Tea plant CsFAAH6 gene and use thereof

Abstract

The present disclosure provides a tea plant CsFAAH6 gene and use thereof. The tea plant CsFAAH6 gene has a nucleotide sequence shown in SEQ ID NO: 1 in sequence listing. A protein coded by the tea plant CsFAAH6 gene has an amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The CsFAAH6 is highly expressed in mature leaves of tea plants; there is significant negative correlation between theanine content in shoots (one bud and two leaves) of different tea cultivars in different months and expression level of the CsFAAH6; instantaneous silencing of CsFAAH6 expression can significantly increase the theanine content, indicating that CsFAAH6 has a physiological function of theanine degradation and a molecular mechanism thereof.

Claims

1. A method for using a tea plant Camellia sinensis fatty acid amide hydrolase 6 (CsFAAH6) gene to lower theanine content in tea shoots and roots, comprising the following steps: cloning a tea plant CsFAAH6 gene sequence encoding the protein of SEQ ID NO:2; constructing a tea plant expression vector comprising the CsFAAH6 gene sequence; and transforming the tea plant expression vector into the tea shoots and roots, thereby expressing the gene sequence and lowering the theanine content in tea shoots and roots.

2. The method according to claim 1, wherein the tea plant CsFAAH6 gene sequence comprises SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A illustrates phenotypes of different tissues and organs of a tea plant; and FIG. 1B illustrates expression patterns of tea plant CsFAAH6 in different tissues of a tea plant;

(2) FIGS. 2A-F illustrates subcellular localization of tea plant CsFAAH6;

(3) FIG. 3A and FIG. 3B illustrate a process of a transient silencing test of CsFAAH6 gene in tea shoots; FIG. 3C illustrates the detection of CsFAAH6 gene expression after the silencing test; and FIG. 3D illustrates the theanine content in the CsFAAH6 gene silenced tea shoots; and

(4) FIG. 4A illustrates the correlation between expression levels of CsFAAH6 in Camellia sinensis cv. Shuchazao and theanine content in tea shoots; FIG. 4B illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Zhongcha 108 and theanine content in tea shoots; FIG. 4C illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Wancha 91 and theanine content in tea shoots; FIG. 4D illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Fuding-dabaicha and theanine content in tea shoots; FIG. 4E illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Shancha 1 and theanine content in tea shoots; FIG. 4F illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Shidacha and theanine content in tea shoots; FIG. 4G illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Tieguanyin and theanine content in tea shoots; FIG. 4H illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Xianyuzao and theanine content in tea shoots; and FIG. 4I illustrates the correlation between expression levels of CsFAAH6 in C. sinensis cv. Yingshuang and theanine content in tea shoots.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(5) Technical solutions in the example of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the example of the present disclosure. Apparently, the described example is only a part of, but not all of, the examples of the present disclosure. Based on the example of the present disclosure, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present disclosure.

(6) 1. Cloning and Sequence Structure Analysis of the CsFAAH6 Gene

(7) The tea plant CsFAAH6 gene is a tea plant fatty acid amide hydrolase encoding gene, and cloning and sequence structure analysis thereof are specifically as follows:

(8) The cultivar C. sinensis cv. Shuchazao was planted in the Nongcuiyuan, Anhui Agricultural University, Anhui Province, and young roots were used for RNA extraction. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen, Beijing, China) in accordance with the instructions. The RNA content and quality were detected by using a spectrophotometer.

