CN114686493B - CsJAZ12 gene and application thereof in regulating and controlling synthesis of tea caffeine - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a CsJAZ12 gene and application thereof in regulating and controlling synthesis of tea caffeine. Wherein the nucleotide sequence of the CsJAZ12 gene is shown as SEQ ID NO.1 in the sequence table; the amino acid sequence of the protein coded by the CsJAZ12 gene is shown as SEQ ID NO.2 in the sequence table. The CsJAZ12 gene and the amino acid protein coded by the same are related to anabolism regulation of tea leaf caffeine, are negative caffeine regulatory factors, and the cloning of the gene is beneficial to analyzing a fine regulatory mechanism of tea leaf caffeine synthesis, is beneficial to cultivating tea varieties with low caffeine content, improves tea quality and has great application value.

Description

CsJAZ12 gene and application thereof in regulating and controlling synthesis of tea caffeine

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a CsJAZ12 gene and application thereof in regulating and controlling the synthesis of tea caffeine.

Background

Caffeine (1, 3, 7-trimethylxanthine) is the most widely used plant-based purine alkaloid with strong nerve stimulating effect. At least 60 plants in the world contain caffeine, and tea (2% -5%), coffee (1% -2%), guarana (4% -7%) and the like with relatively high content are included. Caffeine is known as the core of modern soft drink culture, and the popularity of three nonalcoholic beverages, tea, coffee and cocoa all benefit from the presence of caffeine.

Caffeine has an important contribution to the bitter taste of tea, but complex compounds formed by associating with substances such as theaflavin through hydrogen bonds show fresh taste and special 'muddy after cooling', which also becomes one of indexes for measuring the quality of black tea. In addition, caffeine has been shown to have a wide range of physiological activities, and proper intake of caffeine has important protective effects on human health, including stimulation of the central nervous system, acceleration of release of epinephrine and dopamine, maintenance of cyclic adenosine monophosphate (cAMP) levels to improve cognition, response, memory, promotion of cardiovascular contractions, acceleration of fat degradation, and antioxidant and aging. However, excessive intake of caffeine in the human body over a long period of time induces caffeine poisoning, which is manifested by physiological and psychological problems such as addiction to caffeine, anxiety, dysphoria, irritability, sleep disorders, etc. However, excessive caffeine can also cause gastrointestinal discomfort, increased kidney burden, and accelerated heart rate and heartbeat in children, pregnant women, the elderly, or neurasthenia. Currently, low (no) caffeine has become a new direction for tea tree breeding and tea market development, which is helpful for further expanding tea consumer groups, thereby increasing tea consumption and relieving the problem of excessive tea productivity in China.

The core part of the biosynthesis pathway of the caffeine is that the xanthine nucleoside (xanthosine) is subjected to 3-step methylation and 1-step nucleoside hydrolysis to finally generate the caffeine, and the intermediate products are 7-methylxanthine nucleoside (7-methylxanthosine), 7-methylxanthine (7-methylxanthone), theobromine (3, 7-dimethylxanthine) and the caffeine in sequence. In the tea plant genome, at least 11 NMT genes are involved in these three methylation processes. Among them, TEA tree caffeine synthase 1 (TCS 1, TEA 015791) is most expressed in young leaves, and it catalyzes the 1-N methylation reaction of caffeine from theobromine in the last step, and is the rate-limiting enzyme gene for caffeine synthesis. With the functional analysis of other NMT genes and the research of the mechanism of caffeine degradation, the metabolic pathway of caffeine is relatively clear in tea trees. However, research on the synthesis regulation mechanism of caffeine is slow, and the understanding of the quality formation mechanism of tea leaves and the personalized molecular breeding progress of tea trees are seriously hindered. In recent years, the publication of genome data of tea trees provides theoretical reference for analyzing the caffeine synthesis mechanism in tea tree leaves from the molecular level. The research on the synthesis and regulation mechanism of caffeine can provide excellent gene resources for genetic engineering breeding better. .

Disclosure of Invention

The invention aims at: the CsJAZ12 gene and the application thereof in regulating and controlling the synthesis of tea caffeine are provided, so that the application research of regulating and controlling the synthesis of caffeine in tea leaves and the formation of tea quality is realized, and theoretical and practical reference bases are provided for realizing selective agronomic character breeding of tea.

In order to achieve the above object, the present invention provides the following technical solutions:

a nucleotide sequence of the CsJAZ12 gene is shown in a sequence table SEQ ID NO. 1.

Preferably, the amino acid sequence coded by the CsJAZ12 gene is shown as a sequence table SEQ ID NO. 2.

