Application of transcription factor CsPIF7 gene in regulating theanine synthesis and improving tea quality

Abstract

The present invention provides the application of the transcription factor CsPIF7 gene in regulating theanine synthesis and improving tea quality, belonging to the technical field of agricultural molecular biology. The present invention discovers that overexpression of CsPIF7 can activate the expression of TS1 and GS1, enabling tea leaves to accumulate more theanine; after reducing the expression of CsPIF7 through the VIGS technology of tea plants, the activation and expression of TS1 and GS1 will be reduced, thereby decreasing the theanine content in tea leaves. Further, the present invention uses LUC to verify that the tea plant transcription factor CsPIF7 can positively regulate the expression of TS1 and GS1, and can be used to regulate the theanine content in tea plants and improve tea quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural molecular biology, and specifically relates to the application of the transcription factor CsPIF7 gene in regulating theanine synthesis and improving tea quality. Background Art

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the important cash crops in the world. Its economic value is determined by its leaves, and at the same time, the leaves of the tea plant are also related to its own growth and development and material accumulation. Among them, theanine in tea is the main contributing factor to the fresh taste of tea, and plays a decisive role in the quality and economic value of tea.

[0003] Theanine is a natural acetamide analogue of glutamic acid. It is a characteristic non-protein amino acid of the tea plant. It can not only improve the flavor and quality of tea, but also is very important for the physiological functions of the tea plant. Theanine in the tea plant is mainly synthesized under the action of theanine synthase (TS), which can be roughly divided into two pathways: one is that α-ketoglutaric acid formed by the tricarboxylic acid cycle and other pathways in the tea plant reacts with ammonia to form glutamic acid under the action of glutamate dehydrogenase, and then glutamic acid and ethylamine react under the action of theanine synthase to synthesize theanine; the other is that glutamic acid and ammonia first form glutamine under the action of glutamine synthetase (GS), then glutamine reacts with α-ketoglutaric acid under the action of glutamate synthase to form glutamic acid, and glutamic acid and ethylamine react under the action of theanine synthase to form theanine. Studying the effects of different genes in the tea plant on theanine is beneficial to controlling theanine content in the tea plant and improving tea quality.

[0004] PIF7 is the main regulatory factor in response to shading. Shading stress will reduce the ratio of red light (660 nm) to far-red light (730 nm). The photoreceptor phytochrome B (phyB) that absorbs R / fr plays the most important role during SAS (shade avoidance syndrome). In an open environment under sunlight, most of the phyB moves to the nucleus in the active form of far-infrared absorption (Pfr) and interacts with the basic helix-loop-helix (bHLH) protein, which is called the phytochrome-interacting factor. The photoactivation of phyB induces the rapid phosphorylation of PIF1 / 3 / 4 / 5 before they are degraded. When the plant is under shaded conditions, phyB is mainly in the inactive red light-absorbing (Pr) cytoplasmic form, which promotes the accumulation of PIFs. It has been reported in the literature that the hypocotyl phenotype of the PIF7 mutant in Arabidopsis thaliana is the most obvious, but there is no prior art research on whether the tea plant transcription factor CsPIF7 responds to shading and participates in theanine synthesis. Summary of the Invention

[0005] The present invention aims to provide the application of the transcription factor CsPIF7 gene in regulating theanine synthesis and improving tea quality.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0007] The present invention provides the application of the transcription factor CsPIF7 gene in regulating theanine synthesis.

[0008] Preferably, the nucleotide sequence of the transcription factor CsPIF7 gene is as shown in SEQ ID No.1.

[0009] Preferably, the amino acid sequence of the protein encoded by the transcription factor CsPIF7 gene is as shown in SEQ ID No.2.

[0010] Preferably, the method for regulating theanine synthesis using the transcription factor CsPIF7 gene is: constructing a recombinant vector of CsPIF7, transferring the recombinant vector into tea tree leaves to silence or transiently overexpress CsPIF7, thereby inhibiting or promoting theanine synthesis in tea trees.

[0011] Preferably, the transient overexpression vector is obtained by inserting the transcription factor CsPIF7 gene between the Xba I and Kpn I restriction enzyme sites of the original vector pSH737-35S, and is named: 35S-CsPIF7.

[0012] Preferably, the silencing vector is obtained by inserting the pTRV2 empty vector between the EcoR I and BamH I restriction enzyme sites in the UTR region of the transcription factor CsPIF7 gene to obtain a VIGS silencing vector for silencing the target gene, named: pTRV2-CsPIF7.

