Chrysanthemum indicum aroma regulation gene CiCPS and application thereof

Abstract

The invention discloses an aroma regulation gene CiCPS of dendranthema indicum and application of the aroma regulation gene CiCPS, an overexpression vector for constructing the CiCPS gene is overexpressed in tobacco through an agrobacterium-mediated method, synthesis of tobacco terpenoids and partial aliphatic compounds can be obviously increased, and the aroma of the tobacco is increased. The CiCPS gene can be applied to genetic engineering genetic breeding of plants and cultivation of chrysanthemum varieties rich in fragrance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a chrysanthemum aroma regulating gene CiCPS and its application. Background Technology

[0002] Chrysanthemum indicum var. aromaticum is a variety of Chrysanthemum in the Asteraceae family. It has golden flowers and a strong fragrance. Extracts from its flowers, leaves, and stems are high-quality materials for making perfumes.

[0003] Currently, little is known about the regulatory mechanisms of its typical aromatic components, limiting its development into high-value varieties. This experiment will utilize previously obtained transcriptome data from our research group to clone the CiCPS gene from the leaves of Shennong Xiangju and perform functional verification, contributing to the study of the molecular breeding and metabolic processes of Shennong Xiangju. This will provide theoretical support for better exploring the aromatic application potential of Shennong Xiangju and cultivating new chrysanthemum varieties with aromatic characteristics. Summary of the Invention

[0004] The purpose of this invention is to provide a CiCPS gene of Shennong Xiangju that can promote the synthesis of aroma components and its application, which plays a certain role in the cultivation of new aromatic chrysanthemum varieties.

[0005] This invention utilizes transcriptome analysis of *Chrysanthemum indicum* to screen for the CiCPS gene, which has a full-length reading frame of 1647 bp encoding 548 amino acids. A plant overexpression vector, pBI121-CiCPS-GFP, was constructed. The pBI121-CiCPS-GFP recombinant plasmid was transformed into *Agrobacterium* GV3103 and then into tobacco using the *Agrobacterium*-mediated transformation method. PCR confirmed that the target gene was successfully inserted into the tobacco genome. High-expression lines in the T1 generation of tobacco were screened using qRT-PCR, and the accumulation of volatile metabolites in CiCPS-overexpressing plants was detected. The CiCPS gene promotes the synthesis of terpenes and some aliphatic volatile substances in tobacco, increasing its aroma.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides the application of the above-mentioned gene in enhancing aroma.

[0008] The benefits of this invention are as follows:

[0009] This invention successfully cloned the CiCPS gene of Shennong Xiangju, further demonstrating that the CiCPS gene can enhance the aroma of tobacco and Shennong Xiangju.

[0010] This invention not only enriches the theory of aroma regulation of Shennong chrysanthemum, but can also be used to guide the targeted breeding of aromatic plants. Attached Figure Description

[0011] Appendix Figure 1 This is an agarose gel electrophoresis image of the CiCPS gene clone; where M is the 2000bp marker, and 1 and 2 are the PCR results of the CiCPS gene.

[0012] Appendix Figure 2 This is a phenotypic diagram of wild-type and CiCPS gene-overexpressing tobacco.

[0013] Appendix Figure 3 This is a diagram of volatile metabolites from tobacco of wild type and overexpressing CiCPS gene.

[0014] Appendix Figure 4 This is a map showing the density of tobacco glandular trichomes in wild-type and overexpressing CiCPS genes. Detailed Implementation

[0015] The following examples are provided to further describe the technical solution of the present invention in detail, but are not intended to limit the scope of protection of the present invention.

[0016] Example 1: Construction of an overexpression vector for the CiCPS gene of Shennong Xiangju.

[0017] The overexpression vector used in this invention is pBI121-GFP, into which a CiCPS gene fragment is inserted, resulting in the pBI121-CiCPS-GFP plant overexpression vector. The construction steps of the CiCPS overexpression vector are as follows: Using cDNA from Shennong Xiangju leaves as a template, upstream primers 5'-ATGTCTGCTAAAGAAGAGAAAG-3' and downstream primers 5'-TCATATAGGTATAGGATGAACGAGCCAC-3' were designed based on the CiCPS sequence. The target fragment was obtained after PCR amplification using Sangon Biotech's Dipfu Hotstart DNA Pdymerase (Figure 1). The PCR program annealing temperature was 55 ℃. Detailed instructions for the Dipfu Hotstart DNA Pdymerase were provided in the user manual. The cloning vector used was the pEASY®-Blunt Cloning Kit from TransGen Biotech. 0.8 µL of purified PCR fragment gel-recovered product was mixed with 1 µL of the pEASY®-Blunt Cloning Kit vector and ligated at 25 °C for 10 min. The ligation product was then transformed into competent E. coli DH5α cells at 37 °C.

