CN113201551A - Tobacco delta 7-sterol C5(6) -desaturase gene and application thereof - Google Patents

Abstract

The invention relates to a tobacco delta 7-sterol C5(6) -desaturase gene and application thereof, wherein the nucleotide sequence of the delta 7-sterol C5(6) -desaturase gene is shown as SEQ ID NO. 1. In the application, through preliminary study on specific tobacco sterol delta 7-sterol C5(6) -desaturase gene, the gene is found to be highly related to the sterol content in tobacco leaves, the gene is silenced in Nicotiana benthamiana, the cholesterol, stigmasterol, campesterol and beta-sitosterol content in the tobacco leaves are obviously reduced, and the total sterol content is reduced by 46.3%. Based on the characteristic, a certain application basis and reference are provided for cultivating new tobacco varieties by utilizing genetic engineering and improving the quality of tobacco leaves.

Description

Tobacco delta 7-sterol C5(6) -desaturase gene and application thereof

Technical Field

The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco delta 7-sterol C5(6) -desaturase gene and application thereof.

Background

The phytosterol is an important component of a biological membrane system, and can regulate and control the growth and development of plants. The sterol in the tobacco mainly exists in cell membranes, accounts for 0.1-0.3% of the mass of the tobacco leaves, can promote the growth of the tobacco and has great influence on the safety and the quality of the tobacco. As the structure of the sterol contains hydroxyl, the phenanthrene ring structure of the parent body can form polycyclic aromatic hydrocarbon during pyrolysis, and the sterol in tobacco is reported in documents to be an important precursor compound of cigarette smoke benzopyrene. 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 delta 7-sterol C5(6) -desaturase gene and application thereof, so as to improve sterol content in tobacco, thereby laying a certain foundation for tobacco quality regulation 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 delta 7-sterol C5(6) -desaturase gene has a nucleotide sequence shown in SEQ ID NO.1, contains 816 basic groups and is named as NtSC5 DL.

Furthermore, the amino acid sequence of the encoding protein of the tobacco delta 7-sterol C5(6) -desaturase gene is shown in SEQ ID NO.2 and consists of 271 amino acid residues.

Further, the preparation method of the tobacco delta 7-sterol C5(6) -desaturase gene by PCR amplification comprises 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:

NtSC5DL-F：5’-CATTCCTGCCGGAATCATG-3’，

NtSC5DL-R：5’-ATGTACTCAGAAAGTGATGGAAGG-3’。

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

The application of the tobacco delta 7-sterol C5(6) -desaturase gene in any one of the above plants utilizes a gene silencing technology to regulate and control the sterol content in tobacco leaves by regulating the expression level of the tobacco sterol delta 7-sterol C5(6) -desaturase gene.

Further, a virus-induced silencing vector, an RNAi interference vector and a super-expression vector containing the tobacco sterol delta 7-sterol C5(6) -desaturase gene are constructed by a transgenic technology, a transient expression technology or a genome editing technology, tobacco is transformed, and a new tobacco variety with the content of sterol changed is obtained by screening.

Specific examples thereof include: by using a virus-induced gene silencing (VIGS) technology, the expression of the NtSC5DL gene is interfered to silence, the sterol content in the NtSC5DL gene silenced plant is obviously reduced, and 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 this application, preliminary studies on a specific tobacco sterol Δ 7-sterol C5(6) -desaturase gene NtSC5DL found that it is highly correlated with sterol content in tobacco leaves, and that total sterol content in tobacco leaves was reduced by 46.3% after silencing the gene. 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 showing the relative expression of the gene in plants with NtSC5DL 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 NtSC5DL gene cloning and silencing vector is briefly described as follows.

(1) Cloning of tobacco NtSC5DL Gene

According to the previous research on the tobacco genome and the related NtSC5DL gene, a specific coding sequence is selected as a target segment, and a primer sequence for PCR amplification is designed as follows:

NtSC5DL-F：5’-CATTCCTGCCGGAATCATG-3’，

NtSC5DL-R：5’-ATGTACTCAGAAAGTGATGGAAGG-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 NtSC5DL 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 1min, 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-NtSC5DL 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-NtSC5DL is obtained by combining sequencing verification.

Example 2

On the basis of example 1, the constructed recombinant TRV2-NtSC5DL 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 conditions of related plants, 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-NtSC5DL are respectively transformed into agrobacterium GV3101 competent cells by an electric shock transformation mode, cultured and screened by a YEB plate containing 50mg/L Kan and 50mg/L Rif, and subjected to inverted culture at 28 ℃ for 2 days, and then screened by colony PCR for agrobacterium carrying the target gene.

