Application of CsCAT1 to regulation and control of theanine in tea tree shoot stem-leaf distribution

Abstract

The invention relates to the technical field of gene engineering, in particular to application of CsCAT1 to regulation and control of theanine in tea tree shoot stem-leaf distribution, the stem-leaf ratio of theanine in a tea tree is reduced by reducing the expression quantity of CsCAT1 in tender stems, the base sequence of CsCAT1 is as shown in SEQ ID NO.1, the stem-leaf ratio of theanine in the tea tree is reduced by reducing the stem-leaf ratio of theanine in the tea tree, and the stem-leaf ratio of theanine in the tea tree is reduced by reducing the expression quantity of CsCAT1 in tender stems. Therefore, the content of theanine in the tender leaves of the tea trees is increased, and the quality of the tea leaves is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of CsCAT1 in regulating theanine distribution among new shoots, stems and leaves of tea trees. Background Art

[0002] Theanine, the full name of which is N-ethyl-γ-L-glutamine, is the main ingredient of umami in green tea. It can balance the astringency and bitterness brought by catechins and caffeine. Theanine is a non-protein amino acid unique to tea trees. In the new shoots of tea trees used for tea production, theanine is the most abundant free amino acid, accounting for 40%-70% of the total free amino acids. Theanine accounts for 1%-2% of the dry weight of tea leaves, and in premium green teas, this proportion can even exceed 4%.

[0003] Theanine is mainly synthesized in the roots in 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 part of theanine is distributed in the stems. Therefore, in these new shoots, the theanine content in the young stems is more than twice that in the young leaves compared to the young leaves. However, in the process of tea production, only a small part of the stems are used, or not used at all. For example, the famous green tea brand Lu'an Guapian only uses single leaves from the new shoots. Therefore, a considerable part of theanine in the stems is still not fully utilized. In view of this, increasing the distribution ratio of theanine 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 between different tissues. Membrane-localized amino acid transporters facilitate the transmembrane transport of amino acids. Previous results have shown that six members of amino acid transporters in tea plants, namely CsAAP1, CsAAP3, CsAAP4, CsAAP5, CsAAP6, and CsAAP8, are able to transport theanine. Among these transporters (CsAAPs), CsAAP1 mediates the transport of theanine from roots to branches by recycling theanine from the exoplasmic pathway and facilitating its loading into the xylem. Recently, CsAAP7.2 was shown to have the ability to absorb theanine and promote the long-distance transport of theanine in tea plants. In addition, CsCAT2, a member of the cationic amino acid transporter (CAT) family, is also able to transport theanine and may facilitate its entry into the vacuole for storage in the roots of tea plants. In addition, a member of the LHT family of tea plants exhibits the ability to transport theanine. However, its physiological function in tea plants remains unknown. At the same time, these theanine transporters 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 content of theanine in the stem tissue of tea trees is more than three times that in the leaves. However, only part of the stem is taken during tea picking, or no stem is taken. By studying the mechanism of theanine distribution between the tender stems and tender leaves of tea tree shoots, the accumulation of theanine in tender leaves can be increased. In the new shoots of tea trees, the transporter responsible for the distribution of theanine between tender stems and tender leaves has not yet been identified. In addition, in other plants, including model plants, no amino acid transporter that mediates the distribution of amino acids between stems and leaves has been identified. Therefore, how to increase the content of theanine in tea leaves by regulating the distribution of theanine in the stems and leaves of tea tree shoots has become an urgent problem to be solved.

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

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

[0008] In order to achieve the above object, the present invention discloses the application of CsCAT1 in regulating the distribution of theanine among stems and leaves of new shoots of tea plants, and reduces the stem / leaf ratio of theanine in tea plants by reducing the expression level of CsCAT1 in tender stems. 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 the CsCAT1 is shown in SEQ ID NO.2.

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

[0013] .

