CN119614594A

CN119614594A - Method for regulating and controlling fatty acid content in tobacco by NtMFP gene in tobacco and application

Info

Publication number: CN119614594A
Application number: CN202510140804.XA
Authority: CN
Inventors: 陈彦超; 蒲文宣; 王帅斌; 高军平; 何鑫玺; 黄平俊; 杨小年; 张新要; 彭宇
Original assignee: China Tobacco Hunan Industrial Co Ltd
Current assignee: China Tobacco Hunan Industrial Co Ltd
Priority date: 2025-02-08
Filing date: 2025-02-08
Publication date: 2025-03-14

Abstract

The invention provides a method for regulating and controlling fatty acid content in tobacco, which comprises the step of regulating and controlling the expression level of NtMFP gene in tobacco. Also provided is the use of NtMFP gene in tobacco. The NtMFP gene in the tobacco can regulate and control the fatty acid content of tobacco leaves, and the regulation and control of the gene expression can improve the biomass of tobacco seedlings under the conditions of sugar-free and high-sugar culture and improve the fatty acid content of leaves in the seedling emergence stage of the tobacco, so that the high-fat leaves and excellent growth of tobacco seedlings are realized.

Description

Method for regulating and controlling fatty acid content in tobacco by NtMFP gene in tobacco and application

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a method for regulating and controlling fatty acid content in tobacco by NtMFP gene in tobacco and application thereof.

Background

Tobacco is an important cash crop, widely planted and has a long planting history. As a model plant of Solanaceae, tobacco has important scientific value in aspects of gene function research, molecular breeding and the like. In tobacco, the composition, content and distribution of fatty acids play an important role in regulating various adversity stresses. Fatty acids are not only important components of plant growth and development and cell structure, but also have significant effects on the quality and flavor of tobacco leaves. As the leaves develop, the fatty acid content and composition in the tobacco leaves also change.

Therefore, the research on fatty acid in tobacco to obtain tobacco leaves with high fatty acid content is of great significance in improving stress resistance and quality of tobacco and obtaining good style.

Disclosure of Invention

In view of the above, the present invention provides a method and application of NtMFP gene in tobacco to regulate fatty acid content in tobacco, in order to at least partially solve at least one of the above-mentioned technical problems.

According to an embodiment of one aspect of the present invention, there is provided a method of regulating fatty acid content in tobacco comprising the step of regulating the expression level of NtMFP gene in tobacco.

According to an embodiment of the present invention, the base sequence of NtMFP gene is shown as SEQ ID No. 1.

According to an embodiment of the invention, the NtMFP gene encodes a polypeptide as shown in SEQ ID No. 2.

According to an embodiment of the invention, the modulation comprises increasing or decreasing the expression level of NtMFP gene in tobacco.

According to embodiments of the invention, comprising reducing the expression level of NtMFP gene in tobacco or knocking out NtMFP gene by CRISPR-Cas9, zinc finger nucleases, TALENs or RNAi gene silencing techniques.

According to an embodiment of the invention, reducing the expression level of NtMFP gene in tobacco or knocking out NtMFP gene is performed against the target site shown in SEQ ID No.3 in NtMFP gene.

According to an embodiment of the invention, the NtMFP gene is knocked out by using CRISPR-Cas9 gene editing technology through the sgRNA sequence shown in SEQ ID No. 4.

According to an embodiment of another aspect of the invention there is provided the use of the NtMFP gene in tobacco, the use comprising the use of any one of:

(1) Improving germination rate of tobacco seedlings;

(2) Increasing biomass of tobacco seedlings;

(3) Increasing the short chain and/or long chain fatty acid content of tobacco seedlings in the seedling emergence stage;

(4) Used for tobacco breeding;

(5) Regulating fatty acid content of tobacco leaf, and/or

(6) Changing the tobacco flavor.

According to an embodiment of another aspect of the present invention, there is provided a method for breeding homozygous non-transgenic mutant tobacco of NtMFP gene, comprising:

constructing a knockout vector according to the sgRNA sequence shown as SEQ ID No. 4;

Transferring the constructed knockout vector into agrobacterium competent cells and infecting tobacco leaves;

culturing and screening to obtain homozygous non-transgenic mutant tobacco strain.

