A tea plant ethylamine oxidase gene CsEAO and its application

Abstract

The present invention relates to the field of gene engineering technology, and specifically to a tea tree ethylamine oxidase gene CsEAO and its application. The nucleotide sequence of the tea tree ethylamine oxidase gene CsEAO is shown in SEQ ID NO.1 in the sequence list; the amino acid sequence of the protein encoded by the tea tree ethylamine oxidase gene CsEAO is shown in SEQ ID NO.2 in the sequence list. CsEAO is highly expressed in mature leaves of tea trees; overexpression of CsEAO in tobacco leaves can reduce the ethylamine content, indicating that CsEAO has the function of degrading ethylamine; transient silencing of CsEAO expression in tea leaves can significantly increase the content of ethylamine and theanine, indicating that the expression of CsEAO in leaves negatively regulates the accumulation of theanine. The ethylamine oxidase gene CsEAO disclosed in the present invention is helpful to analyze the molecular mechanism of theanine accumulation in tea leaves, enrich people's understanding of the metabolic mechanism of theanine in tea trees, and provide a theoretical basis and target gene for cultivating new varieties of high-theanine tea trees through molecular-assisted breeding methods.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a tea tree ethylamine oxidase gene CsEAO and an application thereof. Background Art

[0002] Tea, as one of the three major non-alcoholic beverages in the world, is widely loved by consumers for its unique taste and many health properties. Theanine is the most abundant non-protein amino acid in tea, with a content of 1%-2% of the dry weight of tea leaves, accounting for 40%-70% of the total free amino acids. It is a characteristic secondary metabolite that gives green tea its fresh taste and calming effect. At the same time, theanine also has many health-promoting effects, such as reducing stress and anxiety, improving mood and cognition, and protecting the nervous system. Therefore, the content of theanine in tea is highly correlated with the quality of tea.

[0003] The metabolic pathway of theanine in tea plants is divided into two parts: root synthesis and leaf decomposition. The root is the main site of theanine synthesis. Theanine synthesized in the root is transferred to the aboveground leaves in the xylem sap; in the leaves, theanine forms glutamate and ethylamine under the catalysis of theanine hydrolase, and ethylamine forms acetaldehyde under the catalysis of amine oxidase, which then participates in the biosynthesis of catechins. At present, the coding gene of amine oxidase that degrades ethylamine has not been reported.

[0004] The accumulation of theanine in tea plants is dynamically regulated by factors such as theanine synthesis, transport and degradation. At present, the genes related to theanine synthesis, transport and degradation metabolic pathways have been identified one after another, providing important functional gene resources for understanding the molecular mechanism of theanine accumulation in tea leaves. Ethylamine is both a precursor for theanine synthesis and a degradation product of theanine. The key gene that catalyzes the synthesis of ethylamine has been identified, but the gene encoding ethylamine oxidase that degrades ethylamine has not yet been found, which greatly limits the elucidation of the molecular mechanism of theanine dynamic accumulation in tea leaves.

[0005] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of unclear molecular mechanism of theanine accumulation in tea leaves in the prior art, and provide a tea tree ethylamine oxidase gene CsEAO and its application.

[0007] In order to achieve the above object, the present invention discloses a tea tree ethylamine oxidase gene CsEAO, the nucleotide sequence of the tea tree ethylamine oxidase gene CsEAO is shown in SEQ ID NO.1.

[0008] The protein sequence encoded by the tea tree ethylamine oxidase gene CsEAO is shown in SEQ ID NO.2.

[0009] The present invention also discloses the application of the tea tree ethylamine oxidase gene CsEAO in regulating the accumulation of theanine in tea leaves, increasing the theanine content in tea leaves by reducing the expression of CsEAO, and increasing the theanine content in tea leaves by reducing the expression of CsEAO.

[0010] The invention also discloses the application of the tea tree ethylamine oxidase gene CsEAO in the screening or breeding system of tea tree varieties with high theanine content.

[0011] The invention also discloses the application of the tea tree ethylamine oxidase gene CsEAO in constructing a transgenic tea tree system for increasing the content of theanine in tea leaves.

