Application of LcTCP transcription factor in regulation and control of synthesis of terpenoids of lauraceae plants

Abstract

The invention provides an application of an LcTCP transcription factor in regulation and control of synthesis of lauraceae plant terpenoids, and relates to the field of plant molecular biology. The research of the inventor finds that the transcription factor LcTCP can significantly improve the synthesis of the terpenoids of the lauraceae plants by promoting the expression of LcDXS, can be used for regulating and controlling the metabolism of the terpenoids of the lauraceae plants and molecular breeding, can be used for creating high-yield and high-quality new germplasm of the lauraceae plants, and can also be used for detecting and evaluating the synthesis capability of the terpenoids of the lauraceae plants.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to the application of an LcTCP transcription factor in regulating the synthesis of terpenoid compounds in Lauraceae plants. Background Technology

[0002] Litsea cubeba ( Litsea cubeba Litsea cubeba is an important woody aromatic plant, and its fruit essential oil contains a large number of terpenoid compounds (such as citral and limonene), which are widely used in the food, pharmaceutical, fragrance, and cosmetic industries. However, the yield and quality of Litsea cubeba essential oil are currently limited, and there is a lack of clear molecular regulatory targets to improve the efficiency of terpenoid synthesis.

[0003] Limited research exists on the molecular basis for improving the yield and quality of Litsea cubeba essential oil, with a lack of clearly defined molecular regulatory targets. It is known that transcription factors can regulate the expression of metabolic pathway genes by binding to the promoter regions of downstream key enzyme genes, thereby affecting the content of secondary metabolites. However, in Litsea cubeba, no TCP-type transcription factors have been reported to be directly related to terpene synthesis.

[0004] Transcription factor regulation is a highly efficient mechanism for controlling plant secondary metabolism. The TCP (Teosintebranched1 / Cincinnata / Proliferating cell factor) gene family is a class of plant-specific transcription factors that play a crucial role in plant growth and development, environmental stress response, and secondary metabolic regulation. TCP transcription factors all contain a TCP domain composed of 59 amino acid residues, which can bind to and interact with DNA and proteins by forming a basic helix-loop-helix (bHLH) motif. Currently, TCP transcription factors have been found to play an important role in regulating the synthesis of artemisinin and ginsenosides. However, in Lauraceae plants such as Litsea cubeba, no TCP transcription factors have been reported to be directly related to terpene synthesis.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of LcTCP transcription factor in regulating the synthesis of terpenoid compounds in Lauraceae plants, so as to improve the quality and yield of Litsea cubeba essential oil.

[0007] A second objective of this invention is to provide a carrier.

[0008] A third objective of this invention is to provide a genetically engineered bacterium.

[0009] The fourth objective of this invention is to provide a marker related to the characteristics of Lauraceae plants.

[0010] A fifth objective of this invention is to provide the application of reagents for detecting said markers in detecting the ability of Lauraceae plants to synthesize terpenoid compounds.

[0011] The sixth objective of this invention is to provide a reagent for detecting the ability of Lauraceae plants to synthesize terpenoid compounds.

[0012] The seventh objective of this invention is to provide a method for regulating the synthesis of terpenoid compounds in Lauraceae plants.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of LcTCP transcription factor in regulating the synthesis of terpenoid compounds in Lauraceae plants; the amino acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:2.

[0014] As a further technical solution, the nucleic acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:1.

[0015] As a further technical solution, the Lauraceae plants include Litsea cubeba.

[0016] Secondly, the present invention provides a vector containing the gene of the LcTCP transcription factor.

[0017] Thirdly, the present invention provides a genetically engineered bacterium containing the aforementioned vector.

[0018] Fourthly, the present invention provides a marker related to the traits of Lauraceae plants, wherein the marker is selected from any one of the following: a. The LcTCP transcription factor mentioned above; b. The gene of the LcTCP transcription factor; c. The RNA transcribed from the gene of the LcTCP transcription factor; The properties include the ability to synthesize terpenoids.

[0019] Fifthly, the present invention provides the application of reagents for detecting said markers in detecting the ability of Lauraceae plants to synthesize terpenoid compounds.

