CN117025626A - Tobacco nitrate transporter NtNPF7.4, encoding gene thereof, gene editing vector and application - Google Patents
Tobacco nitrate transporter NtNPF7.4, encoding gene thereof, gene editing vector and application Download PDFInfo
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Abstract
The invention discloses a tobacco nitrate transporter NtNPF7.4, and a coding gene, a gene editing vector and application thereof. The coding gene NtNPF7.4 of the tobacco nitrate transport protein is obtained by cloning, and different tissues and organs of tobacco are detected by fluorescent quantitative PCR, and the result shows that the gene is expressed in roots, stems, leaves and flowers of the tobacco. Subcellular localization analysis shows that the NtNPF7.4 protein is localized on the cell membrane, and the expression level of the NtNPF7.4 gene in the leaf after salt stress is obviously increased, which indicates that the NtNPF7.4 protein participates in the chloride ion transport process. The NtNPF7.4 gene is knocked out by utilizing a gene editing vector, a gene knocked-out plant is constructed, and the content of chloride ions in roots and leaves of the knocked-out plant is obviously increased, so that the cultivation of tobacco enriched with chloride ions is possible, and a new direction is provided for improving the condition of low chlorine content of flue-cured tobacco in China.
Description
Technical Field
The invention relates to a tobacco nitrate transporter NtNPF7.4, a coding gene thereof, a gene editing vector and application thereof, belonging to the technical field of plant genetic engineering.
Background
Plants have two classes of nitrate transporters, NRT1/PRT and NRT2, where NRT1/PRT is again designated NPF. The NPF transport substrate reported so far is NO3 - Cl-, polypeptides, various hormones (IAA, ABA, GA, etc.), glucosinolates, etc. The plant NPF family generally consists of 8 subfamily genes. Studies have shown that NPF7.3, a member of the NPF7 subfamily, may be involved in K in addition to nitrate transport + The transport, atnpf7.3 mutant also had a lateral root development and leaf senescence phenotype. Furthermore, it is mentioned that NPF7.3 may also be involved in Cl-transport, but no definitive evidence has been available to date.
Chlorine is one of the essential nutrient elements for the growth and development of flue-cured tobacco, and too high and too low chlorine content of flue-cured tobacco can reduce the quality and yield of tobacco leaves. The chlorine content of tobacco leaves is generally related to the chlorine content of soil, so in areas where the chlorine content of soil is low, such as: the chlorine content of tobacco leaves in southwest area, southern Anhui province and Fujian province is relatively low. The general research shows that the chlorine content in the high-quality tobacco leaves is preferably 0.3-0.8%, but the chlorine content of most tobacco leaves in the tobacco region in China is lower than 0.3%, so that the quality of the tobacco leaves is affected to a certain extent (reference document: flue-cured tobacco chlorine content characteristic comparison research in main tobacco region in China. Guizhou agricultural science 2008,36 (1): 106-107). Analysis of regional characteristics and distribution conditions of chlorine content of flue-cured tobacco in main tobacco areas in China shows that the chlorine content of flue-cured tobacco in China is generally low, wherein high-chlorine flue-cured tobacco is mainly distributed in north (77.32%), low-chlorine flue-cured tobacco is mainly distributed in south (78.23%), in 2712 flue-cured tobacco samples investigated, the chlorine content of samples of 53.32% is lower than 0.30%, the chlorine content of samples of 43.10% is 0.30% -0.80%, and the chlorine content of samples of 3.43% is higher than 0.80% (reference: regional characteristic study of chlorine content of flue-cured tobacco in main tobacco areas in China, chinese soil and fertilizer, 2010 (2): 49-54). The application of the chlorine-containing fertilizer can increase the chlorine content of the soil, but the increase of the chlorine-containing fertilizer has close relation with the chlorine application amount, the rainfall and the water permeability of the soil, and the improper use of the chlorine-containing fertilizer can cause pollution of chlorine ions of the soil.
Along with the increasing maturity of molecular biotechnology and the achievement of chlorine nutrition molecular biology in other crops, research on the transport mechanism of chlorine, chloride ion channels, the effective genes of the tobacco leaf on chlorine absorption and the like from the molecular level on tobacco has important significance for solving the condition of low chlorine content of flue-cured tobacco in China. Therefore, in order to further understand the molecular regulation of chloride ion transport and realize more accurate tobacco breeding, the excavation and screening of proteins and coding genes involved in the chloride ion transport process is a problem to be solved urgently.
Disclosure of Invention
To solve the above problems, a first object of the present invention is to provide a gene encoding a tobacco nitrate transporter, ntnpf7.4, which is capable of encoding a tobacco nitrate transporter, ntnpf7.4, enriching a regulatory network of a tobacco chloride ion transport-related gene.
