Application of HbCBF1 gene in regulating ethylene content of rubber tree

Abstract

This invention discloses the application of the HbCBF1 gene in regulating ethylene content in rubber trees, belonging to the field of agricultural biotechnology. By overexpressing the HbCBF1 gene (nucleotide sequence shown in SEQ ID NO.1), this invention significantly enhances the biosynthesis of ethylene in transgenic plants, manifested as an increased ethylene release rate, elevated content of the ethylene precursor ACC, and enhanced activity of key enzymes in the ethylene synthesis pathway, SAMS, ACS, and ACO. It also exhibits dwarfing phenotypes associated with excessive ethylene accumulation, including significantly reduced plant height, thinner stems, shorter internodes, and accelerated leaf senescence. Molecular mechanism studies show that HbCBF1 directly activates the transcriptional expression of SAMS1 by binding to the CRT / DRE cis-acting element in the SAMS1 gene promoter, thereby upregulating ethylene biosynthesis. This invention provides a new target gene and regulatory method for the genetic engineering improvement of rubber trees.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of the HbCBF1 gene in regulating ethylene content in rubber trees. Background Technology

[0002] Natural rubber is an important renewable industrial raw material, mainly produced by rubber trees ( Hevea brasiliensis Ethylene is a key plant hormone regulating plant growth, development, and stress response. In rubber trees, ethylene has been proven to be a key signaling molecule stimulating increased rubber yield. Exogenous application of ethephon can effectively increase latex yield by prolonging latex expulsion time and accelerating latex regeneration. Therefore, regulating the ethylene content in rubber trees is of great significance for rubber production.

[0003] C-repetition binding factors (CBFs) are an important family of transcription factors that, in addition to regulating plant responses to cold stress, participate in a wide range of physiological processes, including the regulation of carbohydrate and lipid metabolism and hormone signal transduction. Studies have shown that CBF transcription factors can regulate plant growth, development, and morphogenesis; for example, AmCBF1 induces dwarfing in cotton by inhibiting GhPP2C1 or GhPP2C2. Furthermore, CBFs have a network of mutual regulation with other transcription factors involved in hormone signaling pathways.

[0004] The biosynthetic pathway of ethylene is as follows: methionine is catalyzed by S-adenosylmethionine synthase (SAMS) to produce S-adenosylmethionine (SAM), which is further converted into 1-aminocyclopropane-1-carboxylic acid (ACC). ACC is then oxidized to ethylene by ACC synthase (ACS) and ACC oxidase (ACO). Elevated ethylene levels promote leaf senescence, while inhibiting ethylene biosynthesis slows down the senescence process.

[0005] Currently, regulating plant ethylene content through genetic engineering has become a hot research area in plant biotechnology. However, the direct molecular regulatory mechanism between CBF transcription factors and ethylene biosynthesis remains unclear in existing technologies, necessitating the development of new technical solutions to achieve precise regulation of ethylene metabolism in rubber trees. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the HbCBF1 gene in regulating the ethylene content of plants, so as to solve the problem of the lack of effective gene methods for regulating the ethylene content of rubber trees in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of the HbCBF1 gene in regulating plant ethylene content, wherein the nucleotides of the HbCBF1 gene are shown in SEQ ID NO.1; overexpression of the HbCBF1 gene can significantly increase the biosynthesis level of ethylene in plants.

[0008] Furthermore, the plant in question is a rubber tree.

[0009] Furthermore, the overexpression is achieved by constructing the coding sequence of the HbCBF1 gene into a eukaryotic expression vector.

[0010] Furthermore, the eukaryotic expression vector is pCAMBIA-2301.

[0011] The present invention also provides a method for constructing a recombinant expression vector, wherein the nucleotide sequence of the HbCBF1 gene shown in SEQ ID NO.1 is inserted into the eukaryotic expression vector pCAMBIA-2301 through XbaI and EcoRI restriction sites to obtain the recombinant expression vector pCAMBIA2301-35S::HbCBF1.

[0012] The present invention also provides a method for preparing transgenic plant cells, wherein the recombinant expression vector is transformed into Agrobacterium, and Agrobacterium-mediated genetic transformation is performed using plant explants or callus tissue as recipients to obtain transgenic plant cells containing the HbCBF1 gene.

[0013] Furthermore, the Agrobacterium is Agrobacterium tumefaciens strain EHA105; the plant explant is a somatic embryo derived from the anthers of a rubber tree.

[0014] This invention also provides a method for regulating the ethylene content of rubber trees using the HbCBF1 gene, comprising the following steps: (1) Construct an HbCBF1 gene overexpression vector; (2) Transform the overexpression vector into Agrobacterium; (3) Genetic transformation was carried out using rubber tree explants or callus tissue as recipients via Agrobacterium-mediated transformation. (4) Screening and identifying transgenic plants; (5) Transgenic plants obtained through cultivation.

[0015] Furthermore, the method significantly improves the biosynthesis level of ethylene in transgenic plants, manifested by an increased ethylene release rate, higher content of the ethylene precursor ACC, and enhanced activity of key enzymes in the ethylene synthesis pathway, namely SAMS, ACS, and ACO.

[0016] Furthermore, the method induces transgenic plants to exhibit dwarfing phenotypes associated with excessive ethylene accumulation, including: significantly reduced plant height, thinner stems, shorter internode distances, accelerated leaf senescence, and premature leaf drop.

