Camellia sinensis fez gene and its encoded protein and application

Abstract

This invention discloses a tea plant CsFEZ gene, its encoded protein, and its applications, specifically relating to the field of tea plant breeding technology. The CDS sequence of the tea plant CsFEZ gene is shown in SEQ ID No: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID No: 2. By controlling the expression of the CsFEZ gene and its encoded protein in tea and rice somatic embryos, the occurrence of somatic embryos in tea and rice can be effectively regulated to obtain somatic embryos with high regeneration efficiency. This can provide a new solution for the genetic transformation of varieties with low regeneration efficiency. Furthermore, the CsFEZ gene can serve as an important molecular mechanism in the process of plant somatic embryo regeneration, providing a foundation for research and application in plant gene synthesis and customized plant modification.

Description

Technical Field

[0001] This invention relates to the field of tea tree breeding technology, specifically to the CsFEZ gene of tea trees, its encoded protein, and its applications. Background Technology

[0002] Somatic embryogenesis is a key manifestation of plant cell totipotency and a core technological foundation for plant genetic transformation, gene function research, and efficient seedling propagation. However, for important economic crops such as tea, somatic embryogenesis faces technical bottlenecks such as low efficiency and difficulty in regeneration, severely restricting its progress in biotechnology breeding and large-scale application. Therefore, identifying and elucidating the key genes regulating somatic embryogenesis and elucidating their mechanisms of action is of urgent theoretical and practical significance for establishing an efficient and stable plant regeneration and genetic transformation system.

[0003] The specific function of the FEZ gene in somatic embryogenesis in model plants, especially its regulatory role in non-model economic crops, remains unclear. As an important economic crop, tea has relatively few studies on genes regulating somatic embryogenesis, and its genetic background and regulatory network may differ significantly from model plants such as Arabidopsis thaliana, further increasing the difficulty of elucidating the relevant mechanisms.

[0004] Although existing research suggests that FEZ may play a potential role in plant regeneration, direct experimental evidence is still lacking to support whether it directly participates in and positively regulates somatic embryogenesis, and whether this function is conserved across different plant species. Therefore, cloning and functionally identifying the CsFEZ gene in tea plants to clarify its regulatory role as a transcription factor in somatic embryogenesis will not only provide key genes and theoretical basis for a deeper understanding of the molecular mechanisms of somatic embryogenesis in tea plants, but will also provide new gene resources and technical pathways for developing cross-species applicable plant regeneration and genetic improvement technologies. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to provide a tea tree CsFEZ gene, its encoded protein, and its applications.

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

[0007] The first aspect of this invention proposes a CsFEZ gene for tea plants, the CDS sequence of which is shown in SEQ ID No: 1.

[0008] The second aspect of this invention provides a tea plant CsFEZ protein, the amino acid sequence of which is shown in SEQ ID No: 2.

[0009] The third invention proposes a recombinant vector containing the CsFEZ gene.

[0010] Preferably, the skeletal carrier of the recombinant vector includes, but is not limited to, one of pCAMBIA2300, pCAMBIA1305, and pBI121.

[0011] In this invention, "pCAMBIA2300", "pCAMBIA1305", and "pBI121" are all overexpression vectors containing a 35S promoter. It should be noted that, given the known CsFEZ protein or CsFEZ gene of this invention, fusion plasmids can be obtained using conventional methods in the art. Introducing the obtained expression vector into plant cells allows for the overexpression of the CsFEZ gene.

[0012] More preferably, the scaffold carrier of the recombinant vector is pCAMBIA2300; The recombinant vector is pCAMBIA2300-CsFEZ; The method for constructing the recombinant vector is as follows: the CsFEZ gene sequence is inserted into the restriction site of the vector pCAMBIA2300 using homologous recombination. The enzyme cleavage site is Bam HI and Xba I.

[0013] The fourth aspect of this invention proposes the application of the aforementioned tea tree CsFEZ gene in enhancing plant somatic embryogenesis.

[0014] Preferably, the application is to overexpress the CsFEZ gene in plant tissues to increase the somatic embryogenesis rate of plants.

