.

[0058] Comparison of the ThEIN3 sequence of Metasequoia glyptostroboides with EIN3 protein sequences from 10 other plant sources revealed that they all possess a conserved EIN3 domain. Figure 2 This indicates that ThEIN3 may play a core function similar to EIN3 in other plants within the ethylene signaling pathway. To clarify the phylogenetic characteristics of EIN3, EIN3 proteins from 10 species, including Arabidopsis thaliana, Populus tomentosa, japonica rice, and Hickory, were selected, and a phylogenetic tree was constructed using the nearest neighbor linkage method in MEGA 12.0 software. Figure 3 The results showed that ThEIN3 of Metasequoia glyptostroboides clustered with OsEIN3 of Japonica rice, indicating a close genetic distance, while it was much more distant from Arabidopsis thaliana and Populus tomentosa, revealing the differentiation of EIN3 in different species during adaptive evolution.

[0059] Example 2

[0060] Subcellular localization analysis of ThEIN3

[0061] Using Gateway technology, the ORF fragment of ThEIN3 was ligated into the pCAMBIA1302-GFP vector according to the instructions of the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.). The subcellular localization vector was obtained from the multiple cloning sites EcoR I and HindIII of the ThEIN3 gene insertion backbone vector, and the plasmid was stored at -20℃. Several tobacco seeds were sown and cultured under 12-hour light for one month before being used in experiments. The pCAMBIA1302-ThEIN3-GFP recombinant plasmid was transformed into Agrobacterium GV3101 via electroporation and cultured at 30℃ for 2 days. Agrobacterium GV3101 cells were scraped from a solid culture dish using an inoculation loop and inoculated into 10 mL of YEB liquid medium with the corresponding antibiotic, and cultured at 170 rpm for 1 hour. The cells were then centrifuged at 4000 rpm for 4 minutes, and the supernatant was discarded to collect the bacterial cells. The cells were resuspended in a MgCl2 (10 mmol / L) suspension containing 120 μmol / L acetosyringone (AS), and the OD was adjusted. 600 To a concentration of approximately 0.6, select healthy tobacco plants and inject the lower epidermis of the leaves using a 1mL syringe (without the syringe tip), labeling the injection site. After culturing the injected tobacco plants under low light for 48 hours, prepare temporary water-sealed slides of mesophyll cells and observe the fluorescence signal using a laser confocal microscope. A parallel control group was set up, using Agrobacterium GV3101 bacterial suspension containing an empty plasmid under the same conditions for infection.

[0062] The results showed that green and red fluorescence were observed in the nucleus of cells containing 35S::ThEIN3::GFP, indicating that the ThEIN3 protein is located in the nucleus. Figure 4 This suggests that it may exert its effects by directly regulating the transcription of target genes, consistent with the function of EIN3 family proteins as core transcription factors in the ethylene signaling pathway.

[0063] Example 3

[0064] Expression pattern analysis of ThEIN3

[0065] Based on transcriptome data from early adventitious root development, the expression level of ThEIN3 during key developmental stages of adventitious roots in Metasequoia glyptostroboides was analyzed. RNA was extracted from mature cuttings and juvenile cuttings (branches obtained through grafting onto young rootstocks), and ThEIN3 expression was detected using qRT-PCR.

[0066] Internal reference gene primers:

[0067] ThAPRT-F: 5'-TCCACAGGTTCTTGAATCGCT-3' (SEQ ID NO: 7);

[0068] ThAPRT-R: 5'-TGACTTGAGCCTCATTCGCTC-3' (SEQ ID NO: 8);

[0069] ThEIN3 gene primers:

[0070] qThEIN3-F: 5'-CTGAGAAGGGGAAGCCTGTG-3' (SEQ ID NO: 3);

[0071] qThEIN3-R: 5'-GGAGAGTATGCGGGGTAGGA-3' (SEQ ID NO: 4).

[0072] Primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd. qRT-PCR was performed using the HiScriptIII 1st Strand cDNA Synthesis Kit (+gDNAwiper) and ChamQUniversal SYBR qPCR MasterMix from Nanjing Novizan Biotechnology Co., Ltd. for reverse transcription and premixing, respectively. The qRT-PCR amplification system contained 10 μL of 2×ChamQUniversal SYBR qPCRMasterMix, 2 μL of 100 ng / μL cDNA, 0.4 μL of 10 μmol / L upstream primer, 0.4 μL of 10 μmol / L downstream primer, and 7.2 μL of ddH2O. The qRT-PCR amplification program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 40 cycles. Melting curve analysis used the instrument's default acquisition program, with three biological replicates for each treatment. -ΔΔCt The relative expression level of the target gene is calculated using this method.

[0073] The results showed that as the adventitious roots of Metasequoia glyptostroboides developed, the transcription level of ThEIN3 gradually decreased. Figure 5 qRT-PCR results showed that the expression level of ThEIN3 in mature Metasequoia cuttings was significantly higher than that in rejuvenated cuttings. Figure 6 This indicates that ThEIN3 may be involved in regulating the regeneration of adventitious roots in Metasequoia glyptostroboides.

