CsDTX29 gene and application of CsDTX29 gene in regulation and control of fluorine distribution in tea trees

Abstract

The invention discloses a tea tree CsDTX29 gene and application thereof to regulation and control of fluorine distribution in tea trees, and belongs to the technical field of biology, and the sequence of the gene is shown as SEQ ID No.1. Specific expression of the CsDTX29 gene in the tea tree root is provided, and the expression level of the CsDTX29 gene in the root is remarkably positively correlated with the fluorine content of the tea tree root and is remarkably negatively correlated with the fluorine content of the tea tree leaf. The expression of the CsDTX29 gene instantaneously silenced by antisense oligonucleotide in the root system of the tea tree obviously reduces the fluorine content of the root system of the tea tree and increases the fluorine content of stems and leaves of the tea tree at the same time. Therefore, the improvement of the expression level of the CsDTX29 gene in the root system of the tea tree is an important way for reducing the transfer of fluorine to the overground part. Cloning and function verification of the gene help to enrich people's understanding of tea tree fluorine absorption and transport mechanisms, and a certain theoretical basis is provided for cultivation of low-fluorine tea tree varieties and reduction of the fluorine content of tea tree leaves.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a CsDTX29 gene and an application thereof in regulating fluorine distribution in tea plants. Background Art

[0002] Tea (Camellia sinensis) is an important economic crop in my country. Tea, processed from its leaves, is rich in various beneficial ingredients, such as tea polyphenols, amino acids, and caffeine, and possesses important nutritional and health benefits. However, tea is a fluoride hyperaccumulator, with fluoride levels 10-100 times higher than other plants grown in the same area. Fluoride accumulates primarily in the leaves, with higher levels in mature and older leaves. Long-term consumption of high-fluoride tea can lead to excessive fluoride intake, potentially causing health problems such as dental fluorosis and skeletal fluorosis.

[0003] Tea trees absorb fluoride from the soil primarily through their roots, which then transport it to the aboveground parts, where it eventually accumulates in the leaves. The roots are the primary organ for fluoride absorption in tea trees, and they can store some fluoride. If more fluoride could be stored in the roots, the transport of fluoride to the aboveground parts would be reduced, thereby reducing the fluoride content in tea leaves. Currently, research on the molecular mechanisms of fluoride transport and storage in tea roots is relatively limited. Exploring the molecular mechanisms of fluoride storage in tea roots could help cultivate low-fluoride tea varieties, reduce the fluoride content in tea leaves, and provide a theoretical basis for safe tea production.

[0004] Research has shown that the absorption and transport of fluoride in tea plants primarily depends on a number of transporter proteins, such as CsFEX, CsABCB9, CsNPF2.3, and CsALMT6 / 14. These proteins participate in fluoride transmembrane transport, thereby regulating fluoride accumulation in the tea plant. These reported transporters primarily participate in fluoride transmembrane transport in leaves, while no reports have been found of transporters primarily involved in fluoride accumulation and storage in the tea root system. Recent studies have demonstrated that MATE transporters play a crucial role in regulating the uptake and transport of chloride ions in plants. Chlorine and fluorine belong to the same main group in the periodic table and share certain similarities, leading us to speculate that MATE transporters may also be involved in fluoride transmembrane transport in tea plants. However, to date, the involvement of MATE transporters in fluoride absorption and transport in tea plants has not been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to regulate the storage of fluorine in the roots of tea plants and thereby reduce the transport of fluorine to the aboveground parts. The application of the CsDTX29 gene in regulating the distribution of fluorine in tea plants is provided.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a tea plant CsDTX29 gene, whose CDS sequence is shown in SEQ ID NO.1.

[0008] The second aspect of the present invention provides the protein encoded by the above-mentioned tea plant CsDTX29 gene, whose amino acid sequence is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention proposes the use of the above-mentioned tea plant CsDTX29 gene in regulating fluoride distribution in tea plants.

[0010] Preferably, by promoting the expression of the CsDTX29 gene of the tea tree in the root system of the tea tree, fluorine is stored in the roots of the tea tree, thereby reducing the transport of fluorine to the aboveground part, thereby reducing the fluorine content in the tea leaves; by inhibiting the expression of the CsDTX29 gene of the tea tree in the root system of the tea tree, the storage of fluorine in the roots of the tea tree is reduced, and fluorine is transported to the aboveground part, thereby increasing the fluorine content in the tea leaves.

