CN111690661A - Tobacco NtbHLH13 gene mutant and molecular identification method and application - Google Patents
Tobacco NtbHLH13 gene mutant and molecular identification method and application Download PDFInfo
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- CN111690661A CN111690661A CN202010486421.5A CN202010486421A CN111690661A CN 111690661 A CN111690661 A CN 111690661A CN 202010486421 A CN202010486421 A CN 202010486421A CN 111690661 A CN111690661 A CN 111690661A
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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Abstract
A tobacco NtbHLH13 gene mutant and a molecular identification method and application thereof are provided, the tobacco NtbHLH13 gene mutant is Ntbhlh13, which is a termination codon formed by mutating G at the 215 th position of a tobacco Ntbhlh13 gene to A, and the nucleotide sequence of the gene is shown as SEQ ID No. 1. After the tobacco NtbHLH13 gene mutant Ntbhlh13 is mutated, the nicotine content of tobacco can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtbHLH13 gene mutant has great value in tobacco breeding.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to a tobacco NtbHLH13 gene mutant Ntbhlh13-1 and a molecular identification method and application thereof.
Background
Research on the metabolic regulation of tobacco nicotine is a very significant work, and tobacco varieties with different nicotine contents can be provided through gene regulation, so that raw materials are provided for the commercial production of personalized nicotine tobacco products by tobacco. The nicotine has strong physiological stimulation to human body and is the material basis for the commercial use of tobacco. Many top-grade tobacco companies in the world, such as Philippines, empire tobacco, Japanese tobacco, and Yinmei tobacco, have invested huge investment in the research on the metabolic pathways and regulation mechanisms of tobacco nicotine.
Nicotine is a pyridine alkaloid, mainly exists in plants of Nicotiana (Nicotiana) of solanaceae, and is an important secondary metabolite in tobacco bodies. The synthesis and transport of tobacco nicotine are regulated by a plurality of factors, and some key genes in nicotine synthesis pathways, such as QPT, PMT, MPO, JAZ, MYC2a and the like, have been identified and cloned at present.
The anabolic pathway of nicotine has not been completely studied from a molecular biology perspective. The research of regulating nicotine synthesis gene through chloride ion channel to affect nicotine content has not been reported. The nicotine regulation gene is important for the commercial production of tobacco, and most of the related patents of the nicotine synthesis gene are mastered in foreign tobacco companies at present. Therefore, the research of the related regulation and control gene of the nicotine synthesis pathway has important significance for improving the nicotine content in the tobacco products of Chinese tobacco enterprises. It is worth noting that many genes for regulating nicotine are mainly subjected to gene function verification by using RNAi, but the method has the defect that homologous genes can be knocked out simultaneously, and in addition, transgenic materials are not allowed to be used in tobacco breeding, so that in order to breed high-nicotine tobacco materials, gene mutation materials need to be obtained by an EMS knocking-out method.
Disclosure of Invention
The invention aims to provide a tobacco NtbHLH13 gene mutant Ntbhlh13-1 and a molecular identification method thereof. The invention also provides application of the tobacco NtbHLH13 gene mutant Ntbhlh 13-1.
The technical scheme adopted by the invention is as follows:
a tobacco NtbHLH13 gene mutant is Ntbhlh13, which is a tobacco Ntbhlh13 gene with the 215 th G mutated into A to form a stop codon, so that the gene is terminated in advance, and the nucleotide sequence is shown as SEQ ID No. 1.
The molecular identification method of the tobacco NtbHLH13 gene mutant NtbHLH13-1 comprises the step of amplifying a DNA fragment of the mutant NtbHLH13 by using a primer pair, wherein an upstream primer of the primer pair is NtbHLH 13F, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.2, and a downstream primer of the primer pair is NtbHLH 13R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N0.3.
The tobacco NtbHLH13 gene mutant Ntbhlh13-1 is applied to preparation of high nicotine materials.
After the tobacco NtbHLH13 gene mutant Ntbhlh13-1 is mutated, the tobacco nicotine content can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtbHLH13 gene mutant has great value in tobacco breeding.
