CN114958906B - Genes, promoters and applications related to low potassium stress in tobacco - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to a NtIAA13 gene and a promoter, in particular to a gene, a promoter and an application thereof related to tobacco low-potassium stress. The application of the NtIAA13 gene in regulating the growth and development of tobacco tolerance to low potassium stress, the sequence of the NtIAA13 gene is shown in SEQ ID No.1. A promoter used to activate genes related to tobacco growth and development under low potassium stress. By constructing the low-potassium-inducible specific promoter proNtIAA13 to promote the expression vector of the NtIAA13 gene, the specific high expression of the NtIAA13 gene under low-potassium stress conditions can be achieved. On the one hand, the potassium content of tobacco leaves is increased, and on the other hand, the low-potassium tolerance of tobacco is improved. . The invention can be widely used in the related theory of suppressing the expression of tobacco IAA13 gene and the application research of increasing the potassium content of tobacco and resisting low potassium stress.

Description

Genes, promoters and applications related to low potassium stress in tobacco

technical field

The invention belongs to the field of plant genetic engineering and relates to NtIAA13 gene, in particular to a gene related to tobacco low potassium stress, a promoter and application thereof.

Background technique

Potassium is a mineral nutrient element necessary for plant growth and development. It participates in many life activities such as carbon and nitrogen metabolism in plants. It is closely related to plant disease resistance and stress resistance, and affects plant yield and quality. In order to improve crop yield and quality, a large amount of potassium fertilizer is used in agricultural production. On the one hand, due to the shortage of potassium mineral resources in my country and the high cost of imported potassium fertilizer, the use of potassium fertilizer increases production costs; on the other hand, the use of a large amount of potassium fertilizer brings environmental pollution. , has adverse effects on the ecological environment, and does not conform to the "double reduction" policy of my country's agricultural development. Therefore, how to improve crop potassium use efficiency while reducing the use of potassium fertilizer is an important research direction in the development of modern agriculture. Patent CN 103031331 A discloses a protein that improves the ability of rice to tolerate low potassium stress, and is used for the cultivation of new varieties of rice resistant to low potassium stress. Patent CN103965308A discloses the gene StWRKY6 resistant to low potassium stress in potatoes; in different plants The pathways and genes for tolerance to low potassium stress were all different.

Tobacco is an important economic crop in my country, and it is also an important model plant for studying the molecular mechanism of crops. Current bioinformatic analyzes of tobacco genome data suggest that there are at least 77 Aux/IAA members in tobacco, but the functions of these members remain unclear. In the early stage, we discovered an auxin suppressor gene NtIAA13 with unknown gene function from tobacco. The expression of this gene was enhanced by low potassium stress. Therefore, in-depth study of the regulatory mechanism of tobacco NtIAA13 under low potassium stress is crucial for improving the potassium uptake and utilization efficiency of plants. It is very important and can provide a theoretical basis for breeding tobacco varieties with high potassium utilization.

In addition, since the overexpression constructed by the 35S strong promoter is likely to affect some physiological development of tobacco, the use of an inducible promoter will be more conducive to the growth and development of plants. Tobacco is a potassium-loving crop. Potassium can significantly improve the combustibility and safety of tobacco leaves, enhance the color and filling properties, and improve the quality of tobacco leaves. The potassium content of tobacco leaves has been used as an important indicator for evaluating the quality of flue-cured tobacco at home and abroad. Potassium content in tobacco leaves in my country is generally low, and there is a big gap with international high-quality tobacco leaves, which has become one of the main factors restricting the production of high-quality tobacco leaves in my country. Therefore, it is an urgent and significant task to search for a gene and promoter that can improve the potassium content and potassium utilization efficiency of flue-cured tobacco, and to conduct in-depth research on the gene.