[0011] The fourth aspect of the present invention provides a biological material containing the above-mentioned tea plant CsDTX29 gene, wherein the biological material is a recombinant DNA, an expression vector, a host bacteria or a plant material.

[0012] The fifth aspect of the present invention proposes a method for regulating the fluorine content in tea leaves, comprising the following steps: reducing or increasing the fluorine content in tea leaves by promoting or inhibiting the expression of the tea plant CsDTX29 gene in the tea plant root system.

[0013] The sixth aspect of the present invention proposes the use of the above-mentioned tea plant CsDTX29 gene in breeding tea plant varieties with high or low fluorine content in leaves.

[0014] A seventh aspect of the present invention provides a method for breeding tea varieties with low fluorine content in leaves, the method comprising:

[0015] (1) making the tea plant contain the above-mentioned tea plant CsDTX29 gene;

[0016] (2) Overexpressing the tea plant CsDTX29 gene.

[0017] Preferably, the method includes but is not limited to cloning the tea plant CsDTX29 gene, constructing the tea plant CsDTX29 gene sequence into an overexpression vector, transforming the recombinant vector into a strain, genetically modifying, and propagating the transgenic material.

[0018] An eighth aspect of the present invention provides a method for breeding tea varieties with high fluorine content in leaves, the method comprising silencing and inhibiting the expression of the CsDTX29 gene in the roots of the tea trees.

[0019] Preferably, the CsDTX29 gene is suppressed by antisense oligonucleotide gene silencing.

[0020] The present invention has the following beneficial effects:

[0021] 1. The present invention newly discovered a tea plant fluoride transporter gene, CsDTX29, which is specifically expressed in tea plant roots. By silencing the CsDTX29 gene in tea plant roots using antisense oligonucleotides (asODNs), the fluoride content in the roots was significantly reduced, while that in the stems and leaves was significantly increased. This allows CsDTX29 to regulate fluoride storage in tea plant roots and reduce fluoride content in tea leaves.

[0022] 2. The tea plant CsDTX29 gene proposed in the present invention is specifically expressed in the tea plant root system. The expression level of the CsDTX29 gene in the tea plant root system is significantly positively correlated with the fluoride content in the tea plant root system and significantly negatively correlated with the fluoride content in the tea leaf system. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the growth of the CsDTX29 overexpressing yeast strain on YPD solid medium in Example 1 of the present invention;

[0024] Figure 2 This is a graph showing the fluorine content of CsDTX29 in different yeasts in Example 1 of the present invention;

[0025] Figure 3 This is a diagram showing the expression pattern of CsDTX29 in different tissues of tea plants in Example 1 of the present invention;

[0026] Figure 4 Figure 1 is a graph showing the fluorine content in the roots and stems of tea plants after silencing the expression of CsDTX29 in the roots of tea plants in Example 1 of the present invention, wherein A is a graph showing the silencing pattern of the CsDTX29 gene in the roots of tea plants, B is a graph showing the gene expression level after silencing CsDTX29 in the roots, and C is a graph showing the fluorine content in the roots, stems, and leaves after silencing CsDTX29 in the roots of tea plants.

[0027] Figure 5 This is an analysis of CsDTX29 expression and F content in different tea varieties in Example 1 of the present invention, wherein A is a graph showing the fluorine content in leaves of different tea varieties, B is a graph showing the fluorine content in roots of different tea varieties, C is a graph showing the relative expression levels of the CsDTX29 gene in roots of different tea varieties, D is a graph showing the correlation between the relative expression levels of the CsDTX29 gene in roots of different tea varieties and the fluorine content in leaves of different tea varieties, and E is a graph showing the correlation between the relative expression levels of the CsDTX29 gene in roots of different tea varieties and the fluorine content in roots of different tea varieties. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0029] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0030] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0031] Example 1:

[0032] 1. Gene cloning: Total RNA was extracted from the young roots of Shucha, a national tea variety. The total RNA was extracted using the FastPure Universal plant Total RNA Isolation Kit (Vazyme, Nanjing, China) according to the instructions, and the RNA content and quality were detected using a spectrophotometer. The first-strand cDNA of the reverse transcription was obtained according to the instructions of the HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Vazyme, Nanjing, China). The first-strand cDNA was used as the RT-PCR template, and PCR was performed using the conventional method to amplify the CsDTX29 gene. The upstream primers are: (5'-ATGACGATGACGATGGCGGA-3'), (SEQ ID NO.3)

[0033] Downstream primer: (5'-TCAAATTTCTCTGTCATCTG-3'). (SEQ ID NO.4)