[0013] Preferably, the primer pair for the transcription factor CsPIF7 gene is:

[0014] PIF7-F: ATGAAAGGAATCATGAATC (SEQ ID No.6);

[0015] PIF7-R: TCACTTACTTGAGCTAG (SEQ ID No.7).

[0016] The present invention also provides the application of the transcription factor CsPIF7 gene in improving the quality of tea tree leaves.

[0017] Through the construction of an instantaneous expression vector, the present invention discovers that highly expressing the transcription factor CsPIF7 can activate the expression of TS1 and GS1, enabling tea leaves to accumulate more theanine. After reducing the expression of CsPIF7 through the VIGS technology of tea trees, the activation and expression of TS1 and GS1 will be reduced, thereby decreasing the theanine content in tea leaves. Further, the present invention uses LUC to verify that the tea tree transcription factor CsPIF7 can positively regulate the expression of TS1 and GS1, which can be used to regulate the theanine content in tea trees and improve the quality of tea leaves. Description of the Drawings

[0018] Figure 1 It is a subcellular localization map of CsPIF7.

[0019] Figure 2 It is a schematic diagram of the domain of the transcription factor CsPIF7.

[0020] Figure 3 It is a schematic diagram of the construction of the CsPIF7 overexpression vector.

[0021] Figure 4 It is a map of the theanine content in tea trees after transiently expressing the CsPIF7 gene.

[0022] Figure 5 It is a map of the expression levels of CsPIF7, GS1, and TS1 in tea tree leaves after transient expression of tea trees.

[0023] Figure 6 It is a schematic diagram of the construction of the VIGS CsPIF7 silencing vector.

[0024] Figure 7 It is a map of the theanine content in tea trees after silencing the CsPIF7 gene by VIGS.

[0025] Figure 8 It is a map of the expression levels of CsPIF7, GS1, and TS1 in tea tree leaves after silencing the CsPIF7 gene in tea trees by VIGS.

[0026] Figure 9 It is the result of transcriptional activation (where Figure A is the fluorescence detection result of TS1; Figure B is the fluorescence detection result of GS1; Figure C is the statistical result of the fluorescence intensity of TS1; Figure D is the statistical result of the fluorescence intensity of GS1). Detailed Implementation Modes

[0027] The nucleotide sequence of the CsPIF7 gene SEQ ID NO.1 (CSS0002807.1)

