Application of tea tree CsGA2ox5 gene in regulation and control of gibberellin in tea tree leaves

Abstract

The invention discloses application of a tea tree CsGA2ox5 gene in regulation of gibberellin in tea tree leaves, and is characterized in that the nucleotide sequence of the tea tree CsGA2ox5 gene is as shown in SEQ ID NO.1 in a sequence table, and the tea tree CsGA2ox5 gene is used for negative regulation of the content of active gibberellin. The amino acid sequence of the protein coded by the tea tree CsGA2ox5 gene is as shown in SEQ ID NO. 2 in a sequence table. Cloning of the gene not only facilitates analysis of a related mechanism of regulating the plant height by gibberellin metabolism of tea tree leaves, but also facilitates cultivation of tea tree varieties suitable for mechanical picking, improves the efficiency and quality of mechanical picking, and has great application value.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of a tea tree CsGA2ox5 gene in regulating gibberellins in tea leaves. Background Art

[0002] Tea (Camellia sinensis) is a major cash crop in my country and a significant component of the country's agricultural output. Tea, one of the world's most consumed non-alcoholic beverages, is beloved for its rich secondary metabolites, which offer numerous health benefits, including antioxidant, anti-tumor, blood pressure-lowering, and cardiovascular disease prevention. China, the origin of tea, boasts the world's largest tea cultivation area (2.8 million hectares) and tea production (2.41 million tons in 2017). Tea's highest economic value is in spring, when the trees are at their peak growth. High-quality tea leaves must be harvested within a month of early spring, a significant amount of work within a short period of time.

[0003] Traditionally, tea leaves have been picked by hand. Manual plucking reduces leaf breakage and ensures the harvest of the most economically significant parts. However, traditional manual plucking is too time-consuming and labor-intensive. As tea producers continue to increase their demands for labor costs and production quality, mechanized bud plucking is an inevitable trend for the sustainable development of the tea industry. However, mechanical plucking has a significant drawback: it damages the integrity of the tea leaves, reducing the economic value of high-quality tea. Internode length is a key criterion for determining whether a tea plant is suitable for mechanical plucking.

[0004] Growth-promoting plant hormones play a key role in cell growth and internode elongation. Gibberellins (GAs) can promote stem internode elongation by promoting cell division and elongation. Gibberellins were first discovered in the pathogenic fungus Gibberella nigra, which causes a rice disease known as "stupid seedling." Excessive production of GAs causes plants to become spindly, unable to support their own weight, and to appear green and partially sterile. Further research has established GAs as an important class of plant hormones, playing crucial roles in plant growth and development. They regulate seed germination, stem elongation, leaf formation, bolting and flowering, dormancy breaking, and ripening inhibition. To date, over 130 GAs have been discovered in plants, but only a small fraction of these are biologically active. GA1, GA3, GA4, and GA7 are common active GAs in plants, functioning and participating in plant growth processes. Other GAs are precursors of active GAs or inactive metabolites. Previous studies have reported that tea varieties with longer internodes, such as Echa No. 1 and Zhongnong No. 111, exhibit higher levels of GA1 and GA3, while the opposite is true for varieties with shorter internodes. Therefore, the content of active gibberellins is an important factor affecting internode length in tea plants.

[0005] The concentration of active gibberellins in plants is regulated by balancing the rates of synthesis and metabolism. The inactivation of active GA plays a crucial role in regulating GA homeostasis within the plant. Gibberellin 2-oxidase (GA2ox), a dioxygenase encoded by the GA2ox gene family, is involved in the biodegradation of gibberellins. With the continuous advancement of tea genomics and molecular biology techniques, in-depth research on the synthesis and regulatory mechanisms of gibberellins in tea will provide important theoretical foundations and technical support for molecular breeding and precision cultivation of tea, helping to cultivate high-yield, high-quality tea varieties suitable for mechanical harvesting. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of the tea plant CsGA2ox5 gene in regulating gibberellins in tea leaves, so as to negatively regulate the content of active gibberellins.

