阅读说明：本技术 一种烟草NtARF6基因突变体及分子鉴定方法和应用 (Tobacco NtARF6 gene mutant and molecular identification method and application ) 是由 白戈 谢贺 李勇 杨大海 姚恒 逄涛 费明亮 蔺忠龙 张谊寒 于 2020-06-01 设计创作，主要内容包括：一种烟草NtARF6基因突变体及分子鉴定方法和应用,所述烟草NtARF6基因突变体为Ntarf6-1,其为烟草Ntarf6基因第261位的G突变为A,形成终止密码子,使该基因提前终止,其核苷酸序列如SEQ ID No.1所示。本发明的烟草NtARF6基因突变体Ntarf6-1在基因发生突变后,烟草烟碱含量会明显上调,显著提高烟草品质,在烟草育种上具有极大的价值。(A tobacco NtARF6 gene mutant and a molecular identification method and application thereof are disclosed, wherein the tobacco NtARF6 gene mutant is Ntarf6-1, which is a termination codon formed by mutating G at the 261 th position of a tobacco Ntarf6 gene to A, and the nucleotide sequence of the gene is shown as SEQ ID No. 1. After the tobacco NtARF6 gene mutant Ntarf6-1 is subjected to gene mutation, the nicotine content of tobacco can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtARF6 gene mutant has great value in tobacco breeding.)

1. The tobacco NtARF6 gene mutant is characterized in that the tobacco NtARF6 gene mutant is Ntarf6-1, the 261 th G of the tobacco Ntarf6 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 NtARF6 gene mutant NtARF6-1, as claimed in claim 1, wherein the DNA fragment of the mutant NtARF6-1 is amplified by a primer pair, the upstream primer of the primer pair is NtARF6F, the nucleotide sequence of which is shown in SEQ ID N0.2, and the downstream primer of which is NtARF 6R, the nucleotide sequence of which is shown in SEQ ID N0.3.

3. Use of the tobacco NtARF6 gene mutant NtARF6-1 of claim 1 in the preparation of high nicotine material.

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a tobacco NtARF6 gene mutant Ntarf6-1 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 NtARF6 gene mutant Ntarf6-1 and a molecular identification method thereof, and also provides application of the tobacco NtARF6 gene mutant Ntarf 6-1.

The technical scheme adopted by the invention is as follows:

a tobacco NtARF6 gene mutant is Ntarf6-1, wherein the 261 th G of the tobacco NtaRF6 gene is mutated into A to form a stop codon, so that the gene is terminated in advance, and the nucleotide sequence of the gene is shown as SEQID No. 1.

According to the molecular identification method of the tobacco NtARF6 gene mutant Ntarf6-1, a DNA fragment of the mutant Ntarf6-1 is obtained by amplifying the following primer pair, wherein the upstream primer of the primer pair is NtARF6F, the nucleotide sequence of the primer pair is shown as SEQ ID N0.2, the downstream primer is NtARF 6R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N0.3.

The tobacco NtARF6 gene mutant Ntarf6-1 is used for preparing high nicotine materials.

After the tobacco NtARF6 gene mutant Ntarf6-1 is subjected to gene mutation, the nicotine content of tobacco can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtARF6 gene mutant has great value in tobacco breeding.

Drawings

FIG. 1 is an amplification band of the tobacco NtARF6 gene mutant Ntarf 6-1;

FIG. 2 shows the sequencing result of the tobacco NtARF6 gene mutant Ntarf 6-1;

FIG. 3 shows the nicotine content of the tobacco NtARF6 gene mutant Ntarf6-1 individual plant and the wild type individual 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 with the NtARF6 gene of the tobacco by using sodium hypochlorite, and then washing the tobacco seeds with distilled water;

(2) soaking tobacco seeds in a phosphate buffer solution to increase the germination rate of the seeds;

(3) soaking the soaked tobacco seeds in 0.5% EMS (ethyl methane sulfonate) solution for 10-15 hours, and then centrifuging and draining 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 Ntarf 6-1:

(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 NtARF6F, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.2, a downstream primer of the primer pair is NtARF 6R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N0.3.