[0028] ATGAAAGGAATCATGAATCAGTGCATAGTGCATAATTGGAATCCAAGGCACCAAAG

[0029] ACAAGAACAAGTTGAAGGTGAAGAGGAGAAAACATCCTCTCACGTGCACAGCCAT

[0030] CTTCACAACCTCACTACTACTCATATTGTCCCTATGTCTAATTTTGAAGTTACAGAGC

[0031] TAACATGGGAAAATGGGCAGTTAGCCATGCATGGGCTTGGTGGGCTCCTTCCCACA

[0032] GCCCCGACAAAGCCCACATGGGGTAGGACCGGCGACACGCTGGAATCCATAGTCCA

[0033] TCTAGCTGCATGCCACCAACAAAATCTAAATCTACTTCCTCCACATGACCATCTGCAT

[0034] CATCATAATCCGGCTCCAAACAGGACGACCTCCTCCGCCAAAAAATGTGCCGAAAC

[0035] CGTGGGACACATGCCGATGTCTCCAGGATTCTCGAAGAAGCGGATGTGGTCAGAAT

[0036] CCGAAAAAAAATTTGCTAGTGGTATTCAAGACGATAGAAGTGCTTGTGCTAGTGCG

[0037] ACAATTTTTAAGGATAGTGGCACAACTATGATGACATGGGCTTCTTTCGAATCCCCTC

[0038] CTCAAAGCTTGAAGACTAAAACCACGGATGAGGATTCAGCTTGTAATGGTGAATCG

[0039] GAAAACCAAGATGAAGAGCGGGAAATGAAAGGGGAAACAGGCCGGTCCCACTCA

[0040] ACAAGGCGATGTCGAGCTGCTGCTGTACATAATCAGTCTGAAAGGAGACGTAGAGA

[0041] TAGGATCAATCAGAAAATGAAAGCTCTGCAGAAGCTTGTACCAAATGCAAGTAAGA

[0042] CTGATAAAGCATCAATGCTTGATGAGGTGATTGCGTACTTGAAGCAACTACAAGCAC

[0043] AAGTTCAGATGATGAGTAGTGCAAGAAACATGCCCCAACAGATGATGATGCCACTA

[0044] GGAATGCAGCAACACATCCACATGTCTCTCCTGGCCCGTATGGGCCTTGGTGTTGGG

[0045] CTTGGGCTGGGAATGGGCCCAATGGGGATGCTCGACATGAGTACCGTGGCTCGGCC

[0046] TCAGTCTCTCTCTCCCCTCCTCCATCCGGGCACCGTGGCTGCAGCGGTCACAGCCG

[0047] GTCCCACATTTGTTCCCCCTCCCTTTGTGGTGCCTCCCATGATGCCAAGCCACACTC

[0048] AGGCACAGGCAACTTCAGATGCAGCTATCAACAATTCAGTTCCTTTTAACAATCCCT

[0049] ACTTGTGCATTTCTAGCTCAAGTAAGTGAC Amino acid sequence encoded by the sPIF7 gene, SEQ ID NO.2 (CSS0002807.1)

[0050] MKGIMNQCIVHNWNPRHQRQEQVEGEEEKTSSHVHSHLHNLTTTHIVPMSNFEVTELT

[0051] WENGQLAMHGLGGLLPTAPTKPTWGRTGDTLESIVHLAACHQQNLNLLPPHDHLHHH

[0052] NPAPNRTTSSAKKCAETVGHMPMSPGFSKKRMWSESEKKFASGIQDDRSACASATIFK

[0053] DSGTTMMTWASFESPPQSLKTKTTDEDSACNGESENQDEEREMKGETGRSHSTRRCR

[0054] AAAVHNQSERRRRDRINQKMKALQKLVPNASKTDKASMLDEVIAYLKQLQAQVQMM

[0055] SSARNMPQQMMMPLGMQQHIHMSLLARMGLGVGLGLGMGPMGMLDMSTVARPQS

[0056] LSPLLHPGTVAAAVTAGPTFVPPPFVVPPMMPSHTQAQATSDAAINNSVPFNNPYLCISS

[0057] UTR region of the SSKNCsPIF7 gene (SEQ ID NO.3)

[0058] CGAGCTGCTGCTGTACATAATCAGTCTGAAAGGAGACGTAGAGATAGGATCAATCA

[0059] GAAAATGAAAGCTCTGCAGAAGCTTGTACCAAATGCAAGTAAGACTGATAAAGCAT

[0060] CAATGCTTGATGAGGTGATTGCGTACTTGAAGCAACTACAAGCACAAGTTCAGATG

[0061] ATGAGTAGTGCAAGAAACATGCC

[0062] 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.4 and SEQ ID No.5 respectively:

[0063] CsTS1 promoter (SEQ ID No.4):

