Application of CsSPX3 and CsPHL7 genes in regulating theanine synthesis in tea plants and improving the quality of tea leaves of tea plants

Abstract

The present invention provides the applications of CsSPX3 and CsPHL7 genes in regulating theanine synthesis in tea plants and improving the quality of tea leaves of tea plants, belonging to the field of plant molecular biotechnology. The present invention provides the application of the CsPHL7 gene in regulating theanine synthesis in tea plants, as well as the application of the CsSPX3 gene in synergistically regulating theanine synthesis in tea plants with the CsPHL7 gene. The tea plant gene CsPHL7 screened in the present invention can bind to the promoters of theanine synthase TS1 and GS1 genes alone or interact with CsSPX3 to inhibit the accumulation of theanine content in tea plants. After verification, silencing the above genes by virus-induced gene silencing technology can increase the theanine content in tea plants, and overexpressing the above genes by Agrobacterium-mediated transient transformation technology can reduce the theanine content in tea plants, which can be used to regulate theanine synthesis in tea plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology, and particularly relates to the application of CsSPX3 and CsPHL7 genes in regulating theanine synthesis in tea plants and improving tea quality. Background Art

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is an important economic crop in my country, and its growth is closely linked to the essential element phosphorus. Phosphorus plays a crucial regulatory role in the formation of numerous compounds within the tea plant, including theanine, catechins, tea polyphenols, caffeine, and anthocyanins. Theanine, a characteristic amino acid in tea, exists in free form, accounting for 40% to 70% of the total free amino acids in tea. It is the primary component responsible for the sweetness and lubricating properties of tea. It can be used to inhibit the stimulant effects of caffeine, improve learning and memory, and also has anti-blood pressure, anti-tumor, and anti-fatigue effects. Theanine, a key secondary metabolite in tea, directly impacts tea quality. Both phosphorus deficiency and excess are correlated with metabolites. Phosphorus stress reduces the synthesis of flavonoids and phosphorylated metabolites, while excess phosphorus can lead to an imbalance in the chemical composition of tea, impairing the accumulation of beneficial components such as theanine.

[0003] Existing studies have shown that SPX proteins occupy a very important position in the phosphorus signaling network and are closely related to phosphorus uptake, transport, storage and homeostasis. SPX genes can affect the transcription of downstream low-phosphorus response genes PSI (PSR) by regulating PHR activity. For example, maize ZmSPX genes (except ZmSPX3) and wheat TaSPX genes are significantly induced by low-phosphorus stress, Arabidopsis AtSPX1 and AtSPX2 accumulate in branches and roots under phosphorus starvation, and rice OsSPX1 is a suppressor of PHR2 transcriptional activity. Most studies focus on the relationship between SPX proteins and phosphorus stress and their related genes. There are no studies on the joint regulation of theanine synthesis in tea plants by SPX proteins and other proteins. The molecular regulatory network mechanism behind theanine synthesis in tea plants under inorganic phosphate limitation is also unclear. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of two genes, CsSPX3 and CsPHL7, in regulating theanine synthesis in tea plants.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of the CsPHL7 gene in regulating theanine synthesis in tea plants.

[0007] The present invention provides the application of CsSPX3 gene in coordinating with CsPHL7 gene in regulating theanine synthesis in tea plants.

[0008] Preferably, the nucleotide sequence of the CsSPX3 gene is shown in SEQ ID NO.1;

[0009] The nucleotide sequence of the CsPHL7 gene is shown in SEQ ID NO.2.

[0010] The amino acid sequence of the protein encoded by the CsSPX3 gene is shown in SEQ ID No. 3;

[0011] The amino acid sequence of the protein encoded by the CsPHL7 gene is shown in SEQ ID No.4.

[0012] Preferably, the method for regulating theanine synthesis in tea plants using the CsSPX3 gene and / or the CsPHL7 gene comprises: constructing a recombinant vector of CsSPX3 and / or CsPHL7, and transferring the recombinant vector into tea leaves to silence or transiently overexpress the CsSPX3 gene and / or CsPHL7, thereby promoting or inhibiting theanine synthesis in tea plants;

[0013] Among them, the CsSPX3 gene and CsPHL7 gene inhibit the expression of the CsTS1 gene and CsGS1 gene by binding to the CsTS1 and CsGS1 gene promoters, thereby inhibiting the synthesis of theanine in tea trees.

[0014] Preferably, the gene silencing vector is based on pTRV2 as the original vector, and the CsSPX3 gene or CsPHL7 gene is inserted into the multiple cloning site of pTRV2, and the insertion position is located between the EcoRI and BamHI restriction sites on the original vector pTRV2. The resulting gene silencing vectors are named pTRV2-CsSPX3 and pTRV2-CsPHL7, respectively;

[0015] The overexpression recombinant vector is pSH737-35S as the original vector, and CsSPX3 or CsPHL7 is inserted into the multiple cloning site of pSH737-35S. The insertion position is located between the Xba I and Kpn I restriction sites on the original vector pSH737-35S. The inserted overexpression recombinant vectors are named pSH737-35S-CsSPX3 and pSH737-35S-CsPHL7, respectively.

[0016] Preferably, in the process of constructing the recombinant vector, the primer pairs for the CsSPX3 gene and the CsPHL7 gene are:

[0017] CsSPX3:primer F:GCGTTCACTACTCACACCGA;

[0018] primer R: ACATGAACTCAGCCTCAGCC;

[0019] CsPHL7: primer F: AGCTGATGGTTCTAAAGATCAGAAG;

[0020] Primer R: GGAACTGTCTCGGAGGCTTT.

[0021] The present invention also provides the application of the CsPHL7 gene in improving the quality of tea leaves from tea trees.

[0022] The present invention also provides the use of the CsSPX3 gene in conjunction with the CsPHL7 gene in improving the quality of tea leaves from tea trees.

[0023] Beneficial effects:

[0024] The present invention provides two tea tree genes CsSPX3 and CsPHL7 that are involved in the regulation of theanine synthesis in tea trees. The present invention uses Agrobacterium-mediated transient transformation technology to overexpress CsSPX3 and CsPHL7 to reduce the theanine content of tea trees, and through dual luciferase (LUC) and electrophoretic mobility shift assay (EMSA) experiments, it is proved that SPX3 interacts with PHL7 and binds to theanine synthase TS1 and GS1 gene promoters to inhibit the expression of TS1 and GS1 by additive effect, thereby inhibiting theanine synthesis and accumulation; the present invention uses virus-induced gene silencing technology to silence CsSPX3 and CsPHL7 to increase the theanine content of tea trees. Compared with the control plants, the theanine content in the silenced plants increased by 1.58 to 236 times. In summary, the present invention found that the CsPHL7 gene can inhibit theanine synthesis alone or by interacting with the CsSPX3 gene. After silencing the CsSPX3 and CsPHL7 genes, the theanine content of tea trees can be significantly increased, and can be used to regulate the synthesis of theanine in tea trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The content of theanine in the roots and leaves of tea plants treated with phosphorus for 20 days was detected by HPLC.

[0026] Figure 2 The phosphorus content in the roots and leaves of tea plants was determined by the molybdenum antimony colorimetric method after the tea plants were treated with phosphorus for 20 days.

