Application of CsCAT1 in Regulating Distribution of Theanine between New Shoots and Leaves of Tea Plant

Abstract

The application relates to the technical field of genetic engineering, in particular to application of CsCAT1 in regulating distribution of theanine between new shoots and leaves of tea trees, and to reduction of the stem / leaf ratio of theanine in tea trees by reducing the expression amount of CsCAT1 in tender stems, wherein the base sequence of the CsCAT1 is shown as SEQ ID NO. 1, the stem / leaf ratio of theanine in the tea trees is reduced, the content of theanine in tender leaves of the tea trees is increased, and the quality of tea leaves is further improved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of CsCAT1 in regulating the distribution of theanine among the stems and leaves of tea shoots. Background Technology

[0002] Theanine, also known as N-ethyl-γ-L-glutamine, is the main umami component in green tea. It can neutralize the astringency and bitterness brought by catechins and caffeine. Theanine is a non-protein amino acid unique to tea plants, and it is the most abundant free amino acid in the new shoots of tea plants used for tea production, accounting for 40%-70% of the total free amino acids. Theanine accounts for 1%-2% of the dry weight of tea leaves, and in high-quality green teas, this proportion can even exceed 4%.

[0003] Theanine is mainly synthesized in the roots during late autumn and winter, and then transported from the roots to different tissues of the new shoots in spring. After being transferred to the new shoots, a large portion of theanine is distributed in the stems. Therefore, the theanine content in young stems is more than twice that in young leaves compared to young leaves. However, in tea production, only a small portion of the stems are utilized, or not at all. For example, the famous green tea brand Lu'an Melon Seed Tea only uses single leaves from the new shoots. Therefore, a considerable portion of the theanine in the stems remains underutilized. In view of this, increasing the proportion of theanine allocated from young stems to young leaves is an important way to increase the theanine content in young leaves.

[0004] Studies in model plants have shown that amino acid transporters regulate the distribution ratio of amino acids among different tissues. Membrane-localized amino acid transporters promote transmembrane transport of amino acids. Previous results have shown that six members of the amino acid transporter family in tea, namely CsAAP1, CsAAP3, CsAAP4, CsAAP5, CsAAP6, and CsAAP8, are capable of transporting theanine. Among these transporters (CsAAPs), CsAAP1 mediates theanine transport from roots to branches by recovering theanine from the ectoplast and promoting its loading into the xylem. Recently, CsAAP7.2 has been shown to have theanine uptake capacity and promote long-distance transport of theanine in tea plants. In addition, CsCAT2, a member of the cationic amino acid transporter (CAT) family, is also capable of transporting theanine and may promote its entry into vacuoles for storage in tea plant roots. Furthermore, a member of the tea plant LHT family has shown the ability to transport theanine. However, their physiological functions in tea plants remain unknown. These theanine transporters also have the ability to transport many other amino acids. The specific functions of these theanine transporters may be achieved through their spatiotemporal expression and localization.

[0005] The theanine content in the stem tissue of tea plants is more than three times that in the leaves. However, during tea harvesting, only part of the stem is harvested, or the stem is not harvested at all. By studying the mechanism of theanine distribution between young stems and leaves of tea plant shoots, it is possible to increase the accumulation of theanine in young leaves. In tea plant shoots, the transport proteins responsible for theanine distribution between young stems and leaves have not yet been identified. Furthermore, in other plants, including model plants, no amino acid transport proteins mediating the distribution of amino acids between stems and leaves have been identified. Therefore, how to regulate theanine distribution between the stems and leaves of tea plant shoots, thereby increasing the theanine content in tea leaves, is an urgent problem to be solved.

[0006] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0007] The purpose of this invention is to solve the problem of how to regulate the distribution of theanine in the stems and leaves of tea tree shoots, thereby increasing the theanine content in tea tree leaves, and provides the application of CsCAT1 in regulating the distribution of theanine in the stems and leaves of tea tree shoots.