[0034] A 50 μl PCR reaction system consisted of: 5 μl 10× buffer, 5 μl 2 mM dNTPs, 3 μl 25 mM MgSO₄, 1 μl each of upstream and downstream primers, 1 μl KOD-Plus, 2 μl template cDNA, and 32 μl ddH₂O. The reaction procedure was as follows: 94°C for 2 min, 98°C for 10 sec, 58°C for 30 sec, 68°C for 54 sec, and 68°C for 10 min, for 29 cycles. The PCR product, CsDTX29, was purified and ligated into the pEASY-Blunt vector (Promega, Shanghai, China) to generate the pEASY-Blunt::CsDTX29 plasmid. This plasmid was then transformed into competent E. coli DH5α cells and sent to a biotechnology company for sequencing. The nucleotide sequence of the CsDTX29 gene is shown in SEQ ID NO. 1. The protein sequence encoded by the CsDTX29 gene is shown in SEQ ID NO. 2.

[0035] 2. Functional verification of the CsDTX29 gene in yeast

[0036] Using pEASY-Blunt::CsDTX29 plasmid as template,

[0037] Upstream primer: 5'-ccgggctgcaggaattcATGACGATGACGATGGCGGA-3' (SEQ ID NO. 5),

[0038] PCR amplification was performed using the downstream primer: 5'-gccccccctcgaggtcgacTCAAATTTCTCTGTCATCTG-3' (SEQ ID NO. 6). The vector plasmid pDR196-EGFP was then double-digested with enzymes. The digested and PCR products were recovered by agarose gel electrophoresis. Recombination was performed using a one-step rapid cloning enzyme ligation technique, with 2 μl of double-digested vector and 1 μl of PCR gel-recovered product added. Recombination was then performed using 10 μl of 2× Hieff cloning enzyme premix and 7 μl of ddH2O. After recombination, the recombinant plasmid was transformed into Escherichia coli DH5α and sent to a biotechnology company for sequencing.

[0039] 2) CsDTX29 transformed yeast

[0040] CsDTX29 was transferred into yeast for overexpression to verify the inhibitory effect of fluoride on yeast growth. The BY4743 yeast strain was streaked on YPD solid culture medium and cultured in a constant temperature incubator at 28°C for 2 days. Single colonies with good growth were picked and cultured in liquid YPD culture medium. When the OD value grew to about 0.8, the bacteria were centrifuged and collected, and then washed with clean water. Then, the vector plasmid DNA carrying the target gene was added, and the target gene was transferred into the yeast using a chemical method. The transformed yeast was cultured on YNB-U solid culture medium for 3-5 days, single colonies were picked, and colony PCR was performed to verify the positive colonies.

[0041] 3) Yeast dotting experiment

[0042] The positive yeast strains screened were cultured in YPD medium to an OD value of about 0.8, centrifuged and collected the precipitate, washed once with sterile water, and the bacterial solution was resuspended in sterile water to an OD value of 0.8±0.02. Then the bacterial solution was diluted to 10 0 , 10 -1 , 10 -2 , 10 -3 Using the transformed pDR196-EGFP as a negative control, yeast strains in each dilution gradient were cultured on YPD plates containing different concentrations of fluoride at 28°C.

[0043] like Figure 1 The growth phenotype of CsDTX29 in a heterologous yeast system treated with different concentrations of fluoride was analyzed. The results showed that when no fluoride was added, there was no significant growth difference between transgenic CsDTX29 yeast and yeast expressing the empty vector. However, under 40 mM fluoride treatment, yeast expressing the CsDTX29 gene exhibited a significant growth advantage compared to yeast expressing the empty vector, demonstrating improved tolerance to fluoride.

[0044] 4) Determination of fluoride accumulation in yeast

[0045] The yeast strain was streaked on YPD medium and cultured at 28°C for 2 days to obtain a single colony. A single colony was picked and cultured in YPD liquid medium at 28°C until the OD 600 The yeast cell pellet was collected by centrifugation and washed three times with 15 ml of ultrapure water. The yeast pellet was then resuspended in 10 ml of ultrapure water. Finally, the fluoride content in the yeast was extracted using a freeze-thaw method (boiling at 100°C for 1 hour, freezing at -20°C for 2-3 hours, repeated three times). The fluoride content of the extracted solution was measured using a fluoride ion electrode.

[0046] The fluoride content in yeast cells is shown in Figure 2. Figure 2As shown in the Figure 3, the fluoride content in yeast overexpressing CsDTX29 was significantly higher than that in yeast transfected with empty vector, indicating that yeast overexpressing CsDTX29 accumulated more fluoride. These results indicate that CsDTX29 is involved in fluoride transport in yeast.