[0017] This invention also reveals the molecular mechanism by which HbCBF1 regulates ethylene biosynthesis: HbCBF1, as an AP2 / ERF type transcription factor, can recognize and bind to the CRT / DRE cis-acting element (CCGAC); HbCBF1 directly activates the transcriptional expression of SAMS1 by binding to the CRT / DRE cis-acting element in the SAMS1 gene promoter, thereby upregulating the ethylene biosynthesis pathway.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention reveals for the first time the function of the HbCBF1 gene in regulating plant ethylene biosynthesis and finds that overexpression of the HbCBF1 gene can significantly increase the level of ethylene synthesis in plants.

[0019] (2) This invention elucidates the molecular mechanism by which HbCBF1 directly activates SAMS1 transcription to regulate ethylene biosynthesis, establishes a direct regulatory link between CBF transcription factors and ethylene homeostasis, and provides new insights into the trade-off regulation between plant growth and stress response.

[0020] (3) The technical solution provided by the present invention can effectively regulate the ethylene content of rubber trees, and provides new target genes and operation methods for the genetic engineering improvement of rubber trees.

[0021] (4) The transgenic rubber trees obtained by overexpressing HbCBF1 in this invention exhibit a typical ethylene overexpression phenotype, providing an important material basis for studying ethylene signal transduction pathways. Attached Figure Description

[0022] Figure 1This study demonstrates the creation process of HbCBF1 overexpressing transgenic rubber trees. (A) Hygromycin-resistant callus formed after Agrobacterium-mediated transformation; (B) Resistant primary somatic embryos formed on selection medium; (C) Secondary somatic embryos verified by GUS positivity, confirming transgene expression; (D) Acclimation and hardening of tissue-cultured seedlings regenerated from GUS-positive embryoids; (E) PCR analysis showing stable integration of the HbCBF1 transgene; OE1-OE3 are transgenic plants; P is the HbCBF1 overexpression plasmid as a positive control; WT is a wild-type plant; water is a negative control; M is a 5000 bp DNA marker; (F) Histochemical GUS staining of leaves from regenerated plants; (G) qRT-PCR analysis of the relative expression levels of HbCBF1 in the transgenic line and wild type. Data are presented as mean ± standard deviation, n=3; Student's t-test was used for statistical analysis; ***P<0.0001.

[0023] Figure 2 Phenotypic differences between 7-year-old OE-HbCBF1 transgenic rubber trees and wild-type trees are shown; (AB) Whole-plant morphological observation of WT and OE-HbCBF1 transgenic lines, scale bar = 1.5 m; (C) Branch morphological comparison, WT on the left and OE-HbCBF1 on the right, scale bar = 5 cm; (D) Measurement data of plant height, stem diameter, branch height and internode distance; (E) Longitudinal sections of leaves of WT and OE-HbCBF1; (F) Transverse sections of apical meristems of lateral branches of WT and OE-HbCBF1; (GH) Transmission electron micrographs of the cuticle of leaf surface, arrows indicating: w, epidermal wax; o, outer cuticle; i, inner cuticle; (I) Cell number and cell diameter measured in transverse sections of apical meristems of lateral branches of WT and OE-HbCBF1; (J) Thickness of each layer of cuticle; Data are mean ± standard deviation (n=3); Student's... Statistical analysis was performed using t-tests; *P<0.05, **P<0.001, ***P<0.0001.

[0024] Figure 3This study demonstrates how HbCBF1 overexpression leads to constitutive ethylene response and imbalances in multiple plant hormones; (A) Leaf morphology of WT and OE-HbCBF1, scale bar = 2 cm; (BD) Morphological characteristics of branch tips of wild-type (B) and OE-HbCBF1 lines (C, D); (EG) Leaf physiological parameters, including relative chlorophyll content (SPAD value), chlorophyll fluorescence parameters, and stomatal conductance; (H) Ethylene release rate; (I) Content of ethylene precursor ACC; (JL) Activities of key enzymes in the ethylene synthesis pathway: SAMS synthase, ACS, and ACO; Data are presented as mean ± standard deviation (n=3); Statistical analysis was performed using Student's t-test; *P<0.05, **P<0.001, ***P<0.0001.

[0025] Figure 4 The following data are presented: (A) Differential expression analysis between OE-HbCBF1 and WT; (B) Venn diagram analysis of transcript differences; (C) Statistical analysis of the number of differentially expressed genes (DEGs) that are upregulated and downregulated; (D) Volcano plot showing differential expression and key DEGs; (E) GO enrichment analysis of upregulated and downregulated DEGs; (F) Schematic diagram of the ethylene biosynthesis pathway, showing changes in metabolites and genes; * indicates Log2Fold change >1; ** indicates Log2Fold change >2.