The invention provides a tobacco nitrate transporter NtNPF7.4, and experiments prove that the NtNPF7.4 protein participates in absorption and transport of chloride ions in tobacco, lays a foundation for regulating and controlling the content of the chloride ions in the tobacco, and has important significance for accurate breeding of the tobacco.
The third object of the invention is to provide a gene editing vector which can realize the effective knockout of the NtNPF7.4 gene, successfully obtain the tobacco plant with the NtNPF7.4 gene knocked out, inhibit the expression of the NtNPF7.4 gene and further improve the content of chloride ions in the roots and leaves of the knocked-out tobacco plant.
The fourth object of the invention is to provide the application of the tobacco nitrate transporter encoding gene NtNPF7.4 gene or the gene editing vector in the cultivation of the chloride ion enriched tobacco variety, and the CRISPR/Cas9 gene editing technology is used for knocking out the NtNPF7.4 gene in tobacco plants, so that the content of chloride ions in roots and leaves of the knocked-out plant is obviously increased, and the tobacco variety with accumulated chloride ion content is obtained.
The fifth object of the invention is to provide the application of the NtNPF7.4 gene knockout tobacco plant in improving the low chlorine content of flue-cured tobacco in soil with low chlorine ion content.
In order to achieve the aim, the tobacco nitrate transporter encoding gene NtNPF7.4 adopts the following technical scheme:
the nucleotide sequence of the tobacco nitrate transporter encoding gene NtNPF7.4 is as follows:
(1) A nucleotide sequence shown as SEQ ID NO. 1;
(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.
The beneficial effects of the technical scheme are that: according to the invention, a specific primer is designed, a tobacco nitrate transporter coding gene NtNPF7.4 gene is obtained through cloning, and the NtNPF7.4 gene is mainly expressed in roots, stems, leaves and flowers in normal tobacco plants through fluorescent quantitative PCR analysis.
In order to achieve the purpose, the tobacco nitrate transporter NtNPF7.4 adopts the following technical scheme:
the amino acid sequence of the tobacco nitrate transporter NtNPF7.4 is as follows:
(1) An amino acid sequence shown in SEQ ID NO. 2;
(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.
The beneficial effects of the technical scheme are that: proved by verification, the tobacco nitrate transporter NtNPF7.4 is closely related to the absorption and transport of chloride ions in tobacco.
In order to achieve the above purpose, the technical scheme adopted by the gene editing vector of the invention is as follows:
and the gene editing vector comprises a target site knockout sequence designed according to the NtNPF7.4 gene, and the nucleotide sequence of the NtNPF7.4 gene is shown as SEQ ID NO. 1.
The beneficial effects of the technical scheme are that: compared with a VIGS strain, the tobacco with the gene knocked out obtained by using the CRISPR/Cas9 technology has better effect of inhibiting the gene expression quantity, is more stable and can be transferred to the next generation.
As a further improvement, the sequence of the knockout primer designed based on the target site knockout sequence is as follows:
ntnpf7.4-t1_f: GATTGATGGAAGTGTGGATAAGCA (SEQ ID NO. 12);
ntnpf7.4-t1_r: AAAC TGCTTATCCACACTTCCATC (SEQ ID NO. 13).
In order to achieve the above purpose, the application of the tobacco nitrate transporter encoding gene NtNPF7.4 gene or the gene editing vector in the cultivation of chloride ion enriched tobacco varieties adopts the following technical scheme:
the application of the tobacco nitrate transporter encoding gene NtNPF7.4 gene or gene editing vector in the cultivation of chloride ion enriched tobacco varieties.
The beneficial effects of the technical scheme are that: according to the invention, the NtNPF7.4 gene is knocked out from tobacco by using a CRISPR/Cas9 technology, the expression of the NtNPF7.4 gene is inhibited, a tobacco plant with the NtNPF7.4 gene knocked out is obtained, the content of chloride ions in roots and leaves of the obtained NtNPF7.4 gene knocked out plant is obviously increased by detection, a new research object is provided for regulating and controlling the content of the chloride ions in tobacco and other plants, and a gene network for regulating and controlling the chloride ions in tobacco is enriched.
As a further improvement, the ntnpf7.4 gene was knocked out, and the chloride ion content in tobacco roots and leaves was significantly increased.
The beneficial effects of the technical scheme are that: according to the invention, compared with a control tobacco plant, the detection of the NtNPF7.4 gene knockout plant shows that the content of chloride ions in roots and leaves of the NtNPF7.4 gene knockout plant is obviously increased, so that a tobacco variety enriched with chloride ions is obtained, and a foundation is laid for accurate breeding of the tobacco enriched with chloride ions.
