Tea tree CsCUL1 gene, tea tree CsCUL1 protein and application thereof

Abstract

The invention relates to a tea tree CsCUL1 gene, a tea tree CsCUL1 protein and application of the tea tree CsCUL1 gene and the tea tree CsCUL1 protein. The CDS nucleotide sequence of the tea tree CsCUL1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the coded tea tree CsCUL1 protein is as shown in SEQ ID NO.2. A VIGS gene silencing technology is utilized, vacuum infection is utilized, the function of the CsCUL1 gene is verified, an experimental material is an annual branch of tea tree Longjing 43, and a result shows that the content of chlorophyll and carotenoid in tea tree leaves in a silencing strain of CsCUL1 is remarkably reduced. The method can be applied to tea leaf color character molecular genetic breeding, is beneficial to regulation and breeding of tea tree varieties with different leaf colors in production, and has important significance for improving the sensory quality of tea products.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, in particular to a tea plant CsCUL1 gene, a tea plant CsCUL1 protein and applications thereof. Background Art

[0002] The tea plant (Camellia sinensis (L.) O.Ktze.) is a shrub or small tree in the genus Camellia, in the family Theaceae. Originating in the Yunnan-Guizhou Plateau of China, it is a perennial evergreen leaf crop. Leaves not only provide energy for plant growth and development through photosynthesis but also serve as a site for the exchange of soil and atmospheric water, playing an irreplaceable role in the plant's evolution from germination to aging and death. The pigments that determine tea leaf color are primarily chlorophyll and carotenoids, with chlorophyll primarily composed of chlorophyll a (Chl. a) and chlorophyll b (Chl. b). The abundant chlorophyll-binding proteins decompose during degradation to form nitrogen, which is reused. This recycled nitrogen is of great importance to the growth, development, and metabolism of the tea plant. Carotenoids have numerous physiological functions, including photoprotection and antioxidant properties. They are also precursors for the synthesis of plant hormones such as abscisic acid, strigolactones, and carotenoids, playing a crucial role in tea growth and development. Carotenoids are also important components of the appearance and color of tea leaves, and are also precursors of important aroma substances in tea. Their types and content play a vital role in the quality of tea.

[0003] Chlorophyll metabolism has been well characterized in the model plant Arabidopsis thaliana, including all enzymatic pathways, the intracellular locations of encoding genes and enzymes, and potential transcription factors (TFs). In Arabidopsis, MAX2 encodes an F-box protein that participates in protein ubiquitination and degradation by forming the SCF (skp1-cullin-F-box) protein complex. Strigolactones promote the degradation of MAX2-binding proteins, thereby enhancing strigolactone signaling. While the chlorophyll content of max2 mutants is significantly lower than that of wild-type plants, the chlorophyll a / b ratio remains unchanged. This suggests that the MAX2 protein itself may be involved in regulating plant light and pigment synthesis, and that max2 mutants are unable to alter chlorophyll composition by modulating strigolactone signaling. Arabidopsis plants overexpressing the soybean F-box protein GmFBX176 exhibit reduced chlorophyll content compared to wild-type plants. From the above, we can infer that the SCF protein complex is an important component in the chlorophyll synthesis or metabolic pathway in plants, and the cullin-1 protein expressed by the CsCUL1 gene is a part of the SCF protein complex.

[0004] However, there are few studies on the regulation of tea leaf color, and there are few reports on how to regulate tea leaf color in production through biotechnology. Summary of the Invention

[0005] Based on this, it is necessary to provide a nucleic acid molecule that can be used to regulate the color of tea leaves.

[0006] A CsCUL1 nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a tea plant CsCUL1 protein, the amino acid sequence of the tea plant CsCUL1 protein is shown as SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector, which contains the above-mentioned CsCUL1 nucleic acid molecule or the UTR sequence of the CsCUL1 gene, and the UTR sequence is shown in SEQ ID NO.3.

[0009] In one embodiment, the backbone vector of the recombinant vector is a pTRV2 vector.

[0010] The present invention also provides a host cell, the genome of which contains the above-mentioned CsCUL1 nucleic acid molecule or the above-mentioned recombinant vector.

[0011] In one embodiment, the host cell is an Agrobacterium competent cell.

[0012] Use of the CsCUL1 nucleic acid molecule, tea plant CsCUL1 protein, recombinant vector or host cell as described above in the preparation of a product for regulating tea plant leaf color.