[0007] In one aspect of the present invention, the present invention provides the use of the tea plant CsGA2ox5 gene for regulating gibberellins in tea leaves. According to an embodiment of the present invention, the nucleotide sequence of the tea plant CsGA2ox5 gene is shown in the sequence listing as SEQ ID NO. 1, and the tea plant CsGA2ox5 gene is used to negatively regulate the content of active gibberellins in tea leaves.

[0008] In addition, the application of the tea plant CsGA2ox5 gene in regulating gibberellins in tea leaves according to the above embodiment of the present invention may also have the following additional technical features:

[0009] In some embodiments of the present invention, the expression pattern of the tea plant CsGA2ox5 gene is highly negatively correlated with the growth of tea plant stems. CsGA2ox5 reduces the level of active gibberellins in cells, negatively regulating the active gibberellins content, resulting in dwarfing of the plant.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] 1) This study cloned and verified, for the first time, the key gene CsGA2ox5, which regulates gibberellin metabolism in tea plants. This gene precisely regulates gibberellin levels in tea plants by catalyzing the conversion of active gibberellins to inactive forms, thereby influencing the growth, development, and quality of tea. Specifically, by regulating CsGA2ox5 expression, targeted improvements in tea plant architecture (e.g., cultivating tea varieties with longer internodes) can be achieved, improving the adaptability of tea gardens to mechanical harvesting.

[0012] 2) The present invention also provides a recombinant plasmid, transgenic bacteria, and transgenic plants containing the CsGA2ox5 gene. This invention enriches our understanding of tea plant growth and development and provides a theoretical and practical basis for selective agronomic trait breeding in tea plants.

[0013] 3) The expression pattern of CsGA2ox5 is closely related to the growth and development of tea plants. Subcellular localization reveals the association between the functional activity of the molecule and cell structure at the cellular level. The protein participates in gibberellin metabolic reactions in the nucleus and cell membrane. The enzyme activity of CsGA2ox5 was determined to be important in the gibberellin degradation pathway, participating in the conversion of active gibberellins to inactive gibberellins. The construction of tobacco plants overexpressing CsGA2ox5 further confirmed its biological significance, showing that it can affect plant height by degrading active gibberellins. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the difference in expression of the CsGA2ox5 gene in different tissues of the tea plant in Example 2 of the present invention. In the figure, L1-one leaf, L2-two leaves, L3-three leaves, L4-four leaves, L5-five leaves, S1-one stem, S2-two stems, S3-three stems, S4-four stems;

[0015] Figure 2 This is a localization map of the YFP-CsGA2ox5 fusion protein in tobacco epidermal cells in Example 3 of the present invention;

[0016] Figure 3This is the in vitro enzyme activity assay of CsGA2ox5 in Example 4 of the present invention, (A) Schematic diagram of the oxidative inactivation of active gibberellins GA1 and GA4, (B) Chromatogram of inactive GA8 produced by incubation of recombinant MBP-fused CsGA2ox5 protein with GA1, with boiled protein used as a control reaction, (C) Chromatogram of inactive GA34 produced by incubation of recombinant MBP-fused CsGA2ox5 protein with GA4, with boiled protein used as a control reaction;

[0017] Figure 4 This is the functional verification of CsGA2ox5 in tobacco in Example 4 of the present invention, (A) RT-PCR verification of the positive transformation of CsGA2ox5 in tobacco plants, (B) comparison of growth phenotypes of CsGA2ox5 overexpressing lines (OE-1 and OE-2) and empty vector overexpressing plants (EV), (C) measurement of the growth phenotype height of CsGA2ox5 overexpressing lines (OE-1 and OE-2) and empty vector overexpressing plants (EV), (D) morphological examination of the parenchyma tissue of longitudinal sections of transgenic tobacco stems and wild type under a microscope, (E) statistics of the parenchyma cell lengths of longitudinal sections of transgenic tobacco (OE-CsGA2ox5) and wild type (WT) tobacco stems. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0019] Example 1