As a further improvement, the chloride ion enriched tobacco variety is obtained by the following method: and (3) transforming agrobacterium tumefaciens serving as an invader solution by using the gene editing vector, transforming tobacco, and screening and identifying to obtain the chloride ion enriched tobacco variety.
The beneficial effects of the technical scheme are that: the knocked-out tobacco strain obtained by the agrobacterium transformation method has strong operability, short time and high success rate.
In order to achieve the purpose, the application of the NtNPF7.4 gene knockout tobacco plant in improving the low chlorine content of flue-cured tobacco in the soil with low chlorine ion content is as follows:
the application of the NtNPF7.4 gene knockout tobacco plant in improving the low chlorine content of flue-cured tobacco in soil with low chlorine ion content.
The beneficial effects of the technical scheme are that: the invention discovers that the NtNPF7.4 gene is knocked out from tobacco, and can obviously improve the chloride ion content in roots and leaves, thereby laying a theoretical foundation for improving the condition of low chloride ion content in flue-cured tobacco in southern areas of China and providing a new direction for improving the condition of low chloride content in flue-cured tobacco in China.
The invention discovers that the gene NtNPF7.4 of the tobacco nitrate transporter is expressed in the root, stem, leaf and flower tissues of tobacco through real-time PCR. To further confirm the function of the ntnpf7.4 gene, a gene editing vector for knocking out the ntnpf7.4 gene was constructed by CRISPR/Cas9 technology, and a knockout strain for inhibiting the expression of the ntnpf7.4 gene was successfully obtained after transformation. The detection result shows that compared with a control plant, the content of chloride ions in roots and leaves in the NtNPF7.4 gene knockout plant is obviously increased, so that the NtNPF7.4 gene is closely related to absorption and transportation of the chloride ions in tobacco, the functional system of the tobacco nitrate transport protein is enriched, an important reference is provided for plant chloride ion transportation regulation and control, and a foundation is laid for cultivating new plant varieties enriched with new chloride ions.
Drawings
FIG. 1 is a graph showing the expression characteristics of the NtNPF7.4 gene in different tissues in experimental example 1 of the present invention (the histogram does not mark the significant difference in the same lower case letters);
FIG. 2 is a graph showing subcellular localization of tobacco NtNPF7.4 protein in experimental example 1 of the present invention;
FIG. 3 is a graph showing the expression profile of the NtNPF7.4 gene under salt stress in experimental example 2 of the present invention (the histogram does not mark a significant difference in the same lowercase letters);
FIG. 4 is a schematic diagram showing selection of target sites for NtNPF7.4 gene knockout in experimental example 3 of the present invention;
FIG. 5 is a diagram showing T in Experimental example 4 of the present invention 0 Sequencing result diagram of target site knocked out by the generation gene editing plant;
FIG. 6 is a graph of plant root and leaf chloride ion content of the NtNPF7.4 gene editing plant of experimental example 4 of the present invention (representing p < 0.01).
Detailed Description
The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples, experimental examples and comparative examples were all commercially available, except for the specific descriptions.
The tobacco NtNPF7.4 gene codes a tobacco nitrate transporter NtNPF7.4, and the tobacco nitrate transporter comprises an amino acid sequence shown in SEQ ID: no. 2. The tobacco nitrate transporter may also be represented by SEQ ID No:2 via substitution and/or deletion and/or addition of one or more amino acid residues, and has derivative polypeptide affecting tobacco chloride ion transport. The substitution and/or deletion and/or addition of one or several amino acid residues refers to substitution and/or deletion and/or addition of not more than 10 amino acid residues.
The coding gene NtNPF7.4 of the tobacco nitrate transporter comprises a nucleotide sequence shown in SEQ ID: no. 1. Or can be matched with SEQ ID No in a sequence table under high-stringency conditions: 1, a nucleotide sequence which hybridizes to the DNA sequence defined in 1; or with SEQ ID No:1, and the DNA sequence which has more than 90 percent of homology and codes the same functional protein.
The application of the gene NtNPF7.4 of the tobacco nitrate transporter coding gene in the invention is to inhibit the expression of the gene NtNPF7.4 in tobacco plants, so that the content of chloride ions in roots and leaves can be improved. Expression of the ntnpf7.4 gene can be inhibited by a variety of methods, such as: an agrobacterium-mediated transformation gene editing vector, a plant virus vector mediated gene silencing method, an agrobacterium-mediated transformation RNAi interference vector, a method for optimizing and modifying a gene coding frame, a method for optimizing a gene promoter and the like. The method of inhibiting gene expression according to the present invention is not limited to the above-mentioned methods, as long as it can inhibit the expression of ntnpf 7.4.
The following examples and experimental examples are briefly described below for some of the biological materials, experimental reagents, experimental facilities, and the like:
biological material:
tobacco variety: k326, the seeds used were supplied by the national tobacco gene research center.
