CN110862445A

CN110862445A - NtOEP1 gene influencing tobacco pigment content and application thereof

Info

Publication number: CN110862445A
Application number: CN201911316510.9A
Authority: CN
Inventors: 徐国云; 刘萍萍; 周会娜; 郑庆霞; 张慧; 翟妞; 陈千思; 金立锋
Original assignee: Zhengzhou Tobacco Research Institute of CNTC
Current assignee: Zhengzhou Tobacco Research Institute of CNTC
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2020-03-06
Anticipated expiration: 2039-12-19
Also published as: CN110862445B

Abstract

The invention belongs to the technical field of tobacco genetic engineering, and particularly relates to a method for influencing the content of tobacco pigmentNtOEP1Genes and patent applications of the genes. The gene comprises 999 bp bases, wherein the 153 th-522 th nucleotide is a specific core fragment thereof, and the base sequence is shown as SEQ ID NO. 1; the coding sequence has 332 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2. In this application, by treating tobaccoNtOEP1The gene has important application in the regulation and control of tobacco pigment synthesis path and is highly related to the content of pigment substances in plant leaves through cloning and analysis of the gene. Further by virusMediated Gene silencing techniques, the inventors have found thatNtOEP1After gene silencing, the content of pigment substances in the new transgenic plant is obviously reduced. By utilizing the characteristic, a new strategy and a new path can be provided for the genetic engineering breeding of tobacco or the breeding of other new plant varieties.

Description

NtOEP1 gene influencing tobacco pigment content and application thereof

Technical Field

The invention belongs to the technical field of tobacco genetic engineering, and particularly relates to a method for influencing the content of tobacco pigmentNtOEP1Genes and patent applications of the genes.

Background

The tobacco is used as a leaf economic crop, the cultivation technology of the tobacco is different from other field crops, a certain tobacco yield is required, and the quality of the tobacco is emphasized. The quality of the tobacco leaves determines the availability of the tobacco leaves, directly influences the color, the fragrance, the taste and the commodity value of the finished cigarette products, is also related to the economic benefit of tobacco growers, and is the life and the starting point of the tobacco industry.

The pigment is an important compound in the tobacco growth process and mainly comprises chlorophyll and carotenoid substances. Chlorophyll can be degraded and disappeared in a large amount in the processes of tobacco maturation and tobacco leaf modulation, and the main degradation modes are two types: one way is that chlorophyll is degraded by a porphyrin ring to generate pyrrole compounds, thereby increasing the aging fragrance of tobacco leaves; alternatively, phytol produced by hydrolysis of chlorophyll can be further degraded into neophytadiene and then into phytofurans, which are converted into the sweet components of tobacco leaves. Generally speaking, chlorophyll is beneficial to improving the quality of tobacco leaves after being fully degraded, but if the chlorophyll is not fully degraded, the chlorophyll becomes an unfavorable chemical component in dry tobacco leaves, and the tobacco leaves are obviously green and impure. If the chlorophyll is not fully degraded in the process of modulation treatment, the tobacco leaves are baked, and green and yellow tobacco with different degrees can be baked. The green and yellow tobacco has poor appearance quality and has obvious influence on the quality of tobacco leaves. Therefore, whether the chlorophyll is degraded sufficiently is one of the indexes which are strictly controlled in the tobacco leaf grading.

The yellow pigment in the tobacco leaves is mainly carotenoid, and generally, the carotenoid content in the tobacco leaves has positive correlation with the tobacco leaf quality, on one hand, the appearance quality of the tobacco leaves is directly correlated with the content of the components; on the other hand, the carotenoid is an important precursor of the tobacco aroma component, and has a positive correlation with the aroma quantity and the aroma quality of the tobacco. Studies show that a large part of flavor components in tobacco leaves are degradation products of carotenoids, and a plurality of compounds are key flavor components in the tobacco, such as ionone, damascone, isophorone and the like.