[0020] Cloning and sequence structure analysis of the CsGA2ox5 gene:

[0021] Shucha, a nationally recognized tea variety, was cultivated at the Agricultural Industrial Park of Anhui Agricultural University in Hefei, Luyang District, Anhui Province. Young leaves were used for RNA extraction. Total RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions, and RNA content and quality were determined using a spectrophotometer. Reverse transcription to generate the first strand: 1 μg of RNA was used as template. Following the instructions for the Promega M-MLV1 reverse transcriptase reagent, 0.5 μg of each downstream primer was added, the volume was adjusted to 15 μL, and denatured at 70°C for 5 minutes. The mixture was then immediately placed on ice for 5 minutes. Then, 5 μL of M-MLV 5× buffer, 5 μL of 25 mM dNTPs, 25 units of rRNasin Ribonuclease Inhibitor, and 200 units of M-MLV reverse transcriptase were added, with DEPC water making up the volume to 25 μL. The mixture was incubated at 42°C for 1 hour, and the reverse transcriptase was inactivated at 95°C for 5 minutes. After optimization, an appropriate amount of reverse transcription product was used for subsequent PCR. Conventional PCR was performed using the first-strand cDNA as the RT-PCR template to amplify the CsJAZ12 gene. The upstream primer was (5'-ATGGTGGTTCTCGCTAAACC-3'), and the downstream primer was (5'-TTATGAGGCTGCAATTTTCTCAAAC-3'). A 25 μL PCR reaction system consisted of 2.5 μL of 10× Ex Taq buffer, 2.0 μL of dNTPs, 1.5 μL of Mg₂+, 1 μL each of the upstream and downstream primers, 0.2 μL of Ex Taq, 1 μL of template, and 15.8 μL of ddH₂O. The reaction program was as follows: 95°C for 5 min, 95°C for 50 sec, 58°C for 50 sec, 72°C for 1 min, and 72°C for 10 min, for 35 cycles. The PCR product CsJAZ12 gene was purified and recovered, and then ligated into the pMDTM19-TSimple Vector (Takara, Japan) to obtain the pMDTM19-T-CsJAZ12 plasmid. The plasmid was transformed into Escherichia coli DH5α competent cells and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The following nucleotide sequence was obtained (SEQ ID NO. 1):

[0022]

[0023] The protein sequence encoded by the CsGA2ox5 gene (SEQ ID NO. 2) is as follows:

[0024] MVVLAKPAIEQLSAIKTCKLTTTFFSGIPIIDLSKPDSKNLLVKACEEFGFFKVINHGVPTECITKLESEAVSFFSLPLSEKEKAGPPNPFGYGNSIGSNGDVGWVEYLLLTTNPEFNYQGFESIYGKTPETIRCAVDDYVLAVKRMACEVLELLADGLKIQQRN VFSKLLMDEQSDSVFRLNHYPPYPELQEMNGSNFIGFGEHTDPQIISVLRSNNTSGLQISLKDGNWISVPPDQNSFFINVGDSLQVMTNGRFKSVRHRVIANSLKSRVSMIYFGGPPLSEKIAPLPSLMEGEDSLYKEFTWFEYKKSAYKSRLADNRLGLFEKIAAS

[0025] Example 2

[0026] Analysis of CsGA2ox5 gene expression in different tissues of tea plants:

[0027] The Shucha Zao tea variety, a nationally recognized fine tea variety, was cultivated at the Agricultural Industrial Park of Anhui Agricultural University in Hefei, Luyang District, Anhui Province. Eight tissues and organs were used to analyze gene expression. These included one leaf, two leaves, three leaves, four leaves, one stem, two stems, three stems, and four stems. Next-generation transcriptome sequencing was performed by BGI, a Chinese biotechnology company, with three biological replicates for each sample. Gene expression levels were calculated using a standard transcriptome sequencing analysis pipeline and expressed as FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).

