CN117025626B - Tobacco nitrate transporter NtNPF7.4 and its encoding gene, gene editing vector and application - Google Patents
Tobacco nitrate transporter NtNPF7.4 and its encoding gene, gene editing vector and application Download PDFInfo
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Abstract
本发明公开了烟草硝酸盐转运蛋白NtNPF7.4及其编码基因、基因编辑载体和应用。本发明克隆获得了烟草硝酸盐转运蛋白的编码基因NtNPF7.4基因，并通过荧光定量PCR对烟草不同组织和器官进行检测，结果表明该基因在烟草的根、茎、叶和花中均有表达。亚细胞定位分析发现，NtNPF7.4蛋白定位在细胞膜上，盐胁迫后NtNPF7.4基因在叶片中的表达量明显升高，说明NtNPF7.4蛋白参与氯离子转运过程。利用基因编辑载体对NtNPF7.4基因进行敲除，构建基因敲除株，敲除株根和叶片中氯离子含量显著升高，这使培育氯离子富集的烟草成为可能，为改善我国烤烟氯含量偏低的情况提供一个新的方向。
The present invention discloses a tobacco nitrate transporter NtNPF7.4 and its encoding gene, gene editing vector and application. The present invention cloned the encoding gene NtNPF7.4 gene of the tobacco nitrate transporter, and detected different tobacco tissues and organs by fluorescent quantitative PCR. The results showed that the gene was expressed in the roots, stems, leaves and flowers of tobacco. Subcellular localization analysis found that the NtNPF7.4 protein was located on the cell membrane, and the expression level of the NtNPF7.4 gene in the leaves was significantly increased after salt stress, indicating that the NtNPF7.4 protein was involved in the chloride ion transport process. The NtNPF7.4 gene was knocked out using a gene editing vector to construct a gene knockout strain. The chloride ion content in the roots and leaves of the knockout strain was significantly increased, which made it possible to cultivate tobacco enriched in chloride ions, providing a new direction for improving the low chloride content of flue-cured tobacco in my country.
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. NPF transport substrates that have been reported to date are 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 + transport in addition to nitrate transport, and that the atnpf7.3 mutant has 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 that the chlorine content of tobacco leaves in areas with low chlorine content of soil, such as southwest area, southwest jaw area, southern Anhui area and Fujian province area, 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 the chlorine content of the flue-cured tobacco in the main tobacco region in China shows that the chlorine content of the flue-cured tobacco in China is generally low, wherein the high-chlorine flue-cured tobacco is mainly distributed in the north (77.32%), the low-chlorine flue-cured tobacco is mainly distributed in the south (78.23%), in the investigated 2712 flue-cured tobacco samples, the chlorine content of a 53.32% sample is lower than 0.30%, the chlorine content of a 43.10% sample is 0.30% -0.80%, and the chlorine content of a 3.43% sample is higher than 0.80% (reference: regional characteristics research of the chlorine content of the flue-cured tobacco in the main tobacco region 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 technical scheme has the beneficial effects that the tobacco nitrate transporter coding gene NtNPF7.4 is obtained through cloning by designing a specific primer, 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 technical scheme has the beneficial effects that through verification, the tobacco nitrate transporter NtNPF7.4 is closely related to absorption and transportation 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 technical scheme has the beneficial effects that compared with a VIGS strain, the tobacco with the gene knocked out obtained by using the CRISPR/Cas9 technology has better and more stable effect of inhibiting the gene expression quantity 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 (shown as SEQ ID NO. 12);
NtNPF7.4-T1_R AAAC TGCTTATCCACACTTCCATC (shown as 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 technical scheme has the advantages that the NtNPF7.4 gene is knocked out from tobacco through the CRISPR/Cas9 technology, the expression of the NtNPF7.4 gene is restrained, 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 through detection, a new research object is provided for regulating and controlling the content of the chloride ions of the tobacco and other plants, and a gene network for regulating and controlling the chloride ions in the 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 technical scheme has the beneficial effects that 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 transforming agrobacterium with a gene editing vector as an invader solution, transforming tobacco, and obtaining the chloride ion enriched tobacco variety through screening and identification.
