CN103805576B - Tobacco squalene epoxidase protein, tobacco squalene epoxidase gene and applications of tobacco squalene epoxidase gene - Google Patents
Tobacco squalene epoxidase protein, tobacco squalene epoxidase gene and applications of tobacco squalene epoxidase gene Download PDFInfo
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- CN103805576B CN103805576B CN201410062817.1A CN201410062817A CN103805576B CN 103805576 B CN103805576 B CN 103805576B CN 201410062817 A CN201410062817 A CN 201410062817A CN 103805576 B CN103805576 B CN 103805576B
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- tobacco
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- squalene epoxidase
- nicotiana tabacum
- sterol
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- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 108
- 108020003891 Squalene monooxygenase Proteins 0.000 title claims abstract description 42
- 241000208125 Nicotiana Species 0.000 title claims abstract description 19
- 102000005782 Squalene Monooxygenase Human genes 0.000 title abstract description 17
- 229930182558 Sterol Natural products 0.000 claims abstract description 41
- 150000003432 sterols Chemical class 0.000 claims abstract description 41
- 235000003702 sterols Nutrition 0.000 claims abstract description 41
- 241000196324 Embryophyta Species 0.000 claims abstract description 26
- 241000700605 Viruses Species 0.000 claims abstract description 5
- 244000061176 Nicotiana tabacum Species 0.000 claims description 92
- 238000000034 method Methods 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 10
- 230000028604 virus induced gene silencing Effects 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 239000000725 suspension Substances 0.000 claims description 7
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical class CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 claims description 6
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- 229930006000 Sucrose Natural products 0.000 claims description 5
- 230000009368 gene silencing by RNA Effects 0.000 claims description 5
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- 238000013461 design Methods 0.000 claims description 3
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- ZGBSOTLWHZQNLH-UHFFFAOYSA-N [Mg].S(O)(O)(=O)=O Chemical compound [Mg].S(O)(O)(=O)=O ZGBSOTLWHZQNLH-UHFFFAOYSA-N 0.000 claims 1
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- 125000000539 amino acid group Chemical group 0.000 description 5
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
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- 230000033228 biological regulation Effects 0.000 description 2
- OILXMJHPFNGGTO-ZAUYPBDWSA-N brassicasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZAUYPBDWSA-N 0.000 description 2
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- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 2
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- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 2
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 2
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000208134 Nicotiana rustica Species 0.000 description 1
- PPZALDOGKPNJAC-UHFFFAOYSA-N [C].C1=CC=C2C3=CC=CC=C3C=CC2=C1 Chemical group [C].C1=CC=C2C3=CC=CC=C3C=CC2=C1 PPZALDOGKPNJAC-UHFFFAOYSA-N 0.000 description 1
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- ILYCWAKSDCYMBB-OPCMSESCSA-N dihydrotachysterol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1/C[C@@H](O)CC[C@@H]1C ILYCWAKSDCYMBB-OPCMSESCSA-N 0.000 description 1
- 229960000465 dihydrotachysterol Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
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- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
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- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- RGSFGYAAUTVSQA-UHFFFAOYSA-N pentamethylene Natural products C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 1
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical group C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
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- 239000011780 sodium chloride Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
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- 150000003648 triterpenes Chemical class 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/99—Miscellaneous (1.14.99)
- C12Y114/99007—Squalene monooxygenase (1.14.99.7)
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- Genetics & Genomics (AREA)
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- Engineering & Computer Science (AREA)
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- Zoology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a tobacco squalene epoxidase protein with an amino acid sequence shown in SEQ ID NO:1. A tobacco squalene epoxidase gene NtSE is a key control gene in the synthetic route of sterol in tobaccos. The gene is specifically highly expressed in young tissues, a built virus mediated gene silenced carrier TRV-NtSE is inoculated to a Ben's tobacco, and then a plant with a high silencing efficiency is obtained; through detecting the content of phytosterol in the Ben's tobacco, a situation that the sterol content of the gene silenced plant is significantly reduced by 21.37% is found, which shows that the silencing of the gene can significantly reduce the sterol content of the plant. The tobacco squalene epoxidase gene NtSE is a key control gene in the synthetic route of sterol in tobaccos, can be used for regulating the sterol content of tobaccos, and facilitates the genetic engineering breeding of low-sterol high-quality tobaccos.
Description
Technical field
The present invention relates to tabacum sterol synthesis and regulation and control key gene, more particularly to Nicotiana tabacum L. squalene epoxidase enzyme geneNtSE
Application on tabacum sterol content is lowered.