[0018] Construction of the plant overexpression vector pBI121-CiCPS-GFP; analysis and design of homologous arm sequences for seamless cloning of the pBI121-GFP vector; and design of upstream homologous arm primers based on the correctly cloned CiCPS sequence: 5'- CCCGGGATGTCTGCTAAAGAAGAGAAAG ATG TCTGCTAAAGAAGAGAAAG -3', Downstream homologous arm primer: 5'- ACTAGTTATAGGTATAGGATGAACGAGC T CATATAGGTATAGGATGAACGAGCCAC- 3', the above primers retain the SmaI / SpeI restriction sites of pBI121-GFP.

[0019] Using the correctly cloned plasmid identified by CiCPS sequencing of *Chrysanthemum indicum* as a template, PCR amplification with Dipfu Hotstart DNAPdymerase yielded the target gene fragment with added homologous arms of the pBI121-GFP vector. Double digestion with Thermo Scientific restriction endonucleases SmaI and SpeI yielded the pBI121-CiCPS-GFP vector. The pBI121-CiCPS-GFP plant overexpression vector was constructed, ligated at 25 ℃ for 10 min using a PCR instrument, and transformed into competent *E. coli* DH5α cells. Single clones were selected for colony PCR and sequencing verification. The upstream verification primer was 5'-TTCATTTCATTTGGAGAGAACAC-3', and the downstream verification primer was 5'-TTGCCAAATG TTTGAACGATC-3', ultimately obtaining the correct plant overexpression vector pBI121-CiCPS-GFP. The recombinant plasmid pBI 121-CiCPS-GFP was transformed into Agrobacterium GV3103 competent cells using a freeze-transfer method.

[0020] SEQ ID NO:1

[0021] MSAKEEKVIRPTVHFPPSVWADQFLIFDNEQAKQANVEQVVNELREDVRKDLMSYLDVQAEHTNLLKLIDAIQRLSIAYHFEEEIEQALQHIYDTYGDDWKGKSPSLWFRILRQQGFYVSCDIFKNYKEEDGSFKESLTN DVEGLLELYEATYLRLQGEGILDDALVFTRTCLEKIAKDLVHSNPTLSTRIQEALKQPLHKRLTRLEALRY IPMYEQLASHNESLLKLAKLGFNLLQSLHRKELSEVSRWWKGLDVPNNLPYARDRMVECYFWALGVYF

[0022] EPKYSRARIFLAKVISLATVLDDTYDAYGTYEELKIFTEAIQRWSITCIDMLPEYMKLLYQGVLDIYKEME EIMGKGGKAHHLSYAKESMKEFIRSYMMEAKWANEGYVPTAEEHMSVAFVSSGYSMLATTCFVGMGD IVTDEAFKWAMTNPPIVKASCAIARLMDDIHSQKEEKERIHVASSVESYMKQYDVTEEHVHKVFHKKIE DAWKDITRESLACKNIMPLMMRVINLARVMDVLYKHKDGFTNVGEELKDHIKSLLVHPIPI-

[0023] Example 2: Genetic transformation of tobacco using the CiCPS gene of Shennong Xiangju.

[0024] The Agrobacterium containing the pBI121-CiCPS-GFP plant overexpression vector obtained in Example 1 was used to genetically transform tobacco using the leaf disc method. The specific method is as follows:

[0025] (1) Take out Agrobacterium GV3103 with recombinant plasmid from the -80 ℃ freezer, activate it, and culture it in the dark in the 28 ℃ incubator for 36 ~ 48 h. Pick single clones for PCR identification, and then expand the culture of the correctly identified bacterial solution in YEP liquid medium.

[0026] (2) After shaking Agrobacterium to OD600 to reach 0.6~0.8, it is used for infection.

[0027] (3) Select healthy, sterile tobacco seedlings, cut the tender leaves into 1 cm2 square leaves, and then place them on MS solid medium (MS medium + 0.5 mg / L 6-BA + 0.05 mg / L NAA) and incubate in the dark for 2 days.

