Application of transcription factor CsPIF7 gene in regulation and control of theanine synthesis and improvement of tea quality

Abstract

The invention provides application of a transcription factor CsPIF7 gene in regulation and control of theanine synthesis and improvement of tea quality, and belongs to the technical field of agricultural molecular biology. The invention finds that high expression of CsPIF7 can activate expression of TS1 and GS1, so that more theanine can be accumulated in tea leaves; after the expression of CsPIF7 is reduced by a tea tree VIGS technology, the activation and expression of TS1 and GS1 can be reduced, so that the content of theanine in tea leaves is reduced. Furthermore, the tea tree transcription factor CsPIF7 is found to be capable of positively regulating and controlling expression of TS1 and GS1 by utilizing LUC verification, and can be used for regulating and controlling the content of theanine in tea trees and improving the quality of tea leaves.

Description

Technical Field

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

[0002] Tea tree (Camellia sinensis (L.) O. Kuntze) is one of the most important economic crops in the world. Its economic value is determined by its leaves, which are also related to its growth and development and material accumulation. Among them, theanine in tea leaves is the main contributing factor to the umami taste of tea, and plays a decisive role in the quality and economic value of tea leaves.

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

[0004] PIF7 is the main regulator of response to shade. Shade stress reduces the ratio of red light (660nm) to far-red light (730nm). 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, phyB mostly moves to the nucleus in the active form of far-red absorption (Pfr) and interacts with basic helix-loop-helix (bHLH) proteins, which are called photolytic pigment interacting factors (PHYTOCHROME-INTERACTING FACTOR). The light activation of phyB induces rapid phosphorylation of PIF1 / 3 / 4 / 5 before they are degraded. When plants are under shade conditions, phyB is mainly in the inactive red light absorbing (Pr) cytoplasmic form, which promotes the accumulation of PIFs. It has been reported that the hypocotyl phenotype of PIF7 mutants in Arabidopsis is the most obvious, but the prior art has not studied whether the tea tree transcription factor CsPIF7 responds to shade and participates in theanine synthesis. Summary of the invention

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of 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 shown in SEQ ID No.2.

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

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

[0012] Preferably, the silencing vector is a pTRV2 empty vector inserted between the restriction sites EcoR I and BamH I 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 leaves from tea trees.

[0017] The present invention constructs a transient expression vector and finds that high expression of the transcription factor CsPIF7 can activate the expression of TS1 and GS1, allowing tea leaves to accumulate more theanine; after reducing the expression of CsPIF7 through the tea tree VIGS technology, the activation and expression of TS1 and GS1 will be reduced, thereby reducing the theanine content in the tea leaves. Furthermore, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the subcellular localization map of CsPIF7.

[0019] Figure 2 Schematic diagram of the domain structure of transcription factor CsPIF7.

[0020] Figure 3 Schematic diagram of the construction of CsPIF7 overexpression vector.

[0021] Figure 4 This is a graph showing the theanine content in tea plants after transient expression of the CsPIF7 gene.

[0022] Figure 5 This is the expression level diagram of CsPIF7, GS1 and TS1 in tea leaves after transient expression in tea trees.

[0023] Figure 6 Schematic diagram of the construction of the VIGS CsPIF7 silencing vector.

[0024] Figure 7 This is the theanine content in tea plants after VIGS silencing the CsPIF7 gene.

[0025] Figure 8 This is the expression level diagram of CsPIF7, GS1 and TS1 in tea leaves after VIGS silencing the CsPIF7 gene.

[0026] Fig. 9 The results of transcriptional activation (Figure A is the TS1 fluorescence detection result; Figure B is the GS1 fluorescence detection result; Figure C is the TS1 fluorescence intensity statistical result; Figure D is the GS1 fluorescence intensity statistical result). DETAILED DESCRIPTION

[0027] Nucleotide sequence of 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 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:

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

[0064] ATCTAATAGGTAAGGTCTTGTTTGGGGATATAATCAAAAAGTTTTTATAAATTGTCAC

[0065] AATATTATTTTATTTATATTTTTATAAAAATTAAGAAAGTAACTAAAATAATTAATTTCC

[0066] AAACGAGTTATTAATTTTGACTTATTATGAAAATAAGAAAAAAAATCAACTTTTTAAT

[0067] TTTTTTATAATTTTTTTATAAATAAAAATAAATAATATATAATGATCCAAATAAAATAATAA

[0068] ACTTATTTTTTTTTAGTTGTCAAAATGTGGGTACACAGTAGGGATGTAAGAAAAATC

[0069] GAAAAAATCGACCAAATCGATTGAACCGAACCAAACCGGCTATATTTGGTCTGTTTT

[0070] TTAACAAGAATCGGTCTGTGCGGTTTTCAAATTTGAATTTTTTGGATTTTCGGTTCGG

[0071] TATCGGTTTGAGCACCTTGTGCACCGATCTAAACCAAAACCGACTGCTTCAACATAT

[0072] ATTGTATTTATAATATATATATATATATATATAATCTATTAATCTAATGATATTAATAAGTAT

[0073] AATAAATTATTAACCTTAATTAATTAACCTTATCATATTCATAACCTAAGTTTGTAAAC

[0074] TCTTATTATATACTTCATCCGTCCCAAAATATTAGTCCCTTATTCCATTTTTCATTGTCC

[0075] CAAAATATTAGTCTCTCTTCAAAGATCAAGTACAAAAATAATATAAATTTCCTATTTT

[0076] GTCTTCTTTTAAATTAATAAACAATATAAAGGTACAATCATGACATTGATATTCCCA

[0077] AAAAATGTACTATCAAAAGGGTAATTTTGGAAAATCAATTTTTTTCAAATGCAATAAT

[0078] TGTACTACCAAAAAAAGTTAGATTCCCCAAAAGGGACCATTATTTTGGGACGGAGG

[0079] GAGTATAATCTTAACTCTAACTCATTATTCTTTGAACCTCAGTATTTTTAAACTTTTT

[0080] TTTTTGTTTTTATTTTTTAATTTAGGAATTAAAAGTATGTATTAAATGGATGAATTG

[0081] GATACGTTTGAAATGGTAAACAAATAACTAATTTAATATTTTTATATAGATTAATTTA

[0082] TATTTAAAAATGACTCAAGTTTGAAAAATTTTAAACAGATTAAGGCCAAGTTTGGCA

[0083] TAATTAAAAAAAGACAAAAAGTCAAAAAGTCACTATCCTATTTAAGTCACATCATCT

[0084] TATTTAAGTCACATCACCTCTCTAACACATCAATTCAAACCTAACCCAAACCCAACC

[0085] CATATTTTCATAAAAGTTAAAAAGTGCACTTATTTAGTCTTCCCAAACTAGGCCTAAA

[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] CGTTGAGTGTATAAAATAGAGCATTTTTTAAGATTAATTTAGGCTTCATTTGGC

[0103] ATTACKTTTTGTGCTAAATTATTCCAAAAAAAAAAAAAAAAGATTTAT

[0104] TAAAATACCAAATCAATCATTTTTTTTCTTTCAAAAAATTAAAAAAA

[0105] ACTAAAAAAAATCAACCCATACGAACTTTTTAGCCGAAGCTAAAATAGTCAAAAAA

[0106] TTAAAAAGTHELPAAAAAGTCACATGCCAAAACATTAGTCTGATTTTT

[0107] TATTTTTTTATTTTTAACCAGCAAAAGTTTTTTAGGATAAATTTTGAAT

[0108] TTTCTAATAGTATTTGATGGTTAAAAAAAAAATTATTTATTTATTGA

[0109] TGTGACTTTTTTTTTTATTGACTAAATTAATTATTATTATTTATTTAAAAAAAAAAAAAAAAT

[0110] CTCATGTCAAGCCTACCCAAAAAGATACGAAACAATAAGGGGTGTTTGGGA

[0111] AGATAGTTGAGTTAAAAACTCATTAAAATAAATATATACTAATAATTACTAA

[0112] GATAATATTGCATCTAACCTATAACCGTTTTTTTTTCTTTGATAACTATCAAATCA

[0113] GGGGTGTTTTGAGAGATGATTAAGTTATTTTGACTTTTTGTGAGAGAGGGA

[0114] AATGGGTTAAAAGCTGATAAGTGACTTTTAACACACTTTTCAAAATAGCCCTCAATT

[0115] ACAAAAAAGCTAATAAACTATTTTAGTCAGATTAAAATAGTCAAAAAAATTAAATAGT

[0116] TAACTAAAATAGTCAACTTCCCAAATAAGGCCATAGTAAATTAGGGAAATACTAATAT

[0117] ACATACTCTTAAAAATGATATACTTTATACATTTTTAATTATGTCATTTTCTTCTTTAATT

[0118] ATGATATTTATTTATTTTTAATTTTTTTAGTAATGACTTAAAAAATATAAAATTATATTAT

[0119] TTTTTTAATTAGAATGTGCATAAGATATACATCTTTTTAAGAGTATACATAGTGTACAT

[0120] CACTTTTTTTACACTTTAAAAAAAAAAATCAAAAATTTAACTTTCAACTTACTTTTC

[0121] AAAAAAATTCTAATATCTATTTTGGGGACGAAGTTAAAAATATATTTTTTTTAACTTTT

[0122] AATCTCTTCTCAGTCCCATCTAAATTTTTTTAACTCTTTTTAAAAAAATATATAAAAAAA

[0123] ATAAAAAATAATGTAACTCGAAATAAATTAAAAATTAAAAAATAAAATTTAACAACC

[0124] ATTCTAAACTAGTGCAATGTAAAACACCTCAACCACTATAAGGACGAACTCATCC

[0125] CTGGTTCTACAGCACATTACTCCTCGCCACAGACTCCAACACAAACCACGTATAGTA

[0126] CGGAGAGAAAGAGGAGAGAGGGAGAGTAAAAGCATCGTGTGGTTTGTTTGGAGTG