The PCR amplification reaction conditions were as follows:

the amplified band is shown in FIG. 1;

(2) 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 according to the product instruction by adopting a PCR product purification kit of Qiagen company, 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 NtARF6F (nucleotide sequence is shown as SEQ ID No. 0.2) and NtARF 6R (nucleotide sequence is shown as SEQ ID No. 0.3) are used for identifying the mutant, and finally, homozygous mutant plants of which the mutant is Ntarf6-1 are obtained. Compared with the nucleotide sequence of the wild tobacco NtARF6 gene, the G at the position 261 of the NtARF6 gene sequence is mutated into A, so that the base is changed from tryptophan to a stop mutation to form a stop codon, thereby causing the gene to be terminated early. The nucleotide sequence of the mutant Ntarf6-1 is shown as 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) (allence, france);

preparing a reagent: brij35 solution (polyethoxy lauryl ether): adding 5 drops of 22% Brij35 into water, and stirring uniformly;

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. Filtering with 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. Filtering with qualitative filter paper before use;

chloramine T solution (N-chloro-4-methylphenylsulfonamide sodium salt) [ CH₃C₆H₄SO₂N(Na)Cl·3H₂O]: 8.65g of chloramine T is taken, dissolved in water and then transferredPut into a 500mL volumetric flask and water is added to the volume to the scale mark.

Filtering with qualitative filter paper before use;

0.22mol/L NaOH buffer: NaOH 8.8g, Na₂HPO₄26.0g，C₆H₈O₇·H₂10.4g of O (citric acid monohydrate), dissolving with water and metering to 1000 mL;

buffer solution of sulfanilic acid: weighing C₆H₇NO₃S (sulfanilic acid) 7g, Na₂HPO₄26.0g，C₆H₈O₇·H₂10.4g of O (citric acid monohydrate), dissolving with water and metering 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: 0.4g of KCN, and dissolving with pure water to a constant volume of 100 mL;

NaCO₃solution: 10g NaCO₃Dissolving with distilled water and fixing the volume to 1000 mL;

(3) and (3) an analysis step:

weigh 0.3g of tobacco sample into a 150mL Erlenmeyer flask 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, and filtering with filter paper and processing on the machine (if the concentration of the sample solution exceeds the concentration range of the working standard solution, diluting should be carried out).

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 times of detection, the nicotine content of the Ntarf6-1 mutant tobacco is 1.63%, the nicotine content of the wild type tobacco is 1.18% (see figure 3), and the nicotine content of the mutant material is improved by nearly 38.1%, so that the nicotine content of the tobacco is greatly improved after gene mutation, the material has great value for tobacco breeding, and the mutant Ntarf6-1 tobacco can be used for preparing a high-nicotine material.

Sequence listing

<110> research institute of tobacco agricultural science in Yunnan province

<120> tobacco NtARF6 gene mutant, molecular identification method and application

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>831

<212>DNA

<213> tobacco (Ntarf6-1)

<400>1

atgtctatac cattcgaaca tgattatata ggtttatcag aagcttcttt aatggaaaga 60

aattctgata aaaatagtga tgttcttaac cttaaggaga ctgagctaag acttgggtta 120

ccgggtactg aggaaacaaa tacgggtctt aacccttcaa acaattttat atcaagaacc 180

aaaaggggtt tttctgatgc cattgatgct tctggaaaat gggatttgtc cattaattgc 240

agatcagaaa ctgattgaag aaaagaagac ttgttatttt cccccaaagg aagtaatgga 300

agctcaaagc caactccatc aattgaaaat agtgctcctc agacttcaaa ggcacaagta 360

gtaggatggc caccaattag atcattccgc aaaaatacac tggccaccaa aaagaatgat 420

gctgaaggaa aatcaggttc aggttgcctt tatgtgaaag ttagaatgga tggtgctcca 480

tatttgagaa aggttgatat caaaacttac agcgactatg gggagctctc atcagcactt 540

gaaaagatgt tcagctgctt tagtattggg cagtgcgcca gcgatggact tccggggcaa 600

gaggaactta gtgaaagtca cttgatggat cttctcaatg gttctgagta tgttctgact 660

tatgaagaca aagatggtga ttggatgctt gttggcgatg ttccttggga gatgttcata 720

gactcatgca agagattgcg gatcatgaaa agctcagagg caattgggct aggtatgtcc 780

tatcactgtg atcctgtcca tgctgtattt tatgtctata cttaccattg a 831

<210>2

<211>29

<212>DNA

<213>NtARF6 F

<400>2

tctataccat tcgaacatga ttatatagg 29

<210>3

<211>21

<212>DNA

<213>NtARF6 R

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cttcctttrg gggaaaataa c 21