Genes and promoters related to tobacco low iron stress and their applications

Abstract

The present invention belongs to the technical field of plant genetic engineering and relates to NtPIN3 The invention relates to genes and promoters, in particular to genes and promoters related to low iron stress in tobacco and their application. NtPIN3 Application of genes in regulating tobacco growth and development under low iron stress, the NtPIN3 The sequence of the gene is shown in SEQ ID No. 1. A promoter for activating a gene related to tobacco growth and development under low iron stress. By constructing a low iron-induced specific promoter proNtPIN3 to activate NtPIN3 Gene expression vectors to achieve NtPIN3 The gene is specifically highly expressed under low iron stress conditions, which, on the one hand, increases the iron content of tobacco leaves, and on the other hand, improves the low iron tolerance of tobacco. The present invention can be widely used in tobacco NtPIN3 Related theories of gene expression inhibition and applied research on increasing iron content in tobacco and resisting low iron stress.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering and relates to NtPIN3 The invention relates to genes, in particular genes and promoters related to low iron stress in tobacco and applications thereof. Background Art

[0002] Iron is an essential nutrient that participates in all aspects of plant growth and development. It is widely involved in plant photosynthesis, respiration, chlorophyll synthesis, and some metabolic enzymatic reactions, etc., and plays a vital role in plant growth. Although the content of iron in the soil is very rich, mainly in the form of Fe 2+ and Fe 3+ In the form of iron, it exists in the oxygen-rich environment, but in the environment with sufficient oxygen, it will form stable oxides or hydroxides that are difficult to be absorbed and utilized by plants, so it is difficult to meet the normal growth and development needs of plants. Studies have shown that about 40% of the soil in the world is in a state of iron deficiency, and my country still has iron deficiency. For a populous country like China, most areas from south to north (Sichuan Basin to Inner Mongolia Plateau) and from east to west (Huaibei Plain to Xinjiang Uygur Autonomous Region) are seriously iron deficient, causing iron deficiency and chlorosis of plants, which will inevitably have an important impact on agricultural production. Iron deficiency will not only cause a decrease in crop yield and quality, but also reduce the iron content in crops, affect human iron intake, and will inevitably lead to some related diseases such as iron deficiency anemia, which will seriously affect human health. Therefore, the impact of plant iron deficiency and chlorosis is a global problem and one of the hot spots in the current research on plant nutritional stress. Cultivating and breeding crop varieties resistant to iron stress is the key to solving the problem urgently.

[0003] Polar Auxin Transport Family Genes (PINs) are mainly involved in regulating the distribution of auxin in plant tissues, thus affecting plant growth and development. So far, studies on the functions of this family of genes have been conducted in rice and Arabidopsis, and the studies have shown that members of this family are mainly involved in regulating the development of the root system, but there have been no reports on whether members of this family are involved in regulating the absorption of nutrient elements. Current bioinformatics analysis of tobacco genome data shows that there are at least 20 PINs family members in tobacco, but the functions of these members have rarely been reported. Our research group discovered a polar auxin transport family gene in tobacco. NtPIN3 The expression of this gene is enhanced by low iron stress, but the function of this gene has not been documented. Therefore, in-depth research on the effect of low iron stress on tobacco NtPIN3 The regulatory mechanism is crucial to improving the iron absorption and utilization efficiency of plants, and can provide a theoretical basis for breeding tobacco varieties with high iron utilization efficiency. Summary of the invention

[0004] The present invention proposes a gene and a promoter related to tobacco tolerance to low iron stress and their application, creates a new flue-cured tobacco strain resistant to low iron stress, has important application value in reducing the application of iron fertilizer, reducing costs and environmental pollution, and provides ideas and methods for breeding new iron-efficient varieties.

[0005] The technical solution of the present invention is achieved in this way:

[0006] NtPIN3 Application of genes in regulating tobacco growth and development under low iron stress, the NtPIN3 The sequence of the gene is shown in SEQ ID No.1.

[0007] A promoter for driving genes related to tobacco growth and development under low iron stress.

[0008] Furthermore, the sequence of the promoter is shown as SEQ ID No.2.