[0028] like Figure 1 As shown in the study, cluster analysis of gibberellin metabolism gene expression in different tissues of the representative small-leaf tea cultivar Shuchazao revealed that CsGA2ox5 is highly expressed in the stem. The expression pattern of CsGA2ox5 is highly negatively correlated with stem growth, particularly in the four stems, where expression increases with increasing stem development. CsGA2ox5 is the most highly expressed gene, suggesting that it plays a crucial role in tea stem development.

[0029] Example 3

[0030] Functional verification of the CsGA2ox5 gene in tea plants:

[0031] Localization of the CsGA2ox5-YFP fusion protein in tobacco epidermal cells: CsGA2ox5 was cloned into a subcellular localization vector to construct the CsGA2ox5-YFP fusion protein vector. The upstream primer used was (5-CATATGGGATCTACTAGTGAATTCATGGTGGTTCTCGCTAAACC-3') and the downstream primer was (5'-CCCGGGGGTACCGTCGACGGATCCTGAGGCTGCAATTTTCTCAAAC-3'). The specific method was as follows: an appropriate amount of tobacco seeds were vernalized in a 4°C refrigerator with deionized water for 72 hours before sowing. After sowing, the seeds were covered with plastic wrap and placed under appropriate conditions (60% humidity, 23°C, 16 hours light / 8 hours dark) to allow germination. After germination, seedlings of uniform size were selected for transplanting and cultured as usual. CsGA2ox5-YFP was transformed into Agrobacterium GV3101 by electroporation, and positive clones were identified by conventional PCR. Positive colonies harboring the target gene were selected and cultured in 5 mL of LB medium containing the appropriate antibiotics at 28°C and 200 rpm for approximately 24 hours. Aspirate 2 mL of the culture medium and add it to 50 mL of fresh LB medium containing the appropriate antibiotics. Continue shaking the culture until the OD600 is approximately 1.0. Collect the cells by centrifugation and resuspend them in a suitable amount of transformation solution (MS + 0.3 mg / L 6-BA + 150 g / L sucrose + 15 g / L LEMS + 0.06% Silwet L-77, pH 5.7) to a final OD600 concentration of approximately 0.8. The prepared transformation solutions were mixed in equal proportions, placed in a syringe, and gently injected into the dorsal surface of tobacco leaves. The mixture was then left in the dark for 24 hours. Three days later, the injected leaves were examined under a laser confocal microscope.

[0032] like Figure 2 As shown, the subcellular localization of a protein often determines its mechanism of action. To clarify the site of action of CsGA2ox5 in cells, YFP-CsGA2ox5 was transiently expressed in tobacco. The fluorescence signal showed that CsGA2ox5 was localized in the cell nucleus and cell membrane.

[0033] Example 4

[0034] Functional verification of CsGA2ox5 gene in tea plant in vitro:

[0035] 1. Enzyme activity assay

[0036] The reaction mixture (150 μL) contained 10 μg of recombinant CsGA2ox5 protein, 50 μM GA1 or GA4, 5 mM DTT, 13.3 mM α-ketoglutarate, 13.3 mM ascorbic acid, 0.67 mM iron (II), and 5 μg μL-1 BSA in 50 mM Tris-HCl (pH 8.0). The reaction mixture was incubated in a 30°C incubator for 1 hour, terminated by the addition of 1 / 5 volume of 1 M HCl, and then extracted with 180 μL of ethyl acetate. After centrifugation at 14,000 x g for 1 minute, the upper organic phase was dried using a vacuum concentrator and dissolved in 200 μL of methanol prior to LC-MS / MS analysis.