[0064] ATCTAATAGGTAAGGTCTTGTTTGGGGATATAATCAAAAAGTTTTTATAAATTGTCAC

[0065] AATATTATTTTATTTATATTTTTATAAAAATTAAGAAAGTAACTAAAATAATTAATTTCC

[0066] AAACGAGTTATTAATTTTGACTTATTATGAAAATAAGAAAAAAAATCAACTTTTTAAT

[0067] TTTTTTATAATTTTTTTATAATAAAAATAAAAATATAATATGATCCAAATAAAATAATAA

[0068] ACTTATTTTTTTTTAGTTGTCAAAATGTGGGTACACAGTAGGGATGTAAGAAAAATC

[0069] GAAAAAATCGACCAAATCGATTGAACCGAACCAAACCGGCTATATTTGGTCTGTTTT

[0070] TTAACAAGAATCGGTCTGTGCGGTTTTCAAATTTGAATTTTTTGGATTTTCGGTTCGG

[0071] TATCGGTTTGAGCACCTTGTGCACCGATCTAAACCAAAACCGACTGCTTCAACATAT

[0072] ATTGTATTTATAATATATATATATATATATATAATCTATTAATCTAATGATATTAATAAGTAT

[0073] AATAAATTATTAACCTTAATTAATTAACCTTATCATATTCATAACCTAAGTTTGTAAAC

[0074] TCTTATTATATACTTCATCCGTCCCAAAATATTAGTCCCTTATTCCATTTTTCATTGTCC

[0075] CAAAATATTAGTCTCTCTTCAAAGATCAAGTACAAAAATAATATAAATTTCCTATTTT

[0076] GTCTTTCTTTTAAATTAATAAACAATATAAAAAGTACAATCATGACATTGATATTCCCA

[0077] AAAAATGTACTATCAAAAGGGTAATTTTGGAAAATCAATTTTTTTCAAATGCAATAAT

[0078] TGTACTACCAAAAAAAGTTAGATTCCCCAAAAGGGACCATTATTTTGGGACGGAGG

[0079] GAGTATAATCTTAACTCTAACTCATTATTCTTGAAACCTCAGTATTTTTAAACTTTTTT

[0080] TTTTTGTTTTTATTTTTATTAATTTAGGAATTAAAAAGTATGTATTAAATGGATGAATTG

[0081] GATACGTTTGAAATGGTAAACAAATAACTAATTTAATATTTTTATATAGATTAATTATTA

[0082] TATTTAAAAATGACTCAAGTTTGAAAAATTTTAAACAGATTAAGGCCAAGTTTGGCA

[0083] TAATTAAAAAAAGACAAAAAGTCAAAAAGTCACTATCCTATTTAAGTCACATCATCT

[0084] TATTTAAGTCACATCACCTCTCTAACACATCAATTCAAACCTAACCCAAACCCAACC

[0085] CATATTTCATAAAAAGTTAAAAAGTGCACTTATTTAGTCTTCCCAAACTAGGCCTAAA

[0086] TCACACCAAACAAAACTGTTTCAATTGGTTTGATTCAGTGTAATTTGATGTGAGAAC

[0087] TGTATAAATATTTAAAATAATAAAATCGACATTAACAATTCAATATAATTTTTTATTAAA

[0088] ATACCGAACATACCAAACCGATTACATCGTGCTACACAGACTCGTAAAAACAGAAG

[0089] GCATTAATTCGAGGGGGCTGCTCTGCTCTGGACTCTATAAAACCCACTCATGTCCTC

[0090] TGCTCACGGCTCTGCATTAAAAGCACAGACAGAGAGAGAGAGAGAAGCAGAGCGA

[0091] GAGAGACAGAGAGAGCC

[0092] CsGS1 promoter sequence (SEQ ID No.5):