[0127] GGTGTTTTTGATCGATCGTCACT

[0128] The relevant culture medium preparation in the present invention is:

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

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

[0133] 1. Experimental methods

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

[0135] 2. Construction of fusion expression vector

[0136] Primers with restriction sites were designed based on CsPIF7 CDS (Table 1), plasmids were extracted from cloned products with correct CsPIF7 gene CDS, genes were digested with Xba I and Kpn I, vectors were digested with Xba I and Kpn I, and connected to PCAMBIA1300-35S-GFP vectors using T4 DNA ligase, and the obtained ligation products were transformed into DH5a competent cells. After PCR amplification, restriction screening, and sequencing verification, positive clones were screened and plasmids were extracted to obtain GFP and target gene fusion expression vector PCAMBIA1300-35S-CsPIF7-GFP. Subcellular localization vector construction was synthesized by Wuhan Transduction Biological Laboratory Co., Ltd.

[0137] Table 1 Amplification primer names and sequences

[0138]

[0139] 3. Tobacco transient transformation steps

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

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

[0142] (3) 8000 rpm, 2 min, precipitate the bacteria (room temperature), remove the supernatant, add 1 ml of permeate, and suspend the bacteria;

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

[0144] ⑸Take a small amount of suspended bacterial solution, dilute it 10 times, and measure the OD 600 The value was multiplied by 10 and used as the OD of the bacterial suspension. 600 value;

[0145] ⑹ Determine the titer of the suspension to the infiltration solution and calculate the dilution factor so that the final suspension (for infection) is 5.0 ml, OD 600 0.4 (0.1-0.8 as needed, not exceeding 1), usually 0.5-1.0 ml of the final suspension solution is sufficient for infection;

[0146] ⑺ Prepare the final bacterial suspension in a 1.5 ml centrifuge tube and let it stand at room temperature for 2 hours to prepare for infection;

[0147] ⑻ Before infection, place the tobacco under a white fluorescent light for 1 hour to open its stomata;

[0148] ⑼ Select the leaves with three or four leaves in the opposite direction for infection (infect between two leaf veins). Select two leaves from one plant and infect with one bacterial solution.

[0149] ⑽Use a syringe without a needle to gently rub the back of the leaf to be turned, or pierce it with a small needle to remove its wax layer;

[0150] ⑾Before infection, mark the area to be transferred with a marker;

[0151] ⑿ Aspirate the final bacterial suspension in step (7) into a 1 ml syringe without a needle;

[0152] ⒀Point the syringe to the area to be transferred on the back of the blade, press the top of the blade 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] ⒁ Spray the leaves with water, put them in a fresh-keeping bag, put the infected tobacco back into the culture room, and leave them in the dark overnight;

[0154] ⒂Open the fresh-keeping bag on the second day. The expression level is highest 2 days after injection.

