Application of HbCBF1 gene in improving latex yield of rubber tree

Abstract

The application discloses application of HbCBF1 gene in improving tapping ability of rubber trees, and belongs to the field of plant genetic engineering.A coding sequence of HbCBF1 gene shown in SEQ ID NO.1 is cloned into a eukaryotic expression vector to construct a recombinant expression vector pCAMBIA2301-35S::HbCBF1.Agrobacterium tumefaciens EHA105 strain carrying the recombinant vector is used to infect embryogenic calli from anther of a rubber tree, and through callus induction, differentiation screening and plant regeneration, a transgenic rubber tree stably overexpressing HbCBF1 is obtained.Result of tapping ability analysis shows that, compared with a wild type rubber tree, initial tapping rate, tapping volume and dry rubber yield of the OE-HbCBF1 transgenic rubber tree are all significantly improved.The application provides a new gene resource and technical route for molecular breeding of rubber trees.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and specifically relates to the application of the HbCBF1 gene in improving the latex excretion capacity of 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 that regulates plant growth, development, and stress response. In rubber trees, ethylene has been shown to be a key signaling molecule that stimulates increased rubber yield, and exogenous application of ethephon can increase latex production.

[0003] The latex excretion capacity of rubber trees is a complex quantitative trait regulated by multiple factors: the developmental status of the latex duct system, the latex regeneration rate, the patency of the latex excretion channels, the hormone signaling network (not only ethylene, but also auxin, abscisic acid, etc.), carbon source supply, and energy metabolism. Therefore, latex excretion capacity cannot be determined by a single factor, and simply increasing the level of a certain hormone does not necessarily lead to an overall improvement in latex excretion capacity.

[0004] Furthermore, there are fundamental biological differences between exogenous application of ethephon (a compound that releases ethylene) and the regulation of endogenous ethylene expression. The effective concentration of exogenous ethephon is far higher than the physiological concentration of endogenous ethylene, constituting a pharmacological dose. Its site of action and timing of action differ from the precise signal transduction of endogenous ethylene. More importantly, the synthesis of endogenous ethylene is strictly regulated by negative feedback, while high concentrations of exogenous ethylene disrupt this balance. Therefore, the effect of exogenous ethephon on increasing latex yield cannot be simply extrapolated to the conclusion that increasing endogenous ethylene levels through genetic engineering will necessarily improve latex expulsion capacity (expulsion rate).

[0005] 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.

[0006] The role of the CBF gene in latex synthesis and latex excretion regulation in rubber trees remains unclear, and new technical solutions are urgently needed to achieve precise regulation of latex excretion capacity in rubber trees. Summary of the Invention

[0007] This invention provides the application of the HbCBF1 gene in improving the latex excretion capacity of rubber trees, aiming to solve the technical problem of the bottleneck in increasing rubber latex yield due to latex excretion characteristics in the existing technology.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the HbCBF1 gene in improving the latex excretion capacity of rubber trees, wherein the nucleotides of the HbCBF1 gene are shown in SEQ ID NO.1; overexpression of the HbCBF1 gene significantly increases the latex excretion rate, latex volume and / or dry rubber yield of transgenic rubber trees compared with wild type.

[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] Secondly, the present invention 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] Thirdly, the present invention provides a method for improving the latex-excreting capacity of rubber trees, comprising the following steps: (1) The nucleotide sequence shown in SEQ ID NO.1 was constructed into an expression vector to obtain an HbCBF1 overexpression vector; (2) Infect rubber tree explants with Agrobacterium tumefaciens containing the HbCBF1 overexpression vector; (3) The infected explants were cultured to induce the production of transgenic rubber trees.

[0013] Furthermore, the Agrobacterium tumefaciens mentioned in step (2) is strain EHA105.

[0014] Furthermore, the method increases the latex discharge rate, latex discharge volume, and / or dry rubber yield of the transgenic rubber tree.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to demonstrate that overexpression of HbCBF1 through genetic engineering can significantly improve the latex excretion rate (latex excretion capacity) of rubber trees, specifically as follows: (1) The rate of latex expulsion was greatly improved: the initial latex expulsion rate of OE-HbCBF1 transgenic rubber trees was 98.34% higher than that of wild-type control, with the highest increase reaching 193.03% in multiple tapping tests (P=0.0264). (2) Significantly increased latex volume: The latex volume of OE-HbCBF1 transgenic rubber trees was 2.69 times that of the wild-type control (P=0.0377). (3) Significantly increased dry rubber yield: The dry rubber yield of OE-HbCBF1 transgenic rubber trees was more than twice that of the wild-type control on average, with the highest being 2.24 times (P=0.0303). (4) Enhanced continuous latex discharge: Transgenic rubber trees exhibit a more stable continuous latex discharge capacity during multiple tapping processes.

