Tobacco calcium regulatory nerve phosphatase B-like gene and application thereof

Abstract

The invention provides a tobacco calcium modulated nerve phosphatase B-like gene and application of the tobacco calcium modulated nerve phosphatase B-like gene. The tobacco calcium modulated nerve phosphatase B-like gene comprises NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a or NtCBL3b; the nucleotide of the gene comprises: (1) a nucleotide sequence as shown in any one of SEQ ID NO: 2-6; or (2) a nucleotide sequence which is obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence as shown in any one of SEQ ID NO: 2-6 and expresses the same functional protein. Experiments prove that the NtCBL gene is a key gene related to tobacco potassium loss, the potassium content of tobacco can be improved, high-quality tobacco varieties can be cultivated, the quality of tobacco products can be improved, economic benefits can be improved, and the NtCBL gene has good and wide market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco genes, and in particular relates to a tobacco calcineurin B-like gene and an application thereof. Background Art

[0002] Potassium is an essential element for plant growth and development, and plays an important role in tobacco leaf maturity, aroma, flavor, combustibility, tar content, etc. A large number of studies have shown that potassium ions have the greatest impact on tobacco leaf combustibility among tobacco leaf chemical components, and tobacco leaf potassium is an important factor affecting tobacco leaf tar content. Adding potassium to tobacco leaves significantly reduces the tar content. The content of organic potassium in tobacco leaves of different varieties is closely related to the amount of tar, and the potassium ion content in tobacco leaves is extremely significantly negatively correlated with the release of total particulate matter. The increase in organic potassium content can reduce the release of tar. Potassium content has been used as one of the important indicators for evaluating tobacco leaf quality, and plays an important guiding role in breeding and production to improve tobacco leaf quality.

[0003] There are many factors that affect the potassium content of tobacco leaves, including soil, climate, water, fertilizer, and varieties. Compared with foreign countries, the potassium content of tobacco leaves in my country is generally low, especially in the northern tobacco-growing areas, where there is a large gap. Based on the close relationship between potassium ions and tar content, potassium-rich tobacco varieties are selected and bred. Flue-cured tobacco varieties with high potassium content, such as ZT99 and Zhongyan 98, have been bred at home and abroad. However, these tobacco varieties have limited potassium extraction effects, unclear genetic backgrounds, and have not been widely promoted and applied. Therefore, the use of biotechnology to breed high-potassium genotype varieties will be one of the key ways to solve the potassium content of tobacco raw materials in my country.

[0004] Potassium ions (K + ) has strong mobility, and its utilization efficiency depends on the coordination between the absorption, transportation, storage and redistribution of potassium ions in the plant body. Plants mediate the transmembrane absorption of potassium through potassium ion channels and potassium ion transporters on the root cell plasma membrane. After the root absorbs potassium from the external environment, the potassium ions are transported and distributed to various tissues and organs through the vascular system. A large amount of potassium ions are stored in the vacuole. When the external environment is potassium-deficient, the potassium ions stored in the plant vacuole flow back to the cytoplasm to provide for various biochemical reactions and cell metabolism. The growth and development of the double mutant cbl2 cbl3 of two members of the CBL family located in the vacuole membrane in Arabidopsis is extremely sensitive to low potassium environments under various conditions. Under low potassium conditions, the potassium content of the cbl2 cbl3 mutant is significantly higher than that of the wild-type plant, and the process of potassium ion transportation from the vacuole cavity to the cytoplasm in the mutant is blocked. Therefore, blocking the loss of potassium in the vacuole can effectively increase the potassium content of the plant, but there are few reports in the prior art that use genetic means to reduce the loss of tobacco potassium, thereby increasing the potassium content. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a tobacco calcineurin B-like gene and its application. The present invention proves through experiments that the NtCBL gene provided is a key gene related to the loss of potassium in tobacco. By regulating the expression of the NtCBL gene, the potassium content of tobacco can be increased, high-quality tobacco varieties can be cultivated, the quality of tobacco products can be improved, and the economic benefits can be improved, which has good and broad market application prospects.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tobacco calcineurin B-like gene, the gene comprising: NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a or NtCBL3b; the nucleotide sequence of the gene comprises:

[0008] (1) the nucleotide sequence shown in any one of SEQ ID NOs: 2-6;

[0009] or (2) a nucleotide sequence in which one or more nucleotides are replaced and / or deleted and / or added to the nucleotide sequence shown in any one of SEQ ID NOs: 2-6 and the nucleotide sequence expresses a protein with the same function.