Background technology
Nicotiana tabacum L. is Dicotyledoneae Tubiflorae Solanaceae Nicotiana plant.Nicotiana (Nicotiana) has more than 60 to plant, and 2
In individual cultigen, Nicotiana tabacum (also known as safflower tobacco, Nicotiana tabacum) occupies significant area, Folium Nicotianae rusticae
The area very little of (Nicotiana rustica).Cultivation Nicotiana tabacum L. presses quality of tobacco feature, biological character and cultivation modulation methods
Method etc. can be divided into flue-cured tobacco, suncured tabacco, air-curing of tobacco leaves, burley tobaccos, six types of Turkish tobaccos and Nicotiniana rustica, and wherein flue-cured tobacco is to cultivate most wide general
Logical Nicotiana tabacum L..China's tobacco planting area and total output rank first in the world.As a kind of leaf industrial crops, the cultivation of flue-cured tobacco
Technology is different from other field crops, does not require nothing more than certain yield of tobacco, and more focuses on quality of tobacco.Quality of tobacco is determined
Determine the availability of Nicotiana tabacum L., directly affect the color and commodity value of Medicated cigarette commodity, be also related to the economic benefit of tobacco grower, be
The life and starting point of tobacco business.To make to establish oneself in an unassailable position in following domestic and international market competition, meet and roll up both at home and abroad
Cigarette enterprise is to sound tobacco increasing need, it is necessary to improve quality of tobacco and safety.
Plant sterol is the important compound of a class in Nicotiana tabacum L., belongs to lipoid substance, and its basic structure is Pentamethylene.
Many phenanthrene carbon skeletons.Cholesterol, brassicasterol, stigmasterol and cupreol, ergosterol and campesterol are mainly contained in Nicotiana tabacum L.
It is a small amount of to exist.Research shows the hexane extract of Nicotiana tabacum L.（Sterol, triterpene etc.）It is the main of the carcinogenic thing polycyclic aromatic hydrocarbon of cigarette smoke
One of precursor.Due to all containing hydroxyl in the structure of sterol, during pyrolysis, four-wheel cyclopentenes [α] the phenanthrene ring structure of its parent can shape
Into condensed-nuclei aromatics, therefore the sterol in Nicotiana tabacum L. is a kind of potential material for affecting health, so reducing Nicotiana tabacum L. climax leaves
In piece, sterol content is also one of effective way of Tar, there is no at present
Efficacious prescriptions method.
The content of the invention
It is an object of the invention to find the functional gene of regulation and control tabacum sterol content, and then provide a kind of reduction Nicotiana tabacum L. steroid
The method of alcohol content, provides technological means for low sterol tobacco breeding.
The technical scheme is that：One grows tobacco squalene epoxidase albumen, its aminoacid sequence such as SEQ ID NO：1 institute
Show.
One grows tobacco squalene epoxidase gene, encodes the gene of Nicotiana tabacum L. squalene epoxidase pheron described in claim 1.
One grows tobacco squalene epoxidase gene, its base sequence such as SEQ ID NO：Shown in 2.
The nucleic acid specific fragment of the Nicotiana tabacum L. squalene epoxidase enzyme gene is SEQ ID NO in sequence table：2 629-
1127 nucleotide sequences.
Application of the described Nicotiana tabacum L. squalene epoxidase enzyme gene in tabacum sterol content is lowered.By transgenic technology or wink
When expression technology interference, silence or knock out the Nicotiana tabacum L. squalene epoxidase, obtain the Transformation of tobacco plant that sterol content lowers.
The virus induction silent carrier or RNAi interference vectors transformation of tobacco of the Nicotiana tabacum L. squalene epoxidase enzyme gene are built, sterol is obtained
The transgene tobacco that content lowers.The Gene Silencing carrier and RNAi carrier of the Nicotiana tabacum L. squalene epoxidase enzyme gene leads to
Cross following methods to build：Using the specific nucleotide acid fragment of the Nicotiana tabacum L. squalene epoxidase protein gene as homing sequence, will
The nucleic acid specific fragment is inserted in plant expression vector with positive and negative both direction.
For cultivation grows tobacco squalene epoxidase geneNtSECloned, and utilized virus induced gene silencing handss
Duan Jinhang functional analyses, the genetic engineering breeding for High Quality Tobacco provide new strategy and instrument.
Present invention research finds that squalene epoxidase enzyme gene is the key controlling gene of tabacum sterol route of synthesis in Nicotiana tabacum L.,
By virus induced gene silencing（VIGS）Technology, in Nicotiana tabacum L. disturb which to express, obtain gene silencing plant, detection send out
In existing these gene silencing plant, sterol content is relative to being remarkably decreased to sample size.In view of the using value of the gene and its
The application prospect of Exploitative potential, it is necessary to be protected by by patent.