[0013] The present invention also provides a method for regulating tea tree leaf color, which regulates the tea tree leaf color phenotype by increasing or decreasing the level of tea tree CsCUL1 protein, that is, regulating the content and related ratios of chlorophyll a, b and carotenoids.

[0014] In one embodiment, the method for reducing the level of CsCUL1 protein in tea plants is to silence the CsCUL1 gene using VIGS gene silencing technology, and the method for increasing the level of CsCUL1 protein in tea plants is to overexpress the CsCUL1 gene.

[0015] The present invention discloses the use of the tea plant CsCUL1 gene and its encoded CsCUL1 protein in regulating tea leaf color. The function of the gene and its encoded protein was verified using VIGS gene silencing technology with vacuum infection. The experimental materials used were one-year-old branches of the tea variety Longjing 43. The results showed that the chlorophyll a, chlorophyll b, chlorophyll a / chlorophyll b, total chlorophyll, carotenoid content, and total chlorophyll / carotenoid ratio in tea leaves were significantly reduced in the CsCUL1-silenced strain. When lighter-leaf shoots are desired, the CsCUL1 gene in the tea plant is silenced or knocked out; when darker-leaf shoots are desired, the CsCUL1 gene is overexpressed. This invention can be applied to molecular genetic breeding for tea leaf color traits, facilitating the regulation and selection of tea varieties with different leaf colors in production, and is of great significance for improving the sensory quality of tea products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a map of the pTRV2 vector constructed in Example 2 of the present invention;

[0017] Figure 2 This is a comparison of the leaf color phenotypes of the control group (pTRV2) and the CsCUL1 silenced strain (pTRV2-CsCUL1) grown for about 40 days in Example 2 of the present invention;

[0018] Figure 3 Figure 2 shows the results of measuring the total chlorophyll content in the second leaf of the new shoots of tea plants in the control group (pTRV2) and the CsCUL1-silenced strains (pTRV2-CUL1-1, pTRV2-CUL1-2, and pTRV2-CUL1-3) in Example 2 of the present invention; error bars represent the standard error of the mean, and letters represent significant differences compared with those before treatment, p < 0.05;

[0019] Figure 4 Figure 2 shows the results of measuring the chlorophyll a and chlorophyll b contents in the second leaf of the new shoots of tea plants in the control group (pTRV2) and the CsCUL1-silenced strains (pTRV2-CUL1-1, pTRV2-CUL1-2, and pTRV2-CUL1-3) in Example 2 of the present invention; error bars represent the standard error of the mean, and letters represent significant differences compared with pre-treatment values, p < 0.05;

[0020] Figure 5 Figure 2 shows the results of measuring the carotenoid content in the second leaf of the new shoots of tea plants in the control group (pTRV2) and the CsCUL1-silenced strains (pTRV2-CUL1-1, pTRV2-CUL1-2, and pTRV2-CUL1-3) in Example 2 of the present invention; error bars represent the standard error of the mean, and letters represent significant differences compared with those before treatment, p < 0.05;

[0021] Figure 6 Schematic diagram of the changes in the ratio of total chlorophyll to carotenoids in the second leaf of the new shoots of tea plants in the control group (pTRV2) and the CsCUL1-silenced strains (pTRV2-CUL1-1, pTRV2-CUL1-2, pTRV2-CUL1-3) in Example 2 of the present invention; error bars represent the standard error of the mean, and letters represent significant differences compared with before treatment, p<0.05. DETAILED DESCRIPTION

[0022] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments thereof. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0024] Explanation of terms

[0025] "Vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0026] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0027] Changes in tea plant leaf color reflect the plant's growth status and environmental adaptability. Chlorophyll and carotenoids are key pigments that contribute to leaf color phenotype. High chlorophyll content results in greener leaves, while high carotenoid content results in a yellowish-green hue. Chlorophyll is also a key pigment in tea, primarily found in green tea. The shade of green in leaves reflects the chlorophyll content, a key pigment for photosynthesis. Manipulating leaf color can optimize the plant's photosynthetic efficiency, thereby improving its growth rate and quality. Tea leaf color changes are also closely related to tea quality: generally, bright, emerald green tea is preferred by consumers, and it also imparts a unique, refreshing flavor to green tea. In addition to green tea, black tea also experiences a decrease in chlorophyll during fermentation, resulting in varying colors and flavors. In production, leaf color manipulation can enhance tea's appeal and market competitiveness. Carotenoids not only influence tea's color but also serve as precursors for the synthesis of many aromatic compounds.

