CN115838407A

CN115838407A - Application of CsHO1 in regulating and controlling accumulation of theanine in tea

Info

Publication number: CN115838407A
Application number: CN202211402333.8A
Authority: CN
Inventors: 张照亮; 张书沛; 陈子平; 林世嘉; 陈婷婷
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-03-24
Anticipated expiration: 2042-11-09
Also published as: CN115838407B

Abstract

The invention relates to the technical field of regulation and control of theanine content in tea, in particular to application of CsHO1 in regulation and control of accumulation of theanine in tea, wherein the theanine content in tea is improved by reducing CsHO1 expression quantity, a base sequence of the CsHO1 is shown in SEQ ID NO.1, and the expression level of the CsHO1 is highly negatively correlated with the theanine content, so that the theanine in tea is regulated and controlled by regulating and controlling the CsHO1, and the quality of the tea is improved.

Description

Application of CsHO1 in regulating and controlling accumulation of theanine in tea

Technical Field

The invention relates to the technical field of regulation and control of theanine content in tea, and particularly relates to application of CsHO1 in regulation and control of accumulation of theanine in tea.

Background

Tea tree is one of the most important economic crops in the world. It contains abundant secondary metabolites which confer sensory quality and health benefits to tea. Wherein theanine (gamma-glutamyl acetamide) is a unique non-protein amino acid in tea tree, and is a component for imparting unique delicate flavor to green tea. In addition, theanine has a number of health benefits including promoting relaxation and sedation, improving cognitive function (learning and memory), and improving sleep patterns in humans and animals caused by anxiety. Theanine is presumed to increase the levels of inhibitory neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin and dopamine. Therefore, theanine content largely determines the quality and price of green tea.

Theanine biosynthesis takes glutamic acid (Glu) and Ethylamine (EA) as precursors, and is catalyzed by theanine synthase (CsTSI). The Jurisdictyamide synthetases CsGS1.1 and CsGS2 also contribute to theanine synthesis in young shoots. Glutamate Synthases (GOGATs) and Glutamate Dehydrogenases (GDHs) catalyze Glu biosynthesis in tea, while EA is synthesized from alanine by alanine decarboxylase (CsAlaDC). Candidate genes encoding GOGATs, GDHs and CsLaDC have recently been identified. The catabolism of theanine may be catalysed by cspdx2.1 in tea plant. Theanine biosynthesis and catabolic processes are regulated by a number of environmental factors, particularly nutrient levels, light intensity, salt stress, and the like.

Albino and etiolated tea plant varieties, such as 'Huangshan white tea' and 'Huangkui', are tea plant germplasm resources with important value for high-quality green tea production, and the produced albino or etiolated tea leaves are deeply welcomed by the tea market. This is because whitened or yellowed tea contains high theanine and low tea polyphenol, and the tea soup is fresh and refreshing without significant bitterness and astringency. The molecular mechanism behind why high levels of theanine accumulate in albino and etiolated tea leaves is then largely unknown.

In view of the above problems, the inventors of the present invention have finally obtained the present invention through a long period of research and practice.

Disclosure of Invention

The invention aims to solve the problem of how to apply CsHO1 which influences whitening and yellowing to regulate and control the accumulation of theanine in tea and provides application of CsHO1 in regulating and controlling the accumulation level of theanine in tea.

In order to achieve the purpose, the invention discloses application of CsHO1 in regulating and controlling the accumulation level of theanine in tea, and the content of the theanine in the tea is improved by reducing the expression quantity of the CsHO 1. The base sequence of the CsHO1 is shown in SEQ ID NO. 1.

The expression of CsHO1 promotes the degradation of theanine to glutamate and ethylamine in young shoots.

The CsHO1 regulates and controls the accumulation of theanine in each organ of the young shoots of the tea trees and young shoots in different seasons.

The tea is young sprout of tea tree.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, the expression quantity of CsHO1 in yellowing and whitening tea varieties is far lower than that of normal green varieties, so that the relation between the expression quantity of CsHO1 and the accumulation of theanine in tea is researched, the CsHO1 is used for regulating and controlling the accumulation of the theanine in the tea, and the accumulation quantity of the theanine in the tea is increased by reducing the expression quantity of the CsHO 1.

