CsRBE gene for regulating lip form of cymbidium sinense, application of CsRBE gene, protein and method

Abstract

The invention relates to the technical field of biology, in particular to a CsRBE gene for regulating and controlling lip form of cymbidium sinense, application of the CsRBE gene, protein and a method. The nucleotide sequence of the CsRBE gene is as shown in SEQ ID No. 1. The CsRBE gene provided by the invention can regulate and control the lip form of cymbidium sinense, after the CsRBE gene is silenced, the lip form of cymbidium sinense is flat without bending, the lip width and the fold gap in the middle of the lip are increased, and an important basis is provided for cymbidium sinense breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a CsRBE gene for regulating the morphology of the labellum of Cymbidium orchid, and an application, protein and method thereof. Background Art

[0002] Orchids are one of the largest and most diverse families of flowering plants. Their highly specialized labellums hold crucial biological significance in pollination biology, species divergence, and adaptive evolution. As specialized structures within the tepals of orchids, the labellums exhibit a complex and diverse range of morphology, pattern, color, texture, and accessory structures. This directly influences the preferences of insect pollinators and is crucial for pollination strategies and reproductive success. The diversity of labellum morphology and the unique phenotypic pattern of the flower mottled pattern are key factors in attracting consumers and enhancing product value. Among ornamental orchids, the Molan orchid, a representative example of the Chinese orchid, exhibits extensive phenotypic variation in labellum development, including typical morphologies such as a single labellum (standard), multiple labellums (odd flower type), and three labellums (butterfly type). The odd flower and butterfly types are highly valued for their greater ornamental value. However, apart from the CsAP3-2 gene, no genes involved in labellum development and morphological differences in Molan orchids have been reported. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a CsRBE gene for regulating the morphology of the labellum of Cymbidium indica, and its application, protein and method. The CsRBE gene provided by the present invention can regulate the morphology of the labellum of Cymbidium indica. After silencing the CsRBE gene, the morphology of the labellum of Cymbidium indica is flat and without bending, and the width of the labellum and the wrinkled gap in the middle of the labellum are increased.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a CsRBE gene for regulating the morphology of the labellum of Cymbidium sinensis. The nucleotide sequence of the CsRBE gene is shown in SEQ ID No. 1.

[0006] The present invention also provides the application of the CsRBE gene described in the above technical solution in regulating the morphology of the labellum of Cymbidium sinensis.

[0007] Preferably, the morphology of the labellum of Cymbidium orchid is regulated by silencing the CsRBE gene.

[0008] Preferably, the CsRBE gene is silenced, the lip morphology of Cymbidium orchid is flat and without bending, and the lip width and the wrinkled gap in the middle of the lip are increased.

[0009] The present invention also provides a protein encoded by the CsRBE gene described in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0010] The present invention also provides a method for regulating the morphology of the labellum of Cymbidium sinense, comprising the following steps:

[0011] 1) ligating the 1-300 bp sequence of the CsRBE gene described in the above technical solution into the pTRV2 vector to obtain the pTRV2-CsRBE vector;

[0012] 2) transforming Agrobacterium with the pTRV2-CsRBE vector obtained in step 1) and culturing the resulting bacterial solution containing the pTRV2-CsRBE vector;

[0013] 3) The bacterial solution containing the pTRV2-CsRBE vector obtained in step 2) was centrifuged, and the obtained bacteria were mixed with a buffer solution to obtain a CsRBE bacterial solution. The OD 600 The value is 0.5;

[0014] 4) The pTRV1 vector was transformed into Agrobacterium, and the bacterial solution containing the pTRV1 vector was obtained by culture. The bacterial cells obtained by centrifugation were mixed with the buffer solution to obtain the pTRV1 bacterial solution. The OD 600 The value is 0.5;

[0015] 5) mixing equal volumes of the CsRBE bacterial solution obtained in step 3) and the pTRV1 bacterial solution obtained in step 4) to obtain an infection solution;

[0016] 6) Infecting the orchid buds of Cymbidium sinense with the infection solution obtained in step 5).

[0017] Preferably, in step 2) the OD value of the bacterial solution containing the pTRV2-CsRBE vector is 600 The value is 1; the Agrobacterium is Agrobacterium GV3101.

[0018] Preferably, the components of the buffer in step 3) are: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride;

[0019] The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 8 minutes.

[0020] Preferably, the Agrobacterium in step 4) is Agrobacterium GV3101;

[0021] The OD of the bacterial solution containing the pTRV1 vector 600 The value is 1;

[0022] The components of the buffer are: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride;

[0023] The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 8 minutes.