In a word, because the relation between the pigment substances in the tobacco leaves and the quality of the tobacco leaves is very close, the full and deep research on the genes related to the pigment substances of the tobacco leaves is realized, so that the pigment substances are controlled in a targeted manner, and the method has very important theoretical and application significance for improving the quality of the tobacco leaves and improving the varieties of the tobacco leaves.

Disclosure of Invention

The invention aims to provide a method for influencing the pigment content of tobaccoNtOEP1The gene can regulate and control the content of pigment substances in the tobacco, thereby laying a foundation for improving the quality of the tobacco.

The technical solution adopted in the present application is detailed as follows.

Influencing the pigment content of tobaccoNtOEP1The gene comprises 999 bp bases, wherein the 153 th-522 th nucleotide is a specific core fragment thereof, and the base sequence thereof is shown as SEQ ID NO. 1.

Said influencing of tobacco pigment contentNtOEP1Application of gene in regulation of leaf pigment content by regulating gene silencing technology or gene overexpression methodNtOEP1The gene expression quantity is used for regulating and controlling the content of the pigment substances in the leaves.

Said influencing of tobacco pigment contentNtOEP1The PCR amplification preparation method of the gene comprises the following steps:

(1) extracting (for example, taking tobacco K326 leaves as a sample) genome, and carrying out reverse transcription to obtain 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:

NtOEP1-F: 5'- ATGGCTGCCTCTCTACAAGC -3'，

NtOEP1-R: 5'- TCATTCAAGTTGGGCATACC -3'。

silencing said effect on tobacco pigment contentNtOEP1Silencing vector TRV2-NtOEP1To do so byNTOEP1153 th-522 th nucleotide in the gene is used as a guide sequence of VIGS, and the nucleic acid fragment is inserted into a TRV2-LIC empty vector to construct and obtain TRV2-NTOEP1A carrier, in particular:

(1) PCR amplification

To be provided withNTOEP1153 th-522 th nucleotide in the gene is used as a guide sequence of VIGS, and a primer sequence for designing and amplifying is designed as follows:

NtOEP1-VI-F: 5'-TCGACGACAAGACCCTGCAGGCAAACTGAACTCAAGGACT -3'，

NtOEP1-VI-R: 5'-TGAGGAGAAGAGCCCTGCAGAATCTCATCAAGGGTGTAGG-3'；

among the above sequences, "TCGACGACAAGACCCTGCAG" partial sequence at the 5 'end of the F primer portion and "TGAGGAGAAGAGCCCTGCAG" at the 5' end of the R primer portion are the linker sequence of TRV2-LIC vector;

(2) enzyme digestion and connection

Using restriction endonucleasesPstI, carrying out single enzyme digestion on the TRV2-LIC plasmid, and then utilizing Infusion ligase to connect the single enzyme digestion product of the TRV2-LIC with the PCR amplification fragment in the step (1);

(3) transformation and screening

Transforming the ligation product in the step (2) into an Escherichia coli competent cell DH5 α, coating the Escherichia coli competent cell DH5 α on LB medium containing kanamycin (50 mg/L), carrying out overnight inverted culture at 37 ℃ for screening to form a single colony, and determining the correct recombinant single clone as the TRV2-NtOEP1A recombinant vector.

Influencing the pigment content of tobaccoNTOEP1The protein coded by the gene comprises 332 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

Said influencing of tobacco pigment contentNTOEP1GeneThe coded protein is related to the content of the pigments in the plant leaves, and the content of the pigments in the leaves is obviously reduced after the expression content of the protein is reduced.

Using said influencing of tobacco pigment contentNtOEP1The new tobacco variety of gene is bred through interference, silencing, knockout or over expression of transgenic technology, instantaneous expression technology, genome editing technology, etcNtOEP1The gene can obtain a new variety of tobacco transformation plants with changed pigment content. Specific examples thereof include:

using virus-mediated gene silencing technology (VIGS) toNtOEP1153 th-522 th nucleotide in the gene is used as a guide sequence of VIGS, the nucleic acid fragment is inserted into a TRV2-LIC vector to construct and obtain TRV2-NtOEP1The vector is transformed into a plant and is obtained by screening and identifyingNtOEP1A gene-silenced plant, theNtOEP1The content of pigment substances in the gene silencing plants is obviously reduced.