[0037] 2. Chromatographic mass spectrometry acquisition conditions

[0038] The data acquisition instrument system uses liquid chromatography-tandem mass spectrometry LC-MS / MS (SCIEX Triple Quad5500). The liquid phase conditions mainly include:

[0039] (1) Chromatographic column: C18 column (Phenomenex, 4.6 × 250 mm, 5 μm);

[0040] (2) Mobile phase: Phase A, 0.1% formic acid + 5 mM ammonium formate; Phase B, methanol;

[0041] (3) Gradient elution program: 0-3 min, A / B ratio 80:20 (V / V); 3-6.5 min, A / B ratio 5:95 (V / V), 6.5-8 min, 5:95 (V / V), 8-10 min, 80:20 (V / V);

[0042] (4) Flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0043] Mass spectrometry conditions mainly include: electrospray ionization (ESI), negative ion scanning. Each ion pair is scanned and detected based on the optimized fragmentor voltage and collision energy (CE).

[0044] 3. Leaf disc method for transgenic tobacco

[0045] (1) The PBI121-GA2ox5 overexpression vector was constructed using recombinant cloning technology. The upstream primer used was (5'-ACGGGGGACTCTAGAGGATCCATGGTGGTTCTCGCTAAACC-3') and the downstream primer was (5'-CGATCGGGGAAATTCGAGCTCTTATGAGGCTGCAATTTTCTCAAAC-3'). The vector was transformed into the EHA105 strain, and a single colony containing the target gene was picked and inoculated into 3 ml of LB liquid medium (50 mg / L Rif + 100 mg / L Kan antibiotic: medium = 1:1000) and cultured overnight at 28°C in a shaker at 180 rpm.

[0046] (2) Transfer 1%-2% of the overnight culture into freshly prepared LB medium without antibiotics. Cultivate under the same conditions as above for about 6 hours and adjust the OD600 to 0.2-0.5 (0.4 is optimal) for transformation.

[0047] (3) On a clean bench, pour the bacterial solution into a small, sterile Petri dish. Take young, strong leaves from non-resistant, sterile tobacco seedlings, remove the veins, and cut them into 1x1cm pieces. Place them in the bacterial solution and soak for an appropriate time (usually 5-10 minutes). Remove the leaves and place them on sterile filter paper to remove the attached bacterial solution.

[0048] (4) Place the infected tobacco leaves on the co-culture medium (place filter paper on the co-culture medium), seal the culture dish with sealing film, and culture in the dark at 28℃ for 3 days.

[0049] (5) Transfer the tobacco leaves cultured for 3 days to the selective culture medium, seal the culture dish with a sealing film, and select the culture medium under the light and dark conditions of 2000-10000 lux, 25-28℃, 16h / 8h / 1d.

[0050] (6) After about 2-3 weeks, when the adventitious roots grow to about 1 cm, cut the adventitious buds and transfer them to the rooting medium for rooting culture. After 5-10 days, adventitious roots will grow. After growing 4-5 leaves and a certain amount of root system, transfer them to soil for culture.

[0051] (7) Take the leaves of tobacco grown through tissue culture, extract genomic DNA, and identify whether the tobacco is an overexpression plant.

[0052] The reagent formula involved in this experiment is as follows: LB liquid culture medium: 5 g of trypsin Chen, 10 g of yeast extract, 10 g of NaCl, add pure water to 1 L, add 1 g of agar powder for every 100 mL of solid culture medium, and sterilize at 121°C for 15 min.

[0053] Leaf disc tobacco transformation reagent formula: co-culture medium (1L): MS + sucrose 30g + agar 8g + 6-BA 2.25mg + NAA 0.3mg, pH = 6;

[0054] Selection medium (1L): MS + sucrose 30g + agar 8g + 6-BA 2.25mg + NAA 0.3mg + Cef 400mg + K + 2.5mg, pH=6;

[0055] Rooting medium (1 L): MS + sucrose 30 g + agar 8 g + Cef 400 mg + K + 2.5 mg, pH = 6;

[0056] 4. Observation of plant tissue morphology

[0057] For internode sections, samples were collected from the first internode (located between the first and second leaves) and fixed overnight in FAA solution (50% ethanol, 5% formaldehyde, 5% acetic acid, and 40% 865 distilled water). After dehydration with varying concentrations of ethanol, the samples were covered with 5% agar. Sections approximately 50 to 100 microns thick were cut using a vibratome (VT1200S, Leica, Germany). Sections were then imaged using a microscope (DM3000, Leica, Germany), and cell length was measured using ImageJ software.