[0027] Figure 3 Transcriptome sequencing was used to determine the transcription levels of phosphorus-responsive genes and theanine-related genes in tea plants.

[0028] Figure 4 Real-time fluorescence quantitative PCR (qRT-PCR) analysis of leaves.

[0029] Figure 5 Root quantitative real-time PCR (qRT-PCR) analysis.

[0030] Figure 6 Subcellular localization of CsSPX3 and CsPHL7 proteins in tobacco leaf cells.

[0031] Figure 7 Figure 4 shows the interaction detection between CsSPX3 and CsPHL7 (BIFC).

[0032] Figure 8 Figure 4 shows the interaction detection (LCI) between CsSPX3 and CsPHL7 under high-P and low-P conditions, respectively.

[0033] Figure 9 This is a diagram of the protein expression, purification and identification of tea plant CsPHL7.

[0034] Figure 10 This is the interaction detection result between tea plant CsPHL7 and TS1 promoter (EMSA).

[0035] Figure 11 This is the interaction detection result between tea plant CsPHL7 and GS1 promoter (EMSA).

[0036] Figure 12 Schematic diagram of the vectors for tea tree dual luciferases CsSPX3, CsPHL7 and CsTS1, CsGS1.

[0037] Figure 13 This is the interaction detection between tea plant PHL7 and TS1 promoter after adding SPX3.

[0038] Figure 14 This is the interaction detection between tea plant PHL7 and GS1 promoter after adding SPX3.

[0039] Figure 15 Theanine content and relative expression levels of different genes were measured after silencing CsSPX3 by virus-induced gene silencing (Figure a shows the growth status of plants in each group, Figure b shows the theanine content, Figure c shows the CsSPX3 gene, Figure d shows the CsPHL7 gene, Figure e shows the CsTS1 gene, and Figure f shows the CsGS1 gene).

[0040] Figure 16 Theanine content and relative expression levels of different genes were measured after silencing CsPHL7 by virus-induced gene silencing (Figure a shows the growth status of plants in each group, Figure b shows theanine content, Figure c shows the CsPHL7 gene, Figure d shows the CsSPX3 gene, Figure e shows the CsTS1 gene, and Figure f shows the CsGS1 gene).

[0041] Figure 17The theanine content and relative expression level were determined after overexpression of CsSPX3, CsPHL7, and CsSPX3&CsPHL7 using Agrobacterium-mediated transient transformation technology. DETAILED DESCRIPTION

[0042] The present invention treats tea seedlings with phosphorus and screens out CsSPX3 containing an SPX domain and a phosphorus starvation-responsive transcription factor CsPHL7 that significantly respond to phosphorus signals from transcriptome data. The CsSPX3 and CsPHL7 belong to the SPX family and the MYB-CC family, respectively.

[0043] The nucleotide sequence of the CsSPX3 gene is shown in SEQ ID NO.1:

[0044] SEQ ID NO.1

[0045] ATGCTCTCTAAATATTCTATCTCCTCCTTTTTAAACTCCTCCACACCTTATTTTCACTG

[0046] CGTTCACTACTCACACCGACAAATCTTCATTCTTGTTAGGCTTTTTCCTTTCAACTTT

[0047] TTCACTTCCAATTCTGGGTTTAGAGAGAGAATAAAGAAAAAGGGTTGGATGAAATT

[0048] TGGGAAGAGATTGAAGCAACAAGTTCAAGAAACTTTGCCGGGATGGCACGACAAG

[0049] TTTCTGCCTACAAGGATTTGAAAAAGCTTTTGAGGTTAATTTCTTCGGCTCCTCCG

[0050] ATGAATGAATGGATCGCTTGAGTTTGGGAAGGCTGAGGCTGAGTTCATGTACTTGTTG

[0051] AACAATGAGATCGAGAAATTTAACTCTTTTTTTTATGGAACAAGAGGAGGATTTCGTT

[0052] ATCCGCCATAAGGAGTTGCAACAGAGGATTGAGAGAGTGACTGAGATTTGGGGACC

[0053] AAATGGAAGTCAACCATTAGAGATGGATTACAAAGAGGAGATGGGTAAAATCAGAA

[0054] AAGAGATTGTCAATTTCCATGGTGAAATGGTGCTTTTAATGAACTACAGCAATATTA

[0055] ACTACACAGGGTTGGCCAAGATCTTGAAGAAATATGACAAGCGAACTGGTGGGTTG

[0056] TTGCGCTCACCCTTCATTCAGAATGTGTTGCAACAACCTTTTTACACAACTGATCTC

[0057] ATATCAAAACTTGTCAAAGAATGTGAAAGCACCATTGATGCAATGTTCCCGGTGGTT

[0058] GAAGAAGAAGAAAGAATAATCCGCAGAGAAAGAGAGGCGATTATGGTGTCGGGAG

[0059] AAGGAGTTTTTAGGAATACAATTGCAGCTCTGCTGACTATGCAAGAGATTAGAAGA

[0060] GGAAGCTCTACATACAGCCACTTCTCTCTACCGCCTCTCAACTTGCCGGATTTTGATT

[0061] TCATCCAAGCCTTTCAACTCAATTCACCCATACCAATTCCCTAA

[0062] The nucleotide sequence of the CsPHL7 gene is shown in SEQ ID NO.2:

[0063] SEQ ID NO.2

[0064] ATGTATCACGCCAAGAAATTTTCAACTGCAAGCTTAGTGCCACATAAATCTCCAGCC

[0065] CCTGAACAACTTGCATTAGTTGGAGTTTTGGGTGGATCTGCAGCTAGAAGTACTGCT

[0066] CCTGCAGGGGGAGGTGGGGGGAAGCAACGGTTGCGGTGGACTTCAGATCTTCATG

[0067] ATCGCTTCGTAGACGCTATTACTCAACTTGGCGGACCAGATAGAGCAACACCTAAA

[0068] GGTGTTTTACGAGTGATGGGTGAACCTGGACTTACCATATATCACGTGAAAAGCCAT

[0069] TTACAGAAATATCGCCTTGCAAAGTACCTGCCAGAGTCGCCAGCTGATGGTTCTAAA

[0070] GATCAGAAGAAAGGTTCTGGAGACAGCCTAAATAGCTTGGATTCTTCCCAGGGAGT

[0071] GCAAATTAATGATGCGCTGAGGATGCAGATGGAGGTACAGAAGCGTCTTCATGAAC

[0072] AACTTGAGGTACAAAGACAGTTACAAATGAGGATAGAGGCTCAGGGGAAATACTTG

[0073] CAGAAGATAATTGAGGAACAGCAAAAACTAAGTGGTGTTCTGAAAGCCTCCGAGA

[0074] CAGTTCCATTACCCAAGGATCAGCCGCTAGCTCCATCTCATTCACACCCACCTCCCC

[0075] CTCCCAATGCCTCCATGAGTTCCTTCTCTCCTCATAAGAAGCAAAAACTGGATGACG

[0076] GGTCCACAGAAGGCGGCCTGCCTGACTTTGTCCCACCAGAAGAGGACTAG

[0077] The present invention provides proteins encoded by the CsSPX3 and CsPHL7 genes. The amino acid sequence of the protein encoded by the CsSPX3 gene is shown in SEQ ID No. 3, and the amino acid sequence of the protein encoded by the CsPHL7 gene is shown in SEQ ID No. 4. The two proteins contain 316 and 241 amino acids, respectively, and have an SPX domain and Myb_CC_LHEQLE and Myb_DNA-binding_SHAQKYF domains, respectively.