Drawings
FIG. 1 is an amplified band of the tobacco NtbHLH13 gene mutant Ntbhlh 13-1;
FIG. 2 shows the sequencing result of the tobacco NtbHLH13 gene mutant Ntbhlh 13-1;
FIG. 3 shows the nicotine content of the tobacco NtbHLH13 gene mutant Ntbhlh13-1 and wild type single plant.
Detailed Description
The present invention is further described with reference to the following examples and accompanying drawings, but the present invention is not limited in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
1. Obtaining of tobacco mutant material
(1) Cleaning and disinfecting tobacco seeds containing the tobacco NtbHLH13 gene by using sodium hypochlorite, and then washing and drying by using distilled water;
(2) soaking tobacco plants in a phosphate buffer solution to increase the germination rate of seeds;
(3) soaking the tobacco seeds obtained by soaking in 0.5% EMS (ethyl methane sulfonate) solution for 10-15 hours, and then centrifuging and filtering to dry the seeds;
(4) the seeds were rinsed 50 times with distilled water, and EMS solution was sufficiently washed away as tobacco mutant material.
2. Screening to obtain a mutant Ntbhlh 13-1:
(1) the DNA of the mutant material is used as a template to design a specific primer pair for PCR amplification, wherein an upstream primer of the primer pair is NtbHLH 13F, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.2, and a downstream primer of the primer pair is NtbHLH 13R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N0.3.
The PCR reaction conditions were as follows:
the amplified band is shown in FIG. 1;
(2) and (3) carrying out electrophoresis on the PCR product obtained by amplification in 0.8% agarose gel, after the electrophoresis is finished, recovering and purifying the PCR product by using a PCR product purification kit of Qiagen company according to the product instruction, sending the PCR product to Invitrogen for sequencing, and verifying the sequence result, wherein the sequencing result is shown in figure 2.
(3) Self-crossing candidate mutant material to obtain M2 seeds;
(4) m2 seeds are planted to obtain M2 mutant plants, a primer pair NtbHLH 13F (nucleotide sequence is shown as SEQ ID No. 0.2) and a downstream primer NtbHLH 13R (nucleotide sequence is shown as SEQ ID No. 0.3) are used for identifying the mutant, and finally, homozygous mutant plants with the mutant Ntbhlh13-1 are obtained. Compared with the nucleotide sequence of the wild tobacco NtbHLH13 gene, the G at the 215 th position of the NtbHLH13 gene sequence is mutated into A, so that the base is changed from tryptophan to a stop mutation to form a stop codon, and the gene is stopped early. The nucleotide sequence of the mutant Ntbhlh13-1 is shown in SEQ ID No. 1.
3. And (3) nicotine content determination:
(1) the nicotine content of the tobacco material was determined according to standard YC/T160-. The selected tobacco materials are non-transgenic tobacco plants and transgenic tobacco plants which are close in development phenotype in the vigorous growth period as processing objects, and wild tobacco K326 is used as a reference. Taking 5 non-transgenic tobacco plants and the upper, middle and lower leaves of the transgenic tobacco plants. For the other group, 5 non-transgenic tobacco plants and transgenic tobacco plants are subjected to topping treatment, and then the upper leaves, the middle leaves and the lower leaves of the non-transgenic tobacco plants and the transgenic tobacco plants are adopted;
(2) tobacco samples were extracted with 5% aqueous acetic acid and the total plant alkaloids (based on nicotine) in the extract were reacted with sulfanilic acid and cyanogen chloride, which is generated by the on-line reaction of potassium cyanide and chloramine T. The reaction product was measured at 460nm using a colorimeter.
The main apparatus comprises: continuous flow Analyzer (American API) (SEAL AA3, Germany) (ALLIANCE, France).
Preparing a reagent: brij35 solution (polyethoxy lauryl ether): 5 drops of 22% Brij35 are added into water and stirred evenly.
