CN115838407A - Application of CsHO1 in Regulating Theanine Accumulation in Tea - Google Patents

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 Theanine Accumulation in Tea

technical field

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

Background technique

Tea tree is one of the most important economic crops in the world. It is rich in secondary metabolites that impart the organoleptic qualities and health benefits of tea. Among them, theanine (γ-glutamyl acetamide) is a unique non-protein amino acid in tea tree, and it is a component that gives green tea a unique umami taste. Additionally, theanine has many health benefits, including promoting relaxation and calm, improving cognitive function (learning and memory), and improving anxiety-induced sleep in humans and animals. It is hypothesized that theanine increases levels of inhibitory neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin, and dopamine. Therefore, the content of theanine largely determines the quality and price of green tea.

Theanine biosynthesis is catalyzed by theanine synthase (CsTSI) using glutamic acid (Glu) and ethylamine (EA) as precursors. The governing amide 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 tree, while EA is synthesized from alanine by alanine decarboxylase (CsAlaDC). Candidate genes encoding GOGATs, GDHs and CsLaDC were recently identified. The catabolism of theanine may be catalyzed by CsPDX2.1 in tea tree. Theanine biosynthesis and catabolism processes are regulated by many environmental factors, especially nutrient levels, light intensity and salt stress, etc.

Albino and yellow tea tree varieties, such as "Huangshan White Tea" and "Huang Kui", are tea tree germplasm resources that are of great value to the production of high-quality green tea, and the produced albino or yellow tea leaves are very popular in the tea market. This is because albino or yellowed tea contains high theanine and low tea polyphenols, and the tea soup has a prominent fresh taste without obvious bitterness. However, the molecular mechanisms behind why high levels of theanine accumulate in whitened and yellowed tea leaves remain largely unknown.

In view of the above problems, the inventor of the present invention has finally obtained the present invention through long-term research and practice.

Contents of the invention

The purpose of the present invention is to solve the problem of how to apply CsHO1, which affects whitening and yellowing, to regulate the accumulation of theanine in tea, and provides the application of CsHO1 in regulating the accumulation of theanine in tea.

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

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

The CsHO1 regulates the accumulation of theanine in various organs of tea shoots and shoots in different seasons.

The tea leaves are new shoots of tea trees.

Compared with the prior art, the beneficial effect of the present invention is that: the present invention finds that the expression of CsHO1 in yellowed and albino tea tree varieties is much lower than that of normal green tea varieties, so that the relationship between the expression of CsHO1 and the accumulation of theanine in tea The relationship between them was studied, CsHO1 was used to regulate the accumulation of theanine in tea, and the accumulation of theanine in tea was increased by reducing the expression of CsHO1.

Description of drawings

Fig. 1 is glutamine, theanine, chlorophyll content in the Arabidopsis thaliana of yellowing and tea tree seedling; (A) the phenotype of Arabidopsis wild type (WT) and hy1-100 mutant; (B) chlorophyll content; (C) glutamine content; (D) chemical formula of glutamine; (E) chemical formula of theanine; (F) normal green tea cultivar “Shuchazao” (SCZ) and yellowish cultivar “Huangkui” (HK ) leaf color; (G) chlorophyll content; (H) theanine content;

Figure 2 shows the phylogenetic tree, multiple sequence alignment and subcellular localization of CsHO1; (A) Camellia (Cs), Arabidopsis (At), cabbage (Bo), tobacco (Nt), tomato Solanum (Sl), grape Phylogenetic relationships of HOs in (Vv), tremula (Pt), cocoa (Tc), soybean (Gm), rice (Os), and maize (Zm); (B) Alignment of CsHO1 with other plant HO1 proteins; (C ) Subcellular localization of CsHO1 protein in Arabidopsis protoplasts;

Fig. 3 is the expression and theanine content of CsHO1 in normal, etiolated and albino tea tree varieties;

Figure 4 is the relationship between CsHO1 expression and theanine content in different tissues and at different time points;

Figure 5 is the theanine accumulation in hy1-100 and CsHO1 transgenic hy1-100 mutants; (A) wild type (WT), hy1-100 and CsHO1 transgenic hy1-100 mutants (COM1 and COM2) in MS medium Growth phenotypes of 14 days with or without adding 0, 5, 10 or 15 mM theanine; (B) theanine content of Arabidopsis lines; (C) root length of Arabidopsis lines;

Figure 6 shows the effect of inhibiting the expression of CsHO1 in new shoots of tea trees on the accumulation of theanine in tea leaves; (A) After the new shoots were treated with 40 μM sDON or asODN for 24 hours, the expression of CsHO1 was analyzed by real-time PCR; (B) Theanine in new shoots content;

Detailed ways

The above and other technical features and advantages of the present invention will be described in more detail below in conjunction with the accompanying drawings.

The 8-year-old tea tree variety that present embodiment uses. These varieties include "Shu Chazao" (SCZ), "Zhe Nong 113" (ZN113), "Zhongcha 302" (ZC302), "Huangkui" (HK) and "Huangshan White Tea" (HSBC), which were produced in Guohe Tea Garden ( 31°N, 117°E, China) planting. Tissues include roots, stems, leaf buds and the 1st, 2nd, 3rd, 4th, 5th leaves of new shoots.