[0020] In a sixth aspect, the present invention provides a reagent for detecting the ability of Lauraceae plants to synthesize terpenoid compounds, the reagent being used to detect the gene of the LcTCP transcription factor; The reagents include primer pairs for amplifying the LcTCP transcription factor gene, the nucleic acid sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0021] In a seventh aspect, the present invention provides a method for regulating the synthesis of terpenoid compounds in Lauraceae plants, including overexpressing the gene of the LcTCP transcription factor described above.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of the LcTCP transcription factor in regulating terpene synthesis in Lauraceae plants. The inventors' research has found that the transcription factor LcTCP can significantly enhance the synthesis of terpenoid compounds in Lauraceae plants by promoting the expression of LcDXS. It can be used for the regulation of terpene metabolism in Lauraceae plants and molecular breeding, creating new high-yield and high-quality Lauraceae germplasm, and can also be used for the detection and evaluation of terpene synthesis capacity in Lauraceae plants. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 Phylogenetic tree of LcTCP protein and other plant TCP proteins; Figure 2 This is a diagram illustrating the expression pattern of LcTCP during the fruit development of Litsea cubeba; Figure 3 The graph shows the change in LcDXS expression level after transient overexpression of LcTCP in Litsea cubeba. Figure 4 The graph shows the change in monoterpene content after transient overexpression of LcTCP in Litsea cubeba. Figure 5 The results of the yeast monohybrid experiment show that LcTCP can bind to the TCP recognition element on the LcDXS promoter. Detailed Implementation

[0025] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In a first aspect, the present invention provides the application of LcTCP transcription factor in regulating the synthesis of terpenoids in Lauraceae plants; the amino acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:2: MQAVSDITGSLGWEEEEEEQQQQGGEKPVRKRPRNQNLEERIRRRTALDPLKKTIQPFLNSITQVEIEEEDEDEDEEEEEEQQAYKHPPPPKKRSFFQPSSSSEHAHFDMGENHRQNQ SSAAATMAVRNAGVGEIVEVEGGHIVRSTGRKDRHSKVCTAKGPRDRRVRLSAHTAIKFYDVQDRLGYDRPSKAVDWLIKNAKPAIDELEQLPPWKPTAANANEVAQQVFDDGRMREIE ANDVGKKAFEAAGYPFQANPSAENSSFLPPSLDSFFPIGSSSTSSSSSASIHLHSRQNQDLRLSLQSFQDPLLLQHQHHHSTPSGEQALFAGSAPMAFDVPAASVGWSQQEMSRFQRLI AWNAAEGSGGGGGGGGGYVFPASSQSPMPLMLQNPFFSQRGPLQSSNSPSVRAWADLPPVSAGEHQLHQPTASSVPAVRFAPGGFSGFHVPARIQGDEERDGGDKLSSVSSAPHH (SEQ ID NO.2).

[0027] This invention is the first to discover and verify that the LcTCP transcription factor can bind to and activate the promoter of LcDXS, a key gene for terpene synthesis, thereby regulating the synthesis of terpenes in Litsea cubeba, providing a new molecular tool for improving the quality and yield of essential oils from Litsea cubeba and other Lauraceae plants.

[0028] In some optional embodiments, the nucleic acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:1:

[0029] Secondly, the present invention provides a vector containing the gene of the LcTCP transcription factor.

[0030] When this vector is introduced into recipient cells, it can express the LcTCP transcription factor.

[0031] Thirdly, the present invention provides a genetically engineered bacterium containing the aforementioned vector.

[0032] For example, the above-mentioned vector can be introduced into Agrobacterium, and then Agrobacterium can be used to infect tobacco leaves. The expression location of the gene can be observed under a laser confocal microscope. If Litsea cubeba tissue culture seedlings are infected, the ability of the gene to promote terpene synthesis can be detected.

[0033] Fourthly, the present invention provides a marker related to the traits of Lauraceae plants, wherein the marker is selected from any one of the following: a. The LcTCP transcription factor mentioned above; b. The gene of the LcTCP transcription factor; c. The RNA transcribed from the gene of the LcTCP transcription factor; The properties include the ability to synthesize terpenoids.

[0034] Since the LcTCP transcription factor can enhance terpene synthesis in Lauraceae plants such as Litsea cubeba by promoting the expression of LcDXS, the terpene synthesis capacity of Lauraceae plants can be determined based on the LcTCP transcription factor, the LcTCP transcription factor gene, or the RNA transcribed from the LcTCP transcription factor gene.

[0035] Fifthly, the present invention provides the application of reagents for detecting said markers in detecting the ability of Lauraceae plants to synthesize terpenoid compounds.

[0036] Since the transcription factor LcTCP is related to the terpene synthesis ability of Lauraceae plants such as Litsea cubeba, the ability of Lauraceae plants to synthesize terpenes can be detected by detecting the transcription factor LcTCP.