[0074] Example 4

[0075] Genetic transformation of ThEIN3 in tobacco

[0076] The ThEIN3 gene was cloned into the pCAMBIA1305 vector using Gateway technology, and the recombinant vector pCAMBIA1305-ThEIN3 was transformed into Agrobacterium GV3101 via liquid nitrogen freeze-thaw conversion. Successfully detected recombinant bacteria were picked and placed in the infection medium to prepare OD (Organic Dioxide) culture. 600 =0.5% Agrobacterium resuspension. Sterile tobacco leaves were cut into 1cm pieces using a sterile scalpel. 2 Small pieces of the leaves were immersed in the prepared Agrobacterium tumefaciens solution for 15 minutes. After the time was up, the leaves were placed on sterile filter paper to dry and then inoculated onto co-culture medium (MS + 0.1 mg / L NAA + 1.5 mg / L 6-BA) and incubated in the dark at 23°C for 48 hours. After co-culture, the material was inoculated onto callus induction medium (MS + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 0.3 mg / L 6-KT) and cultured for 10 days. Vigorous callus tissue was selected and inoculated onto selection medium (induction medium + 400 mg / L cephalosporin) for 15 days of selection, followed by a second selection. After the second screening, the callus tissue was inoculated into differentiation medium (MS + 0.6 mg / L LBA + 0.6 mg / L 6-BA + 0.5 mg / L 6-KT + 400 mg / L cephalosporin). If any callus tissue formed seedlings, they were subcultured in seedling strengthening medium (MS + 0.01 mg / L NAA + 0.1 mg / L 6-BA + 400 mg / L cephalosporin) to obtain transgenic tobacco plants.

[0077] Genomic DNA was extracted from transgenic plants using a genome extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), and the transgenic plants were identified by gel electrophoresis (the primers, system, and reaction procedure used for identification were the same as in Example 1), resulting in transgenic tobacco plants.

[0078] Example 5

[0079] Phenotypic observation and hormone level detection of transgenic tobacco plants

[0080] 1. Phenotypic observation of transgenic tobacco plants

[0081] Terminal shoots, 4–5 cm in length, were taken from wild-type and transgenic tobacco plants and placed in rooting medium (1 / 2 MS + 0.2 mg / L NAA). The medium for wild-type plants did not contain antibiotics, while the medium for transgenic plants contained 400 mg / mL cephalosporins. The growth of adventitious roots in the medium was observed every 3 days. Once rooting phenotypes appeared, photographs were taken and recorded. Samples were also taken from the basal 1–2 cm of the tobacco plant, flash-frozen in liquid nitrogen, and stored at -80°C.

[0082] The results showed that, compared with WT, transgenic tobacco plants overexpressing pCAMBIA1305::ThEIN3 significantly inhibited adventitious root development in transgenic tobacco. Figure 7 ).

[0083] 2. Hormone level detection in transgenic plants

[0084] Endogenous plant hormones in the samples were extracted using an isopropanol-water-hydrochloric acid mixed extraction buffer (with internal standard solution added). The mixture was centrifuged at 4°C for 5 min, the organic phase was dried under nitrogen, and then reconstituted with methanol (0.1% formic acid). After centrifugation at 4°C for 10 min, the supernatant was filtered through a 0.22 μm filter membrane. The contents of indole-3-acetic acid (IAA), trans-zeatin riboside (TZR), gibberellic acid (GA3), indole-3-butyric acid (IBA), abscisic acid (ABA), and cis-zeatin riboside (CZR) were determined by high performance liquid chromatography (HPLC) (Agilent 1290, https: / / www.agilent.com / ) and tandem mass spectrometry (MS / MS) (Applied Biosystems 6500 Quadrupole Trap, https: / / sciex.com.cn / ).

[0085] The results showed that, compared with WT, the GA3 concentration at the base of transgenic tobacco was significantly increased, while the IAA and TZR concentrations were significantly decreased. Figure 8 ).

[0086] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Metasequoia glyptostroboides EIN3 protein, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the EIN3 protein of *Taxus chinensis*, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

2.

3. A gene derivative comprising the gene of claim 2, characterized in that, It includes at least one of the following products: expression cassette, recombinant vector, and recombinant bacteria.

4. The use of the Metasequoia glyptostroboides EIN3 protein of claim 1, the gene of claim 2, or the gene derivative of claim 3 in at least one of the following: regulating plant root development, regulating plant hormone levels, and cultivating transgenic plant varieties; The plant is Metasequoia glyptostroboides and / or tobacco; The application of regulating plant root development is that the EIN3 protein of Metasequoia glyptostroboides, the gene or the gene derivative negatively regulates plant root development. The root system is adventitious; The plant hormone is at least one of indoleacetic acid, trans-zeatin nucleoside, and gibberellin. The application of regulating plant hormone levels includes at least one of the following: The EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative positively regulates the gibberellin level in plants; The EIN3 protein of Metasequoia glyptostroboides, the gene, or the gene derivative negatively regulates the levels of indoleacetic acid and / or trans-zeatin nucleotides in plants.

5. A method for negatively regulating the development of adventitious roots in plants, characterized in that, To increase the expression level of the EIN3 protein of Metasequoia glyptostroboides as described in claim 1 or the gene as described in claim 2 in plants; The plants mentioned are Metasequoia glyptostroboides and / or tobacco.

6. A method for assessing the adventitious root development capacity of plants, characterized in that, The relative expression level of the EIN3 protein of Metasequoia glyptostroboides as described in claim 1 or the gene as described in claim 2 was detected in plants, wherein the relative expression level was inversely proportional to the adventitious root regeneration capacity of the plant; The plants mentioned are Metasequoia glyptostroboides and / or tobacco.

7. A method for plant propagation by cuttings, characterized in that, The relative expression level of the EIN3 protein of Metasequoia glyptostroboides as described in claim 1 or the gene as described in claim 2 was detected in the plant, and cuttings with a relatively low expression level were selected for propagation. The plants mentioned are Metasequoia glyptostroboides and / or tobacco.

8. The method according to claim 7, characterized in that, The reagents for detecting the relative expression level include a forward primer with a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:4.

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