CN107099540B - NtFERL gene influencing tobacco pigment content and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for influencing the pigment content of tobaccoNtFERLGenes and patent applications of the genes. The gene comprises 2667bp base, and the base sequence is shown as SEQ ID NO. 1; wherein the 2017-2405 nucleotide is a specific core fragment thereof. This application deals with tobaccoNtFERLThe 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 thatNtFERLAfter 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

NtFERL gene influencing tobacco pigment content and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for influencing the pigment content of tobaccoIs/are as followsNtFERLGenes and patent applications of the genes.

Background

Tobacco is a plant of the genus Nicotiana of the family Solanaceae of the order tubuliformes of the class Dicotyledoneae, genus Nicotiana (A)Nicotiana) There are more than 60 species, of which 2 of the cultivars common tobacco (also known as nicotiana tabacum,Nicotiana tabacum) Tobacco yellow flower (occupying major area)Nicotiana rustica) Is small. The cultivated tobacco can be divided into six types of flue-cured tobacco, sun-cured tobacco, air-cured tobacco, burley tobacco, aromatic tobacco and yellow flower tobacco according to the quality characteristics, biological characters, cultivation modulation methods and the like of the tobacco leaves, wherein the flue-cured tobacco is the most widely cultivated common tobacco. The flue-cured tobacco planting area and the total yield of China are in the first place in the world.

As a leaf economic crop, the cultivation technology of flue-cured tobacco is different from other field crops, and not only a certain tobacco yield is required, but also the quality of the tobacco is emphasized. The tobacco leaf quality determines the availability of the tobacco leaves, directly influences the color, the fragrance, the taste and the commodity value of cigarette commodities, is also related to the economic benefit of tobacco growers, and is the life and the starting point of the tobacco industry. In order to stand in a field in future market competition at home and abroad and meet the increasing demand of cigarette enterprises at home and abroad on high-quality tobacco leaves, the quality and the safety of the tobacco leaves must be improved.

Plant pigments are an important class of compounds in tobacco, mainly including chlorophyll and carotenoids. 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, chlorophyll is beneficial to the tobacco quality after it is sufficiently degraded, but if not completely degraded, it becomes an undesirable chemical component in dry tobacco leaves, which in turn carries significant green and miscellaneous gases. If the chlorophyll is not fully degraded in the modulation treatment process, 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, the carotenoid content in the tobacco leaves has positive correlation with the tobacco leaf quality, on one hand, the tobacco leaf appearance quality 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. The flavor components in tobacco leaves are mostly degradation products of carotenoids, and many of the compounds are key aroma components in tobacco, such as ionone, damascone, isophorone and the like.

Because the relationship 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 pigments of the tobacco leaves is carried out, 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 tobaccoNtFERLThe 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 tobaccoNtFERLThe gene comprises 2667bp base, and the base sequence is shown as SEQ ID NO. 1; wherein the 2017-2405 nucleotide is a specific core fragment thereof.

Said influencing of tobacco pigment contentNtFERLThe protein coded by the gene comprises 888 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

Said influencing of tobacco pigment contentNtFERLThe application of the gene in tobacco, the gene is highly related to the content of tobacco pigments, and the content of pigments in tobacco is obviously reduced after the gene is silenced.

For silencing and influencing tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLThe construction method comprises the following steps: to be provided withNtFERLThe 2017-2405 nucleotide in the gene is used as a guide sequence of VIGS, and the nucleic acid fragment is inserted into a TRV2-LIC vector to construct and obtain TRV 2-oneNtFERLAnd (3) a carrier.

The method for silencing influences tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLUse in plants for silencingNtFERLThe gene can further reduce the expression level of the pigment substances in the plant.