According to an embodiment of a further aspect of the present invention, there is provided a mutant gene of tobacco NtMFP gene, the mutant gene sequence of tobacco NtMFP gene is GC base deletion at 15bp and 16bp of the base sequence of the target site shown in SEQ ID No. 3.

According to the embodiment of the invention, ntMFP genes in tobacco can regulate and control the fatty acid content of tobacco leaves, and the regulation and control of the gene expression can improve the biomass of tobacco seedlings under the sugar-free and high-sugar culture conditions and the fatty acid content of leaves in the seedling stage of the tobacco, so that the high-fat leaves and excellent growth of tobacco seedlings are realized.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the present invention with reference to the accompanying drawings, in which:

FIG. 1 is a diagram showing the electrophoresis of gene amplification according to example NtMFP of the present invention;

FIG. 2 is a graph showing the comparison of the expression of the different tissues in example NtMFP of the present invention;

FIG. 3 is a schematic diagram showing the construction of a NtMFP gene knockout vector according to example NtMFP of the present invention;

FIG. 4 is a schematic diagram showing the editing pattern of the target site NtMFP according to example NtMFP of the present invention;

FIG. 5 is a chart showing germination rate statistics of NtMFP mutants under different culture mediums according to an example of the present invention;

FIG. 6 is a chart showing biomass statistics of NtMFP mutants under different media according to the example of the present invention;

FIG. 7 is a graph showing the phenotype identification result of the short-chain fatty acid in the seedling stage of the NtMFP mutant of the example NtMFP;

FIG. 8 shows the results of phenotype identification of long chain fatty acids in the seedlings of the mutant NtMFP of the present invention, which shows the cluster heat of medium-long chain fatty acids, the histogram of the medium-long chain fatty acid content of NtMFP2 down-regulated compared with K326, and the histogram of the medium-long chain fatty acid content of NtMFP2 up-regulated compared with K326.

Detailed Description

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the invention. It may be evident, however, that one or more embodiments may be practiced without these specific details. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" as used herein indicates the presence of a feature, step, operation, but does not preclude the presence or addition of one or more other features.

Where a convention analogous to "at least one of A, B and C, etc." is used, in general such a convention should be interpreted in accordance with the meaning of one of skill in the art having generally understood the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, systems having a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.). Where a formulation similar to at least one of "A, B or C, etc." is used, in general such a formulation should be interpreted in accordance with the ordinary understanding of one skilled in the art (e.g. "a system with at least one of A, B or C" would include but not be limited to systems with a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.).

The term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide position identity (i.e., sequence similarity or identity). The term homology as used herein also refers to the concept of similar functional properties between different polynucleotide molecules, e.g. promoters with similar functions may have homologous cis-elements. Polynucleotide molecules are homologous when they hybridize specifically under specific conditions to form duplex molecules. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule may be used as a probe or primer to identify another polynucleotide molecule that shares homology.

The term "promoter" refers to a polynucleotide molecule that is located in its natural state upstream or 5' to the translation initiation codon of the open reading frame (or protein coding region) and is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule, and more preferably are contiguous. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

The term "recombinant plant expression vector" means one or more DNA vectors used to effect transformation of plants, and these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "transformation" is a method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" is the transcription and/or translation of an endogenous gene or transgene in a plant cell.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, e.g., direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

In the course of implementing the inventive concept, it was found in the related art that oxidized fatty acid β -oxidized multifunctional protein (Multifunctional protein, MFP) in rice can regulate mRNA localization or translation by interaction with microtubules in addition to its peroxisome function, seedlings of MFP2 mutant in arabidopsis need exogenous sucrose supply to grow normally, and mature plants exhibit abnormal inflorescence meristem phenotype in mutation of abnormal inflorescence meristem gene AIM1 (AtMFP homologous gene). The effect of the different MFP genes was shown to be different, and the Arabidopsis MFP2-1 and aim-1 double mutants stopped developing at the early stages of embryo development. A MFP gene was also identified in tobacco, ntMFP, which when inhibited by RNAi, exhibited a phenotype of dwarfing, early senescence and reduced expression of jasmonic acid responsive genes.