[0026] Figure 5 The diagram illustrates the binding of HbCBF1 to the HbSAMS1 promoter; (A) a schematic diagram of the location of the CRT / DRE (CCGAC) element in the HbSAMS1 promoter, with numbers indicating distances (bp) relative to the start codon (+1); (B) a yeast one-hybrid assay (Y1H) verifying the interaction between HbCBF1 and the HbSAMS1 promoter, using selective medium: SD / -Leu medium supplemented with AbA (100 ng / ml), positive control: Bait 53 + AD 53, negative control: Bait EV + AD. EV; (C) Electrophoretic mobility shift analysis (EMSA) showed the binding of recombinant HbCBF1 protein to a biotin-labeled probe containing a CRT / DRE element in the HbSAMS1 promoter. The competition experiment was performed using an unlabeled cold probe or a mutant probe. * indicates a mutant probe in lane 8. "Bound" and "Unbound" represent the protein-DNA complex and the free probe, respectively; (D) Luminescent imaging for dual-luciferase activity detection. Tobacco leaves were divided into four regions, and the plasmid combination described in Example 4 was injected into each region; (E) Dual-luciferase reporter gene activity assay of tobacco leaves in each treatment group; Statistical analysis was performed using Student's t-test; *P<0.05, **P<0.001. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0028] The plant material used in this invention is the rubber tree ( Hevea brasiliensis The clone “CATAS-73397” and the transgenic lines derived from this clone were used. Wild-type (WT) plants and transgenic lines were planted in an isolated nursery of a rubber plantation in Danzhou City, Hainan Province, under natural conditions and with routine field management. After one year of growth in sand beds, budded seedlings were used for all experimental measurements.

[0029] Example 1: Creation of HbCBF1 overexpression transgenic rubber trees 1. Construction of HbCBF1 gene overexpression vector Using cDNA from rubber tree leaves as a template, the HbCBF1 gene (HbAchr17G0017054, SEQ ID NO.1) was amplified using high-fidelity enzymes. XbaI and EcoRI sites were introduced at the 5′ end of the primers (primer sequences are shown in Table 1). PCR conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, 30 cycles; final extension at 72℃ for 5 min. The product was recovered by gel extraction after 1.5% agarose gel electrophoresis. The pCAMBIA-2301 vector (2 μg) was double-digested with XbaI and EcoRI at 37℃ for 2 h, and the linearized vector was recovered.

[0030] Table 1 Primer sequence information for amplifying the HbCBF1 gene

[0031] The recovered HbCBF1 fragment and the linearized vector were ligated using homologous recombinase at 50°C for 30 min. The ligation system consisted of 50 ng of the target fragment, 30 ng of the vector, and 5 μL of 2×Hieff Clone® Premix, for a total volume of 10 μL. 5 μL of the ligation product was transformed into *E. coli* DH5α and plated on LB agar plates containing 50 mg / L kanamycin, incubated overnight at 37°C. Single colonies were picked, amplified, and the plasmid was extracted. PCR identification and bidirectional sequencing confirmed the correctness of the recombinant plasmid sequence.

[0032] The correctly sequenced pCAMBIA-2301-HbCBF1 plasmid was transformed into Agrobacterium tumefaciens using a freeze-thaw method. Agrobacterium tumefaciensThe recombinant plasmid was added to 100 μL of Agrobacterium competent cells. The specific steps were as follows: 1 μg of recombinant plasmid was added to 100 μL of Agrobacterium competent cells, mixed, and incubated on ice for 30 min; then flash-frozen in liquid nitrogen for 1 min, heat-shocked in a 37℃ water bath for 5 min, and immediately incubated on ice for 2 min; 500 μL of YEB liquid medium was added, and the cells were incubated at 28℃ and 200 rpm for 3 h; 100 μL of the bacterial culture was spread onto YEB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubated upside down at 28℃ for 2 days. Single colonies were picked for PCR identification. After confirming positive clones, the plasmid was extracted and digested again for verification. The verified Agrobacterium strain was used for subsequent genetic transformation of rubber trees.

[0033] 2. Genetic transformation of rubber trees Well-developed, yellowish-green rubber tree flower buds were selected as explant materials. The flower buds were surface-sterilized with 75% ethanol for 1 min, sterilized with 0.1% mercuric chloride (HgCl2) solution for 10 min, rinsed three times with sterile water, and then the stamens were removed in a clean bench. The anthers were removed with forceps and inoculated into callus induction medium (MS medium supplemented with 3.0 mmol / L CaCl2, 7.0 μmol / L KT, 8.1 μmol / L NAA, 6.8 μmol / L 2,4-D, 204.5 mmol / L sucrose and 2.2 g / L Phytagel). After 40 days of dark culture, the induced callus was transferred to somatic embryogenesis medium (MS medium supplemented with 2.2 μmol / L 6-BA, 14.0 μmol / L KT, 1.4 μmol / L GA3, 0.1 μmol / L NAA, 3.8 μmol / L ABA, 204.5 mmol / L sucrose, and 2.2 g / L Phytagel). Cultured under light (photoperiod 16 h / 8 h, light intensity 2000 lx), subcultured every 15 days until mature embryogenic callus was obtained. Agrobacterium tumefaciens strain EHA105 containing the recombinant plasmid pCAMBIA-2301-HbCBF1 was inoculated into YEB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin) and cultured at 28°C with shaking at 200 rpm until the logarithmic growth phase (OD2). 600 The bacterial culture was centrifuged at 5000 rpm for 10 min at 4℃ to collect the cells, which were then resuspended in infection medium (MS liquid medium supplemented with 100 μmol / L acetylsuccine, 30 g / L sucrose, pH 5.2). The OD was adjusted. 600 Up to 0.6-0.8.