[0028] (4) Collect the bacteria from the shaken bacterial solution, resuspend the bacterial cells in sterile water to make the bacterial solution OD600 about 0.3~0.4. Then soak the pre-cultured tobacco leaves in the resuspension and shake continuously for 5 minutes. After infection, place them on filter paper to dry, and then put them back on the culture medium for co-culture for 2~3 days.

[0029] (5) Place the leaves on the screening medium and culture them under light. Change the medium every 15 days. The medium composition is MS medium + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 50 mg / L Kana + 200 mg / L Tim.

[0030] (6) When adventitious buds differentiate from the leaves, cut off the adventitious buds and transfer them to MS solid medium (MS medium + 0.05 mg / L NAA + 50 mg / L Kana + 200 mg / L Tim) for rooting culture.

[0031] (7) The rooted tobacco seedlings were transferred to nutrient soil for further cultivation, and the leaves were cut off to extract DNA for PCR verification. The positive tobacco seedlings were further cultivated until the seeds were harvested, and the next generation of positive plants were screened.

[0032] Example 3: morphological observation and volatile component analysis of CiCPS overexpressing tobacco and control tobacco.

[0033] The harvested tobacco seeds were disinfected and sown on 1 / 2 MS + 50 mg / L Kana selection medium. The disinfection method was as follows: disinfect with 75% alcohol for 1 min, then rinse 3 times with sterile water, then disinfect with 2% sodium hypochlorite solution with shaking for 10 min, and finally rinse 5 times with sterile water. After vernalization at 4 ℃ for 2 days, the seeds were cultured normally in the tissue culture room. When the tobacco plants had grown four leaves and were slightly larger, they were transferred to nutrient soil (nutrient soil: vermiculite: perlite = 3:1:1) for cultivation. The light environment was light / dark 14 h / 10 h, and the plants were watered once a week. The morphology of the tobacco plants was observed. The volatile components of CiCPS-overexpressing tobacco and control tobacco were analyzed using gas chromatography (GC) with an SPME extraction head (DVB / CAR / PDMS, 50 / 30 μm), 20 mL headspace vials. GC operating conditions were as follows: a 50 μm C18 column (60 m × 0.25 mm × 0.25 μm), initial temperature 45℃, held for 2 min, then ramped to 200℃ at a rate of 5℃ / min, held for 2 min, and finally ramped to 250℃ at a rate of 15℃ / min, held for 5 min. MS operating conditions were: EI source ionization; electron energy 70 eV; voltage multiplier tube 1.2 kV; ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 280℃; scan quality range 30–450 m / z. Before sample loading, 1 g of tobacco leaves were ground into powder in liquid nitrogen, and 40 μL of 2-octanol (500 mg·L⁻¹) was added as an internal standard. The secreted substances were then subjected to headspace sampling for 40 min under constant temperature water bath conditions at 80 ℃.

[0034] Extraction. SPME needles were aged for 20 minutes before each use to remove residual impurities from the previous experiment and reduce error. All experiments were performed in triplicate. Results showed that compared to wild-type WT and empty-vectored EV, transgenic tobacco CiCPS-1, CiCPS-2, and CiCPS-3 plants were taller, had smaller leaf area, increased leaf length, smaller leaf width, and a darker green leaf color; stems were thinner, and plant morphology was slightly tilted and curved; flowering was earlier. Overexpression of the CiCPS gene in tobacco significantly altered the density and secretion content of glandular trichomes in tobacco leaves. The number of long-stalked and short-stalked glandular trichomes detected in different parts of the upper and lower epidermis of CiCPS lines was significantly higher than that in wild-type WT and empty-vectored EV. GC-MS was used to detect volatile substances in tobacco leaves. The results showed that the main volatile substances in transgenic tobacco leaves were terpenes, alcohols, esters, and hydrocarbons. The types and contents of leaf secretions varied among different strains. Compared with WT and EV strains, the contents of terpenes, ketones, aldehydes, and esters in transgenic tobacco leaves increased, and phenolic substances (3-methoxyphenol, phenol) were unique to transgenic tobacco. Furthermore, the contents of alcohols, hydrocarbons, and ethers decreased in CiCPS transgenic strains (Figure 4).

[0035] The description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A CiCPS gene, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The CiCPS gene according to claim 1, characterized in that, An overexpression vector for the CiCPS gene of *Chrysanthemum indicum* was constructed and transformed into the recipient plant, tobacco, through stable genetic transformation.

3. The application according to claim 2, wherein the Shennong Xiangju aroma regulating gene CiCPS increases tobacco terpenes and some aliphatic volatile substances, characterized in that... The CiCPS gene was overexpressed in recipient plants.

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