[0078] SEQ ID No. 3

[0079] MLSKYSISSFLNSSTPYFHCVHYSHRQIFILVRLFPFNFFTSNSGFRERIKKKGWMKFGK

[0080] RLKQQVQETLPGWHDKFLSYKDLKKLLRLISSAPPMMNGSLEFGKAEAEFMYLLNNE

[0081] IEKFNSFFMEQEEDFVIRHKELQQRIERVTEIWGPNGSQPLEMDYKEEMGKIRKEIVNF

[0082] HGEMVLLMNYSNINYTGLAKILKKYDKRTGGLLRSPFIQNVLQQPFYTTDLISKLVKEC

[0083] ESTIDAMFPVVEEEERIIRREREAIMVSGEGVFRNTIAALLTMQEIRRGSSTYSHFSLPPL

[0084] NLPDFDFIQAFQLNSPIPIP

[0085] SEQ ID No.4

[0086] MYHAKKFSTASLVPHKSPAPEQLALVGVLGGSAARSTAPAGGGGGKQRLRWTSDLHD

[0087] RFVDAITQLGGPDRATPKGVLRVMGEPGLTIYHVKSHLQKYRLAKYLPESPADGSKDQ

[0088] KKGSGDSLNSLDSSQGVQINDALRMQMEVQKRLHEQLEVQRQLQMRIEAQGKYLQKI

[0089] IEEQQKLSGVLKASETVPLPKDQPLAPSHSHPPPPPNASMSSFSPHKKQKLDDGSTEGG

[0090] LPDFVPPEED

[0091] The sequences of the CsTS1 promoter and the CsGS1 promoter used in the following examples of the present invention are shown in SEQ ID No. 5 and SEQ ID No. 6:

[0092] CsTS1 promoter (SEQ ID No. 5):

[0093] ATCTAATAGGTAAGGTCTTGTTTGGGGATATAATCAAAAAGTTTTTATAAATTGTCAC

[0094] AATATTATTTTATTTATATTTTTATAAAAATTAAGAAAGTAACTAAAATAATTAATTTCC

[0095] AAACGAGTTATTAATTTTGACTTATTATGAAAATAAGAAAAAAAATCAACTTTTTAAT

[0096] TTTTTTATAATTTTTTTATAATAAAAATAAAAATATAATATGATCCAAATAAAAATAATAA

[0097] ACTTATTTTTTTTTAGTTGTCAAAATGTGGGTACACAGTAGGGATGTAAGAAAAAATC

[0098] GAAAAAATCGACCAAATCGATTGAACCGAACCAAACCGGCTATATTTGGTCTGTTTT

[0099] TTAACAAGAATCGGTCTGTGCGGTTTTCAAATTTGAATTTTTTGGATTTTCGGTTCGG

[0100] TATCGGTTTGAGCACCTTGTGCACCGATCTAAACCAAAACCGACTGCTTCAACATAT

[0101] ATTGTATTTATAATATATATATATATATATATAATCTATTAATCTAATGATATTAATAAGTAT

[0102] AATAAATTATTAACCTTAATTAATTAACCTTATCATATTCATAACCTAAGTTTGTAAAC

[0103] TCTTATTATATACTTTCATCCGTCCCAAAATATTAGTCCCTTATTCCATTTTTCATTGTCC

[0104] CAAAAATTTAGTCTCTCTTCAAAGATCAAGTACAAAAATAATAAATTTCCTATTTT

[0105] GTCTTTCTTTTAAATTAATAAACAATATAAAAGTACAATCATGACATTGATATTCCCA

[0106] AAAAATGTACTATCAAAAGGGTAATTTTGGAAAATCAATTTTTTTCAAATGCAATAAT

[0107] TGTACTACCAAAAAAAGTTAGATTCCCCAAAAGGGACCATTATTTTGGGACGGAGG

[0108] GAGTATAATCTTAACTCTAACTCATTATTCTTTGAACCTCAGTATTTTTAAACTTTTT

[0109] TTTTTGTTTTTATTTTTTAATTTAGGAATTAAAAGTATGTATTAAATGGATGAATTG

[0110] GATACGTTTGAAATGGTAAACAAATAACTAATTTAATATTTTTATATAGATTAATTTA

[0111] TATTTAAAAATGACTCAAGTTTGAAAAATTTTAAACAGATTAAGGCCAAGTTTGGCA

[0112] TAATTAAAAAAAGACAAAAAGTCAAAAAGTCACTATCCTATTTAAGTCACATCATCT

[0113] TATTTAAGTCACATCACCTCTCTAACACATCAATTCAAACCTAACCCAAACCCAACC

[0114] CATATTTCATAAAAAGTTAAAAAGTGCACTTATTTAGTCTTCCCAAACTAGGCCTAAA

[0115] TCACACCAAACAAAACTGTTTCAATTGGTTTGATTCAGTGTAATTTGATGTGAGAAC

[0116] TGTATAAATATTTAAAATAATAAAATCGACATTAACAATTCAATATAATTTTTTATTAAA

[0117] ATACCGAACATACCAAACCGATTACATCGTGCTACACAGACTCGTAAAAACAGAAG

[0118] GCATTAATTCGAGGGGGCTGCTCTGCTCTGGACTCTATAAAACCCACTCATGTCCTC

[0119] TGCTCACGGCTCTGCATTAAAAGCACAGACAGAGAGAGAGAGAGAAGCAGAGCGA

[0120] GAGAGACAGAGAGAGCC

[0121] CsGS1 promoter sequence (SEQ ID No.6):