Preferably, the CsJAZ12 gene is applied to the synthesis of tea caffeine, and can inhibit the synthesis of caffeine in tea leaves and improve the quality of tea.

The invention has the beneficial effects that:

in the invention, a key transcription factor CsJAZ12 for negatively regulating and controlling the synthesis of tea tree caffeine is cloned and verified for the first time. The transcription factor can inhibit caffeine synthesis in tea leaf, and affect tea quality. The invention also provides a recombinant plasmid, transgenic engineering bacteria and transgenic plants containing the CsJAZ12 gene. The invention enriches the cognition of tea tree secondary metabolism and the genetic mechanism of tea quality formation, especially tea bitter taste formation. The invention provides theoretical and practical reference bases for realizing selective agronomic character breeding of tea trees.

Drawings

Fig. 1: is an expression difference graph of CsJAZ12 and caffeine synthetic genes in different tissues of tea trees;

fig. 2: regulating and controlling a caffeine synthesis accumulation graph in tea leaves for CsJAZ12 genes;

fig. 3: molecular mechanism drawing for CsJAZ12 gene to regulate and control caffeine biosynthesis in tea leaves.

Detailed Description

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

The present invention will be described in further detail with reference to the following specific preparation examples and application examples. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The primers used are all indicated at the first occurrence, and the same primers used thereafter are all identical to the first indicated ones.

Example 1:

1. cloning and sequence structural analysis of CsJAZ12 gene

Tea tree national grade fine variety Shucha is planted in an agricultural industry garden of an Anhui agricultural university in the Guangxi province of Hefei, and young leaves are taken for extracting RNA. Total RNA was extracted using Trizol reagent (Invitrogen, USA) and assayed for RNA content and quality using a spectroscope according to the instructions. Reverse transcription generates the first strand: 1. Mu.g of RNA was used as a template, 0.5. Mu.g of the downstream primer was added respectively according to the instructions of the Promega M-MLV1 reverse transcriptase kit, the volume was fixed to 15. Mu.L, the denaturation was carried out at 70℃for 5min, and the mixture was immediately left on ice for 5min. Then, 5. Mu.L of M-MLV 5 Xbuffer, 5. Mu.L of dNTP (25 mM), rRNasin Ribonuclease Inhibitor U, 200U of M-MLV reverse transcriptase, and 25. Mu.L of DEPC water were added, respectively. The reverse transcriptase is inactivated by incubation at 42℃for 1h and at 95℃for 5min. After optimization, appropriate amounts of reverse transcription products were taken for subsequent PCR. The first strand of cDNA is used as RT-PCR template, PCR is performed in conventional method, and CsJAZ12 gene is amplified. An upstream primer: (5'-ATGTCGAGTTCGTCTGGT-3'), downstream primer: (5'-CTACAAATGGTGCTCAAA-3'). The 25. Mu.L PCR reaction system was: 10 XEx taq buffer 2.5. Mu.L, dNTP 2.0. Mu.L, mg2+1.5. Mu.L, 1. Mu.L each of the upstream and downstream primers, 0.2. Mu.L of Ex taq, 1. Mu.L of template, and ddH 20.8. Mu.L. The reaction procedure was as follows for 95℃5min,95℃50sec,58℃50sec,72℃1min,72℃10min,35 cycles. After the PCR product CsJAZ12 gene is purified and recovered, the PCR product CsJAZ12 gene is connected to a pMDTM19-T Simple Vector (Takara, japan) to obtain a pMDTM19-T-CsJAZ12 plasmid, and the pMDTM19-T-CsJAZ12 plasmid is transformed into escherichia coli competent cells DH5 alpha and sent to Shanghai industrial company for sequencing, and the nucleotide sequence of the obtained CsJAZ12 gene is shown as a sequence table SEQ ID NO.1 and is specifically as follows:

ATGTCGAGTTCGTCTGGTTCTGCTGATTCCGGGAGGTTTTCCGGCCATAGACACTCCTCCAGAGTGCCCGAGAAGTCGGCGTCGTCGAGCTTCTCTCAGACGTGTAGTTTGTTGAGCCAGTACCTGAAGGAGAAGAAAGGCACATTCGGAGATCTCAGTCTCGGCATGCCAACCGCAACGACAACTATGAATTTGTTCCCCGTTGCTATGAAATCGAGTGAGGTTTCCCGAAATGGAACTCCGGTGACGAGAAACCTCACGTCTATGGACTTATTTCCACAGCAGTCTGGTTTTGGTTCCAACATTTCCGTGGAAGAGTCCCTCAACAAGGTTGATTCGAGTGCCATAAACAAGTCAGAGCCAGAAACTGCACAAATGACCATATTCTATGCCGGGCAAGTGATTGTGTTTAATGATTTCCCGGCTGATAAGGCAAAGGAAATCATGCTCTTAGCTAGCAAGGGAAGTTCCCATCACAACCCCGGCACGCTTGCTTCAACACCGGTCCAAAAACCAATCGAACCCACTAATTTGATTCCCACTAGCTCAGCGAGTGCTATTGTCCCTAATTCAGGCAACAATACGATCTCAATCGAACCCATTAGTTTGATTCCCACTACCTCGAGTGTTGCTGCCCCTAATTTTAGCAACAATATGATTCAAGAACGTGTCCAGGTCCAACGGGCTTCTCAACCCACTGCTACCGATCTACCAATTGCTAGAAAAGCGTCGCTCACCCGGTTCTTGGAGAAGAGAAAAGATAGGATCACATCAAGAGCACCATACCAAATAAGCAGCTCAACTTCTCCTCCCAAGCCGACTGAAAGCAAGTCATGGCTCGGCTTGGCAGCTCAATCTCCGGTGCAGTTTGAGCACCATTTGTAG。

the amino acid protein sequence coded by the CsJAZ12 gene is shown in a sequence table SEQ ID NO.2 and is specifically as follows:

MSSSSGSADSGRFSGHRHSSRVPEKSASSSFSQTCSLLSQYLKEKKGTFGDLSLGMPTATTTMNLFPVAMKSSEVSRNGTPVTRNLTSMDLFPQQSGFGSNISVEESLNKVDSSAINKSEPETAQMTIFYAGQVIVFNDFPADKAKEIMLLASKGSSHHNPGTLASTPVQKPIEPTNLIPTSSASAIVPNSGNNTISIEPISLIPTTSSVAAPNFSNNMIQERVQVQRASQPTATDLPIARKASLTRFLEKRKDRITSRAPYQISSSTSPPKPTESKSWLGLAAQSPVQFEHHL。

2. analysis of CsJAZ12 gene expression of different tissues of tea tree

The tea plant national grade fine variety Shucha early variety is planted in an agricultural industry garden of Anhui agricultural university in the Guangxi province of Guangxi province, and 8 tissues and organs are used for analyzing related gene expression. The 8 tissues and organs comprise terminal buds, tender leaves, mature leaves, old leaves, tender stems, flowers, fruits and roots. The samples were submitted to China Huada for second generation transcriptome sequencing, three biological replicates per sample. The gene expression calculations were generated by standard transcriptome sequencing analysis procedures, and the gene expression was expressed as FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values.

FIG. 1 is a graph showing the difference in expression of CsJAZ12 and caffeine synthetic gene in different tissues of tea tree. As shown in FIG. 1, the expression pattern of CsJAZ12 is highly positively correlated with the expression pattern of structural genes related to caffeine synthesis by taking a representative small leaf tea variety Shucha as a research material. The gene expression cluster analysis of different tissues of the tea tree shows that CsJAZ12 and some caffeine metabolic pathway structural genes, especially TCS1, have similar expression tendencies and are highly expressed in tender tissues of the tea tree. This suggests that CsJAZ12 is involved in the process of regulating the synthesis of caffeine. RT, root; OL, old leaves; FL, flowers; FR, fruit; ML, mature leaves; ST, stems; AB, shoots; YL, young leaves.

3. Functional verification of CsJAZ12 gene in tea tree body

1. In vitro oligonucleotide antisense inhibition experiments

The primer for synthesizing the oligonucleotide antisense is designed according to the CsJAZ12 predicted sequence, and the design is completed on the website http:// sfold, wasth, org/cgi-bin/sol, and the primer sequence is as follows:

P1，(5’-TTCTCCTTCAGGTACTGGCT-3’)；

P2，(5’-CAACGGGGAACAAATTCATA-3’)；

P3，(5’-ATTTCATAGCAACGGGGAAC-3’)；

dissolving with 80mM sucrose solution, preparing and obtaining in vitro oligonucleotide antisense inhibition buffer solution, blank is sucrose solution; the scissors are used for cutting the buds and leaves with basically consistent size, bright color and healthy color, and the buds and leaves without insects and diseases are inserted into a 96-well plate with buffer solution, so that the tails of the buds and leaves are immersed into the buffer solution. The 96-well plate is placed in an illumination incubator for illumination culture according to 16h of illumination/8 h of darkness, and the temperature of the incubator is 28 ℃. The post-treatment 3d primer treated samples and the blank samples were sampled for metabolic and gene expression analysis, respectively.