(9) The first strand was generated by reverse transcription: with 1 g of RNA as a template, a reaction buffer was prepared according to the instructions of PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Biotech, China), where 0.6 L of Oligo dT Primer (50 M), 0.4 L of Random 6mers (50 M), and 1 L of dNTP Mixture (10 mM each) were added, and the reaction system was made up to 10 L with RNase Free ddH.sub.2O; the RNA was denatured at 65 C. for 5 min and immediately placed on ice. Subsequently, the above reaction buffer was added with 4 L of 5 PrimerScript Buffer, 0.5 L of RNase Inhibitor (40 U), and 1 L of PrimerScript RTase (200 U), made up to 20 L with ddH.sub.2O, and incubated at 42 C. for 45 min. and reverse transcriptase was inactivated at 95 C. for 5 min. After optimization, a quantity of reverse transcription product was taken for subsequent PCR. The CsFAAH6 gene was amplified by conventional PCR using the first-strand cDNA as an RT-PCR template. The upstream primer was 5-ATGGGCATTTTCAAGGCCAA-3 (SEQ ID NO: 3), and the downstream primer was 5-TCAATCCTITITGAGCAGATCA-3 (SEQ ID NO: 4). The 20 L PCR system was: 2.5 L of 10Ex Taq buffer, 2.0 L of dNTP, 1 L each of upstream and downstream primers, 0.2 L of Ex Taq, 1 L of template, and 15.8 L of ddH.sub.2O.

(10) The reaction program was as follows: initial denaturation at 98 C. for 10 s, 35 cycles of denaturation at 98 C. for 10 s, annealing at 57 C. for 30 s, and extension at 72 C. for 2 min; and extension at 72 C. for 10 min. The PCR product CsFAAH6 gene was purified, recovered, and ligated to the pEASY-Blunt Vector (Promega, Shanghai, China) to obtain a pEASY-Blunt::CsFAAH6 plasmid, which was transformed into E. coli DH5a Competent Cells and sent to GM for sequencing. The nucleotide sequence of the resulting CsFAAH6 gene obtained is shown in SEQ ID NO: 1 in the sequence listing, which was specifically shown as follows:

(11) TABLE-US-00001 ATGGGCATTTTCAAGGCCAAAGGCGTAGTCTACAAGCCTGTCGACGAT GTCGATCTCGGTCCTCACAGCGATGAGTTTTATCTCCGTGCTAACGTCA AAGCTCCTCGCATGGCTGGATTGCTGGTTAAAATTTTTGTTTGGTTCCT CGAGTCGCGGATTTTCGGGGGTATTTTGTTGTACATGTTGAAGAGAAAC AACCTAATTCACAAGCTTGTTTCATATGCAGAGTTGGAAGAGTCACCTG TATTTGTTCCTTCACACCCTTATGAAGGCCTTAAAGAACAAGAAGTCAA ATTAGTAGAGGATGATCTCTCTCCATCTGACAAAATTCAGAAGGCCATG GAATGCATACAATGCTCAGAAAGTATACAAGAAAATTCGGAGCTTAGTT TCCATCGCTGGACAGTATTGGATTATTCAAGAGCTTACATTTCAGGAGA GATTACTCCTCTCATGGTGGCGGAGCGATTTATAGCTGCTGTCCATGAA TCGTCTGAACCTGCATTGCACATGTCATTCTTTATTGATTATAATGTTG GAGACATATTAAGGCAAGCTACTGAGTCAACTCAGCGGTACAAACAAGG AGAACCATTATCACCTCTAGATGGAGTCCCAATCGCAATCAAAGACGAA ATAGATTGTATGCCCTATCCAACTACAGGGGGTACAAAGTGGTTGCAAA AGGTAAGACATTGTGCAGATGATGCATGCTGTGTTAAGCGCCTGAGATT ATGTGGTGCCATACTTGTTGGGAAGACAAATATGCATGAGCTCGGGGCT GGAACCAGTGGTATCAATCCTCATTATGGGGTACCTAGAAATCCATATG ATCCCAACAAGGTCTCTGGGGGTTCTTCTAGTGGATCTGCAGCTGTGGT TTCTGCAGGGTTGTGCCCTGTTGCCCTAGGTGTTGATGGGGGAGGATCT GTGAGAATGCCTGCTGCTCTTTGTGGTGTTGTTGGTCTGAAGCCAACTT TTGGACGTGTGCCCCATTCTGGTGTTATTCCTCTGAACTGGACAGTTGG GATGGTCGGTATCCTAGCAGGCACAGTTGAAGATGCATTTATTACTTAT GCAGCTATCAGTGGTCAATTTCCATCATGCCAACCCACAGATGCAGTGA AAAAAATTAATTTCCCACTCCTGAAGACACCAAACTGTATATCTAACAT CAAGATGGCTAAATATGGGGAGTGGTTTAATGATTGCACCGACGACATC AGAGTCTGTTGTTCCCATGCTCTGGACCAGCTTCACAAGCATTATGGAT GGGAGACCATGGACGTGACCATACCAGAGATAGAGGTGATGCGCCTGGC GCATTATTCAACAATTGGATCGGAGTGTAGCAATTCAATTGCTTGTCAT CTTGAAAACATGAATGTGGCAGAAATAGGGTTGGATGCAAGAGTAGCAC TCTCTGTTTATGGTTCTTTCAGCAGCAGGGAGTATTTGAATGCCCAGAA AATTAGGAACCGACAGATGCAGTTTCATAAGAAAATATTTGCCATGGCA GATGTTATTGTTACACCAACGACAGGTGTGACTGCCTACCCAATATTCG ATGATGCTTTGAAAACTGGGGAACTTGACTACATAAATGGAGCTGCACT TGTTCGGTATCAGATATCAGGAAATTTCTTGGGATTGCCAGCAGTAACC ATACCTATTGGATACGACAAAGTTGGCTTGCCTATAGGCCTTCAATTTA TTGGGAAGCCATGGTCCGAAGCTACGCTGATCCACATAGCGTTCGCAAT GCAGGCCATCTCGGACTCAAAAAAACCACAGATTTTCTATGATCTGCTC AAAAAGGATTGA

