CN112980809A

CN112980809A - Tobacco farnesyl pyrophosphate synthase gene and application thereof

Info

Publication number: CN112980809A
Application number: CN202110287865.0A
Authority: CN
Inventors: 曾婉俐; 李雪梅; 向海英; 高茜; 阴耕云; 蒋佳芮; 许力; 邓乐乐; 李晶; 米其利; 王晋; 陈千思
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2021-03-17
Filing date: 2021-03-17
Publication date: 2021-06-18
Anticipated expiration: 2041-03-17
Also published as: CN112980809B

Abstract

The invention relates to a tobacco farnesyl pyrophosphate synthase gene and application thereof, wherein the nucleotide sequence of the farnesyl pyrophosphate synthase gene is shown as SEQ ID No. 1. Based on the important effects of sterol on plant growth and development and on tobacco safety, the tobacco sterol regulation and control gene is deeply researched, a new tobacco variety is constructed by using genetic engineering, and a good application foundation is laid for improving the tobacco variety. In the application, through preliminary research on a specific tobacco sterol farnesyl pyrophosphate synthase NtFDPS, the high correlation with the sterol content in tobacco leaves is found, and after the gene is silenced, the sterol content in the tobacco leaves is obviously reduced. Based on the characteristic, a certain application basis and reference can be provided for the quality control of tobacco leaves and the cultivation of new tobacco varieties.

Description

Tobacco farnesyl pyrophosphate synthase gene and application thereof

Technical Field

The invention belongs to the field of tobacco gene engineering, and particularly relates to a farnesyl pyrophosphate synthase gene for tobacco sterol anabolism and application thereof.

Background

The phytosterol is an important component of a biological membrane system, can regulate and control the growth and development of plants and responds to various biotic and abiotic stresses. The sterol substance accounts for 0.1-0.3% of the tobacco leaf by weight. Because the structure of the sterol contains hydroxyl, the phenanthrene ring structure of the parent body can form carcinogenic polycyclic aromatic hydrocarbon during pyrolysis, and the sterol in tobacco is proved to be an important precursor compound of cigarette smoke benzopyrene by the literature. The sterol compounds in tobacco mainly comprise cholesterol (cholestrol), stigmasterol (stigmasterol), campesterol (campasterol), beta-sitosterol (beta-sitosterol) and the like, so that the content of sterol in mature tobacco leaves can be effectively reduced, and the content of benzopyrene in cigarette smoke can be effectively reduced.

At present, anabolism of sterol in plants has been studied, but genes for regulating sterol synthesis in tobacco cultivation are rarely reported. The research on the gene function influencing the sterol content in the tobacco provides theoretical support for the improvement of the safety of tobacco leaves and the genetic improvement of tobacco varieties, and has important significance for improving the safety of tobacco products in China.

Disclosure of Invention

The invention aims to provide a tobacco farnesyl pyrophosphate synthase gene and application thereof to improve the sterol content in tobacco, thereby laying a certain foundation for tobacco quality regulation and control and new tobacco variety cultivation.

In order to achieve the purpose of the invention, the following technical scheme is adopted in the application:

a tobacco farnesyl pyrophosphate synthase gene has a nucleotide sequence shown in SEQ ID NO.1, contains 1029 bases and is named as NtFDPS.

Furthermore, the amino acid sequence of the coding protein of the tobacco farnesyl pyrophosphate synthase gene is shown in SEQ ID NO.2 and consists of 342 amino acid residues.

Further, the tobacco farnesyl pyrophosphate synthase gene NtFDPS and the PCR amplification preparation method of the tobacco farnesyl pyrophosphate synthase gene NtFDPS comprise the following steps:

(1) extracting genome and reverse transcribing into cDNA for later use;

(2) designing a primer for PCR amplification, and carrying out PCR amplification, wherein the specific primer sequence is designed as follows:

NtFDPS-F：5’-ATGTATTTGGTACTTGCAACAAG-3’，

NtFDPS-R：5’-CACCATCACATATACCAGCC-3’。

further, in the step (1), when the genome is extracted, tobacco variety Honghuadajinyuan leaf is taken as a sample.

The application of the tobacco farnesyl pyrophosphate synthase gene NtFDPS utilizes a gene silencing technology to regulate and control the sterol content in tobacco leaves by regulating the expression level of tobacco sterol farnesyl pyrophosphate synthase.

