CN113278600A - Tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof - Google Patents

Tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof Download PDF

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CN113278600A
CN113278600A CN202110580251.1A CN202110580251A CN113278600A CN 113278600 A CN113278600 A CN 113278600A CN 202110580251 A CN202110580251 A CN 202110580251A CN 113278600 A CN113278600 A CN 113278600A
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tobacco
hydroxysteroid dehydrogenase
decarboxylase gene
gene
ntnsdhl
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CN113278600B (en
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曾婉俐
宋春满
高茜
陈千思
许力
向海英
黄海涛
邓乐乐
李雪梅
蒋佳芮
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China Tobacco Yunnan Industrial Co Ltd
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    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/0117—3Beta-hydroxy-4alpha-carboxy-sterol 3-dehydrogenase (decarboxylating) (1.1.1.170)

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Abstract

The invention relates to a tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof, wherein the nucleotide sequence of the 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene is shown as SEQ ID No. 1. In the application, preliminary study on a specific tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene shows that the gene is highly related to the sterol content in tobacco leaves, the gene is silenced in Nicotiana benthamiana, the stigmasterol and campesterol content in the tobacco leaves is obviously reduced, and the total sterol content is reduced by 17.9%. Based on the characteristic, a genetic engineering means can be utilized to provide a certain application basis and reference for tobacco leaf quality regulation and new tobacco variety cultivation.

Description

Tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof
Technical Field
The invention belongs to the field of tobacco genetic engineering, and particularly relates to a tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene 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. The sterol compounds in tobacco mainly comprise cholesterol (cholestrol), stigmasterol (stigmasterol), campesterol (campasterol), beta-sitosterol (beta-sitosterol) and the like.
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 3 beta-hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof (a tobacco 3 beta-hydroxysteroid dehydrogenase/C-4 decarboxylase gene and application thereof) 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 3 beta-hydroxysteroid dehydrogenase/C4 decarboxylase gene has the nucleotide sequence shown in SEQ ID No.1, contains 1167 basic groups and is named NtNSDHL.
Furthermore, the amino acid sequence of the coding protein of the tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene is shown in SEQ ID NO.2 and consists of 388 amino acid residues.
Further, the PCR amplification preparation method of the tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene 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:
NtNSDHL-F：5’-AACAAATCCCAAGTCCTCAAAG-3’，
NtNSDHL-R：5’-CTACCTTAGCAGGATATGGC-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 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene disclosed by any one of the above is to regulate and control the sterol content in tobacco leaves by regulating the expression level of the synthesis of tobacco sterol ergosterol by using a gene silencing technology.
Further, a virus-induced silencing vector, an RNAi interference vector and an overexpression vector containing the tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase 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 NtNSDHL gene is interfered and silenced by utilizing the virus-induced gene silencing (VIGS) technology, the sterol content in the NtNSDHL gene silencing plant 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 the synthesis of NtNSDHL from specific tobacco sterol ergosterol, the NtNSDHL is found to be highly related to the sterol content in tobacco leaves, and the sterol content in the tobacco leaves is obviously reduced after the gene is silenced. 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 NtNSDHL gene-silenced plants 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 NtNSDHL gene cloning and silencing vector is briefly described as follows.
(1) Tobacco NtNSDHL gene cloning
According to the previous research on the tobacco genome and the related NtNSDHL gene, a specific coding sequence is selected as a target segment, and a primer sequence for PCR amplification is designed as follows:
NtNSDHL-F：5’-AACAAATCCCAAGTCCTCAAAG-3’，
NtNSDHL-R：5’-CTACCTTAGCAGGATATGGC-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 NtNSDHL 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-NtNSDHL 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-NtNSDHL is obtained by combining sequencing verification.
Example 2
On the basis of example 1, the constructed recombinant TRV2-NtNSDHL 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-NtNSDHL 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 the overnight culture was inoculated into 50mL of YEB liquid medium (containing 50mg/L Kan), and cultured 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 NtNSDHL gene is detected by qRT-PCR, and the result is shown in FIG. 1, and it can be seen that the expression level of NtNSDHL is remarkably reduced in the infected plant of TRV2-NtNSDHL, and the qRT-PCR primers are as follows:
NtNSDHL-F：5’-ACGGTTCCACAGTTGACTCC-3’，
NtNSDHL-R：5’-CTGGTCCTCGTTCAGCTCTC-3’。
further, the sterol content in leaf was measured in the experimental group (TRV 2-NtNSDHL-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 is significantly reduced compared to the control group, the total sterol content is reduced by 17.9%, and stigmasterol and campesterol are significantly reduced compared to the control group. The further indication shows that the silencing of the NtNSDHL gene can regulate and control the content of the phytosterol in the tobacco leaves, and further can lay a certain technical foundation 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 NtNSDHL 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
<110> tobacco industry Limited liability company in Yunnan
<120> tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene and application thereof
<130> WPC211447
<141> 2021-05-25
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atgggggaag gagaagaaga gaaatggtgt gtggtgactg gtggaagagg ctttgctgct 60
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ggtccgtcca ttaaacttga cccgactgag gaaaagggta tacttggtca agccctccaa 180
tcaggccgtg ctgtatatgt atccatggat cttcgtaaca aatcccaagt cctcaaagct 240
tgtgaaggag ctgaggttgt cttccacatg gctgctccag attcatcaat caacaaccac 300
cagctccact attcagttaa tgtgcaagga acccagaata taattgatgc ttgcattgag 360
ctgaaagtga aaagacttat ttacaccagc tctcccagtg tggtgtttga tggagttcat 420
ggaattctaa atggggatga atcactgcca tatcctgcta agcataatga ttcctactct 480
gcaaccaaag ctgaaggaga ggcacttgtt atcaagtcaa atggtaccaa agggctgctg 540
acatgctgca ttagacctag cagtcttttt ggccctggtg ataggctgct cgttccttca 600
ctagttgcag ctgcaaaggc aggaaaatca aagttcatta ttggtgatgg caacaacatg 660
tatgatttca cttacgtgga gaatgtagca catgctcatg tgtgtgcaga acgagctcta 720
gcatcaggag gagcagttgc agagaaagct gctgggaatg catattttgt cacgaacatg 780
gagcccatta agttttggga gtttgtctca cttattcttg aaggtcttgg ctatgacagg 840
ccaaatatta agattcctgc atctgttatg atgccaattg cacatttggt ggagcttagt 900
tataagctgt tagctcctta tggaatgaag gtcccacagt tgactccttc aagaatcaga 960
ctcctgtccc gtagcagaac atttagttgt tcaaaagcaa gtgatcgaat aggatacaca 1020
cctattatct cacttcagga gggccttcgg aggacaattg agtcctatcc acatttgaga 1080
gctgaacatg ggcctggaaa ggaaggtcct tctaaatcat ctgcatctct ttggatgttt 1140
ttcctcatgg taatttctaa tacataa 1167
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Lys Gly Leu Leu Thr Cys Cys Ile Arg Pro Ser Ser Leu Phe Gly Pro
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Claims (7)