Drawings

FIG. 1 shows the glutamine, theanine, chlorophyll content in etiolated Arabidopsis and tea tree seedlings; (A) Phenotypes of Arabidopsis Wild Type (WT) and hy1-100 mutants; (B) chlorophyll content; (C) glutamine content; (D) the formula of glutamine; (E) the chemical formula of theanine; (F) Leaf color of normal green tea plant variety "Shuchazao" (SCZ) and yellowing variety "Huangkui" (HK); (G) chlorophyll content; (H) theanine content;

FIG. 2 is a phylogenetic tree, multiple sequence alignment and subcellular localization of CsHO 1; (A) Phylogenetic relationship of HOs in camellia japonica (Cs), arabidopsis thaliana (At), cabbage (Bo), tobacco (Nt), tomato eggplant (Sl), grape (Vv), trememula (Pt), cocoa (Tc), soybean (Gm), rice (Os) and corn (Zm); (B) CsHO1 and other plant HO1 protein alignment; (C) Subcellular localization of CsHO1 protein in Arabidopsis protoplasts;

FIG. 3 shows CsHO1 expression and theanine content in normal, etiolated and albino tea varieties;

FIG. 4 is a graph of CsHO1 expression versus theanine content for different tissues at different time points;

FIG. 5 is the accumulation of theanine in hy1-100 and CsHO1 transgenic hy1-100 mutants; (A) Wild Type (WT), hy1-100 and CsHO1 transgenic hy1-100 mutants (COM 1 and COM 2) with or without 0, 5, 10 or 15mM theanine for 14 days of growth phenotype in MS medium; (B) theanine content of arabidopsis thaliana lines; (C) root length of Arabidopsis lines;

FIG. 6 shows the effect of suppressing CsHO1 expression in tea tree piggyback on the accumulation of theanine in tea; (A) Carrying out real-time PCR analysis on CsHO1 expression after the young shoots are treated by 40 mu M sDON or asODN for 24 h; (B) theanine content in young shoots;

Detailed Description

The above and further features and advantages of the present invention are described in more detail below with reference to the accompanying drawings.

The 8-year-old variety of tea used in this example. These varieties include "Shucha Zao" (SCZ), "Zhe nong 113" (ZN 113), "Zhongcha 302" (ZC 302), "Huangkui" (HK) and "Huangshan Bai cha" (HSBC), and are planted in Guo He tea garden (31 ° N,117 ° E, china). The tissue comprises 1, 2, 3, 4 and 5 leaves of roots, stems, leaf buds and young shoots.

Arabidopsis thaliana hy1-100 mutants are from Shenwenbiao and Schwanger professor of the university of agriculture, nanjing, china. Seeds were sterilized and cultured on solid (MS) medium as usual. The seeds are transferred into a growth chamber after being cultured for 2 days in a layering way at 4 ℃. The conditions are set as day and night illumination period of 16h/8h and illumination intensity of 150 μmol m ^-2 s ^-1 Irradiance, 23 ℃/18 ℃, relative humidity of 70%. Arabidopsis Wild Type (WT), hy1-100 mutant and transgenic lines were grown for 14d in MS medium with or without 0, 5, 10 or 15mM theanine.

1. Hyper-accumulation of Gln or theanine in arabidopsis yellowing mutant hy1-100 and tea tree variety' Huangkui

Haem Oxygenase 1 (HO 1/HY 1) plays a crucial role in the synthesis of arabidopsis chlorophyll, and chlorophyll of seedlings is extracted for 24h in the dark by using 95% (v/v) ethanol, then the chlorophyll content is determined by detecting absorbance meters at 649nm and 665nm, and the arabidopsis HO1 mutant HY1-100 shows a albino phenotype and has a much lower chlorophyll content by verification (figure 1A, B). hy1-100 accumulated more Gln, an analog of theanine, than Wild Type (WT) seedlings (FIGS. 1C-E). Also, the yellowing tea variety "yellow kui" (HK) accumulated lower levels of chlorophyll and higher levels of theanine than the normal green tea variety "shuchazao" (SCZ) (fig. 1F-H). These results indicate that HO1 regulates the accumulation of Gln in arabidopsis thaliana.

2. CsHO1 in sequence and subcellular localization

The gene was amplified from a cDNA library of the tea variety "Shuchazao" using CsHO1 specific primers. The PCR fragment without the stop codon was inserted into the pAN580 vector and fused with Green Fluorescent Protein (GFP). The obtained pAN580-CsHO1-GFP and the empty vector pAN580-GFP were transformed into Arabidopsis protoplasts by polyethylene glycol (PEG) -mediated gene transformation. After incubation for 16 hours at room temperature in the dark, fluorescence was observed using a laser scanning confocal microscope.