[0016] The actual effect of overexpressing the HbCBF1 gene in this application is to increase the gel excretion rate (gel excretion capacity), while the actual effect of existing technology (exogenous application of ethephon) is to increase yield. Therefore, the technical solution of exogenous application of ethephon and the technical solution of this application are fundamentally different in terms of technical effect.

[0017] In summary, this application experimentally verifies that overexpression of the HbCBF1 gene can lead to an overall improvement in rubber tree excretion capacity, providing new gene resources and technical routes for molecular breeding of rubber trees. Attached Figure Description

[0018] Figure 1 This 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 mean ± standard deviation, n=3; Student's t-test was used for statistical analysis; ***P<0.0001.

[0019] Figure 2This study monitored latex flow dynamics during seven consecutive tapping events in OE-HbCBF1 and 73397 (WT) transgenic rubber trees. The data included: (a) latex volume recorded in the first 5 minutes after tapping, and initial latex flow rate calculated; (b) latex flow duration (time from start to finish); (c) latex volume collected at each tapping event; and (d) dry rubber yield obtained from the latex. Except for latex flow duration, all collected data were standardized to per 1000 cm² of rubber tree stem surface area. Data are expressed as mean ± standard deviation (n ≥ 3). An asterisk (*) indicates statistical significance (P < 0.05); Student's t-test was used for statistical analysis. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] Table 1 Primer sequence information for amplifying the HbCBF1 gene

[0023] 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.

[0024] The correctly sequenced pCAMBIA-2301-HbCBF1 plasmid was transformed into Agrobacterium tumefaciens using a freeze-thaw method. Agrobacterium tumefaciens The 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Table 2 Primer sequence information for PCR identification

[0029] (2) The expression level of HbCBF1 gene was detected by real-time quantitative RT-PCR.

[0030] 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.

[0031] Table 3 Primer sequence information for detecting HbCBF1 gene expression level

[0032] (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.

[0033] 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 in the text). 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 and D in the text).

[0034] 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 in the text). GUS staining showed a blue signal in the leaves of all three transgenic lines, indicating successful expression of the reporter gene. Figure 1 (F in the text). 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 (F). Figure 1 (G in the middle).

[0035] 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.

[0036] Example 2: Analysis of glue removal ability Rubber trees (WT) of type 73397, which have similar stem circumference to the OE-HbCBF1 transgenic rubber trees obtained in Example 1, are untapped, and are 5-6 years old. Lactation tests were conducted in mid-April. Tapping was performed every three days for seven consecutive times. The latex volume, total latex drainage time, and final latex volume were recorded for the first 5 minutes after each tap. The initial latex drainage velocity was calculated by dividing the latex volume in the first 5 minutes by 5 minutes. The total latex drainage time and total latex volume were measured and recorded on-site. The latex was completely dried in a 65°C oven and then weighed; the weight was recorded as the dry latex yield. Except for the latex flow duration, all values ​​were calculated based on the tapped surface area (1000 cm²). 2 Based on this, all glue discharge data were homogenized.

[0037] The results showed that the initial latex expulsion rate of OE-HbCBF1 transgenic rubber trees was 98.34% higher than that of the control, with the most significant differences at T6 and T7 time points, increasing by 193.03% (P = 0.0264) and 148% (P = 0.0424), respectively. Furthermore, the increase in latex expulsion rate of OE-HbCBF1 transgenic rubber trees after each cut (average 45.24%) was significantly higher than that of 73397 rubber trees (WT, average 20.85%). Figure 2 (a) in the text. Regarding the duration of glue removal ( Figure 2 In (b) of the study, the difference between OE-HbCBF1 transgenic rubber trees and 73397 rubber trees (WT) was small from T1 to T4; after T4, the latex expulsion time of OE-HbCBF1 transgenic rubber trees gradually increased, and then decreased at T7. From T2 onwards, the latex expulsion volume of OE-HbCBF1 transgenic rubber trees exceeded that of the control by more than 50%, and the highest occurred at T6, which was 2.69 times higher than the control (P = 0.0377); the increase in latex expulsion volume of OE-HbCBF1 transgenic rubber trees at T5 and T6 both exceeded 90%, which was much higher than the 30% increase of 73397 rubber trees (WT). Figure 2 (c) in the middle). In terms of dry rubber production ( Figure 2 In (d), the OE-HbCBF1 transgenic rubber tree also had a much higher yield than the 73397 rubber tree (WT), with an average yield more than double that of the 73397 rubber tree (WT); among them, the T6 and T7 lines had the largest increases, which were 2.24 times (P = 0.0303) and 1.71 times (P = 0.0314) higher than the 73397 rubber tree (WT), respectively.

[0038] The above results indicate that after tapping (T1-T7), the OE-HbCBF1 transgenic rubber tree consistently exhibited superior latex discharge capacity, with faster flow rate, larger latex volume, and higher dry rubber yield.

Claims

1. The application of the HbCBF1 gene in improving the latex excretion capacity of 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 latex excretion rate, latex volume, and / or dry rubber yield of transgenic rubber trees compared with wild type.

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.

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