[0008] To achieve the above objectives, this invention discloses the application of CsCAT1 in regulating the distribution of theanine among the stems and leaves of tea shoots. By reducing the expression level of CsCAT1 in tender stems, the stem / leaf ratio of theanine in tea plants is reduced. The base sequence of CsCAT1 is shown in SEQ ID NO.1.

[0009] The base sequence of CsCAT1 (SEQ ID NO.1):

[0010]

[0011] The protein sequence encoded by CsCAT1 is shown in SEQ ID NO.2.

[0012] The protein sequence encoded by CsCAT1 (SEQ ID NO.2):

[0013] .

[0014] The expression level of CsCAT1 in young stems was significantly negatively correlated with the theanine content in young leaves and stems, and significantly positively correlated with the theanine ratio between stems and leaves.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention significantly reduces the expression of CsCAT1 in tender stems through asODN treatment, the content of theanine in tender stems decreases, the content of theanine in tender leaves increases significantly, and the ratio of theanine in stems to leaves decreases extremely significantly. Thus, the distribution of theanine in the stems and leaves of tea tree shoots can be regulated by CsCAT1, thereby increasing the content of amines produced in tea tree leaves. Attached Figure Description

[0016] Figure 1 Subcellular localization of CsCAT1 in tobacco epidermal cells. Note: CsCAT1-GFP and pm-rK-CD3-1007 are transiently co-expressed. Green fluorescence represents the CsCAT1-GFP fusion protein, and red fluorescence represents the mCherry fusion plasma membrane marker protein. The merged image shows the superposition of green and red fluorescence.

[0017] Figure 2 To reconstruct the growth phenotype of CsCAT1 yeast on theanine and control media, note: CsCAT1 transgenic yeast was grown on YNB medium with 2 mM theanine, (NH4)2SO4, and ON as the sole nitrogen source. 22Δ10α is an amino acid uptake-deficient yeast mutant, EV / 22Δ10α is a strain containing the empty vector 22Δ10α, and CsCAT1 / 22Δ10α is a transgenic 22Δ10α strain.

[0018] Figure 3 To compensate for the growth rate of CsCAT1 yeast with theanine as the nitrogen source, note: the OD of EV / 22Δ10α and CsCAT1 / 22Δ10α was measured by shaking in liquid medium with 0.2 and 2 mM theanine as the sole nitrogen source. 600 bacterial concentration;

[0019] Figure 4 Tissue samples were collected for theanine analysis, including (a) tea garden tea trees used for sampling; (b) tea tree shoots and sampling sites; and (c) abbreviations and full names of 12 tea tree varieties.

[0020] Figure 5 The theanine content in the tender leaves of 12 tea varieties;

[0021] Figure 6 The theanine content in the tender stems of 12 tea varieties;

[0022] Figure 7 The study investigated the ratio of theanine content in stems and leaves of different tea varieties and its correlation. (a) The ratio of theanine content in stems and leaves of 12 tea varieties; (b) Correlation analysis between the ratio of theanine content in stems and leaves and the theanine content in leaves and stems. r refers to the Pearson correlation coefficient, and "*", "**", and "***" indicate significance at the P<0.05, P<0.01, and P<0.001 levels, respectively, based on the Tukey significance test.

[0023] Figure 8 Expression of CsCAT1 in the tender stems of different tea varieties;

[0024] Figure 9Tissue localization of CsCAT1 in young tea stems. Note: Tea stems were used for in situ PCR. Blue signals indicate the expression sites of CsCAT1. A control without reverse transcription primers was used. Scale bar: upper layer 250 μm, lower layer 600 μm;

[0025] Figure 10 To transiently inhibit the effect of CsCAT1 expression in stems on theanine distribution, the following experiments were conducted: (a) treatment of the first stem of tea plants with sODN-CsCAT1 and asODN-CsCAT1; (b) CsCAT1 expression in tea plant stems treated with sODN-CsCAT1 and asODN-CsCAT1; (c) changes in theanine content in tea plant stems and leaves after treatment with sODN-CsCAT1 and asODN-CsCAT1. Asterisks above the error lines indicate significant differences according to Student's t-test (*, p≤0.05; **, p≤0.01). Detailed Implementation