The Arabidopsis hy1-100 mutant was obtained from Prof. Shen Wenbiao and Xie Yanjie, School of Life Sciences, Nanjing Agricultural University, China. Seeds were routinely sterilized and grown on solid state (MS) medium. Seeds were cultured in layers at 4°C for 2 days and then transferred to the growth chamber. The conditions were set as diurnal light period of 16h/8h, light intensity of 150μmol m ^-2 s ^-1 irradiance, 23°C/18°C, 70% relative humidity. Arabidopsis wild-type (WT), hy1-100 mutant lines and transgenic lines were grown on MS medium with or without 0, 5, 10 or 15 mM theanine for 14 days.

1. Hyperaccumulation of Gln or theanine in Arabidopsis thaliana yellow mutant hy1-100 and tea tree cultivar “Huangkui”

Haem Oxygenase 1 (HO1/HY1) plays a vital role in the synthesis of chlorophyll in Arabidopsis. The chlorophyll of the seedlings was extracted with 95% (v/v) ethanol in the dark for 24 hours, and then detected by the absorbance meter at 649nm and 665nm Chlorophyll content was measured, and it was verified that the Arabidopsis HO1 mutant hy1-100 exhibited an albino phenotype with much lower chlorophyll content (Fig. 1A,B). hy1-100 accumulated more Gln, an analog of theanine, than wild-type (WT) seedlings (Fig. 1C-E). Likewise, the etiolated tea cultivar 'Huangkui' (HK) accumulated lower levels of chlorophyll and higher levels of theanine than the normal green tea cultivar 'Shuchazao' (SCZ) (Fig. 1F–H). These results suggest that HO1 regulates the accumulation of Gln in Arabidopsis.

2. Sequence and subcellular localization of CsHO1

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

We searched for HO homologues in tea tree through the tea tree information archive and identified 3 possible CsHOs. The phylogenetic tree, multiple sequence alignment, and subcellular localization of CsHO1 are shown in Figure 2. It can be seen from the figure that CsHOs belong to two subfamilies: HO1 subfamily and HO2 subfamily. CsHO1 and CsHO3 were clearly classified into the HO1 subfamily, among which HO1, HO3 and HO4 were found in tobacco, tomato, Arabidopsis, cabbage, grape, Populus euphratica, cocoa, glycine, rice and maize (Fig. 2A). CsHO1 encoded 289 amino acid residues, of which 58 amino acid transit peptides were recognized by the ChloroP algorithm. Several highly conserved HO1s domains were identified in various plant species (Fig. 2B), suggesting that CsHO1 is conserved from HO1s in other plants.

To assess the subcellular localization of CsHO1, we fused CsHO1 to green fluorescent protein (GFP) driven by the CaMV 35S promoter (35S::CsHO1-GFP). 35S::CsHO1-GFP and control 35S::GFP were transformed into Arabidopsis protoplasts. As shown in Figure 2C, free GFP fluorescence was visible in both the cytoplasm and nucleus. In contrast, the fluorescence signal of CsHO1-GFP overlaps with the autofluorescence signal of chlorophyll in chloroplasts. These results indicated that CsHO1 localized in chloroplasts. This localization is consistent with HO1 in alfalfa, tomato, wheat and cucumber.

3. Expression of CsHO1 in leaves of yellowed and albino tea varieties

The expression of CsHO1 in new shoots of three normal tea cultivars (SCZ, ZN113, ZC302), etiolated cultivar HK and albino cultivar HSBC was compared. The results showed that the expression levels of HK and HSBC were much lower than that of normal green varieties (Fig. 3). Both HK and HSBC had significantly lower chlorophyll content than green varieties in this and previous studies (Fig. 1G). Thus, this result suggests that CsHO1 has an active role in chlorophyll biosynthesis in tea plant, like its homologues in other plants.

The content of theanine in the shoots of these varieties was determined, as shown in Figure 3, the content of theanine in these varieties showed the opposite pattern to that of CsHO1 expression, and the content of theanine was higher in HK and HSBC, further analysis Correlation coefficient between CsHO1 expression and theanine content. The results showed that the expression of CsHO1 was highly negatively correlated with the content of theanine (r=-0.98, p<0.001). This result supports the idea that CsHO1 negatively regulates theanine accumulation in tea trees.

4. Relationship between CsHO1 expression and theanine content

The expression of CsHO1 in different organs and the accumulation level of theanine in tea plant varieties were detected. Tissues examined included ^roots , young stems, leaf buds, 1st leaf, ^2nd leaf, ^3rd leaf, ^4th leaf, and ^5th leaf (Fig. 4). In these organs, the expression of CsHO1 gradually increased; conversely, the content of theanine gradually decreased (Fig. 4B). Further correlation analysis showed that CsHO1 expression was significantly negatively correlated with theanine accumulation in these tissues.