In other words, a method for breeding a new variety of tobacco with low content of pigment substances by interfering with virus-induced gene silencing (VIGS)NtOEP1The expression of the gene causes it to be silenced,NtOEP1the pigment content in the plants of the new tobacco variety with the silent gene is obviously reduced.

In this application, by treating tobaccoNtOEP1The cloning of the gene and the analysis of the corresponding protein, the inventor finds that the gene has important application in the regulation of the tobacco pigment synthesis pathway and is highly related to the content of pigment substances in plant leaves. Further by the virus-mediated gene silencing technique, the inventors have found thatNtOEP1After gene silencing, the content of pigment substances in the new transgenic plant is obviously reduced. By utilizing the characteristic, a new strategy and a new path can be provided for the genetic engineering breeding of tobacco or the breeding of other new plant varieties.

Drawings

FIG. 1 compares to empty vector control plantsNtOEP1Relative expression of the gene in the gene-silenced plant;

FIG. 2 Virus induced silencingNtOEP1Post-gene tableAnd (4) type analysis.

Detailed Description

The present application is further illustrated by the following examples, and prior to describing the specific examples, the basic aspects of the biological materials, reagents, instruments, etc. involved in the examples described below are briefly described as follows.

Biological material:

tobacco material, cultivated tobacco: (Nicotiana tabacum) Variety K326, available from yuxi tobacco seeds llc; ben's cigarette (Ben's cigarette)Nicotiana benthamiana) The seeds are presented by the tobacco institute of Chinese academy of agricultural sciences;

vector plasmids TRV2-LIC (empty vector control), TRV1 and TRV2-PDSThe vector is purchased from China plasmid vector strain cell line gene collection center.

Gene sequencing and primer synthesis were performed by Shanghai Bioengineering Co., Ltd.

Experimental reagent:

restriction enzymes, dATP, dTTP, PrimeSTAR GXL DNA polymerase, DNA Gel recovery Kit MiniBEST Agarose Gel DNA Extraction Kit, plasmid DNA miniprep Kit MiniBEST plasmid purification Kit, acetosyringone, TAKARA biotechnology Limited products;

TRIZOL reagent for RNA extraction, manufactured by Invitrogen corporation;

reverse transcription kit, product of Roche company;

dnase enzyme, product of Fermentas corporation;

MES, product of Sigma;

insusion ligase, product of Clontech;

LB liquid medium (1L): 10 g bacterial peptone (bacteriological peptone), 10 g sodium chloride (NaCl), 5 g yeast extract (yeast extract), autoclaving;

1M 2- (N-morpholine) ethanesulfonic acid (MES) stock: ddH₂Dissolving MES in O, filtering, sterilizing, and storing at-20 deg.C;

200 mM Acetosyringone (Acetosyringone) stock solution: dissolving acetosyringone with anhydrous ethanol, and storing at-20 deg.C;

an experimental instrument:

the PCR instrument has the following model: mastercycler ep gradient, Eppendorf, Germany.

Example 1

This example affects primarily the tobacco pigment contentNtOEP1The process of obtaining the gene is briefly described below.

Tobacco RNA extraction and cDNA synthesis

(1) Total RNA extraction

Taking young leaves of tobacco K326 growing for about 3 weeks as samples, and fully grinding the samples into powder by liquid nitrogen;

placing about 100 mg of the powdery material into a 1.5 ml centrifuge tube containing 1.0 ml of TRIZOL reagent, adding 200 μ l of chloroform, shaking, mixing, centrifuging, and carefully transferring the upper aqueous phase into another centrifuge tube;

adding 500 μ l isopropanol, precipitating, centrifuging to separate RNA, washing with 75% ethanol, slightly drying at room temperature, adding appropriate volume of RNase free water, and dissolving completely;

finally, the extracted total RNA is treated by DNase I for the subsequent preparation of cDNA, and when the total RNA is treated by DNase I digestion, the reference of a 10-mu L reaction system is as follows:

1 μ g of total RNA extracted;

10×reaction buffer with MgCl₂，1 μl；

DNase I（RNase-free），1 μl （1U）；

DEPC-treated water to 10. mu.l;

placing in water bath at 37 deg.C for 30 min.