[0015] Preferably, the application can be achieved by contacting plant tissue with Agrobacterium containing the CsFEZ recombinant vector or gold powder.

[0016] Preferably, the application involves overexpressing the CsFEZ gene in plant tissues to cultivate plant varieties with high somatic embryogenesis rates.

[0017] More preferably, the plant tissue includes at least one selected from callus tissue, injured plant tissue, and ex vivo plant tissue.

[0018] Preferably, the plant is a tea tree or rice.

[0019] Preferably, the plant somatic embryogenesis includes indirect or direct occurrence by plant somatic cells.

[0020] Preferably, the indirect occurrence refers to the development of plant tissue from callus tissue into a somatic embryo.

[0021] Preferably, the direct occurrence refers to the direct development of plant tissue from explant somatic cells into somatic embryos.

[0022] The fifth aspect of this invention provides a method for promoting somatic embryogenesis in plants, the method comprising: introducing the above-mentioned CsFEZ gene into a target plant to obtain a transgenic plant with increased somatic embryogenesis rate, wherein the plant is a tea tree or a rice plant.

[0023] The specific steps of the method are as follows: (1) Cloning the CsFEZ gene in tea plants; (2) Construct a CsFEZ gene overexpression vector for tea plants; (3) The target plant was transformed by the CsFEZ gene overexpression vector of tea tree and the transgenic plant with increased somatic embryogenesis rate was obtained after identification.

[0024] The beneficial effects of this invention are as follows: 1. This invention provides an application of the CsFEZ gene and its encoded protein in regulating plant somatic embryogenesis. By controlling the expression of the CsFEZ gene and its encoded protein in the somatic embryos of tea and rice, the occurrence of somatic embryos in tea and rice can be effectively improved, so as to obtain somatic embryos with high regeneration efficiency.

[0025] 2. This invention discovers that applying the CsFEZ gene and its encoded protein and other regulatory factors to improve the efficiency of plant somatic embryo formation can provide a new solution for the genetic transformation of varieties with low regeneration efficiency; furthermore, the CsFEZ gene can serve as an important molecular mechanism in the process of plant somatic embryo regeneration, providing a foundation for research and application in the fields of plant gene synthesis and plant customization.

[0026] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0027] Figure 1 The pCAMBIA2300-CsFEZ vector spectrum in Example 2 of this invention; Figure 2 For the identification of the expression level of the CsFEZ overexpression lines in Example 2 of the present invention, OE 1# is the CsFEZ overexpression line CsFEZ-OE 1, OE 2# is the CsFEZ overexpression line CsFEZ-OE 2, and OE 3# is the CsFEZ overexpression line CsFEZ-OE 3; Figure 3 This is an observation of somatic cell embryo induction culture of tea plant lines overexpressing CsFEZ in Example 2 of the present invention; Figure 4 This is a statistical analysis of the number of tea tree somatic embryo regenerations in Example 2 of the present invention; Figure 5 This is an observation of somatic embryogenesis induced by CsFEZ overexpression in rice callus in Example 3 of the present invention; Figure 6 This is a statistical analysis of the number of rice somatic embryo regenerations in Example 3 of the present invention; Figure 7 This is an observation of shoot induction in rice somatic embryos with CsFEZ overexpression in Example 3 of the present invention; Figure 8 This is a statistical analysis of the number of rice shoot regenerations in Example 3 of the present invention; Appendix Figure 3-8 CsFEZ-OE indicates CsFEZ overexpression; The T-test was used to analyze the significance of differences in the attached figure. P < 0.05 was considered significant. P < 0.01 indicates a highly significant difference. P > 0.05 indicates that the difference is not significant. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0029] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0030] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, the features defined by "first" and "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to the time or condition described below that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] In this paper, the term “regeneration” refers to the process by which a damaged plant or plant tissue undergoes somatic embryogenesis.

[0035] According to embodiments of the present invention, the damaged plant tissue includes, but is not limited to, at least one of injured plant tissue, detached plant tissue, and callus tissue.