Use of said for silencing to affect tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLConstructed byNtFERLThe method for cultivating the new variety of the gene silencing plant comprises the following specific steps:

interfering, silencing, knocking out by transgenic technology, transient expression technology or genome editing technologyNtFERLThe gene can obtain a new variety of tobacco transformation plants with changed pigment content (other vectors can also be adopted to carry out the transformation on the new varietyNtFERLOverexpression of the Gene, constructionNtFERLNew varieties of gene-overexpressing transgenic plants); specifically, the method comprises the following steps:

the RNAi vector TRV2-NtFERLTransformed plants, screening and identifyingNtFERLA gene-silenced plant, theNtFERLThe phenotype of the gene-silenced plant is that the content of pigment substances in the gene-silenced transgenic plant is obviously reduced compared with that of a normal plant.

In this application, by treating tobaccoNtFERLThe 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 thatNtFERLAfter 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 control plantsNtFERLRelative expression of the gene in the gene-silenced plant;

FIG. 2 VirusInduction of silencingNtFERLPost-genetic phenotypic analysis;

FIG. 3 Virus InductionNtFERLThe main pigment content in the gene-silenced tobacco leaves and the control tobacco leaves is compared.

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) K326 variety, available from Yuxi tobacco seeds, Inc.;

ben's cigarette (Ben's cigarette)Nicotiana benthamiana) Seeds are presented by the tobacco institute of Chinese academy of agricultural sciences;

vector plasmids TRV2-LIC, TRV1, TRV2 and TRV2 used for silencing tobacco genePDSVectors and the like, purchased from China plasmid vector strain cell line gene collection center;

the gene sequencing and the primer synthesis are completed by Shanghai biological engineering Co., Ltd;

experimental reagent:

restriction enzymes, dATP, dTTP, PrimeSTAR GXL DNA polymerase, DNA Gel recovery Kit MiniBEST Agarose Gel DNA extraction Kit, plasmid DNA minification Kit MiniBESTplasmid purification Kit, acetosyringone and the like, all of which are products of TAKARA biotechnology Limited;

t4 DNA polymerase, TRIZOL reagent extracted from RNA, etc., which is a product of Invitrogen corporation;

reverse transcription kit, product of Roche company;

dnase enzyme, product of Fermentas corporation;

MES, product of Sigma;

antibiotics such as kanamycin and rifampicin are purchased from Shanghai biological engineering Co., Ltd;

the formula and preparation method of part of the reagents are briefly described as follows:

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

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

(3) 200 mM Acetosyringone (Acetosyringone) stock solution: dissolving acetosyringone with anhydrous ethanol, and storing at-20 deg.C;

an experimental instrument:

gradient PCR apparatus Mastercycler ep gradient (for cloning the target gene fragment), product of eppendorf, Germany.

Example 1

This example affects primarily the tobacco pigment contentNtFERLThe process of obtaining the gene is briefly described as follows:

(1) tobacco RNA extraction and cDNA Synthesis

The RNA extraction can be specifically referred to the following steps:

taking a young leaf of cultivated tobacco K326 growing for 4 weeks as a sample, fully grinding the young leaf into powder by liquid nitrogen, putting about 100 mg of the powder material into a 1.5 ml centrifugal tube containing 1.0 ml of TRIZOL reagent, and adding 200 mu L of chloroform; oscillating, mixing, centrifuging, carefully taking out the upper water phase, and transferring 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;

DNase I treatment is carried out on the extracted total RNA, and a 10-mu-L digestion reaction system is designed as follows:

1. mu.g of the extracted RNA;

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.

The RNA extract after DNase I treatment is subjected to cDNA reverse transcription, and the following operation steps can be specifically referred.

First strand synthesis: the following template RNA/primer mixture (7 μ L system) was placed in a sterile 0.2 mL centrifuge tube:

template RN A (100 ng/. mu.l), 1. mu.l;

Oligo (dT) Primer (50 μM)，1 μl；

RNase free dH₂O，5 μl；

after the temperature is preserved for 10 min at 70 ℃, rapidly quenching for more than 2min on ice, and centrifuging for several seconds to lead the denatured solution of the template RNA/primer to gather at the bottom of a centrifuge tube;

the following 10. mu.L reverse transcription reaction solution system was prepared in the centrifuge tube:

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 (RNaseH-) (200 U/μl)，0.25 μl；

preserving heat for 1 h at 42 ℃; the temperature is kept at 70 ℃ for 15 min, then the mixture is cooled on ice, and the obtained cDNA is used for subsequent PCR amplification.