[0028] In summary, regulating tea leaf color is of great significance for improving tea quality. The present invention can be applied to the molecular genetic breeding of tea leaf color traits, facilitating the selection and breeding of tea varieties with different leaf colors, thereby adjusting the sensory quality of tea products and ultimately obtaining products with different flavors based on demand.

[0029] Through screening and VIGS (virus-induced gene silencing) technology, the present invention discovered that the tea plant CsCUL1 gene and its encoded CsCUL1 protein are associated with tea leaf color. CsCUL1-silenced tea plant strains exhibit reduced chlorophyll and carotenoid content. Therefore, to produce tea shoots with low chlorophyll and carotenoid content, the CsCUL1 gene in the tea plant is silenced or knocked out. To produce tea shoots with high chlorophyll and carotenoid content, the CsCUL1 gene is overexpressed.

[0030] The CsCUL1 nucleic acid molecule of one embodiment of the present invention is the tea plant CsCUL1 gene, which encodes the tea plant CsCUL1 protein.

[0031] In a specific example, the CDS nucleotide sequence of the tea plant CsCUL1 gene is shown in SEQ ID NO. 1. It is understood that due to the degeneracy of codons, there are multiple forms of nucleic acid sequences that can express the same protein, so the specific sequence is not limited thereto.

[0032] The amino acid sequence of the tea plant CsCUL1 protein according to one embodiment of the present invention is shown in SEQ ID NO.2.

[0033] The recombinant vector of one embodiment of the present invention contains the CsCUL1 gene of tea plant or the UTR sequence of the CsCUL1 gene of tea plant. The UTR sequence is shown in SEQ ID NO.3.

[0034] In one specific example, the backbone vector of the above-mentioned recombinant vector is a pTRV2 vector, but this is not limited thereto. Those skilled in the art may also select other types of backbone vectors according to functional requirements. It is understood that the vector may also contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, and other expression control elements (such as transcription termination signals, polyadenylation signals, and poly-U sequences).

[0035] The host cell of one embodiment of the present invention contains the CsCUL1 nucleic acid molecule or recombinant vector described above in its genome.

[0036] In a specific example, the host cell is an Agrobacterium competent cell such as GV3101 Agrobacterium competent cell, but is not limited thereto. The specific type can be selected as needed.

[0037] In some embodiments, obtaining the host cells comprises transfecting or transforming the recombinant vector into a recipient cell. Non-limiting examples of such transformation methods include chemical transformation and electroporation; and such transfection methods include, but are not limited to, calcium phosphate coprecipitation, artificial liposomes, and viral transfection.

[0038] The method for regulating tea leaf color according to one embodiment of the present invention regulates the tea leaf color phenotype by increasing or decreasing the level of CsCUL1 protein in tea plants.

[0039] In a specific example, the method for reducing the level of CsCUL1 protein in tea plants is to silence the CsCUL1 gene using VIGS gene silencing technology, and the method for increasing the level of CsCUL1 protein in tea plants is to overexpress the CsCUL1 gene.

[0040] VIGS (Virus-Induced Gene Silencing) is a gene silencing technology that primarily utilizes viral vectors to carry target gene fragments. Through viral infection and replication, it achieves specific silencing of endogenous plant genes. First, a suitable viral vector is selected and the target gene fragment is inserted into it. These viral vectors are typically modified RNA viruses, such as tobacco rattle virus (TRV), tobacco mosaic virus (TMV), and potato virus X (PVX). The virus carrying the target gene fragment is then inoculated into the plant, where it infects and replicates within the plant cells. During replication, the virus transcribes RNA containing the target gene fragment. The Dicer enzyme within the plant cells recognizes and cleaves this RNA, forming small interfering RNAs (siRNAs). These siRNAs form a silencing effector complex (RISC) with the Ago1 protein and its associated proteins. RISC specifically binds to and degrades mRNA sequences complementary to the target gene fragment, thereby silencing the target gene. It is understood that methods for reducing CsCUL1 expression in tea plants are not limited to this method; other gene silencing or knockout methods can also be employed or combined.

[0041] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0042] Example 1 Discovery of SNP sites associated with tea leaf color traits

[0043] Differences in leaf color were found among different strains of the Guiding Bird King population. A genome-wide association mapping GWAS analysis was conducted. The results suggested that the CsCUL1 gene may be a regulatory gene affecting tea leaf color.