2. In vitro oligonucleotide antisense inhibition of metabolites and related gene expression assays of samples

The treated sample and the control sample were used for total RNA extraction and cDNA first strand synthesis, respectively. The reverse transcription product (cDNA first strand) was diluted 80-fold as a template to prepare 20. Mu.l of a reaction system using SYBR Realtime Mix (TOYOBO, osaka, japan): 8.4. Mu.l of 80-fold diluted reverse transcription product, 0.8. Mu.l (10 pmol/. Mu.l) of each of the upstream and downstream primers, 10. Mu.l of 2X SYBR Green PCR Master Mix, 3 replicates per reaction. The procedure was then followed on bio-rad CFX-96: (1) 95℃for 3min, (2) 95℃for 10s,60℃for 15s,72℃for 30s for 45 cycles, (3) from 65℃to 95℃the melting curve was plotted at 0.1℃per second. An upstream primer: (5'-TGATTCCGGGAGGTTTTCCG-3'), downstream primer: (5'-GGTTTCTCGTCACCGGAGTT-3') with tea tree ACTIN gene as reference, upstream primer: (5'-GCCATATTTGATTGGAATGG-3'), downstream primer: (5'-GGTGCCACAACCTTGATCTT-3') the relative expression value of CsJAZ12 was calculated by the instrument's own analysis software. Similarly, the relative expression level of the caffeine synthase gene TCS1 (upstream primer 5'-GATGGGAGTAGCGGGGTCTT-3', downstream primer 5'-TGGTGCCTGAGTAAGCCAAT-3') was calculated. The results show that the antisense inhibition of CsJAZ12 in vitro oligonucleotides can significantly interfere with the expression level of the target gene. Analysis of the expression of the key gene for caffeine synthesis showed significant down-regulation of TCS1 gene expression. Analysis of the metabolite content indicated a significant reduction in caffeine content. Samples were analyzed for caffeine content using an Agilent High Performance Liquid Chromatograph (HPLC). About 0.05g of freshly ground sample powder was taken, 1ml of extract (80% methanol) was added and mixed well and treated with an ultrasonic at room temperature for 1-2h. Centrifuging at 12000r/min for 10min, collecting supernatant, filtering with 0.2 μm filter membrane, and packaging into sample bottle for sample analysis. HPLC parameters were set as follows: c18 chromatographic column (5 um 4.6X1250 mm), column temperature 39 ℃, sample injection amount 10 μl, detection wavelength 280nm, binary mobile phase with flow rate 1.0mL/min, gradient elution (0.1-5 min,95% A+5% B;5min-8min,75% A+25% B;8min-15min,70% A+30% B;15min-25min,60% A+40% B;25min-45min,55% A+45% B;45min-60min,45% A+55% B;60min-65min,30% A+70% B;65min-70min,100% B;70min-75min,95% A+5% B. Wherein A is 0.1% glacial acetic acid and B is 100% methanol). The detected substances are both qualitatively and quantitatively determined by an internal standard method.

FIG. 2 is a graph showing the synthesis and accumulation of caffeine in tea leaves regulated by CsJAZ12 gene. Wherein, the expression of the A, csJAZ12 gene is significantly inhibited in the treated sample. One bud and one leaf material is obtained from Shucha early variety, sense ODN, blank control; csJAZ12-KD, csJAZ12 oligonucleotide antisense strand inhibition treatment. B, key gene expression changes in the caffeine metabolic pathway. And C, changing the content of caffeine in tea leaves.

In fig. 2, it can be seen that the expression level of CsJAZ12 can be significantly inhibited compared with the control by an in vitro oligonucleotide antisense inhibition experiment, and the expression level of key structural genes in the caffeine synthesis pathway can be significantly increased by inhibiting the expression of CsJAZ12 through the in vitro oligonucleotide antisense inhibition of the expression of CsJAZ12 gene for 3d and then the metabolic substance measurement result can be seen that the synthesis and accumulation of caffeine in tea leaves can be significantly improved by inhibiting the expression of CsJAZ12.