[0026] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0027] I. Construction of the Expression Carrier

[0028] 1. Gene Cloning. Based on the CsCAT1 ORF sequence provided by the Tea Plant Genome Website (http: / / tpia.teaplants.cn), and the multiple cloning restriction sites of the yeast expression vector pDR196, the subcellular localization vector pCAMBIA1305.1, and the Arabidopsis overexpression vector pCAMBIA1305.1 (vector map shown in the attached figure), primers containing the corresponding restriction sites were designed (as shown in Table 1). Using cDNA from the 'Shuchazao' tea variety as a template, PCR amplification was performed using the high-fidelity enzyme Primerstar. PCR reaction system: 12.5 μL GoldPfu PCR SuperMix, 1 μL cDNA template, 0.5 μL upstream and downstream primers, 5.5 μL sterile water; PCR reaction program: 94℃ denaturation pre-denaturation for 2 min, 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 2 min, 30 cycles, 72℃ extension for 5 min, 16℃ forever.

[0029] 2. Gel product recovery. Refer to the instruction manual for the "Gel Recovery Kit (AxyGEN, Hangzhou)" for experimental methods.

[0030] 3. pEASY carrier ligation.

[0031] (a) The gel recovery product of the cloned gene was mixed with pEASY-Blunt Simple Cloning Vector at a ratio of 4:1, with a total volume of 5 μL.

[0032] (b) React at room temperature (20℃-37℃) for 5 min.

[0033] (c) Finally, the reaction was carried out in a PCR instrument at 25°C for 15 min.

[0034] 4. Escherichia coli transformation.

[0035] (a) Gently mix 5 μL of the ligated vector with 50 μL of competent cells.

[0036] (b) Incubate the mixture in an ice bath for 30 minutes.

[0037] (c) Heat shock for 45 seconds in a 42°C water bath.

[0038] (d) Gently remove and place on ice for 5 minutes.

[0039] (e) Add 500 μL of antibiotic-free LB medium and incubate in a shaker at 37°C for 1 h.

[0040] (f) Take 50 μL of the mixture and spread it onto a plate. The plate should contain Amp at this point. + Or Kan + .

[0041] (g) Incubate overnight in a 37°C constant temperature incubator.

[0042] 5. Colony PCR verification.

[0043] 6. Digest the empty vector and sequencing plasmid with enzymes.

[0044] 7. Ligate the enzyme-digested fragments with the empty vector.

[0045] 8. Transform Escherichia coli competent cells.

[0046] 9. Colony PCR, enzyme digestion, and sequencing verification.

[0047] Table 1 Primers for vector construction

[0048]

[0049]

[0050] II. Subcellular localization of transient expression in tobacco epidermis

[0051] Amino acid transporters mediate the transmembrane transport of amino acids. To explore the role of CsCAT1 in the transport of theanine, the subcellular localization of CsCAT1 was first examined.

[0052] 1. Instantaneous tobacco conversion:

[0053] (a) Pick a single colony of Agrobacterium and place it in 20 μg / mL Rif, 50 μg / mL Kan 500 μL LB liquid. Incubate at 28°C until turbid. Then, expand the culture by shaking at a ratio of 1:100 for 2 days. Take an appropriate amount of the bacterial culture and keep it in an equal volume of 50% glycerol at -80°C.

[0054] (b) Take 5 mL of bacterial culture (6000 g), centrifuge at room temperature for 10 min, discard the liquid and collect the bacterial cells, resuspend in MMA and centrifuge again.

[0055] (c) Resuspend the bacterial cell mass in freshly prepared MMA suspension until the final concentration OD is reached. 600 At 0.6-0.8; add the corresponding volume of AS solution (volume ratio of 1:2000) to the bacterial solution according to the volume of each bacterial solution, and activate at room temperature in the dark for 2 hours.