[0044] Example 2 Verification of CsCUL1 gene function using VIGS gene silencing technology

[0045] To further verify whether the tea leaf color phenotype is regulated by the CsCUL1 gene, the pTRV vector was constructed. The vector map is shown in Figure 2. Figure 1 As shown, it carries the UTR sequence of the CsCUL1 gene (SEQ ID NO. 3). The transformed Longjing 43 tea tree branches (Cscul1) are different from the green leaves of the wild type. The leaves of Cscul1 are white, as shown in FIG. Figure 2 As shown, the results further verified that the reduced transcription level of CsCUL1 caused the leaf albino phenotype of Cscul1. The specific steps are as follows:

[0046] (1) pTRV2-CsCUL1 recombinant plasmid was introduced into Agrobacterium

[0047] Thaw the GV3101 Agrobacterium competent cells (preserved by our group, volume 100 μL) stored in a -80°C ultra-low temperature freezer on ice;

[0048] Add 20-40 μL of the recombinant plasmid pTRV2-CsCUL1 to the competent cells and mix gently with a pipette;

[0049] Place the competent cells containing the recombinant plasmid on ice for 30 minutes, then transfer to liquid nitrogen and freeze for 5 minutes;

[0050] Then place in a 28°C water bath for 5 minutes to lyse the cells;

[0051] Add 1 mL of YEP liquid medium without any antibiotics to the tube, and then place it in a constant temperature shaking incubator for 5 hours (28°C, 200 rpm / min);

[0052] The cultured bacterial suspension was centrifuged for 1 minute (4°C, 5000 rpm / min), the supernatant was discarded, and 0.1 mL of YEP liquid medium (Rif: 100 mg / L and Kan: 100 mg / L) was added to rehydrate the bacterial cells.

[0053] The above bacteria were evenly spread on a YEP solid plate containing Rif (100 mg / L) and Kan (50 mg / L) using a sterilized applicator. After standing for several minutes until the bacterial solution was completely absorbed, the culture dish was sealed with a sealing film and then placed in a 28°C biochemical incubator for 2 to 3 days; a single colony was picked and preserved for later use.

[0054] (2) Tea virus-induced gene silencing (VIGS) infects tea tree branches

[0055] The constructed plasmid was transformed into Agrobacterium competent cells, and the positive clone transformants of pTRV1, pTRV2, and pTRV2-CsCUL1 were picked and inoculated into 3 mL to 5 mL of YEP liquid culture medium, respectively, and cultured at 28°C and 200 rpm for 24 h.

[0056] 3 mL of the cultured bacterial solution was inoculated into 250 mL of YEP liquid medium and cultured at 28°C and 200 rpm for 12 h until the OD600 of the bacterial solution reached about 0.9.

[0057] The bacterial cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in an appropriate volume of resuspension solution to a final concentration of 0.8 (OD600).

[0058] The resuspension was allowed to stand at room temperature for 0.5 h, and the resuspensions containing pTRV2 and pTRV2-CsCUL1 were mixed with the resuspension containing pTRV1 at a volume ratio of 1:1, and then vacuum infiltrated into wounded Longjing 43 cuttings.

[0059] Infected tea tree branches were cultured in an artificial climate chamber with a day / night temperature of 25°C / 15°C and a 16h / 8h light / dark cycle. Negative control and experimental group branches were cultured under the same conditions. Samples were collected after 6-7 weeks, snap-frozen in liquid nitrogen, and then stored in a -80°C freezer until testing.

[0060] (3) Detection of chlorophyll and carotenoid content in tea leaves

[0061] The mass fraction of photosynthetic pigments in plant leaves was determined by referring to the spectrophotometric method. 50-100 mg of the second true leaf of a new shoot of a tea plant was cut and extracted with 95% ethanol in the dark at room temperature until the leaves turned completely white (24 h). The absorbance at wavelengths of 665 nm, 649 nm, and 470 nm was measured using a UV-2600 (SHIMADZU) spectrophotometer. The mass fractions of chlorophyll a, chlorophyll a, total chlorophyll, and carotenoids, as well as the chlorophyll a / b and total chlorophyll / carotenoid ratio were calculated using the formula. The results are shown in Figure 2. Figures 3 to 6 As shown in the results, after silencing the CsCUL1 gene, the total chlorophyll content, chlorophyll a, chlorophyll b content, and carotenoid content in the second true leaf of the tea shoots were significantly reduced, while the chlorophyll a / b ratio had no significant difference.

[0062] In summary, by regulating the expression of the CsCUL1 gene, the color changes of tea leaves can be regulated, thereby regulating the quality of tea products, which is helpful for breeding tea varieties with different leaf colors according to production needs.