There is no related art at present that effectively changes the fatty acid content in tobacco leaves by regulating the expression of MFP gene. Therefore, the novel NtMFP gene which is newly identified is researched and applied, so that not only can the tobacco with high fatty acid content be obtained, but also a novel strategy can be provided for tobacco quality breeding. The invention is put forward under the background, and aims to provide a technical scheme for improving fatty acid of tobacco leaves by regulating NtMFP gene expression so as to improve the quality of tobacco, obtain high-fat and excellent-style tobacco leaves and further improve economic benefits.

In some embodiments of the invention, the NtMFP gene editing vector may be constructed by conventional gene editing techniques or construction methods of gene knockout vectors or recombinant plant expression vectors containing NtMFP gene may be constructed according to conventional methods in the art, which are well known to those skilled in the art, e.g., by operably linking the NtMFP gene to expression regulatory elements to obtain a recombinant plant expression vector that can express the gene in plants, which recombinant plant expression vector comprises a promoter, the CDS sequence of NtMFP2 gene and a terminator, which promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter, and the terminator sequence may be taken from Ti-plasmids of Agrobacterium tumefaciens, such as octopine synthase and nopaline synthase termination regions. The vector may also contain a selectable marker gene for selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, genes conferring herbicide resistance, and the like.

In some embodiments of the invention, a kit is provided for controlling fatty acid content in tobacco, comprising constructing NtMFP an editing vector for a gene by conventional gene editing techniques or construction methods for gene knockout vectors or constructing a recombinant plant expression vector containing NtMFP gene according to conventional methods in the art, as well as other conventional optional components, such as marker genes, etc., or necessary components for selection of gene editing methods, etc.

The sequence of SEQ ID No.1 is shown below:

ATGAGCTCAAAGAGTAGAAGTACCATTGAGGTTGGAGCTGATGGAGTTGCTGTTATCACCATTGTTAACCCTCCTGTCAATTCTCTTTCCTTAGATGTTTTGTACAGCTTGGAAGAGAAATTACAGGAAGGCTTAAGGAGAGATGATGTGAAGGCAATTGTTGTGATAGGTTATCAAGGAAATTTCTCGGGTGGTTTCCATATCTCTTCCTTTGCTGACTTGCAACAAGGAAAAGTTGCCCAACCAAAACCTGGTTATGTATCAGTTGATATTCTCACCGACACTGTGGAAGCTGCGCGAAAACCTTTTGTGGCAGCCATTGATGGTCATGCTTTAGGTGGAGGACTAGAAATTGCAATGTGTTGTCATGCACGGATTTCCACTCCGAATGCACAACTTGGATTGCCAGAACTTCACCTTGGTATAATTCCTGGATTTGGAGGTACTCAAAGACTTCCACGCCTTACAGCAAAACTTGCTAAAGGGGAGGAAGCTCTTGATCTTGGCCTAGTTGATGCTATAGTTTCACCTAACCAATTATTGGAAACTGCTCGTAAATGGGCACTCGATATTTGGGAGCGCAAAAGACCTTGGATTCCCACTCTTAACAGAAATGACAAGGTAGAGTCTGTTAGTGATGCAAAGGATATTCTAAAGTTTGCTAGAGCTCAAGCCATCAAACAGGCTCCAAATCTTTATCATCCTTTAGCCTACATAGATGTTATTGAAGAGGGCGTAGTGTCCGGTCCACGTGCTGGACTTATGAAGGAGTATGAAACATTCGAAGTTCTTCTTCGTTCAGACACTTGTAAGGCCTTAGTCCACATTTTCTTTGCTCGTCGTGGAACGACTAAGGTTCCAGGGGTGACTGATCTGGGATTAGTGCCACGACACGTTAAGAAAGTTGCGATTGTTGGAGGTGGATTAATGGGTTCTGGAATTGCAACTGCATTTCTTCTTAGTAATTATGCTGTGATCTTAAAAGAAGTTAATGACAAATTTCTGGAGGCTGGGATTGATAGACAAATCTGCAACGCCACGTTAAAAGGAAAATTGAGTGAAGAGAAGTTTGAAAAAGCTCTCTCCCTACTCAAGGGTACTCTTGATTATGAAAGCTTTAAAGATGTTGACATGGCTGTCACTGAAGACGTACCTTTAAAGCAACAAATATTTATTGATCTAGAGAAGTTTTGCCCTCCACATTGTATACTTGCTAGTAATACCTCTACAATTGACTTGAATTTGATTGGAGAAAGGACCAAATCTCAAGATCGTATTATTGGAGCTCACTTTTTCAGTCCGGCTCATGTGATCCCACTTCTGGAAATTGTTCGCGCTCAACAGACATCTCCACAAGTAATAGTAGACTTGCTTGATGTTGGAAAGAAGATAAAGAAAACCCCTGTAGTAGTAAGGAATTGCACTGGTTTTGCTGTCAACAGAATGTTCTTCCCTTATACCCAAGCTGCTCTTTTGCTTGTTGAACATGGAACTGATATGTATTGCATTGATAGAGCTTTTACTAAATTTGGAATGCATATGGGTCCCTTCAGACTGTGTGATCTTATTGGAATTGGTGTTGCAATGGCTACTGAAGCTCAGTTTATATTGAATATGCCCGATAGAACTTACAAGTCAATGCTTATTCCACTCATGCAACAAGATAAAAGATTAGGTGAAACGACTCAGAGAGGGTTCTATATATACGATGAAAGATGCAAAGCCAAGCCTGATCCAGAAATTAAGAAATACATTGAGAAGGCAAGAGATATTTCTGGTGTCAGTACTGATACTAAGCTGGAAAAACTCTCGGACAAGGATATCGTCGAAATGATATCATTCCCTGTAGTTAATGAAGCATGCAGATTACTTGCTGAAGGCATTGCTGTCAAAGCTGCTGATCTTGACATTGCTTCTGTTATGGGCACATCTGTTATGTTCTGA.