[0014] The expression level of CsCAT1 in tender stems is significantly negatively correlated with the content of theanine in tender leaves and tender stems, and is significantly positively correlated with the ratio of theanine 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 theanine content in tender stems decreases, the theanine content in tender leaves significantly increases, and the theanine stem-to-leaf ratio extremely significantly decreases, thereby regulating the distribution of theanine between the stems and leaves of new shoots of tea trees through CsCAT1, and increasing the content of amino acids in tea leaves. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 To complement the growth phenotype of CsCAT1 yeast on theanine and control medium, Note: CsCAT1 transgenic yeast was grown on YNB medium with 2mM theanine, (NH4)2SO4 and 0N as the sole nitrogen source. 22Δ10α is a yeast mutant deficient in amino acid absorption, EV / 22Δ10α is a 22Δ10α strain containing no vector, and CsCAT1 / 22Δ10α is a transgenic 22Δ10α strain;

[0018] Figure 3 To complement the growth rate of CsCAT1 yeast on theanine as the nitrogen source, note: The OD values ​​of EV / 22Δ10α and CsCAT1 / 22Δ10α were calculated by shaking the culture medium with 0.2 and 2 mM theanine as the sole nitrogen source. 600 Bacterial solution concentration;

[0019] Figure 4 To collect tissue samples for theanine analysis, including: (a) tea trees in the tea garden used for sampling; (b) new shoots of tea trees and sampling parts; (c) abbreviations and full names of 12 tea tree varieties;

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

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

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

[0023] Figure 8 The expression of CsCAT1 in tender stems of different tea varieties;

[0024] Fig. 9This is the tissue localization of CsCAT1 in the tender stems of tea plants. Note: The stems of tea plants were taken for in situ PCR experiments. The blue signal indicates the expression position of CsCAT1. The control was made with no reverse transcription primer. Scale bar: 250μm for the upper layer and 600μm for the lower layer;

[0025] Fig.10 The effect of transient inhibition of CsCAT1 expression in stems on theanine distribution, including: (a) treatment of the first stem of tea plants with sODN-CsCAT1 and asODN-CsCAT1; (b) expression of CsCAT1 in stems of tea plants treated with sODN-CsCAT1 and asODN-CsCAT1; (c) changes in theanine content in stems and leaves of tea plants after treatment with sODN-CsCAT1 and asODN-CsCAT1; asterisks above the error bars indicate significant differences by Student's t test (*, p≤0.05; **, p≤0.01). DETAILED DESCRIPTION

[0026] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0027] 1. Construction of expression vector

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

[0029] 2. Gel product recovery: For the experimental method, refer to the instruction manual of the “Gel Recovery Kit (AxyGEN, Hangzhou)”.

[0030] 3. pEASY vector ligation.

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

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

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

[0034] 4. E. coli transformation.

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

[0036] (b) The mixture was placed in an ice bath for 30 min.

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

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

[0039] (e) Add 500 μL of LB medium without antibiotics and incubate at 37°C in a shaker for 1 h.

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

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

[0042] 5. Colony PCR verification.

[0043] 6. Enzyme digestion of empty vector and sequencing plasmid.

[0044] 7. Connect the enzyme-digested fragment to the empty vector.

[0045] 8. Transform competent E. coli.

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

[0047] Table 2 Vector construction primers

[0048]

[0049]

[0050] 2. Subcellular localization detection of transient expression in tobacco epidermis

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

[0052] 1. Instant tobacco:

[0053] (a) Pick a single colony of Agrobacterium tumefaciens in 500 μL LB liquid containing 20 μg / mL Rif and 50 μg / mL Kan, and culture at 28°C with shaking until turbidity. Then, expand the culture liquid at a ratio of 1:100 for 2 days, take an appropriate amount of the culture liquid and store it at -80°C with an equal amount of 50% glycerol.

[0054] (b) Take 5 mL of bacterial solution and centrifuge at 6000g for 10 min at room temperature. Pour off the liquid to collect the bacteria, resuspend them in MMA and centrifuge them again.

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

[0056] (d) When injecting Nicotiana benthamiana, be sure to inject a whole leaf at a time. No more than three leaves should be injected per Nicotiana benthamiana plant.

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

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

[0059] (a) Preparation. Take a clean glass slide and drop a drop of pure water in the middle of the slide. Cut a small piece of the leaf where the injection site is located and place it in the pure water on the slide. Use tweezers to pick up a cover glass and cover the leaf from one end to remove excess water and air.

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

[0061] Given that the homologous gene AtCAT1 of CsCAT1 in Arabidopsis is localized to the plasma membrane, it is speculated that CsCAT1 is also likely to be localized to the plasma membrane. In order to observe the subcellular localization, CsCAT1-eGFP and the plasma membrane marker pm-rK-CD3-1007 were expressed in tobacco epidermal cells. It was observed that CsCAT1-eGFP exhibited green fluorescence, which almost overlapped with the red fluorescence of the plasma membrane marker ( Figure 1 ). This result confirmed the localization of CsCAT1 on the plasma membrane and suggested that CsCAT1 was involved in the trans-plasma membrane transport of amino acids in tea plants.