[0063] The following is the specific sequence information:

[0064] SEQ ID NO.1

[0065] >TEA028016Scaffold5480:781471-786199+

[0066]

[0067] SEQ ID NO.2

[0068] MEKGITKLKNILEGLPEPQFSSEDYEMFYTTIYYMCTQNPHDYSQQLYDKYRESFEEYITSTVLPSLREKHDEFMMRELVNRWSNHKVMVRWLSRFFHYLSRYFIPQRSLPALNDVRLTCFQYLVYRELNGKVRDAVISLIDREGEQIDRALLKNVLDIFVEIGMGQMDHYENDFEADMLKDTEAYYSRKASNWILEDSCPDYMLKAEECLKREKDRVAHYLHFSSELKLLEKVQHELLYVYATQLRENKHSGCHALLRDDKVEDLSRMYRLFSKIGLDPVSSIFKQHVTAEVTALVKQAEDAASNKKADKKDVLGLPEQVFVRKVIELLHAKYMAYVSNYFMNPTLFHE ALKEAFEVFCNKGVGGSSSAELLATFCDNILKKGGSEIEKKLVRRLLFDKSANDEHERSILTKLKQQCGGQFTSKMEGMVTDLTLARENQTNFEEYLKTNPASPGIDLTVLTTGFWPSYKSFDLNLPAEMMSCLFTSALVKCVEVFREFYQTKTKHRKLTWIYSLGTCNINGKFEPKTMELIVTTYQAFVLLLFNASDRWSYQEIMTQLNLTDDDVVRLLHSLSCEKYKILNKEPNNKTISPTDHFEFNSKLTDKMRRIKIPLPPVDEKKVIEDVNKDRQYAIDASIVHIMKSRKVLGHQQLVMECVEQLGRMFKPHFKAIKKRIEDLITRDYLERDKDNPNLFRYLA

[0069] SEQ ID NO.3

[0070] TGAATAATGAAGTGGTGACAGTGGCTGCTGCAATTTGCATTGAAAGAGAGGCTGTAGACAATTTGAAGAAGCACAAGAATATGTATAAATGCCATTAATTATGCTCTGCTTTTGGTGTTTCCTTAGAAGCAGGAGCTGAGGCTGCTTGTACATTTTGCCTTCAGAGCCAGGTATGAGCAACAACTGTAAGATT GTGCACCCGAGCCGGAGAATTGTAATTGATTCCATAAAGAGGCCCAAGCACCAATAGGACCATTTTGTATCTCGTCTTCTCATTGCTGCAAATCCCATCACATCACTTGGTGCCTCTTTGTTAATTGTTTATTTACCGCCCCTCTGGAAGAAGAGAAGAAAATGTAATTGGATTTTACTTGCACTATGTTTTG

[0071] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A CsCUL1 nucleic acid molecule, characterized in that The nucleotide sequence of the CsCUL1 nucleic acid molecule is shown in SEQ ID NO.

1.

2. A tea plant CsCUL1 protein, characterized in that The amino acid sequence of the tea plant CsCUL1 protein is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the CsCUL1 nucleic acid molecule or the UTR sequence of the CsCUL1 gene according to claim 1, and the UTR sequence is shown as SEQ ID NO.

3.

4. The recombinant vector according to claim 3, characterized in that The backbone vector of the recombinant vector is a pTRV2 vector.

5. A host cell, characterized in that The genome of the host cell contains the CsCUL1 nucleic acid molecule according to claim 1 or the recombinant vector according to any one of claims 3 to 4.

6. The host cell according to claim 5, characterized in that The host cell is Agrobacterium GV3101 competent cell.

7. Use of the CsCUL1 nucleic acid molecule of claim 1, the tea plant CsCUL1 protein of claim 2, the recombinant vector of any one of claims 3 to 4, or the host cell of any one of claims 5 to 6 in the preparation of a product for regulating the color of tea leaves.

8. A method for regulating the color of tea leaves, characterized in that: The leaf color phenotype of tea leaves is regulated by increasing or decreasing the level of the tea plant CsCUL1 protein as described in claim 2.

9. The method according to claim 8, characterized in that The method for reducing the level of the CsCUL1 protein in the tea plant is to silence the CsCUL1 gene using the VIGS gene silencing technology, and the method for increasing the level of the CsCUL1 protein in the tea plant is to overexpress the CsCUL1 gene.

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