Further, a virus-induced silencing vector, an RNAi interference vector and an overexpression vector containing the NtFDPS gene are constructed by a transgenic technology, a transient expression technology or a genome editing technology, the tobacco is transformed, and a new tobacco variety with the changed sterol content is obtained by screening.

Specific examples thereof include: the expression of NtFDPS genes is interfered and silenced by utilizing a virus-induced gene silencing (VIGS) technology, the sterol content in NtFDPS gene silencing plants is obviously reduced, and then a new plant variety with reduced sterol content is obtained.

The invention has the beneficial effects that:

based on the important effects of sterol on plant growth and development and on tobacco safety, the tobacco sterol regulation and control gene is deeply researched, a new tobacco variety is constructed by using genetic engineering, and a good application foundation is laid for improving the tobacco variety. In the application, through preliminary research on a specific tobacco sterol farnesyl pyrophosphate synthase NtFDPS, the high correlation with the sterol content in tobacco leaves is found, and after the gene is silenced, the sterol content in the tobacco leaves is obviously reduced. Based on the characteristic, a certain application basis and reference can be provided for the quality control of tobacco leaves and the cultivation of new tobacco varieties.

Drawings

FIG. 1 is a graph of the relative expression of the gene in plants with NtFDPS gene silencing compared to control plants;

FIG. 2 is a comparison of sterol content in virus-induced gene-silenced tobacco leaves and control tobacco leaves.

Detailed Description

The technical solutions of the present invention are described in detail by the following specific examples, which are only exemplary and can be used for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.

In the embodiments of the present application, those who do not specify a specific technique or condition, and those who do follow the existing techniques or conditions in the field, and those who do not specify a manufacturer or a material used, are general products that can be obtained by purchasing.

The percentage numbers are volume percentages and the ratios are volume ratios unless otherwise specified.

Biological material:

the Nicotiana benthamiana, a common tobacco material, is used for seedling cultivation in a seedling cultivation pot, seedling division is carried out two weeks after germination, and the Nicotiana benthamiana is planted in a plastic pot (10cm multiplied by 10cm) and is subjected to cultivation management such as daily fertilizer and water management under the dark condition of 16h light/8 h at the temperature of 22 ℃.

The VIGS vector used in the following examples is a viral vector derived from Tobacco Rattle Virus (TRV), specifically using TRV2 (a commonly used vector) carrying kanamycin selection marker and 35S promoter, and TRV2 carrying multiple cloning sites such as EcoR I and BamH I, which can be used to carry and transform foreign genes.

Experimental reagent:

LB liquid medium, 1L content contains: 10g bacterial peptone (bacteriological peptone); 10g sodium chloride (NaCl); 5g of yeast extract (yeast extract) and autoclaved.

YEB liquid culture medium, 1L content contains: 5g beef extract (beef extract); 5g bacterial peptone (bacteriological peptone); 5g sucrose (sucrose); 1g yeast extract (yeast extract); 2mL of 1M magnesium sulfate (MgSO4), autoclaved.

1M 2- (N-morpholine) ethanesulfonic acid (MES) stock: ddH₂Dissolving O, filtering, sterilizing, and storing at-20 deg.C.

200mM Acetosyringone (Acetosyringone, As) stock solution: dimethyl Sulfoxide (DSMO) was dissolved and stored at-20 ℃ until use.

Example 1

The construction process of the tobacco NtFDPS gene cloning and silencing vector is briefly described as follows.

(1) Cloning of tobacco NtFDPS Gene

According to the previous research on tobacco genomes and related NtFDPS genes, a specific coding sequence is selected as a target segment, and a primer sequence for PCR amplification is designed as follows:

NtFDPS-F：5’-ATGTATTTGGTACTTGCAACAAG-3’，

NtFDPS-R：5’-CACCATCACATATACCAGCC-3’。

taking cDNA of tobacco safflower gold leaf (firstly extracting genome, then reverse transcribing into cDNA) as a template, and carrying out PCR amplification to obtain NtFDPS gene;

the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 30s, and complete extension at 72 ℃ for 5min after 34 cycles;

and carrying out agarose gel electrophoresis detection on the PCR amplification product, and recovering the electrophoresis product for later use.

