A crisper gene activation system and a method for increasing theanine content in camellia sinensis by using the system

Abstract

This invention discloses a CRISPR gene activation system and a method for increasing the theanine content in tea plants using this system, belonging to the field of plant genetic engineering. The method for increasing the theanine content in tea plants includes: constructing a CRISPR gene activation system comprising an expression element of a dCas9-transcriptional activator fusion protein and at least one sgRNA expression element targeting the promoter region of a key gene in the theanine biosynthesis pathway; introducing this system into tea plant cells or tissues to specifically activate the transcription of endogenous target genes, thereby increasing the synthesis of theanine. Compared with existing technologies, this invention establishes a CRISPR-Act platform in tea plants for the first time, achieving synchronous, stable, and efficient activation of multiple genes in the theanine synthesis pathway through the use of highly efficient endogenous promoters, providing a powerful and precise technical means for cultivating new tea varieties with high theanine content and for the biomanufacturing of theanine.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and metabolic engineering, specifically relating to a method for using the CRISPR-dCas9 gene activation system (CRISPR-Act) in tea plants (… Camellia sinensis Methods and related applications for simultaneously activating endogenous gene expression in [a specific organism] to increase the content of the key secondary metabolite L-theanine. Background Technology

[0002] Theanine is a unique non-protein amino acid found in tea and is a core component determining its flavor and health benefits. Traditional breeding methods are time-consuming and inefficient, while conventional transgenic overexpression methods suffer from insertion site effects, gene silencing, and transgenic safety issues. Therefore, developing a technology that can precisely, efficiently, and stably regulate endogenous gene expression to improve tea plant traits without introducing exogenous functional genes has significant application value.

[0003] CRISPR / Cas9 technology has seen initial applications in tea plant gene editing, but these applications mainly focus on gene knockout. More powerful gene activation technologies, such as the CRISPR-Act system, can target and activate the expression of endogenous genes under the guidance of sgRNA by fusing inactivated Cas9 protein (dCas9) with a transcriptional activation domain, achieving "seamless" functional gain. However, prior to this invention application, this technology has not been successfully established and applied in tea plants.

[0004] Furthermore, the expression efficiency of sgRNA in the CRISPR system directly determines the success or failure of editing or activation. Existing studies mostly rely on heterologous promoters (such as Arabidopsis thaliana AtU6), whose activity and universality in tea plants have not been systematically evaluated, which may become a bottleneck limiting the efficiency of the technology.

[0005] Therefore, there is an urgent need in this field for a gene activation technology that can operate efficiently in tea plants in order to achieve precise regulation of important metabolic pathways such as theanine. Summary of the Invention

[0006] This invention aims to address the problems of insufficient activation capacity of endogenous genes and low efficiency due to reliance on heterologous promoters in existing tea plant genetic improvement technologies. It provides a method that can efficiently, synchronously, and stably activate key genes in the endogenous theanine synthesis pathway of tea plants, thereby significantly increasing the theanine content.

[0007] The present invention is implemented using the following technical solutions:

[0008] In a first aspect, this invention provides a method for increasing the theanine content in tea plants using a CRISPR gene activation system. The method is characterized by introducing a specially constructed CRISPR gene activation system into tea plant cells, tissues, or plants, said CRISPR gene activation system comprising:

[0009] (a) An expression element encoding a dCas9-transcriptional activator fusion protein; and

[0010] (b) At least one expression element encoding an sgRNA that targets the promoter region of at least one key gene in the theanine biosynthesis pathway;

[0011] The system can specifically bind to and activate the transcription of one or more endogenous key genes in the theanine biosynthesis pathway, thereby increasing the amount of theanine biosynthesis.

[0012] In a preferred embodiment, the key gene of the present invention is selected from the alanine decarboxylase gene. Cheat and / or theanine synthase gene CsTSI By simultaneously targeting and activating Cheat and CsTSI Two genes synergistically enhance the theanine synthesis pathway.

[0013] In a preferred embodiment, the system of the present invention comprises at least two expression elements encoding sgRNA, each targeting... Cheat The promoter region of a gene and CsTSI The promoter region of a gene.

[0014] In a preferred embodiment, the expression element encoding sgRNA of the present invention is driven by a plant RNA polymerase III promoter.

[0015] In a preferred embodiment, the plant RNA polymerase III promoter of the present invention is the tea plant endogenous U3 promoter.

[0016] In a preferred embodiment, the endogenous U3 promoter of the tea plant described in this invention is a CsU3a promoter or a CsU3b promoter.

[0017] In a preferred embodiment, the introduction step of the present invention is achieved by an Agrobacterium tumefaciens-mediated transformation method.