The accumulation of theanine in tea tree twigs is highly season-dependent, therefore, the accumulation of theanine in winter (December 12, spring (March 1, March 23, Arp 13) and summer (June 14 ) The expression of CsHO1 and the content of theanine in tea buds were detected at five time points. Again, CsHO1 expression showed an opposite trend with theanine content, with a correlation coefficient of -0.97, p<0.01 (Fig. 4C). These results further indicated that CsHO1 negatively regulates theanine accumulation in tea plants.

The expression levels of CsHO1 were different in different organs and also responded to seasons (Fig. 4), and more importantly, the expression levels of CsHO1 were highly negatively correlated with theanine content. These results suggest that CsHO1 may regulate the accumulation of theanine in bud organs and in different seasons. Considering that theanine is mainly synthesized in roots and decomposed in shoots, CsHO1 may promote the degradation of theanine to glutamate and ethylamine in shoots.

5. Effects of AtHO1 and CsHO1 on theanine accumulation in Arabidopsis fed with theanine

The CsHO1 coding region was amplified by RT-PCR with gene-specific primers and cloned into the pCAMBIA1302 vector containing the CaMV35S promoter. The recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101, and then the Arabidopsis hy1-100 mutant was transformed by flower dipping method.

tested whether AtHO1 regulates theanine accumulation in theanine-fed Arabidopsis. Different concentrations of theanine (0, 5, 10, and 15 mM) were added to MS medium to feed 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 under the same conditions, the hy1-100 mutant The accumulation of theanine in the body was also significantly higher than that in the WT (Fig. 5B). These results suggest that AtHO1 regulates theanine accumulation in Arabidopsis fed theanine, and also suggest that Arabidopsis can be used as a model plant to study the regulation of theanine accumulation.

To verify the role of CsHO1 in theanine accumulation, 35S promoter-driven CsHO1 was transferred into Arabidopsis hy1-100 mutant. Expression of CsHO1 in hy1-100 mutants restored the yellowish phenotype of hy1-100 in two independent transgenic lines (COM1 and COM2) (Fig. 5A). This result suggests that CsHO1 is functionally conserved with AtHO1 in regulating chlorophyll biosynthesis. Furthermore, expression of CsHO1 in hy1-100 mutants also restored the theanine hyperaccumulation phenotype under 10 and 15 mM feeding conditions (Fig. 5B). This result provides genetic evidence for a negative regulatory role of CsHO1 in theanine accumulation.

6. Effect of CsHO1 expression in tea buds on theanine

Candidate antisense oligonucleotides (AsODN) were designed using SOLIGO software, and the CsHO1 cDNA sequence was used as input. The corresponding sense oligonucleotide (sODN) was used as a control. New shoots with one shoot and 1 ^st leaf were treated with 0.1 ml of 40 μM AsODN or sODN solution. After 24 hours of incubation, the shoots were harvested and stored at -80°C.

To provide in vivo genetic evidence for the negative role of CsHO1 in tea tree theanine accumulation, SCZ shoots were treated with CsHO1-specific sense oligonucleotides (sODN) or antisense oligonucleotides (asODN) for 24 h (Fig. 6A ). Compared with control sODN treatment, asODN treatment significantly inhibited the expression level of CsHO1 (Fig. 6B). Meanwhile, theanine accumulation was significantly increased in asODN-treated shoots (Fig. 6B). These results suggest that CsHO1 genetically regulates theanine accumulation in tea plants.

To sum up, in the present invention, by finding that the expression level of CsHO1 in etiolated and albino tea tree varieties is much lower than that in normal green varieties (Figure 3), more importantly, when CsHO1 is expressed in hy1-100 mutant , the yellowing phenotype and chlorophyll content of the mutant were restored (Fig. 5), and the phenotype of higher theanine accumulation in the mutant was also restored by feeding the mutant with theanine. More importantly, transient reduction of CsHO1 expression in tea tree seedlings using asODN significantly increased theanine accumulation (Fig. 6), and these genetic analyzes provide strong support for the role of CsHO1 in regulating theanine accumulation in tea evidence. Therefore, the low expression of CsHO1 may be responsible for the low chlorophyll content and high theanine content in these yellowed or albino tea plants.

Heme accumulation in hy1-100 mutant feedback inhibits chlorophyll biosynthesis from glutamate. Therefore, reduced chloroplast biosynthesis is likely to lead to increased glutamate accumulation in etiolated and albino plants. In tea plants, glutamate is both a substrate for theanine biosynthesis and a product of theanine degradation. Over-accumulation of glutamate in etiolated and albino tea tree cultivars may feedback to inhibit theanine degradation or promote theanine biosynthesis, thereby increasing theanine accumulation in these cultivars. This study reveals a novel regulatory mechanism for theanine accumulation in tea plants and also provides target genes for the creation of etiolated or albino tea plant germplasm that is much needed by the tea industry.

The above descriptions are only preferred embodiments of the present invention, and are only illustrative rather than restrictive to the present invention. Those skilled in the art understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, but all will fall within the protection scope of the present invention.

Claims (4)

1. 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. 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. 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. 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.

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