[0034] Embryogenic callus was used as recipient material and infected with Agrobacterium suspension for 5 min. It was then transferred to co-culture medium (MS medium supplemented with 100 μmol / L AS, 10 mg / L AgNO3, 30 g / L sucrose, pH 5.8) and cultured in the dark at 25°C for 1–9 days. After co-culture, the callus was transferred to callus induction medium supplemented with 10 mg / L AgNO3 and 500 mg / L termethin for recovery culture, and cultured in the dark at 25°C for 7–10 days. The recovered callus was then transferred to callus induction medium supplemented with 10 mg / L AgNO3, 50 mg / L kanamycin, and 500 mg / L termethin for resistant callus selection. Subculture was performed every 20 days, and after two subcultures, the resistant callus was transferred to differentiation medium supplemented with 500 mg / L termethin and 50 mg / L kanamycin to induce somatic embryogenesis. Finally, the proliferated somatic cells were transferred to a plant regeneration medium containing 50 mg / L kanamycin (MS medium supplemented with 0.23 μmol / L KT, 0.11 μmol / L IAA, 8.7 mmol / L GA3, and 4.5 μol / L 2,4-D) to induce plant regeneration. After the embryoids germinated and formed small plantlets with complete roots, stems, and leaves, the plants underwent hardening-off. First, the culture medium bottle caps were gradually opened to allow the plants to gradually adapt to the external environment. After 5-8 days of adaptation, the plants were removed, cleaned and disinfected with 0.1% carbendazim solution, and then transplanted into substrate soil (watered thoroughly with 1 / 2 MS nutrient solution) and cultured in an artificial climate greenhouse. Leaves from healthy plants were removed for positive identification. Afterward, the plants were transferred to a sand bed for further culture.

[0035] 3. Molecular identification of transgenic lines (1) PCR identification Young leaves (approximately 100 mg) from candidate transgenic lines and non-transgenic control (wild-type) plants were collected, and genomic DNA was extracted using the CTAB method. Using the extracted genomic DNA as a template, the GUS reporter gene, NptII selection marker gene, and 35S:HbCBF1 fusion fragment of the transgenic materials were detected. Primer sequences, expected product sizes, and annealing temperatures are shown in Table 2. PCR reaction system (20 μL): template DNA 50 ng, 2× Taq Master Mix 10 μL, forward and reverse primers 0.5 μL each (10 μM), and ddH2O added to 20 μL. Reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, (annealing temperature depends on primers) for 30 s, extension at 72℃ (calculated at 1 kb / min), for a total of 35 cycles; final extension at 72℃ for 10 min. The amplified products were separated by 1.5% agarose gel electrophoresis. Wild-type plant DNA and water were used as negative controls, and recombinant plasmid pCAMBIA-2301-HbCBF1 was used as a positive control. Primer sequences are shown in Table 2.

[0036] Table 2 Primer sequence information for PCR identification

[0037] (2) The expression level of HbCBF1 gene was detected by real-time quantitative RT-PCR.

[0038] Total RNA was extracted from rubber tree leaves using a plant RNA extraction kit. After screening to confirm RNA quality, it was reverse transcribed into cDNA using the PrimeScript™ RT kit according to the appropriate protocol. Amplification was performed using a SYBR® Premix Ex Taq™ II (TaKaRa, Japan) real-time quantitative PCR instrument. The reaction procedure was as follows: preheating at 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for 10 s. The relative expression level of HbCBF1 was calculated using the 2-ΔΔCt method with the rubber tree YL8 gene as an internal control. Each sample was tested in triplicate (3 technical replicates and 3 biological replicates). Primer sequences are shown in Table 3.

[0039] Table 3 Primer sequence information for detecting HbCBF1 gene expression level

[0040] (3) GUS histochemical staining Prepare X-Gluc staining solution: 0.1 M sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide (K3Fe(CN)2) buffer. )60.5 mM potassium ferrocyanide (K4Fe(CN)6), 0.1% Triton X-100, 1.0 mg / mL X-Gluc (5-bromo-4-chloro-3-indole-β-D-glucuronic acid, soluble in dimethylformamide). Prepare fresh and use immediately, store away from light. Take leaves (approximately 0.5 cm) from transgenic plants. 2 The tissue was immersed in X-Gluc staining solution. A vacuum pump was used to create a vacuum for 5–10 min to promote penetration of the staining solution into the tissue. It was then transferred to a 37°C incubator and incubated overnight (12–16 h) in the dark. The same tissue from the wild-type material was used as a negative control. After staining, the staining solution was discarded, and 70% ethanol was added 2–3 times (30 min each time) to decolorize and remove chlorophyll until the background was clear. The tissue was observed and photographed under a stereomicroscope. GUS-positive tissue appeared blue, while the negative control did not.

[0041] 4. Experimental Results and Analysis Embryogenic callus derived from the anthers of the cultivar “CATAS-73397” (wild type, WT) was used as explants and co-cultured with Agrobacterium strain EHA105 carrying the recombinant vector pCAMBIA2301-35S::HbCBF1. Figure 1 A). After screening on a kanamycin-containing medium, resistant callus tissue was subcultured twice to ensure stability, and then transferred to differentiation medium to induce somatic embryogenesis, with an induction efficiency of 9% ( Figure 1 B). After obtaining resistant embryos, the plants were regenerated, and the seedlings were gradually acclimatized and transplanted into a sand bed, with a survival rate of 40%. Figure 1 C, D).