[0093] CGAAACTCATTTTTTATGATTGAATTTACGAGATAAATGATTTAAATTATTGTTATAGA

[0094] AAAAATTATATCCAATTATGTAATTAGGTTAGAAATGGTTGCCCAATGTATCACTATCG

[0095] TCTATACTTTTTCATATTCTATTTTGTAAAATTTGTAGGCCAGTATCATCATCGTTCATA

[0096] TTTCTTCATATTCTAATTTGTAGAATTCATTTTTATTACTAGACTTATAAGATAAATGGT

[0097] TGATATCCTTTTTGTAGGTCCAAAATAGAGGCAAAAAGTAAAATATGAAAGAATATA

[0098] TCATCCATACTTTTTTATATTCTATTTTCAGAATATTTCATCATTGAAAAATGATTTGCA

[0099] CCCCTAATTTTGAAGACCCCAGTGACCCCCAACTGATGTGGCGGCATGCAATTGGTT

[0100] GAATTTTTTTTTTTTCCCTTGGTCCACAGCTTCCCACGTGGAGTGGGGGTGCTAGGG

[0101] GTGCCCAGCACAGCTCATTCATTATTATTACTATTTATGAAATAAATGATTCAAATTAT

[0102] CGTTGAGTGTATAAAATAGAGCATTTTTTAAGATAGACTAATTTAGGCTTCATTTGGC

[0103] ATTGACATTTTGTGCTAAATTATTCACAAAAAAAAAGAAGATAAAAAGTTAATAAGA

[0104] TAAAATACCACATCCAAATCATAATCATTTTATTTTTCTTTTACAAAAATTAAAAAAA

[0105] ACTAAAAAAAATCAACCCATACGAACTTTTTAGCCGAAGCTAAAATAGTCAAAAAA

[0106] TTAAAAAGTCAATTAAAAAAGTCACATGCCAAACAACAACTTAGTCTGATTATTTTT

[0107] TATTTTTTTATTTATTTTTAACCAGACCAAAAAAGTTATTTTTAGGATAAATTTTGAAT

[0108] TTTTTCTAATAGTTAATTTGATGGTTATAAAAGAAAAAATAAAATGATTATAATTTGAA

[0109] TGTGACTTTTTATTTTATTGACTAAATTAATCTATTTTATAAATAATTTAACCAAAATAA

[0110] CTCATGTCAAAGCCTACCCAAAATAAGATACGAAACAATATAAGGGGTGTTTGGGA

[0111] AGATAGTTGAGTTAAAATAACTCACTAAAAGATAAAATAGACTAAAATAATCAATAA

[0112] GATAATATATTGCATCTAACCTATAACCGTTTTATTTTTTCTTTGATAACTATCAAATCA

[0113] GGGGTGTTTTGAGAAGATGATTAAAAGTTATTTTTTGACTTTTTTGTGAGAGAGGGA

[0114] AATGGGTTAAAAGCTGATAAGTGACTTTTAACACACTTTTCAAAATAGCCCTCAATT

[0115] ACAAAAAAGCTAATAAACTATTTTAGTCAGATTAAAATAGTCAAAAAATTAAATAGT

[0116] TAACTAAAATAGTCAACTTCCCAAATAAGGCCATAGTAAATTAGGGAAATACTAATAT

[0117] ACATACTCTTAAAAATGATATACTTTATACATTTTTAATTATGTCATTTTCTTCTTTAATT

[0118] ATGATATTTATTTATTTTTAATTTTTTTAGTAATGACTTAAAAAATATAAAATTATATTAT

[0119] TTTTTTAATTAGAATGTGCATAAGATATACATCTTTTTAAGAGTATACATAGTGTACAT

[0120] CACTTTTTTTACACTTTAAAAAAAAAAATCAAAAAATTTAACTTTCAACTTACTTTTC

[0121] AAAAAAATTCTAATATCTATTTTGGGGACGAAGTTAAAAATATATTTTTTTTAACTTTT

[0122] AATCTCTTCTCAGTCCCATCTAAATTTTTTTAACTCTTTTTAAAAAAATATATAAAAAA

[0123] ATAAAAAATAATGTAACTCGAAATAAATTAAAAATTAAAAAATAAAATTTAACAACC

[0124] ATTCTAAACTAGTGCAATGTAAAACACCTCAACCACTATAAAAGGACGAACTCATCC

[0125] CTGGTTCTACAGCACATTACTCCTCGCCACAGACTCCAACACAAACCACGTATAGTA

[0126] CGGAGAGAAAGAGGAGAGAGGGAGAGTAAAAGCATCGTGTGGTTTGTTTGGAGTG

[0127] GGTGTTTTTGATCGATCGTCACT

[0128] Preparation of related culture media in the present invention:

[0129] (1) YEP solid medium: 10 g / L peptone + 10 g / L yeast extract + 5 g / L sodium chloride + 7.5 g / L agar powder.

[0130] (2) YEP liquid medium: 10 g / L yeast extract + 10 g / L peptone + 5 g / L sodium chloride.

[0131] All biomaterials involved in the following examples are commercially available.

[0132] Example 1 Subcellular localization of CsPIF7 in tea plants

[0133] (I) Experimental method

[0134] 1. The tobacco used is Nicotiana benthamiana.

[0135] 2. Construction of fusion expression vector

[0136] Primers with restriction sites were designed through CsPIF7 CDS (Table 1). The plasmid was extracted from the correct cloning product of CsPIF7 gene CDS. The gene was digested with Xba I and Kpn I, and the vector was digested with Xba I and Kpn I. It was ligated to the PCAMBIA1300-35S-GFP vector through T4 DNA ligase. The obtained ligation product was transformed into DH5a competent cells. After PCR amplification, restriction enzyme digestion screening, and correct sequencing verification, positive clones were screened and plasmids were extracted to obtain the fusion expression vector PCAMBIA1300-35S-CsPIF7-GFP of GFP and the target gene. The construction of the subcellular localization vector was synthesized by Wuhan Transduction Biotechnology Laboratory Co., Ltd.

[0137] Table 1 Names and sequences of amplification primers

[0138]

[0139] 3. Steps of tobacco transient transformation

[0140] ⑴ Shake the successfully detected Agrobacterium liquid overnight at 28 °C and 200 rpm;

[0141] ⑵ Take 1 ml of the bacterial liquid and add it to a sterilized 1.5 ml centrifuge tube;

[0142] (3) 8000 rpm, for 2 min, precipitate the bacterial cells (at room temperature), discard the supernatant, add 1 ml of permeation solution, and resuspend the bacterial cells;

[0143] (4) Repeat step 3 to further remove a small amount of antibiotics;

[0144] (5) Take a small amount of the resuspended bacterial solution, dilute it 10-fold, measure the OD 600 value, and multiply it by 10 to obtain the OD 600 value of the resuspended bacterial solution;

[0145] (6) Determine the titer of the resuspended bacterial solution against the permeation solution, calculate the dilution factor, and make the final resuspended bacterial solution (for infection) 5.0 ml, with an OD 600 of 0.4 (0.1 - 0.8 as needed, not exceeding 1). Usually, 0.5 - 1.0 ml of the final resuspended bacterial solution can meet the infection requirements;

[0146] (7) In a 1.5 ml centrifuge tube, prepare the final resuspended bacterial solution and let it stand at room temperature for 2 hours to prepare for infection;