(12) The protein sequence encoded by the CsFAAH6 gene was specifically shown in SEQ ID NO: 2 in the sequence listing:

(13) TABLE-US-00002 MGIFKAKGVVYKPVDDVDLGPHSDEFYLRANVKAPRMAGLLVKIFVWF LESRIFGGILLYMLKRNNLIHKLVSYAELEESPVFVPSHPYEGLKEQEV KLVEDDLSPSDKIQKAMECIQCSESIQENSELSFHRWTVLDYSRAYISG EITPLMVAERFIAAVHESSEPALHMSFFIDYNVGDILRQATESTQRYKQ GEPLSPLDGVPIAIKDEIDCMPYPTTGGTKWLQKVRHCADDACCVKRLR LCGAILVGKTNMHELGAGTSGINPHYGVPRNPYDPNKVSGGSSSGSAAV VSAGLCPVALGVDGGGSVRMPAALCGVVGLKPTFGRVPHSGVIPLNWTV GMVGILAGTVEDAFITYAAISGQFPSCQPTDAVKKINFPLLKTPNCISN IKMAKYGEWFNDCTDDIRVCCSHALDQLHKHYGWETMDVTIPEIEVMRL AHYSTIGSECSNSIACHLENMNVAEIGLDARVALSVYGSFSSREYLNAQ KIRNRQMQFHKKIFAMADVIVTPTTGVTAYPIFDDALKTGELDYINGAA LVRYQISGNFLGLPAVTIPIGYDKVGLPIGLQFIGKPWSEATLIHIAFA MQAISDSKKPQIFYDLLKKD
2. Analysis of Differential Expression of CsFAAH6 Gene
(1) Expression of CsFAAH6 Gene in Different Tissues of Tea Plant