(2) cDNA Synthesis

Preparing a template RNA/primer mixed solution in a sterile 0.2 ml centrifuge tube, rapidly quenching for more than 2 min on ice after preserving heat for 10 min at 70 ℃, and centrifuging for several seconds to enable a template RNA/primer denatured solution to be gathered at the bottom of the centrifuge tube;

the template RNA/primer mixed solution (7 mu l system) specifically comprises:

RNA (100 ng/. mu.l) digested by DNase I in the step (1), and 1. mu.l;

Oligo (dT) Primer (50 μM)，1 μl；

RNase free dH₂O，5 μl；

preparing a reverse transcription reaction solution in the centrifuge tube, and then preserving heat for 1 h at 42 ℃; keeping the temperature at 70 ℃ for 15 min, and cooling on ice to obtain cDNA;

the specific reverse transcription reaction solution system (10. mu.l) was:

7. mu.l of the above template RNA/primer denaturing solution;

5×M-MLV buffer，2 μl；

dNTP mix (10 mM each), 0.5. mu.l;

RNase Inhibitor （40 U/μl），0.25 μl；

RTase M-MLV (RNase H-) (200 U/μl)，0.25 μl。

(3) PCR amplification

First, primer sequences for PCR amplification were designed as follows:

NtOEP1-F: 5'- ATGGCTGCCTCTCTACAAGC -3'，

NtOEP1-R: 5'- TCATTCAAGTTGGGCATACC -3'；

subsequently, PCR amplification was performed using the cDNA prepared in step (2) as a template, and a 50. mu.l amplification system was designed as follows:

cDNA template, 1. mu.l;

GXL polymerase，1 μl；

5×GXL buffer，10 μl；

dNTP Mixture (10 mM)，4 μl；

Primer-F/R，8 μl；

ddH₂O，26 μl；

the PCR reaction procedure is shown in the table below: at 98 ℃ for 10 sec; 55 deg.C, 10sec, 68 deg.C, 1min, 35 cycles, 72 deg.C extension for 5 min.

And (3) carrying out 1% agarose gel electrophoresis detection on the PCR product obtained by amplification, and then, recovering and purifying the PCR amplification product, and sequencing.

Sequencing results show that the clone of the application obtainsNtOEP1The gene comprises 999 bp bases, wherein the 153 th-522 th nucleotide is a specific core fragment thereof, and the base sequence is shown as SEQ ID NO.1 and specifically comprises the following steps:

ATGGCTGCCTCTCTACAAGCAGCTGCTACTCTAATGCAACCAACAAAGGTTGGTGTTGCCCCAGCTAGAAACAACCTGCAGTTGAGGTCTGCTCAAAGTGTGAGCAAAGCATTTGGTGTTGAACCAGCTGCAGCTAGGCTTACTTGCTCTTTGCAAACTGAACTCAAGGACTTGGCTCAAAAGTGCACTGATGCTGCCAAGGTTGCTGGTTTTGCTCTGGCCACTTCTGCCCTTGTCGTCTCAGGAGCAAATGCTGAAGGAGTTCCAAAACGTCTAACCTTCGACGAAATTCAAAGCAAGACATACATGGAAGTAAAGGGAACTGGAACTGCTAACCAGTGCCCTACGATAGAAGGAGGTGTTGCCAGCTTTGCCTTCAAGCCAGGCAAATACAATGCCAAGAAATTCTGCTTAGAGCCCACATCATTCACAGTCAAGGCAGAGAGTGTGAACAAGAATGCACCCCCAGATTTCCAGAAAACCAAGCTCATGACACGCTTAACCTACACCCTTGATGAGATTGAGGGACCATTCGAAGTGTCTTCTGATGGCACTGTTAAGTTTGAGGAGAAGGATGGAATTGATTATGCTGCTGTTACAGTTCAGCTTCCTGGTGGTGAGCGTGTGCCCTTCCTCTTCACTATCAAACAGCTAGTGGCAAGCGGCAAACCAGAAAGCTTTAGCGGTGAATTCCTTGTGCCATCATACAGAGGTTCATCCTTCCTTGACCCAAAGGGACGGGGTGGATCTACTGGCTATGACAACGCTGTTGCATTGCCTGCTGGAGGGAGAGGAGACGAGGAGGAGCTTGAGAAGGAGAACGTAAAGAATACTGCATCTTCTACAGGAAAGATCACCCTGAGTGTTACCCAGTGCAAGCCAGAGACCGGTGAGGTCATTGGAGTATTTGAGAGCATCCAGCCATCTGATACTGATCTCGGTGCAAAGGTCCCCAAGGATGTGAAAATCCAGGGTATCTGGTATGCCCAACTTGAATGA。

to pairNtOEP1After the gene is analyzed, the gene codes 332 amino acids, and the amino acid sequence is shown as SEQ ID NO.2, and specifically comprises the following steps:

MAASLQAAATLMQPTKVGVAPARNNLQLRSAQSVSKAFGVEPAAARLTCSLQTELKDLAQKCTDAAKVAGFALATSALVVSGANAEGVPKRLTFDEIQSKTYMEVKGTGTANQCPTIEGGVASFAFKPGKYNAKKFCLEPTSFTVKAESVNKNAPPDFQKTKLMTRLTYTLDEIEGPFEVSSDGTVKFEEKDGIDYAAVTVQLPGGERVPFLFTIKQLVASGKPESFSGEFLVPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRGDEEELEKENVKNTASSTGKITLSVTQCKPETGEVIGVFESIQPSDTDLGAKVPKDVKIQGIWYAQLE。

example 2

Using the tobacco pigment content obtained in example 1NtOEP1Gene, the inventors further constructed a VIGS interference vector TRV2 for gene silencingNtOEP1The related construction process is briefly described as follows.

(1) Obtaining the fragment of interest

SelectingNTOEP1A specific nucleic acid segment (nucleotide sequence 153-522 of SEQ ID NO. 1) in the gene is a guide sequence of VIGS, a primer is designed, and the cDNA obtained in the implementation 1 is used as a template for PCR amplification to obtain a target gene segment.

NtOEP1When the gene silencing vector is constructed, the primer sequence used for PCR amplification is designed as follows:

NtOEP1-VI-F: 5'-TCGACGACAAGACCCTGCAGGCAAACTGAACTCAAGGACT -3'，

NtOEP1-VI-R: 5'-TGAGGAGAAGAGCCCTGCAGAATCTCATCAAGGGTGTAGG-3'；

the relevant amplification system and the amplification procedure were as described in example 1, and the final amplified fragment length was 370 bp.

(2) Enzyme digestion and connection

Using restriction endonucleasesPstI, carrying out single enzyme digestion on TRV2-LIC plasmid, then utilizing Infusion ligase to connect the single enzyme digestion product of TRV2-LIC with the PCR amplification fragment in the step (1), and designing a 10 mu l treatment system as follows:

2 x Infusion ligase, 2 μ l;

TRV2-LIC Single restriction fragment, 2. mu.l

PCR amplified fragment, 6. mu.l;

at 50 deg.C for 15 min, and then on ice for 1-2 min.

(3) Transformation and screening

Transforming the ligation product in the step (2) into an Escherichia coli competent cell DH5 α, coating the Escherichia coli competent cell DH5 α on LB medium containing kanamycin (50 mg/L), carrying out overnight inverted culture at 37 ℃ for screening to form a single colony, and further carrying out sequencing identification to determine whether the recombination is correct, namely the recombination is interference TRV2-NtOEP1A recombinant vector.