[0009] Furthermore, the gene sequence is shown as SEQ ID No.1.

[0010] A recombinant vector comprising the above NtPIN3 Gene and promoter as described above.

[0011] The application of the above-mentioned recombinant vector in regulating the growth and development of tobacco under low iron stress.

[0012] The application of the above-mentioned recombinant vector in cultivating new flue-cured tobacco varieties that efficiently utilize iron.

[0013] Furthermore, the step is: genetically transforming the recombinant vector into the tobacco to be transformed, so as to cultivate superior tobacco with efficient iron utilization.

[0014] Preferably, the tobacco to be transformed is a flue-cured tobacco variety.

[0015] Preferably, the tobacco to be transformed is the flue-cured tobacco variety Yunyan 87.

[0016] The present invention has the following beneficial effects:

[0017] 1. The present invention utilizes Agrobacterium transformation to obtain NtPIN3 Overexpression plants were found NtPIN3 Under low iron stress, the gene increases the iron content and iron utilization efficiency of tobacco plants by regulating the expression of genes related to the high-affinity iron transporter NtHAK gene family, thereby regulating the growth and development of tobacco plants under low iron stress and improving the tolerance of tobacco plants to low iron stress, which can provide a theoretical basis for breeding new low-iron resistant flue-cured tobacco varieties.

[0018] 2. This application found NtPIN3The expression level was highest in roots, followed by stems, and lowest in leaves ( Figure 4 A). We further tested the effects of different stresses (N deficiency, P deficiency, K deficiency, Ca deficiency, and Mg deficiency) on the whole tobacco seedlings after 21 days of cultivation. NtPIN3 The results showed that low iron stress significantly upregulated the expression of NtPIN3 Gene expression ( Figure 4 B). These findings suggest that NtPIN3 Genes involved in tobacco growth and development and can regulate low iron stress NtPIN3 Gene expression; Initiated by constructing a low iron-induced specific promoter NtPIN3 Gene, create a new flue-cured tobacco strain resistant to low iron stress, and provide ideas and methods for breeding new iron-efficient varieties. This application uses the low iron-induced specific promoter proNtPIN3 to construct an overexpression vector and create a new flue-cured tobacco strain resistant to low iron stress, which has important application value in reducing the application of iron fertilizer, reducing costs and environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 for NtPIN3 Electrophoresis diagram of gene cloning.

[0021] Figure 2 for NtPIN3 Subcellular localization map in tobacco epidermal cells.

[0022] Figure 3 The embodiment of the present invention provides NtPIN3 Expression patterns in different parts of tobacco and under different nutrient deficiency stresses.

[0023] Figure 4 The phenotypic characteristics of wild-type tobacco (WT) and proNtPIN3::NtPIN3 transgenic materials provided in the embodiments of the present invention include biomass, chlorophyll content and antioxidant capacity under different iron supply conditions.

[0024] Figure 5 The iron content and iron accumulation of wild-type tobacco (WT) and proNtPIN3::NtPIN3 transgenic materials provided in the embodiments of the present invention.

[0025] Figure 6The expression of genes related to the iron absorption and utilization pathway in wild-type tobacco (WT) and proNtPIN3::NtPIN3 transgenic materials provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0026] The technical solution 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] The present invention cloned from tobacco Yunyan 87 NtPIN3 Gene and promoter sequences, construction NtPIN3 Promoter start NtPIN3 Gene overexpression vector and genetic transformation were performed to obtain NtPIN3 Overexpression transgenic plants, using wild-type tobacco Yunyan 87 and NtPIN3 The biomass, antioxidant capacity, iron content and iron accumulation, and expression levels of high-affinity iron transporter-related genes of the overexpressing transgenic plants under low iron stress were analyzed. NtPIN3 Gene overexpression can regulate the growth and development of tobacco plants under low iron stress and improve the tolerance of tobacco plants to low iron stress.

[0029] Tobacco auxin repressor gene NtPIN3 The sequence is shown as SEQ ID No.1, and the promoter sequence is shown as SEQ ID No.2.

