Key gene NtOS for controlling ocimene biosynthesis in tobacco and application of key gene NtOS

Abstract

The invention discloses a key gene NtOS for controlling ocimene biosynthesis in tobacco and application of the key gene NtOS. The nucleotide sequence of the key gene NtOS for controlling ocimene biosynthesis in tobacco comprises a sequence as shown in SEQ ID No. 1, and the coded amino acid sequence comprises a sequence as shown in SEQ ID No. 2. The NtOS gene is transformed into tobacco through amplification, enzyme digestion, expression vector construction and agrobacterium infection, the ocimene release amount of an obtained NtOS gene transformed tobacco plant is remarkably increased, it is proved that the NtOS gene is a key gene for controlling ocimene biosynthesis, the function identification of the tobacco NtOS gene enriches the gene resources of tobacco quality breeding, and the tobacco NtOS gene has a wide application prospect. The method has important theoretical significance and application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to NtOS, a key gene controlling ocimene biosynthesis in tobacco, and its application. Background Technology

[0002] Tobacco (Nicotiana tabacum L.) is an annual herbaceous plant belonging to the Solanaceae family and the Nicotiana genus. The aroma components and their content in tobacco leaves have a significant impact on the quality of tobacco products. Previous studies have extensively investigated the composition and content of volatile substances in fresh and cured tobacco leaves, identifying over 700 secondary metabolites that influence tobacco quality. Existing research indicates that terpenes and their derivatives are major components of the volatile aroma compounds in tobacco. Terpenes are compounds and their derivatives formed using isoprene units (C5 units) as their basic structural unit. Based on the number of isoprene units they contain, terpenes can be classified into different types, including hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes.

[0003] Ocimene, a monoterpene compound, possesses a unique green leafy and fruity aroma and is a core ingredient in high-end flavorings and tobacco fragrances. Global annual demand exceeds 800 tons, and it is widely used in food additives, daily chemical products, and pharmaceutical excipients. Currently, ocimene synthase genes have been isolated and identified from species such as basil, citrus, tea, and mint. Some genes have been successfully expressed transgenic in model plants, increasing ocimene yield.

[0004] However, research on terpene synthases in tobacco remains insufficient. Existing literature shows that the number of identified terpene synthesis genes in tobacco is limited, and the understanding of the ocimene biosynthesis pathway is relatively vague. This limits the possibility of precisely regulating tobacco aroma through molecular breeding. This invention is the first to discover a key gene in tobacco controlling ocimene biosynthesis, namely the tobacco NtROS gene, providing genetic resources and a theoretical basis for further in-depth exploration of the molecular mechanisms of volatile aroma compound formation in tobacco, as well as for tobacco quality breeding.

[0005] Therefore, there is an urgent need to provide a key gene for promoting ocimene biosynthesis in tobacco. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a key gene, NtOS, that controls ocimene biosynthesis in tobacco and its application. Transgenic lines obtained by transforming tobacco with the NtOS gene show a significant increase in ocimene release, demonstrating that this gene can promote ocimene biosynthesis in tobacco.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a key gene NtOS in tobacco that promotes ocimene biosynthesis, the nucleic acid sequence of the key gene NtOS including the sequence shown in SEQ ID No. 1.

[0009] In this invention, transgenic lines obtained by transforming tobacco with the NtOS gene showed a significant increase in ocimene release, demonstrating that the gene can promote the biosynthesis of ocimene in tobacco. The functional identification of the tobacco NtOS gene enriches the genetic resources for tobacco quality breeding and has significant theoretical and practical value.

[0010] Preferably, the amino acid sequence of the protein encoded by the key gene NtOS includes the sequence shown in SEQ ID No. 2.

[0011] SEQ ID No. 1:

[0012]

[0013] SEQ ID No. 2:

[0014] MKGLHNYSCIFSTSIIPILCPNIVSLFPTMQTNKLHSTIKKQSLPERKICTYKPNIWKYDHLLTLTSEYSEEKYKVEAEKLKEEVSCTFSNSTISPLDLLELIDSIDKLGLSCYFEVETKEALEK IIMSVKTSSSSKEENLYATALCFRLLREHGHHASQDMLKDFFDGKGKLKVSDVKTLLEVLEGSYLSMEGENNLLDDTRLFTTKNLKSLVSKSDYTFTNKEYNLSNSLSFPLAWRVKWYDVRKHICAQ ELECNNTNPMLLKLAKLNFNIVQATHQKDLKHLLRWWRNLSIVEDLSFTRDRIVESFFCAVGVAPEPRHGNMRKWLTKVIELVLIIDDVYDIYGSLAQVQQFTRAIEKWDPNEVEGLPECMQICFR SLHDTVKEISVEIQQQKGGLSALPYLKQVAELERGDVASSILCYMQQENVSEDVAREHIKSIILDSWKKINYHFNTVSTSHRKLVKHVVNEAQMAHVMYQFGDGFGVQDGETRDQVLFNLVHPIT.

[0015] Secondly, the present invention provides the application of NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in the first aspect, in regulating ocimene biosynthesis in plants.