The sequence of SEQ ID No.2 is shown below:

MSSKSRSTIEVGADGVAVITIVNPPVNSLSLDVLYSLEEKLQEGLRRDDVKAIVVIGYQGNFSGGFHISSFADLQQGKVAQPKPGYVSVDILTDTVEAARKPFVAAIDGHALGGGLEIAMCCHARISTPNAQLGLPELHLGIIPGFGGTQRLPRLTAKLAKGEEALDLGLVDAIVSPNQLLETARKWALDIWERKRPWIPTLNRNDKVESVSDAKDILKFARAQAIKQAPNLYHPLAYIDVIEEGVVSGPRAGLMKEYETFEVLLRSDTCKALVHIFFARRGTTKVPGVTDLGLVPRHVKKVAIVGGGLMGSGIATAFLLSNYAVILKEVNDKFLEAGIDRQICNATLKGKLSEEKFEKALSLLKGTLDYESFKDVDMAVTEDVPLKQQIFIDLEKFCPPHCILASNTSTIDLNLIGERTKSQDRIIGAHFFSPAHVIPLLEIVRAQQTSPQVIVDLLDVGKKIKKTPVVVRNCTGFAVNRMFFPYTQAALLLVEHGTDMYCIDRAFTKFGMHMGPFRLCDLIGIGVAMATEAQFILNMPDRTYKSMLIPLMQQDKRLGETTQRGFYIYDERCKAKPDPEIKKYIEKARDISGVSTDTKLEKLSDKDIVEMISFPVVNEACRLLAEGIAVKAADLDIASVMGTSVMF*.

NtMFP 2A gene encoding a peroxisome β -oxidized multifunctional protein (Multifunctional protein, MFP) according to an embodiment of the present invention. Catabolism of fatty acids proceeds mainly through the beta-oxidation cycle, with the multifunctional protein MFP having hydratase, dehydrogenase, isomerase, and epimerase activities, catalyzing the hydration step of beta-oxidation. MFPs are mainly localized to peroxisomes and are induced during development and growth.