[0122] CGAAACTCATTTTTTATGATTGAATTTACGAGATAAATGATTTAAATTATTGTTATAGA

[0123] AAAAATTATATCCAATTATGTAATTAGGTTAGAAATGGTTGCCCAATGTATCACTATCG

[0124] TCTATACTTTTTCATATTCTATTTTGTAAAATTTGTAGGCCAGTATCATCATCGTTCATA

[0125] TTTCTTCATATTCTAATTTGTAGAATTCATTTTTATTACTAGACTTATAAGATAAATGGT

[0126] TGATATCCTTTTTGTAGGTCCAAAATAGAGGCAAAAAGTAAAATATGAAAGAATATA

[0127] TCATCCATACTTTTTTATATTCTATTTTCAGAATATTTCATCATTGAAAAATGATTTGCA

[0128] CCCCTAATTTTGAAGACCCCAGTGACCCCCAACTGATGTGGCGGCATGCAATTGGTT

[0129] GAATTTTTTTTTTTTCCCTTGGTCCACAGCTTCCCACGTGGAGTGGGGGTGCTAGGG

[0130] GTGCCCAGCACAGCTCATTCATTATTATTACTATTTATGAAATAAATGATTCAAATTAT

[0131] CGTTGAGTGTATAAAATAGAGCATTTTTTAAGATAGACTAATTTAGGCTTCATTTGGC

[0132] ATTGACATTTTGTGCTAAATTATTCACAAAAAAAAAGAAGATAAAAAGTTAATAAGA

[0133] TAAAATACCACATCCAAATCATAATCATTTTATTTTTCTTTTACAAAAATTAAAAAAA

[0134] ACTAAAAAAAATCAACCCATACGAACTTTTTAGCCGAAGCTAAAATAGTCAAAAAA

[0135] TTAAAAAGTCAATTAAAAAAGTCACATGCCAAACAACAACTTAGTCTGATTATTTTT

[0136] TATTTTTTTATTTATTTTTAACCAGACCAAAAAAGTTATTTTTAGGATAAATTTTGAAT

[0137] TTTTTCTAATAGTTAATTTGATGGTTATAAAAGAAAAAATAAAATGATTATAATTTGAA

[0138] TGTGACTTTTTTTTTTATTGACTAAATTAATTATTATTATTTATTTAAAAAAAAAAAAAAAAT

[0139] CTCATGTCAAGCCTACCCAAAAAGATACGAAACAATAAGGGGTGTTTGGGA

[0140] AGATAGTTGAGTTAAAAACTCATTAAAATAAATATATACTAATAATTACTAA

[0141] GATAATATTGCATCTAACCTATAACCGTTTTTTTTTCTTTGATAACTATCAAATCA

[0142] GGGGTGTTTTGAGAGATGATTAAGTTATTTTGACTTTTTGTGAGAGAGGGA

[0143] AATGGGTTAAAAGCTGATAGTGACTTTTAACACTTTTCAAAATAGCCCTCAAT

[0144] ACAAAAAAACTTATATACTTATTTAGTTAGTATAAATAGTCAAAAAAATATAGTTAGT

[0145] TACTAAAATAGTCAACTTCCCAAATAAGGCCATAGTAAATTAGGGAATTACTAT

[0146] ACATACTCTTAAAAATGATACTTTATACATTTTTATGTCATTTTCTTCTTTATT

[0147] ATGATATTTTTATTTTATTTTTTTAGTATTGACTTAAAAATAAAATTATTAT

[0148] TTTTTTAATTAGATGTGCATAAGATACATCTTTTTAAGTATACATAGTGTACAT

[0149] CACTTTTTTTACTTTAAAAAAAAAAATCAAAAAATTACTTTCAACTTACTTTTC

[0150] AAAAAAATTCTAATATCTATTTTGGGGACGAAGTTAAAAATATATTTTTTTAACTTTT

[0151] AATCTCTTCTCAGTCCCATCTAAATTTTTTTAACTCTTTTTAAAAAAATATATAAAAAA

[0152] ATAAAAAAAATGTAACTCGAAATAAATTAAAAATTAAAAAATAAAATTTAACAACC

[0153] ATTCTAAACTAGTGCAATGTAAAACACCTCAACCACTATAAAAGGACGAACTCATCC

[0154] CTGGTTCTACAGCACATTACTCCTCGCCACAGACTCCAACACAAACCACGTATAGTA

[0155] CGGAGAGAAAGAGGAGAGAGGGAGAGTAAAAGCATCGTGTGGTTTGTTTGGAGTG

[0156] GGTGTTTTTGATCGATCGTCACT

[0157] Related culture medium preparation:

[0158] (1) LB solid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract + 7.5 g / L agar powder

[0159] (2) LB liquid medium: 10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract

[0160] (3) YEP solid medium: 10g / L peptone + 10g / L yeast extract + 5g / L sodium chloride + 7.5g / L agar powder

[0161] (4) YEP liquid medium: 10g / L yeast extract + 10g / L peptone + 5g / L sodium chloride

[0162] (5) Resuspension: 4.74g / L MS + 30g / L sucrose

[0163] The biological materials involved in the following experiments are all commercially available.

[0164] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0165] Example 1 Determination of theanine content in tea seedlings treated with phosphorus and analysis of transcriptome data

[0166] (1) Experimental methods

[0167] 1. Phosphorus treatment of tea seedlings, growth conditions and material collection

[0168] One-year-old Fuding Dabaicha tea seedlings, grown uniformly from the same seedbed, were collected from the Meitan Tea Factory in Zunyi, Guizhou Province, China. The soil around the seedlings' roots was cleaned and then hydroponically cultivated. The seedlings were tightly wrapped with sponges and planted in 10-liter polyethylene plastic pots covered with perforated foam board. The hydroponics were carried out in an artificial climate chamber at the School of Tea, Guizhou University. After two weeks of incubation in an untreated nutrient solution, various concentrations of potassium dihydrogen phosphate were added to the normal nutrient solution. The phosphorus concentrations were 0, 0.5, 1, 2, 4, 8, 16, and 32 mg·L. -1 The pH of the nutrient solution was maintained at 5.5 and replaced every 5 days. After 20 days of cultivation, the first mature leaf and the main lateral root were collected from the top to the bottom. To reduce individual differences between tea seedlings, three samples were collected as a composite sample, and three groups were used as three biological replicates. After collection, the samples were immediately frozen in liquid nitrogen and stored at -80°C for further study.

[0169] 2. HPLC Analysis of Theanine Content in Tea Roots and Leaves

[0170] Freeze-dry tea samples removed from -80°C and prepare the test solution according to the method specified in GB / T 23193-2017, "Determination of Theanine in Tea - High Performance Liquid Chromatography." Weigh 1.0 g (accurate to 0.01 g) of ground sample into a 200 mL beaker and add 100 mL of boiling distilled water. Soak in a 100°C water bath for 30 minutes, filter, and transfer to a 100 mL volumetric flask. After cooling, dilute to the mark with water and mix thoroughly. Filter 1 mL of the sample through a 0.45 μm aqueous filter membrane before analysis by liquid chromatography.

[0171] Chromatographic conditions: mobile phase A was 100% pure water, mobile phase B was acetonitrile, detection wavelength was 210 nm, flow rate was 1 mL / min, injection volume was 10 μL, and column temperature was 35°C.

[0172] 3. Determination of Phosphorus in Tea Roots and Leaves by Molybdenum-Antimony Antimony Colorimetry

[0173] Plant samples were dried at 65°C until easily grinded. After cooling, they were immediately pulverized and passed through a 0.25 mm sieve for later use. Total phosphorus in plants was determined using the molybdenum antimony colorimetric method, according to NY / T 2421-2013, "Molybdenum antimony colorimetric method for the determination of total phosphorus in plants." The specific steps are as follows: Weigh 0.2 g of sample and place it at the bottom of a digestion tube. Moisten the sample with water, then add 8 mL of concentrated sulfuric acid. Heat in a digestion oven at 250°C until the digestion solution is a uniform brown-brown color. After cooling slightly, add 2 mL of hydrogen peroxide dropwise. Repeat the digestion process until the solution is clear. Transfer the digestion solution to a 50 mL volumetric flask, cool, and dilute to volume. Then, transfer 1 mL of the resulting solution to a 50 mL volumetric flask, add water to 30 mL, add 2 drops of dinitrophenol indicator, adjust the pH until the solution is just slightly yellow, and then add 5 mL of molybdenum antimony colorimetric reagent and dilute to volume. Place the sample in an environment above 20°C for 30 minutes, and perform colorimetric determination after adjusting the spectrophotometer to the zero point of the standard curve at a wavelength of 700 nm.