4. Molecular mechanism verification of CsJAZ12 for regulating and controlling caffeine synthesis

1. Interaction verification of CsJAZ12 and CsMYB184 protein

CsMYB184 is a core transcription factor that positively regulates caffeine synthesis in tea trees. To explore the molecular mechanism by which CsJAZ12 negatively regulates caffeine synthesis, it was examined whether CsMYB184 interacted with CsJAZ12 in the presence of protein. CsMYB184 was cloned into the yeast vector pGADT7 using the upstream primer: (5-CATATGGCCATGGAGGCCGAATTCACATATGGCTCCGAAGAGCAGTGA-3'), downstream primer: (5-CTGCAGCTCGAGCTCGATGGATCCAGGATCCTTACCATTTATCGGTAAGTGCC-3'). CsJAZ12 was cloned into the yeast vector pGBKT7 using the upstream primers: (5-CATATGGCCATGGAGGCCGAATTCATGTCGAGTTCGTCTGGT-3'), downstream primer: (5-CGGCCGCTGCAGGTCGACGGATCCCTACAAATGGTGCTCAAA-3'). pGADT7-CsMYB184 and pGBKT7-CsJAZ12 were then co-transformed into the yeast strain AH 109. The specific method comprises the following steps: preparation of Yeast competent cells: (1) Selecting AH109 yeast, adding 3mLYPDA liquid culture medium, and shake culturing at 30deg.C in shaking table at 230r/min overnight; (2) After shaking and mixing, 5 mu L of the bacterial liquid is sucked into a 250mL conical flask, 50mL of YPDA is added, and shaking is carried out for 16-20h at 230r/min until the OD600 is 0.15-0.3; (3) Centrifuging at 2500r/min for 5min at room temperature, discarding supernatant, re-suspending with 100mL YPDA, and continuing culturing until OD600 is 0.4-0.5 (3-5 h); (4) Split charging with 250mL centrifuge tubes, centrifuging at 2500r/min for 5min at room temperature, discarding supernatant, and re-suspending with 30mL ddH 2O; (5) The supernatant was discarded by centrifugation again, resuspended in 1.5mL of 1.1 XSTE/LiAc, transferred to a 1.5mL centrifuge tube, and centrifuged at high speed for 15s; (6) The supernatant was discarded and resuspended in 600. Mu.L of 1.1 XSTE/LiAc for later use. Plasmid transformation: (1) Adding 1ug of AD plasmid and BD plasmid containing target gene fragment, and 10 μl of denatured salmon sperm DNA (salmon sperm DNA is heated at 95-100deg.C for 5min, and rapidly cooled on ice); (2) adding 600. Mu.L of yeast competent cells, and gently mixing; (3) adding 2.5ml of 50% PEG, and mixing gently; (4) Culturing in a constant temperature incubator at 30deg.C for 45min, shaking the mixed cells every 10-15 min; (5) 160. Mu.L DMSO was added and gently mixed; (6) heat shock at 42 ℃ for 20min, shaking and uniformly mixing the cells every 5-10 min; (7) centrifuging at 2500r/min for 5min at room temperature, and discarding the supernatant; (8) Resuspension was performed with 3mL YPDA and shaking at 30℃for 90min at low speed; (9) Centrifuging at 2500r/min for 5min at room temperature, discarding supernatant, and re-suspending with 10mL of 0.9% NaCl solution; (10) After dilution by a certain multiple, the mixture is evenly coated on a culture medium of SD/-leu-trp, and is cultured for 2 to 3 days in a constant temperature incubator at 30 ℃; (11) The monoclonal is selected and diluted by sterile water for 1000 times, then evenly spotted on SD/-leu-trp-His culture medium, cultured for 2-3 days in a constant temperature incubator at 30 ℃, and the growth condition of yeast is observed.

Cloning CsMYB184 into a plant bimolecular fluorescence complementation experiment (BiFC) vector pFGC-YN173, constructing a YN-CsMYB184 vector, using the upstream primer: (5'-TCTCTCTCGAGCTTTCGCGAGCTCATGGCTCCGAAGAGCAGTGA-3'), downstream primer: (5'-CATGGTGGCGATGGATCTTCTAGACCATTTATCGGTAAGTGCC-3'). Cloning CsJAZ12 into plant BiFC vector pFGC-YC155, constructing YC-CsJAZ12 vector, using the upstream primer: (5'-TCTCTCTCGAGCTTTCGCGAGCTCATGTCGAGTTCGTCTGGT-3'), downstream primer: (5'-GGTACCGGATCCCTCGAGTCTAGACAAATGGTGCTCAAACTG-3'). The specific method comprises the following steps: proper amount of the tobacco seeds are taken, added with deionized water, placed in a refrigerator at 4 ℃ for vernalization treatment for 72 hours and sown. After seeding, the plastic wrap is covered, and the plastic wrap is placed under proper conditions (humidity is 60 percent, temperature is 23 ℃,16 hours of illumination/8 hours of darkness) to wait for germination. After the seeds bud, selecting the seedlings with consistent sizes as much as possible for transplanting and normally culturing. The YN-CsMYB184 and YC-CsJAZ12 vectors were transformed into GV3101 Agrobacterium by electric excitation, respectively, and positive clones were identified by conventional PCR methods. Selecting a positive colony containing a target gene, and culturing for about 24 hours at 28 ℃ and 200r/min in 5mL of LB liquid medium containing corresponding antibiotics; 2mL of the cultured bacterial liquid is sucked, added into 50mL of fresh LB liquid culture medium containing corresponding antibiotics, continuously shake-cultured until the OD600 is about 1.0, centrifugally collected, and resuspended in a proper amount of transformation liquid (MS+0.3 mg/L6-BA+150 g/L sucrose+15 g/L EMS+0.06%silwet L-77, PH=5.7) to obtain the final concentration OD600 of about 0.8. The prepared conversion solution was mixed in equal proportions and loaded into a syringe, gently injected into the back of tobacco leaves, and then placed in darkness for 24H. Three days later, the injected leaves were taken for laser confocal microscopy.