[0010] The present invention screens out key genes NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b related to the loss of potassium in tobacco vacuoles through bioinformatics analysis combined with molecular biological experiments, and further conducts in-depth research on the functions of the genes through gene editing, proving that the NtCBL gene is a key gene related to the loss of potassium in tobacco.

[0011] In a second aspect, the present invention provides a tobacco calcineurin B-like protein, the protein comprising: NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a or NtCBL3b; the amino acid sequence of the protein comprises:

[0012] (I) the amino acid sequence shown in any one of SEQ ID NOs: 8-12;

[0013] or (II) a derivative protein having the same function as that of any one of SEQ ID NOs: 8-12, wherein one or more amino acid residues are substituted and / or deleted and / or added.

[0014] In a third aspect, the present invention provides the use of the tobacco calcineurin B-like gene described in the first aspect as a target for increasing the potassium content of tobacco.

[0015] In a fourth aspect, the present invention provides a method for increasing the potassium content of tobacco, the method comprising: reducing the expression level of tobacco calcineurin B-like gene, reducing the loss of potassium in tobacco vacuoles, and thus increasing the potassium content of tobacco.

[0016] The present invention firstly finds that by regulating the expression amount of the NtCBL gene, the potassium content of tobacco can be increased, high-quality tobacco varieties can be cultivated, the quality of tobacco products can be improved, and the economic benefits can be improved.

[0017] Preferably, the method of reducing the expression level of tobacco calcineurin B-like gene comprises:

[0018] (a) inserting, deleting or replacing one or more bases in the target gene;

[0019] (b) replacing the transcriptional or translational regulatory element of the target gene with a regulatory element with lower activity;

[0020] (c) Inactivation or reduction of the activity of the target gene through CRIPSRi gene inhibition or antisense RNA technology.

[0021] Preferably, the step of inserting, deleting or replacing one or more bases in the target gene comprises: using the CRISPR / Cas9 gene editing system to knock out tobacco calcineurin B-like genes in the tobacco genome, including: NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b.

[0022] Preferably, the CRISPR / Cas9 gene editing system comprises: sgRNA and Cas9.

[0023] Preferably, the sgRNA comprises: sgRNA1 and sgRNA2, wherein the sgRNA1 targets NtCBL1a, NtCBL1b, NtCBL3a and NtCBL3b genes; and the sgRNA2 targets NtCBL2a and NtCBL2b genes.

[0024] Preferably, the target site sequence of the sgRNA1 includes SEQ ID NO:13.

[0025] Preferably, the target site sequence of the sgRNA2 includes SEQ ID NO:14.

[0026] In a fifth aspect, the present invention provides a method for breeding potassium-rich tobacco varieties, the method comprising: constructing a CRISPR / Cas9 gene editing vector for knocking out the tobacco calcineurin B-like gene described in the first aspect, transforming tobacco through Agrobacterium-mediated transformation to obtain T0 generation positive plants, collecting leaf samples and extracting genomic DNA; sequencing the products after PCR amplification, screening homozygous plants with 6 genes knocked out at the same time, and obtaining potassium-rich tobacco varieties.

[0027] Preferably, the CRISPR / Cas9 gene editing vector is pORE-Cas9 / gRNA.

[0028] Preferably, the vector comprises sgRNA and Cas9.

[0029] Preferably, the promoter connected to the sgRNA is a U6 promoter; the promoter connected to the Cas9 is a 35S strong promoter.