[0062] 3. Yeast Transformation and Analysis

[0063] In order to study the transport activity of CsCAT1 for theanine, CsCAT1 was expressed in the amino acid transport-deficient yeast mutant 22Δ10α, which lacks 10 amino acid transport genes and cannot grow with amino acids as the sole nitrogen source. Yeast strains including wild type (23344c), 22Δ10α, yeast 22Δ10α mutant transformed with 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 yeast competent cell preparation kit Frozen-EZ was purchased from Zymo Research.

[0066] (a) The 22Δ10α yeast cells stored in a -80°C ultra-low temperature freezer were taken out, streaked onto YNB+U solid medium, and cultured at 30°C for 3 days.

[0067] (b) Pick a single colony and place it on 10 mL of YNB+U liquid medium and shake at 200 rpm and 30°C until the OD 600 It is 0.8-1.0.

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

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

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

[0071] 2. Yeast transformation.

[0072] The yeast transformation kit Frozen-EZ Yeast TransformationⅡ was purchased from Zymo Research.

[0073] (a) Completely thaw the competent yeast cells stored in a -80°C freezer in ice.

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

[0075] (c) The competent cells were cultured at 30°C for 45 min and mixed every 15 min.

[0076] (d) 200 μL of activated bacterial solution was evenly spread on the solid screening medium of YNB-U and cultured at 30°C for 3 days.

[0077] 3. Yeast dot analysis.

[0078] (a) A positive single clone of CsCAT1-pDR196 was picked and placed in 2 mL of YNB-U liquid culture medium and shaken at 200 rpm and 30°C for 3 days.

[0079] (b) Pipette 500 μL of bacterial solution into 10 mL of fresh YNB-U liquid medium and shake until OD 600 =0.8.

[0080] (c) Transfer 1 mL of the above bacterial solution 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 three times. Then add 1 mL of sterile water to resuspend the 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 solution and spot it on YNB-U solid culture medium with 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, and culture at 30°C inverted for 2-8 days. Observe and take pictures.

[0084] (g) Take 2 μL of bacterial solution and spot it on YNB-U solid medium with amino acids as the only nitrogen source, incubate it upside down at 30°C for 2-7 days, and observe and take pictures on different days according to the growth status.

[0085] 4. Shake the yeast liquid to detect its growth.

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

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

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

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

[0090] Similarly, 22Δ10α / CsCAT1 also showed good growth ability in liquid medium with 0.2 and 2 mM theanine as the sole nitrogen source (e.g. Figure 3 ). Regardless of theanine concentration of 0.2mM or 2mM, CsCAT1 / 22Δ10α showed an exponential growth curve from the first to the eighth day, while EV / 22Δ10α barely grew. The 2mM concentration was superior to the 0.2mM concentration, and better growth was observed at higher concentrations of theanine, indicating that the growth of CsCAT1 / 22Δ10α was dependent on theanine concentration. In summary, CsCAT1 was able to restore the amino acid absorption defect phenotype of the 22Δ10α yeast strain, indicating that CsCAT1 has the function of transporting theanine in tea plants.

[0091] 4. Relationship between CsCAT1 expression in tea leaves and theanine distribution in stems and leaves

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

[0093] Place the tea tree tissue sample in a mortar, add liquid nitrogen and grind thoroughly. Place the ground sample in an ultra-low temperature freeze dryer for 2 days and freeze-dry to constant weight. Weigh 0.05g of freeze-dried sample, add 3mL of Watsons purified water, and mix thoroughly by turning upside down. Soak the sample in a 98℃ water bath for 20 minutes, turning it upside down every 5 minutes. Centrifuge the extracted liquid at 13000rpm for 10 minutes. Take out the supernatant and repeat the centrifugation once. Take 2mL of supernatant, filter with a 0.22μm aqueous filter membrane, and use for HPLC detection.

[0094] A Waters e2695 HPLC system with a 2489 UV / Vis detector was used, with a reverse C18 column (5 μm, 250 mm×4.6 mm), a column temperature of 28°C, and a detection wavelength of 210 nm. 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, and the excitation wavelength for theanine detection was 210 nm.

