阅读说明：本技术 一种烟草NtIAA27基因突变体及分子鉴定方法和应用 (Tobacco NtIAA27 gene mutant and molecular identification method and application ) 是由 谢贺 白戈 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NtIAA27基因突变体及分子鉴定方法和应用,所述烟草NtIAA27基因突变体为Ntiaa27-1,其为烟草NtIAA27基因第576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NtIAA27基因突变体Ntiaa27-1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 NtIAA27 gene mutant and a molecular identification method and application thereof are disclosed, wherein the tobacco NtIAA27 gene mutant is Ntiaa27-1, which is obtained by mutating the 576 th G of the tobacco NtIAA27 gene 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 SEQ ID No. 1. After the tobacco NtIAA27 gene mutant Ntiaa27-1 is mutated, the nicotine content of tobacco can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtIAA27 gene mutant has great value in tobacco breeding.)

1. A tobacco NtIAA27 gene mutant is characterized in that the tobacco NtIAA27 gene mutant is Ntiaa27-1, wherein the 576-th G of the tobacco NtIAA27 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 SEQ ID No. 1.

2. The molecular identification method of the tobacco NtiiA 27 gene mutant Ntiaa27-1 of claim 1, wherein the DNA fragment of the mutant Ntiaa27-1 is obtained by amplifying a primer pair, wherein an upstream primer of the primer pair is NtiiA 27F, the nucleotide sequence of the primer pair is shown as SEQ ID N0.2, and a downstream primer of the primer pair is NtiiA 27R, the nucleotide sequence of the primer pair is shown as SEQ ID N0.3.

3. The use of the tobacco NtIAA27 gene mutant NtIAA27-1 of claim 1 in the preparation of a high nicotine material.

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a tobacco NtIAA27 gene mutant Ntiaa27-1 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 NtIAA27 gene mutant Ntiaa27-1 and a molecular identification method thereof, and also provides application of the tobacco NtIAA27 gene mutant Ntiaa 27-1.

The technical scheme adopted by the invention is as follows:

a tobacco NtIAA27 gene mutant is a tobacco Ntiaa27-1 gene, wherein the 576-th G of the tobacco NtIAA27 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 SEQ ID No. 1.

The molecular identification method of the tobacco NtiaA27 gene mutant Ntiaa27-1 comprises the step of amplifying a DNA fragment of the mutant Ntiaa27-1 by using a primer pair, wherein an upstream primer of the primer pair is NtiaA 27F, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.2, a downstream primer of the primer pair is NtiaA 27R, and the nucleotide sequence of the downstream primer is shown as SEQ ID N0.3.

The tobacco NtIAA27 gene mutant Ntiaa27-1 is used for preparing high nicotine materials.

After the tobacco NtIAA27 gene mutant Ntiaa27-1 is mutated, the nicotine content of tobacco can be obviously increased, the tobacco quality is obviously improved, and the tobacco mutant NtIAA27 gene mutant has great value in tobacco breeding.

Drawings

FIG. 1 is an amplification band of the tobacco NtIAA27 gene mutant Ntiaa 27-1;

FIG. 2 shows the sequencing result of the tobacco NtIAA27 gene mutant Ntiaa 27-1;

FIG. 3 shows the nicotine content of the tobacco NtIAA27 gene mutant Ntiaa27-1 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 tobacco NtIAA27 gene with sodium hypochlorite, and then washing with 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 Ntiaa 27-1:

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 NtIAA 27F, the nucleotide sequence of the upstream primer is shown as SEQ ID N0.2, a downstream primer of the primer pair is NtIAA 27R, 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) 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 NtIAA 27F (nucleotide sequence is shown as SEQ ID No. 0.2) and NtIAA 27R (nucleotide sequence is shown as SEQ ID No. 0.3) are used for identifying mutants, and finally homozygous mutant plants of which the mutants are Ntiaa27-1 are obtained. Compared with the nucleotide sequence of the wild tobacco NtIAA27 gene, the mutant contains NtIAA27 gene sequence, wherein G at 576 position 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 Ntiaa27-1 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 T solution (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:

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, 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 detections, the nicotine content of the tobacco Ntiaa27-1 mutant tobacco is 1.65%, while the nicotine content of the wild type tobacco is 1.18% (see fig. 3), and the nicotine content of the mutant material is improved by nearly 39.8%, which proves 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 Ntiaa27-1 tobacco can be used for preparing high-nicotine materials.

Sequence listing

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

<120> tobacco NtIAA27 gene mutant, molecular identification method and application

<141>2020-06-01

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>3177

<212>DNA

<213> tobacco (Ntiaa27-1)