(2) Obtained by PCR amplificationNtFERLGene

Designed for PCR amplificationNtFERLThe primer sequences of the full-length gene are as follows:

NtFERL-F: 5'-ATGACAGAAGGCAGTAAATTC-3'，

NtFERL-R: 5'-TTAGCGTCCTTTTGGATTCAT-3'；

with the cDNA prepared in the step (1) as a template, a 50 μ L amplification system was designed as follows:

GXL polymerase，1 μL；

cDNA，1 μL；

5×GXL buffer，10 μL；

dNTP Mixture (10 mM)，4 μL；

Primer-F/R，8 μL；

ddH₂O，26 μL；

the PCR reaction program is: at 98 ℃ for 10 sec; 55 deg.C, 15 sec, 68 deg.C, 3 min; 35 cycles.

After PCR amplification products are recovered and purified, sequencing is carried out to obtain the product which influences the tobacco pigment contentNtFERLThe gene sequence has the base sequence shown in SEQ ID NO.1 and includes 2667bp base, with the 2017-position 2405 nucleotide as the specific core segment.

After the gene sequence is translated, the coded protein sequence is shown as SEQ ID No.2 and comprises 888 amino acids.

Example 2

Using the tobacco pigment content obtained in example 1NtFERLGene, the inventors further constructed an RNAi interference vector TRV2 for gene silencingNtFERLThe related construction process is briefly described as follows.

A specific nucleic acid fragment (the 2017-2405 nucleotide sequence of the sequence table SEQ ID NO. 1) in the gene is selected as a guide sequence of the VIGS, and a primer is designed to obtain the sequence. Then the nucleic acid fragment is inserted into the VIGS vector to construct TRV2-NtFERLThe vector has the specific construction process that:

(1) obtaining a target fragment, selecting a specific nucleic acid fragment (the 2017-2405 nucleotide sequence of the sequence table SEQ ID NO. 1) in the gene as a guide sequence of the VIGS, designing a primer, and carrying out PCR amplification by using the full length of the gene obtained in the step 1 as a template to obtain the target gene fragment, wherein the sequence of the primer is designed as follows:

NtFERL-VI-F：5’- CGACGACAAGACCCTTCTGATTTCGGGTTGTCTAA -3’，

NtFERL-VI-R：5’- GAGGAGAAGAGCCCTAGGTTCCAAAGCACATCTCC -3’；

NtFERL- -VI-F primer 5' end portion sequence "CGACGACAAGACCCT" andNtFERLthe 5' -end part sequence "GAGGAGAAGAGCCCT" of the VI-R primer is the linker sequence of the TRV2-LIC vector;

length of PCR amplified fragment: 389 bp.

(2) Construction of TRV2-NtFERLVector, first using restriction endonucleasePstI TRV2-LIC plasmid is subjected to single enzyme digestion and enzyme recoveryCutting the product, and performing single-enzyme digestion on the TRV2-LIC single-enzyme digestion product fragment and the target fragment (namely the target fragment obtained in the step (1)) by using T4 DNA polymeraseNtFERLGene specific fragments) are subjected to ligation treatment;

the 10 μ L ligation system was designed as follows:

t4 DNA polymerase, 1. mu.L;

10 x T4 DNA polymerase buffer, 1 μ L;

TRV2-LIC single enzyme cutting fragment (orNtFERLGene specific fragment), 7 μ L;

dTTP (or dATP), 1. mu.L;

connecting at 22 deg.C for 30 min, and then at 70 deg.C for 20 min;

then, the TRV2-LIC plasmid vector fragment and the gene fragment which are respectively treated are fully and uniformly mixed, and are subjected to heat preservation treatment at 65 ℃ for 2min and at 22 ℃ for 10 min;

transforming the ligation product into Escherichia coli competent cell DH5 alpha, coating on LB culture medium containing kanamycin (50 mg/L), performing overnight inverted culture at 37 ℃ to form a single colony, detecting the single colony to obtain TRV2-NtFERLA recombinant vector.