[0030] Preferably, the primers used in the PCR amplification include:

[0031] The upstream and downstream primers for amplifying the NtCBL1a gene include SEQ ID NO: 15 and SEQ ID NO: 16;

[0032] The upstream and downstream primers for amplifying the NtCBL1b gene include SEQ ID NO: 17 and SEQ ID NO: 18;

[0033] The upstream and downstream primers for amplifying the NtCBL2a gene include SEQ ID NO: 19 and SEQ ID NO: 20;

[0034] The upstream and downstream primers for amplifying the NtCBL2b gene include SEQ ID NO: 21 and SEQ ID NO: 22;

[0035] The upstream and downstream primers for amplifying the NtCBL3a gene include SEQ ID NO: 23 and SEQ ID NO: 24;

[0036] The upstream and downstream primers for amplifying the NtCBL3b gene include SEQ ID NO:25 and SEQ ID NO:26.

[0037] In a sixth aspect, the present invention provides an sgRNA, comprising: sgRNA1 and sgRNA2, wherein the sgRNA1 targets NtCBL1a, NtCBL1b, NtCBL3a, and NtCBL3b genes; and the sgRNA2 targets NtCBL2a and NtCBL2b genes.

[0038] Preferably, the target site sequence of the sgRNA1 includes SEQ ID NO:13.

[0039] Preferably, the target site sequence of the sgRNA2 includes SEQ ID NO:14.

[0040] In a seventh aspect, the present invention provides a CRISPR / Cas9 gene editing system, comprising: the sgRNA and Cas9 described in the sixth aspect.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The present invention screened out the key genes NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b related to the loss of potassium in tobacco vacuoles through bioinformatics analysis combined with molecular biological experiments.

[0043] (2) The present invention first discovered that by regulating the expression level of the NtCBL gene, the potassium content of tobacco can be increased, high-quality tobacco varieties can be cultivated, the quality of tobacco products can be improved, and economic benefits can be improved, which has good and broad market application prospects.

[0044] (3) The present invention has developed a breeding method for potassium-rich tobacco varieties through gene editing technology. The NtCBL-CRISPR / Cas9 recombinant plasmid is constructed and transformed into tobacco through Agrobacterium-mediated transformation. Homozygous plants with six genes knocked out simultaneously are screened. Compared with the control plants, the dry matter potassium content is significantly increased, by 46.2% and 71.2%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 These are the test results of potassium content in tobacco leaves of NtCBL gene-edited multi-gene homozygous knockout strains and wild-type control plants. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] Informatics analysis of tobacco NtCBL genes.

[0050] Using the protein sequences of Arabidopsis cbl2 and cbl3 genes as reference sequences, the tobacco genome database (http: / / lifenglab.hzau.edu.cn / Nicomics / Download / index.php) was searched homologously using the blastP program, and the gene and protein sequences of six tobacco Ntcbl (Nta11g22530, Nta21g09270, Nta08g02350, Nta15g25960, Nta18g11150 and Nta17g11900) were obtained and named NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b, respectively.

[0051] Tobacco calcineurin B-like genes and their numbers: NtCBL1a (SEQ ID NO: 1), NtCBL1b (SEQ ID NO: 2), NtCBL2a (SEQ ID NO: 3), NtCBL2b (SEQ ID NO: 4), NtCBL3a (SEQ ID NO: 5) or NtCBL3b (SEQ ID NO: 6).

[0052] Tobacco calcineurin B-like protein and its number: NtCBL1a (SEQ ID NO: 7), NtCBL1b (SEQ ID NO: 8), NtCBL2a (SEQ ID NO: 9), NtCBL2b (10), NtCBL3a (SEQ ID NO: 11) or NtCBL3b (SEQ ID NO: 12).

[0053] Example 2

[0054] Editing of the tobacco NtCBL gene.

[0055] According to the sequences of tobacco NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b6 genes (shown in SEQ ID NO: 1-6), gene editing knockout targets were designed. The same target sequence was designed for the four genes NtCBL1a, NtCBL1b, NtCBL3a and NtCBL3b, and the same target sequence was designed for the two genes NtCBL2a and NtCBL2b (Table 1). The CRISPR / Cas expression vector backbone was linearized and connected to the annealing product, which was introduced into the downstream of the U6 promoter and the upstream region of the gRNA. The vector also enhances the expression of the Cas9 protein through two tandem 35S strong promoters.