(2) Construction of recombinant TRV2-NtFDPS vector

Carrying out EcoRI and BamHI double enzyme digestion on the PCR amplification product in the step (1), simultaneously carrying out EcoRI and BamHI double enzyme digestion on an empty vector TRV2, respectively recovering enzyme digestion products, and utilizing T4 DNA ligase to carry out ligation.

The ligation product was transformed into E.coli competent DH 5. alpha. and after the transformation, the transformation product was spread on LB solid medium containing 50mg/L Kan and incubated overnight at 37 ℃.

And selecting positive single colonies, amplifying, and then further performing PCR identification, and ensuring that a correctly constructed recombinant vector TRV2-NtFDPS is obtained by combining sequencing verification.

Example 2

On the basis of example 1, the constructed recombinant TRV2-NtFDPS vector is further transformed into a tobacco plant by utilizing the agrobacterium-mediated VIGS technology, and verification analysis is carried out on the phenotype change condition of the related plant, and the specific experimental process is briefly described as follows.

(1) Transformation of Agrobacterium

It should be noted that, referring to the operation of example 1 and the prior art, TRV2-GFP recombinant vector was prepared as a control, and the specific transformation process was:

positive cloning plasmids of TRV2-GFP (vector control) and TRV2-NtFDPS are respectively transformed into competent cells of agrobacterium GV3101 by an electric shock transformation mode, cultured and screened by a YEB plate containing 50mg/L Kan and 50mg/L Rif, and the agrobacterium carrying the target gene is screened by colony PCR after inverted culture for 2 days at 28 ℃.

(2) Preparation of a bacterial solution for transfection

Culturing the positive Agrobacterium clones screened in step (1) in 5mL YEB liquid medium (containing 50mg/L Kan and 50mg/L Rif) at 28 ℃ and 250rpm overnight;

50uL of overnight culture was inoculated into 50mL of YEB liquidCulturing in culture medium (containing 50mg/L Kan) to OD₆₀₀Centrifuging at 4000g for 5min, collecting thallus, resuspending with MMA, and adjusting OD₆₀₀About 1.0;

finally, the mixture is placed at room temperature for about 3 hours and then used as a bacterial liquid for transfection.

(3) Transient transformation

And (3) taking 3-4w (week) of seedling-age tobacco leaves as an experimental material, injecting the bacterial liquid for transfection prepared in the step (2) into the tobacco leaves by using a 1 mL-specification injector, continuously culturing the injected tobacco in an artificial incubator, and observing the phenotypic change.

Further, the expression condition of the NtFDPS gene is detected through qRT-PCR, and the result is shown in figure 1, so that the expression quantity of the NtFDPS is remarkably reduced in the infected plant of TRV2-NtFDPS, and the qRT-PCR primers are as follows:

NtFDPS-F：5’-CTCCTACTCTGAAAGAAACGAACA-3’，

NtFDPS-R：5’-CGAGCCTCATCAGTAAACTCG-3’。

further, the sterol content in leaf was measured in the experimental group (TRV 2-NtFDPS-impregnated plants) and the control group (TRV 2-GFP-impregnated plants) (the measurement method was referred to "metabonomics analysis procedure of fresh tobacco leaves based on combined use of gas chromatography and liquid chromatography-mass spectrometry" (zhengqingxia et al, tobacco science and technology, 2019)), and the results are shown in fig. 2.

As can be seen from the results of fig. 2, the sterol content in the experimental group was significantly reduced compared to the control group, wherein the cholesterol (cholestrol), stigmasterol (stigmasterol) and β -sitosterol (β -sitosterol) content were significantly reduced compared to the control group. The further indication shows that the silencing of the NtFDPS gene can regulate and control the content of the phytosterol in the tobacco leaves, and further, a certain technical basis can be laid for the regulation and control of the tobacco leaf quality and the cultivation of new tobacco varieties.

Through a transgenic technology, a transient expression technology or a genome editing technology, a virus-induced silencing vector, an RNAi interference vector, a super-expression vector or a genome editing vector containing the NtFDPS gene is constructed, tobacco is transformed, and a new tobacco variety with the changed sterol content in the tobacco leaves is obtained through screening.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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cttatcgaaa tgggaatcta tttccaagtt caggatgatt acttggactg ctttgctgac 720

tccgaggtgc tgggtaagat tggcacggat attcaagatt tcaagtgctc ttggttggta 780

gtgaaagcct tagaatactg caatgaggag caaaagaaat tattatatga gaactatgga 840

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Gln Trp Val Glu Arg Met Leu Asp Tyr Asn Val Pro Gly Gly Lys Leu