[0030] Tobacco auxin response factor NtPIN3 The cloning methods of genes and promoters specifically include:

[0031] Step 1: Extraction of total RNA from tobacco leaves; Using the root system of Yunyan 87 as the material, the total RNA was extracted using the method provided by the Eastep® Super TotalRNA Extraction Kit (Shanghai Promega Bioproducts Co., Ltd.), the OD260 / 280 value of the extracted total RNA was determined, and its integrity was detected by 1.2% agarose gel electrophoresis;

[0032] Step 2: Obtaining total cDNA and genomic DNA from tobacco roots; using 1ug of total RNA sample from young leaves of Yunyan 87 as a template, reverse transcription was performed using the HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novogene Biotechnology Co., Ltd.) to obtain cDNA; the total genomic DNA of tobacco was extracted using the EasyPurePlant Genomic DNA Kit from Beijing Quanshijin Biotechnology Co., Ltd. according to the instructions;

[0033] In a preferred embodiment of the present invention, the reverse transcription conditions are: 42°C for 40 min, 50°C for 30 min, 99°C for 5 min, and 5°C for 5 min.

[0034] Step 3: According to the prediction on NCBI NtPIN3 Design of gene and promoter sequences in Nicotiana tabacum NtPIN3 The primer sequences for gene and promoter cloning and eukaryotic expression vector construction are shown in Table 1:

[0035] Table 1 Primer sequences

[0036]

[0037] In a preferred embodiment of the present invention, the eukaryotic expression vector is pro NtPIN3 The CAMBIA1305 transformation vector was constructed by replacing the 35S constitutive promoter with the promoter.

[0038] Step 4: Tobacco NtPIN3 PCR amplification of genes and promoters. Using 1 μL of tobacco root cDNA and genomic DNA as templates, a standard 50 μL PCR reaction system was used to amplify the genes with Phusion high-fidelity DNA polymerase. NtPIN3 Full-length CDS sequence and promoter sequence of the gene;

[0039] In a preferred embodiment of the present invention, the parameters for PCR amplification of the target fragment are as follows: NtPIN3 CDS sequence (pre-denaturation at 94°C for 4 min, 35 cycles, and incubation at 72°C for 10 min. The cycle process was denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min); NtPIN3 Gene promoter sequence of about 1500 bp (pre-denaturation at 94°C for 4 min, 35 cycles, and insulation at 72°C for 10 min. The cycle process is denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1.5 min);

[0040] The method for constructing an overexpression vector provided in the embodiment of the present invention specifically includes:

[0041] Overexpression vector construction and verification. NtPIN3 -pCAMBIA1305, first, using tobacco genomic cDNA as a template, using primers to amplify NtPIN3 The entire promoter sequence of NtPIN3 The promoter-activated CAMBIA1305 intermediate vector was then cloned NtPIN3 Full-length CDS sequence ( Figure 1 ), constructed by enzyme digestion NtPIN3 Promoter start NtPIN3 expression vector; select positive clones for bacterial liquid PCR identification, and send positive clones for sequencing to verify that the sequence is correct and the vector is successfully constructed;

[0042] Subcellular localization of NtPIN3 in tobacco epidermal cells

[0043] To analyze subcellular localization, NtPIN3 The cDNA 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 Nicotiana benthamiana, and control cells were transformed in the same way with 35S::GFP. The transformed tobacco epidermal samples were then stored in the dark at 25°C for 16 h. Transient expression of GFP was monitored by confocal laser scanning microscopy (Nikon C2-ER). The results are shown in Figure 2 As shown, NtPIN3 is located in the cell membrane and is a membrane protein.

[0044] Sure NtPIN3 is regulated by low iron signaling

[0045] Wild-type (WT) tobacco seeds were sterilized, evenly spread in a sponge seedling tray, and placed in a constant temperature and humidity culture room (28°C for 14 h during the day and 22°C for 10 h at night, with a cycle every 24 h; humidity was set to 60%). NtPIN3 The tissue-specific expression of the gene was determined by sampling the roots, stems, and leaves of tobacco cultured for 10 weeks using the qRT-PCR method (the primers used are shown in Table 1). The results showed that NtPIN3 The expression level was highest in stems, followed by roots, and lowest in leaves ( Figure 3 A). We further tested the effects of different stresses (N deficiency, P deficiency, K deficiency, Ca deficiency, and Mg deficiency) on the whole tobacco seedlings after 21 days of cultivation. NtPIN3 The results showed that low iron stress upregulated the expression of NtPIN3 Gene expression ( Figure 3 B). These findings suggest that NtPIN3Genes involved in plant growth and development and can regulate low iron stress NtPIN3 Gene expression.