[0147] (8) Before infection, place the tobacco under a white fluorescent lamp for 1 h to open its stomata;

[0148] (9) Select the third and fourth leaves from the top and use them for infection (infect between the two leaf veins). Select two leaves for one plant and infect with one bacterial solution;

[0149] (10) Use a syringe without a needle to gently rub the back of the leaf to be transformed, or pierce it with a small needle to remove its waxy layer;

[0150] (11) Before infection, mark the area to be transformed with a marker pen;

[0151] (12) Aspirate the final resuspended bacterial solution in step (7) into a 1 ml syringe without a needle;

[0152] (13) Point the syringe at the area to be transformed on the back of the leaf. Hold the leaf from above 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 pen;

[0153] (14) Spray water on the leaf, put it in a fresh-keeping bag, and place the infected tobacco back in the culture room and keep it in the dark overnight;

[0154] (15) Open the fresh-keeping bag the next day. The expression level is the highest 2 days after injection.

[0155] (16) Cut the infected area, tear the epidermis to make a slide, and observe it under a confocal laser microscope.

[0156] As Figure 1 shown by the subcellular localization results: CsPIF7 is localized in the nucleus and is a transcription factor. Its domain is asFigure 2 as shown

[0157] Example 2 Verification of the function of CsPIF7 in the Agrobacterium-mediated transient overexpression system of tea plants

[0158] (I) Experimental method

[0159] 1. Experimental materials:

[0160] The materials were cuttings of the "Fuding Dabaicha" tea plant from the Tea College of Guizhou University.

[0161] 2. Construction of plant overexpression vector

[0162] The plant overexpression vector was based on the initial vector pSH737, and the pSH737-35S-CsPIF7 vector was designed and constructed (the vector structure is as Figure 3 shown). The recombinant plasmid was transformed into competent Escherichia coli (DH5α), and positive clones were screened using Kan 100 mg·L -1 . The recombinant plasmid with a positive digestion verification result was transformed into competent cells of Agrobacterium tumefaciens strain LBA4404, and Agrobacterium positive strains were screened using Kan 100 mg·L -1 , Rif 20 mg·L -1 . The positive Agrobacterium strains were detected by colony PCR using primers, and the Agrobacterium strains with positive PCR results were expanded and cultured and stored at -80 °C. The plasmid containing the overexpression vector was transformed into Agrobacterium strain LBA4404 by the freeze-thaw method to prepare engineering strains. Take the LBA4404 competent cells stored at -80 °C and place them on ice to thaw for 10 min; add 5 μL of plasmid DNA to each tube of competent cells, gently flick to mix and then incubate on ice for 30 min; after quick-freezing in liquid nitrogen for 5 min, immediately incubate in a water bath at 37 °C for 2 min, add 900 μL of YEP liquid medium preheated at 37 °C, and incubate at 28 °C with shaking at 200 rpm for 3 h; at room temperature, centrifuge at 4000×g for 1 min, discard the supernatant, add 100 μL of YEP liquid medium to the cell pellet, pipette to mix, take an appropriate amount of the bacterial solution and spread it on a YEP plate medium containing 100 mg·L -1 Kan and 20 mg·L - 1 Rif, and invert and culture in an incubator at 28 °C for 2 d, and store the bacterial solution at -80 °C.

[0163] 3. Tea plant infection

[0164] The pSH737-35S-CsPIF7 recombinant plasmid was introduced into competent Agrobacterium tumefaciens LBA4404 by the freeze-thaw method. Each Agrobacterium strain was inoculated into a medium containing 100 mg·L -1 kanamycin and 50 mg·L-1 In the solid YEP medium of rifampicin, it was activated and cultured at 28 °C for 48 h. Single colonies were cultured in the corresponding liquid YEP medium until the OD 600 reached 1.0. The Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial solution was collected and suspended in 4.74 g / L MS, 0.5% (m / v) D-glucose, 150 μmol / L acetosyringone (AS), and 25 μmol / L MES, with a pH of 5.6. Agrobacterium carrying the pSH737 vector with the 35S promoter was injected into the second leaf as a control (CK), and the CsPIF7 overexpression vector was injected and infected into the second leaf of the tea plant respectively. Each experiment was set with at least 5 replicates, and each replicate had 5 leaves. Samples were collected 3 days after injection and divided into two parts. One part was immediately frozen in liquid nitrogen, and the other part was subjected to fixation by heating, drying, grinding, and mixing evenly, and then qRT-PCR and theanine detection were carried out respectively. The detection results are as Figure 4 and Figure 5 shown.