In some embodiments of the invention, the improvement or reduction of the NtMFP gene expression level in tobacco can be achieved by a person skilled in the art through various conventional technical means, such as construction of a NtMFP2 gene over-expression vector, transformation of plants through agrobacterium-mediated genetic transformation to obtain a NtMFP gene over-expression line, or knocking out or interfering with NtMFP2 genes in tobacco or NtMFP homologous genes in other plants through CRISPR and VIGS methods to enable the NtMFP2 genes in tobacco or NtMFP homologous genes in other plants to generate deletion or mutation or reduce NtMFP gene expression level, so as to achieve regulation of fatty acid content in tobacco.

According to the embodiment of the invention, through CRISPR-Cas9, zinc finger nuclease, TALENs or RNAi gene silencing technology, expression of the beta-oxidized multifunctional protein of the fatty acid peroxide can be realized by reducing the expression level of NtMFP genes, so that the regulation of the content of the fatty acid in tobacco can be performed, and the regulation of the content of the fatty acid in tobacco can be realized by knocking out NtMFP genes. The invention does not limit the regulation mode or the knockout mode of the NtMFP gene expression level, the target sites of the regulation mode or the knockout mode of the expression level of various genes can be selected according to various modes, and one or more target sites can be selected to regulate or knockout the expression level of the genes.

According to the embodiment of the invention, the expression level of NtMFP gene or the base sequence of knockout NtMFP gene which is positioned in NtMFP gene is 207 bp-227 bp in the sequence shown in SEQ ID No.1, namely an underlined part.

The sequence of SEQ ID No.3 of the target site is shown below:

TCCTTTGCTGACTTGCAACA。

In accordance with an embodiment of the present invention,

The sgRNA sequence SEQ ID No.4 is shown below:

TCCTTTGCTGACTTGCAACAAGG。

According to the embodiment of the invention, ntMFP genes in tobacco are knocked out by CRISPR-Cas9 gene editing technology, corresponding primers are designed aiming at sgRNA sequences corresponding to target sites, so that efficient gene editing can be realized in various cell types and species rapidly and efficiently, the success rate and efficiency of gene editing are improved, and the target site SEQ ID No.3 sequences in NtMFP genes can be specifically identified and cut through the sgRNA sequences shown in SEQ ID No.4, so that accurate gene knockdown is realized.

(1) Improving germination rate of tobacco seedlings;

(2) Increasing biomass of tobacco seedlings;

(4) Used for tobacco breeding;

(5) Regulating fatty acid content of tobacco leaf, and/or

(6) Changing the tobacco flavor.

According to the embodiment of the invention, the NtMFP gene is knocked out by CRISPR-Cas9 technology, so that the germination rate of tobacco seedlings can be remarkably improved. The method is characterized in that the influence of certain adverse factors in the fatty acid metabolic process is possibly reduced by knocking out NtMFP genes, so that germination of seedlings is promoted, biomass of tobacco seedlings is obviously increased after the NtMFP genes are knocked out, more energy and nutrient substances are distributed in the growth and development process possibly due to the change of fatty acid metabolic pathways, the short chain and long chain fatty acid content of the seedling emergence stage of the tobacco seedlings can be increased by knocking out NtMFP genes through CRISPR-Cas9 technology, stress resistance and quality of the tobacco can be improved, knocking out or regulating of NtMFP genes can be used as an important means of tobacco breeding, tobacco varieties with higher germination rate, larger biomass and better stress resistance can be cultivated through a gene editing technology, the fatty acid content in tobacco leaves can be effectively regulated and controlled through CRISPR-Cas9 technology, quality and economic value of the tobacco can be improved, the flavor of the tobacco can be improved, and the flavor can be changed according to the market demand of the tobacco by knocking out NtMFP genes.

According to the embodiment of the invention, a tobacco plant of a homozygous non-transgenic mutant can be quickly and efficiently obtained by knocking out a NtMFP gene in a tobacco breeding mode, an agrobacterium competent cell is used as a host cell, a strain in which homozygous editing of NtMFP gene occurs can be screened, a T0 generation transgenic positive strain can be obtained, and 1 non-transgenic homozygous strain can be obtained in a T1 generation.