And (3) a carrier: the pFF19 vector is provided by the national tobacco gene research center; pCS1300 was given to wuhan-tian biotechnology limited; the CRISPR/Cas9 vector is provided by the national emphasis laboratory of silkworm genome biology at southwest university.
Strains: trans5 alpha chemically competent cells, purchased from Beijing full gold biotechnology Co., ltd; GV3101 Agrobacterium competent cells, purchased from Shanghai Biotechnology, inc.; primer synthesis and DNA sequencing were performed by Beijing Liuhua macrogene technologies Inc.
Experimental reagent: RNA extraction kit (RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit), genome extraction kit (polysaccharide polyphenol plant genome DNA extraction kit) were purchased from the company of the root biochemistry science and technology (beijing); fluorescent quantification kit, reverse transcription kit (Transcriptor First Strand cDNASynthesis Kit) were purchased from Roche, switzerland; DNA amplification enzymes purchased from beijing full gold biotechnology limited; restriction enzyme BsaI, plasmid extraction kit and DNA gel recovery kit were purchased from Takara Bio Inc.
Experimental facilities: PCR instrument Tprofessional Thermocycler, biomera company; quantitative PCR instrument LightCycler96, roche company.
Example 1 of the tobacco nitrate transporter encoding Gene NtNPF7.4 Gene
The nucleotide sequence of the tobacco nitrate transporter encoding gene ntnpf7.4 in this example is:
(1) A nucleotide sequence shown as SEQ ID NO. 1;
(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.
Example 1 of tobacco nitrate transporter NtNPF7.4
The amino acid sequence of the tobacco nitrate transporter ntnpf7.4 in this example is:
(1) An amino acid sequence shown in SEQ ID NO. 2;
(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.
Example 1 of Gene editing vector
The gene editing vector of this example contains a knockout primer sequence designed according to the target site of the ntnpf7.4 gene, and the nucleotide sequence of the ntnpf7.4 gene is shown in SEQ ID No. 1.
The sequence of the knockout primer designed according to the target site knockout sequence is as follows:
ntnpf7.4-t1_f: GATTGATGGAAGTGTGGATAAGCA (SEQ ID NO. 12);
ntnpf7.4-t1_r: AAAC TGCTTATCCACACTTCCATC (SEQ ID NO. 13).
Example 1 application of tobacco nitrate transporter encoding Gene NtNPF7.4 Gene or Gene editing vector in cultivation of chloride enriched tobacco variety
In the embodiment, after a gene editing vector containing a knockout primer sequence designed according to a target site of the NtNPF7.4 gene is transformed into a tobacco plant, the tobacco plant with the NtNPF7.4 gene knocked out is constructed, compared with a normal tobacco plant, the content of chloride ions in roots and leaves of the plant is obviously increased, and a tobacco variety enriched in chloride ions is obtained.
Example 1 of the use of NtNPF7.4 knockout tobacco plants for improving lower chlorine content flue-cured tobacco in soil with lower chlorine ion content
According to the embodiment, the CRISPR/Cas9 technology is utilized to knock out the NtNPF7.4 gene in the tobacco plant, so that the NtNPF7.4 gene knocked-out plant is obtained, the chlorine ion content in the roots and leaves of the knocked-out plant is increased, and the method is applied to soil with low chlorine ion content in China, so that the condition of low chlorine content in flue-cured tobacco is improved, and the quality of flue-cured tobacco is improved.
Experimental example 1 analysis of expression pattern of NtNPF7.4 Gene and cloning of NtNPF7.4 Gene fragment
In this example, total RNA of roots of K326 tobacco plants is extracted, reverse transcribed into cDNA, and a ntnpf7.4 gene fragment is amplified by PCR, and the expression patterns of the ntnpf7.4 gene in different tissues and organs of tobacco are analyzed by using fluorescent quantitative PCR, and the specific implementation operations are as follows:
1. cloning of the NtNPF7.4 Gene
Cloning and obtaining procedures of the NtNPF7.4 gene are as follows:
(1) Extracting RNA and reverse transcribing cDNA
And (3) inoculating the K326 seeds to an MS culture medium for germination after disinfecting, transplanting seedlings into a pot after two weeks of germination, culturing in a plant culture room with a culture temperature of 23-26 ℃, transferring the seedlings to a 1/2MS liquid culture medium for continuous culture when the tobacco seedlings grow to six leaves and one heart, selecting roots for sampling after two weeks, quick-freezing with liquid nitrogen for later use, and extracting total RNA of tobacco roots by using a plant RNA extraction kit. During RNA extraction, the method is carried out by referring to the instruction of the kit, and then reverse transcription is carried out to obtain cDNA for standby.