[0165] 4. Detection of the expression levels of related genes:

[0166] Specific primers for the CsPIF7, CsTS1, and CsGS1 genes were designed using Primer Premier 5.0 software (the primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. Chongqing Branch), and the tea plant actin gene (Actin) was used as an internal reference (the primer sequences are shown in Table 2). The qRT-PCR experiment was operated on a Bio Rad CFX ConnectTM real-time quantitative PCR instrument (Bio-Rad). The qRT-PCR used a Nanjing Novoprotein universal high-sensitivity dye-based quantitative PCR detection kit. According to the instructions on the kit, the qRT-PCR reaction system was configured, and the 20 μL system is shown in Table 3 below.

[0167] Table 2 Primer names and sequences

[0168] Name Sequence(5’-3’) Serial number PIF7-F ATGAAAGGAATCATGAATC SEQ ID NO.6 PIF7-R TCACTTACTTGAGCTAG SEQ ID NO.7 qPCRPIF7-F CAAGGCACCAAAGACAAGAAC SEQ ID NO.8 qPCRPIF7-R GATGGTCATGTGGAGGAAGTAG SEQ ID NO.9 qPCRTS1-F GACAAGCTCCTGGTGAAGATAG SEQ ID NO.10 qPCRTS1-R CCATTCCAATCACCCTCGATAG SEQ ID NO.11 qPCRGS1-F ATCAGTTGTGGATGGCTCG SEQ ID NO.12 qPCRGS1-R CACTTCGCATGGACTTGGTAC SEQ ID NO.13 qPCRActin-F CAGACCGTATGAGCAAGGAAAT SEQ ID NO.14 qPCRActin-R GTGCTTAGGGATGCAAGGATAG SEQ ID NO.15

[0169] Table 3 Fluorescent quantitative PCR reaction system

[0170] Component Volume (μL) cDNA 3μL Primer F 1μL Primer R 1μL 2×ChamQ Universal SYBR qPCR Master Mix 10μL Nuclease-free H2O 5μL Total volume 20μL

[0171] (2) Experimental results

[0172] As Figure 4 and Figure 5As shown in the figure, after overexpressing CsPIF7, the theanine content increased significantly, rising by 108% compared with the control group (CK). The expression level of CsPIF7 increased by 29.67%, and the expression levels of CsTS1 and CsGS1 also increased by 160% and 95.67% respectively. It can be seen that overexpressing CsPIF7 can activate the expression of CsTS1 and CsGS1, and can significantly increase the theanine content in tea plants.

[0173] Example 3 Verification of the function of CsPIF7 by virus-induced gene silencing (VIGS) technology

[0174] (I) Experimental method

[0175] 1. Experimental materials: Cuttings of "Fuding Dabaicha" tea plants from the Tea College of Guizhou University.

[0176] 2. Construction of plant VIGS vector

[0177] Gene fragment structure: EcoRI-CsPIF7-BamHI

[0178] Construction of pTRV2-CsPIF7 plasmid:

[0179] Obtain the CsPIF7 gene fragment by total gene synthesis method and load it onto the pUC57-Simple vector to obtain the pUC57-Simple-CsPIF7 plasmid. Then double-digest the pUC57-Simple-CsPIF7 plasmid with EcoR I and BamH I to obtain the CsPIF7 gene fragment, run gel electrophoresis and recover the fragment. At the same time, double-digest the pTRV2 vector with EcoR I and BamH I, run gel electrophoresis and recover the vector fragment. Insert the UTR region of CsPIF7 into the empty TRV2 (the vector structure is as Figure 6 shown). The ligation product was transformed into DH5a competent cells. Pick several clones on the transformed plate, extract the plasmid, and perform enzyme digestion identification with XhoI and HindIII. The recombinant plasmid was further sequenced (Note: The above processes were all entrusted to Changsha Kewen Biotechnology Co., Ltd. to complete).