[0036] It should be noted that for damaged plants, injured plant tissues, and isolated plant tissues, somatic embryos will grow on the uninjured or callus surfaces. Callus refers to the new tissue that grows on the surface of the wound after a local injury to the original plant, or it is formed by the dedifferentiation of plant somatic cells under specific induction conditions.

[0037] In this article, the terms "plant body" and "plant" are used synonymously.

[0038] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. All reagents or instruments used, where the manufacturer is specified, are conventional products that can be obtained commercially.

[0039] Example 1: The origin and screening process of the tea plant gene CsFEZ: The CsFEZ gene was identified from the single-cell transcriptome of the somatic embryo of “Shuchazao”. Using DNA obtained from “Shuchazao” as material, PCR amplification was performed using the following primers (F: ATGGATGAGATAGGAAGTGAAA (SEQ ID No: 3), R: GGTATTGCACTTCTTGTTAGC (SEQ ID No: 4)), and the CDS sequence of CsFEZ (SEQ ID No: 1) was obtained.

[0040] Example 2: Somatic embryogenesis of tea plants overexpressing CsFEZ 1. Construction of pCAMBIA2300-CsFEZ recombinant plasmid The pCAMBIA2300 vector was digested with restriction endonucleases BamHI / XbaI to obtain the digested fragments. The CsFEZ coding sequence of the tea plant gene was ligated into the expression vector using a fusion cloning kit (Novizan Nanjing, China). The ligation strategy was as follows: Figure 1 As shown, after confirming the nucleotide sequence of the constructed vector by PCR and DNA sequencing, the recombinant vector pCAMBIA2300-CsFEZ (F: GAGCGTGTACAAGGGATCCATGGATGAGATAGGAAGTGAAA (SEQ ID No: 5), R: CTGCAGGTCGACTCTAGAGGTATTGCACTTCTTGTTAGC (SEQ ID No: 6)) was obtained.

[0041] 2. Obtaining transgenic plants The recombinant vector pCAMBIA2300-CsFEZ was introduced into the somatic embryonic tissue of the tea plant 'Shuchazao' using a gene gun to obtain transgenic plants.

[0042] 3. Identification of expression levels in tea plant somatic embryo overexpression lines RNA was extracted from CsFEZ-overexpressing somatic embryos, reverse transcribed into cDNA, and its expression level was identified by quantitative PCR (F: CAAGTTGCTCATTTTCGTCG (SEQ ID No: 7), R: CCTGCGGTGAGAAACCATTA (SEQ ID No: 8)). The results showed that the expression level of CsFEZ was significantly upregulated in the overexpressing lines CsFEZ-OE 1, CsFEZ-OE 2, and CsFEZ-OE 3. Figure 2 It is known that the present invention obtained CsFEZ somatic embryos of tea plants with CsFEZ gene overexpression.

[0043] 4. Induction of CsFEZ overexpression in tea plant somatic embryos Sterile somatic embryos of the CsFEZ (CsFEZ-OE) overexpressing line were inoculated into somatic embryo induction medium and cultured in the dark at 25°C. After 2 weeks, somatic embryos were induced and morphologically normal. Photographs were taken regularly to record the results, and the number of regenerated somatic embryos from explants was counted. See [link to results]. Figure 3 and Figure 4 .

[0044] The results showed that, through observation and recording of the somatic embryo culture process of the CsFEZ overexpression (CsFEZ-OE) lines, the number of somatic embryos in the CsFEZ overexpression lines was significantly increased after 2 weeks of culture on the medium compared with wild-type tea tree explants (Control), indicating that the somatic embryo regeneration efficiency was significantly improved (see details). Figure 3According to the statistical analysis of the number of somatic embryos regenerated from explants, the number of somatic embryos regenerated from wild-type CsFEZ overexpression lines was significantly different (T-test, P<0.01, see details). Figure 4 Therefore, it was further verified that the ability of plants or plant tissues to undergo somatic embryogenesis can be regulated by controlling the expression or activity of CsFEZ protein and CsFEZ gene.