[0056] (d) When injecting Nicotiana benthamiana, be careful to inject a whole leaf at once, and no more than three leaves should be injected per Nicotiana benthamiana plant.

[0057] (e) After injection, the injected tobacco should be placed back into the plant growth climate chamber for at least 36 hours of incubation. After 2-3 days of normal incubation, the tobacco epidermal cells should be photographed using laser confocal microscopy.

[0058] 2. Laser confocal imaging of tobacco epidermal cells:

[0059] (a) Slide preparation. Take a clean glass slide and place a drop of pure water in the center of the slide. Cut a small piece of the leaf blade at the injection site and place it in the pure water on the glass slide. Use tweezers to take a coverslip and cover the leaf blade from one end to remove excess moisture and air.

[0060] (b) Take a picture. Use a laser confocal imager to find the optimal subcellular localization map within the field of view.

[0061] Given that the homolog of CsCAT1, AtCAT1, in Arabidopsis thaliana is located on the plasma membrane, it is hypothesized that CsCAT1 is also likely located on the plasma membrane. To observe subcellular localization, CsCAT1-eGFP and the plasma membrane marker pm-rK-CD3-1007 were expressed in tobacco epidermal cells. CsCAT1-eGFP exhibited green fluorescence, which almost overlapped with the red fluorescence of the plasma membrane marker. Figure 1 This result confirms that CsCAT1 is located on the plasma membrane and indicates that CsCAT1 is involved in the transmembrane transport of amino acids in tea plants.

[0062] III. Yeast Transformation and Analysis

[0063] To investigate the transport activity of CsCAT1 for theanine, CsCAT1 was transformed into the amino acid transport-deficient yeast mutant 22Δ10α for expression. 22Δ10α lacks 10 amino acid transport genes and cannot grow using amino acids as its sole nitrogen source. Yeast strains including wild-type (23344c), 22Δ10α, yeast 22Δ10α mutant transformed with an empty vector (EV / 22Δ10α), and yeast 22Δ10α mutant transformed with CsCAT1 (CsCAT1 / 22Δ10α) were cultured on solid medium.

[0064] 1. Preparation of yeast competent cells.

[0065] The Frozen-EZ yeast competency preparation kit was purchased from Zymo Research.

[0066] (a) Take out the 22Δ10α yeast cells stored in an ultra-low temperature freezer at -80℃, streak them on YNB+U solid medium, and incubate at 30℃ for 3 days.

[0067] (b) Pick a single colony and place it on 10 mL of YNB+U liquid medium. Incubate at 200 rpm and 30°C until the culture temperature reaches OD. 600 It ranges from 0.8 to 1.0.

[0068] (c) Pipette 10 mL of bacterial culture into a centrifuge tube, centrifuge at 4000 rpm for 10 min, and discard the supernatant.

[0069] (d) Add 10 mL of EZ-1 Solution, vortex to remove the precipitate, centrifuge at 4000 rpm for 5 min, and discard the supernatant.

[0070] (e) Add 1 mL of EZ-2Solution to resuspend the bacterial cells, aliquot 100 μL into each tube, and store in an ultra-low temperature freezer at -80°C for later use.

[0071] 2. Yeast conversion.

[0072] The Frozen-EZ Yeast Transformation II kit was purchased from Zymo Research.

[0073] (a) Completely thaw yeast competent cells frozen at -80°C in ice.

[0074] (b) Add 1 μg plasmid DNA and 500 μL of EZ-3 Solution to 50 μL of competent yeast cells and shake gently.

[0075] (c) Incubate the competent cells at 30°C for 45 min, and take them out and mix them every 15 min.

[0076] (d) Take 200 μL of the activated bacterial solution and spread it evenly on YNB-U solid screening medium, and incubate at 30℃ for 3 days.

[0077] 3. Yeast spot analysis.

[0078] (a) Pick CsCAT1-pDR196 positive monoclonal cells and place them in 2 mL of YNB-U liquid culture medium. Incubate at 200 rpm and 30 °C for 3 days.