[0174] 4. Transcriptome Sequencing and Real-time Quantitative PCR (qRT-PCR) Analysis

[0175] Transcriptome sequencing was performed by Novogene (Beijing, China) using the Illumina NovaSeq 6000 platform on triplicate samples. The FPKM values ​​of key differentially expressed genes were analyzed in this study.

[0176] Total RNA was extracted from the samples using a Trizol kit (TaKaRa, Dalian, China), and reverse transcribed using a Prime ScriptRT kit (Vazyme, Nanjing, China) to synthesize cDNA. The cDNA was diluted 10-fold for fluorescence quantitative PCR detection. The qRT-PCR conditions and system were as follows: one denaturation cycle (95°C, 3 minutes); followed by 40 amplification cycles (95°C, 10 seconds; 60°C, 20 seconds) and signal acquisition (72°C, 30 seconds); 20 μL of the qRT-PCR selection system, including 10 μL of SYBR Green qPCR Mix, 3 μL of template, 1 μL each of upstream and downstream primers, and 5 μL of ddH2O. 2 -ΔCt The relative expression levels of genes were analyzed using the tea leaf actin gene CsActin (XM_028240327.1) as an internal reference (primers are shown in Table 1).

[0177] Table 1 Primer names and sequences

[0178]

[0179]

[0180] (2) Experimental results

[0181] 1. Detection of theanine and phosphorus content in phosphorus-treated tea trees

[0182] After Fuding Dabai was hydroponically treated for 20 days at different phosphorus concentrations, the theanine content in roots and leaves was determined by HPLC. Figure 1 ). It was found that the content of theanine decreased significantly with the increase of phosphorus concentration. At the same time, the phosphorus content of leaves and roots under different phosphorus treatments was detected ( Figure 2 ), and found that it showed an opposite trend to theanine content, increasing with increasing phosphorus treatment concentration. In summary, low phosphorus can promote the formation of theanine in tea plants, while high phosphorus can significantly reduce theanine content in tea plants, affecting the quality of tea leaves.

[0183] 2. Identification of phosphorus-responsive genes CsSPX3 and CsPHL7 by transcriptome sequencing and qRT-PCR analysis

[0184] The SPX domain-containing protein family and phosphate starvation response proteins (PHRs) are widely involved in phosphorus signaling and homeostasis and are central regulators of phosphorus signaling in plants. Four phosphorus treatment groups were selected for transcriptome sequencing. We obtained RNA-seq transcriptome profiles of mature leaves of phosphorus-treated tea plants. Heat map analysis ( Figure 3 ), from which CsSPX3 (CSS0042550) and CsPHL7 (CSS0038558) that significantly responded to phosphorylation signals were screened.

[0185] 3. Further analysis by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) showed that phosphorus treatment promoted the growth of leaves ( Figure 4 ) and roots ( Figure 5 ) The expression levels of CsSPX3 and CsPHL7 were increased under high phosphorus concentration conditions, while the opposite was true under low phosphorus conditions (e.g. Figure 4 、 Figure 5 Furthermore, the expression levels of CsTS1, CsTS2, CsGS1, and CsGS2 were downregulated in both leaves and roots. These data suggest that the expression levels of CsSPX3 and CsPHL7 are negatively correlated with theanine accumulation in tea plant roots and leaves.

[0186] Example 2 Subcellular Localization of Tea Plant CsSPX3 and CsPHL7 Genes

[0187] (1) Experimental methods

[0188] 1. The tobacco used is Nicotiana benthamiana.

[0189] 2. Construction of fusion expression vector

[0190] Primers with restriction sites were designed based on the CsSPX3 and CsPHL7 CDSs (Table 2). Plasmids were extracted from clones containing the correct CDSs for the CsSPX3 and CsPHL7 genes. The genes were digested with Kpn I, and the vectors were digested with Xba I. The resulting ligation products were ligated to the pCAMBIA1300-35S-GFP vector using T4 DNA ligase. The resulting ligation products were transformed into DH5a competent cells. After PCR amplification, restriction enzyme screening, and sequencing verification, positive clones were screened and plasmids were extracted to obtain the GFP-target gene fusion expression vectors, PCAMBIA1300-35S-CsSPX3-GFP and PCAMBIA1300-35S-CsPHL7-GFP. Subcellular localization vectors were synthesized by Wuhan Transduction Biolabs Co., Ltd.

[0191] Table 2 Amplification primer names and sequences

[0192]

[0193] 3. Tobacco Transient Transformation Steps

[0194] (1) Shake the successfully detected Agrobacterium culture overnight at 28°C and 200 rpm;

[0195] ⑵Take 1ml of bacterial solution and add it to a sterilized 1.5ml centrifuge tube;

[0196] (3) 8000 rpm, 2 min, sediment the bacteria (room temperature), remove the supernatant, add 1 ml of permeate, and suspend the bacteria;

[0197] (4) Repeat step 3 to further remove a small amount of antibiotics;

[0198] ⑸ Take a small amount of suspended bacterial solution, dilute it 10 times, and measure the OD 600 The value was multiplied by 10 to obtain the OD value of the bacterial suspension. 600 value;

[0199] ⑹ Determine the titer of the bacterial suspension to the permeate and calculate the dilution factor so that the final bacterial suspension (for infection) is 5.0 ml and the OD 600 The value is 0.4 (0.1-0.8 as needed, not exceeding 1). Usually 0.5-1.0 ml of the final suspension is sufficient for infection.

[0200] ⑺ Prepare the final bacterial suspension in a 1.5ml centrifuge tube and let it stand at room temperature for 2 hours to prepare for infection;

[0201] ⑻ Before infection, place the tobacco under a white fluorescent light for 1 hour to open its stomata;

[0202] (9) Select the third and fourth leaves from the bottom for infection (infect between two leaf veins). Select two leaves from one plant and infect with one bacterial solution.

[0203] ⑽Use a syringe without a needle to gently rub the back of the leaf to be rotated, or pierce it with a small needle to remove its wax layer;

[0204] ⑾Before infection, mark the area to be transferred with a marker;

[0205] ⑿ Aspirate the final bacterial suspension from step (7) into a 1 ml syringe without a needle;

[0206] ⒀Point the syringe at the area to be transferred on the back of the leaf, press the leaf with one hand, and gently push the piston with the other hand until the liquid spreads, then infect other parts. After infection, circle the infected area with a marker;

[0207] ⒁ Spray the leaves with water, put them in a fresh-keeping bag, and return the infected tobacco to the culture room and leave them in the dark overnight;

[0208] ⒂Open the fresh-keeping bag on the second day. The expression level is the highest 2 days after injection.

[0209] ⒃Cut the infected area, tear off the epidermis to prepare a slide, and observe under a confocal laser microscope.

[0210] (2) Experimental results

[0211] like Figure 6 As shown in the figure, the fluorescent signal of the empty vector carrying GFP was detected in the entire tobacco epidermal leaf cells, and the fluorescent signal of the PCAMBIA1300-35S-CsSPX3-GFP and PCAMBIA1300-35S-CsPHL7-GFP fusion proteins was detected in the cell nucleus and cell membrane, proving that CsSPX3 and CsPHL7 proteins function in the cell nucleus and cell membrane.