Is in the plantNtFERLGene silencing, in general, requires the introduction of TRV2-NtFERLTransforming agrobacterium with the recombinant vector, and further transforming TRV2-NtFERLThe recombinant vector is integrated into the plant genome, thereby realizing the aim geneNtFERLSilencing of the gene.

TRV2-NtFERLAfter agrobacterium is transformed by the recombinant vector, further screening and identification are generally needed to obtain engineering bacteria for transformation, and the specific operation process refers to the following steps:

TRV2-NtFERLAfter GV3301 agrobacterium competent cells are transformed, the transformed cells are spread on LB plate (containing 50 mg/L kanamycin and 50 mg/L rifampicin), inverted and dark cultured for 2-3 days at 28 ℃, single colony is picked up, and colony PCR screening and carrying is utilizedNtFERLAgrobacterium of gene fragment is monoclonal; the 10. mu.L colony PCR amplification detection system is designed as follows:

GXL polymerase，0.2 μL；

5×GXL buffer，2 μL；

dNTP Mixture (10 mM)，0.8 μL；

Primer-F/R (in example 1)NtFERL-F/R primer), 1.6. mu.L;

ddH₂O，5.2 μL；

colony, 0.2 μ L;

PCR reaction procedure: at 98 ℃ for 10 sec; 55 deg.C, 15 sec, 68 deg.C, 1min, 35 cycles.

Example 3

Use of the VIGS silencing vector containing TRV2 constructed in example 2NtFERLTaking Nicotiana benthamiana as an example, the inventor further carried out a cultivation test to influence the pigment content of the tobacco in silent plantsNtFERLThe gene and related experimental procedures are briefly described as follows.

(1) Sowing tobacco seeds in a seedling pot for seedling, carrying out seedling division two weeks after germination, planting in a plastic pot (10 cm multiplied by 10 cm), carrying out daily fertilizer and water management under the conditions of 22 ℃, 16 h light/8 h dark, and the like; growing for 4 weeks, selecting seedlings with consistent growth vigor and grouping for later use;

(2) will contain TRV1, TRV2, TRV2-NtFERL、TRV2-PDSInoculating Agrobacterium single colony (other plasmid construction process can refer to the above construction process) into 5 mL LB medium (containing 50 mg/L kanamycin and 50 mg/L rifampicin), shaking and culturing at 28 deg.C and 180 r/min overnight to OD₆₀₀About 1.0;

inoculating the grown bacterial liquid into 30 mL LB liquid culture medium for continuous culture, and carrying out shaking culture at 28 ℃ 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 μ M acetosyringone), adjusting OD value to about 1.0, standing at room temperature for 3-6 hours, and adding into TRV2, TRV2-NtFERL、TRV2-PDSAdding TRV1 suspension into the suspension in a medium volume, and mixing uniformly;

(3) 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 pressure filtration method by using a 1ml pinless sterile injector to ensure that the whole leaves are filled with the bacterium liquid, and culturing in air humidity of 75% at 22 ℃;

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

The analysis result shows that the TRV 2-induced silencing by the virusNtFERLIn plantsNtFERLThe expression level of the gene is only about 30% of that of the control (as shown in 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 from our injection are truly reliable (as shown in fig. 2).

NtFERLAfter gene silencing, the new leaves are yellow and have obvious green losing phenotype, and the negative control TRV2 empty vector control plant new leaves have the same phenotype as the non-injected plants. In addition, TRV 2-induced silencing by virusesNtFERLThe content of neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b and beta carotene in the plants was significantly reduced compared to the control plants (as shown in fig. 3), indicating thatNtFERLThe gene is related to the tobacco pigment content.