[0016] Thirdly, the present invention provides a method for regulating ocimene biosynthesis in plants, the method comprising the following steps:

[0017] (1) Amplify NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in the first aspect;

[0018] (2) The key gene NtOS in tobacco that promotes ocimene biosynthesis described in the first aspect was cloned, and after enzyme digestion and purification, it was ligated into an expression vector to obtain an overexpression recombinant vector;

[0019] (3) Transform the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0020] (4) The overexpressing recombinant strain was transformed into plants, and NtOS transgenic lines were screened and obtained to regulate the biosynthesis of ocimene in plants.

[0021] Preferably, the nucleic acid sequence of the primers used for amplification in step (1) includes the sequences shown in SEQ ID No. 3-SEQ ID No. 4.

[0022] SEQ ID No. 3: GGATCCATGAAAGGTCTCCACAATTATTCCTG.

[0023] SEQ ID No. 4: GAGCCTTATGTAATAGGATGAACCAAGTTGA.

[0024] Preferably, the amplification reaction conditions in step (1) are as follows: (1) Pre-denaturation: 93-98℃ (e.g., 93℃, 95℃ or 98℃), 20-50 s (e.g., 20 s, 40 s or 50 s); (2) Denaturation: 93-98℃ (e.g., 93℃, 95℃ or 98℃), 5-15 s (e.g., 5 s, 10 s or 15 s); (3) Annealing extension: 52-60℃ (e.g., 52℃, 55℃ or 60℃), 5-15 s (e.g., 5 s, 10 s or 15 s); 70-75℃ (e.g., 70℃, 72℃ or 75℃), 30-60 s (e.g., 30 s, 50 s or 60 s). (3) s); 25-35 cycles (e.g., 25 cycles, 30 cycles or 35 cycles); (4) extension: 70-75°C (e.g., 70°C, 72°C or 75°C), 5-15 min (e.g., 5 min, 10 min or 15 min).

[0025] Preferably, the expression vector in step (2) includes the pCambia1300 vector, which includes a 35S promoter and a NOS terminator.

[0026] Preferably, the plant includes tobacco.

[0027] Preferably, the transformation in step (3) includes an instantaneous transformation method.

[0028] Fourthly, the present invention provides the application of NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in the first aspect, in the breeding of new varieties of tobacco with high aroma.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention screened a gene, NtOS, related to ocimene biosynthesis in tobacco through transcriptome sequencing analysis. After cloning, constructing an overexpression vector, and overexpressing the gene in a recombinant strain, the gene was transformed into tobacco to obtain transgenic tobacco plants NtOS-OE. The release of ocimene in these plants was significantly increased compared to the wild type, proving that the NtOS gene is a key gene controlling ocimene biosynthesis. The functional identification of the tobacco NtOS gene enriches the key breeding gene resources for tobacco quality breeding and has important theoretical significance and application value. Attached Figure Description

[0031] Figure 1 This is a graph showing the gene expression levels of NtOS transgenic tobacco lines, where OE2 and OE5 are transgenic tobacco lines and WT is wild-type tobacco without transgenesis.

[0032] Figure 2 The diagram shows the results of NtOS gene controlling ocimene biosynthesis, where OE2 and OE5 are transgenic tobacco lines, and WT is non-transgenic wild-type tobacco. Detailed Implementation

[0033] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Example 1

[0036] This embodiment describes the cloning of NtOS, a key gene for tobacco-controlled ocimene biosynthesis.

[0037] 1. Using the coding region sequence of the tobacco NtOS gene as a reference, specific primers NtOS-F and NtOS-R were designed. The primer sequences are as follows:

[0038] NtOS-F (SEQ ID No. 3): GGATCCATGAAAGGTCTCCACAATTATTCCTG.

[0039] NtOS-R (SEQ ID No. 4):GAGCCTTATGTAATAGGATGAACCAAGTTGA.

[0040] 2. Extract mRNA from seedling leaves of tobacco cultivar "K326", reverse transcribe it into cDNA, and use it as a template for PCR amplification. Perform PCR amplification using the primers from step 1 to obtain the amplified fragment of the NtOS gene.

[0041] The PCR amplification system was: 31 μL ddH2O, containing Mg. 2+ 10 μL of 5×HF buffer; 2 μL of 2.5 mM dNTPs; 2 μL each of NtOS-F and NtOS-R (5 μM each); 0.6 μL of DMSO; 0.5 μL of DNA polymerase; and 2 μL of cDNA template.

[0042] The PCR reaction conditions were: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ for 50 s, for 30 cycles; extension at 72℃ for 10 min.

[0043] The nucleic acid sequence of the NtOS gene was obtained by sequencing the PCR amplification product, as shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 2.

[0044] Example 2

[0045] This embodiment constructs an overexpression vector for NtOS.

[0046] The PCR amplification fragment obtained in Example 1 was ligated into the cloning vector pEASY-Blunt simple. After correct sequencing, the plasmid was extracted, digested with BamHI and SacI, purified, and ligated into the pCambia1300 vector (containing a 35S promoter and a NOS terminator) digested with BamHI and SacI. After correct sequencing, Agrobacterium GV3101 was transformed using the freeze-thaw method, and tobacco was genetically transformed using the infiltration method to obtain the transgenic plant NtOS-OE.