[0042] PCR testing confirmed that the β-glucuronidase (GUS) reporter gene, the NptII selector gene, and the fusion fragment of the CaMV 35S promoter with HbCBF1 were all successfully amplified by PCR, and the fragment size was consistent with the expected fragment, proving that the exogenous gene had been inserted into the transgenic plant. Figure 1 E. GUS staining showed a blue signal in the leaves of all three transgenic lines, indicating successful expression of the reporter gene. Figure 1 F). Consistent with this, qRT-PCR showed that the HbCBF1 transcription level in the transgenic line was more than 800-fold higher than that in the wild type, further confirming its strong overexpression. Figure 1 G).

[0043] In summary, these results demonstrate that we have successfully obtained three OE-HbCBF1 transgenic rubber trees that have grown stably for seven years. These materials provide a robust and reliable platform for subsequent functional studies of HbCBF1.

[0044] Example 2: Phenotypic analysis of HbCBF1 overexpression transgenic rubber trees After seven years of field growth, the OE-HbCBF1 transgenic line showed significant developmental changes.

[0045] 1. Plant morphology observation All three OE-HbCBF1 transgenic lines exhibited a significantly dwarfed phenotype compared to the wild type, with several typical characteristics: a compact overall plant type, severely inhibited longitudinal growth leading to a significant reduction in plant height and leaf canopy contraction; at the organ level, the stems were noticeably thinner and the internodes were significantly shortened, resulting in an overall compact shrub-like morphology. Figure 2 (A~C).

[0046] Quantitative morphological analysis further validated these observations: OE-HbCBF1 showed a 4.3-fold reduction in plant height, a 3.4-fold reduction in main stem thickness, and a reduction of more than 10-fold in both first branch height and internode distance. Figure 2 D).

[0047] 2. Microscopic observation and cytological analysis Wild-type (WT) and OE-HbCBF1 transgenic rubber trees with uniform growth and free from pests and diseases were selected. Leaves from the same leaf position (e.g., the third mature leaf from the top downwards) were used for mesophyll cell observation; the tips of lateral branches (approximately 0.5–1.0 cm long) were used for apical meristem observation. Tissue blocks of 5 mm × 3 mm were cut from both sides of the midrib of the leaf, avoiding the midrib and leaf margin, and quickly immersed in FAA fixative with a ratio of 90:5:5 (70% ethanol: formaldehyde: glacial acetic acid). The fixation was carried out under vacuum at 0.05 MPa for 15 min until the tissue settled, and fixed at room temperature for 24–48 h. Subsequently, the tissue was dehydrated with graded ethanol, cleared with xylene, infiltrated with paraffin at 60°C overnight, and embedded. 8-10 μm continuous sections were prepared from the paraffin blocks using a microtome. After spreading in a 42°C water bath, the sections were transferred to poly-L-lysine slides and dried overnight at 40°C. The sections were dewaxed with xylene, rehydrated with a gradient of ethanol, and then double-stained with safranin and Fast Green: stained with 1% safranin aqueous solution, washed with water, briefly separated by gradient ethanol, and then stained with 0.5% Fast Green ethanol solution. Finally, the sections were dehydrated and cleared, mounted with neutral resin, and air-dried. **0.5-1.0 cm** stem tips from the lateral branches were cut into the fixative and sectioned using the same paraffin method.

[0048] (1) Observation of mesophyll cells: Use an optical microscope (such as Olympus BX53) to observe the cross-sectional structure of the leaf under 10×, 20×, and 40× objectives, focusing on the number of palisade cell layers, cell morphology (long columnar / short columnar), and density of cell arrangement in the mesophyll tissue; as well as the intercellular spaces and cell shape of the spongy tissue. Use ImageJ software to measure the leaf thickness from the abaxial to the adaxial plane.

[0049] (2) Observation of apical meristem: Observe the transverse section structure of the apical meristem under an optical microscope, focusing on the characteristics of meristematic cells, including cell size, diameter, and density of arrangement. Measure the cell layer span and cell diameter at the widest point using ImageJ.

[0050] Microscopic observations further revealed the cellular basis of phenotypic differences. Leaf cell morphology showed almost no change. Figure 2 E), indicating that HbCBF1 may not regulate leaf cell structure. In contrast, the apical meristem of lateral branches in OE-HbCBF1 underwent significant remodeling ( Figure 2 F). Quantitative measurements showed that the cell density in this region increased to 2.56 times that of the WT, while the average cell diameter decreased to approximately half that of the WT. Figure 2 I). These data suggest that HbCBF1 may drive the overall dwarfing phenotype by inhibiting cell elongation in the apical meristem.

[0051] 3. Leaf cuticle analysis Transmission electron microscopy (TEM) observation: Mature leaves from wild-type and OE-HbCBF1 transgenic rubber trees were cut into 1 mm × 2 mm pieces, fixed with 3% glutaraldehyde and 2% osmium tetroxide, dehydrated with graded ethanol, embedded in Spurr resin, and then prepared into 70-90 nm sections using an ultramicrotome. After double staining with uranium acetate and lead citrate, the structure of the outer wax layer of epidermal cells was observed under a TEM microscope. Images were acquired at magnification of ×30,000 to ×50,000, and the thickness of each wax layer was measured using ImageJ software to compare the differences between wild-type and transgenic lines.