Buffer solution a: 2.35g of sodium chloride (NaCl) and 7.60g of sodium borate (Na) were weighed out2B4O3·10H2O), dissolved in water, and transferred to a 1L volumetric flask, 1mL of Brij35 was added and diluted to 1L with distilled water. Filtered through qualitative filter paper before use.
Buffer solution B: 26g disodium hydrogen phosphate (Na) are weighed2HPO4)10.4g citric acid [ COH (COOH) (CH)2COOH)2·H2O]7g of sulfanilic acid (NH)2C6H4SO3H) Dissolved in water, transferred to a 1L volumetric flask, added 1mL Brij35 and diluted to 1L with distilled water. Filtered through qualitative filter paper before use.
Chloramine TSolution (N-chloro-4-methylphenylsulfonamide sodium salt) [ CH3C6H4SO2N(Na)Cl·3H2O]: 8.65g of chloramine T is dissolved in water and transferred to a 500mL volumetric flask, and the volume is fixed to the scale with water. Filtered through qualitative filter paper before use.
0.22mol/L NaOH buffer: NaOH 8.8g, Na2HPO426.0g，C6H8O7·H2O (citric acid monohydrate) 10.4g, dissolved in water and made to 1000 mL.
Buffer solution of sulfanilic acid: weighing C6H7NO3S (sulfanilic acid) 7g, Na2HPO426.0g，C6H8O7·H2O (citric acid monohydrate) 10.4g, dissolved in water and made to 1000 mL.
Chloramine T: weighing chloramine T1.2g, dissolving with pure water to a constant volume of 100mL, and storing with a brown reagent bottle.
Potassium cyanide: KCN 0.4g, dissolved with pure water to a constant volume of 100 mL.
NaCO3Solution: 10g NaCO3And dissolving in distilled water and fixing the volume to 1000 mL.
(3) And (3) an analysis step:
weighing 0.3g of the smoke cell sample in a 150mL triangular or plastic bottle (to the nearest 0.0001 g); adding 50mL of 5% acetic acid solution and covering a plug; shaking and extracting on a common shaking table for 30min, controlling the rotating speed at 170r/min, filtering with filter paper, and loading on a machine. (if the concentration of the sample solution is beyond the concentration range of the working standard solution, the sample solution should be diluted).
The total plant alkaloid content on a dry basis is given by the following formula:
in the formula:
c is the instrument observation value of the total plant alkaloid in the sample liquid, and the unit is mg/mL;
v is volume of extract liquid, unit is mL;
m is the mass of the sample, and the unit is mg;
w-moisture content of the sample in%.
Through three tests, the nicotine content of the mutant tobacco Ntbhlh13-1 is 2.06%, while the nicotine content of the mutant wild type tobacco is 1.18% (see figure 3), the nicotine content of the mutant material is improved by nearly 74.5%, and the result shows that the nicotine content of the tobacco is greatly improved after gene mutation, so that the material has great value for tobacco breeding, and the mutant Ntbhlh13-1 tobacco can be used for preparing high-nicotine materials.
Sequence listing
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<210>2
<211>20
<212>DNA
<213>NtbHLH13 F
<400>2
gaatgttctt taatggcaat 20
<210>3
<211>20
<212>DNA
<213>NtbHLH13 R
<400>3
cgagggaacg aaaagtacat 20
Claims (3)
1. A tobacco NtbHLH13 gene mutant is characterized in that the tobacco NtbHLH13 gene mutant is Ntbhlh13, wherein the 215 th G of the tobacco Ntbhlh13 gene is mutated into A to form a stop codon, so that the gene is stopped in advance, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
2. The molecular identification method of the tobacco NtbHLH13 gene mutant NtbHLH13-1 in claim 1, wherein the DNA fragment of the mutant NtbHLh13 is obtained by amplifying a primer pair, wherein the upstream primer of the primer pair is NtbHLH 13F, the nucleotide sequence of which is shown as SEQ ID N0.2, and the downstream primer is NtbHLH 13R, the nucleotide sequence of which is shown as SEQ ID N0.3.
3. Use of the tobacco NtbHLH13 gene mutant NtbHLH13-1 as defined in claim 1 in the preparation of high nicotine materials.
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