(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 brothCulturing in 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 NtSC5DL gene is detected by qRT-PCR, and the result is shown in figure 1, and it can be seen that in the infected plant of TRV2-NtSC5DL, the expression level of NtSC5DL is significantly reduced, and the qRT-PCR primers are as follows:

NtSC5DL-F：5’-TATAATGGGCGCTGGCTATC-3’，

NtSC5DL-R：5’-CTTGGCTTCCTCTTCAATGG-3’。

further, the sterol content in leaf was measured in the experimental group (TRV2-NtSC5 DL-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 in FIG. 2, the sterol content in the experimental group was significantly reduced compared to the control group, wherein the campesterol was reduced by 74.4% at most. The further indication shows that the silencing of the NtSC5DL 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, an overexpression vector or a genome editing vector containing the NtSC5DL 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 illustrates and describes the principles of the present invention and its advantages. 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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Claims (7)

Translated fromChinese

1.一种烟草δ7-甾醇C5(6)-去饱和酶基因，其特征在于，核苷酸序列如SEQ ID NO.1所示。1. A tobacco δ7-sterol C5(6)-desaturase gene, characterized in that the nucleotide sequence is as shown in SEQ ID NO.1.2.根据权利要求1所述的烟草δ7-甾醇C5(6)-去饱和酶基因，其特征在于，烟草δ7-甾醇C5(6)-去饱和酶基因编码的蛋白的氨基酸序列如SEQ ID NO.2所示。2. tobacco delta 7-sterol C5 (6)-desaturase gene according to claim 1 is characterized in that, the amino acid sequence of the protein encoded by tobacco delta 7-sterol C5 (6)-desaturase gene is as SEQ ID NO .2 shown.3.根据权利要求1或2所述的烟草δ7-甾醇C5(6)-去饱和酶基因，其特征在于，烟草δ7-甾醇C5(6)-去饱和酶基因的PCR扩增制备方法，包括如下步骤：3. tobacco δ7-sterol C5 (6)-desaturase gene according to claim 1 and 2, is characterized in that, the PCR amplification preparation method of tobacco δ7-sterol C5 (6)-desaturase gene, comprising Follow the steps below:(1)提取基因组，并反转录为cDNA备用；(1) Extracting the genome and reverse transcribing into cDNA for subsequent use;(2)设计PCR扩增用引物，并进行PCR扩增，具体引物序列设计如下：(2) Design primers for PCR amplification, and carry out PCR amplification. The specific primer sequences are designed as follows:NtSC5DL-F：5’-CATTCCTGCCGGAATCATG-3’，NtSC5DL-F: 5'-CATTCCTGCCGGAATCATG-3',NtSC5DL-R：5’-ATGTACTCAGAAAGTGATGGAAGG-3’。NtSC5DL-R: 5'-ATGTACTCAGAAAGTGATGGAAGG-3'.4.根据权利要求3所述的烟草δ7-甾醇C5(6)-去饱和酶基因，其特征在于，在步骤(1)中提取基因组时，以烟草品种红花大金元叶片为样品。4 . The tobacco δ7-sterol C5(6)-desaturase gene according to claim 3, characterized in that, when extracting the genome in step (1), the leaves of tobacco variety Safflower Dajinyuan were used as samples.5.一种烟草δ7-甾醇C5(6)-去饱和酶基因的应用，其特征在于，利用上述权利要求1至4中任一项的烟草δ7-甾醇C5(6)-去饱和酶基因，该基因表达的蛋白与植物叶片中甾醇含量相关，降低该蛋白表达后，叶片中总甾醇含量明显降低。5. an application of tobacco δ7-sterol C5(6)-desaturase gene, is characterized in that, utilizes the tobacco δ7-sterol C5(6)-desaturase gene of any one of the above claims 1 to 4, The protein expressed by this gene is related to the sterol content in plant leaves. After reducing the expression of this protein, the total sterol content in leaves is significantly reduced.6.根据权利要求5所述的烟草δ7-甾醇C5(6)-去饱和酶基因的应用，其特征在于，利用基因沉默技术、或者基因超表达方法，通过调节烟草δ7-甾醇C5(6)-去饱和酶基因的表达量，来调节控制烟叶中甾醇含量情况。6. the application of tobacco δ7-sterol C5 (6)-desaturase gene according to claim 5, is characterized in that, utilizes gene silencing technology or gene overexpression method, by regulating tobacco δ7-sterol C5 (6) -Desaturase gene expression to regulate and control sterol content in tobacco leaves.7.根据权利要求6所述的烟草δ7-甾醇C5(6)-去饱和酶基因的应用，其特征在于，通过转基因技术、瞬时表达技术或基因组编辑技术，构建含有烟草δ7-甾醇C5(6)-去饱和酶基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体，转化烟草，筛选获得甾醇含量变化的烟草新品种。7. the application of tobacco δ7-sterol C5(6)-desaturase gene according to claim 6, it is characterized in that, by transgenic technology, transient expression technology or genome editing technology, construct and contain tobacco δ7-sterol C5(6) )-desaturase gene virus-induced silencing vector, RNAi interference vector, overexpression vector or genome editing vector, transform tobacco, and screen to obtain new tobacco varieties with varying sterol content.

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