[0056] Table 1

[0057]

[0058] The constructed NtCBL-CRISPR / Cas9 recombinant plasmid was transformed into tobacco K326 by Agrobacterium-mediated transformation to obtain T0 generation positive plants, and genomic DNA was extracted after collecting leaf samples. According to the differences between the target site and the gene sequence, specific primers were designed, and the products were subjected to Sanger sequencing (Shenzhen BGI Co., Ltd.) after PCR amplification. The specific primer sequences used are shown in Table 2.

[0059] Table 2

[0060]

[0061] By sequencing the specific amplified sequence of each gene, 16 different types of NtCBL gene knockout plants were obtained, of which two were homozygous plants NtCBL-2 and NtCBL-8 with six genes knocked out at the same time. The mutation types are shown in Tables 3 and 4.

[0062] Table 3

[0063]

[0064] Table 4

[0065]

[0066] Example 3

[0067] Detection of potassium content in tobacco leaves of gene-edited materials.

[0068] Yu Wangchang selected the middle leaves of the T0 generation NtCBL gene-edited multi-gene homozygous knockout strains and wild-type control plants, dried them in an oven at 70-80°C, weighed their dry weight and crushed them, and determined the potassium content by hydrochloric acid extraction-flame photometry. The results are as follows Figure 1 As shown, the results showed that the dry matter potassium content of the NtCBL gene knockout strain was significantly increased compared with the control, by 46.2% and 71.2%, respectively.

[0069] Example 4

[0070] The potassium content in tobacco leaves of different types of NtCBL gene knockout plants was tested.

[0071] Includes: NtCBL-1, NtCBL-3, NtCBL-4, NtCBL-5, NtCBL-6, NtCBL-7, NtCBL-9, NtCBL-10, NtCBL-11, NtCBL-12, NtCBL-13, NtCBL-14, NtCBL-15, NtCBL-16.

[0072] The test results are shown in Table 5.

[0073] Table 5

[0074] Plant number Knockout type Average potassium content K326 Control group 4.03％ NtCBL-1 / NtCBL-9 / NtCBL-12 NTCBL1A / 1B double mutation 4.57％ NtCBL-3 / NtCBL-10 / NtCBL-14 NTCBL2A / 2B double mutation 4.62％ NtCBL-6 / NtCBL-15 / NtCBL-16 NTCBL3A / 3B double mutation 3.84％ NtCBL-13 ntcbl1a / 1b / 2a / 2b tetramer 4.51％ NtCBL-4 / NtCBL-5 ntcbl1a / 1b / 3a / 3b tetramer 4.19％ NtCBL-7 / NtCBL-11 NTCBL2A / 2B / 3A / 3B Tetra 3.77％

[0075] From the results of 5 above, it can be seen that there is redundancy between NtCBL genes, and the potassium ion content of the double-mutant and quadruple-mutant strains is not significantly increased compared with the control.

[0076] In summary, the present invention screened out key genes related to the loss of potassium in tobacco vacuoles through bioinformatics analysis combined with molecular biological experiments, and performed gene editing on the above key genes. The results showed that the homozygous plants with 6 genes knocked out at the same time had significantly increased dry matter potassium content compared with the control plants; based on this, a breeding method for potassium-rich tobacco varieties was developed, and NtCBL-CRISPR / Cas9 recombinant plasmids were constructed to transform tobacco through Agrobacterium-mediated transformation. The resulting high-quality tobacco varieties can improve the quality of tobacco products and have broad market application prospects. The applicant declares that the above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and disclosure scope of the present invention.

Claims

1. A tobacco calcineurin B-like gene, characterized in that: The gene includes: NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a or NtCBL3b; the nucleotide sequence of the gene includes: (1) the nucleotide sequence shown in any one of SEQ ID NOs: 2-6; or (2) a nucleotide sequence in which one or more nucleotides are replaced and / or deleted and / or added to the nucleotide sequence shown in any one of SEQ ID NOs: 2-6 and the nucleotide sequence expresses a protein with the same function.