According to the embodiment of the invention, the NtMFP gene is edited by CRISPR-Cas9 technology, so that the 'GC' base deletion of a NtMFP gene target site is realized, and the mutation type is frame shift mutation.

The scheme of the invention will be further illustrated with reference to the following examples, which are conventional commercially available reagents unless specifically stated otherwise.

Example 1 amplification of the NtMFP2 Gene

The genome and expression information of the tobacco genome database and the RNA-seq database are utilized, specific primers are designed, CDS of tobacco flower buds is used as a template, the gene NtMFP is specifically amplified, and an electrophoresis diagram is shown in figure 1.

Amplification primers:

BD-NtMFP F, sequence shown below as SEQ ID No. 5:

GCCATGGAGGCCGAATTCATGAGCTCAAAGAGT。

BD-NtMFP R, sequence shown below in SEQ ID No. 6:

CTGCAGGTCGACGGATCCTCAGAACATAACAGA。

Wherein the first half of the amplification primer is a specific primer of a target gene, and the second half of the amplification primer is a sequencing vector homology arm.

FIG. 1 shows an electrophoresis chart of gene amplification in example NtMFP of the present invention.

As can be seen from FIG. 1, the NtMFP gene was amplified successfully, and the size was about 2000 bp.

After the gene is constructed to the sequencing vector, the sequence information of the gene is obtained through first generation sequencing.

NtMFP2 full length 1944 bp, the sequence is shown as SEQ ID No.1, 647 amino acids are encoded, and the amino acid polypeptide sequence is shown as SEQ ID No. 2. The genes are found to have three homologous genes in tobacco through sequence alignment, and are similar to each other.

Example 2 tissue specific expression detection

Samples of different tissues in different tobacco plants were collected, including young leaves, mature leaves, petioles, veins, stems, roots, and buds.

The extracted RNA was sequenced using high throughput sequencing techniques to obtain data on the expression of NtMFP genes in different tissues, the results are shown in FIG. 2.

FIG. 2 is a graph showing the comparison of the expression of the different tissues in example NtMFP of the present invention.

As can be seen from FIG. 2, ntMFP gene was expressed in the highest amount in the bud, and expressed in the other part was relatively low, i.e., TPM <1.

EXAMPLE 3 construction of NtMFP2 Gene knockout vector

Primers were designed based on the sgRNA sequence shown in SEQ ID No. 4.

The double stranded sgRNA primers are shown below:

BsaI-sgRNA-F sequence is shown in SEQ ID No.7:

5’-TGCATCCTTTGCTGACTTGCAACA。

BsaI-sgRNA-R sequence is shown in SEQ ID No.8:

5’-AAACTGTTGCAAGTCAGCAAAGGA。

And (3) annealing to obtain a double-chain sgRNA target product with a connector, and obtaining a purified target product. The linearized gene editing vector and the target product are subjected to ligation reaction by using T4 DNA ligase, DH5 alpha E.coli is transformed into the ligated product, the transformed E.coli is spread on LB resistance screening plates containing antibiotics (kanamycin, kan), and the plates are cultured upside down at 37 ℃ overnight to screen positive clones. Positive monoclonal colonies with accurate sequencing are selected for culture, and plasmids are extracted to complete construction as shown in figure 3.

FIG. 3 is a schematic diagram showing the construction of a gene knockout vector of example NtMFP of the present invention.

EXAMPLE 4 NtMFP2 homozygous non-transgenic mutant

The NtMFP gene knockout vector plasmid obtained in example 3 was transferred into agrobacterium competent LBA4404 by using an electric shock transformation method, after transformation, SOC medium was added to shake culture 1h at 30 ℃ 100 rpm, bacterial liquid was spread on a resistant LB plate containing rifampicin (Rif) and kanamycin (Kan), after culture, single colony was selected after inversion culture 48h at 30 ℃, 16 h was cultured in LB liquid medium containing the same resistance at 28 ℃ 200 rpm, bacteria were preserved (600 μl bacterial liquid+600 μl 50% glycerol) after culture, and after PCR verification was correct, bacterial liquid was expanded and cultured and tobacco genetic transformation was performed.