[0012] The invention also discloses the application of the tea tree ethylamine oxidase gene CsEAO in an industrial theanine production system.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a tea tree ethylamine oxidase gene CsEAO and its application, and finds that CsEAO catalyzes the degradation of ethylamine in tea leaves, and the reduction of ethylamine inhibits the degradation of theanine, thereby increasing the content of theanine in the leaves. The present invention enriches the theoretical knowledge of theanine metabolism regulation in tea trees, provides important functional gene resources for screening high-theanine tea varieties and constructing transgenic tea trees, and provides a theoretical basis for the industrial production of theanine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Analysis of the expression pattern of CsEAO in different tissues of tea plants. (A) Phenotype diagram of different tissues of tea plants. (B) Expression level of CsEAO in different tissues of tea plants. MV, vein; VB, vascular bundle. CsGAPDH was used as an internal reference. Data are expressed as mean ± SD (n = 3). Different letters are used to mark significant differences (P < 0.05) according to Duncan's multiple range test.

[0015] Figure 2 Enzyme activity analysis of CsEAO in N. benthamiana, (A) transient expression of CsEAO in N. benthamiana; (B) ethylamine content in N. benthamiana leaves. GFP, empty vector control, transient overexpression of pCAMBIA1305 (with GFP tag) empty vector in N. benthamiana leaves; CsEAO-GFP, transient overexpression of CsEAO and pCAMBIA1305 (with GFP tag) recombinant vector in N. benthamiana leaves. Data are expressed as mean ± SD (n = 3), * indicates statistical significance determined by Student's t test (*P < 0.05);

[0016] Figure 3 The effect of transient silencing of CsEAO in tea leaves on theanine accumulation. (A) Tea leaves were treated with sOND-CsEAO and AsOND-CsEAO. (B) Expression of CsEAO in tea leaves after gene silencing. (C) Ethylamine content in tea leaves after gene silencing. (D) Theanine content in tea leaves after gene silencing. Data are expressed as mean ± SD (n = 3), * indicates statistical significance determined by Student's t test (*P < 0.05). DETAILED DESCRIPTION

[0017] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0018] 1. Cloning of CsEAO gene

[0019] The CsEAO gene of tea tree is a gene encoding tea tree ethylamine oxidase. Its cloning and sequence structure analysis are as follows:

[0020] The national tea tree variety Shuchazao was planted in the Agricultural Garden of Anhui Agricultural University, and young leaves were taken for RNA extraction. Total RNA extraction was performed according to the instructions of the RNAprep Pure Plant Kit of Tiangen Biochemical Technology Co., Ltd., and the integrity of RNA was detected by agarose gel electrophoresis, and the concentration of RNA was detected by a nucleic acid quantifier.

[0021] cDNA synthesis: cDNA synthesis was performed using the Hiscript III RT SuperMixfor qPCR (+gDNAwiper) reverse transcription kit from Novizan Biotechnology Co., Ltd. According to the kit method, 1 μg RNA and 4 μL 4×gDNAwiper Mix were added to an RNase-free centrifuge tube, and the mixture was supplemented to 16 μL with RNase free ddH2O. The mixture was gently pipetted and mixed, and the mixture was reacted at 42°C for 2 min on a PCR instrument. After the above reaction, 4 μL Hiscript III RTSuperMix was added to the centrifuge tube, and the mixture was gently pipetted and mixed, and the mixture was reacted at 37°C for 15 min on a PCR instrument, and the reaction was terminated at 85°C for 5 s. After the reaction, the cDNA solution was diluted to 200 ng / μL with RNase free ddH2O, and an appropriate amount of reverse transcription product was taken for subsequent PCR reactions. The CsEAO gene was amplified using cDNA as a PCR template. The upstream primer is (5'-ATGGAAGAAAAGAGCCTCCTCCG-3'), and the downstream primer is (5'-TCAGGCAGAAGCAGAAGCCTTG-3'). The 20μl PCR reaction system is: KOD One TMPCR Master Mix 10μL, upstream and downstream primers 1μL each, cDNA 1μL, ddH2O 7μL.

[0022] The reaction procedure is as follows: denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 68°C for 15s, 35 cycles, and extension at 72°C for 5min. PCR amplification was detected by agarose gel electrophoresis, and the bands that matched the size of the target gene were cut out and the products were recovered using a gel recovery kit (Nanjing Novizan Biotechnology Co., Ltd.). The recovered product fragments were connected to the pEASY-Blunt CloningVector cloning vector (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the pEASY-CsEAO plasmid, which was transformed into DH5α competent cells (Shanghai Weidi Biotechnology Co., Ltd.) by heat shock method and evenly coated on LB solid plates containing 50μg / mL kanamycin. Inverted culture was carried out in a 37°C constant temperature incubator for 12h, and single colonies were picked for PCR verification. The single colonies with the correct band position were sent to Shenggong Biological Company for gene sequencing. The nucleotide sequence of the CsEAO gene obtained is shown in the sequence list SEQ ID NO.1, and the protein sequence encoded by the CsEAO gene is shown in the sequence list SEQ ID NO.2.