2. A tobacco calcineurin B-like protein, characterized in that: The protein includes: NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a or NtCBL3b; the amino acid sequence of the protein includes: (I) the amino acid sequence shown in any one of SEQ ID NOs: 8-12; or (II) a derivative protein having the same function as that of any one of SEQ ID NOs: 8-12, wherein one or more amino acid residues are substituted and / or deleted and / or added.

3. Use of the tobacco calcineurin B-like gene according to claim 1 as a target for increasing the potassium content of tobacco.

4. A method for increasing the potassium content of tobacco, characterized in that: The method comprises: reducing the expression amount of tobacco calcineurin B-like gene, reducing the loss of potassium in tobacco vacuoles, and thus increasing the potassium content of tobacco.

5. The method for increasing potassium content in tobacco according to claim 4, characterized in that: The method of reducing the expression level of tobacco calcineurin B-like gene includes: (a) inserting, deleting or replacing one or more bases in the target gene; (b) replacing the transcriptional or translational regulatory element of the target gene with a regulatory element with lower activity; (c) Inactivation or reduction of the activity of the target gene through CRIPSRi gene inhibition or antisense RNA technology.

6. The method for increasing potassium content in tobacco according to claim 5, characterized in that: The step of inserting, deleting or replacing one or more bases in the target gene comprises: using CRISPR / Cas9 gene editing system to knock out tobacco calcineurin B-like genes in the tobacco genome, including: NtCBL1a, NtCBL1b, NtCBL2a, NtCBL2b, NtCBL3a and NtCBL3b; Preferably, the CRISPR / Cas9 gene editing system comprises: sgRNA and Cas9; Preferably, the sgRNA comprises: sgRNA1 and sgRNA2, wherein the sgRNA1 targets NtCBL1a, NtCBL1b, NtCBL3a and NtCBL3b genes; and the sgRNA2 targets NtCBL2a and NtCBL2b genes; Preferably, the target site sequence of the sgRNA1 includes SEQ ID NO: 13; Preferably, the target site sequence of the sgRNA2 includes SEQ ID NO:

14.

7. A method for breeding potassium-rich tobacco varieties, characterized in that: The method comprises: constructing a CRISPR / Cas9 gene editing vector for knocking out the tobacco calcineurin B-like gene according to claim 1, transforming tobacco through Agrobacterium-mediated transformation to obtain T0 generation positive plants, collecting leaf samples and extracting genomic DNA; sequencing the products after PCR amplification, screening homozygous plants with six genes knocked out at the same time, and obtaining potassium-rich tobacco varieties.

8. The method for breeding potassium-rich tobacco varieties according to claim 7, characterized in that: The CRISPR / Cas9 gene editing vector is pORE-Cas9 / gRNA; Preferably, the vector comprises sgRNA and Cas9; Preferably, the promoter connected to the sgRNA is a U6 promoter; the promoter connected to the Cas9 is a 35S strong promoter; Preferably, the primers used in the PCR amplification include: The upstream and downstream primers for amplifying the NtCBL1a gene include SEQ ID NO: 15 and SEQ ID NO: 16; The upstream and downstream primers for amplifying the NtCBL1b gene include SEQ ID NO: 17 and SEQ ID NO: 18; The upstream and downstream primers for amplifying the NtCBL2a gene include SEQ ID NO: 19 and SEQ ID NO: 20; The upstream and downstream primers for amplifying the NtCBL2b gene include SEQ ID NO: 21 and SEQ ID NO: 22; The upstream and downstream primers for amplifying the NtCBL3a gene include SEQ ID NO: 23 and SEQ ID NO: 24 The upstream and downstream primers for amplifying the NtCBL3b gene include SEQ ID NO:25 and SEQ ID NO:

26.

9. An sgRNA, characterized in that The sgRNA includes: sgRNA1 and sgRNA2, wherein the sgRNA1 targets NtCBL1a, NtCBL1b, NtCBL3a and NtCBL3b genes; the sgRNA2 targets NtCBL2a and NtCBL2b genes; Preferably, the target site sequence of the sgRNA1 includes SEQ ID NO: 13; Preferably, the target site sequence of the sgRNA2 includes SEQ ID NO:

14.

10. A CRISPR / Cas9 gene editing system, characterized in that: The system comprises: the sgRNA and Cas9 described in claim 9.

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