[0212] Example 3: LCI and BiFC methods verify the interaction between CsSPX3 and CsPHL7

[0213] (1) Experimental methods

[0214] 1. Bimolecular Fluorescence Complementation (BiFC)

[0215] The CsSPX3 and CsPHL7 coding sequences, excluding the stop codon, were amplified using PCR with specific primers (Table 3) and ligated to the N-terminus or C-terminus of the yellow fluorescent protein (YFP), respectively. The constructed vector plasmids were transformed into Agrobacterium tumefaciens LBA4404 via the freeze-thaw method. One-month-old tobacco plants (nicotinoids) were selected and transformed by injecting 1 ml of the solution into the back of the leaves using a disposable syringe (see Example 2 for transient tobacco transformation). After 36 hours of culture, the fluorescence signal was detected using a confocal laser scanning microscope (Leica, TCS, SP8).

[0216] Table 3 Primer names and sequences

[0217]

[0218]

[0219] 2. Firefly Luciferase Complementation (LCI)

[0220] The full-length coding sequence of SPX3 was ligated to the N-terminal fragment of luciferase (nLUC), and the full-length coding sequence of PHL7 was fused to the C-terminal fragment. The resulting plasmids were transferred into Agrobacterium tumefaciens strain GV3101. Using Agrobacterium-mediated transient expression in tobacco, tobacco leaves were infiltrated with the empty vector, negative control, and experimental groups. After overnight incubation in the dark and normal growth, 48 hours later, the leaves were smeared with 1 mM D-luciferin potassium salt. The reaction was allowed to stand in the dark for 10 minutes, and then observed using a Fusion FX7 chemiluminescence analyzer (VILBER, France). The fluorescent signal was captured and photographed.

[0221] (2) Experimental results

[0222] like Figure 7 As shown, laser confocal microscopy revealed that the co-infected pCAMBIA1300-35S-YFP C and pCAMBIA1300-35S-YFP N No fluorescence was observed in the empty group. N and pCAMBIA1300-35S-Y C 、pCAMBIA1300-35S-CsPHL7-Y C and pCAMBIA1300-35S-Y N No fluorescence signal was observed when co-infecting the lower epidermis of tobacco. N and pCAMBIA1300-35S-CsPHL7-Y CCo-infected tobacco epidermal cells revealed yellow fluorescence on the cell membrane under excitation light, indicating that CsSPX3 and CsPHL7 proteins can interact in tobacco epidermal cells, with the interaction site located at the cell membrane.

[0223] LCI experiments were performed in tobacco seedlings grown under high-phosphorus (HP; 200 μM Pi) and low-phosphorus (LP; 5 μM Pi) conditions. We performed SPX3-PHL7 interaction assays by adding different concentrations of inositol polyphosphates (InsPs) and found that strong fluorescence signals were observed for both CsSPX3-nLUC and cLUC-CsPHL7 combinations regardless of whether InsP6 was present at high or low concentrations ( Figure 8 ), indicating that CsSPX3 and CsPHL7 interact in a phosphate-independent manner.

[0224] Example 4 EMSA and LUC methods to detect the binding of CsPHL7 to the promoters of CsTS1 and CsGS1

[0225] (1) Experimental methods

[0226] 1. Electrophoretic Mobility Shift Assay (EMSA)

[0227] The cDNA of CsPHL7 was introduced into pET32a, and recombinant His-CsPHL7 was purified using a Ni-NTA His Bind purification kit (Novagen, Beijing, China) according to the manufacturer's instructions. EMSA was performed using a Lightshift™ chemiluminescent EMSA kit (Thermo Scientific, USA). The sequence of the biotinylated probe (Zoonbio Biotechnology, Nanjing, China) is shown in Table 4.

[0228] Table 4 Probe names and sequences

[0229]

[0230]

[0231] 2. Dual luciferase reporter gene assay (LUC)

[0232] The transcriptional activity in tobacco was analyzed by a dual luciferase assay system. The CDS of CsSPX3 and CsPHL7 were cloned into the pGreenII 62-SK vector as effectors, and the promoter fragments of CsTS1 and CsGS1 were introduced into the pGreenII0800-LUC vector as reporter genes. They were used to transform the Agrobacterium tumefaciens EHA105 strain, and then the recombinant plasmids were transiently expressed in the leaf cells infected with EHA105. After 3 days, the cells were treated with 0.2 mg mL-1 Infected leaves were sprayed with sodium D-luciferin and incubated at 37°C for 10 minutes. Fluorescence images were acquired using a Fusion FX7 chemiluminescence instrument (VILBER, France). LUC / REN-based transcriptional activity was measured using a dual-luciferase reporter gene assay kit.

[0233] (2) Experimental results

[0234] First, the purified CsPHL7 protein ( Figure 9 ) were incubated with different DNA fragments of the TS1 promoter labeled with biotin, and then subjected to electrophoresis gel shift assay. After CsPHL7 protein bound to nucleic acid fragments of different lengths, the protein + nucleic acid lane bands lagged significantly behind the lane. In lanes 3-6, the gel electrophoresis results of the interaction between probes of different lengths and proteins were different, but it clearly showed that CsPHL7 protein could bind to the TS1 promoter fragment ( Figure 10 The results showed that CsPHL7 and CsTS1 promoters have specific binding. In addition, EMSA proved that CsPHL7 and CsGS1 promoters also have specific binding ability ( Figure 11 ).

[0235] The promoters of CsTS1 and CsGS1 were integrated into the 0800-LUC plasmid, while the CDS of CsSPX3 and CsPHL7 were integrated into the 62-SK plasmid ( Figure 12 ). These plasmids were co-transfected into Agrobacterium and transformed into tobacco. Given the coexistence of CsSPX3, CsPHL7 and CsTS1 and CsGS1 promoters, when CsPHL7 was co-expressed with CsTS1 and CsGS1 promoters, respectively, the LUC / REN ratios were lower than those in the control group. However, when CsSPX3 was added, the LUC / REN ratios decreased significantly. The co-expression of CsSPX3 and CsPHL7 inhibited the luciferase reporter gene driven by the CsTS1 and CsGS1 promoters. The results showed that the co-expression of CsSPX3 increased the ability of CsPHL7 to inhibit the expression of CsTS1 and CsGS1 (e.g. Figure 13 、 Figure 14 shown).

[0236] Example 5 Virus-induced gene silencing (VIGS) technology verifies the functions of CsSPX3 and CsPHL7

[0237] (1) Experimental methods

[0238] 1. Experimental Materials

[0239] The materials were cuttings of the "Fuding Dabai" tea tree from the School of Tea at Guizhou University, and the virus-induced gene silencing (VIGS) technology was used to verify the functions of CsSPX3 and CsPHL7.

[0240] 2. Construction of Plant VIGS Vector

[0241] The pTRV2-CsSPX3 vector was constructed from a 444-bp fragment of the CsSPX3 gene, and the pTRV2-CsPHL7 vector was constructed from a 284-bp fragment of the CsPHL7 gene. Both vectors contain EcoRI and BamHI restriction sites. The PCR product was ligated with the pTRV2 vector, which had been digested with EcoRI and BamHI, using T4 DNA ligase (Kewen Biotechnology Co., Ltd.). Ligation was performed overnight at 16°C and then transformed into Escherichia coli DH5α. Insertion of the products was verified by bacterial culture PCR using primers specific for pTRV2-CsSPX3-F and pTRV2-CsSPX3-R, and pTRV2-CsPHL7-F and pTRV2-CsPHL7-R. The CsSPX3 and CsPHL7 gene fragments were obtained by whole gene synthesis and loaded into the pUC57-Simple vector to obtain the pUC57-Simple-CsSPX3 and pUC57-Simple-CsPHL7 plasmids.