[0058] like Figure 3 As shown, the MBP-fused CsGA2ox5 protein oxidizes the H at the C-2 position of the active gibberellins to -OH, converting the active gibberellins GA1 and GA4 into inactive GA8 and GA34, respectively. This indicates that CsGA2ox5 plays a role in the catabolism of bioactive GAs. (A) Schematic diagram of the enzymatic reaction mechanism. CsGA2ox5 catalyzes the oxidative inactivation of the active gibberellins GA1 and GA4 through C-2β-hydroxylation, generating the inactive metabolites GA8 and GA34, respectively. (B) LC-MS analysis confirmed that CsGA2ox5 efficiently catalyzes the C-2β-hydroxylation of GA1 to generate inactive GA8. A boiled protein control showed no conversion, confirming its specific enzymatic function. (C) LC-MS analysis showed that CsGA2ox5 efficiently catalyzes the C-2β-hydroxylation of GA4 to convert it into inactive GA34. A boiled protein control showed no product formation, confirming its strong degradation activity.

[0059] like Figure 4As shown, compared to wild-type tobacco (WT), tobacco plants overexpressing CsGA2ox5 (OE-1 and OE-2) exhibited a significant dwarf phenotype. Morphological examination of the parenchyma tissue of the first internode (between the first and second leaves) in OE-1 plants revealed a significantly shorter average cell length compared to WT plants. This experiment demonstrated that overexpression of CsGA2ox5 inhibited the elongation of parenchyma cells in the stem, reducing plant height. (A) RT-PCR analysis confirmed the successful integration of CsGA2ox5 into the tobacco genome, while no amplified bands were observed in the empty vector control (EV) and wild-type (WT) lines (Lanes 1-2), indicating that the transgenic lines OE-1 / OE-2 were effectively constructed. (B) Phenotypic comparison: OE lines (OE-1 / OE-2) showed significantly shorter plant height than EV, with compact leaves and shortened internodes, visually verifying the dwarfing function of CsGA2ox5. (C) The plant heights of OE-1 and OE-2 lines were 24.3±1.2 cm and 25.1±0.9 cm, respectively, approximately 50% lower than the EV control (48.7±2.1 cm), confirming that CsGA2ox5 negatively regulates stem elongation. (D) Stem morphology: Longitudinal sections of OE stems revealed densely packed parenchyma cells (Figure D2), while EV cells were longitudinally elongated (Figure D1), suggesting that CsGA2ox5 inhibits growth by inhibiting cell elongation. (E) Cell length statistics: The average length of OE parenchyma cells was significantly shorter than that of EV, directly correlated with the dwarf phenotype, revealing that CsGA2ox5 reduces plant height by inhibiting cell elongation.

[0060] In summary, the CsGA2ox5 protein is related to the metabolic regulation of gibberellins in tea trees. It affects the elongation of tea stems by degrading active gibberellins (such as GA1 and GA4), and has important applications in cultivating tea varieties suitable for mechanical picking.

[0061] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they shall fall within the scope of protection of the present invention.

Claims

1. The application of the tea plant CsGA2ox5 gene in regulating gibberellins in tea leaves is characterized by: The nucleotide sequence of the tea plant CsGA2ox5 gene is shown in the sequence table SEQ ID NO.

1. The tea plant CsGA2ox5 gene is used to negatively regulate the content of active gibberellins in tea leaves.

2. The use of the tea plant CsGA2ox5 gene according to claim 1 in regulating gibberellins in tea leaves, characterized in that The expression pattern of the tea plant CsGA2ox5 gene is highly negatively correlated with the growth of the tea plant stem.

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