(14) The national elite tea cultivar C. sinensis cv. Shuchazao was planted in Nongcuiyuan. Anhui Agricultural University, Luyang District, Hefei, Anhui Province, and 16 tissues and organs were used to analyze gene expression. The 16 tissues and organs included bud, 1.sup.st leaf, 1.sup.st main vein, 2.sup.nd leaf, 2.sup.nd main vein, 3.sup.rd leaf, 3.sup.rd main vein, 4.sup.th leaf, 4.sup.th main vein, 5.sup.th leaf, 5.sup.th main vein, 6.sup.th leaf, 6.sup.th main vein, vascular bundle, shoot between 2.sup.nd and 3.sup.rd leaves (stem), and roots. Also, these samples were used for total RNA extraction and first-strand cDNA synthesis. The reverse transcription product (first-strand cDNA) was diluted 5-fold as a template, and a 10 L reaction system was prepared using 2AceQ Universal qPCR SYBR9@ Master Mix (Vazyme, Nanjing, China): 1.0 L of 5-fold diluted reverse transcription product, 0.4 L each of upstream and downstream primers (10 mol/L), 5 L of 2AceQ Universal qPCR SYBR Master Mix, and 3.2 L of ddH.sub.2O. Three replicates were prepared for each reaction. Subsequently, the following program was run on the Bio-rad CFX-384 Touch System: i) initial denaturation at 95 C. for 5 min; ii) 40 cycles of denaturation at 95 C. for 10 s, annealing at 60 C. for 30 s, and extension at 72 C. for 30 s; and iii) from 65 C. to 95 C., to plot the melting curve at 0.1 C./s. The upstream primer was 5-GTTCTITCAGCAGCAGGGAG-3 (SEQ ID NO: 5), and the downstream primer was 5-CGAACAAGTGCAGCTCCATT-3 (SEQ ID NO: 6). With tea plant CsGADPH gene as internal reference, based on the upstream primer (5-TTGGCATCGTTGAGGGTCT-3) (SEQ ID NO: 7) and the downstream primer (5-CAGTGGGAACACGGAAAGC-3) (SEQ ID NO: 8), the relative expression levels of CsFAAH6 in different tissues were calculated through the analysis software of the instrument.

(15) FIGS. 1A-B illustrates the expression of the CsFAAH6 gene in different tissues of tea plant. As can be seen from FIGS. 1A-B, the results of qRT-PCR showed that CsFAAH6 was expressed in various tissues, and the expression level in mature leaves was significantly higher than that in other parts.

(16) 3. Subcellular Localization of CsFAAH6 in Tea Plant Protoplasts

(17) (1) Construction of pCAMBIA1305.1-CsFAAH6 Vector

(18) Using pEASY-Blunt::CsFAAH6 plasmid as a template, based on the upstream primer (5-GGACTAGTATGGGCATTTTCAAGGCC-3) (SEQ ID NO: 9) and the downstream primer (5-CGGGATCCATCCITTGAGCAGATCATAGAA-3) (SEQ ID NO: 10), PCR amplification was conducted. PCR products were recovered with 1.2% agarose gel electrophoresis bands. First, the recovered gene PCR product and the vector plasmid were double digested, and the digested product was recovered with a 1.2% agarose gel electrophoresis band. Using T4 DNA Ligase ligation technology, the band was digested with 2 L of the vector and 6 L of the gene, and the product was recovered. 1 L of T4 DNA Ligase Mix and 1 L of T4 DNA Ligase Buffer were left to stand overnight at 4 C., transformed into E. coli DH5. Competent Cells, and sent to Sangon for sequencing.

(19) (2) Preparation of Tea Plant Protoplasts

(20) i) The petals of well-grown C. sinensis cv. Shuchazao plants were collected. ii) Using a sharp razor blade, the petals were cut into about 1 mm and put in a Petri dish (901.5 mm) containing 20 mL of enzymatic hydrolyzate. iii) The Petri dish was placed in a shaker at 20-25 C. (40 r/min) for 30-90 min to allow the protoplast cells to be fully enzymatically hydrolyzed from the petals. iv) The enzymatic hydrolyzate containing protoplasts were transferred into a 50 mL centrifuge tube, and centrifuged at 100g for 3 min to collect protoplasts. The protoplasts were washed twice with 10 mL of pre-cooled W5 solution. The operation should be gentle to avoid cell disruption. The protoplasts were resuspended with an appropriate volume of W5 solution and placed on ice for a minimum of 30 min. During this period, the protoplasts were counted using a hemocytometer and the protoplast concentration was measured (if the concentration is too high, it can be diluted 10 to 20 fold and then determined).
(3) Transformation of Protoplasts i) The protoplasts were centrifuged at 100g for 1 min, the supernatant was discarded, the protoplasts were resuspended with freshly prepared MMg solution, and the concentration was adjusted to 3-510.sup.5 cells/mL. ii) 20 L of pCAMBIA1305.1-CsFAAH6 plasmids were added to the bottom of a 5 mL centrifuge tube (if multiple plasmids are used for co-transformation, the transformed plasmids need to be premixed), 200 L of MMg solution (containing 6-1010.sup.4 protoplast cells), and the bottom of the centrifuge tube was flicked to mix the mixture thoroughly. iii) 220 L of the freshly prepared PEG4000 solution was added, and the centrifuge tube was slowly inverted to mix well and left to stand at room temperature for 5-30 min. iv) The protoplasts were collected by centrifugation at 100g for 1 min, and rinsed twice with 2 mL of W5 solution. v) The protoplasts were resuspended with 0.6 mL of W5 solution and transferred to a 24-well plate for culture (the plate wells were soaked with 0.5-0.8 mL of 1% BSA for at least 30 min, and the BSA solution was discarded for use). After culturing at 20-23 C. for 14-18 h, the fluorescence signal was detected under a laser scanning confocal microscope.