The technical scheme has the beneficial effects 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 technical scheme has the beneficial effects that the NtNPF7.4 gene is knocked out from tobacco, so that the chloride ion content in roots and leaves can be obviously improved, a theoretical basis is laid for improving the condition of low chloride ion content in flue-cured tobacco in southern areas of China, and a new direction is provided 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 the sequencing result of the knockout target site of the T 0 generation gene editing plant in experimental example 4 of the present invention;
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 amino acid sequence of the tobacco nitrate transporter is shown as SEQ ID No. 2. The tobacco nitrate transporter is also formed by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No.2, and has derivative polypeptide for affecting the transport of tobacco chloride ions. 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 nucleotide sequence contained in the tobacco nitrate transporter coding gene NtNPF7.4 gene is shown as SEQ ID No. 1. Or a nucleotide sequence which can be hybridized with a DNA sequence limited by SEQ ID No.1 in a sequence table under high strict conditions, or a DNA sequence which has more than 90 percent of homology with the DNA sequence limited by SEQ ID No.1 in the sequence table 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. The expression of the NtNPF7.4 gene can be inhibited by various methods, such as agrobacterium-mediated transformation gene editing vector, plant virus vector mediated gene silencing method, agrobacterium-mediated transformation RNAi interference vector, optimized modification of gene coding frame, optimized 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, seed used was supplied by the national tobacco Gene research center.
The vector pFF19 is provided by the national tobacco gene research center, pCS1300 is obtained from the biological technology limited company of Wuhan, and the CRISPR/Cas9 vector is provided by the national emphasis laboratory of the silkworm genome biology of southwest university.
The strain is Trans5 alpha chemically competent cells purchased from Beijing full gold biotechnology Co., ltd., GV3101 Agrobacterium competent cells purchased from Shanghai Weidi biotechnology Co., ltd., and the synthesis of primers and DNA sequencing were completed by Beijing Liuhua big Gene technologies Co., ltd.
The experimental reagent comprises an RNA extraction kit (RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit), a genome extraction kit (polysaccharide polyphenol plant genome DNA extraction kit) which are purchased from Tiangen biochemical technology (Beijing) limited company, a fluorescence quantitative kit, a reverse transcription kit (Transcriptor FIRST STRAND CDNASYNTHESIS KIT) which are purchased from Roche Switzerland company, DNA amplification enzyme which is purchased from Beijing full-scale gold biotechnology limited company, restriction enzyme BsaI, a plasmid extraction kit and a DNA gel recovery kit which are purchased from Bao biotechnology limited company.
The experimental equipment was a PCR instrument Tprofessional Thermocycler, a Biometra company, a 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 (shown as SEQ ID NO. 12);
NtNPF7.4-T1_R AAAC TGCTTATCCACACTTCCATC (shown as 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 on 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-F5'-ATGGCTTGCTTAAACATTG-3' (shown as SEQ ID NO. 3);
NtNPF7.4-R5'-TTAGACCTTGAAATCTCCTT-3' (shown as 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 was 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 was added to 50. Mu.L. The PCR amplification conditions were 98℃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 to pFF19 vector in a system comprising 6. Mu.L of DNA amplification product, 1. Mu.L of pFF19 vector, and after mixing, ligated at 25℃for 25min.
The ligation products were transformed into E.coli DH 5. Alpha. Competent cells as follows:
Taking out competent cells from-80 ℃ refrigerator, dissolving them on ice, adding the connection product into 100 mu L DH5 alpha competent cells, flicking and mixing them uniformly, ice-bathing for 20min, heating for 90s in 42 ℃ water bath, immediately placing them on ice for 2min, adding 900 mu L LB (without antibiotics) liquid culture medium balanced to room temperature, shaking and culturing for 1h at 37 ℃ and centrifuging for 3min at 4000rpm, removing part of supernatant, leaving 100 mu L of supernatant, mixing the precipitate uniformly, coating on LB solid plate (containing 50 mu g/mu L kanamycin), inverting the culture dish, culturing at 37 ℃ overnight.
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, and the analysis of the gene shows that the amino acid sequence of the coded 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-F5'-CAGTCTCAGGCTTTCAT-3' (shown as SEQ ID NO. 5);
RT-NtNPF7.4-R5'-TCAAGAACTCTTCTATAG-3' (shown as SEQ ID NO. 6).