HO homologues were searched in tea trees through tea tree information files, and 3 possible CsHOs were determined. Phylogenetic trees, multiple sequence alignments and subcellular localization of CsHO1 are shown in FIG. 2, from which it can be seen that CsHOs belong to two subfamilies: the HO1 subfamily and the HO2 subfamily. CsHO1 and CsHO3 clearly fall into the HO1 subfamily, with HO1, HO3 and HO4 in tobacco, tomato eggplant, arabidopsis, cabbage, grape, populus euphratica, cocoa, glycine, rice and maize (fig. 2A). CsHO1 encodes 289 amino acid residues, of which 58 amino acid transit peptides are recognized by the ChloroP algorithm. Several highly conserved HO1s domains were identified in various plant species (FIG. 2B), suggesting that CsHO1 is conserved among other plants relative to HO1s.

To assess the subcellular localization of CsHO1, we fused CsHO1 to a CaMV35S promoter (35S:: csHO 1-GFP) driven Green Fluorescent Protein (GFP). 35S:: csHO1-GFP and control 35S:: GFP was transformed into Arabidopsis protoplasts. As shown in FIG. 2C, free GFP fluorescence was visible in both cytoplasm and nucleus. In contrast, the fluorescence signal of CsHO1-GFP overlaps with the autofluorescence signal of chlorophyll in chloroplasts. These results indicate that CsHO1 is localized in chloroplasts. This localization is consistent with HO1 in alfalfa, tomato, wheat, and cucumber.

3. Expression quantity of CsHO1 in etiolated and whitened tea variety leaves

The expression of CsHO1 in three normal tea tree varieties (SCZ, ZN113, ZC 302), etiolated variety HK and albino variety HSBC new branches was compared. The results showed that the expression levels of HK and HSBC were much lower than the normal green variety (fig. 3). In both this and previous studies, the chlorophyll content of HK and HSBC was significantly lower than that of the green variety (fig. 1G). Thus, this result indicates that CsHO1 has a positive role in chlorophyll biosynthesis in tea plant, just like its homolog in other plants.

The theanine content in the sprouts of the varieties is measured, as shown in figure 3, the theanine content shows a reverse mode with the CsHO1 expression in the varieties, the theanine content in HK and HSBC is higher, and the correlation coefficient of the CsHO1 expression and the theanine content is further analyzed. The results showed that the CsHO1 expression level was highly inversely correlated with the theanine content (r = -0.98, p-straw bundle 0.001). This result supports the view that CsHO1 negatively regulates the accumulation of theanine in tea plants.

4. CsHO1 expression and theanine content relation

The expression of CsHO1 in different organs and the accumulation level of theanine in the tea variety are detected. The tissue to be detected includes root, tender stem, leaf bud, 1 ^st Leaf, 2 ^nd Leaf, 3 ^rd Leaf, 4 ^th Leaf, 5 ^th Leaves (fig. 4). In these organs, csHO1 expression gradually increased; in contrast, the theanine content gradually decreased (fig. 4B). Further correlation analysis showed that CsHO1 expression was significantly inversely correlated with accumulation of theanine in these tissues.

Accumulation of theanine in tea tree shoots is highly seasonally dependent, and therefore, expression of CsHO1 and theanine content in tea tree leaf buds were examined at 5 time points of 12 months in winter, 12 days in spring (3 months 1 day, 3 months 23 days, arp 13 days) and summer (6 months 14 days), respectively. Again, csHO1 expression showed an opposite trend to theanine content with a correlation coefficient of-0.97 and p-straw of 0.01 (FIG. 4C). These results further indicate that CsHO1 has a negative regulatory effect on the accumulation of theanine in tea.

The expression levels of CsHO1 in different organs were different and also responsive to seasons (FIG. 4), and more importantly, the expression levels of CsHO1 were highly inversely correlated with theanine content. These results indicate that CsHO1 may regulate shoot organs and accumulation of theanine during different seasons. Considering that theanine is mainly synthesized in roots and decomposed in shoots, csHO1 may promote the degradation of theanine to glutamic acid and ethylamine in shoots.

5. Effect of AtHO1 and CsHO1 on the accumulation of theanine in theanine-fed Arabidopsis thaliana

The CsHO1 coding region was amplified by gene-specific primer RT-PCR and cloned into pCAMBIA1302 vector containing CaMV35S promoter. The recombinant plasmid is converted into agrobacterium tumefaciens GV3101, and then the arabidopsis hy1-100 mutant is converted by a flower dipping method.