Contents of the invention

The present invention proposes a gene, a promoter and its application related to tobacco tolerance to low potassium stress, and creates a new strain of flue-cured tobacco resistant to low potassium stress, which has important application value for reducing potassium fertilizer application, reducing cost and environmental pollution, and is useful for cultivating potassium High-efficiency new varieties provide ideas and methods.

Technical scheme of the present invention is realized like this:

The application of NtIAA13 gene in regulating the growth and development of tobacco tolerance to low potassium stress, the sequence of the NtIAA13 gene is shown in SEQ ID No.1.

A promoter used for starting genes related to tobacco growth and development under low potassium stress.

Further, the sequence of the promoter is shown in SEQ ID No.2.

Further, the gene sequence is shown as SEQ ID No.1.

A recombinant vector, the recombinant vector contains the above-mentioned NtIAA13 gene and the above-mentioned promoter.

The application of the above-mentioned recombinant vector in regulating the growth and development of tobacco resistant to low potassium stress.

The application of the above-mentioned recombinant vector in cultivating new varieties of flue-cured tobacco with high potassium utilization.

Further, the step is: genetically transforming the recombinant vector into the tobacco to be transformed, so as to cultivate the dominant tobacco with high potassium utilization.

Preferably, the tobacco to be modified is a variety of flue-cured tobacco.

Preferably, the tobacco to be modified is flue-cured tobacco variety K326.

The present invention has the following beneficial effects:

1. The present invention utilizes the Agrobacterium transformation method to obtain NtIAA13 overexpression plants, and finds that the NtIAA13 gene increases the potassium content and potassium content of tobacco plants by regulating the expression of genes related to the high-affinity potassium transporter NtHAK gene family under low potassium stress. Therefore, it can regulate the growth and development of tobacco plants under low-potassium stress, improve the tolerance of tobacco plants to low-potassium stress, and provide a theoretical basis for cultivating new low-potassium-tolerant flue-cured tobacco varieties.

2. The present application found that the expression level of NtIAA13 was the highest in roots, followed by stems, and lowest in leaves ( FIG. 4A ). The relative expression level of NtIAA13 gene in whole tobacco seedlings cultured for 21 days under different stresses (N deficiency, P deficiency, K deficiency, Ca deficiency and Mg deficiency) was further determined. The results showed that low potassium stress up-regulated the expression of NtIAA13 gene in tobacco (Fig. 4B). These findings indicate that the NtIAA13 gene is involved in the growth and development of tobacco, and can regulate the expression of the NtIAA13 gene under low potassium stress; by constructing a low potassium-inducible specific promoter to activate the NtIAA13 gene, a new flue-cured tobacco line that is tolerant to low potassium stress can be created, and it can be used for the cultivation of new potassium-efficient tobacco plants. Varieties provide ideas and methods. This application uses the low-potassium-induced specific promoter proNtIAA13 to construct an overexpression vector and create a new strain of flue-cured tobacco resistant to low-potassium stress, which has important application value for reducing potassium fertilizer application, cost and environmental pollution.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Figure 1 is the electrophoresis of NtIAA13 gene clone.

Fig. 2 is a subcellular localization map of NtIAA13 in tobacco epidermal cells.

Fig. 3 is the expression pattern of NtIAA13 in different parts of tobacco and under different starvation stress provided by the embodiment of the present invention.

Fig. 4 shows the phenotypic characteristics biomass, chlorophyll content and antioxidant capacity of wild-type tobacco (WT) and proNtIAA13::NtIAA13 transgenic material under different potassium supply conditions provided by the embodiment of the present invention.

Fig. 5 shows the potassium content and potassium accumulation of wild-type tobacco (WT) and proNtIAA13::NtIAA13 transgenic materials provided in the examples of the present invention.

Fig. 6 shows the gene expression of high-affinity potassium transporter in wild-type tobacco (WT) and proNtIAA13::NtIAA13 transgenic material provided by the embodiment of the present invention.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The application of NtIAA13 gene in regulating the growth and development of tobacco tolerance to low potassium stress, the sequence of the NtIAA13 gene is shown in SEQ ID No.1.