The detection of the pigment content in the tobacco leaves is shown in the following table (the control sample represents a blank carrier control):

。

as can be seen from a combination of the above results,NtFERLthe gene is highly related to the pigment content of the plant tobacco leaves through silencingNtFERLThe gene can obviously adjust the pigment content in the tobacco leaves, and further lay a foundation for tobacco improvement or cultivation of other new plant varieties.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

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

<130>none

<160>2

<170>PatentIn version 3.5

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<212>DNA

<213>Nicotiana tabacum K326

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caatacaatt tgacttgggt tttttcagtt gattcggggt tctcttatct tgttaggctc 960

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ggagtagaga tttttaaagt gaatgacacc aacggaaacc tcgcagggcc taatcctgtt 1260

ccagtgccag aaccagatca tattgaccct ctgccgtcga aaaaaggtca atcaaaaagt 1320

aataaatcag ctattggtgg aggtgttgga ggtggaattg ctgccataat tcttatcggt 1380

ttggttgtat gccttgtcac ccgccgccgg aaacatggga aggtccaaag tccaagtgat 1440

ggaccatcag gctggctccc tttatcttta tatggaaatt cacatacttc tggttctgct 1500

aagacaaaca ctacaggcag ttatgcttcg tccctcccat caaacctttg tcgtcacttt 1560

tcatttgctg agatcaaggc agccactaat aactttgatg aatctctgct tcttggggtg 1620

ggtggttttg gcaaagtata caagggagaa attgatggtg gcacaaaagt tgctatcaaa 1680

cgtgggaatc ctctctctga gcaaggtgtt catgaatttc aaactgaaat tgaaatgctc 1740

tctaaacttc gccatcgcca ccttgtttct ttgattggtt attgcgagga gaactgtgag 1800

atgatccttg tatatgacta catggctcat ggtacccttc gcgagcatct ctacaagacc 1860

cagaagcctc ctttaccttg gaagcagagg cttgatattg gcattggtgc tgctcgggga 1920

ttgcactatc ttcatactgg tgccaagcat actattatcc accgtgatgt gaagaccact 1980

aatatcctct tggatgagaa gtgggtggca aaggtttctg atttcgggtt gtctaagaca 2040

ggtcctacat tggatcacac ccatgtcagc accgtggtga agggcagttt tggatatctg 2100

gatccagaat acttcagaag gcagcaactc acagacaaat ctgatgtata ctcatttggt 2160

gtagtgctgt ttgagctttt gtgtgctcgg ccagcattga acccaactct tcccaaggag 2220

caagtgagct tagctgagtg ggcattccat tgctacaaga aaggcacttt tgaccagata 2280

attgatccat atctgaaagg gaagattgca ccagaatgct tgaagaaatt tacagagaca 2340

gcagtgaagt gtgtgtctga tgttggtgtt gacaggccct ccatgggaga tgtgctttgg 2400

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ggcggccgca gccttgccag tgacgactca gacgggttaa cacctagtgc tgttttctct 2640

caaatcatga atccaaaagg acgctaa 2667

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Gly Asp Asp Ile Leu Leu Asn Cys Gly Gly Pro Asp Gly Leu Lys Asp

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Ala Asp Gly Arg Lys Trp Gly Ser Asp Ile Gly Ser Thr Tyr Leu Lys

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

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Pro Gln Val Pro Phe Met Ser Ala Arg Val Phe Glu Ser Glu Phe Thr

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Tyr Ser Phe Pro Val Ala Pro Gly Arg Lys Phe Val Arg Leu Tyr Phe

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Tyr Pro Ser Ser Tyr Asn Lys Leu Asn Ala Thr Asn Ala Ile Phe Ser

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Asn Gly Val Thr Asp Thr Ala Asp Asp Glu Asn Leu Thr Val Ser Tyr

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Pro Glu Gly Thr Pro Ser Asp Ile Ala Pro Leu Asp Val Tyr Lys Thr

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Thr Trp Val Phe Ser Val Asp Ser Gly Phe Ser Tyr Leu Val Arg Leu

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Phe Val Ile Tyr Met Asn Asn Gln Thr Ala Glu Pro Ala Ala Asp Val

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Ile Ala Trp Thr Gly Ser Asn Gly Val Pro Phe His Lys Asp Tyr Val

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Val Ser Val Asn Ser Gly Ala Pro Gln Gln Asp Leu Trp Leu Ala Leu