Example 3

Based on example 2, the inventors further utilizedAgrobacterium mediated transgenic system and screeningNtOEP1Transgenic plants with gene silencing, combining plant phenotypes toNtOEP1Further analysis of gene function was performed. The specific process is briefly described as follows.

(1) Transforming agrobacterium and preparing infection liquid

The TRV2 prepared in example 2NtOEP1After GV3301 Agrobacterium-infected cells are transformed by the recombinant vector, LB plate (containing 50 mg/L kanamycin and 50 mg/L rifampicin) is coated, inverted and dark culture is carried out at 28 ℃ for 2-3 days, single colony is picked up, and colony PCR screening and carrying is carried outNtOEP1Agrobacterium of gene fragment is monoclonal;

as controls, TRV1, TRV2-LIC, and TRV2-PDS were also transformed with Agrobacterium, respectively.

Respectively contain TRV1, TRV2-LIC and TRV2-NtOEP1TRV2-PDS Agrobacterium tumefaciens single colony is inoculated into 5 ml LB culture medium (containing 50 mg/L kanamycin and 50 mg/L rifampicin), and is shake-cultured at 28 ℃ and 180 r/min overnight until OD is about 1.0; inoculating into 30 ml LB liquid culture medium for continuous culture, and shaking culturing at 28 deg.C overnight until OD is about 2.0;

centrifuging at 3900 r/min for 15 min to collect Agrobacterium, discarding the supernatant, adding injection buffer (10 mM MES, 10 mM MgCl) to each cell₂250 mu M acetosyringone), adjusting the OD value to about 1.0, and standing at room temperature for 3-6 hours;

further to TRV2-LIC, TRV2-NtOEP1、TRV2-PDSAdding TRV1 suspension into the suspension in a medium volume, and mixing to obtain the final product.

(2) Plant infection

Selecting Bunsen tobacco seedlings growing for about 4 weeks (tobacco seeds are sown in seedling pots in advance for seedling culture, seedling division is carried out two weeks after germination, the seedlings are planted in plastic pots (10 cm multiplied by 10 cm) and are grown and cultured under the conditions of 22 ℃, 16 h illumination/8 h darkness), after grouping, selecting tobacco plants with consistent growth vigor and about 4-5 leaves, pressing agrobacterium tumefaciens suspension liquid containing different TRV recombinant plasmids into all unfolded leaves from the back of the leaves by a 1ml pinless sterile injector through a pressure filtration method, filling the whole leaves with bacterial liquid, and culturing in air humidity of 22 ℃ and 75%.

After inoculating for 2 weeks, collecting leaves and extracting RNA, detecting gene silencing efficiency by using real-time PCR, and measuring the content of pigment substances in the tobacco leaves by using high performance liquid chromatography.

The results of the gene expression level detection after gene silencing are shown in FIG. 1, and the results of the partial phenotype are shown in FIG. 2. The specific pigment content measurements are shown in the following table (unit: μ g/g):

。

as can be seen by comprehensive analysis, TRV2-NtOEP1In plantsNtOEP1The expression level of the gene is only about 15% of that of the control (FIG. 1), indicating that the silencing effect is significant. Phytoene Dehydrogenase (PDS) is one of rate-limiting enzymes affecting carotenoid synthesis, and tobacco silencing of the gene can cause leaf chlorosis and bleaching phenotype, and the gene is also used as a marker gene for detecting silencing efficiency at present. Positive control TRV2-PDSThe fresh leaves were completely bleached and completely green, indicating that the results obtained with our injection are truly reliable (fig. 2).NtOEP1After gene silencing, the new leaves are light yellow and have obvious green losing phenotype, and the negative control TRV2-LIC empty vector control plant new leaves have the same phenotype as the non-injected plants. In addition, TRV 2-induced silencing by virusesNtOEP1The plant content of neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b and β carotene was significantly reduced compared to the control plants (table 1), indicating thatNtOEP1The gene is related to the tobacco pigment content. These experimental results show that the pigment content in tobacco leaves can be adjusted significantly by a virus-induced gene silencing method.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> NtOEP1 gene influencing tobacco pigment content and application thereof