2. Caffeine synthase TCS1 promoter cloning

The method is characterized in that young leaves of Shucha early are used as materials, DNA extraction is carried out by using a DNA extraction kit special for root plants, and the method is strictly referred to a kit instruction. Measurement of total DNA concentration was measured using a GeneQuantil spectrophotometer (Fisher Scientific, CA, USA) and then adjusted to 50 ng/. Mu.l with TE (10mM Tris,1mM EDTAPH 8.0) solution and stored at-20℃for use. Then, DNA is used as RT-PCR template, PCR is performed in conventional method to amplify TCS1 promoter sequence. An upstream primer: (5'-CCGGGCCCCCCCTCGAGGTCGACGTGAATCCTGAAAATTCAAACC-3'), downstream primer: (5'-GCCGCTCTAGAACTAGTGGATCCCACCTTCCCCGTAGTAGCTA-3'). The 25. Mu.L PCR reaction system was: 10 XEx taq buffer 2.5. Mu.L, dNTP 2.0. Mu.L, mg2+1.5. Mu.L, 1. Mu.L each of the upstream and downstream primers, ex taq 0.2. Mu.L, template 1. Mu.L, ddH2015.8. Mu.L. The reaction procedure was as follows for 5min at 95 ℃, 50sec at 58 ℃, 2min at 72 ℃,10 min at 72 ℃ for 35 cycles. After the PCR product was purified and recovered, it was ligated to pMDTM19-T Simple Vector (Takara, japan) to obtain pMDTM19-T-CsTCS1 pro plasmid, which was transformed into E.coli competent cell DH 5. Alpha. And sent to sequencing company for sequence confirmation.

3. Promoter activation/inhibition experiments

The CsTCS1 pro promoter fragment was recombined onto the promoter-activating vector pGreen 0800. CsTCS1 pro upstream primer: (5'-CCGGGCCCCCCCTCGAGGTCGACGTGAATCCTGAAAATTCAAACC-3'); csTCS1 pro downstream primer: (5'-GCCGCTCTAGAACTAGTGGATCCCACCTTCCCCGTAGTAGCTA-3'). Then handleCsMYB184 was cloned into the promoter activating vector pGreen0800-Sk by homologous recombination using the upstream primer: (5'-GCCGCTCTAGAACTAGTGGATCCATGGCTCCGAAGAGCAGTGA-3'), downstream primer: (5'-TCGATAAGCTTGATATCGAATTCTTACCATTTATCGGTAAGTGCC-3'); csJAZ12 was cloned by homologous recombination into the promoter activating vector pGreen0800-Sk using the upstream primer: (5'-GGCCGCTCTAGAACTAGTGGATCCATGTCGAGTTCGTCTGGT-3'), downstream primer: (5'-ATCGATAAGCTTGATATCGAATTCCTACAAATGGTGCTCAAA-3'). Then, the promoter activation activity was verified in the respective transformed Arabidopsis protoplasts. The specific method comprises the following steps: (1) Preparing enzymolysis liquid (generally, 20 blades are needed for 10mL enzymolysis liquid, and 20-30 conversion can be performed). (2) Pouring the treated enzymolysis liquid into a clean and dry plate. (3) Selecting Arabidopsis leaves with good growth condition and before flowering, cutting into filaments with the width of 0.5-1mm, putting into enzymolysis solution with forceps, and immersing both sides. (4) vacuum pumping in the dark for 30min. (5) Standing in dark for enzymolysis for 3h, and gently shaking to release protoplast, wherein the enzymolysis liquid should turn green. (6) The enzymatic hydrolysis reaction was terminated by adding an equal volume of pre-chilled W5 solution, sucking the enzymatic hydrolysis product with a syringe, and filtering into a round bottom centrifuge tube with a 30-75 μm nylon mesh. (7) 100g was centrifuged for 1-2min, the supernatant carefully aspirated, and the protoplasts resuspended in 10mL of pre-chilled W5 solution (to a number of protoplasts of approximately 2X 105 mL-1). (8) an ice bath for 30min. The solution was centrifuged at 100g for 8-10min, the W5 solution was removed as much as possible, and the protoplasts were resuspended in an appropriate amount of MMG solution (the number of protoplasts was approximately 2X 105 mL-1). Plasmid transformation step: (1) The plasmid concentration was adjusted to 1-1.5. Mu.g/. Mu.L, 10. Mu.L plasmid and 100. Mu.L protoplast were added to a 2mL centrifuge tube, the tube wall was tapped and mixed well, and then 110. Mu.L PEG was added and mixed well gently. (2) left at room temperature for about 15 min. (3) diluted with 440 μ L W solution, gently mixed upside down. (4) centrifuging at 100g at room temperature for 1-2min, and carefully removing the supernatant. (5) Protoplasts were resuspended with 1mL of W5 and induced at room temperature in the dark for about 12 h. The enzyme activity determination method comprises the following steps: (1) Taking protoplast induced to transform, centrifuging for 2min at 100g, discarding supernatant, and collecting protoplast sediment. (2) Adding 140 mu L of 1 XPLB lysate, shaking and mixing well, and cracking at room temperature for about 15 min. (3) Centrifuging at 4deg.C for 10min at high speed, and sucking supernatant to anotherThe centrifuge tube is cleaned for standby. (4) setting of a microplate reader: the measurement delay time was set to 2s and the measurement time was set to 10 to 20s. (5) Transfer 40 μl of PLB lysate to the assay tube, 3 replicates per sample. (6) 40. Mu.L of LAR II was added to the tube, mixed well, and the activity of firefly luciferase was detected and read as RLU1. (7) Immediately add 40 μl of 1× to the test tube