[0242] The sequence of the CsSPX3 gene containing restriction enzyme cleavage sites on both sides is shown in SEQ ID NO.43:

[0243] CGGAATTCCG AAATTTGGGAAGAGATTGAAGCAACAAGTTCAAGAAACTTTGCCG

[0244] GGATGGCACGACAAGTTTCTGTCCTACAAGGATTTGAAAAGCTTTTGAGGTTAATT

[0245] TCTTCGGCTCCTCCGATGATGAATGGATCGCTTGAGTTTGGGAAGGCTGAGGCTGA

[0246] GTTCATGTACTTGTTGAACAATGAGATCGAGAAATTTAACTCTTTTTTTATGGAACAA

[0247] GAGGAGGATTTCGTTATCCGCCATAAGGAGTTGCAACAGAGGATTGAGAGAGTGAC

[0248] TGAGATTTGGGGACCAAATGGAAGTCAACCATTAGAGATGGATTACAAAGAGGAGA

[0249] TGGGTAAAATCAGAAAAGAGATTGTCAATTTCCATGGTGAAATGGTGCTTTTAATGA

[0250] ACTACAGCAATATTAACTACACAGGGTTGGCCAAGATCTTGAAGAAATATGACAAGC

[0251] GA CGGGATCCCG

[0252] The sequence of the CsPHL7 gene containing restriction enzyme cleavage sites on both sides is shown in SEQ ID NO.44:

[0253] CGGAATTCCG AATCTTGGGCGTGTCAATCAACAGTCCATATATGCTGATGATGAAATT

[0254] GTGTTTGGATTAAATATGTCAAAAGAGTTGAAAGGATAGGAGGATGATTGTAATGGG

[0255] TATAGATGGTTTTCTTGGGATGATTGTTAATGTAGTGATAAACCCGTTTTTCATATAGA

[0256] TCATGTACTGGTAATCCATTTGTAATTATAATTAAACACGTATGTAGTGGTTATTCCTA

[0257] TTGTTGTTGGTAGTCTATTGGTTACATTTGTTGGAAAATAGAGTTAAGGGTTTGAGA

[0258] GAAA CGGGATCCCG

[0259] Gene fragment structure: EcoRI-CsSPX3-BamHI, EcoRI-CsPHL7-BamHI.

[0260] Construction of the pTRV2-CsSPX3 and pTRV2-CsPHL7 plasmids: Double-digest the pUC57-Simple-CsSPX3 and pUC57-Simple-CsPHL7 plasmids with EcoRI and BamHI to obtain the CsSPX3 and CsPHL7 gene fragments, which were then run on a gel and recovered. Simultaneously, double-digest the pTRV2 vector with EcoRI and BamHI, run on a gel, and recover the vector fragments. Ligate the digested CsSPX3 and CsPHL7 gene fragments with the pTRV2 vector fragment. Transform the ligation products into DH5a competent cells. Select several colonies from the transformed plates, extract the plasmids, and verify by digestion with XhoI and HindIII. The recombinant plasmids were further verified by sequencing. (Note: All the above procedures were commissioned by Changsha Kewen Biotechnology Co., Ltd.)

[0261] 3. Tea tree infection

[0262] pTRV1, pTRV2, pTRV2-CsSPX3, and pTRV2-CsPHL7 were introduced into Agrobacterium GV3101 competent cells using the freeze-thaw method. Each Agrobacterium strain was inoculated into a medium containing 100 mg·L -1 Kanamycin and 50 mg·L -1 Rifampicin was activated in solid YEP medium at 28℃ for 48h. Single colonies were cultured in corresponding liquid YEP medium until OD 600 The Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial suspension was collected and suspended in 4.74 g / L MS, 2 mol / L 6-BA, 2 mol / L acetosyringone (AS), 100 umol / L naphthaleneacetic acid (NAA), pH 5.6. The two tea tree materials were vacuum infiltrated. The OD 600 Adjust to 1.2. At room temperature, pTRV1 was mixed with pTRV2, pTRV2-CsSPX3, and pTRV2-CsPHL7 bacterial solutions in a 1:1 ratio (pTRV1+pTRV2, pTRV1+pTRV2-CsSPX3, pTRV1+pTRV2-CsPHL7), and the tea tree cuttings were vacuum infiltrated. Cut the tea tree cuttings to a length of 20 cm with pruning shears. Keep two mature leaves, and then place the tea tree cuttings in a Buchner flask filled with the mixed bacterial solution for vacuum infiltration. Keep them in the dark for three days and then grow them in a greenhouse at 25°C with a light / dark cycle of 16h / 8h.

[0263] Vacuum-infected pTRV2-CsSPX3, pTRV2-CsPHL7-silenced plants, pTRV2 plants, and wild-type plants were cultured for 30–45 days after lateral shoots emerged. Samples were collected, sterilized, dried to a constant weight, and ground into powder. Theanine content was determined by high-performance liquid chromatography.

[0264] 4. Detection of related gene expression

[0265] Primer Premier 5.0 software was used to design specific primers for the CsSPX3, CsPHL7, CsTS1, and CsGS1 genes (primers were synthesized by the Chongqing Branch of Beijing Qingke Biotechnology Co., Ltd.). The tea plant actin gene (Actin) was used as an internal reference (primer sequences are shown in Table 1). qRT-PCR experiments were performed on a Bio-Rad CFX Connect™ Real-Time PCR Instrument (Bio-Rad). The qRT-PCR reaction system was configured using the Nanjing Novozymes Universal High-Sensitivity Dye-Based Quantitative PCR Detection Kit according to the kit's instructions. The 20 μL system is shown in Table 5 below.

[0266] Table 5 Fluorescence quantitative PCR reaction system

[0267] Components Volume (μL) cDNA 3μL Primer F 1 μL Primer R 1 μL 2×ChamQ Universal SYBR qPCR Master Mix 10 μL Nuclease-free H2O 5μL Total volume 20 μL

[0268] (2) Experimental results

[0269] like Figure 15 As shown, CsSPX3 was successfully silenced in tea plants, with its expression levels reduced to 63%, 53%, and 39%, respectively, compared to wild-type and empty vector controls. Meanwhile, the expression levels of CsTS1 and CsGS1 were significantly increased in CsSPX3-silenced plants. Furthermore, CsPHL7 expression was significantly reduced in CsSPX3-silenced plants compared to control plants (WT and pTRV2). Examination of theanine content in CsSPX3-silenced tea cuttings revealed an increase in theanine content compared to control plants (pTRV1+pTRV2). Theanine content increased by 1.89-fold (pTRV2-CsSPX3-1), 2.36-fold (pTRV2-CsSPX3-2), and 1.80-fold (pTRV2-CsSPX3-3), respectively. These data indicate that silencing CsSPX3 promotes theanine formation in Fuding Dabaicha.