[0023] 2. Analysis of tissue expression patterns of CsEAO genes

[0024] The national tea tree variety Shucha was planted in the Agricultural Garden of Anhui Agricultural University. 17 tissue organs were used to analyze gene expression. These 17 tissue organs include buds, leaves, and st leaf), two leaves (2 nd leaf), three leaves (3 rd leaf), four leaves (4 th leaf), five leaves th leaf), one vein(1 st MV), two veins (2 nd MV), three veins (3 rd MV), four veins (4 th MV), five veins (5 th MV), stem between one or two leaves (1 st stem), stem between the second and third leaves (2 nd stem), the stem between the third and fourth leaves (3 rd stem), stem between four or five leaves (4 th stem), stem between five or six leaves (5 thstem), vascular bundle (VB) and root (Root). The above samples were also used for total RNA extraction and cDNA first-strand synthesis. After the reaction, the cDNA solution was diluted to 200ng / μL with RNase free ddH2O as a template for qRT-PCR reaction. The qRT-PCR reaction system was 20μL, 0.8μL of 10μM upstream and downstream primers, 2μL of cDNA template, 10μL of 2×AceQ Universal SYBRqPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd.), and ddH2O was added to make the volume 20μL. Among them, the upstream primer of CsEAO: (5'-TCGTCCTCACCTGGCAAAAG-3'), the downstream primer: (5'-GCAATCGGTAAGCAAGCGAG-3'). Then amplification was performed on the ABIQuantStudio 6 quantitative PCR instrument. The PCR program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s, with 40 cycles; melting curve was 95.0°C for 15 s; 60.0°C for 60 s; 95.0°C for 15 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene, with the CsGAPDH upstream primer: (5'-TTGGCATCGTTGAGGGTCT-3'), and the downstream primer: (5'-CAGTGGGAACACGGAAAGC-3'). According to formula 2 -ΔΔCt The relative expression of genes was calculated by the method, and 3 technical replicates and 3 biological replicates were set for each sample.

[0025] Figure 1 is the expression level of CsEAO gene in different tissues of tea plant. Figure 1 It can be seen that the qRT-PCR detection results showed that CsEAO was expressed in various tissues, and the expression level was higher in relatively mature leaves.

[0026] 3. Analysis of enzyme activity of CsEAO in Nicotiana benthamiana

[0027] Nicotiana benthamiana was grown in an artificial climate chamber (16 h photoperiod, 8 h light on, ambient temperature 25 ± 1 °C, light intensity 150-200 μmol m-2s-1) at Anhui Agricultural University, and five-week-old tobacco leaves were taken for enzyme activity analysis.

[0028] Construction of pCAMBIA1305-CsEAO vector: pEASY-CsEAO plasmid was used as template, upstream primer (5'-AGGACAGCCCAGATCACTAGTATGGAAGAAAAGAGCCTCCTCC-3'), downstream primer (5'-GCCCTTGCTCACCATGGATCCGGCAGAAGCAGAAGCCT-3'), PCR amplification was performed. PCR amplification was detected by agarose gel electrophoresis, and the band that matched the size of the target gene was cut to recover the product. The plant expression vector pCAMBIA1305 was double-digested and linearized with restriction endonucleases SpeI and BamHI from Takara Company. The reaction system was: 5μL Buffer, 1000ng plasmid, 2μL of restriction endonuclease each, and ddH2O was added to make up to 50μL. Enzyme digestion was performed at 37℃ for 3h, and the digestion product was recovered by gel. The target gene was constructed into the linearized vector using the cloning and recombination kit ClonExpress II One Step Cloning Kit. The reaction system was as follows: 20 ng of the target gene product, 10 ng of the restriction plasmid, 2 μL of 5×CEII Buffer, 1 μL of ExnaseII enzyme, and ddH2O was added to 10 μL. Use a pipette to gently blow and mix, and centrifuge briefly to collect the reaction solution to the bottom of the centrifuge tube. After reacting at 37°C for 30 minutes, transform DH5α competent cells, and incubate inverted in a 37°C constant temperature incubator for 12 hours. Pick a single colony for PCR verification, and send the single colony with the correct band position to Sangon Biotech for gene sequencing.