<130>none

<160>2

<170>PatentIn version 3.5

<210>1

<211>999

<212>DNA

<213>Nicotiana tabacum

<400>1

atggctgcct ctctacaagc agctgctact ctaatgcaac caacaaaggt tggtgttgcc 60

ccagctagaa acaacctgca gttgaggtct gctcaaagtg tgagcaaagc atttggtgtt 120

gaaccagctg cagctaggct tacttgctct ttgcaaactg aactcaagga cttggctcaa 180

aagtgcactg atgctgccaa ggttgctggt tttgctctgg ccacttctgc ccttgtcgtc 240

tcaggagcaa atgctgaagg agttccaaaa cgtctaacct tcgacgaaat tcaaagcaag 300

acatacatgg aagtaaaggg aactggaact gctaaccagt gccctacgat agaaggaggt 360

gttgccagct ttgccttcaa gccaggcaaa tacaatgcca agaaattctg cttagagccc 420

acatcattca cagtcaaggc agagagtgtg aacaagaatg cacccccaga tttccagaaa 480

accaagctca tgacacgctt aacctacacc cttgatgaga ttgagggacc attcgaagtg 540

tcttctgatg gcactgttaa gtttgaggag aaggatggaa ttgattatgc tgctgttaca 600

gttcagcttc ctggtggtga gcgtgtgccc ttcctcttca ctatcaaaca gctagtggca 660

agcggcaaac cagaaagctt tagcggtgaa ttccttgtgc catcatacag aggttcatcc 720

ttccttgacc caaagggacg gggtggatct actggctatg acaacgctgt tgcattgcct 780

gctggaggga gaggagacga ggaggagctt gagaaggaga acgtaaagaa tactgcatct 840

tctacaggaa agatcaccct gagtgttacc cagtgcaagc cagagaccgg tgaggtcatt 900

ggagtatttg agagcatcca gccatctgat actgatctcg gtgcaaaggt ccccaaggat 960

gtgaaaatcc agggtatctg gtatgcccaa cttgaatga 999

<210>2

<211>332

<212>PRT

<213>Nicotiana tabacum

<400>2

Met Ala Ala Ser Leu Gln Ala Ala Ala Thr Leu Met Gln Pro Thr Lys

1 5 10 15

Val Gly Val Ala Pro Ala Arg Asn Asn Leu Gln Leu Arg Ser Ala Gln

20 25 30

Ser Val Ser Lys Ala Phe Gly Val Glu Pro Ala Ala Ala Arg Leu Thr

35 40 45

Cys Ser Leu Gln Thr Glu Leu Lys Asp Leu Ala Gln Lys Cys Thr Asp

50 55 60

Ala Ala Lys Val Ala Gly Phe Ala Leu Ala Thr Ser Ala Leu Val Val

65 70 75 80

Ser Gly Ala Asn Ala Glu Gly Val Pro Lys Arg Leu Thr Phe Asp Glu

85 90 95

Ile Gln Ser Lys Thr Tyr Met Glu Val Lys Gly Thr Gly Thr Ala Asn

100 105 110

Gln Cys Pro Thr Ile Glu Gly Gly Val Ala Ser Phe Ala Phe Lys Pro

115 120 125

Gly Lys Tyr Asn Ala Lys Lys Phe Cys Leu Glu Pro Thr Ser Phe Thr

130 135 140

Val Lys Ala Glu Ser Val Asn Lys Asn Ala Pro Pro Asp Phe Gln Lys

145 150 155 160

Thr Lys Leu Met Thr Arg Leu Thr Tyr Thr Leu Asp Glu Ile Glu Gly

165 170 175

Pro Phe Glu Val Ser Ser Asp Gly Thr Val Lys Phe Glu Glu Lys Asp

180 185 190

Gly Ile Asp Tyr Ala Ala Val Thr Val Gln Leu Pro Gly Gly Glu Arg

195 200 205

Val Pro Phe Leu Phe Thr Ile Lys Gln Leu Val Ala Ser Gly Lys Pro

210 215 220

Glu Ser Phe Ser Gly Glu Phe Leu Val Pro Ser Tyr Arg Gly Ser Ser

225 230 235 240

Phe Leu Asp Pro Lys Gly Arg Gly Gly Ser Thr Gly Tyr Asp Asn Ala

245 250 255

Val Ala Leu Pro Ala Gly Gly Arg Gly Asp Glu Glu Glu Leu Glu Lys

260 265 270

Glu Asn Val Lys Asn Thr Ala Ser Ser Thr Gly Lys Ile Thr Leu Ser

275 280 285

Val Thr Gln Cys Lys Pro Glu Thr Gly Glu Val Ile Gly Val Phe Glu

290 295 300

Ser Ile Gln Pro Ser Asp Thr Asp Leu Gly Ala Lys Val Pro Lys Asp