[0079] (b) Transfer 500 μL of bacterial culture to 10 mL of fresh YNB-U liquid medium and shake until OD is reached. 600 =0.8.

[0080] (c) Take 1 mL of the above bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 7000 g for 2 min, and discard the supernatant.

[0081] (d) Add 1 mL of sterile water to the centrifuge tube and wash 3 times. Then add 1 mL of sterile water to resuspend the bacterial cells.

[0082] (e) Dilute the bacterial solution with sterile water by 1, 10, 100 and 1000 times respectively.

[0083] (f) Take 2 μL of bacterial culture and spot it onto YNB-U solid medium containing 2 mM Thea, Gln, Ala, Glu, Asn, Leu, Phe, Ser, Thr, Asp, Trp, Gly, Lle, Tyr, Val, GABA, and Pro amino acids as the sole nitrogen source. Add ammonium sulfate as a control. Incubate at 30℃ upside down for 2-8 days and observe and take pictures.

[0084] (g) Take 2 μL of bacterial culture and spot it onto YNB-U solid medium with amino acids as the sole nitrogen source. Incubate at 30℃ with the medium inverted for 2-7 days. Observe and take pictures at different times according to the growth status.

[0085] 4. Yeast liquid culture and growth detection.

[0086] (a) Transgenic yeasts CsCAT1-pDR196 and pDR196 were cultured in YNB+NU liquid medium and cultured on a shaker at 200 rpm and 30°C until OD. 600 =0.8.

[0087] (b) Centrifuge the shaken yeast culture at 8000g for 2 minutes, discard the supernatant, wash three times with sterile water, and finally resuspend the precipitated yeast cells with sterile water. Adjust the concentration of the culture to OD using a UV spectrophotometer. 600 =1.5.

[0088] (c) YNB-NU liquid culture medium was used, with 0.2 mM and 2 mM theanine added as the sole nitrogen source. 100 μL of bacterial culture was added to 10 mL of fresh culture medium and incubated at 30°C and 200 rpm. OD was measured every 24 hours. 600 Once, the growth curve was recorded and fitted.

[0089] like Figure 2 As shown, the growth of CsCAT1 / 22Δ10α on solid medium with 2 mM theanine as the sole nitrogen source recovered to a level similar to that of the wild type. Both the control 22Δ10α and EV / 22Δ10α could grow normally on solid medium with 2 mM (NH4)2SO4 as the nitrogen source, and could not grow under conditions with 0N or theanine as the sole nitrogen source. This indicates that CsCAT1 has the activity of uptake and transport of theanine in yeast.

[0090] Similarly, 22Δ10α / CsCAT1 also showed good growth ability in liquid media with 0.2 and 2 mM theanine as the sole nitrogen source (e.g., Figure 3 (As shown). Regardless of the theanine concentration (0.2 mM or 2 mM), CsCAT1 / 22Δ10α exhibited exponential growth from day one to day eight, while EV / 22Δ10α showed almost no growth. The 2 mM concentration was superior to the 0.2 mM concentration, and better growth was observed at higher theanine concentrations, indicating that the growth of CsCAT1 / 22Δ10α is concentration-dependent. In conclusion, CsCAT1 can restore the phenotype of amino acid uptake deficiency in the 22Δ10α yeast strain, suggesting that CsCAT1 has the function of transporting theanine in tea plants.

[0091] IV. Relationship between CsCAT1 expression level in young tea leaves and the allocation of theanine between stems and leaves

[0092] 1. Analysis of theanine content in tender leaves and stems of different tea varieties

[0093] Tea plant tissue samples were placed in a mortar, liquid nitrogen was added, and the samples were ground thoroughly. The ground samples were then placed in an ultra-low temperature freeze dryer for 2 days to achieve constant weight. 0.05 g of the freeze-dried sample was weighed, and 3 mL of Watson's purified water was added. The mixture was thoroughly mixed by inverting the container. The sample was then extracted in a 98°C water bath for 20 min, inverting the container every 5 min. The extracted liquid was centrifuged at 13000 rpm for 10 min. The supernatant was collected, and the centrifugation was repeated once. 2 mL of the supernatant was collected, filtered through a 0.22 μm aqueous filter membrane, and used for HPLC analysis.