A promoter used for starting genes related to tobacco growth and development under low potassium stress.

Further, the sequence of the promoter is shown in SEQ ID No.2.

Further, the gene sequence is shown as SEQ ID No.1.

A recombinant vector, the recombinant vector contains the above-mentioned NtIAA13 gene and the above-mentioned promoter.

The application of the above-mentioned recombinant vector in regulating the growth and development of tobacco resistant to low potassium stress.

The application of the above-mentioned recombinant vector in cultivating new varieties of flue-cured tobacco with high potassium utilization.

Further, the step is: genetically transforming the recombinant vector into the tobacco to be transformed, so as to cultivate the dominant tobacco with high potassium utilization.

Preferably, the tobacco to be modified is a tobacco variety.

Preferably, the tobacco to be modified is flue-cured tobacco variety K326.

Example 1

In view of the lack of cloning and expression of NtIAA13 in the prior art, the present invention clones the NtIAA13 gene and promoter sequence from tobacco K326, constructs an NtIAA13 promoter to activate the overexpression vector of the NtIAA13 gene, and performs genetic transformation to obtain NtIAA13 overexpression transgenic plants, Using wild-type tobacco K326 and NtIAA13 overexpression transgenic plants, the biomass, antioxidant capacity, potassium content and potassium accumulation, and the expression levels of genes related to high-affinity potassium transporters were analyzed under low potassium stress. After analysis, it was found that the NtIAA13 gene Overexpression can regulate the growth and development of tobacco plants under low potassium stress, improve the tolerance of tobacco plants to low potassium stress,

The sequence of the tobacco auxin suppressor gene NtIAA13 is shown in SEQ ID No.1, and the promoter sequence is shown in SEQ ID No.2.

The cloning method of tobacco auxin response factor NtIAA13 gene and promoter specifically includes:

The first step: extraction of total RNA from tobacco leaves; using the K326 root system as a material, the total RNA was extracted using the method provided by the Eastep® Super TotalRNA Extraction Kit (Shanghai Promega Biological Products Co., Ltd.), and the concentration of the total RNA was determined. OD260/280 value, and use 1.2% agarose gel electrophoresis to detect its integrity;

Step 2: Acquisition of total cDNA and genomic total DNA from tobacco roots; using 1ug of total RNA sample of K326 young leaves as a template, use the HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) kit (Nanjing Novizan Biotechnology Co., Ltd. Company) was reverse-transcribed to obtain cDNA; the extraction of the total DNA of the tobacco genome was performed using the EasyPure Plant Genomic DNA Kit of Beijing Quanshijin Biotechnology Co., Ltd. with reference to the instructions;

In a preferred embodiment of the present invention, the reverse transcription conditions are: 42°C for 40 minutes, 50°C for 30 minutes, 99°C for 5 minutes, and 5°C for 5 minutes.

The third step: according to the design of the NtIAA13 gene and promoter sequence predicted on NCBI and the primers for cloning the tobacco NtIAA13 gene and the promoter, the primer sequences constructed by the eukaryotic expression vector are shown in Table 1:

Table 1 Primer sequences

In a preferred embodiment of the present invention, the eukaryotic expression vector is a CAMBIA1305 transformation vector in which the pro NtIAA13 promoter replaces the 35S constitutive promoter.