[0024] Preferably, the infection conditions in step 6) include: infection for 10 minutes under vacuum conditions, and the flower buds are flower buds with wounds.

[0025] Beneficial effects of the present invention:

[0026] The CsRBE gene provided by the present invention can regulate the morphology of the labellum of Mo Orchid. After silencing the CsRBE gene, the morphology of the labellum of Mo Orchid is flat and without bending, and the width of the labellum and the wrinkle gap in the middle of the labellum are increased, providing an important basis for Mo Orchid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0028] Figure 1 This is the phylogenetic tree analysis of CsRBE of Cymbidium sinense;

[0029] Figure 2 is an amino acid sequence alignment diagram of CsRBE of Cynanchum indica;

[0030] Figure 3 The expression levels of single flowers of Cymbidium orchid cultivars 'Xiaoxiang' and 'Yushizi' and the CsRBE gene during the five stages of flower development are shown.

[0031] Figure 4 This is the map of the constructed Cymbidium orchid gene silencing vector pTRV2-CsRBE;

[0032] Figure 5 The inflorescence phenotypes of the silenced strain and the empty vector control after treatment with pTRV2-CsRBE are shown;

[0033] Figure 6 The phenotypes of the flower lip of the silenced strain and the empty vector control after treatment with pTRV2-CsRBE are shown in Figure 2.

[0034] Figure 7 The expression levels in the lip petals of the silenced strain and the empty control after treatment with pTRV2-CsRBE. DETAILED DESCRIPTION

[0035] The present invention provides a CsRBE gene for regulating the morphology of the labellum of Cymbidium sinensis. The nucleotide sequence of the CsRBE gene is shown in SEQ ID No. 1, and is specifically as follows:

[0036] .

[0037] The present invention also provides the use of the CsRBE gene described in the above technical solution to regulate the morphology of the labellum of Cymbidium indica. The present invention preferably regulates the morphology of the labellum of Cymbidium indica by silencing the CsRBE gene. In the present invention, silencing the CsRBE gene results in a flat, uncurved labellum morphology, an increase in the width of the labellum, and an increase in the folded gap between the labellums.

[0038] The present invention also provides a protein encoded by the CsRBE gene described in the above technical solution, the amino acid sequence of the protein is shown in SEQ ID No. 2, and is as follows:

[0039] MRAERNFLCRPLPTFILSSSTNVSWEEQAFAKDSSGDLGGCVWPPRSYTCSFCREFRSAQALGGHMNVHRRDRAKLKEASSHSRETDEDIQNFDPFSSFIYNPNPNSDALG FEVASFLSPEEEICSPKSVVSVSLEIGRENWKAGELDHRRIRDFFDGDDEILCKKVKTDLMLTMDRMDPPDNQQTSNSKVIKLSHSPVDLDLELRLGDSPKVKESRNIIVIE*.

[0040] The present invention also provides a method for VIGS (virus-induced gene silencing) based on TRV (Tobacco Rattle Virus), which regulates the morphology of the labellum of Cymbidium sinensis by silencing the CsRBE gene, comprising the following steps:

[0041] 1) ligating the 1-300 bp sequence of the CsRBE gene described in the above technical solution into the pTRV2 vector to obtain the pTRV2-CsRBE vector;

[0042] 2) transforming Agrobacterium with the pTRV2-CsRBE vector obtained in step 1) and culturing the resulting bacterial solution containing the pTRV2-CsRBE vector;

[0043] 3) The bacterial solution containing the pTRV2-CsRBE vector obtained in step 2) was centrifuged, and the obtained bacteria were mixed with a buffer solution to obtain a CsRBE bacterial solution. The OD 600 The value is 0.5;

[0044] 4) The pTRV1 vector was transformed into Agrobacterium, and the bacterial solution containing the pTRV1 vector was obtained by culture. The bacterial cells obtained by centrifugation were mixed with the buffer solution to obtain the pTRV1 bacterial solution. The OD 600 The value is 0.5;

[0045] 5) mixing equal volumes of the CsRBE bacterial solution obtained in step 3) and the pTRV1 bacterial solution obtained in step 4) to obtain an infection solution;

[0046] 6) Infecting the orchid buds of Cymbidium sinense with the infection solution obtained in step 5).

[0047] The present invention connects the 1-300 bp sequence of the CsRBE gene described in the above technical solution into the pTRV2 vector to obtain the pTRV2-CsRBE vector. The present invention does not specifically limit the method of connecting the CsRBE gene into the pTRV2 vector, and those skilled in the art can use conventional methods.