And (3) taking the K326 variety as a chassis, and infecting tobacco leaves by a leaf disc method. T0 generation transgenic positive lines were screened by kanamycin resistance screening and transgenic molecular identification, and were sequenced using target site detection primers, the sequences of which were as follows.

The sgRNA JC-F sequence is shown as SEQ ID No. 9:

5’-TTAGGTTTACCCGCCAATA-3’。

the sgRNA JC-R sequence is shown as SEQ ID No. 10:

5’-CGGTGCCACTTTTTCAAGTT-3’。

Screening NtMFP lines subjected to homozygous editing of genes to obtain T0 generation transgenic positive lines, identifying the T1 generation transgenic positive lines through transgenic and target site sequencing, screening to obtain 1 non-transgenic homozygous lines, and determining that the target site of NtMFP genes of the lines is subjected to GC base deletion and the mutation type is frame shift mutation through first generation sequencing, wherein the GC base deletion is shown in the following figure 4.

FIG. 4 is a schematic diagram showing the editing pattern of the target site according to example NtMFP of the present invention.

As can be seen from FIG. 4, the 15 th bp and 16 th bp of NtMFP target sites are "GC" base deleted.

EXAMPLE 5 NtMFP2 mutant phenotyping

The germination rate of NtMFP mutants was determined by preparing different MS media, i.e.M 519.43. 4.43 g, sucrose 25 g, pH 5.8 was adjusted and agar 9 g was added to the 1000 mL media to analyze the change in germination rate under different culture conditions.

Wherein normal MS medium was used as a control group, high sugar MS medium with twice sucrose (sucrose 50 g) and sugarless MS medium without sucrose was used as an experimental group, respectively. Seeds were all spring-treated at 4℃for three days and then dibbled into different media and placed in a 25℃constant temperature long-day tissue culture chamber for cultivation, the results are shown in FIG. 5.

Analysis and statistics are carried out on phenotypes of 18-day-old seedlings of NtMFP mutants under different culture mediums, and the results are shown in FIG. 6.

FIG. 5 is a chart showing germination rate statistics of NtMFP mutants under different culture mediums according to an embodiment of the present invention, and FIG. 6 is a chart showing biomass statistics of NtMFP mutants under different culture mediums according to an embodiment of the present invention.

As can be seen from FIG. 5, the germination rates of NtMFP2 and control group K326 (CK) in normal medium were similar, K326 was slightly higher than NtMFP2, control and NtMFP2 were germinated in advance under sugarless culture conditions, ntMFP2 was germinated substantially all over the fifth day and NtMFP was higher than the control, control and NtMFP2 were inhibited in 2-fold sucrose medium, and NtMFP was germinated much lower than the control on the fourth day. The NtMFP mutant was shown to have a phenotype of increased germination under sugarless and high sugar culture conditions.

As can be seen from fig. 6, the leaf area of the mutant was significantly higher than that of the control group under the culture condition of 2-fold sugar, and the maximum leaf length of the mutant was significantly higher than that of the control group under the sugar-free culture condition. The NtMFP mutant was shown to have a phenotype of increased biomass under sugarless and 2-fold sugar culture conditions.

Example 6 short chain fatty acid phenotype identification of NtMFP2 mutant at seedling stage

The short chain fatty acid content of the two cotyledons in the fully developed state was determined for the seedling stage NtMFP mutant and the seedling leaf of the control chassis material K326, i.e., 10-day-old seedlings after germination, respectively. Short chain fatty acids and medium-long chain fatty acids were detected separately using targeted metabonomics, and the detection method used a multi-reaction monitoring mode (MRM) based LC/MS platform to detect content. The results of total detection of 10 short chain fatty acids by sample collection, target metabolite extraction, standard curve establishment, methodological validation, LC-MS/MS detection steps followed by data analysis are shown in fig. 7.

FIG. 7 is a graph showing the phenotype identification result of the short-chain fatty acid in the seedling stage of the NtMFP mutant of the present invention.