[0018] In a second aspect, the present invention provides a CRISPR gene activation system for efficiently driving gene activation in tea plants, the system comprising:

[0019] (a) An expression element encoding a dCas9-transcriptional activator fusion protein; and

[0020] (b) At least one expression element encoding an sgRNA that targets the alanine decarboxylase gene. Cheat and / or theanine synthase gene CsTSI The promoter region of the sgRNA is defined, and the expression of the sgRNA is driven by the tea plant endogenous U3 promoter.

[0021] Specifically, to achieve efficient activation, sgRNA expression in the sgRNA expression cassette is driven by the tea plant's endogenous U3 promoter. In a more preferred embodiment, the tea plant's endogenous U3 promoter is either the CsU3a promoter or the CsU3b promoter, which exhibit high transcriptional activity. The application of this highly efficient endogenous promoter ensures adequate sgRNA expression and is crucial for achieving efficient gene activation.

[0022] In a third aspect, the present invention provides a kit comprising the CRISPR gene activation system described herein. The kit is designed for stable or transient expression in tea plant cells. In a preferred embodiment, the vector is a binary expression vector suitable for Agrobacterium tumefaciens-mediated transformation, wherein it contains [amount of vectors for simultaneous activation]. Cheat and CsTSI The gene contains two or more sgRNA expression cassettes, and each sgRNA expression cassette is driven by the tea plant endogenous CsU3 promoter.

[0023] In a fourth aspect, the present invention provides the application of the CRISPR gene activation system or kit in the cultivation of new tea varieties with high theanine content or the production of high value-added tea products.

[0024] Compared with the prior art, the present invention has the following significant advantages:

[0025] (1) The CRISPR-Act system was successfully applied to tea trees for the first time: This invention established a gene activation platform in tea trees, an important economic crop, for the first time, filling a key gap in the toolbox of functional genomics research on tea trees.

[0026] (2) Extremely high activation efficiency: By screening and using highly efficient tea plant endogenous CsU3 promoters (especially CsU3) to drive sgRNA expression, the gene activation efficiency of this invention is far higher than that of systems using conventional heterologous promoters (such as AtU3). Experiments show that the CsU3-driven system can upregulate target gene expression by more than 20-fold.

[0027] (3) Achieved simultaneous activation of multiple genes and synergistic enhancement of metabolic flux: This invention can simultaneously activate two key rate-limiting enzyme genes in the theanine synthesis pathway ( Family C and CsTSI), which synergistically enhances metabolic flux, thereby greatly increasing the theanine content (up to 8.5 times higher in positively transformed roots).

[0028] (4) The system has broad application prospects: Through the Agrobacterium tumefaciens transformation system, this invention has verified the long-term stable expression and effect of the gene activation system. This method provides a powerful and precise advanced technical means for cultivating new tea varieties with high theanine content and producing high-value-added tea cell lines or positively transformed root cultures. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of a tea tree gene activation vector.

[0030] Figure 2 Transient expression and functional validation of the CRISPR-Act system in tea roots. (A) Positive roots transiently expressing CRISPR-Act. (B) Positive root samples. Cheat Gene expression levels. (C) In positive root samples CsTSI Gene expression levels. (D) Theanine content in positive root samples.

[0031] Figure 3 Stable expression and functional validation of the CRISPR-Act system in induced hairy roots of tea plants. (A) Stable positive hairy roots induced by Agrobacterium rhizogenes. (B) Positive hairy root samples. Cheat Gene expression levels. (C) In positive hairy root samples CsTSI Gene expression levels. (D) Theanine content in positive hairy root samples. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be noted that the following embodiments are only for explaining the invention and are not intended to limit the scope of protection of the invention. Any modifications, equivalent substitutions, and improvements made based on the content of this invention should be included within the scope of protection of this invention.

[0033] Experimental materials and general methods used:

[0034] Plant material: tea tree ( Camellia sinensis cv. Longjing 43) Two-year-old cuttings, planted in a greenhouse.

[0035] Strains: Agrobacterium tumefaciens ( Agrobacterium rhizogenes ) K599 strain.

[0036] Main reagents and vectors: Seamless cloning kit (TransGen Biotech), Golden Gate assembly kit (NEB), Gateway LR enzyme (Thermo Fisher Scientific), pYLCRISPR / Cas9P35S-H, pYPQ series vectors (pYPQ131, pYPQ132, pYPQ142, pYPQ202).

[0037] Example 1: Construction of CRISPR-Act gene activation vector

[0038] This embodiment focuses on the simultaneous activation of key genes in the theanine synthesis pathway of tea plants. Cheat and CsTSI To achieve this goal, we constructed a multi-gene activation vector.