[0045] Example 3: Somatic embryo regeneration of callus from rice lines overexpressing CsFEZ 1. Induction of somatic embryos from rice callus overexpressing CsFEZ Callus was obtained by culturing rice seeds ("Xiushui 134"). The recombinant plasmid pCAMBIA2300-CsFEZ was introduced into Agrobacterium tumefaciens (EHA105), and the callus was infected to obtain CsFEZ-overexpressing rice callus tissue. The CsFEZ-overexpressing rice callus was inoculated into somatic embryo induction medium and cultured under light at 25℃. After 7 days, somatic embryos were induced and morphologically normal. Photographs were taken regularly, and the number of regenerated somatic embryos from explants was counted. The results are shown in [link to results]. Figure 5 and Figure 6 .

[0046] The results showed that, through observation and recording of the somatic embryo culture process of CsFEZ overexpression (CsFEZ-OE) lines, the number of somatic embryos in the CsFEZ overexpression lines was significantly increased after one week of culture on the medium compared with wild-type rice callus (Control), indicating that the somatic embryo regeneration efficiency was significantly improved (see details). Figure 5 ). Statistical analysis of the number of somatic embryos regenerated from rice callus revealed a highly significant difference in the number of somatic embryos regenerated between wild-type and CsFEZ overexpression lines (T-test, P<0.01, see details). Figure 6 Therefore, it was further verified that the ability of plants or plant tissues to undergo somatic embryogenesis can be regulated by controlling the expression or activity of CsFEZ protein and CsFEZ gene.

[0047] 2. Induction of shoot regeneration in somatic embryos of rice overexpressing CsFEZ. Rice callus overexpressing CsFEZ was inoculated onto hormone-free MS medium and cultured under light at 25°C. After 2 weeks, morphologically normal shoots appeared in the callus tissue. Photographs were taken regularly to record the results, and the number of regenerated shoots from the explants was counted. (See attached table for details.) Figure 7 and Figure 8 .

[0048] The results showed that, through observation and recording of the somatic embryo culture process of CsFEZ overexpression (CsFEZ-OE) lines, the number of shoots in the CsFEZ overexpression lines was significantly increased after 2 weeks of culture on the medium compared with wild-type rice somatic embryos (Control), indicating a significant improvement in shoot regeneration efficiency (see details). Figure 7 ). Statistical analysis of the number of regenerated shoots from rice somatic embryos revealed a highly significant difference in the number of shoots regenerated between wild-type and CsFEZ overexpression lines (T-test, P<0.01, see details). Figure 8 Therefore, it was further verified that the regeneration capacity of plant or plant tissue somatic embryos can be regulated by controlling the expression or activity of CsFEZ protein and CsFEZ gene.

[0049] This invention proposes the use of CsFEZ protein or CsFEZ gene in regulating plant tissue regeneration. Experiments have shown that expressing CsFEZ protein or CsFEZ gene in tea and rice somatic embryos can effectively regulate somatic embryogenesis. Therefore, regulating CsFEZ protein or CsFEZ gene can provide a new solution for the genetic transformation of tea varieties or other species with low regeneration efficiency; furthermore, the CsFEZ gene can serve as an important molecular mechanism in the process of plant somatic embryo regeneration, providing a foundation for research and application in plant gene synthesis and customized plant modification.

[0050] According to embodiments of the present invention, the regulation of plant or plant tissue regeneration promotes the regeneration of plants or plant tissues. Experiments have shown that overexpression of the CsFEZ gene or increasing the expression and activity of the CsFEZ gene and CsFEZ protein may increase the regeneration efficiency of plants or plant tissues. This provides a foundation for studying the important molecular mechanisms of the response regulator CsFEZ in plant regeneration and is expected to play a key role in the research and application of plant genome synthesis and customized plant modification. Furthermore, by controlling the expression or activity of the regulator CsFEZ gene or CsFEZ protein, the somatic embryo regeneration efficiency of plants or plant tissues can be regulated to obtain plants or plant tissues with high somatic embryo regeneration efficiency.