[0270] like Figure 16As shown, compared with the wild-type and empty vector controls, CsPHL7 was successfully silenced in tea plants, with its expression levels reduced to 45%, 55%, and 49%, respectively, while the expression of CsTS1 and CsGS1 was elevated, and theanine content was significantly increased by 1.58-fold (pTRV2-CsPHL7-1), 2.10-fold (pTRV2-CsPHL7-2), and 2.12-fold (pTRV2-CsPHL7-3), respectively. This indicates that silencing CsPHL7 promotes theanine formation in "Fuding Dabaicha", and therefore CsPHL7 is a negative regulator of theanine biosynthesis in tea plants.

[0271] Example 6: Verification of the functions of CsSPX3 and CsPHL7 using Agrobacterium-mediated transient overexpression system in tea plants

[0272] (1) Experimental methods

[0273] 1. Experimental materials: Same as Example 5.

[0274] 2. Construction of plant overexpression vector

[0275] Plant overexpression vectors were designed and constructed based on the original vector pSH737. The recombinant plasmids were transformed into competent Escherichia coli (DH5α) and 100 mg·L Kan was used. -1 Positive clones were screened, and the recombinant plasmids were extracted and double-digested with XbaI and KpnI for verification. The recombinant plasmids with positive results were transformed into Agrobacterium strain LBA4404 competent cells and cultured with Kan 100 mg·L -1 、Rif20 mg·L -1 Agrobacterium-positive strains were screened, and colony PCR was performed on the positive strains using primers. Agrobacterium strains with positive PCR results were expanded and stored at -80°C. The plasmid containing the overexpression vector was transformed into Agrobacterium strain LBA4404 by freeze-thaw method to prepare the engineered strain. Take the LBA4404 competent cells stored at -80°C and thaw them in ice for 10 minutes; add 5μL of plasmid DNA to each competent cell, gently flick to mix, and then ice bath for 30 minutes; after liquid nitrogen quick freezing for 5 minutes, immediately water bath at 37°C for 2 minutes, add 900μL of 37°C preheated YEP liquid medium, and shake and culture at 28°C and 200rpm for 3 hours; after centrifugation at 4000×g for 1 minute at room temperature, discard the supernatant, add 100μL YEP liquid medium to the bacteria, mix it with a pipette tip, and take an appropriate amount of bacterial liquid to spread on a plate containing 100mg·L -1 Kan and 20mg·L -1 The culture medium was placed on the YEP plate of Rif, inverted and cultured in a constant temperature incubator at 28℃ for 2 days, and the bacterial solution was stored at -80℃.

[0276] 3. Tea tree infection

[0277] The pSH737-35S-CsSPX3 and pSH737-35S-CsPHL7 recombinant plasmids were introduced into Agrobacterium tumefaciens LBA4404 competent cells by freeze-thaw method. Each Agrobacterium strain was inoculated into a medium containing 100 mg·L -1 Kanamycin and 50 mg·L -1 Rifampicin was activated in solid YEP medium at 28℃ for 48h. Single colonies were cultured in corresponding liquid YEP medium until OD 600 is 1.0. The Agrobacterium cells were centrifuged at 6000rpm for 6min. The bacterial solution was collected and suspended in 4.74g / L MS, 0.5% (m / v) D-glucose, 150μmol / L acetosyringone (AS), 25μmol / LMES, pH 5.6. Agrobacterium carrying the pSH737 vector with the 35S promoter was injected into the second leaf as a control (Control), and the CsSPX3, CsPHL7 and CsSPX3&CsPHL7 overexpression vectors were injected into the second leaf of the tea tree respectively. Each experiment was repeated at least 5 times, with 5 leaves in each repeat. Samples were collected 3 days after injection, divided into two parts, and immediately quick-frozen in liquid nitrogen and dried, ground, mixed evenly, and subjected to qRT-PCR and theanine detection respectively.

[0278] 4. Detection of related gene expression levels: The detection method is similar to that in Example 5.

[0279] (2) Experimental results

[0280] like Figure 17 As shown, theanine content in tea leaves overexpressing the target gene was measured by high-performance liquid chromatography. The results showed that compared with the control, gene expression levels were significantly upregulated in leaves of tea plants transiently overexpressing the target gene. Theanine content in leaves expressing single transgenic CsSPX3 and single transgenic CsPHL7 decreased by 23% and 19%, respectively, compared with the control, while theanine content in leaves co-expressing CsSPX3 and CsPHL7 decreased by 37%. This suggests that the CsSPX3 and CsPHL7 genes can reduce theanine content in tea plants.

[0281] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. CsPHL7 The application of the gene in regulating theanine synthesis in tea trees is characterized in that: Said CsPHL7 The nucleotide sequence of the gene is shown in SEQ ID NO.2; Using the CsPHL7 The method of genetically regulating theanine synthesis in tea trees is: CsPHL7 Gene silencing promotes the synthesis of theanine in tea plants; or through the CsPHL7 Transient overexpression of the gene inhibits theanine synthesis in tea plants.

2. The use according to claim 1, characterized in that: Said CsPHL7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

4.

3. The use according to claim 2, characterized in that: Using the CsPHL7 The method for genetically regulating theanine synthesis in tea trees is as follows: CsPHL7 The recombinant vector is transferred into tea leaves to make the CsPHL7 Silencing or transient overexpression can promote or inhibit theanine synthesis in tea plants.

4. CsSPX3 Gene Synergy CsPHL7 The application of the gene in regulating theanine synthesis in tea trees is characterized by: Said CsSPX3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Said CsPHL7 The nucleotide sequence of the gene is shown in SEQ ID NO.2; CsSPX3 Gene Synergy CsPHL7 The method for genetically regulating theanine synthesis in tea trees is as follows: CsPHL7 Genes and CsSPX3 Gene silencing promotes the synthesis of theanine in tea plants; or through the CsSPX3 Genes and CsPHL7 Transient overexpression of the gene inhibits theanine synthesis in tea plants.

5. The use according to claim 4, characterized in that: Said CsSPX3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 3; Said CsPHL7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

4.

6. The use according to claim 5, characterized in that Using the CsSPX3 Genes and CsPHL7 The method for genetically regulating theanine synthesis in tea trees is as follows: CsSPX3 and CsPHL7 The recombinant vector is transferred into tea leaves to make the CsSPX3 Genes and CsPHL7 Silencing or transient overexpression, thereby promoting or inhibiting theanine synthesis in tea plants; Among them, the CsSPX3 Genes and CsPHL7 Genes through binding CsTS1 and CsGS1 Gene promoter, repression CsTS1 and CsGS1 The expression of theanine gene is inhibited, thereby inhibiting the synthesis of theanine in tea trees.

7. CsPHL7 The application of genes in improving the quality of tea leaves from tea trees is characterized in that: Using the CsPHL7 The method of genetically improving the quality of tea leaves from tea trees is: CsPHL7 Gene silencing promotes the synthesis of theanine in tea trees, thereby improving the quality of tea leaves; CsPHL7 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

8. CsSPX3 Gene Synergy CsPHL7 The application of genes in improving the quality of tea leaves from tea trees is characterized in that: Using the CsSPX3 Genes and CsPHL7 The method for improving the quality of tea leaves by gene is as follows: CsPHL7 Genes and CsSPX3 Gene silencing promotes the synthesis of theanine in tea trees, thereby improving the quality of tea leaves; CsSPX3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CsPHL7 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

Images