[0048] The present invention transforms the obtained pTRV2-CsRBE vector into Agrobacterium, and cultivates to obtain a bacterial solution containing the pTRV2-CsRBE vector. The present invention does not specifically limit the method for transforming Agrobacterium with the pTRV2-CsRBE vector, and a person skilled in the art can use conventional methods. The present invention does not specifically limit the culture conditions, and a person skilled in the art can use conventional culture conditions for culture. In the present invention, the OD value of the bacterial solution containing the pTRV2-CsRBE vector is 600 The value is preferably 1. In the present invention, the Agrobacterium is preferably Agrobacterium GV3101.

[0049] The present invention centrifuges the bacterial solution containing the pTRV2-CsRBE vector, mixes the obtained bacterial cells with a buffer solution, and obtains a CsRBE bacterial solution. The OD 600 The value is 0.5. In the present invention, the components of the buffer solution are preferably: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride. In the present invention, the centrifugal conditions preferably include: a rotation speed of 5000 rpm and a time of 8 min.

[0050] The present invention transforms the pTRV1 vector into Agrobacterium, cultures the bacterial solution containing the pTRV1 vector, and mixes the bacterial cells obtained by centrifugation with a buffer solution to obtain a pTRV1 bacterial solution. The OD 600 The present invention does not specifically limit the method for transforming Agrobacterium with the pTRV1 vector, and those skilled in the art can use conventional methods. The present invention does not specifically limit the conditions for the culture, and those skilled in the art can use conventional culture methods. In the present invention, the Agrobacterium is preferably Agrobacterium GV3101. In the present invention, the OD value of the bacterial solution containing the pTRV1 vector is 600 The value is preferably 1. In the present invention, the components of the buffer are preferably: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride. In the present invention, the centrifugal conditions preferably include: a rotation speed of 5000 rpm and a time of 8 min.

[0051] The present invention mixes the obtained CsRBE bacterial solution and the obtained pTRV1 bacterial solution in equal volumes to obtain an infection solution.

[0052] The present invention uses the obtained infection liquid to infect the buds of Cymbidium orchid. In the present invention, the infection conditions preferably include: infecting for 10 minutes under vacuum conditions, and the flower buds are flower buds with wounds.

[0053] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] Cloning of the CsRBE gene from Cymbidium sinense

[0056] (1) Total RNA was extracted from the flowers of the Molan variety 'Xiaoxiang' using an RNA extraction kit (commercially available). The total RNA was reverse transcribed into cDNA using a reverse transcription kit (commercially available). The operation method was carried out according to the kit instructions.

[0057] (2) Primers were designed based on the CDS sequence of the CsRBE (RABBIT EARS) gene in the Cymbidium orchid genome. The primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0058] SEQ ID No.3: ATGCGTGCAGAAAGGAATT;

[0059] SEQ ID No. 4: TTACTCTATCACAATGATGTTACGAC.

[0060] (3) PCR amplification was performed using the cDNA obtained in step (1) as a template. The amplified product was purified and recovered using a gel recovery kit (commercially available, operated according to the instructions). The recovered product was ligated to a TA / Blunt-Zero vector using a 5-min TA / Blunt-Zero Cloning Kit (Vazyme, commercially available, operated according to the instructions), and the vector was transformed into DH5α Escherichia coli competent cells (commercially available, operated according to the instructions). Positive single clones were selected and Sanger sequenced. After analyzing and comparing the sequencing results, the complete CDS sequence of the target gene CsRBE was obtained. The sequence contains 687 bases, the nucleotide sequence is shown in SEQ ID No. 1, and consists of 229 amino acid residues, the amino acid sequence is shown in SEQ ID No. 2.

[0061] Example 2

[0062] Characterization Analysis of CsRBE Gene in Cymbidium sinense

[0063] (1) The amino acid sequences of CsRBE were compared using the https: / / www.ncbi.nlm.nih.gov website, and 14 sequences with high homology were found.

[0064] (2) MAGA11 software was used to perform evolutionary analysis and mapping of the 14 sequences in step (1), as shown in the following example: Figure 1 As shown, Cymbidium sinense mainly clusters with monocots and has a high homology with Dendrobium officinale and Phalaenopsis.

[0065] (3) The amino acid sequences of CsRBE were aligned using DNAMAN software. Figure 2The CsRBE gene contains a C2H2-type zinc finger conserved domain and is a zinc finger protein transcription factor.

[0066] Example 3

[0067] Verification of expression profiles of Cymbidium orchid at different developmental stages

[0068] (1) Using the single-lip variety of Mo Lan 'Xiao Xiang' and the multi-lip variety 'Yu Shi Zi' as materials, RNA was extracted from the five flower development stages of the two varieties using an RNA extraction kit (commercially available). The total RNA was reverse transcribed into cDNA using a reverse transcription kit. The operation methods were carried out according to the kit instructions.