(21) As shown in FIGS. 2A-F, the CsFAAH6-fused green fluorescent protein (GFP) signal was specifically expressed in mitochondria of tea plant protoplast cells, while the empty vector GFP signal filled the whole tea plant protoplast cells.

(22) 4. Inhibition of CsFAAH6 Gene Expression can Significantly Increase the Theanine Content in Tea Leaves

(23) To verify whether CsFAAH6 has the function of degrading theanine in tea plants, antisense oligonucleotides were used to transiently silence CsFAAH6 in leaves. First, 250 L of ddH.sub.2O was added to four tubes each of sense (sODN) and antisense (AsODN) oligonucleotide primers of CsFAAH6 and mixed into one tube. The final concentration of the primers was 40 M, and 330 L was added to a 1.5 mL centrifuge tube for five replicates. The first, second, and third leaves of the tea shoot that had been dark-treated in advance were obliquely cut to an appropriate height and inserted into centrifuge tubes with primers, and the tubes were sealed with parafilm. The processed samples were inserted on the plate and put in a foam box with a small amount of water, sealed with plastic wrap, and placed in a phytotron for 24 h for sampling. RNA was extracted for fluorescence quantitative PCR analysis of gene expression, and the rest of the samples were freeze-dried. The theanine was extracted and the theanine content was detected by high performance liquid chromatography (HPLC).

(24) As shown in FIGS. 3A-D, the leaves of the four biological replicates had different degrees of CsFAAH6 gene silencing; the detection results of theanine content in the leaves showed that the theanine content in the leaves of the four biological replicates significantly increased in the treatment group compared with the control (sODN). Combined with the high expression of CsFAAH6 in mature leaves and the mitochondrial subcellular localization of CsFAAH6, these results indicated that CsFAAH6 was involved in the cytological process of theanine degradation in tea shoots.

(25) 5. The Expression Level of CsFAAH6 in Tea Plant is Significantly Negatively Correlated with the Theanine Content in Tea Shoots

(26) The theanine content and the expression levels of CsFAAH6 in shoots (one bud and two leaves) of nine tea cultivars during three different periods in spring (on March 24, April 8, and April 22) were detected. As shown in FIGS. 4A-I, one-bud-two-leaf tea plant samples of the nine different cultivars during three periods (on March 24, April 8, and Apr. 22, 2020) were ground. RNA was extracted from the samples of the nine tea cultivars for fluorescence quantitative PCR analysis of CsFAAH6 gene expression; the theanine content in shoots of the nine different tea cultivars was detected by HPLC, and the correlation between gene expression level and theanine content of different cultivars during different periods was analyzed.

(27) As shown in FIGS. 4A-I, the theanine content decreased significantly with the increase of the expression level of CsFAAH6, showing a significant negative correlation trend. Except for C. sinensis cv. Xianyuzao, the correlation coefficients of eight cultivars all exceeded 0.9. The correlation between the theanine content in shoots of different tea cultivars and CsFAAH6 further verified that CsFAAH6 has the function of theanine degradation.

(28) The above content is only an example and description of the structure of the present disclosure. Those skilled in the art can make various modifications or supplements to the specific example described or replace them in a similar manner, as long as they do not depart from the structure of the present disclosure or go beyond the scope defined by the claims, all of which fall within the protection scope of the present disclosure.