[0052] The results showed a unique macroscopic morphological feature observed in OE-HbCBF1: its leaf surface was significantly more glossy and reflective. Transmission electron microscopy (TEM) revealed the ultrastructural basis of this feature, confirming that the OE-HbCBF1 leaf surface was covered with significantly thickened epidermal wax crystals and an underlying cuticle layer (…). Figure 2 (G, H, J). The outer cuticle showed the greatest increase, being 2.34 times thicker than WT. This indicates that HbCBF1 overexpression can regulate cuticle development in rubber trees.

[0053] 4. Phenotypic analysis of leaf senescence SPAD values ​​showed a highly significant positive correlation with chlorophyll a, chlorophyll b, and total chlorophyll content, and can be used for rapid and non-destructive estimation of relative chlorophyll content. The SPAD-502 Plus chlorophyll meter was used to clamp the middle region of the leaf (avoiding the veins) and record the SPAD value. Chlorophyll fluorescence parameters and stomatal conductance of rubber tree leaves were simultaneously measured using a portable modulated chlorophyll fluorescence meter (MINI-PAM-II). Before measurement, the leaves were dark-adapted for 30 min to ensure that all photosystem II (PSII) reaction centers were fully open. Subsequently, the dark-adapted leaves were clamped in a leaf chamber, and measurements were started after the fluorescence signal and stomatal conductance parameters stabilized. The instrument parameters were set as follows: saturated pulsed light intensity of 800 μmol·m⁻¹. -2 ·s -1 Record the following metrics: maximum photochemical quantum efficiency of PSII (Fv / Fm) and porosity (Gs, unit: mol·m). -2 ·s -1 ).

[0054] The results showed that, in addition to significant dwarfing, the OE-HbCBF1 line also exhibited premature leaf senescence. The transgenic plants showed smaller leaves, thicker veins, accompanied by significant chlorosis and gradual wilting. Figure 3 A). Furthermore, during the non-leaf-falling season, the OE-HbCBF1 series often exhibits terminal bud dieback and premature leaf drop at the branch tips. In contrast, WT plants maintain vigorous growth throughout the same developmental stage, with fully expanded and green leaves. Figure 3 BD).

[0055] Multiple physiological indicators further support the senescence phenotype of OE-HbCBF1. Compared with WT, its leaves showed significantly reduced relative chlorophyll content, chlorophyll fluorescence parameters, and stomatal conductance. Figure 3 (EG) indicates impaired photosynthetic capacity and accelerated leaf senescence.

[0056] 5. Determination of ethylene content and related indicators (1) Determination of ethylene release: Ethylene concentration was measured using an electrochemical sensor with a PolarCept™ filter. Healthy, fully expanded leaves of similar age were collected from OE-HbCBF1 and wild-type plants for testing. Before analysis, each leaf was quickly wiped 15 times with a chloroform-saturated cotton swab to remove epidermal wax. The leaves were then sprayed with distilled water, allowed to equilibrate at room temperature for 1 hour, and then dried. The samples were placed in sealed 100 mL glass bottles and incubated for 1 hour to allow ethylene to equilibrate in the headspace. Ethylene concentration was measured using an F-900 portable ethylene analyzer. Ethylene release per unit leaf area was calculated based on the measured concentration, container volume, and leaf area.

[0057] (2) Determination of key enzyme activities in ethylene synthesis: The enzyme activities of ethylene synthase, plant S-adenosylmethionine synthase, 1-aminocyclopropane-1-carboxylic acid synthase, and 1-aminocyclopropane-1-carboxylic acid oxidase were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Enzyme activities in leaves were determined by ELISA using commercial assay kits, following the manufacturer's specifications. Standard curves were generated using serial dilutions of standards provided with each kit. Enzyme activities for each sample were determined by interpolation using the optical density values ​​of the samples to the corresponding standard curves. Activities were reported in units per gram of leaf, where the definition of one unit followed the manufacturer's instructions. Each sample was subjected to three technical replicates and six biological replicates for independent replicates.

[0058] The results showed that, compared with WT, the ethylene yield of the OE-HbCBF1 series (increased by 40.8%, P = 0.0036) and its precursor ACC (increased by 22.14%, P = 0.0021) were significantly increased. Figure 3 HJ).

[0059] Enzyme activity analysis further revealed that key enzymes in the ethylene biosynthesis pathway—SAMS, ACS, and ACO—were all enhanced in the OE-HbCBF1 system. Among them, SAMS activity showed the largest increase (50.31%, P = 0.0012). Figure 3 J), followed by ACS (23.29% increase, P = 0.0299), Figure 3 K) and ACO (increased by 16.45%, P = 0.0146, Figure 3 L).

[0060] In summary, these data provide consistent physiological evidence that the entire ethylene biosynthesis pathway in the OE-HbCBF1 system is systematically enhanced, which directly leads to excessive ethylene accumulation and consequently premature aging.

[0061] Example 3: Transcriptome analysis reveals overactivation of the ethylene biosynthesis pathway in HbCBF1 transgenic lines To investigate the genome-wide transcriptional changes induced by HbCBF1 overexpression, RNA sequencing (RNA-seq) was performed on the leaves of WT and OE-HbCBF1 lines.