305 310 315 320

Val Lys Ile Gln Gly Ile Trp Tyr Ala Gln Leu Glu

325 330

Claims

1. Influencing the pigment content of tobaccoNtOEP1The gene is characterized by comprising 999 bp bases, wherein the 153 th-522 th nucleotide is a specific core fragment thereof, and the base sequence is shown as SEQ ID NO. 1.

2. The method of claim 1 for affecting tobacco color contentNtOEP1The application of gene in the regulation of leaf pigment content is characterized by utilizing gene silencing technology or gene overexpression method to regulateNtOEP1The gene expression quantity is used for regulating and controlling the content of the pigment substances in the leaves.

3. The method of claim 1 for affecting tobacco color contentNtOEP1The PCR amplification preparation method of the gene is characterized by comprising the following steps:

(1) extracting a tobacco sample genome, and carrying out reverse transcription to obtain cDNA for later use;

NtOEP1-F: 5'- ATGGCTGCCTCTCTACAAGC -3'，

NtOEP1-R: 5'- TCATTCAAGTTGGGCATACC -3'。

4. silencing of the effects on tobacco pigment content of claim 1NtOEP1Silencing vector TRV2-NtOEP1The vector is characterized by being constructed and obtained through the following steps:

(1) PCR amplification

NtOEP1-VI-F: 5'-TCGACGACAAGACCCTGCAGGCAAACTGAACTCAAGGACT -3'，

NtOEP1-VI-R: 5'-TGAGGAGAAGAGCCCTGCAGAATCTCATCAAGGGTGTAGG-3'；

(2) enzyme digestion and connection

(3) transformation and screening

Transforming the ligation product in the step (2) into an Escherichia coli competent cell DH5 α, coating the Escherichia coli competent cell DH5 α on an LB culture medium containing kanamycin, carrying out overnight inverted culture at 37 ℃ for screening to form a single colony, and further identifying to determine that the correct recombinant single clone is the TRV2-NtOEP1A recombinant vector.

5. The method of claim 1 for affecting tobacco color contentNtOEP1The protein coded by the gene is characterized by comprising 332 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

6. The method of claim 5 for affecting tobacco color contentNtOEP1The application of the protein coded by the gene in regulating and controlling the content of the leaf pigments is characterized in that the protein is related to the content of the pigments in the plant leaves, and the content of the pigments in the leaves is obviously reduced after the expression content of the protein is reduced.

7. Use of the composition of claim 1 for influencing tobacco pigment contentNtOEP1A method for breeding a new tobacco variety of a gene, characterized in that the gene is disrupted, silenced, knocked out or overexpressed by a transgenic technique, a transient expression technique or a genome editing techniqueNtOEP1The gene can obtain a new variety of tobacco transformation plants with changed pigment content.