(2) Designing primer for PCR amplification
The PCR amplification primer sequences were as follows:
ntnpf7.4-F:5'-ATGGCTTGCTTAAACATTG-3' (SEQ ID NO. 3);
ntnpf7.4-R:5'-TTAGACCTTGAAATCTCCTT-3' (SEQ ID NO. 4).
PCR amplification was performed using the cDNA reverse transcribed in step (1) as a template and the above primers. The PCR amplification system is as follows: 5 XGCL buffer 10. Mu.L, cDNA 2. Mu.L, 1. Mu.L each of the upstream and downstream primers, dNTP 6. Mu.L, GXL DNAPolymerase. Mu.L, and sterilized water to 50. Mu.L. The PCR amplification conditions were: 98 ℃ for 10sec;55 ℃, 15sec,68 ℃, 2min,30 cycles. The PCR products were subjected to agarose electrophoresis detection and analysis, and the PCR amplified products were purified and recovered by referring to the DNA gel recovery kit instructions.
The purified product was then ligated onto pFF19 vector as follows: DNA amplification product, 6. Mu.L; pFF19 vector, 1. Mu.L; after mixing, the mixture was connected at 25℃for 25min.
The ligation products were transformed into E.coli DH 5. Alpha. Competent cells as follows:
taking out competent cells from a refrigerator at-80 ℃, putting the competent cells on ice to dissolve the competent cells, adding a connection product into 100 mu L DH5 alpha competent cells, flicking and mixing the competent cells uniformly, and carrying out ice bath for 20min; heat shock in a water bath at 42 ℃ for 90s, and immediately placing on ice for 2min; 900. Mu.L of LB (without antibiotics) liquid medium equilibrated to room temperature was added, and cultured with shaking at 37℃for 1h; centrifugation at 4000rpm for 3min, removing part of the supernatant, leaving 100. Mu.L of the pellet, mixing well, spreading evenly on LB solid plates (containing 50. Mu.g/. Mu.L kanamycin), inverting the dishes, and culturing overnight at 37 ℃.
The next day, the monoclonal was picked and sequenced.
Sequencing results and analysis results show that the length of the coding region of the NtNPF7.4 gene is 1788bp nucleotides; after analysis of the gene, the amino acid sequence of the encoded NtNPF7.4 protein is shown as SEQ ID NO. 2.
2. Analysis of expression patterns of the NtNPF7.4 Gene in different tissues and organs of Nicotiana tabacum
And (3) taking the root, stem, leaf, flower and other tissues of the Wanglong-term K326 tobacco plant, extracting RNA reverse transcription cDNA, and detecting the expression quantity of the NtNPF7.4 gene in each tissue organ by using a real-time quantitative PCR method. Taking the tobacco Nt26S gene as an internal reference, carrying out fluorescent quantitative PCR detection, wherein the primer sequences are as follows:
the fluorescent quantitative primer for detecting the NtNPF7.4 gene has the following primer sequence:
RT-NtNPF7.4-F:5'-CAGTCTCAGGCTTTCAT-3' (SEQ ID NO. 5);
RT-NtNPF7.4-R:5'-TCAAGAACTCTTCTATAG-3' (SEQ ID NO. 6).
When detecting the tobacco Nt26S gene, specific primers are as follows:
nt26S-F:5'-GAAGAAGGTCCCAAGGGTTC-3' (SEQ ID NO. 7);
nt26S-R:5'-TCTCCCTTTAACACCAACGG-3' (SEQ ID NO. 8).
The reaction system of the fluorescent quantitative PCR is as follows: 10. Mu.L of 2 XSYBR I Master, 0.5. Mu.L of each of the upstream and downstream primers, 50ng of cDNA, and ddH were added 2 O to 20. Mu.L.
The reaction conditions of the fluorescent quantitative PCR are as follows: the first step of pre-denaturation, at 95 ℃ for 10s; the second step of PCR reaction, 95 ℃ for 5s,60 ℃ for 30s,39 cycles; and a third step of melting curve.
Each sample was subjected to 3 biological replicates byThe method analyzes the relative gene expression differences.
The results of fluorescent quantitative PCR are shown in FIG. 1 (bar graph does not show significant differences in the same lowercase letters), and the results indicate that the NtNPF7.4 gene is expressed in roots, stems, leaves and flowers of tobacco.
3. Subcellular localization of tobacco NtNPF7.4 protein
Primers pCS1300-NPF7.4F and pCS1300-NPF7.4R were designed and NtNPF7.4 was cloned into the pCS1300 vector containing the GFP tag, the primer sequences were:
pCS1300-NPF7.4F:5'-GCTTTCGCGAGCTCGGTACCATGGCTTGCTTAAACATTG-3' (SEQ ID NO. 9);
pCS1300-NPF7.4R:5'-CCCTTGCTCACCATGGATCCGACCTTGAAATCTCCTTGC-3' (SEQ ID NO. 10).