[0069] (2) Real-time fluorescence quantitative PCR (RT-qPCR) primers were designed based on the coding region sequence of the CsRBE gene. The primer sequences are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0070] SEQ ID No.5:TTTCGCAAAAGACTCGTCGG;

[0071] SEQ ID No. 6: CCTAGAGCTTGGGCTGATCG.

[0072] (3) RT-qPCR analysis was performed using the Taq Pro Universal SYBR qPCR Master Mix Kit (Vazyme, commercially available). The reaction system was 20 μl and the method in the instructions was followed. The amplification program was 95°C for 2 min, (95°C for 10 sec, 60°C for 30 sec), 40 cycles of the steps in brackets, and then melting for 15 sec. Actin was used as the internal reference, and the comparative Ct value method was used to analyze the data. The relative expression levels of CsRBE in the five different stages of flower development are shown in Figure 2. Figure 3 As shown, the CsRBE gene was highly expressed in the early stage of flower development in both varieties and gradually decreased significantly with flower development. However, the expression level of the CsRBE gene in the multi-lip variant variety 'Yushizi' was significantly higher than that in the normal flower variety 'Xiaoxiang' at all five stages, suggesting that this gene may be involved in regulating the development of the lip of Mo Orchid.

[0073] Example 4

[0074] Tobacco rattle virus (TRV)-induced VIGS silencing of the CsRBE gene in Cymbidium sinense

[0075] (1) Homologous recombination primers were designed using the online primer design software CE Design https: / / tool.vazyme.com:18002 / cetool / singlefragment.html on the official website of Vazyme. The primer sequences are shown in SEQ ID No. 7 and SEQ ID No. 8.

[0076] SEQ ID No.7: agaaggcctccatggggatccATGCGTGCAGAAAGGAATTTTC;

[0077] SEQ ID No.8:

[0078] gagacgcgtgagctcggtaccAGAGCTAAATGGATCAAAATTTTGAA.

[0079] (2) BamHI and KpnI were selected as double enzyme digestion sites, and the pTRV2 plasmid was double enzyme digested. The product was recovered using a gel recovery kit (commercially available, according to the instructions).

[0080] (3) Select the CsRBE gene, i.e., the sequence of 1-300 bp in SEQ ID No. 1, and use the cDNA of Cymbidium sinense'Xiaoxiang' as a template. PCR amplification is performed with the primers in step (1), and the PCR product is recovered by gel recovery kit (commercially available, according to the instructions).

[0081] (4) CloneExpress Ultra One Step Cloning Kit V2 (commercially available, follow the instructions) was used to connect the 300 bp sequence in step (3) to the pTRV2 vector to form the pTRV2-CsRBE vector containing the CsRBE gene fragment. The vector map is shown in FIG. Figure 4 The size of the inserted fragment was verified by PCR using universal primers SEQ ID No. 9 and SEQ ID No. 10 for the pTRV2 vector, and the bacterial solution was subjected to Sanger sequencing to verify the accuracy of the inserted CsRBE gene fragment.

[0082] SEQ ID No.9: TTAGTTCAGGCGGTTCTT;

[0083] SEQ ID No. 10: TTGCCTTTGTAACCATCATC.

[0084] (5) The pTRV2 empty vector, the pTRV1 empty vector, and the pTRV2-CsRBE vector in step (4) were respectively transformed into Agrobacterium GV3101 (commercially available). The experimental operation was carried out according to the steps in the instruction manual, and the plates were spread on LB solid plates containing 50 mg / L kanamycin and 25 mg / L rifampicin. The plates were cultured in the dark at 28°C for 2-3 days. The positive single clones containing the pTRV2 empty vector or pTRV2-CsRBE obtained by screening were verified by bacterial liquid PCR using pTRV2 vector primers SEQ ID No. 9 and SEQ ID No. 10. The positive single clones containing the pTRV1 empty vector obtained by screening were verified by bacterial liquid PCR using pTRV1 vector primers SEQ ID NO. 11 and SEQ ID NO. 12. Agrobacterium bacterial liquids containing the pTRV2 empty vector, the pTRV1 empty vector, and the pTRV2-CsRBE vector were obtained, respectively.

[0085] SEQ ID No.11: GCGATAGCTCTTCACAGCCT;

[0086] SEQ ID No. 12: CATGACAGGTCTCGCCACTT.

[0087] (6) Take the three correctly identified Agrobacterium cultures from step (5) and inoculate them into LB liquid culture medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and culture them overnight at 28°C and 200 rpm in the dark until the OD of the Agrobacterium culture reaches 0. 600 Both are 1.0.