When detecting the tobacco Nt26S gene, specific primers are as follows:
Nt 26S-F5'-GAAGAAGGTCCCAAGGGTTC-3' (shown as SEQ ID NO. 7);
nt 26S-R5'-TCTCCCTTTAACACCAACGG-3' (shown as SEQ ID NO. 8).
The reaction system of the fluorescent quantitative PCR was 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 2 O to 20. Mu.L.
The reaction conditions of the fluorescent quantitative PCR are that the first step of pre-denaturation is carried out at 95 ℃ for 10s, the second step of PCR reaction is carried out at 95 ℃ for 5s,60 ℃ for 30s and 39 cycles, and the third step of melting curve is carried out.
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 pCS1300 vector containing GFP tag, the primer sequences were:
pCS1300-NPF7.4F:5'-GCTTTCGCGAGCTCGGTACCATGGCTTGCTTAAACATTG-3' (shown as SEQ ID NO. 9);
pCS1300-NPF7.4R:5'-CCCTTGCTCACCATGGATCCGACCTTGAAATCTCCTTGC-3' (shown as SEQ ID NO. 10).
The PCR amplification system and reaction procedure were the same as those used In cloning of the NtNPF7.4 gene of Experimental example 1, and the obtained PCR product was ligated to pCS1300 vector using In-Fusion enzyme In a manner such that the DNA amplification product was 2. Mu.L, the pCS1300 vector was 2. Mu.L and the In-Fusion enzyme was 1. Mu.L. After mixing, 50 ℃ connection 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 the competent cells of Agrobacterium GV3101 from a refrigerator at-80 ℃, freezing and thawing on ice, adding 5 mu L of the established GFP-NtNPF7.4 fusion carrier when thawing is to be performed, mixing the mixture slightly and uniformly, freezing the mixture in ice bath for 30min, freezing the mixture in liquid nitrogen for 5min, immediately placing the mixture on ice after water bath at 37 ℃ for 5min, adding 900 mu L of LB liquid medium without antibiotics, shaking and culturing the mixture for 4h at 200rpm, centrifuging the bacterial liquid at 4500rpm for 3min, discarding half volume of supernatant, re-suspending the supernatant, uniformly coating the supernatant on LB solid medium containing Rif (100 mu g/mL) and Kan (50 mu g/mL), culturing the supernatant for about 2-3 d at 28 ℃ in an inverted mode until single colony is formed, picking the single colony, performing PCR identification on the bacterial liquid after the culture expansion, and identifying the correct positive clone bacterial strain, namely the correct engineering bacteria are converted.
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 under the culture conditions of temperature (23+/-1 ℃) and relative humidity (60+/-2%), 16h light culture and 8h dark culture. And after the tobacco seedlings grow to a six-leaf stage, transferring the tobacco seedlings to Hoagland's nutrient solution for continuous culture, adding 300mM NaCl when the nutrient solution is replaced after 1 week, respectively collecting roots and leaves after NaCl treatment for 0h, 12h, 3d and 7d, flushing the roots with distilled water when sampling, sucking water with water-absorbing paper, quick-freezing the water with liquid nitrogen, and storing the water in a refrigerator at-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, a target site is designed according to the recognition characteristic of a CRISPR/Cas9 system, a 20bp sgRNA target sequence is designed in the 1 st exon region of the NtNPF7.4 gene, as shown in FIG. 4, the sgRNA target sequence is GATGGAAGTGTGGATAAGCA (shown as SEQ ID NO. 11), and knockout primer sequences NtNPF7.4-T1_F and NtNPF7.4-T1_R are designed as follows:
NtNPF7.4-T1_F GATTGATGGAAGTGTGGATAAGCA (shown as SEQ ID NO. 12);
NtNPF7.4-T1_R AAACTGCTTATCCACACTTCCATC (shown as SEQ ID NO. 13).
The reaction system was designed to obtain a double strand (annealing) of the target site DNA, and 20. Mu.L of the reaction system was designed as ANNEALING BUFFER FOR DNAOLIGOS (5X), 4. Mu.L, upstream and downstream primers (NtNPF7.4-T1_F, ntNPF7.4-T1_R), 4. Mu.L each (50. Mu.mol/. Mu.L), and nucleic-FREE WATER was supplemented to 20. Mu.L.