It was tested whether AtHO1 regulates theanine accumulation in theanine-fed Arabidopsis thaliana. Various concentrations of theanine (0, 5, 10 and 15 mM) were added to MS medium and fed Arabidopsis Wild Type (WT) and hy1-100 mutants. From the inhibitory effect of theanine on root growth, the hy1-100 mutant showed high sensitivity to high concentrations of theanine (10 and 15 mM) (fig. 5a, c), and the accumulation of theanine was also significantly higher for the hy1-100 mutant than for the WT under the same conditions (fig. 5B). These results indicate that AtHO1 regulates the accumulation of theanine in theanine-fed Arabidopsis thaliana, and also suggest that Arabidopsis thaliana can be used as a model plant for studying the regulation of theanine accumulation.

To verify the role of CsHO1 in theanine accumulation, csHO1 driven by the 35S promoter was transferred into Arabidopsis thaliana hy1-100 mutants. Expression of CsHO1 in the hy1-100 mutant restored the etiolating phenotype of hy1-100 in two independent transgenic lines (COM 1 and COM 2) (FIG. 5A). This result indicates that CsHO1 and atoho 1 are functionally conserved in regulating chlorophyll biosynthesis. In addition, csHO1 expression in hy1-100 mutants also restored the theanine hyper-accumulation phenotype under 10 and 15mM feeding conditions (FIG. 5B). This result provides genetic evidence for the negative regulatory role of CsHO1 in theanine accumulation.

6. Influence of CsHO1 expression on theanine in tea tree sprout

Candidate antisense oligonucleotides (AsODN) were designed using SOLIGO software with the CsHO1 cDNA sequence as input. The corresponding sense oligonucleotide (sODN) was used as a control. The young shoot has a bud and 1 ^st The leaves were treated with 0.1ml of 40. Mu.M solution of AsODN or sODN. After 24h incubation, young shoots were harvested and stored at-80 ℃.

To provide in vivo genetic evidence for the negative role of CsHO1 in tea plant theanine accumulation, young shoots of SCZ were treated with CsHO 1-specific sense oligonucleotides (sODN) or antisense oligonucleotides (asODN) for 24h (fig. 6A). asODN treatment significantly inhibited CsHO1 expression levels compared to control sODN treatment (fig. 6B). Meanwhile, theanine accumulation was significantly increased in asODN-treated shoots (fig. 6B). These results indicate that CsHO1 genetically regulates the accumulation of theanine in tea.

In conclusion, in the present invention, it was found that the expression level of CsHO1 in yellowing and whitening tea plant varieties was much lower than that of normal green varieties (FIG. 3), and more importantly, when CsHO1 was expressed in hy1-100 mutant, the yellowing phenotype and chlorophyll content of the mutant were recovered (FIG. 5), and when theanine was fed to the mutant, the phenotype with higher accumulation of the mutant theanine was also recovered. More importantly, transient reduction of CsHO1 expression in tea seedlings by asODN significantly increased the accumulation of theanine (fig. 6), and these genetic analyses provided strong evidence to support the role of CsHO1 in regulating theanine accumulation in tea. Therefore, low expression of CsHO1 in these yellow or whitened tea plants may be responsible for low chlorophyll and high theanine content.

Heme accumulation in the feedback of hy1-100 mutants inhibits the chlorophyll biosynthesis of glutamate. Thus, a decrease in chloroplast biosynthesis is likely to result in an increase in accumulation of etiolated and albino plant glutamate in tea plants, which is both a substrate for theanine biosynthesis and a product of theanine degradation. Excessive accumulation of glutamic acid in yellowing and whitening tea varieties may feedback inhibition of theanine degradation or promotion of theanine biosynthesis, thereby increasing accumulation of theanine in these varieties. The research reveals a new regulation mechanism of theanine accumulation in tea trees, and provides a target gene for creating yellowing or whitening tea tree germplasm which is urgently needed by the tea industry.

The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. It will be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

The application of CsHO1 in regulating and controlling the accumulation of theanine in tea is characterized in that the content of the theanine in the tea is improved by reducing the expression quantity of CsHO1, and the base sequence of the CsHO1 is shown in SEQ ID NO. 1.
2. The use of CsHO1 for regulating the accumulation of theanine in tea as claimed in claim 1, wherein said CsHO1 expression promotes the degradation of theanine into glutamic acid and ethylamine in young shoots.
3. The use of CsHO1 to regulate the accumulation of theanine in tea leaves, as claimed in claim 1, wherein said CsHO1 regulates the accumulation of theanine in shoot organs and in different seasons.
4. The use of CsHO1 to regulate the accumulation of theanine in tea leaves according to claim 1, wherein said tea leaves are tea tree sprouts.