[0094] The Waters e2695 HPLC system with a 2489 UV / Vis detector, a reverse-flow C18 column (5 μm, 250 mm × 4.6 mm), a column temperature of 28 °C, and a detection wavelength of 210 nm was used. Mobile phase A was water, and mobile phase B was 100% acetonitrile. The gradient elution conditions were: 0 min, 100% A, 0% B; 7 min, 100% A, 0% B; 9 min, 40% A, 60% B; 15 min, 100% A, 0% B; 20 min, 100% A, 0% B, with a flow rate of 1 mL / min. The excitation wavelength for theanine detection was 210 nm.

[0095] To study the allocation of theanine in stems and leaves, the first leaf and the first stem below the first leaf of 12 tea varieties were first collected from the Guohe Tea Germplasm Resource Garden. Figure 4 (a) Figure 4 (b) The full names and abbreviations of these varieties are as follows: Figure 4 (c) is shown. Subsequently, the theanine levels in the young leaves of these 12 varieties were measured, as shown... Figure 5 As shown, the theanine content in the tender leaves of different tea varieties varies significantly, ranging from 2.8 mg / g to 10.5 mg / g. Among all tested varieties, 'Shuchazao' (SCZ) showed the lowest content at only 2.82 mg / g, while 'Benshan' (BS) had the highest at 10.47 mg / g. Furthermore, the theanine content in the tender leaves of these varieties differed by as much as 3.7 times.

[0096] Next, the theanine content in the first segment of tender stem tissue corresponding to the first leaf taken from these 12 varieties was tested. The results are as follows... Figure 6 As shown, there was little difference in the theanine content among the tender stems of different tea varieties, ranging from a minimum of 20.8 mg / g for 'Zhongcha102' (ZC102) to a maximum of 40.5 mg / g for 'Benshan' (BS), with a difference of less than 2 times. Meanwhile, the theanine content in the tender stems was significantly higher than that in the tender leaves; even the variety with the lowest theanine content in tender stems (ZC102) at 20.8 mg / g was much higher than the variety with the highest theanine content in tender leaves (BS) at 10.5 mg / g.

[0097] To more accurately assess and quantify the distribution of theanine between young stems and leaves of tea plants, a new indicator called the "stem-leaf content ratio" or "theanine stem-leaf ratio" has been introduced. Figure 7As shown in (a), the theanine stem-to-leaf ratio exhibited significant variability among different tea varieties, ranging from 8.8 to 3.3. Among all tested varieties, 'Shuchazao' (SCZ) showed an unusually high ratio of 8.83, while 'Sidacha 1' (SDC1) had the lowest ratio at only 3.29. Therefore, the difference between their respective ratios was as high as 2.7 times. Correlation analysis was also performed on the theanine stem-to-leaf ratio and the theanine content in leaves and stems of the 12 tea varieties. The results are as follows... Figure 7 As shown in (b), the theanine content in young leaves was significantly negatively correlated with the theanine ratio between stem and leaf, with a correlation coefficient of -0.89, which was significantly higher than the correlation coefficient of -0.70 between theanine in stem and the stem-leaf ratio.

[0098] 2. Correlation analysis between amino acid transporter expression levels and theanine content

[0099] like Figure 8 As shown, the expression trend of CsCAT1 in the tender stems of different varieties is very similar to that of theanine in the stems and leaves of these varieties. That is, as the ratio of theanine in stems and leaves decreases among different varieties, the expression level of CsCAT1 also shows a decreasing trend.