As can be seen from FIG. 7, 9 short chain fatty acids out of 10 short chain fatty acids were significantly up-regulated at NtMFP compared to K326 (FC >1.2 or FC <0.833 and P-value < 0.05), K326 isovalerate content was 0.06 μg/g, ntMFP2 mutant was 0.08 μg/g, increased to 1.33 fold. The NtMFP mutant has a phenotype of high leaf short-chain fatty acid content in seedling stage, and the NtMFP gene can be proved to be capable of regulating and controlling the short-chain fatty acid content in tobacco leaves.

EXAMPLE 7 phenotype identification of long chain fatty acids in NtMFP2 mutant seedlings

In the same manner as in example 6, the medium-long chain fatty acid content was measured, and the results of 39 fatty acids measured are shown in FIG. 8.

In fig. 8, the C number is a designation for fatty acids, reflecting the number of carbon atoms, the number of double bonds, and the position thereof.

As can be seen from FIG. 8, of the 39 fatty acids tested, ntMFP had 2 fatty acids significantly down-regulated compared to K326, 0 down-regulated by 0.74 times, C18:1 (n-7) T down-regulated by 0.43 times, 24 substances significantly up-regulated by 1.23-7.80 times, wherein the 4 substances up-regulated to the highest extent were up-regulated by 4.07 times, C19:1 (n-9) T up-regulated by 4.73 times, C22:2 up-regulated by 6.83 times, and C20:5 up-regulated by 7.80 times, respectively. The cluster analysis shows that the content of the medium-long chain fatty acid of NtMFP except two fatty acids is higher than K326, and the NtMFP gene can be proved to be capable of regulating the content of the long chain fatty acid in tobacco leaves.

The above embodiments are further described in detail with respect to the objects, technical solutions and advantageous effects of the present invention, and it should be understood that the above are only embodiments of the present invention, and not as an attempt to limit the application of the doctrine of equivalents to the details of the invention, the invention should be construed in light of the number of equivalents and alternatives falling within the spirit and scope of the invention.

Claims

1. A method for regulating the fatty acid content in tobacco, comprising the step of regulating the expression level of NtMFP2 gene in tobacco.

2. The method according to claim 1, wherein:

The base sequence of the NtMFP2 gene is shown in SEQ ID No.1.

3. The method according to claim 1, wherein:

The NtMFP2 gene encodes the polypeptide shown in SEQ ID No.2.

4. The method according to any one of claims 1 to 3, wherein

The regulation includes increasing or decreasing the expression level of the NtMFP2 gene in tobacco.

5. The method according to any one of claims 1 to 3, wherein:

The method comprises reducing the expression level of the NtMFP2 gene in tobacco or knocking out the NtMFP2 gene by using CRISPR-Cas9, zinc finger nuclease, TALENs or RNAi gene silencing technology.

6 . The method according to claim 5 , wherein reducing the expression level of the NtMFP2 gene in tobacco or knocking out the NtMFP2 gene is performed by targeting the target site shown in SEQ ID No. 3 in the NtMFP2 gene.

7. The method according to claim 6, wherein:

The NtMFP2 gene was knocked out using CRISPR-Cas9 gene editing technology using the sgRNA sequence shown in SEQ ID No.4.

8. Application of the NtMFP2 gene in tobacco, wherein the application includes any of the following applications:

(1) Improve the germination rate of tobacco seedlings;

(2) Increase the biomass of tobacco seedlings;

(3) Increase the content of short-chain and/or long-chain fatty acids in tobacco seedlings at the emergence stage;

(4) Used in tobacco breeding;

(5) regulating the fatty acid content of tobacco leaves; and/or

(6) Change the flavor of tobacco.

9. A method for breeding homozygous non-transgenic mutant tobacco of NtMFP2 gene, comprising:

Construct a knockout vector according to the sgRNA sequence shown in SEQ ID No. 4;

The constructed knockout vector was transferred into Agrobacterium competent cells and infected tobacco leaves;

Homozygous non-transgenic mutant tobacco strains were obtained by cultivation and screening.

10. A mutant gene of tobacco NtMFP2 gene, wherein:

The mutant gene sequence of the tobacco NtMFP2 gene is a GC base deletion at the 15th bp and the 16th bp of the target site base sequence shown in SEQ ID No.3.