[0029] Agrobacterium-mediated transient expression in tobacco: The correctly sequenced pCAMBIA1305-CsEAO recombinant plasmid was transferred into Agrobacterium tumefaciens competent cells GV3101 (pSoup-p19) by chemical transformation; the positive Agrobacterium containing the pCAMBIA1305-CsEAO plasmid was inoculated into LB liquid medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and cultured overnight at 28°C and 200 rpm until OD 600 When the OD value reached about 1.0, the cells were collected by centrifugation at 6,000 g for 10 min. The cells were resuspended in MMA buffer (10 mM MgCl2, 10 mM 4-morpholineethanesulfonic acid, 0.1 mM acetosyringone) and the OD value was adjusted to 600 The value was adjusted to about 0.8 and incubated at room temperature in the dark for 3 hours; tobacco of appropriate age was selected and the bacterial solution was injected into the back of the tobacco leaves with a syringe. After culturing in the dark for 2 days in a climate room, 0.5 mL of 1 mM ethylamine solution was injected into the tobacco leaves. After culturing in the dark for another 1 day, the leaves were collected for detecting the ethylamine content.

[0030] Figure 2 This is an analysis of the enzyme activity of CsEAO in Nicotiana benthamiana. Figure 2 It can be seen that after the ethylamine solution was injected into the tobacco leaves expressing the pCAMBIA1305 empty vector, the ethylamine content in the leaves of Nicotiana benthamiana did not decrease after 24 hours of reaction; after the ethylamine solution was injected into the tobacco leaves expressing the pCAMBIA1305-CsEAO recombinant plasmid, the ethylamine content in the leaves decreased significantly after 24 hours. The above results show that CsEAO has the enzyme activity of degrading ethylamine in tobacco.

[0031] 4. Effect of transient inhibition of CsEAO expression on theanine content in tea plants

[0032] The nucleotide sequence of the CsEAO gene was used as the input sequence, and the candidate antisense oligonucleotide (AsODN) sequence was designed using Soligo software and sent to Sangon Biotech for sequence synthesis. The AsODN sequence was diluted to 500 μM with 80 mM sucrose solution. The tea shoots were treated with 500 μL of 20 μM AsODN solution, and the sense oligonucleotide (sODN) was used as a control. After 24 hours of cultivation, tea leaves were collected for gene expression, ethylamine content, and theanine content detection. The sODN sequence is 5'-GGACCCCACTGATGCATGGT-3', and the AsODN sequence is 5'-ACCATGCATCAGTGGGGTCC-3'.

[0033] Figure 3 The effect of transient silencing of CsEAO on theanine accumulation in tea leaves. Figure 3 It can be seen that after the tea leaves were treated with AsODN primers for 24 hours, the expression level of CsEAO gene in the AsOND treatment group was significantly reduced compared with that in the control group ( Figure 3 A). Subsequently, GC-MS was used to detect the ethylamine content in tea leaves. The results showed that the ethylamine content in the treatment group AsOND was significantly higher than that in the control group soND, indicating that transient inhibition of CsEAO expression reduced the degradation of ethylamine. On this basis, the content of theanine in the leaves was detected, and it was found that the content of theanine in the treatment group AsOND was higher than that in the control group soND, indicating that inhibiting CsEAO expression can increase the content of theanine in tea leaves by inhibiting the degradation of ethylamine.

[0034] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A tea tree ethylamine oxidase gene CsEAO, characterized in that: The nucleotide sequence of the tea tree ethylamine oxidase gene CsEAO is shown in SEQ ID NO.

1.

2. An application of reducing the expression level of the tea plant ethylamine oxidase gene CsEAO as claimed in claim 1 in increasing the content of theanine in tea leaves.

3. An application of the tea tree ethylamine oxidase gene CsEAO as claimed in claim 1 in a high theanine content tea variety screening or breeding system.

4. Use of the tea tree ethylamine oxidase gene CsEAO as claimed in claim 1 in constructing a transgenic tea tree system for increasing theanine content in tea leaves.

5. Use of the tea tree ethylamine oxidase gene CsEAO as claimed in claim 1 in an industrial theanine production system.

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