[0100] Subsequently, correlation analysis was performed on the gene expression levels of differentially expressed amino acid transporters in these 11 varieties with theanine content and stem-to-leaf ratio in young stems and leaves to reveal the relationship between gene expression in young stems and theanine content in new shoots. The results showed that CsCAT1 expression was significantly positively correlated with the theanine content in young stems and leaves (r = 0.97; p < 0.0001). Simultaneously, CsCAT1 expression was also significantly negatively correlated with theanine content in young leaves (r = -0.79; p < 0.01) and significantly negatively correlated with theanine content in young stems (r = -0.64; p < 0.05). These results suggest that CsCAT1 may play a role in regulating theanine distribution between young leaves and young stems.

[0101] V. In situ PCR of tea trees

[0102] To further elucidate the role of CsCAT1 in the transport and distribution of theanine, its expression in different cell types within young stems was investigated. Because its expression in young stem tissues showed a strong correlation with the theanine content in the stem / leaf ratio, young stems were chosen as the research subject. In situ RT-PCR was used to observe the expression of CsCAT1 in young stems. These young stem tissue sections underwent reverse transcription and amplification using CsCAT1 gene-specific primers to detect CsCAT1 expression, with a negative control used for those without reverse transcription primers.

[0103] 1. Organizational preparation.

[0104] Perform the procedure on ice. Take a 1cm tender stem from a tea seedling and immediately place it into a fresh, ice-cold Eppendorf tube containing 5mL of FAA fixative. To improve tissue penetration of the fixative, puncture the Eppendorf tube cap with a syringe needle, insert it into the air gap, and gently aspirate for 3 minutes. Seal the Eppendorf tube cap with sealing film and incubate at 4°C for 12 hours.

[0105] 2. Tissue embedding section.

[0106] Operate on ice. No rnase or treated with DEPC.

[0107] (a) Remove the fixative and replace it with 70% ethanol and 5% acetic acid three times, 10 minutes each time. Wash the paper towel twice with 1×PBS, 5 minutes each time.

[0108] (b) Dissolve low melting point agarose (VWR) in 1×PBS to prepare a 5% agar solution. Heat the solution in a microwave oven until boiling and mix well. Place the tissue material in an embedding cassette. When the agarose solution is about to solidify, pour it into the embedding cassette. The material can be stored at 4°C for 24 hours.

[0109] (c) Using a Leica RM2255 microscope (Germany), cut 50 μm thick sections of the material, place them on coverslips, and cover the material with a drop of Sample Protector for RNA / DNA containing RNase inhibitors and 1% PVP solution. Remove the agar, select intact tissues under a stereomicroscope, and transfer the selected tissues and solution to 50 μL Eppendorf tubes using a pipette tip with the tip removed.

[0110] 3. In situ RT-PCR.

[0111] Operate on ice. No RNase or treated with DEPC.

[0112] (a) Wash the sections twice with PBS.

[0113] (b) Aspirate the solution, add 15 μg / mL proteinase K, heat at 55°C for 1 hour, and then heat at 95°C for 2 minutes to inactivate the proteinase K.

[0114] (c) Aspirate the solution. Using the PrimeScript™ II Reverse Transcription Kit, perform reverse transcription with random primers according to the manufacturer's instructions. Remove the reaction solution and wash once with water. The reaction without reverse transcription primers serves as a control (ultrapure water is used instead of primers here).

[0115] (d) Amplify PCR using Taq DNA polymerase, using slide material as a template. The Taq DNA polymerase reaction mixture should be prepared in EP tubes. The mixture contains: 2 μL 10X ThermoPol buffer, 1.6 μL 2.5 mM dNTPs, 0.1 μL Taq DNA polymerase, 0.32 μL 25 mM digoxigenin 11-duTP, 1 μL each of the 10 μM primers, and 14 μL water. Primer design should follow the guidelines for quantitative expression primer design.

[0116] (e) Cyclic reaction settings: 95℃, 5 min; 35 PCR cycles (95℃ 30 s, 57℃ 60 s, 68℃ 30 s); finally extended at 68℃ for 5 min. Primers are the same as the CsCAT1 primers in Table 1.

[0117] 4. Testing.

[0118] All steps were performed at room temperature using freshly prepared buffer solutions.