Gene code Nicotiana tabacum L. squalene epoxidase provided by the present invention for lowering sterol content, from Nicotiana tabacum L.
（Nicotiana tabacum）, NtSE is named as, following proteins are encoded（i）Or（ii）：
（i）SEQ ID NO in sequence table：The protein of aminoacid sequence shown in 1；
（ii）SEQ ID NO in sequence table：Aminoacid shown in 1 arrange through one to ten amino acid residue replacement,
Lack or add and derivative protein, and derivative protein with（i）The protein identical function.
SEQ ID NO in sequence table：1 sequence is made up of 531 amino acid residues, wherein the 5th to the 27th is amino
Sour residue is membrane spaning domain, during the replacement, one to ten amino acid residue for lacking or adding can be said structure domain
Amino acid residue, which changes and will not produce impact to the function of the albumen.Amino acid residue is replaced, lacked or is added
Plus, and the detection of relative function can be realized by the ordinary skill in the art.
The Nicotiana tabacum L. squalene epoxidase SE genes NtSE of the present invention can be cDNA sequence, or genomic dna sequence,
Or there is the DNA sequence of more than 90% homology and coding identical function albumen with these sequences.Such as in sequence table SEQ
ID NO：DNA sequence shown in 2.
By the method for homologous clone, according to the squalene epoxidase gene order in higher plant, with cultigen tobacco leaf
The cDNA of the total serum IgE reverse transcription of piece is material, designs the ORF total length 1596bp sequences that primer has cloned NtSE（SEQ ID NO：
2）, the SEQ ID NO in the protein sequence such as sequence table of its coding：Shown in 1, sequence results analysis shows, the albumen contain together
Source property is very high, highly conserved.Secondly find that the gene is all table during each is organized in Nicotiana tabacum L. using by real-time PCR
Reach, the special high expression in Nicotiana tabacum L. tender tissue.
The invention has the beneficial effects as follows：By virus induced gene silencing technology（VIGS）Technique construction NtSE genes
VIGS carriers, have successfully been obtained suppression silence plant of the NtSE in Ben's Nicotiana tabacum L., the silence plant for being obtained possesses sterol
The specific phenotypes that content relative comparison plant substantially reduces.It can be seen that, using gene silent technology or knock out NtSE genes and can obtain
The gene silencing plant that sterol content is reduced is obtained, this causes low sterol content tobacco breeding to be possibly realized, be to reduce tabacum sterol
Content provides a kind of effective technological means.
Nicotiana tabacum L. squalene epoxidase enzyme geneNtSEIt is the key controlling gene of tabacum sterol route of synthesis.It is special in tender tissue
Different high expression, by the virus-mediated gene silencing vector TRV-NtSE inoculation Ben's Nicotiana tabacum L.s for building, and obtains high silence effect
The plant of rate, detects the content of plant sterol in Ben's Nicotiana tabacum L., and in finding gene silencing plant, sterol content is remarkably decreased, and declines
21.37%, show that the silence gene can significantly reduce the sterol content of plant.Nicotiana tabacum L. squalene epoxidase enzyme geneNtSEIt is
The key controlling gene of tabacum sterol route of synthesis, can be used to adjust the sterol content of Nicotiana tabacum L., contributes to low sterol High Quality Tobacco
Genetic engineering breeding.
Description of the drawings
Fig. 1 is the relative expression quantity of NtSE in different tissues；
Relative expression quantities of the Fig. 2 for the NtSE genes in gene silencing plant；
Fig. 3 is the main sterol content in the Nicotiana tabacum L. and control Nicotiana tabacum L. of Gene Silencing.
Specific embodiment
One grows tobacco squalene epoxidase albumen, its aminoacid sequence such as SEQ ID NO：Shown in 1.
One grows tobacco squalene epoxidase gene, encodes the gene of Nicotiana tabacum L. squalene epoxidase pheron described in claim 1.
One grows tobacco squalene epoxidase gene, its base sequence such as SEQ ID NO：Shown in 2.
The nucleic acid specific fragment of the Nicotiana tabacum L. squalene epoxidase enzyme gene is SEQ ID NO in sequence table：2 629-
1127 nucleotide sequences.
Application of the described Nicotiana tabacum L. squalene epoxidase enzyme gene in tabacum sterol content is lowered.By transgenic technology or wink
When expression technology interference, silence or knock out the Nicotiana tabacum L. squalene epoxidase, obtain the Transformation of tobacco plant that sterol content lowers.