PCR amplification System and reaction procedure the same as In cloning of the NtNPF7.4 Gene of Experimental example 1, the obtained PCR product was ligated to the pCS1300 vector with In-Fusion enzyme, the ligation system was as follows: 2. Mu.L of DNA amplification product; 2. Mu.L of pCS1300 vector; in-Fusion enzyme 1. Mu.L. After mixing, the mixture was connected at 50℃for 15min. The ligation product was transformed into E.coli DH 5. Alpha. Competence and single colony was picked for sequencing.
The fusion plasmid GFP-NtNPF7.4 was extracted from the correctly sequenced colonies and transformed into Agrobacterium, which was transformed as follows:
taking out competent cells of Agrobacterium GV3101 from a refrigerator at-80 ℃, freezing and thawing on ice, adding 5 mu L of constructed GFP-NtNPF7.4 fusion vector when thawing is about to occur, and flicking and uniformly mixing; ice-bath for 30min, freezing in liquid nitrogen for 5min, and immediately placing on ice after water bath at 37 ℃ for 5 min; adding 900 mu L of LB liquid medium without antibiotics, and culturing for 4h at 200rpm with shaking; centrifuging the bacterial liquid at 4500rpm for 3min, discarding half of the volume of supernatant, re-suspending, uniformly coating on LB solid medium containing Rif (100 mug/mL) and Kan (50 mug/mL), and inversely culturing at 28 ℃ for about 2-3 d until single colony is formed; and (3) selecting single bacterial colony, carrying out PCR identification on bacterial liquid after amplification and culture, and identifying the correct positive clone bacterial strain, namely the engineering bacteria with correct transformation.
The correctly transformed agrobacteria were injected into healthy leaf-tobacco flakes and after 2d the distribution of the signal in the cells was observed using a laser confocal microscope. The results are shown in FIG. 2, which illustrates that the NtNPF7.4 protein is localized on the cell membrane (where FM is a membrane localization dye).
Experimental example 2 salt stress test of tobacco
In order to determine the specific response situation of the NtNPF7.4 gene in salt stress, the common cultivated tobacco K326 is treated by salt stress, roots are collected at different treatment times, and the expression of the NtNPF7.4 gene is analyzed by a fluorescence quantitative PCR method, and the specific implementation operation is as follows:
(1) Salt stress experiments
Tobacco K326 is planted in a plant culture room of a national tobacco gene research center, and the culture conditions are as follows: the temperature (23+/-1) DEG C, the relative humidity (60% +/-2%), the light culture for 16 hours and the dark culture for 8 hours. When the tobacco seedlings grow to six true leaf periods, transferring the tobacco seedlings to Hoagland's nutrient solution for continuous culture, adding 300mM NaCl when changing the nutrient solution after 1 week, respectively collecting roots and leaves after NaCl treatment for 0h, 12h, 3d and 7d, flushing the roots by distilled water when sampling, sucking water by using water absorbing paper, quick-freezing the water by liquid nitrogen, and storing the water in a refrigerator at the temperature of minus 80 ℃. Each strain is provided with 3 independent repeats, and at least 3 tobacco seedlings with consistent growth vigor are selected from each repeat.
(2) qPCR detection of expression level of NtNPF7.4 Gene
The preserved material is extracted with RNA, cDNA is synthesized by using a reverse transcription kit, and then fluorescent quantitative PCR detection is carried out by taking the tobacco Nt26S gene as an internal reference, wherein the specific operation process is as described in the step 2 of the experimental example 1.
The fluorescent quantitative PCR result is shown in figure 3 (the bar graph does not mark the obvious difference of the same lower case letters), which shows that the expression level of the NtNPF7.4 gene in the leaf is obviously increased after the tobacco is subjected to salt stress, and the NtNPF7.4 gene plays a certain role in the salt stress and possibly participates in the chloride ion transportation process.
Experimental example 3 construction of Gene editing vector
In order to further understand the function of the NtNPF7.4 gene in the absorption and transportation of tobacco chloride ions, a gene editing vector for knocking out the NtNPF7.4 gene is constructed, and the specific implementation operation is as follows:
firstly, designing a target site according to the recognition characteristic of a CRISPR/Cas9 system, and designing a 20bp sgRNA target sequence in the 1 st exon region of the NtNPF7.4 gene, wherein the sgRNA target sequence is as shown in figure 4: GATGGAAGTGTGGATAAGCA (SEQ ID NO. 11) the knockout primer sequences NtNPF7.4-T1_F and NtNPF7.4-T1_R were designed as follows:
ntnpf7.4-t1_f: GATTGATGGAAGTGTGGATAAGCA (SEQ ID NO. 12);
ntnpf7.4-t1_r: AAACTGCTTATCCACACTTCCATC (SEQ ID NO. 13).