[0047] Example 3

[0048] This embodiment analyzes and identifies the key functions of NtOS in controlling ocimene biosynthesis.

[0049] The T3 generation seeds of the transgenic plant NtOS-OE obtained in Example 2 were sterilized and grown on normal MS solid medium for one week. Then, tobacco seedlings with similar growth were selected and transferred to new MS solid medium for one week. After that, they were transplanted into flower pots, and the key role of the ocimene biosynthesis gene was identified at the tobacco plant maturity stage. Wild-type tobacco (WT) was used as a control.

[0050] The expression level of the NtOS gene in transgenic plants was analyzed using quantitative real-time PCR.

[0051] The primer sequences for real-time PCR are as follows:

[0052] NtOS-qF (SEQ ID No. 5): CTTCAGGCTTCTCAGGGAACAT.

[0053] NtOS-qR (SEQ ID No. 6): TGTTGTGAACAATCGTGTGTCG.

[0054] The results are as follows Figure 1 As shown, the expression level of NtOS gene in transgenic plants increased significantly, by 10-20 times.

[0055] Volatile substances from wild-type tobacco (WT) and transgenic tobacco (NtOS-OE2 and NtOS-OE5) were collected using headspace extraction. Perforated Erlenmeyer flasks (pre-treated with silanization to reduce adsorption of volatile substances by active sites) were used as sampling devices. Static headspace extraction was employed to collect secondary metabolites from the tobacco plants, with three biological replicates for each material. The procedure was as follows: The extraction head was pre-aged at 250°C for 15 min at the gas chromatograph inlet. Two to three terminal or lateral buds from healthy, uniformly growing tobacco plants free from pests, diseases, and physical damage were selected. The Erlenmeyer flask was inverted over the buds, ensuring the tobacco leaves were completely inside. The extraction head was inserted into the flask through the small hole at the top, and the handle was pushed to extend the fiber tip from the syringe. Headspace extraction was performed for 1 h. After extraction, the fiber tip was retrieved, and the samples were analyzed by GC-MS. The results are shown in Table 1.

[0056] Table 1

[0057]

[0058] In this context, ND means that the substance was not detected.

[0059] The release of ocimene from transgenic tobacco plants NtOS-OE2 and NtOS-OE5 was significantly increased compared to wild-type tobacco (WT), indicating that the biosynthetic capacity of ocimene in transgenic tobacco is significantly better than that in wild-type tobacco. This means that the NtOS gene can control the biosynthesis of ocimene in transgenic tobacco and significantly improve the biosynthetic capacity of ocimene.

[0060] In summary, this invention amplifies, digests, constructs an expression vector, and transfers the NtOS gene into tobacco through Agrobacterium infection. The resulting NtOS-transgenic tobacco plants exhibit significantly increased ocimene release, demonstrating that the NtOS gene is a key gene controlling ocimene biosynthesis. The functional identification of the tobacco NtOS gene enriches the gene resources for tobacco quality breeding and has significant theoretical and practical value.

[0061] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A key gene NtOS in tobacco that promotes ocimene biosynthesis, characterized in that, The nucleic acid sequence of the key gene NtOS includes the sequence shown in SEQ ID No.

1.

2. The key gene NtOS in tobacco that promotes ocimene biosynthesis according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the key gene NtOS includes the sequence shown in SEQ ID No.

2.

3. The application of NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in claim 1 or 2, in regulating ocimene biosynthesis in plants.

4. A method for regulating ocimene biosynthesis in plants, characterized in that, The method includes the following steps: (1) Amplify NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in claim 1 or 2; (2) Cloning the key gene NtOS in tobacco that promotes ocimene biosynthesis as described in claim 1 or 2, and ligating it into an expression vector after enzyme digestion and purification to obtain an overexpression recombinant vector; (3) Transform the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) The overexpressing recombinant strain was transformed into plants, and NtOS transgenic lines were screened and obtained to regulate the biosynthesis of ocimene in plants.

5. The method according to claim 4, characterized in that, The nucleic acid sequences of the primers used for amplification in step (1) include those shown in SEQ ID No. 3-SEQ ID No.

4.

6. The method according to claim 4 or 5, characterized in that, The amplification reaction conditions described in step (1) are as follows: (1) Pre-denaturation: 93-98℃, 20-50 s; (2) Denaturation: 93-98℃, 5-15 s; (3) Annealing extension: 52-60℃, 5-15 s; 70-75℃, 30-60 s; 25-35 cycles; (4) Extension: 70-75℃, 5-15 min.

7. The method according to any one of claims 4-6, characterized in that, The expression vector mentioned in step (2) includes the pCambia1300 vector, which includes a 35S promoter and a NOS terminator.

8. The method according to any one of claims 4-7, characterized in that, The plant mentioned includes tobacco.

9. The method according to any one of claims 4-8, characterized in that, The transformation described in step (3) includes the instantaneous transformation method.

10. The application of NtOS, a key gene in tobacco that promotes ocimene biosynthesis as described in claim 1 or 2, in the breeding of new varieties of tobacco with high aroma.

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