[0039] 1.1 sgRNA Design and Synthesis

[0040] against Cheat and CsTSI Design specific sgRNAs (sgRNA4 targeting) for gene promoter regions Cheat sgRNA5 targeting CsTSI The nucleotide sequence of sgRNA4 is shown in SEQ ID NO.1, and the nucleotide sequence of sgRNA5 is shown in SEQ ID NO.2.

[0041] 1.2 Construction of gene activation expression vector

[0042] The CRISPR-Act3.0 system expression vector was constructed using the Golden Gate assembly strategy.

[0043] (1) The sgRNA4 expression cassette (CsU3a-sgRNA4) driven by the CsU3a promoter was cloned into Bsa Linear pYPQ131 vector was digested with enzyme I. The sgRNA5 expression cassette (CsU3b-sgRNA5) driven by the CsU3b promoter was cloned into... Bsa In the pYPQ132 vector digested with enzyme I.

[0044] (2) Using Golden Gate assembly technology, the two modules (CsU3a-sgRNA4-gR2.0 and CsU3b-sgRNA5-gR2.0) obtained in step (1) are simultaneously assembled into the pYPQ142 backbone vector to form a multi-target module containing two sgRNA expression cassettes.

[0045] (3) Using Gateway LR recombinase, the complete multi-target expression cassette assembled in step (2) was transferred from the pYPQ142 vector to the final plant binary expression vector pYPQ202. This vector also contains the dCas9-VPR fusion protein expression cassette driven by the 35S promoter.

[0046] (4) Using the same molecular strategy, construct a gene activation vector that drives sgRNA expression via the CsU6 promoter.

[0047] (5) As a control, the same molecular strategy was used to construct a gene activation vector with AtU3 promoter-driven sgRNA expression.

[0048] The results are as follows Figure 1 As shown, all the finally constructed vectors were verified by enzyme digestion and sequencing to ensure that the sequences were correct.

[0049] Example 2: Transient expression and functional verification of the CRISPR-Act system in tea roots

[0050] 2.1 Instantaneous transformation of tea tree root system

[0051] The CRISPR-Act plasmids containing different promoters (CsU3, AtU3, CsU6) constructed in Example 1 were transformed into Agrobacterium tumefaciens K599. The cultured bacterial culture was resuspended in infection buffer containing 150 μM acetylsyringone (AS), and OD... 600 The value was adjusted to 0.6, and the mixture was induced at 28°C in the dark for 4 hours.

[0052] The roots of tea tree cuttings were immersed in the bacterial solution and subjected to a vacuum permeation at -0.5 MPa for 15 minutes. After infection, the plants were transferred to 1 / 2 MS liquid medium and cultured at 25°C for 5 days (with continuous oxygen supply).

[0053] 2.2 Analysis of gene activation effect

[0054] GFP-positive transformed root segments were screened using a handheld fluorescent lamp and then analyzed.

[0055] qRT-PCR analysis: Total RNA was extracted from positive root segments, reverse transcribed into cDNA, and then subjected to quantitative real-time PCR. CsPTB As an internal reference gene, it is used to detect target genes. Cheat and CsTSI The relative expression level. Cheat The qRT-PCR detection primers include qAlaDC-F and qAlaDC-R. The nucleotide sequence of qAlaDC-F is shown in SEQ ID NO.3, and the nucleotide sequence of qAlaDC-R is shown in SEQ ID NO.4. CsTSI The qRT-PCR detection primers include qTSI-F and qTSI-R. The nucleotide sequence of qTSI-F is shown in SEQ ID NO.5, and the nucleotide sequence of qTSI-R is shown in SEQ ID NO.6.

[0056] Theanine content determination: 0.5 g of positive root segment was weighed, extracted in a boiling water bath for 30 minutes, centrifuged and filtered, and the extract was pre-column derivatized using a Waters AccQ-Tag kit. Detection was performed using a Waters e2695 HPLC system equipped with an AccQ-Tag Ultra column and a fluorescence detector (excitation wavelength 260 nm). Theanine content was calculated based on the standard curve. Results are as follows. Figure 2 As shown, the system driven by the CsU3 bootstrap exhibits the strongest activation effect, making Cheat and CsTSI The expression level was upregulated by up to 10.5-fold, with a corresponding increase in theanine content of approximately 5.8-fold. This effect was significantly superior to systems driven by AtU3 (4.8-fold upregulation) and CsU6 (5.3-fold upregulation).

[0057] Example 3: Stable expression and functional verification of the CRISPR-Act system in the hairy roots of tea trees

[0058] To overcome the instability of transient expression, this embodiment uses the induction of stable transgenic hairy roots to verify the long-term effects of the system.