The reaction system was designed to obtain a DNA double strand (annealing) of the target site, and 20. Mu.L of the reaction system was designed as follows: annealing Buffer for DNAOLigos (5×), 4 μl; upstream and downstream primers (NtNPF7.4-T1_F, ntNPF7.4-T1_R), 4. Mu.L each (50. Mu. MoL/. Mu.L); nuclease-free water was supplemented to 20. Mu.L.
The reaction procedure is: 5min at 95 ℃, 0.1 ℃ every 8s, and 25 ℃; the reaction product is stored at 4 ℃ for standby or directly subjected to subsequent reaction.
The annealing product is connected with a BsaI digested CRISPR/Cas9 vector, and the CRISPR/Cas9 expression vector for knocking out the NtNPF7.4 gene is obtained by screening, and a 20 mu L connecting system is designed as follows: annealing product, 6 μl; 3 mu L of enzyme digestion product (BsaI enzyme digested CRISPR/Cas9 vector); 10×T DNA Ligase Buffer, 2. Mu.L; t4 DNALigase, 1. Mu.L; sterilized water was added to 20. Mu.L and connected at 37℃for 3 hours.
The connection product is transformed into competent cells of the escherichia coli, positive cloning is selected, amplification culture is carried out, plasmids are extracted, and after the construction success of the vectors is confirmed by PCR, the vectors are preserved at low temperature and used for agrobacterium transformation.
Experimental example 4 acquisition of transgenic plants and detection of chloride ion content
The experimental example adopts the gene editing vector constructed in experimental example 3 to transform agrobacterium and further transform tobacco plants to construct transgenic plants with NtNPF7.4 gene knocked out, and the specific implementation operation is as follows:
(1) Transformation of Agrobacterium
The specific method is the same as that of the step of Agrobacterium transformation in the subcellular localization of cells in Experimental example 1.
(2) Transformation of tobacco plants
Taking K326 tobacco aseptic seedling leaves which grow for about one month, processing the leaves into leaf discs with the diameter of 0.5cm by using a puncher, and pre-culturing the leaf discs after processing on an MS solid culture medium for 3d; culturing the transformed agrobacteria engineering bacteria to OD 600 About=0.6, centrifuging at 4000rpm for 5min to collect the thalli, and suspending the thalli with 20mL of MS liquid medium; then placing the pre-cultured leaf discs in bacterial liquid, and infecting for 10min; the excess bacterial liquid around the leaf disc after dip-dyeing is sucked by sterile filter paper, and the leaf disc is subjected to dark culture for 3d on a solid culture medium of MS+6-BA (2 mg/L) +NAA (0.5 mg/L); washing the leaf disc with sterile water containing Cef (400 mg/L), sucking off the excess liquid with sterile filter paper, transferring the leaf disc to MS solid screening medium containing 6-BA (2 mg/L), NAA (0.5 mg/L), cef (200 mg/L) and Kan (50 mg/L), and culturing at 28deg.C under light; when the adventitious bud length reached 0.5cm, the shoots were transferred to MS solid medium containing Cef (200 mg/L) and Kan (50 mg/L) for rooting.
(3) Identification of Gene-editing Strain
And growing the tobacco plants to be transformed for about one month, taking a small number of leaves, extracting DNA (deoxyribonucleic acid) by referring to a plant genome extraction kit instruction, and detecting positive transgenic lines and mutant forms by using PCR (polymerase chain reaction) amplification, cloning and sequencing methods. The specific identification method comprises the following steps:
on the ntnpf7.4 genome, a pair of detection primers is designed, which are located on both sides of the knockout target site, specifically:
NtNPF7.4-J-F5'-ggagtgagtacggtgtgcCTTAACGGCTAATGCATG-3' (SEQ ID NO. 14);
NtNPF7.4-J-R:5'-gagttggatgctggatggTCAATAACTGTTAAAGTTG-3' (SEQ ID NO. 15).
Note that: the lower case bases are herein linker sequences.
By T 0 Carrying out PCR amplification on the transgenic strain DNA template, and carrying out a reaction system: 1. Mu.L of genomic DNA, 2. Mu.L of 10 Xbuffer, 0.5. Mu.L of each of the upstream and downstream primers, 3. Mu.L of dNTP, 0.5. Mu.L of easy Taq enzyme, and ddH were added 2 O to 20. Mu.L; the PCR conditions were: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 56℃for 30s, extension at 72℃for 40s for 25 cycles; and finally extending at 72 ℃ for 10min. The PCR product was Hi-tom sequenced.