[0062] Leaf samples were collected from 7-year-old transgenic and wild-type rubber trees, with three biological replicates; each replicate consisted of a mixed sample of multiple leaves. Total RNA was extracted and a library was constructed, followed by paired-end 150bp sequencing on an Illumina novaseq 6000 platform (Wuhan Metawell).

[0063] Raw reads were quality checked using FastQC, and clean reads were aligned to the rubber tree telomere-to-telomere reference genome using HISAT2. Transcript abundance was assessed using StringTie, and expression levels were normalized to TPM. Differential expression analysis was performed on the count data using DESeq2; genes with |fold change| > 2 and p < 0.05 were defined as differentially expressed genes. Volcano plots were generated based on the differential analysis results using tidyverse and ggrepel. GO annotation was derived from T2T genome functional annotation, and GO enrichment analysis was performed on upregulated and downregulated differentially expressed genes using TBtools. The GO enrichment results were visualized as bubble plots using ggplot2.

[0064] A total of 21,900 transcripts shared by the two genotypes were detected. In addition, OE-HbCBF1 and WT had 943 and 925 specifically detected transcripts, respectively. Figure 4 A). Differential expression analysis identified a total of 3,603 differentially expressed genes (DEGs). Compared with WT, the OE-HbCBF1 line had 2,198 upregulated genes and 1,405 downregulated genes, with the number of upregulated genes significantly exceeding the number of downregulated genes. Figure 4 B). The volcano plot shows the relationship between fold change and statistical significance, further indicating that the OE-HbCBF1 lineage as a whole exhibits a significant transcriptional activation bias, especially in highly significant DEGs (B). Figure 4 C). Besides the target gene HbCBF1, the most significantly upregulated genes included two UDP-glycosyltransferase genes (UGT74B1 and UGT88F4), the aquaporin gene PIP2-7, and two GA2ox8 homologs encoding gibberellin dioxygenases. Conversely, the most significantly downregulated genes included ACCELERATED CELL DEATH 6 (ACD6), strigolactone esterase D14, CCG-binding protein 1 (MEE14), ADAGIO protein 3 (ADO3), iron-ascorbic acid-dependent oxidoreductase SRG1, and CONSTANS-LIKE protein 16 (COL16). Figure 4 C).

[0065] GO enrichment analysis was performed on the upregulated and downregulated DEGs. The upregulated DEGs were significantly enriched in multiple functional categories, including ethylene biosynthesis (GO:0009693), circadian rhythms (GO:0007623), and hormone responses (GO:0009725), suggesting that HbCBF1 broadly influences ethylene metabolism, circadian rhythm regulation, and hormone-related signaling pathways. Figure 4D). Downregulation of DEGs enriches in processes such as xylem and phloem pattern formation (GO:0010051), meristematic development (GO:0048507), and anatomical development (GO:0048856). Figure 4 D). These functional categories are closely related to plant growth and development, providing a molecular-level explanation for the significant dwarfing phenotype of the OE-HbCBF1 line.

[0066] Consistent with evidence of increased ethylene production in physiological data, genes related to the ethylene biosynthesis pathway were significantly enriched in upregulated DEGs. All members of the ethylene biosynthesis pathway were identified from the rubber tree genome, including 9 SAMS, 13 ACS, and 11 ACO genes. Expression profiling analysis showed differential expression of 8 of these genes in the OE-HbCBF1 line, particularly 5 SAMS family members which were significantly upregulated, while only 1 ACS gene and 3 ACO genes showed increased expression. Figure 4 E). Among them, SAMS1 had the highest expression level and the largest fold induction (4.55-fold, P < 0.0001) in the SAMS family, suggesting that SAMS1 may be a core effector driving ethylene biosynthesis.

[0067] Example 4: Study on the molecular mechanism of HbCBF1 binding to the HbSAMS1 promoter and activating its transcription As an AP2 / ERF type transcription factor, HbCBF1 recognizes CRT / DRE cis-elements (CCGACs) to regulate its downstream target genes. To elucidate the molecular mechanism by which HbCBF1 regulates ethylene biosynthesis, cis-regulatory elements in the promoter regions of all ethylene biosynthesis genes were analyzed using the PlantCARE database. Among these genes, SAMS1 showed the most significant transcriptional induction, with its promoter containing a conserved CRT / DRE element (CCGAC) located 1355 to 1349 bp upstream of the translation initiation site. Figure 5 A).

[0068] 1. Yeast one-hybrid (Y1H) experiment A 222 bp bait fragment from the HbSAMS1 promoter containing the CRT / DRE element was inserted into the pAbAi plasmid and transformed into Y1HGold yeast. Active yeast clones were extracted from a uracil-deficient selection medium. The self-activation of Bait-HbSAMS1 yeast was detected on SD / -ura medium containing the antibiotic basididin A. The optimal selection concentration of Bait-HbSAMS1 was determined to be inhibited on medium containing 100 ng / mL AbA. The CDS of HbCBF1 was inserted into the pGADT7 plasmid. The AD-HbCBF1 plasmid was transformed into Bait-HbSAMS1 yeast, and selection was performed on SD / -Leu medium containing 100 ng / mL AbA. Bait-53 + AD-53 was used as a positive control. The pAbAi empty vector + pGADT7 empty vector was used as a negative control. The primer sequences used are shown in Table 4.