1. A tobacco 3 beta-hydroxysteroid dehydrogenase/C4 decarboxylase gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene as claimed in claim 1, wherein the amino acid sequence of the protein encoded by the tobacco 3 beta hydroxysteroid dehydrogenase/C4 decarboxylase gene is shown as SEQ ID No. 2.
3. The tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene according to claim 1 or 2, wherein the PCR amplification preparation method of the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene 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:
NtNSDHL-F：5’-AACAAATCCCAAGTCCTCAAAG-3’，
NtNSDHL-R：5’-CTACCTTAGCAGGATATGGC-3’。
4. the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene as set forth in claim 3, wherein the tobacco variety Honghuadajinyuan leaf is used as a sample when extracting the genome in step (1).
5. Use of a tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene, wherein the protein expressed by the gene is related to the sterol content in plant leaves, and the sterol content in leaves is significantly reduced after the protein expression is reduced, by using the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene according to any one of claims 1 to 4.
6. The use of the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene as claimed in claim 5, wherein the sterol content in tobacco leaves is controlled by regulating the expression level of the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene by using gene silencing technique or gene overexpression method.
7. The use of the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene as claimed in claim 6, wherein a virus-induced silencing vector, an RNAi interference vector, a overexpression vector or a genome editing vector containing the tobacco 3 β hydroxysteroid dehydrogenase/C4 decarboxylase gene is constructed by a transgenic technique, a transient expression technique or a genome editing technique, and the tobacco is transformed and a new variety of tobacco with varying sterol content is screened.
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CN105349478A (en) * 2015-12-23 2016-02-24 郑州轻工业学院 Method for degrading sterol compounds in tobacco extract with biological method
CN105400794A (en) * 2015-12-30 2016-03-16 中国烟草总公司郑州烟草研究院 Gene bHLH93 for synthesizing related transcription factors through tobacco sterol and application
CN110590723A (en) * 2019-08-29 2019-12-20 云南中烟工业有限责任公司 Sterol compound in tobacco and preparation method and application thereof

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