<400>1

atgttaaagt acctcatgga acgaagtaat gagaaacaac tacaaataca aaggctaggg 60

gatactaacc aaatacaatg ggatactatc caaaggcaag aggcaactat tcttaacctg 120

gaggtacaag tgaggcaatt gtttgaggct ttacatgctc aacaagataa tattatgaat 180

agtagccatg agcagtatgc attagagaca gaaattgagg tgtcaataga agggtccata 240

gtagaacacc aacaatctat caaactagaa tttgaggatg tcgatgttgt agaagagata 300

ctagtatcaa ccaaggacat tgatgataca tatttagttg actataatgt gattggtgtt 360

gagggtgtcg aaaatattga agttcatgta tttgagcgcg ttggacctca ttccaaacac 420

ttatctacct tgtgttctga tggtgaaatg aaaattaatc cacatgagca taaagagggg 480

tcaagaaaaa aggtagatga tccttatatt ttgaaaattt taaggtcgtc tatgcaagca 540

gcaggggaga agaaatgctt gaattcagag ttgtgacacg cgtgtgccgg gccactagtt 600

tcgcttccgc ctgtaggaag cagagttgtg tattttcctc aagggcatag tgaacaggtt 660

gctgcctcga caaacaagga agtagatgct catatcccta actatcctgg tttaccacct 720

cagctaattt gtcagcttca caacctgaca atgcatgcag atgttgagac cgatgaagta 780

tatgctcaaa tgacgttgca gccactaagt gcacaagagc aaaaggatgt gtgcctgcta 840

ccagcagaac ttggcatccc gagtaaacaa ccaaccaact atttctgcaa aaccttgacg 900

gcaagtgaca ccagtactca cggtggattc tctgtccccc gacgtgcagc agaaaaagtt 960

tttccccctc ttgattactc tcagcagccg ccctgtcaag agttgattgc aaaagatctc 1020

catggaaatg aatggaaatt ccggcatatt tttcgtggcc aaccaaagag gcatctattg 1080

acaacaggat ggagtgtgtt cgtaagtgca aagagacttg ttgcgggtga tgcagtcatc 1140

tttatctgga atgaaaataa tcaattgctt ttggggattc gacgtgctaa tcgtcctcaa 1200

accgttatgc cttcttcagt tttgtcaagt gatagcatgc acattggtct ccttgctgcg 1260

gcggctcatg cagctgcaac taatagccgc tttacaatat tttataatcc aagggcaagt 1320

ccatcagagt ttgtcatacc tcttgccaag tatgctaaag cagtttatca tacacggatt 1380

tctgttggta tgaggttccg gatgctgttt gaaacagaag aatcgagtgt ccgtaggtat 1440

atgggcacaa ttaccggtat cagtgattta gatcctgttc gttggccaaa ttcacattgg 1500

cggtctgtga aggttggatg ggatgaatca actgcaggag agaggcagcc cagagtttcg 1560

ctgtgggaaa ttgaacctct gacaactttt cctatgtatc cttctccttt ctctcttagg 1620

ctaaaaaggc cttggccatc tggactacct tctctccctg gttttcccaa tggtgatatg 1680

actatgaatt ctccactctc gtggctgcgt ggtgacatag gagaccaagg gattcagtcg 1740

cttaatttcc agggctatgg tgttactccg tttatgcagc caagaattga tgcttctatg 1800

ttaggtttgc aacctgacat tctgcaaaca atggctgcac tagatccatc gaaacttgca 1860

aatcaatcct ttatgcagtt ccaacaaagt atacctggcg gttcagcatc tttgagtcat 1920

agtcaaattt tgcagccttc tcattcacag caaaatctgc tccacggctt ctccgaaaac 1980

cagttaatat ctcaggcaca gatgcttcag caacaattgc agcgccgtca gaattataat 2040

gatcaacagc aattgctgca gccacagctt cagcaacacc aagaagtgaa ctcctcgcag 2100

tttcaacatc aacagcaaac caaggccatg tccagtctct ctcagatgac ttcggctgcg 2160

cagccccagc tttctcattt gcgagtctta agttcaactg gttctccaca aacattttct 2220

gatatacttg gtaaccatgt caatgcatct agtaattcta ctatgcaaag tctgttgagt 2280

tcattttccc gtgatggagc gtctgctgtc cttaacatgc atgaagctca ccctctagtg 2340

tcttcgtcct catcatcaaa gcgaattgct ctagaatctc agctcccttc tcgggttact 2400

ccattcgctg tgccccagcc tgaggatgtg atatcacaca atactaaagt ttctgatctt 2460

tcctctctgt tgcccccttt tcctggcaga gagtcttttt ctgattatag aggagtagaa 2520

gatagccaaa acaatgcact gtatggattt aataccgact ctttgaacat actgccgaat 2580

ggtatgtcca acatgaagga tagtagtggt gataatggat ctttatctat tccttatgct 2640

acctctacct tcacaaatac tgtgggcaac gagtatccca ttaactcaga catgacaact 2700

tcaagttgtg tagatgaatc aggtttcttg cagtcctccg agaatgggga tcaaggaaac 2760

ccaactaata gaacctttgt gaaggttcat aaatcagggt cctttggacg gtcactcgat 2820

atctccaagt ttagcagcta tcacgaactt cgaagtgagc ttgctcacat gtttgggcta 2880

gaaggcttgt tggaggaccc tgagagatca ggctggcagc ttgtatttgt agaccgagag 2940

aatgatgttc tcctcctcgg tgacgatccc tggcaggagt ttgtgaacaa tgtttggtac 3000

ataaagatac tttctccact cgaagtgcaa cagatgggga aagacggcct tgatcttcca 3060

aatgctggcc tagtacaaag gcttcctagc aatggcgtcg gatgtgatga ctatatgaac 3120

caaaagggct cccaaaatgc catgaatggg atacccttgg ggtcgctcga ctactaa 3177

<210>2

<211>25

<212>DNA

<213>NtIAA27 F

<400>2

cttgaagtga agcattrttt gtagt 25

<210>3

<211>25

<212>DNA

<213>NtIAA27 R

<400>3

tcccgaaatt aatyaaatat gaacc 25