Reagent, detect the activity of Renilla luciferase, read as RLU2. (8) The reaction intensity Ratio of firefly and Renilla luciferase was calculated=RLU1/RLU2.

FIG. 3 is a molecular mechanism diagram of CsJAZ12 gene regulating caffeine biosynthesis in tea leaves. Wherein, A, csJAZ12 and a caffeine positive regulatory factor CsMYB184 generate protein interaction in yeast. B, csJAZ12 protein interactions with the caffeine positive regulatory factor CsMYB184 in tobacco epidermal cells. C, promoter activation and inhibition vector schematic. And D, a promoter activation/inhibition experiment verifies the inhibition activity of CsJAZ12 on the TCS1 promoter. Experiments show that CsJAZ12 can interact with CsMYB184, thereby inhibiting expression of TCS 1.

As shown in FIG. 3, csJAZ12 is capable of protein interaction with the caffeine positive regulatory factor CsMYB184 in both yeast and plant cells. Promoter activation experiments show that CsMYB184 can remarkably activate the expression of the caffeine synthase gene TCS1, and after CsJAZ12 is added, the activation activity of CsMYB184 on the TCS1 gene can be remarkably inhibited.

In conclusion, the expression mode of the CsJAZ12 is highly related to the synthesis of caffeine in tea leaves, the expression of the CsJAZ12 is inhibited by an antisense oligonucleotide technology, the content of caffeine in the tea leaves is obviously increased, the expression of a gene related to the synthesis of caffeine is also obviously increased, and a yeast two-hybrid and two-molecule fluorescence complementation experiment shows that the CsJAZ12 can interact with a known positive control factor CsMYB184, and a promoter activation/inhibition experiment shows that the CsJAZ12 can obviously inhibit the promoter activity of a caffeine synthase gene. The cloning of the gene is not only beneficial to analyzing a fine regulation mechanism of the synthesis of the caffeine of tea leaves, but also beneficial to cultivating tea varieties with low caffeine content, improving the tea quality, and has great application value.

In the invention, a key transcription factor CsJAZ12 for negatively regulating and controlling the synthesis of tea tree caffeine is cloned and verified for the first time. The transcription factor can inhibit caffeine synthesis in tea leaf, and affect tea quality. The invention also provides a recombinant plasmid, transgenic engineering bacteria and transgenic plants containing the CsJAZ12 gene. The invention enriches the cognition of tea tree secondary metabolism and the genetic mechanism of tea quality formation, especially tea bitter taste formation. The invention provides theoretical and practical reference bases for realizing selective agronomic character breeding of tea trees.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Sequence listing

<110> Anhui university of agriculture

<120> CsJAZ12 gene and application thereof in regulating and controlling synthesis of tea caffeine

<130> NO

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 885

<212> DNA

<213> tea tree (Camellia sinensis L.O. Kuntze)