[0069] Table 4 Primer sequence information used in yeast one-hybrid (Y1H) experiments

[0070] Experimental results showed that yeast cells co-expressing AD-HbCBF1 and SAMS1 promoter bait grew well on AbA-containing selective medium, while yeast cells co-expressing empty AD vector and bait constructs failed to grow, indicating that HbCBF1 can interact with the SAMS1 promoter. Figure 5 B).

[0071] 2. Electrophoretic mobility variation analysis (EMSA) The coding sequence of HbCBF1 was cloned into the pET-28 protein expression vector. Simultaneously, a FAM-labeled DNA probe containing the CRT / DRE motif, derived from the SAMS1 promoter, was synthesized; the CRT / DRE core sequence of this probe was mutated to "TTTTT" as a non-specific binding mutant control probe. The recombinantly expressed HbCBF1-His fusion protein and the DNA probe were incubated in binding buffer at 22°C for 30 minutes. Unlabeled cold probes and mutant probes were used in a competition assay. The reaction products were separated by electrophoresis on a 6% non-denaturing polyacrylamide gel at 4°C, and the FAM signal was detected using a fluorescence scanner. The primer sequences used are shown in Table 5.

[0072] Table 5 Primer sequence information used for Electrophoretic Mobility Variation Analysis (EMSA)

[0073] Experimental results showed that the addition of recombinant HbCBF1 protein resulted in a migration lag band, and its intensity increased in a dose-dependent manner with increasing probe concentration (lane 2–4). With incremental addition of unlabeled competitive probes, the migration complex was gradually eliminated by competition (lane 5–7), while the mutant competitive probes failed to produce a competitive effect (lane 8), indicating that HbCBF1 can specifically bind to the CRT / DRE element in vitro. Figure 5 C).

[0074] 3. Dual-luciferase reporter gene assay The 1478-bp promoter sequence of SAMS1 was amplified from the genomic DNA of rubber tree and cloned into the reporter vector pGreenII 0800-LUC. The coding sequence of HbCBF1 was inserted into the expression vector pGreenII 62-SK. These recombinant plasmids were transformed into Agrobacterium tumefaciens strain GV3101, and then the reporter and expression strains were co-injected into tobacco leaves. The injected leaves were divided into four regions for the following co-injection treatments: (1) empty vector EV-SK + EV-LUC; (2) 35S::HbCBF1 + EV-LUC; (3) EV-SK + pro-HbSAMS1-LUC; (4) 35S::HbCBF1 + pro-HbSAMS1-LUC. After 72 hours, the luminescence signal was captured using a multifunctional plant in vivo imaging system. At the same time, the activities of firefly luciferase and Renilla luciferase were quantified using a dual-luciferase reporter gene assay kit, and the LUC / REN ratio was calculated according to the manufacturer's protocol. The primer sequences used are shown in Table 6.

[0075] Table 6 Primer sequence information used for dual-luciferase reporter gene detection

[0076] The experimental results showed that, compared with the empty vector control, the LUC reporter gene co-expressed by the HbCBF1 and HbSAMS1 promoters significantly enhanced luciferase activity, resulting in a significantly increased LUC / REN ratio. Figure 5 (D–E) indicates that HbCBF1, as a transcriptional activator, can positively regulate the promoter activity of SAMS1.

[0077] In summary, yeast one-hybrid assays, electrophoretic mobility shift analysis, and dual-luciferase assays provided mutually corroborating evidence, clearly demonstrating that HbCBF1 can directly bind to the CRT / DRE cis-elements in the SAMS1 promoter and upregulate its expression as a transcriptional activator. This establishes a direct molecular link between the transcription factor HbCBF1 and the activation of ethylene biosynthesis in rubber trees.

Claims

1. The application of the HbCBF1 gene in regulating ethylene content in rubber trees, characterized by, The nucleotides of the HbCBF1 gene are shown in SEQ ID NO.1; overexpression of the HbCBF1 gene can significantly increase the biosynthesis level of ethylene in rubber trees.

2. The application according to claim 1, characterized in that, The overexpression was achieved by constructing the coding sequence of the HbCBF1 gene into a eukaryotic expression vector.

3. The application according to claim 2, characterized in that, The eukaryotic expression vector is pCAMBIA-2301.

4. A method for increasing the ethylene content of rubber trees using the HbCBF1 gene described in claim 1, characterized in that, Includes the following steps: (1) Construct an HbCBF1 gene overexpression vector; (2) Transform the overexpression vector into Agrobacterium; (3) Genetic transformation was carried out using rubber tree explants or callus tissue as recipients via Agrobacterium-mediated transformation. (4) Screening and identifying transgenic plants; (5) Transgenic plants obtained through cultivation.

5. The method according to claim 4, characterized in that, The method significantly improves the biosynthesis level of ethylene in transgenic plants, manifested by an increased ethylene release rate, higher content of the ethylene precursor ACC, and enhanced activity of key enzymes in the ethylene synthesis pathway, namely SAMS, ACS, and ACO.

6. The method according to claim 4, characterized in that, The method induces transgenic plants to exhibit dwarfing phenotypes associated with excessive ethylene accumulation, including: significantly reduced plant height, thinner stems, accelerated leaf senescence, and premature leaf drop.

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