[0095] In order to study the distribution of theanine in stems and leaves, the first leaves and the first stems below the first leaves of 12 tea varieties were collected in the Guohe tea germplasm resource nursery, such as Figure 4 (a) Figure 4 (b) The full names and abbreviations of these varieties are as follows: Figure 4 (c) As shown. Subsequently, the levels of theanine in the young leaves of these 12 varieties were tested. Figure 5 As shown in the results, there are significant differences in the content of theanine in young leaves among different tea varieties, ranging from 2.8 mg / g to 10.5 mg / g. Among all the tested varieties, 'Shuchazao' (SCZ) showed the lowest content, only 2.82 mg / g, while 'Benshan' (BS) was relatively the highest, at 10.47 mg / g. At the same time, the difference in theanine content in young leaves of these varieties was as high as 3.7 times.

[0096] Then, the theanine content in the first section of tender stem tissue corresponding to the first leaf of these 12 varieties was tested. Figure 6 As shown, the performance difference between the tender stems of different tea tree varieties is not large, ranging from the lowest 'Zhongcha102' (ZC102) 20.8 to the highest 'Benshan' (BS) 40.5 mg / g, and the difference between them is less than 2 times. At the same time, it can be seen that the content of theanine in the tender stems is significantly higher than that in the tender leaves. Even the variety with the lowest theanine content in the tender stems (ZC102) 20.8 mg / g is much higher than the variety with the highest theanine content in the tender leaves (BS) 10.5 mg / g.

[0097] In order to more accurately evaluate and quantify the distribution of theanine between young stems and young leaves of tea plants, a new indicator called "stem-to-leaf content ratio" or "theanine stem-to-leaf ratio" was introduced. Figure 7As shown in (a), the theanine stem-to-leaf ratio showed great variability among different tea varieties, ranging from 8.8 to 3.3. Among all the tested varieties, 'Shuchazao' (SCZ) showed an abnormally high ratio of 8.83, while 'Sidacha 1' (SDC1) was relatively the lowest at only 3.29. Therefore, the difference between their respective ratios was as high as 2.7 times. At the same time, the correlation analysis of the theanine stem-to-leaf content ratio and the theanine content of leaves and stems of 12 tea varieties was carried out. The results are shown in Figure 7 As shown in (b), the theanine content in young leaves is extremely significantly negatively correlated with the theanine stem-to-leaf ratio, with a correlation coefficient of -0.89, which is significantly higher than the correlation coefficient of -0.70 between theanine in stems and the stem-to-leaf ratio.

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

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

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

[0101] 5. Tea tree in situ PCR

[0102] To further elucidate the role of CsCAT1 in theanine transport and distribution, its expression in different cell types within tender stems was investigated. Tender stems were chosen as the study object because its expression in tender stem tissues was strongly correlated with the stem / leaf ratio of theanine content. In situ RT-PCR was used to observe CsCAT1 expressing cells within tender stems. These tender stem tissue sections were reverse transcribed and amplified with CsCAT1 gene-specific primers to detect CsCAT1 expression, with no reverse transcription primer added as a negative control.

[0103] 1. Organizational preparation.

[0104] Operate on ice. Take 1cm of young stems of tea seedlings and immediately put them into a fresh, iced Eppendorf tube filled with 5mL FAA fixative and refrigerate. Poke the cap of the Eppendorf tube with a syringe needle, insert it into the air, and gently pump air for 3 minutes to increase the penetration of the fixative into the tissue. Seal the cap of the Eppendorf tube with a sealing film and place it at 4℃ for 12 hours.

[0105] 2. Tissue embedding and sectioning.

[0106] Work on ice. RNase-free or DEPC-treated.

[0107] (a) Remove the fixative and replace 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 make a 5% agar solution. Heat in a microwave oven until boiling and mix well. Place the tissue material in an embedding box. When the agarose solution is about to condense, pour it into the embedding box. The material can be stored at 4°C for 24 hours.

[0109] (c) The material was cut into 50 μm thick slices by a microscope (Leica RM2255, Germany), placed on a cover glass, and covered with a drop of Sample protector for RNA / DNA with 1% pvp solution containing RNase inhibitor. The agar was peeled off, and the intact tissue was selected under a stereomicroscope. The selected tissue and solution were transferred to a 50 μL Eppendorf tube using a pipette tip with the top removed.

[0110] 3. In situ RT-PCR.

[0111] Work on ice. RNase-free or DEPC-treated.

[0112] (a) Sections were washed twice with PBS.

[0113] (b) The solution was aspirated, 15 μg / mL proteinase K was added and heated at 55°C for 1 hour, and then heated at 95°C for 2 minutes to inactivate proteinase K.

[0114] (c) Aspirate the solution. Perform reverse transcription with random primers using the PrimeScriptTMII Reverse Transcription Kit according to its instructions. Remove the reaction solution and wash once with water. The reaction without reverse transcription primer is the control reaction (here ultrapure water is used instead of primer).