[0037] In a sixth aspect, the present invention provides a reagent for detecting the ability of Lauraceae plants to synthesize terpenoid compounds, the reagent being used to detect the gene of the LcTCP transcription factor; The reagent includes primer pairs for amplifying the LcTCP transcription factor gene, and the nucleic acid sequences of the primer pairs are shown in SEQ ID NO:3 and SEQ ID NO:4: Primer F: ATGCAGGCAGTATCAGATATCACA (SEQ ID NO: 3); Primer R: ATGGTGAGGAGCAGAAGACA (SEQ ID NO:4).

[0038] By designing specific primers to detect the RNA of the LcTCP transcription factor gene, the terpene synthesis capacity of plants in the Lauraceae family, such as Litsea cubeba, can be inferred to some extent.

[0039] In a seventh aspect, the present invention provides a method for regulating the synthesis of terpenoid compounds in Lauraceae plants, including overexpressing the gene of the LcTCP transcription factor described above.

[0040] In some alternative implementations, the gene for the transcription factor LcTCP was introduced into Litsea cubeba leaves, resulting in an increase in terpene synthesis in Litsea cubeba.

[0041] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0042] Example 1: Identification and Classification of LcTCPs Transcription Factors (1) Identification of LcTCP transcription factors The Hidden Markov Model (HMM) of the TCP conserved domain (PF03634) was obtained from the PFAM database. Using HMM Search in TBtools v2.085 with a threshold of 1e^-10, and referencing the whole genome data of *Litsea cubeba*, genes containing the TCP conserved domain were searched for in the *Litsea cubeba* genome. *Arabidopsis thaliana* (…) Arabidopsis thaliana Using the TCP transcription factor family sequence as a reference, the BLASTP tool was used with a threshold of 1e^-10 to align with the Litsea cubeba protein sequence. The HMM results and BLASTP results were combined and duplicates were removed to obtain the Litsea cubeba TCP transcription factor gene family sequence.

[0043] (2) Classification of LcTCPs transcription factors The Litsea cubeba and the Arabidopsis thaliana TCP transcription factor gene family were used to construct a phylogenetic tree. The genes that branched off from the Arabidopsis thaliana TCP gene family were designated as the Litsea cubeba TCP gene family.

[0044] Thirty TCP transcription factors were identified from the genome of Litsea cubeba and classified into ( ) Figure 1 The three subfamilies are PCF, CIN, and CYC / TB1.

[0045] Example 2: Gene Cloning of LcTCP (1) RNA extraction Litsea cubeba fruit samples stored at -80℃ were ground into powder using liquid nitrogen and RNA was extracted using the RN38EASY spin plus plant RNA rapid extraction kit. First-strand cDNA was synthesized according to the instructions of the GoldenstarRT6 cDNA Synthesis Kit. Based on the CDS sequence of LcTCP obtained from Litsea cubeba genome sequencing, primers such as SEQ ID NO:3 and SEQ ID NO:4 were designed, and the LcTCP cDNA sequence was amplified using MCLAB high-fidelity enzyme.

[0046] PCR reaction system:

[0047] Reaction program: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 50 s, for a total of 35 cycles; 72°C extension for 5 min. PCR products were agarose gel electrophoresis followed by DNA gel extraction to recover the target fragment.

[0048] Cloning vectors were constructed using MonClone™ Single Assembly Cloning Mix. Reaction system:

[0049] Add the sample to ice, mix gently, and react at 50°C for 30 minutes.

[0050] Add 5 μL of the ligation product to 50 mL of E. coli DH-5α competent cells in an ice-water mixture, gently mix, and incubate on ice for 30 min; heat shock at 42 °C for 60 s; stand on ice for 2-3 min; add 700 μL of antibiotic-free LB liquid medium; shake and incubate at 37 °C for 1 h at 200 rpm; centrifuge at 3000 rpm for 1 min; discard 600 μL of supernatant, resuspend the bacterial cells, and spread an appropriate amount of the bacterial suspension onto LB solid medium containing 50 mg / L Kan; incubate upside down at 37 °C overnight until single colonies grow, and perform positive PCR detection and sequencing to obtain the nucleotide sequence of LcTCP as shown in SEQ ID NO:1 and the amino acid sequence as shown in SEQ ID NO:2.

[0051] Example 3: Analysis of LcTCP expression patterns at different fruit development stages (1) RNA extraction RNA was extracted from fruits at different developmental stages of Litsea cubeba, and the concentration and quality of the samples were measured. The total RNA amount from different tissue samples was standardized during cDNA first-strand synthesis for use in quantitative real-time PCR. The QuantStudio7Flex quantitative real-time PCR detection system was used, with the Litsea cubeba UBC gene as an internal control, following the procedures outlined in the TB GreenPremix Ex Tag II (TliRNaseH Plus) quantitative real-time PCR kit.