[0059] 3.1 Induction of hairy roots

[0060] Agrobacterium tumefaciens K599 bacterial culture (OD) carrying the CRISPR-Act plasmid described in Example 1 was used. 600 = 0.8 (containing 200 μM AS) was used to infect sterile mature tea leaves. The base of the petiole was immersed in the bacterial solution and vacuum-permeated at -0.8 MPa for 30 minutes. After infection, the leaves were vertically inserted into sterilized vermiculite medium and cultured under a 16h / 8h light / dark cycle and a 22℃ / 18℃ day / night temperature. After about 8 weeks, hairy root growth was observed at the base of the petiole.

[0061] 3.2 Analysis of stable activation effect

[0062] GFP-positive hairy roots were screened using a handheld fluorescent lamp, and after being cut off, they were subjected to the same qRT-PCR and theanine content analysis as in Example 2.

[0063] The results are as follows Figure 3 As shown, in stably expressed hairy roots, the CsU3 promoter-driven system exhibits a more robust and stable activation capacity. Target genes Cheat and CsTSIThe expression level was upregulated by up to 20.8-fold, and the theanine concentration increased by 8.5-fold accordingly. In contrast, the AtU3- and CsU6-driven systems increased theanine content by 3.8-fold and 2.6-fold, respectively.

[0064] The above embodiments fully demonstrate that the CRISPR-Act gene activation system constructed in this invention, especially when its sgRNA expression is driven by the highly efficient tea plant endogenous CsU3 promoter, can efficiently, synchronously and stably activate multiple key endogenous genes in the theanine synthesis pathway. Cheat and CsTSI This method significantly increases the theanine content, providing a novel and efficient technical approach for molecular breeding of high-theanine tea varieties and the bioproduction of theanine.

[0065] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the claims of the present invention.

Claims

1. A method for increasing the theanine content in tea plants, characterized in that, This includes the step of introducing a CRISPR gene activation system into tea tree cells or tissues; The tea tree is Camellia sinensis cv. Longjing 43 The CRISPR gene activation system comprises: (a) An expression element encoding a dCas9-transcriptional activator fusion protein; and (b) Two expression elements encoding sgRNAs that target the promoter region of a key gene in the theanine biosynthesis pathway; The key gene is selected from the alanine decarboxylase gene. CsAlaDC and / or theanine synthase gene CsTSI The sgRNA is selected from targeted RNA. CsAlaDC sgRNA4 and targeting CsTSI The sgRNA5, wherein the nucleotide sequence of the sgRNA4 is shown in SEQ ID NO.1, and the nucleotide sequence of the sgRNA5 is shown in SEQ ID NO.2; The expression elements encoding sgRNA target, respectively CsAlaDC The promoter region of a gene and CsTSI The promoter region of the gene, in the expression element encoding sgRNA, the expression of sgRNA is driven by a plant RNA polymerase III promoter, the plant RNA polymerase III promoter is the tea plant endogenous U3 promoter, the tea plant endogenous U3 promoter is the CsU3a promoter or the CsU3b promoter.

2. The method according to claim 1, characterized in that, The importation step is achieved through a transformation method mediated by Agrobacterium tumefaciens.

3. A CRISPR gene activation system, characterized in that, The system includes: (a) An expression element encoding a dCas9-transcriptional activator fusion protein; and (b) Two expression elements encoding sgRNAs that target the promoter region of a key gene in the theanine biosynthesis pathway; The key gene is selected from the alanine decarboxylase gene. CsAlaDC and / or theanine synthase gene CsTSI The sgRNA is selected from targeted RNA. CsAlaDC sgRNA4 and targeting CsTSI The sgRNA5, wherein the nucleotide sequence of the sgRNA4 is shown in SEQ ID NO.1, and the nucleotide sequence of the sgRNA5 is shown in SEQ ID NO.2; The expression elements encoding sgRNA target, respectively CsAlaDC The promoter region of a gene and CsTSI The promoter region of the gene, in the expression element encoding sgRNA, the expression of sgRNA is driven by a plant RNA polymerase III promoter, the plant RNA polymerase III promoter is the tea plant endogenous U3 promoter, the tea plant endogenous U3 promoter is the CsU3a promoter or the CsU3b promoter; The tea tree is Camellia sinensis cv. Longjing 43 .

4. A kit for increasing the theanine content of tea trees, characterized in that, Includes the CRISPR gene activation system as described in claim 3.

5. The application of the CRISPR gene activation system as described in claim 3 or the kit for increasing the theanine content of tea plants as described in claim 4 in the preparation of tea plants, tissues or cell lines with high theanine content.

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