[0155] ⒃Cut the infected area, tear off the epidermis for preparation, and observe under a confocal laser microscope.

[0156] like Figure 1 Subcellular localization results show that CsPIF7 is localized in the cell nucleus and is a transcription factor. Figure 2 shown.

[0157] Example 2: Verification of the function of CsPIF7 by Agrobacterium-mediated tea plant transient overexpression system

[0158] 1. Experimental methods

[0159] 1. Experimental materials:

[0160] The materials are cuttings of "Fuding Dabai" tea trees 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 follows Figure 3 The recombinant plasmid was transformed into competent Escherichia coli (DH5α) and Kan 100 mg·L -1 Positive clones were screened, and recombinant plasmids were extracted and double-digested with XbaI and KpnI for verification. The recombinant plasmids with positive results of enzyme digestion were transformed into competent cells of Agrobacterium strain LBA4404 and incubated 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 cultured at -80°C for seed preservation. 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 place 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 bathe at 37°C for 2 minutes, add 900μL 37°C preheated YEP liquid culture medium, and culture at 28°C, 200rpm shaking for 3 hours; at room temperature, centrifuge at 4000×g for 1 minute, discard the supernatant, add 100μL YEP liquid culture medium to the bacteria, use a pipette tip to mix it, and take an appropriate amount of bacterial liquid to apply 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℃.

[0163] 3. Tea tree infection

[0164] The pSH737-35S-CsPIF7 recombinant plasmid was introduced into the competent cells of Agrobacterium tumefaciens LBA4404 by freeze-thaw method. Each Agrobacterium strain was inoculated into a medium containing 100 mg·L -1 Kanamycin and 50 mg L-1 The single colony was cultured in the corresponding liquid YEP medium until the 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 / L MES, pH 5.6. The 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 to infect the second leaf of the tea tree. 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, immediately frozen in liquid nitrogen and dried, ground, mixed evenly, and qRT-PCR and theanine tests were performed respectively. The test results are shown as follows Figure 4 and Figure 5 shown.

[0165] 4. Detection of related gene expression:

[0166] PrimerPremier 5.0 software was used to design specific primers for CsPIF7, CsTS1, and CsGS1 genes (primers were synthesized by the Chongqing Branch of Beijing Qingke Biotechnology Co., Ltd.), and the tea tree actin gene (Actin) was used as an internal reference (primer sequences are shown in Table 2). The qRT-PCR experiment was performed on a Bio Rad CFX ConnectTM real-time quantitative PCR instrument (Bio-Rad). The qRT-PCR used was the Nanjing Novogene Universal High Sensitivity Dye Method 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 Fluorescence quantitative PCR reaction system

[0170] Components 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] (II) Experimental results

[0172] like Figure 4 and Figure 5As shown in the figure, after overexpression of CsPIF7, the content of theanine increased significantly, which was 108% higher than that of the control group (CK), the expression of CsPIF7 increased by 29.67%, and the expression of CsTS1 and CsGS1 increased by 160% and 95.67%, respectively. It can be seen that overexpression of CsPIF7 can activate the expression of CsTS1 and CsGS1, and can significantly increase the content of theanine in tea plants.

[0173] Example 3 Virus-induced gene silencing (VIGS) technology verifies the function of CsPIF7

[0174] 1. Experimental methods

[0175] 1. Experimental materials: “Fuding Dabai” tea tree cuttings from the School of Tea, Guizhou University.

[0176] 2. Construction of plant VIGS vector

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

[0178] Construction of pTRV2-CsPIF7 plasmid:

[0179] The CsPIF7 gene fragment was obtained by the whole gene synthesis method and loaded into the pUC57-Simple vector to obtain the pUC57-Simple-CsPIF7 plasmid. The pUC57-Simple-CsPIF7 plasmid was then double-digested with EcoR I and BamH I to obtain the CsPIF7 gene fragment, which was run on a gel and recovered. At the same time, the pTRV2 vector was double-digested with EcoR I and BamH I, and the gel was run and the vector fragment was recovered. The UTR region of CsPIF7 was inserted into the TRV2 empty vector (the vector structure is as shown in FIG. Figure 6 The ligation product was transformed into DH5a competent cells. Several clones were picked from the transformed plate, plasmids were extracted, and enzyme digestion was performed with XhoI and HindIII for identification. The recombinant plasmid was further sequenced for identification (Note: The above process was commissioned to Changsha Kewen Biotechnology Co., Ltd.).