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His Pro Asn Val Ala Ser Lys Ser Asn Trp Tyr Asp Ala Ile Leu Asn

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Ala His Gly Thr Leu Arg Glu His Leu Tyr Lys Thr Gln Lys Pro Pro

610 615 620

Leu Pro Trp Lys Gln Arg Leu Asp Ile Gly Ile Gly Ala Ala Arg Gly

625 630 635 640

Leu His Tyr Leu His Thr Gly Ala Lys His Thr Ile Ile His Arg Asp

645 650 655

Val Lys Thr Thr Asn Ile Leu Leu Asp Glu Lys Trp Val Ala Lys Val

660 665 670

Ser Asp Phe Gly Leu Ser Lys Thr Gly Pro Thr Leu Asp His Thr His

675 680 685

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

690 695 700

Phe Arg Arg Gln Gln Leu Thr Asp Lys Ser Asp Val Tyr Ser Phe Gly

705 710 715 720

Val Val Leu Phe Glu Leu Leu Cys Ala Arg Pro Ala Leu Asn Pro Thr

725 730 735

Leu Pro Lys Glu Gln Val Ser Leu Ala Glu Trp Ala Phe His Cys Tyr

740 745 750

Lys Lys Gly Thr Phe Asp Gln Ile Ile Asp Pro Tyr Leu Lys Gly Lys

755 760 765

Ile Ala Pro Glu Cys Leu Lys Lys Phe Thr Glu Thr Ala Val Lys Cys

770 775 780

Val Ser Asp Val Gly Val Asp Arg Pro Ser Met Gly Asp Val Leu Trp

785 790 795 800

Asn Leu Glu Phe Ala Leu Gln Leu Gln Glu Ser Ala Glu Glu Cys Gly

805 810 815

Lys Gly Phe Gly Lys Met Asp Ile Glu Glu Gly Phe Asp Val Thr Cys

820 825 830

Lys Gly Lys Lys Asp Leu Asn Glu Ser Ala Gly Phe Asp Ala Ser Met

835 840 845

Thr Asp Ser Arg Ser Ser Gly Ile Ser Met Ser Ile Gly Gly Arg Ser

850 855 860

Leu Ala Ser Asp Asp Ser Asp Gly Leu Thr Pro Ser Ala Val Phe Ser

865 870 875 880

Gln Ile Met Asn Pro Lys Gly Arg

885

Claims (4)

1. TobaccoNtFERLThe application of the gene in the regulation of the tobacco pigment content is characterized in that the gene is highly related to the tobacco pigment content, and the content of pigment substances in tobacco is obviously reduced after the gene is silenced;

said tobaccoNtFERLThe gene comprises 2667bp base, and the base sequence is shown as SEQ ID NO. 1; wherein the nucleotide at the 2017-2405 th site is the specificity thereofA core fragment;

the tobacco pigment substances are specifically as follows: neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b and beta carotene.

2. For silencing tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLThe method is characterized by comprising the following steps: to be provided withNtFERLThe 2017-2405 nucleotide in the gene is used as a guide sequence of VIGS, and the nucleic acid fragment is inserted into a TRV2-LIC vector to construct and obtain TRV 2-oneNtFERLAnd (3) a carrier.

3. The method of claim 2 for silencing effects on tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLUse in plants for silencingNtFERLThe gene can further reduce the expression level of the pigment substances in the plant;

the tobacco pigment substances are specifically as follows: neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b and beta carotene.

4. Use of the composition of claim 2 for silencing effects on tobacco pigment contentNtFERLRNAi vector TRV2 for genesNtFERLConstructed byNtFERLThe method for cultivating the new variety of the gene silencing plant is characterized by comprising the following steps:

the RNAi vector TRV2-NtFERLTransformed plants, screening and identifyingNtFERLA gene-silenced plant, theNtFERLThe phenotype of the gene-silenced plant is that the content of pigment substances in the gene-silenced transgenic plant is obviously reduced compared with that of a normal plant;

the tobacco pigment substances are specifically as follows: neoxanthin, violaxanthin, lutein, chlorophyll a, chlorophyll b and beta carotene.

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