Step 4: PCR amplification of tobacco NtIAA13 gene and promoter. Using 1 μL of tobacco root cDNA and genomic DNA as templates, a standard 50 μL PCR reaction system was used to amplify the full-length CDS sequence and promoter sequence of the NtIAA13 gene with Phusion ultra-fidelity DNA polymerase;

In a preferred embodiment of the present invention, the parameters for PCR amplification of the target fragment are as follows: NtIAA13 CDS sequence (pre-denatured at 94°C for 4 minutes, 35 cycles, and incubated at 72°C for 10 minutes. The cycle process is denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute , 72°C extension for 1 min); NtIAA13 gene promoter about 1500bp sequence (pre-denaturation at 94°C for 4 minutes, 35 cycles, 72°C for 10 minutes. The cycle process is denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1.5 minutes) ;

The overexpression vector construction method provided by the embodiments of the present invention specifically includes:

Construction and verification of overexpression vectors. In order to construct the overexpression vector pro NtIAA13 -pCAMBIA1305, at first, using tobacco genomic DNA as a template, use primers to amplify the entire promoter sequence of NtIAA13 , construct the CAMBIA1305 intermediate vector initiated by the NtIAA13 promoter, and then clone the full-length CDS sequence of NtIAA13 (Fig. 1 ), the NtIAA13 promoter was constructed by enzyme digestion to start the expression vector of NtIAA13 ; the positive clones were selected for bacterial liquid PCR identification, and the positive clones were sent for sequencing to verify that the sequence was correct and the vector was successfully constructed;

Subcellular localization of NtIAA13 in tobacco epidermal cells

To analyze subcellular localization, the NtIAA13 cDNA without the terminal codon was fused to the N-terminus of the green fluorescent protein (GFP) gene in the pBWA(V)HS vector. Subcellular localization Transient expression was performed in leaf epidermal cells of N. benthamiana, and control cells were similarly transformed with 35S::GFP. The transformed tobacco skin samples were then stored at 25°C in the dark for 16 hours. Transient expression of GFP was monitored by confocal laser scanning microscopy (Nikon C2-ER). The results are shown in Figure 2. NtIAA13 is localized in the nucleus and is a regulatory factor.

Determines that NtIAA13 is regulated by hypopotassium signaling

Wild-type (WT) tobacco seeds were sterilized, evenly sprinkled on sponge seedling trays, and placed in a constant temperature and humidity culture room (28°C during the day, 14h, 22°C at night, 10h, cycle every 24h; humidity set at 60%) . In order to detect the tissue-specific expression of the NtIAA13 gene in different tobacco tissues, the roots, stems, and leaves of tobacco cultured for 10 weeks were sampled in different parts, and determined by qRT-PCR (the primers used are shown in Table 1). The results showed that the expression level of NtIAA13 was the highest in roots, followed by stems, and lowest in leaves (Fig. 3A). The relative expression level of NtIAA13 gene in whole tobacco seedlings cultured for 21 days under different stresses (N deficiency, P deficiency, K deficiency, Ca deficiency and Mg deficiency) was further determined. The results showed that low potassium stress up-regulated the expression of NtIAA13 gene in tobacco (Fig. 3B). These findings indicated that the NtIAA13 gene was involved in plant growth and development and could regulate NtIAA13 gene expression under low potassium stress.

Obtaining NtIAA13 Overexpression Transgenic Tobacco Plants

The CDS sequence of NtIAA13 was amplified from tobacco leaves with the specific primers in Table 1 for the overexpression of NtIAA13 . The PCR product was ligated into the pCAMBIA 1305 vector driven by the proNtIAA13 promoter and nopaline synthase terminator. The construct was transferred by electrophoresis to Agrobacterium tumefaciens strain EHA105, transformed into tobacco (Nicotiana tabacum cv, K326). By introducing the NtIAA13 overexpression construct, 16 transgenic tobacco lines were obtained. The analysis identified two transgenic lines (named OX1 and OX2) with better overexpression effects, which were confirmed and used in this study.