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Asn Arg Gly Ile Ser Val Ile Asp Ser Tyr Ser Leu Leu Lys Glu Gly

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Lys Glu Leu Thr Asn Glu Glu Ile Phe Gln Thr Ser Ser Leu Gly Trp

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Cys Ile Glu Trp Leu Gln Ala Tyr Phe Leu Val Leu Asp Asp Ile Met

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Asp Gly Ser His Thr Arg Arg Gly Gln Pro Cys Trp Phe Arg Leu Pro

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Lys Val Gly Met Ile Ala Ala Asn Asp Gly Ile Leu Leu Arg Asn His

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Ile Pro Arg Ile Leu Lys Asn His Phe Arg Gly Lys Pro Tyr Tyr Val

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Asp Leu Leu Glu Leu Phe Asn Glu Val Glu Phe Gln Thr Ala Ser Gly

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Gln Met Ile Asp Leu Ile Thr Thr Leu Val Gly Glu Lys Asp Leu Ser

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Lys Tyr Leu Leu Pro Ile His Arg Arg Ile Val Gln Tyr Lys Thr Ala

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Tyr Tyr Ser Phe Tyr Leu Pro Val Ala Cys Ala Leu Leu Met Ala Gly

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Glu Ser Leu Asp Asn His Val Asn Val Lys Asn Ile Leu Ile Glu Met

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Gly Ile Tyr Phe Gln Val Gln Asp Asp Tyr Leu Asp Cys Phe Ala Asp

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Ser Glu Val Leu Gly Lys Ile Gly Thr Asp Ile Gln Asp Phe Lys Cys

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Ser Trp Leu Val Val Lys Ala Leu Glu Tyr Cys Asn Glu Glu Gln Lys

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Lys Leu Leu Tyr Glu Asn Tyr Gly Lys Asp Asp Pro Ala Cys Val Ala

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Lys Val Lys Ala Leu Tyr Asn Asp Leu Lys Leu Glu Asp Val Tyr Leu

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Glu Tyr Glu Lys Thr Thr Tyr Glu Lys Leu Ile Asn Ser Ile Glu Ala

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Gln Pro Ser Lys Ala Val Gln Ala Val Leu Lys Ser Phe Leu Ala Lys

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Ile Tyr Lys Arg Gln Lys

340

Claims

1. A tobacco farnesyl pyrophosphate synthase gene is characterized in that a nucleotide sequence is shown in SEQ ID NO. 1.

2. The tobacco farnesyl pyrophosphate synthase gene according to claim 1, characterized in that the coding protein of the tobacco farnesyl pyrophosphate synthase gene has the amino acid sequence shown in SEQ ID No. 2.

3. The tobacco farnesyl pyrophosphate synthase gene according to claim 1 or 2, wherein the method for preparing tobacco farnesyl pyrophosphate synthase gene NtFDPS by PCR amplification comprises the following steps:

(1) extracting genome and reverse transcribing into cDNA for later use;

NtFDPS-F：5’-ATGTATTTGGTACTTGCAACAAG-3’，

NtFDPS-R：5’-CACCATCACATATACCAGCC-3’。

4. the tobacco farnesyl pyrophosphate synthase gene according to claim 3, wherein, in the step (1), a tobacco variety Honghuadajinyuan leaf is used as a sample in extracting a genome.

5. The application of the tobacco farnesyl pyrophosphate synthase gene is characterized in that by using the tobacco farnesyl pyrophosphate synthase gene of any one of the genes, the expressed protein of the gene is related to the sterol content in plant leaves, and after the expression of the protein is reduced, the sterol content in the leaves is obviously reduced.

6. The use of the tobacco farnesyl pyrophosphate synthase gene according to claim 5, wherein the sterol content in tobacco leaves is regulated and controlled by regulating the expression level of the tobacco farnesyl pyrophosphate synthase gene by using a gene silencing technique or a gene overexpression method.

7. The use of the tobacco farnesyl pyrophosphate synthase gene according to claim 5, wherein a viral-induced silencing vector, an RNAi interference vector, an overexpression vector or a genome editing vector containing the tobacco farnesyl pyrophosphate synthase gene is constructed by a transgenic technique, a transient expression technique or a genome editing technique, tobacco is transformed, and a new variety of tobacco with a varied sterol content is screened.