The reaction procedure is 95 ℃ for 5min, 0.1 ℃ is reduced every 8s, the temperature is reduced to 25 ℃, and the reaction product is kept at 4 ℃ for standby or is directly subjected to subsequent reaction.
The annealed product is connected with the BsaI digested CRISPR/Cas9 vector, and the CRISPR/Cas9 expression vector for knocking out the NtNPF7.4 gene is obtained by screening, wherein a 20 mu L connecting system is designed as follows, the annealed product is 6 mu L, the digested product (BsaI digested CRISPR/Cas9 vector) is 3 mu L, 10 xT 4 DNA LIGASE Buffer is 2 mu L, T4 DNALIGASE is 1 mu L, and sterilized water is supplemented to 20 mu L and connected for 3h at 37 ℃.
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 growing for about one month, processing the leaves into leaf discs with the diameter of 0.5cm by a puncher, pre-culturing the leaf discs on MS solid culture medium for 3d, culturing the prepared transformed agrobacterium engineering bacteria to OD 600 =0.6, centrifuging at 4000rpm for 5min to collect thalli, suspending the thalli by using 20mL of MS liquid culture medium, then placing the pre-cultured leaf discs in the bacterial liquid, infecting for 10min, sucking up redundant bacterial liquid around the immersed leaf discs by using sterile filter paper, culturing in dark for 3d on solid culture medium of MS+6-BA (2 mg/L) +NAA (0.5 mg/L), washing the leaf discs by using sterile filter paper, sucking the redundant liquid, transferring the leaf discs to solid culture medium containing 6-BA (2 mg/L), NAA (0.5 mg/L), cef (200 mg/L) and Kan (50 mg/L), and culturing until the solid culture medium containing Cef (200 mg/L) does not grow to root when the solid culture medium containing Cef (0.5 mg/L) is not used 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' (shown as SEQ ID NO. 14);
NtNPF7.4-J-R5'-gagttggatgctggatggTCAATAACTGTTAAAGTTG-3' (shown as SEQ ID NO. 15).
Note that the lower case bases are linker sequences here.
PCR amplification was performed using T 0 generation transgenic line DNA template, the reaction system was 1. Mu.L of genomic DNA, 10 Xbuffer 2. Mu.L of each of the upstream and downstream primers, 0.5. Mu.L of dNTP 3. Mu.L, 0.5. Mu.L of EasyTaq enzyme, and ddH 2 O to 20. Mu.L, under conditions of 94℃pre-denaturation for 4min, 94℃denaturation for 30s,56℃annealing for 30s,72℃extension for 40s, total 25 cycles, and 72℃final extension for 10min. The PCR product was Hi-tom sequenced.
Analytical sequencing results As shown in FIG. 5, in 11T 0 generation plants, 2 forms of mutations were detected in the NtNPF7.4 gene, all of which occurred at the target site of knockout, whereas no mutation was detected in the NtNPF7.4 gene of the wild type plant, indicating that knockout of the NtNPF7.4 gene has been successfully achieved in T 0 generation plants.
(4) Hydroponic test
The NtNPF7.4 gene editing plant T 1 generation and the control K326 plant are planted in a plant culture room of a national tobacco gene research center, wherein the culture conditions are that the temperature is 23+/-1 ℃ and the relative humidity is 60+/-2 percent, the light culture is carried out for 16 hours, and the dark culture is carried out 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 strain is provided with 3 independent repeats, and at least 3 tobacco seedlings with consistent growth vigor are selected from each repeat.
(5) Determination of chloride ion content
The stored root and leaf samples were freeze-dried, ground to powder using a mixed vibration mill, about 0.0500g (accurate to 0.1 mg) of the powder was weighed, placed in 10ml of 5% (volume fraction) acetic acid, extracted with 30 ℃ shaking for 30min, then filtered with qualitative filter paper, and the filtrate was diluted and assayed for chloride ion content using a bench pH of model us An Laili si Alalis MP 6500.
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 NtNPF7.4 gene of tobacco 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 culture, the content of chloride ions in roots and leaves is obviously increased, so that the 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.
It should be understood by those skilled in the art that, although the present invention has been described in detail with reference to the foregoing embodiments, some or all of the technical features may be modified or replaced by other technical features, and the modifications or substitutions do not depart from the scope of the technical features of the embodiments of the present invention.
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