[0180] 3. Infection of tea plants

[0181] Introduce pTRV1, pTRV2, and pTRV2-CsPIF7 into Agrobacterium tumefaciens GV3101 competent cells by the freeze-thaw method. Inoculate each Agrobacterium strain into a solid YEP medium containing 100 mg·L -1 kanamycin and 50 mg·L -1 rifampicin, and activate and culture at 28 °C for 48 h. Single colonies were cultured in the corresponding liquid YEP medium until OD 600It was 1.2. The Agrobacterium cells were centrifuged at 6000 rpm for 6 min. The bacterial solution was collected and suspended in MS at 4.74 g / L, 6-BA at 2 mol / L, acetosyringone (AS) at 2 mol / L, and naphthaleneacetic acid (NAA) at 100 μmol / L, with a pH of 5.6. Two tea tree materials were subjected to vacuum infiltration treatment. The OD 600 was adjusted to 1.2. At room temperature, pTRV1 was mixed with pTRV2 and pTRV2-CsPIF7 bacterial solutions at a ratio of 1:1 (pTRV1 + pTRV2, pTRV1 + pTRV2-CsPIF7), and the tea tree cuttings were subjected to vacuum infiltration treatment. The tea tree cuttings were cut to a length of 20 cm with pruning shears. Two mature leaves were retained, and then the tea tree cuttings were placed in a Buchner flask containing the mixed bacterial solution for vacuum infiltration. They were stored in the dark for three days and then grown in a greenhouse at a temperature of 25 °C with a light / dark cycle of 16 h / 8 h.

[0182] The pTRV2-CsPIF7-silenced plants, pTRV2 plants, and wild-type plants (WT) were vacuum-inoculated. After lateral buds grew out after 45 days of cultivation, samples were collected, blanched, dried to a constant weight, and ground into powder. The theanine content was determined by high-performance liquid chromatography.

[0183] 4. Detection of the expression levels of related genes: The same as in Example 2.

[0184] (2) Experimental results

[0185] As Figure 7 and Figure 8 shown, after VIGS silencing of CsPIF7, the theanine content decreased by 77.73%, 56.06%, and 79.2% compared with the control (pTRV2), respectively. The expression level of CsPIF7 decreased by 92.66%, 54.67%, and 81%, respectively; the expression level of CsTS1 decreased by 31.67%, 53%, and 17%, respectively; the expression level of CsGS1 decreased by 32.97%, 51.44%, and 24.98%, respectively. It can be seen that after reducing the expression of CsPIF7 by VIGS technology, the activation and expression of TS1 and GS1 are reduced, resulting in a decrease in the theanine content in tea leaves.

[0186] Example 4 Verification of the regulatory effects of LUC on CsPIF7, GS1, and TS1

[0187] Analyze the transcriptional activity in tobacco by the dual-luciferase assay system. Clone the CDS of CsPIF7 into the pGreenII 62-SK vector as an effector respectively, and introduce the promoter fragments of CsTS1 and CsGS1 into the pGreenII 0800-LUC vector as reporter genes, which are used to transform the Agrobacterium tumefaciens strain EHA105 respectively, and then perform transient fusion expression of the recombinant plasmids in the leaf cells infected with EHA105. After 3 days, spray the infected leaves with 0.2 mg·mL -1 D-luciferin sodium salt and incubate at 37 °C for 10 minutes. Use a chemiluminescence device named Fusion FX7 (VILBER, France) to create fluorescence images and calculate the fluorescence values.

[0188] As Figure 9 shown: In both figures, the fluorescence intensity in region 4 increased significantly compared to region 3. That is, CsPIF7 can positively activate the promoters of CsTS1 and CsGS1.

[0189] In summary, in the present invention, by highly expressing CsPIF7, the expression of TS1 and GS1 is activated, enabling tea leaves to accumulate more theanine; by using the VIGS technology of tea plants to reduce the expression of CsPIF7, the activation of TS1 and GS1 is reduced, thereby decreasing the theanine content in tea leaves. The tea plant transcription factor CsPIF7 gene can positively regulate the expression of TS1 and GS1.

[0190] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Transcription Factors CsPIF7 The application of a gene in regulating theanine synthesis in tea plants is characterized in that: The transcription factor CsPIF7 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: Using the transcription factor CsPIF7 The method for genetically regulating theanine synthesis in tea plants is as follows: CsPIF7 The recombinant vector is transferred into tea leaves. CsPIF7 Silencing or transient overexpression can inhibit or promote theanine synthesis in tea plants.

3. The use according to claim 2, characterized in that: The transient overexpression vector was constructed by inserting the transcription factor between the Xba I and Kpn I restriction sites of the original vector pSH737-35S. CsPIF7 The gene was obtained and named: 35S-CsPIF7 。 4. The use according to claim 2, characterized in that: Silencing vector is a transcription factor CsPIF7 The pTRV2 empty vector was inserted between the restriction sites EcoR I and BamH I in the UTR region of the gene to obtain a VIGS silencing vector for silencing the target gene, which was named: pTRV2-CsPIF7.

5. The use according to claim 2, characterized in that: Transcription Factors CsPIF7 The primer pairs for the genes are: PIF7-F:ATGAAAGGAATCATGAATC; PIF7-R:TCACTTACTTGAGCTAG。

Images