[0052] Each sample was biologically replicated 1 time and technically replicated 3 times. Utilizing 2 -ΔΔCT The method calculates the relative expression level of the LcTCP gene.

[0053] The results showed that ( Figure 2 LcTCP showed high expression levels in DAF60 during the critical period of fruit essential oil synthesis and had a similar expression pattern to LcDXS, with an expression correlation of 85%.

[0054] Example 4: Transient conversion of Litsea cubeba to verify the role of LcTCP in promoting terpene synthesis The overexpression vector LcTCP-BGV008 was constructed, and Agrobacterium tumefaciens containing LcTCP-BGV008 was added to 50 mL of LB medium and cultured until OD500. 600 When the concentration was 1.0, bacterial cells were collected and resuspended in a suspension (acetylsyleugenol 150 μmol·L, MgCl2 10 mM, MES 10 mM). After incubation for 2 hours, the bacterial cells were infiltrated into the leaves of Litsea cubeba tissue culture seedlings using a syringe through vacuum. The experimental group was infiltrated with Agrobacterium carrying the LcTCP-BGV008 recombinant vector, while the control group was infiltrated with Agrobacterium carrying the empty BGV008 vector.

[0055] Forty-eight hours later, the leaves of *Litsea cubeba* were ground into powder in liquid nitrogen, and RNA was extracted, reverse transcribed, and quantified to determine the expression levels of LcTCP and LcDXS. Terpenoid components were detected using LC-MS, and qualitative analysis was performed by fragment comparison, relevant literature reports, and the relative retention times of each component. Quantitative analysis was performed by calculating the relative content of each peak area using the peak area normalization method. Each sample was tested in triplicate.

[0056] The results are as follows Figure 3 As shown, LcTCP was transiently expressed 17.74 times in Litsea cubeba leaves, while the corresponding expression level of LcDXS increased by 2.64 times; simultaneously, as Figure 4 As shown, the content of monoterpenes in Litsea cubeba leaves was significantly increased, indicating that LcTCP positively regulates the biosynthesis of monoterpenes in Litsea cubeba by regulating LcDXS.

[0057] Example 5: Yeast monohybrid verification of LcTCP combined with LcDXS promoter element The LcTCP-pGADT7 vector was constructed and co-transformed into yeast strains with the AbAi vector containing the LcTCP binding element (5'-GTGGGTCC-3') and its corresponding mutant element (5'-TTTAAGAA-3'). The transformed yeast strains were then plated and inoculated onto auxotrophic medium (SD / -Trp / -Ura) and cultured at 30°C for about 3 days. The growth of yeast colonies on the plates was monitored.

[0058] It should be noted that the sequence of the TCP binding element is 5'-GTGGGTCC-3'. The LcTCP protein can function by binding to this sequence, while its mutant element cannot bind to LcTCP and can be used as a negative control in experiments.

[0059] The results are as follows Figure 5 As shown, yeast was observed to grow normally on culture medium without aureobasidin A (AbA); however, when AbA was added, only yeast containing both the LcTCP binding element and the LcTCP-pGADT7 vector could grow normally on the culture medium. This indicates that LcTCP has a recognition function for the LcDXS promoter.

[0060] This invention demonstrates that LcTCP positively regulates terpene biosynthesis in Litsea cubeba and can be applied to the regulation of terpene synthesis and molecular breeding in Litsea cubeba and other plants.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of LcTCP transcription factor in regulating the synthesis of terpenoids in Lauraceae plants; the amino acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The nucleic acid sequence of the LcTCP transcription factor is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The Lauraceae plants mentioned include Litsea cubeba.

4. A carrier, characterized in that, A gene containing the LcTCP transcription factor as described in any one of claims 1-3.

5. A genetically engineered bacterium, characterized in that, It contains the carrier as described in claim 4.

6. A marker related to traits of Lauraceae plants, characterized in that, The marker is selected from any one of ac: a. The LcTCP transcription factor as described in any one of claims 1-3; b. The gene of the LcTCP transcription factor; c. The RNA transcribed from the gene of the LcTCP transcription factor; The properties include the ability to synthesize terpenoids.

7. The use of the reagent for detecting the marker of claim 6 in detecting the ability of Lauraceae plants to synthesize terpenoid compounds.

8. A reagent for detecting the ability of Lauraceae plants to synthesize terpenoid compounds, characterized in that, The reagent is used to detect the gene of the LcTCP transcription factor as described in claim 6; The reagents include primer pairs for amplifying the LcTCP transcription factor gene, the nucleic acid sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:

4.

9. A method for regulating the synthesis of terpenoid compounds in Lauraceae plants, characterized in that, Genes that overexpress the LcTCP transcription factor as described in any one of claims 1-3.

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