[0088] (7) The Agrobacterium bacterial suspension was transferred to a 50 ml centrifuge tube and centrifuged at 5000 rpm for 8 minutes. The supernatant was discarded and the bacterial pellet was resuspended in a buffer containing 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid, and 10 mM magnesium chloride until the bacterial suspension OD was 0. 600 The value was 0.5, and then equal volumes of pTRV2-CsRBE and pTRV1 resuspensions were mixed, and equal volumes of pTRV1 and pTRV2 resuspensions were mixed (control), and allowed to stand for 3 h to obtain two infection solutions.

[0089] (8) Use a syringe needle to create a wound on the flower bud of the black orchid, soak the flower bud in the infection solution under vacuum conditions for 10 minutes, take it out and plant it back in the flowerpot, and transfer it to the greenhouse for regular cultivation and management.

[0090] (9) After 70-90 days of conventional cultivation, observe the flower phenotype. Figure 5 and Figure 6 As shown in the results, the morphology of the lip of the flowers of the silenced Orchid cymbidium flower changed from the upward curvature on both sides of the control to a flat, uncurved shape. The lip width also widened, and the gap in the middle of the lip also became larger, indicating that the CsRBE gene is involved in regulating the development of the lip of Orchid cymbidium flower.

[0091] (10) Select Figure 6 The labellum of the empty control strain and the gene silenced strain shown in the figure was used to extract RNA using an RNA extraction kit (commercially available), and the total RNA was reverse transcribed into cDNA using a reverse transcription kit according to the kit instructions. Then, RT-qPCR detection and analysis were performed according to the primers in step (2) and the method in step (3) in Example 3, as shown in FIG. Figure 7 As shown in the figure, the expression level of CsRBE gene in the labellum of silenced plants was significantly downregulated, indicating that the silencing efficiency of CsRBE in silenced plants was high.

[0092] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A CsRBE gene for regulating the morphology of the labellum of Cymbidium sinense, characterized in that: The nucleotide sequence of the CsRBE gene is shown in SEQ ID No.

1.

2. Use of the CsRBE gene according to claim 1 in regulating the morphology of the labellum of Cymbidium sinense.

3. The use according to claim 2, characterized in that The morphology of the labellum of Cymbidium orchid is regulated by silencing the CsRBE gene.

4. The use according to claim 3, characterized in that When the CsRBE gene is silenced, the lip of Cymbidium orchid becomes flat and without bending, and the width of the lip and the wrinkled gap in the middle of the lip are increased.

5. A protein encoded by the CsRBE gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

6. A method for regulating the morphology of the labellum of Cymbidium sinense, characterized in that: The following steps are involved: 1) ligating the 1-300 bp sequence of the CsRBE gene of claim 1 into the pTRV2 vector to obtain the pTRV2-CsRBE vector; 2) transforming Agrobacterium with the pTRV2-CsRBE vector obtained in step 1) and culturing the resulting bacterial solution containing the pTRV2-CsRBE vector; 3) The bacterial solution containing the pTRV2-CsRBE vector obtained in step 2) was centrifuged, and the obtained bacteria were mixed with a buffer solution to obtain a CsRBE bacterial solution. The OD 600 The value is 0.5; 4) The pTRV1 vector was transformed into Agrobacterium, and the bacterial solution containing the pTRV1 vector was obtained by culture. The bacterial cells obtained by centrifugation were mixed with the buffer solution to obtain the pTRV1 bacterial solution. The OD 600 The value is 0.5; 5) mixing equal volumes of the CsRBE bacterial solution obtained in step 3) and the pTRV1 bacterial solution obtained in step 4) to obtain an infection solution; 6) Infecting the orchid buds of Cymbidium sinense with the infection solution obtained in step 5).

7. The method according to claim 6, characterized in that Step 2) OD of the bacterial solution containing the pTRV2-CsRBE vector 600 The value is 1; the Agrobacterium is Agrobacterium GV3101.

8. The method according to claim 6, characterized in that The components of the buffer in step 3) are: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride; The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 8 minutes.

9. The method according to claim 6, characterized in that The Agrobacterium in step 4) is Agrobacterium GV3101; The OD of the bacterial solution containing the pTRV1 vector 600 The value is 1; The components of the buffer are: 100 μM acetosyringone, 10 mM morpholineethanesulfonic acid and 10 mM magnesium chloride; The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 8 minutes.

10. The method according to claim 6, characterized in that The infection conditions of step 6) include: infecting for 10 minutes under vacuum conditions, and the flower buds are flower buds with wounds.

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