The virus induction silent carrier or RNAi interference vectors transformation of tobacco of the Nicotiana tabacum L. squalene epoxidase enzyme gene are built, sterol is obtained
The transgene tobacco that content lowers.The Gene Silencing carrier and RNAi carrier of the Nicotiana tabacum L. squalene epoxidase enzyme gene leads to
Cross following methods to build：Using the specific nucleotide acid fragment of the Nicotiana tabacum L. squalene epoxidase protein gene as homing sequence, will
The nucleic acid specific fragment is inserted in plant expression vector with positive and negative both direction.
First, select more special one section of accounting fragment (sequence table SEQ ID NO in this gene：The 629th-the of 2
1127 nucleotide sequences) for the homing sequence of VIGS, design primer obtains this sequence.Then by this accounting fragment insertion VIGS
In carrier, TRV-NtSE carriers are built.
Ben's tobacco planting time and place：In September, 2013, Zhengzhou.
Concrete silence step is as follows：
（1）Tobacco seed is seeded in nursery in pot for growing seedlings, carries out seedling separation, plants in polypots two weeks after to be germinateed（10cm ×
10cm）In, in 22 DEG C, under 16h light/8h dark conditions, carry out daily rich water quality management etc.；
（2）Growth 4-5w, chooses growing way and unanimously will access YEB containing TRV1, TRV2, TRV2-SS Agrobacterium single bacterium colony
（5ml）In culture medium (contain corresponding antibiotic), 28 DEG C, 250 r/min overnight shaking cultures be about 1.0 to OD；
（3）OD values are surveyed, the bacterium solution volume X value that subculture needs is calculated, and is drawn the bacterium solution of X volumes and access 50 ml YEB
In, 10 mmol/L2-N- beautiful jades base ethyl sulfonic acid (MES) and 20 μ l/L second phthalein syringone (acteosyringone, As) are included,
5 ml As are added in 50 mlYEB, 500 ml MES, 50 ml antibiotic, 28 DEG C of overnight shaking cultures are about to OD
0.2-2.0；
（4）OD values are surveyed, MMA (the 10 mmol/L MgCl for adding are calculated2, 10 mmol/L MES and 100mmol/LAS)
Amount Y value, adjusts concentration OD600=2.0 of suspension, and 4000 r/min are centrifuged 8 min collection Agrobacteriums in 50 ml centrifuge tubes,
Supernatant is poured out, MMA is added in each thalline, then is added the MMA of TRV1 to hang in the medium volume of TRV2, TRV2-SS MMA suspensions
Supernatant liquid is mixed, and can be inoculated with after static 1 h of room temperature, selects growing way unanimously, the tobacco plant of about 4-5 piece leaves, with 1 ml needleless
Head asepsis injector is pressed into whole exhibitions from vacuum side of blade the agrobacterium suspension containing difference TRV recombiant plasmid by filter press technique
In the blade opened, bacterium solution is made full of whole blade, in 22 DEG C, cultivate in 75% air humidity.
Preparation of reagents method：
（1）LB fluid mediums（1L）：10 g bacto peptones（bacteriological peptone）；10 g chlorinations
Sodium（NaCl）；5 g yeast extracts（yeast extract）, autoclave sterilization；
（2）YEB fluid mediums（1L）：5 g beef extracts（beef extract）；5 g bacto peptones
（bacteriological peptone）；5 g sucrose（sucrose）；1 g yeast extracts（yeast extract）；2 ml
1M magnesium sulfate（MgSO4）, autoclave sterilization；
（3）1M 2- (N- morpholines) ethyl sulfonic acid（MES）Storing solution：DdH2O dissolves, filtration sterilization, and -20 DEG C store for future use；
（4）200 mM acetosyringones（Acetosyringone）Storing solution：Dimethyl sulfoxide（DSMO）Dissolving, -20 DEG C
Store for future use；
（5）MMA（1L）：20 g sucrose（sucrose）；5 g MS salts（Duchefa Biochemie）；1.95 g
MES；1 ml acetosyringones（200 mM,）；PH：5.6, it is now with the current.
TRV-NtSE carrier informations：
1 squalene epoxidase of table（Squalene monooxygenase, SE）Gene silencing vector builds primer information
Ben's tobacco seed is seeded in nursery in pot for growing seedlings, carries out seedling separation, plants in polypots two weeks after to be germinateed（10cm
× 10cm）In, in 22 DEG C, daily rich water quality management etc. under 16h light/8h dark conditions, is carried out, grow 4-5w, carry out choosing growing way one
12 basin of plant of cause is tested.Wherein 6 basins carry out injection silence using TRV1, TRV2, TRV2-SS carrier, and remaining 6 basin is right
According to.