To verify the tolerance of NtIAA13 overexpression transgenic tobacco to low potassium stress. By introducing the NtIAA13 overexpression construct, wild-type (WT) tobacco seeds and NtIAA13-OX transgenic tobacco seeds (T2 generation) were sterilized, evenly spread in sponge seedling trays, and placed in a constant temperature and humidity culture room (28°C during the day, 14h, 22°C at night, 10h, cycle every 24h; humidity set to 60%). When the seeds germinate and grow into two leaves, start adding 1/4 Hoagland nutrient solution for 4 days, then add 1/2 Hoagland nutrient solution for 4 days, then add Hoagland nutrient solution for 14 days. Tobacco seedlings about 30 days old were subjected to normal potassium supply (2 mmol/L) and low potassium (0.1 mmol/L) stress treatment for 7 days, and the tobacco seedlings were harvested, and the leaves and roots were washed with deionized water.

Example analysis of implementation effect

The following studies were performed on wild-type and transgenic line samples:

1. Study the biomass of tobacco (Fig. 4A); it shows that compared with the control, under low potassium stress, the biomass of NtIAA13-OX transgenic plants is higher than that of wild-type (WT) tobacco, and the growth is relatively better.

2. Measure the chlorophyll content (Figure 4B); it shows that compared with the control, the chlorophyll content of the NtIAA13-OX transgenic plants is significantly higher than that of the control WT under low potassium stress conditions.

3. Measure the activity of antioxidant enzyme SOD and MDA content (Fig. 4C, D); under low potassium stress, the MDA content of NtIAA13-OX transgenic tobacco was significantly lower than that of wild type, while the activity of SOD was significantly higher than that of wild type. This suggests that overexpression of NtIAA13 can enhance the antioxidant capacity of tobacco.

4. Determination of potassium content and potassium accumulation (Figure 5); when treated with low potassium for 7 days, compared with the control WT, the potassium content and potassium accumulation of the NtIAA13 overexpression transgenic lines OX1 and OX2 were significantly higher than those of the wild type . Low potassium treatment decreased the absorption and utilization of potassium in tobacco plants, and overexpression of NtIAA13 could enhance the absorption and accumulation of potassium in tobacco.

5. Determination of the expression levels of nitrate transporter genes NtHAK1 , NtHAK5 , NtHAK6 , and NtNKT1 (Figure 6); under low potassium stress conditions, the expression levels of NtHAK1 and NtHAK5 genes in NtIAA13-OX transgenic tobacco were significantly higher than those of wild type, while NtHAK6 , NtNKT1 gene expression was not significantly different from the wild type.

The above results show that the overexpression of NtIAA13 gene can regulate the growth and development of tobacco plants under low potassium stress, improve the tolerance of tobacco plants to low potassium stress, and provide a basis for the cultivation technology of salt-tolerant tobacco and the comprehensive utilization of tobacco leaves.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

<110> Guizhou Provincial Tobacco Company Bijie City Company

Henan Agricultural University

<120> Genes, promoters and applications related to low potassium stress in tobacco

<130> Sequence Listing

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<170> PatentIn version 3.5

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Claims (7)

1.NtIAA13The application of the gene in regulating the growth and development of tobacco under low potassium stress is characterized in that: overexpression of the tobacco in the tobaccoNtIAA13Genes of the order ofNtIAA13The gene sequence is shown as SEQ ID No. 1.

2. A promoter, characterized in that: the sequence of the promoter is shown as SEQ ID No.2 and is used for promoting genes related to tobacco growth and development under low potassium stress.

3. A recombinant vector, characterized in that: the recombinant vector comprising the recombinant vector according to claim 1NtIAA13A gene and the promoter of claim 2.

4. Use of the recombinant vector of claim 3 for regulating tobacco growth under low potassium stress, characterized in that: overexpression of the tobacco in the tobaccoNtIAA13And (3) a gene.

5. The use of the recombinant vector of claim 4 for breeding new flue-cured tobacco varieties which efficiently utilize potassium.

6. The use according to claim 5, characterized by the steps of: and (3) genetically transforming the recombinant vector into tobacco to be transformed, so that the dominant tobacco with high-efficiency utilization of potassium element can be cultivated.

7. The use according to claim 6, characterized in that: the tobacco to be modified is flue-cured tobacco variety K326.