[0115] (d) Amplify PCR using Taq DNA polymerase, using the slice material as a template. The Taq DNA polymerase reaction mixture should be prepared in an EP tube. The mixture contains: 2 μL of 10X ThermoPol buffer, 1.6 μL of 2.5 mM dNTPs, 0.1 μL of Taq DNA polymerase, 0.32 μL of 25 mM digoxigenin 11-duTP, 1 μL of 10 μM primers, and 14 μL of water. The design of primers refers to the design of expression quantification primers.

[0116] (e) Cycling reaction settings: 95°C, 5 min; 35 PCR cycles (95°C 30 s, 57°C 60 s, 68°C 30 s); final extension at 68°C for 5 min. Primers are the same as those in Table 2.1 for CsCAT1 primers.

[0117] 4. Detection.

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

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

[0120] (b) Alkaline phosphatase antibody was added to the blocking buffer at a dilution of 1:200 and allowed to stand for 1 h.

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

[0122] (d) The samples were stained with BM Puple AP in the dark for 30 min, washed twice with water, and the staining was observed and photographed under a stereo microscope (Zeiss RM2255, Germany).

[0123] like Fig. 9 As shown, CsCAT1 showed high expression levels in xylem parenchyma (especially medullary ray parenchyma) and phloem parenchyma cells, suggesting that CsCAT1 contributes to theanine storage in these tissues and also provides a nitrogen source by importing theanine into these cells.

[0124] VI. Tea Tree Gene Transient Oligonucleotide Inhibition Experiment

[0125] The CsCAT1 antisense probe was designed using the Soligo online website (http: / / sfold.wadsworth.org / cgi-bin / soligo.pl). Nucleotide sequences with low free energy and stable secondary structure were selected, and the target gene sequence was compared with the tea plant genome. The sequence with large differences from other genes was selected as the antisense (asODN) probe, and the reverse complementary nucleic acid sequence was used as the sense (sODN) probe. The sODN sequence was 5'-AACCCGAAAACTTCCGCATC-3', and the asODN sequence was 5'-GATGCGGAAGTTTTCGGGTT-3'. The Oligo specific sequence was sent to Biotech for synthesis and diluted with water to a concentration of 20 μM.

[0126] Select new shoots of tea trees with consistent growth and no pests and diseases, cut only the first leaf and the first stem as experimental materials, and insert them into a 200μL centrifuge tube containing 100μL probe. The control group is treated with a positive chain probe. The centrifuge tubes are marked and placed in a foam box covered with plastic wrap. After 24 hours of treatment, the leaves and stems are collected as materials 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 stem of new shoots was treated with CsCAT1-specific antisense oligonucleotides (asODNs), while its corresponding sense oligonucleotides (sODNs) were used as controls. Fig.10 (a). The results are shown in Fig.10 As shown in (b), asODN treatment significantly reduced CsCAT1 expression in young stems compared with sODN control treatment. Therefore, under these conditions, the stem / leaf ratio of theanine decreased significantly, and was only 60% of that in sODN treatment after asODN treatment. The content of theanine in young leaves increased, and was 1.5 times that in sODN treatment. At the same time, Fig.10 As shown in (c), the theanine content in the young stems showed a downward trend. It can be seen that CsCAT1 can regulate the distribution of theanine between the young leaves and young stems of tea trees. AsODN treatment significantly reduced the expression of CsCAT1 in young stems, the theanine content in young stems decreased, the theanine content in young leaves increased significantly, and the theanine stem-to-leaf ratio decreased significantly.

[0128] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. Application of CsCAT1 in regulating theanine distribution among new shoots, stems and leaves of tea trees, characterized in that: The stem / leaf ratio of theanine in tea plants is reduced by reducing the expression level of CsCAT1 in young stems, wherein the base sequence of CsCAT1 is shown in SEQ ID NO.

1.

2. The use of CsCAT1 to regulate theanine distribution among new shoots, stems and leaves of tea plants as claimed in claim 1, characterized in that: The protein sequence encoded by CsCAT1 is shown in SEQ ID NO.

2.

3. The use of CsCAT1 to regulate theanine distribution among new shoots, stems and leaves of tea plants as claimed in claim 1, characterized in that: The expression level of CsCAT1 in tender stems is significantly negatively correlated with the content of theanine in tender leaves and tender stems, and is significantly positively correlated with the ratio of theanine between stems and leaves.

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