After being inoculated with 3 weeks, the content of main sterol material in detecting silence plant and in adjoining tree（Fig. 1）, as a result show,
Cholesterol, dihydrotachysterol, brassicasterol, campesterol, stigmasterol, β-sitoesterol etc. 6 in Gene Silencing plant
Middle sterol content is remarkably decreased compared with adjoining tree, and 6 kinds of sterol total contents decline 21.37% altogether（Table 2）.
In 2 application invention patent fresh tobacco leaves sample of table, content of phytosterol declines percentage ratio
Relative expression quantities of the Fig. 2 for the NtSE genes in gene silencing plant, Nicotiana tabacum Lves of the Fig. 3 for Gene Silencing
And the main sterol content in control Nicotiana tabacum L., in gene silencing plant, main sterol content is remarkably decreased.Result of the test of the present invention
Show：By the method for virus induced gene silencing, the sterol content in fresh tobacco leaves is significantly reduced.
Claims (1)
1. the method for cultivating the transgene tobacco that sterol content lowers using Nicotiana tabacum L. squalene epoxidase enzyme gene, it is characterised in that logical
Cross and build the virus induction silent carrier or RNAi interference vectors for being loaded with Nicotiana tabacum L. squalene epoxidase enzyme gene, and transformation of tobacco, obtain
The transgene tobacco that sterol content lowers；The base sequence of the Nicotiana tabacum L. squalene epoxidase enzyme gene such as SEQ ID NO：Shown in 2, tool
Body is comprised the following steps：
(1) design primer sequence as follows：
NT-SS-F, 5 ˊ-ATGGTACCGATGTACTGGTCAAAAAGTGCCT-3 ˊ,
NT-SS-R, 5 ˊ-CACTCGAGTCAGCCGGGCTCCAATCCAC-3 ˊ；
629th to the 1127th nucleotide sequence in PCR amplification Nicotiana tabacum L. squalene epoxidase enzyme genes, common 499bp fragments,
(2) by amplified fragments insertion VIGS carriers in step (1), TRV-NtSE carriers are built；
(3) tobacco seed is seeded in nursery in pot for growing seedlings, carries out seedling separation two weeks after to be germinateed, and plants in polypots, in 22 DEG C,
Daily rich water quality management is carried out under 16h light/8h dark conditions；
(4) 4-5w is grown, choose growing way and unanimously YEB culture medium will be accessed containing TRV1, TRV2, TRV2-SS Agrobacterium single bacterium colony
In, 1L YEB culture medium consists of 5g beef extracts, 5g bacto peptones, 5g sucrose, 1g yeast extracts, 2ml 1M sulphuric acid
Magnesium, autoclave sterilization, 28 DEG C, 250r/min overnight shakings culture to OD be 1.0；
(5) OD values are surveyed, calculates the bacterium solution volume X value that subculture needs, and draw the bacterium solution of X volumes and access in 50ml YEB, it is interior
The beautiful jade base ethyl sulfonic acids of 2-N- containing 10mmol/L and 20 μ l/L acetosyringones, i.e., add 5ml acetosyringones in 50ml YEB,
500ml 2-N- beautiful jade base ethyl sulfonic acids, 50ml antibiotic, 28 DEG C of overnight shaking cultures to OD are 0.2-2.0；
(6) OD values are surveyed, calculates the amount Y value of the MMA for adding, MMA is 10mmol/L MgCl2, 10mmol/L 2-N- beautiful jade base second sulphurs
Acid and 100mmol/L acetosyringones, adjust concentration OD600=2.0 of suspension, and 4000r/min centrifugation 8min collect agriculture bar
Bacterium pours out supernatant in 50ml centrifuge tubes, and MMA is added in each thalline, then in TRV2, TRV2-SS MMA suspension medium bodies
The long-pending MMA suspensions for adding TRV1 are mixed, and can be inoculated with after the static 1h of room temperature, select growing way unanimously, and the Nicotiana tabacum L. of 4-5 piece leaves is planted
Strain, carries on the back the agrobacterium suspension containing difference TRV recombiant plasmid by filter press technique with 1ml needlelesss asepsis injector from blade
Face pressure enters in all blades of expansion, makes bacterium solution full of whole blade, in 22 DEG C, cultivates in 75% air humidity.
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| Title |
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| NCBI Reference Sequence: NP_564734.1，probable squalene epoxidase 1 [Arabidopsis thaliana];Swarbreck et al;《NCBI》;20110218;"origin"部分 * |
| Studies on the regulation of the sterol pathway in tobacco BY-2 cells;Bach et al;《CNRS-IBMP & Univ. Louis Pasteur》;20021231;摘要 * |
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