[0051] In another aspect of the invention, the present invention proposes the use of a CSFEZ protein or CsFEZ gene in the process of somatic embryo regeneration in plants or plant tissues. As is known before, by controlling the expression of the CsFEZ protein or CsFEZ gene in plants or plant tissues, the regeneration of somatic embryos in plants or plant tissues can be effectively regulated. Therefore, by employing the above-mentioned overexpression method, the expression of the CsFEZ protein or CsFEZ gene can be promoted, thereby regulating the regeneration of somatic embryos in plants or plant tissues.

[0052] According to an embodiment of the present invention, the CsFEZ gene is used to encode the CsFEZ protein.

[0053] It should be noted that those skilled in the art will know that the nucleotide sequence encoding the CsFEZ protein (i.e., the CsFEZ gene in this invention) can be obtained from the amino acid sequence of the CsFEZ protein using conventional methods or software (such as the online database Tea Plant Information Archive (website: https: / / tpia.teaplants.cn / )). Therefore, in addition to SEQ ID No: 1 as defined in this invention, other genes that can encode the CsFEZ protein are also within the scope of this invention.

[0054] According to an embodiment of the present invention, the CsFEZ gene has a CDS sequence as shown in SEQ ID No: 1.

[0055]

[0056] It should be noted that the CsFEZ gene mentioned in this invention, as understood by those skilled in the art, actually includes any one or both of the complementary double strands. For convenience, although one strand is usually specified herein, the complementary strand is also disclosed. Furthermore, the nucleotide sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0057] According to an embodiment of the present invention, the CsFEZ protein has the amino acid sequence shown in SEQ ID No: 2.

[0058] MDEIGSETDKMDEIMLPGFRFHPTDEELVGFYLKRKVQQRPLSVEVIKQLDIYKHDPWDLPKLATTGEKEWYFYCPRDRKYRNSTRPNRVTGAGFWKATGTDRAIYSSEGSKCIGLKKSLVFYKGRAAKGIKTDWMMHEFRLPSSLTDSTPQKRFMDKNIPANDSWAICRIFKKINTTTQRAISHSWVSPPPPLPQ TTSSETFTQLAPPHNTHHFTSATMSFTPKPTSLSSIQFSTNNDLQHSPINTSFSPLDVLLKDLPTSCSFSSLINTSLGHSKSTVDSSSMFLNLSSSTSIFGDFDGLQEQCNGFSPQEMEAFPGYGQETALMKDSNGAYFDDDQWETMKSIGVPFNLPLNMLESDAWKSNLLWDSFPSLSEMSTSYSANKKCNT (SEQ ID No: 2).

[0059] In the description of this study, the terms "one embodiment," "some embodiments," "example," "specific instance," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0060] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the invention. Those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CsFEZ gene from a tea plant, characterized in that, Its CDS sequence is shown in SEQ ID No:

1.

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

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the CsFEZ gene as described in claim 1.

4. The recombinant vector according to claim 3, characterized in that, The scaffold vector for the recombinant vector is one of pCAMBIA2300, pCAMBIA1305, or pBI121.

5. The application of the tea tree gene CsFEZ as described in claim 1 in improving plant somatic embryogenesis.

6. The application according to claim 5, characterized in that, The application is that overexpression of the CsFEZ gene in plant tissues can enhance somatic embryogenesis in plants.

7. The application according to claim 6, characterized in that, Overexpression of the CsFEZ gene can be achieved by contacting the plant tissue with Agrobacterium containing the CsFEZ recombinant vector or gold powder.

8. A method for promoting plant somatic embryogenesis, characterized in that, The method includes: introducing the CsFEZ gene of claim 1 into a target plant to obtain a transgenic plant with increased somatic embryogenesis rate.

9. The method according to claim 8, characterized in that, The specific steps are as follows: (1) Cloning the CsFEZ gene in tea plants; (2) Construct a CsFEZ gene overexpression vector for tea plants; (3) The target plant was transformed by the CsFEZ gene overexpression vector of tea tree and the transgenic plant with increased somatic embryogenesis rate was obtained after identification.

10. The method according to claim 8, characterized in that, The plants mentioned are tea trees and rice.

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