[0047] 3. Analysis of differential expression of CsDTX29 gene

[0048] 1) CsDTX29 gene expression in different tissues of tea plants

[0049] Nine tissues and organs from the national tea variety Shuchazao were used to analyze gene expression. The nine tissues and organs included root, stem, bud, flower, 1st leaf, 2nd leaf, 3rd leaf, 4th leaf, and 5th leaf. These samples were also used for total RNA extraction and first-strand cDNA synthesis. The reverse transcription product (first-strand cDNA) was diluted 5-fold and used as a template. Using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), a 20μl reaction system was prepared: 1.0μl of the 5-fold diluted reverse transcription product, 0.5μl of each upstream and downstream primer, 10μl ChamQ Universal SYBR qPCR Master Mix, and 8μl ddH2O. Each reaction was replicated four times. Fluorescence quantitative PCR was then performed.

[0050] Upstream primer: 5'-TACATGCAAAGGTCGTGGCT-3' (SEQ ID NO. 7),

[0051] Downstream primer: 5'-GGCCCCACCCTAACTTCATC-3' (SEQ ID NO. 8),

[0052] The CsGADPH gene of tea plant was used as an internal reference.

[0053] Upstream primer: 5'-TTGGCATCGTTGAAGGGTCT-3' (SEQ ID NO. 9),

[0054] Downstream primer: 5'-CAGTGGGAACACGGAAAGC-3' (SEQ ID NO. 10), the relative expression levels of CsDTX29 in different tissues were calculated.

[0055] The expression levels of CsDTX29 gene in different tissues of tea plants are as follows Figure 3 As shown, the results of qRT-PCR detection showed that CsDTX29 was expressed in various tissues, and the expression level in roots was significantly higher than that in other parts.

[0056] 2) Inhibiting CsDTX29 gene expression can significantly change the distribution of fluoride content in tea plants

[0057] To verify whether CsDTX29 transports fluoride within tea plants, we transiently silenced CsDTX29 expression in roots using antisense oligonucleotides. Young Shucha early tea seedlings were treated with approximately 20 ml of CsDTX29 sense (sODN, control) and antisense (AsODN) oligonucleotide primers. After 24 hours of silencing, roots were incubated in a 5 mg / mL aqueous solution of LF for another 24 hours. Roots, stems, and leaves were then harvested and immediately placed in a -80°C freezer for further analysis.

[0058] sODN sequence:

[0059] (5'-ACATATGATGCCTAGTGGGC-3', (SEQ ID NO. 11)

[0060] 5'-AACTCGAAATGCTAGGGATC-3', (SEQ ID NO.12)

[0061] 5'-TGCTAGGGATCTACATGCAA-3', (SEQ ID NO.13)

[0062] 5'-GCACACCCGACAGCTGCAAA-3'(SEQ ID NO.14)),

[0063] asODN sequence:

[0064] (5'-GCCCACTAGGCATCATATGT-3', (SEQ ID NO.15)

[0065] 5'-GATCCCTAGCATTTCGAGTT-3', (SEQ ID NO.16)

[0066] 5'-TTGCATGTAGATCCCTAGCA-3', (SEQ ID NO. 17)

[0067] 5'-TTTGCAGCTGTCGGGTGTGC-3' (SEQ ID NO. 18)).

[0068] Fluoride content in roots and stems after transient silencing of CsDTX29 Figure 4As shown, after silencing the CsDTX29 gene in the roots, the fluoride content in the roots was significantly reduced, and the fluoride content in the stems and leaves was significantly increased. Combined with the functional verification of CsDTX29 in yeast and its high expression in roots, these results indicate that CsDTX29 is involved in the fluoride transport process in the roots of tea trees.

[0069] 3) Correlation analysis between CsDTX29 expression in tea plants and fluoride content in tea leaves

[0070] Fluoride content in leaves 1-3 and CsDTX29 expression in roots of 12 tea cultivars were measured. The 12 tea cultivars included 'Fuding Dabaicha' (FDDBC), 'Fuding Dahaocha' (FDDHC), 'Zhongcha 108' (ZC108), 'Yaoshanxiulv' (YSXL), 'Xinyang 10' (XY10), 'Huangguanyin' (HGY), 'Echa 4' (EC 4), 'Yingshuang' (YS), 'Wuniuzao' (WNZ), 'Jinguanyin' (JGY), 'Shuchazao' (SCZ), and 'Wancha 6' (WC 6). Root samples of 12 tea varieties were ground and RNA was extracted for reverse transcription. Fluorescence quantitative qRT-PCR was used to analyze the expression of CsDTX29. The fluoride content in leaves 1-3 of the 12 different tea varieties was detected using a fluoride ion electrode. The correlation between the expression of CsDTX29 gene and the fluoride content in roots and leaves of different varieties was analyzed.