[0119] (a) Wash sections twice with 1×PBS for 5 minutes. Transfer the material to a 96-well cell culture plate. Add 100 μL of blocking buffer with 1% PVP for 30 minutes.

[0120] (b) Alkaline phosphatase antibody was added to blocking buffer at a ratio of 1:200 and incubated for 1 hour.

[0121] (c) Wash twice with 1×washing buffer, 15 minutes each time.

[0122] (d) Stain with BM Puple AP in the dark for 30 min, wash twice with water, observe the staining and take pictures with a stereomicroscope (Zeiss RM2255, Germany).

[0123] like Figure 9 As shown, CsCAT1 exhibits high expression levels in xylem parenchyma cells (particularly medullary ray parenchyma) and phloem parenchyma cells. This suggests that CsCAT1 contributes to theanine storage in these tissues, while simultaneously providing a nitrogen source by introducing theanine into these cells.

[0124] VI. Transient Oligonucleotide Inhibition Experiment of Tea Tree Genes

[0125] CsCAT1 antisense probes were designed using the Solido online website (http: / / sfold.wadsworth.org / cgi-bin / soligo.pl). Nucleotide sequences with low free energy and stable secondary structures were selected. The target gene sequence was compared with the tea plant genome, and sequences significantly different from other genes were chosen as the antisense strand (asODN) probes, while the reverse complementary nucleic acid sequence was chosen as the sense strand (sODN) probe. The sODN sequence is 5'-AACCCGAAAACTTCCGCATC-3', and the asODN sequence is 5'-GATGCGGAAGTTTTCGGGTT-3'. The Solido-specific sequences were synthesized by Sangon Biotech and diluted with water to a concentration of 20 μM.

[0126] New shoots of tea trees with uniform growth and free from pests and diseases were selected. Only the first leaf and the first stem were cut as experimental material and inserted into 200 μL centrifuge tubes containing 100 μL of probe. The control group was treated with a positive-strand probe. The centrifuge tubes were clearly labeled and placed in a foam box covered with plastic wrap. After 24 hours of treatment, leaves and stems were collected separately for subsequent qRT-PCR and theanine content determination. Six biological replicates were performed.

[0127] To investigate the role of CsCAT1 in the distribution of theanine in tea plants, the first shoot of new shoots was treated with a CsCAT1-specific antisense oligonucleotide (asODN), while the corresponding sense oligonucleotide (sODN) served as a control. Figure 10 As shown in (a). The results are as follows. Figure 10 As shown in (b), compared with the sODN control treatment, the asODN treatment significantly reduced CsCAT1 expression in young stems. Therefore, under these conditions, the stem / leaf ratio of theanine decreased significantly, reaching only 60% of that in the sODN treatment. The theanine content in young leaves increased, reaching 1.5 times that in the sODN treatment. Meanwhile, as... Figure 10 As shown in (c), the theanine content in the tender stems showed a decreasing trend. This indicates that CsCAT1 can regulate the distribution of theanine between the tender leaves and tender stems of tea plants. The asODN treatment significantly reduced the expression of CsCAT1 in the tender stems, resulting in a decrease in theanine content in the tender stems, while the theanine content in the tender leaves significantly increased, and the theanine ratio in the stems to leaves decreased significantly.

[0128] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. Application of CsCAT1 in regulating the distribution of theanine between new shoots and leaves of tea plant, characterized in that, By antisense strand probe asODN treatment, the expression of CsCAT1 in the tender stems is inhibited or reduced, which can make the theanine stem to leaf ratio decrease extremely significantly, the theanine content in the tender stems decrease, and the theanine content in the tender leaves increase significantly, the base sequence of the CsCAT1 is shown as SEQ ID NO. 1; the sequence of the antisense strand probe asODN is 5'-GATGCGGAAGTTTTCGGGTT-3'.

2. The use of CsCATl to regulate the distribution of theanine among the new shoots and leaves of tea plants according to claim 1, characterized in that, The protein sequence of the CsCAT1 is shown as SEQ ID NO. 2.

Images