[0180] 3. Tea tree infection

[0181] pTRV1, pTRV2, and pTRV2-CsPIF7 were introduced into Agrobacterium GV3101 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 The single colony was cultured in the corresponding liquid YEP medium until the OD 600The 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 μmol / 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 and pTRV2-CsPIF7 in a ratio of 1:1 (pTRV1+pTRV2, pTRV1+pTRV2-CsPIF7), 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 bottle containing 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.

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

[0183] 4. Detection of related gene expression levels: Same as Example 2.

[0184] (II) Experimental results

[0185] like Figure 7 and Figure 8 As shown in the figure, after VIGS silencing CsPIF7, the theanine content was reduced by 77.73%, 56.06% and 79.2% compared with the control (pTRV2), respectively. The expression of CsPIF7 was reduced by 92.66%, 54.67% and 81%, respectively; the expression of CsTS1 was reduced by 31.67%, 53% and 17%, respectively; the expression of CsGS1 was reduced 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 will be reduced, and the theanine content in tea leaves will be reduced.

[0186] Example 4 LUC Verification of the Regulatory Effects of CsPIF7, GS1, and TS1

[0187] The transcriptional activity in tobacco was analyzed by dual luciferase assay system. The CDS of CsPIF7 was cloned into pGreenII 62-SK vector as effector, and the promoter fragments of CsTS1 and CsGS1 were introduced into pGreenII 0800-LUC vector as reporter genes, which were used to transform Agrobacterium tumefaciens EHA105 strain, and then transient fusion expression of recombinant plasmids was performed in leaf cells infected with EHA105. After 3 days, 0.2 mg mL -1 Infected leaves were sprayed with sodium D-luciferin and incubated for 10 min at 37° C. A chemiluminescent instrument named Fusion FX7 (VILBER, France) was used to create fluorescent images and calculate the fluorescence values.

[0188] like Fig. 9 As shown in the two figures, the fluorescence intensity in area 4 is significantly increased compared with that in area 3. That is, CsPIF7 can positively activate the promoters of CsTS1 and CsGS1.

[0189] In summary, the present invention activates the expression of TS1 and GS1 by highly expressing CsPIF7, allowing tea leaves to accumulate more theanine; and reduces the expression of CsPIF7 through tea tree VIGS technology, reducing the activation of TS1 and GS1, thereby reducing the theanine content in tea leaves. The tea tree transcription factor CsPIF7 gene can positively regulate the expression of TS1 and GS1.

[0190] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of transcription factor CsPIF7 gene in regulating theanine synthesis.

2. The use as claimed in claim 1, It is characterized in that The nucleotide sequence of the transcription factor CsPIF7 gene is shown as SEQ ID No.

1.

3. The use as claimed in claim 2, It is characterized in that The amino acid sequence of the protein encoded by the transcription factor CsPIF7 gene is shown in SEQ ID No.

2.

4. The use as claimed in claim 3, It is characterized in that The method for regulating theanine synthesis by using the transcription factor CsPIF7 gene is as follows: constructing a recombinant vector of CsPIF7, transferring the recombinant vector into tea leaves to silence or transiently overexpress CsPIF7, thereby inhibiting or promoting the synthesis of theanine in tea trees.

5. The use as claimed in claim 4, It is characterized in that The transient overexpression vector was obtained by inserting the transcription factor CsPIF7 gene between the Xba I and Kpn I restriction sites of the original vector pSH737-35S and was named: 35S-CsPIF7.

6. The use as claimed in claim 4, It is characterized in that The silencing vector is a pTRV2 empty vector inserted between the restriction sites EcoR I and BamH I in the UTR region of the transcription factor CsPIF7 gene to obtain a VIGS silencing vector for silencing the target gene, named: pTRV2-CsPIF7.

7. The use as claimed in claim 4, It is characterized in that The primer pairs for the transcription factor CsPIF7 gene are: PIF7-F: ATGAAAGGAATCATGAATC; PIF7-R:TCACTTACTTGAGCTAG.

8. Application of transcription factor CsPIF7 gene in improving tea quality of tea trees.

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