[0071] like Figure 5 As shown in Figure 2, the fluorine content in tea leaves decreased significantly with the increase of CsDTX29 expression in roots, showing a significant negative correlation trend, with a correlation coefficient of -0.6788 ( Figure 5 D); the fluorine content in tea tree roots increased significantly with the increase in the expression of CsDTX29 in the roots, showing a significant positive correlation trend, with a correlation coefficient of 0.6713 ( Figure 5 E). The correlation between fluoride content in leaves and roots of different tea varieties and CsDTX29 further confirmed that CsDTX29 has the function of transporting fluoride.

[0072] SEQ ID NO.1:

[0073]

[0074] SEQ ID NO.2:

[0075] MTMTMAEGRDVTTKGRDAMAKRDYGHDDVHFIVMVMVSIFLNLDLNPNLNLPLPWVIQVFIMSCILHMMPSGQLCIKTLEIYIGLNGGTNLLNSGPRGIGHSSGIFVVYYSSMEGQTDSTQVQEELRMAGGSSQVQTFFREFYAESKKL WFLASPAIFTSICQYSLGAITQVFAGQLGTSELAAVSVENSIIAGFAYGIMWGMGSALETLCGIAFGAGQLEMLGIYMQRSWLILNTTALMLVFVYIFATHILKIIGQTDQISEDAGKFALWMIPQLFAYAMNYPLAKFLQAQGKFMAM AVIAGLVLVLHAFFSWVLMMKLGWGLAGAAVVLNSSWWFIVVAQLVYVLCGACGGAWNGFSWLAFHNLWGFLKLSVASAVMLALEMWYTMSLTLLFAGYLKDTEVSVDASSICVNILGWTTMVGFGFNAAISVRVSNELGAAHPTAAKFS AMVVAVTSFLIGLFLALILIIGGKEYPSFFSNDAAVKELVYELTPLLGLAIVVYSVQLALAGVAIGAGWQAYIAYVNLGCYYLFGIPLSLLMGFKFNMGIKGIWWGVISGTVLEACVVLWIINRTNWNNEASVTGDKLKQWGGETDDREI

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A tea plant CsDTX29 gene, characterized in that Its CDS sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the tea plant CsDTX29 gene according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. Use of the tea plant CsDTX29 gene according to claim 1 in regulating fluoride distribution in tea plants.

4. The use according to claim 3, characterized in that By promoting the expression of the CsDTX29 gene in the roots of tea trees, fluorine is stored in the roots of tea trees, thereby reducing the transport of fluorine to the aboveground parts, thereby reducing the fluorine content in tea leaves; by inhibiting the expression of the CsDTX29 gene in the roots of tea trees, the storage of fluorine in the roots of tea trees is reduced, and fluorine is transported to the aboveground parts, thereby increasing the fluorine content in tea leaves.

5. A biological material containing the tea plant CsDTX29 gene according to claim 1, characterized in that: The biological material is recombinant DNA, expression vector, host bacteria or plant material.

6. A method for regulating the fluorine content of tea leaves, characterized in that: The following steps are involved: The fluorine content in tea leaves is reduced or increased by promoting or inhibiting the expression of the tea tree CsDTX29 gene according to claim 1 in the tea tree root system.

7. Use of the tea plant CsDTX29 gene according to claim 1 in breeding tea varieties with high or low fluorine content in leaves.

8. A method for breeding tea varieties with low fluorine content in leaves, characterized in that: The method comprises: (1) making the tea plant contain the tea plant CsDTX29 gene according to claim 1; (2) Overexpressing the tea plant CsDTX29 gene according to claim 1 in tea plants.

9. The method according to claim 8, characterized in that The method comprises cloning the tea plant CsDTX29 gene, constructing the tea plant CsDTX29 gene sequence into an overexpression vector, transforming the recombinant vector into a strain, performing genetic modification, and propagating the genetically modified material.

10. A method for breeding tea varieties with high fluorine content in leaves, characterized in that: The method comprises silencing and inhibiting the expression of CsDTX29 gene in the root system of tea plants.

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