As shown in FIG. 5, the sequencing results were analyzed at 11 strain T 0 In the generation plants, 2 forms of mutation were detected in the NtNPF7.4 gene, all of which occurred at the target site of knockout, whereas no mutation was detected in the wild-type plant NtNPF7.4 gene, indicating that at T 0 Knockout of the ntnpf7.4 gene has been successfully achieved in the generation of plants.
(4) Hydroponic test
NtNPF7.4 Gene editing plant T 1 The generation and control K326 plants are planted in a plant culture room of a national tobacco gene research center, and the culture conditions are as follows: the temperature (23+/-1) DEG C, the relative humidity (60% +/-2%), the light culture for 16 hours and the dark culture for 8 hours. And (3) after the tobacco seedlings grow to a six-leaf stage, transferring the tobacco seedlings to Hoagland's nutrient solution for continuous culture, replacing the nutrient solution once after 1 week, and taking roots and leaves for subsequent determination of chloride ion content after 3 days of culture. The root is washed by distilled water and is quickly frozen by liquid nitrogen after the water is absorbed by absorbent paper, and then the root is stored in a refrigerator at-80 ℃. Each strain3 independent replicates were set, and at least 3 tobacco seedlings with consistent growth vigor were selected for each replicate.
(5) Determination of chloride ion content
Freeze-drying preserved root and leaf samples, grinding the root and leaf samples into powder by using a mixed vibration grinder, weighing about 0.0500g (accurate to 0.1 mg) of powder, placing the powder into 10mL of 5% (volume fraction) acetic acid, performing vibration extraction at 30 ℃ for 30min, filtering the powder by using qualitative filter paper, diluting the filtrate, and measuring the chloride ion content by using an Alalis MP6500 table type pH meter in U.S. An Laili.
The results are shown in fig. 6, where the chloride content in roots and leaves increases significantly after the ntnpf7.4 gene knockout (x represents p < 0.01).
In conclusion, according to the invention, the research on the tobacco NtNPF7.4 gene shows that the NtNPF7.4 protein is positioned on a cell membrane; the NtNPF7.4 gene knockout strain is obtained through a gene editing technology, and after water planting, the content of chloride ions in roots and leaves is obviously increased, so that a tobacco plant with accumulated chloride ions is obtained. The NtNPF7.4 gene knockout strain constructed by the invention can be applied to soil with low chloride ion content in China, the condition of low chloride content of flue-cured tobacco is improved, and the quality of flue-cured tobacco is improved.
The last explanation is: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (8)
1. The tobacco nitrate transporter encoding gene NtNPF7.4 is characterized in that: the nucleotide sequence is as follows:
(1) A nucleotide sequence shown as SEQ ID NO. 1;
(2) The nucleotide sequence shown in SEQ ID NO.1 is substituted and/or deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein.
2. The tobacco nitrate transporter NtNPF7.4 is characterized in that: the amino acid sequence is as follows:
(1) An amino acid sequence shown in SEQ ID NO. 2;
(2) The amino acid sequence shown in SEQ ID NO.2 is a derivative protein with identical functions and with one or more amino acid residues replaced and/or deleted and/or added.
3. A gene editing vector, characterized in that: the gene editing vector comprises a target site knockout sequence designed according to the NtNPF7.4 gene, and the nucleotide sequence of the NtNPF7.4 gene is shown as SEQ ID NO. 1.
4. A gene editing vector according to claim 3, characterized in that: the sequence of the knockout primer designed according to the target site knockout sequence is as follows:
ntnpf7.4-t1_f: GATTGATGGAAGTGTGGATAAGCA (SEQ ID NO. 12);
ntnpf7.4-t1_r: AAACTGCTTATCCACACTTCCATC (SEQ ID NO. 13).
5. Use of the tobacco nitrate transporter encoding gene ntnpf7.4 gene of claim 1 or the gene editing vector of claims 3-4 in the cultivation of a chloride enriched tobacco variety.
6. The use of the gene NtNPF7.4 encoding the tobacco nitrate transporter or the gene editing vector in cultivating chloride enriched tobacco varieties according to claim 5, wherein the method comprises the following steps: the NtNPF7.4 gene is knocked out, and the chloride ion content in tobacco roots and leaves is obviously improved.
7. The use of the tobacco nitrate transporter encoding gene ntnpf7.4 or gene editing vector according to claim 5 or 6 in the cultivation of chloride enriched tobacco varieties, characterized in that: the chloride ion enriched tobacco variety is obtained by the following method: and (3) transforming agrobacterium tumefaciens serving as an invader solution by using the gene editing vector, transforming tobacco, and screening and identifying to obtain the chloride ion enriched tobacco variety.
The application of the NtNPF7.4 gene knockout tobacco plant in improving the low chlorine content of flue-cured tobacco in soil with low chlorine ion content.
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