<400> 1

atgtcgagtt cgtctggttc tgctgattcc gggaggtttt ccggccatag acactcctcc 60

agagtgcccg agaagtcggc gtcgtcgagc ttctctcaga cgtgtagttt gttgagccag 120

tacctgaagg agaagaaagg cacattcgga gatctcagtc tcggcatgcc aaccgcaacg 180

acaactatga atttgttccc cgttgctatg aaatcgagtg aggtttcccg aaatggaact 240

ccggtgacga gaaacctcac gtctatggac ttatttccac agcagtctgg ttttggttcc 300

aacatttccg tggaagagtc cctcaacaag gttgattcga gtgccataaa caagtcagag 360

ccagaaactg cacaaatgac catattctat gccgggcaag tgattgtgtt taatgatttc 420

ccggctgata aggcaaagga aatcatgctc ttagctagca agggaagttc ccatcacaac 480

cccggcacgc ttgcttcaac accggtccaa aaaccaatcg aacccactaa tttgattccc 540

actagctcag cgagtgctat tgtccctaat tcaggcaaca atacgatctc aatcgaaccc 600

attagtttga ttcccactac ctcgagtgtt gctgccccta attttagcaa caatatgatt 660

caagaacgtg tccaggtcca acgggcttct caacccactg ctaccgatct accaattgct 720

agaaaagcgt cgctcacccg gttcttggag aagagaaaag ataggatcac atcaagagca 780

ccataccaaa taagcagctc aacttctcct cccaagccga ctgaaagcaa gtcatggctc 840

ggcttggcag ctcaatctcc ggtgcagttt gagcaccatt tgtag 885

<210> 2

<211> 294

<212> PRT

<213> tea tree (Camellia sinensis L.O. Kuntze)

<400> 2

Met Ser Ser Ser Ser Gly Ser Ala Asp Ser Gly Arg Phe Ser Gly His

1 5 10 15

Arg His Ser Ser Arg Val Pro Glu Lys Ser Ala Ser Ser Ser Phe Ser

20 25 30

Gln Thr Cys Ser Leu Leu Ser Gln Tyr Leu Lys Glu Lys Lys Gly Thr

35 40 45

Phe Gly Asp Leu Ser Leu Gly Met Pro Thr Ala Thr Thr Thr Met Asn

50 55 60

Leu Phe Pro Val Ala Met Lys Ser Ser Glu Val Ser Arg Asn Gly Thr

65 70 75 80

Pro Val Thr Arg Asn Leu Thr Ser Met Asp Leu Phe Pro Gln Gln Ser

85 90 95

Gly Phe Gly Ser Asn Ile Ser Val Glu Glu Ser Leu Asn Lys Val Asp

100 105 110

Ser Ser Ala Ile Asn Lys Ser Glu Pro Glu Thr Ala Gln Met Thr Ile

115 120 125

Phe Tyr Ala Gly Gln Val Ile Val Phe Asn Asp Phe Pro Ala Asp Lys

130 135 140

Ala Lys Glu Ile Met Leu Leu Ala Ser Lys Gly Ser Ser His His Asn

145 150 155 160

Pro Gly Thr Leu Ala Ser Thr Pro Val Gln Lys Pro Ile Glu Pro Thr

165 170 175

Asn Leu Ile Pro Thr Ser Ser Ala Ser Ala Ile Val Pro Asn Ser Gly

180 185 190

Asn Asn Thr Ile Ser Ile Glu Pro Ile Ser Leu Ile Pro Thr Thr Ser

195 200 205

Ser Val Ala Ala Pro Asn Phe Ser Asn Asn Met Ile Gln Glu Arg Val

210 215 220

Gln Val Gln Arg Ala Ser Gln Pro Thr Ala Thr Asp Leu Pro Ile Ala

225 230 235 240

Arg Lys Ala Ser Leu Thr Arg Phe Leu Glu Lys Arg Lys Asp Arg Ile

245 250 255

Thr Ser Arg Ala Pro Tyr Gln Ile Ser Ser Ser Thr Ser Pro Pro Lys

260 265 270

Pro Thr Glu Ser Lys Ser Trp Leu Gly Leu Ala Ala Gln Ser Pro Val

275 280 285

Gln Phe Glu His His Leu

290

Claims (1)

1. The application of the CsJAZ12 gene in negative regulation of tea caffeine synthesis is characterized in that the nucleotide sequence of the CsJAZ12 gene is shown in a sequence table SEQ ID NO. 1; the CsJAZ12 gene is applied to the synthesis of tea caffeine, and can inhibit the synthesis of caffeine in tea leaves and improve the formation of tea quality.

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