[0046] get NtPIN3 Overexpression transgenic tobacco plants

[0047] The specific primers in Table 1 were used to amplify the NtPIN3 CDS sequence for NtPIN3 The PCR product was ligated to the pCAMBIA 1305 vector driven by the proNtPIN3 promoter and the nopaline synthase terminator. The construct was transferred to the Agrobacteriumtumefaciens strain EHA105 by electroporation, and 16 transgenic tobacco lines were obtained by Agrobacterium infection of tobacco (Nicotiana tabacum cv, Yunyan 87). Two transgenic lines with better overexpression effects (named OX1 and OX2) were identified and confirmed and used in this study.

[0048] verify NtPIN3 Overexpression of transgenic tobacco toler- ance to low iron stress. NtPIN3 Overexpression constructs, wild-type (WT) tobacco seeds and NtPIN3 - OX Transgenic tobacco seeds (T2 generation) were sterilized, evenly spread in a sponge seedling tray, and placed in a constant temperature and humidity culture room (28°C, 14h during the day, 22°C, 10h at night, cycle every 24h; humidity set to 60%). When the seeds germinated and grew into two leaves, 1 / 4 Hoagland nutrient solution was added for 4 days, then 1 / 2 Hoagland nutrient solution was added for 4 days, and then Hoagland nutrient solution was added for 14 days. About 30-day-old tobacco seedlings were used for normal iron (20μmol / L) and low iron (0.1μmol / L) stress treatments for 7 days, and the tobacco seedlings were harvested and the leaves and roots were rinsed with deionized water.

[0049] Analysis of implementation effect examples

[0050] The following studies were performed on samples of wild-type and transgenic lines:

[0051] 1. Study the biomass of tobacco ( Figure 4 A); Compared with the control, under low iron stress conditions, NtPIN3 - OX The biomass of transgenic plants was higher than that of wild-type (WT) tobacco and their growth was relatively good.

[0052] 2. Determination of chlorophyll content ( Figure 4 B); Compared with the control, under low iron stress conditions, NtPIN3 - OX The chlorophyll content of the transgenic plants was significantly higher than that of the WT control.

[0053] 3. Determination of iron content and iron accumulation ( Figure 4 C, D); After 7 days of normal iron treatment, compared with the control WT, NtPIN3 The iron content and iron accumulation of the overexpressing transgenic lines OX1 and OX2 were significantly higher than those of the wild type. NtPIN3 Overexpression can enhance the absorption and accumulation of iron in tobacco.

[0054] 4. Determination of the activity of antioxidant enzyme SOD and MDA content ( Figure 5 ); Under low iron stress conditions, NtPIN3 - OX H 2 O 2 The content of CAT and SOD in the wild type was significantly lower than that in the wild type, while the activity of CAT and SOD was significantly higher than that in the wild type. NtPIN3 Overexpression of β-catenin can enhance the antioxidant capacity of tobacco.

[0055] 5. Determination of genes related to iron absorption and utilization NtIRT1 , NtFRO , NtNRAMP3 , NtOPT3 The expression level of Figure 6 ); Under normal iron supply and low iron stress conditions, NtPIN3 - OX Genetically modified tobacco NtIRT1 , NtFRO , NtNRAMP3 , NtOPT3 The gene expression level was significantly higher than that of the wild type.

[0056] The above results show that NtPIN3 Overexpression of the gene can regulate the growth and development of tobacco plants under low iron stress, improve the tolerance of tobacco plants to low iron stress, and provide a basis for salt-tolerant tobacco cultivation technology and the comprehensive utilization of tobacco leaves.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Overexpression NtPIN3 The application of the gene in promoting tobacco growth and development under low iron stress is characterized by: Said NtPIN3 The sequence of the gene is shown in SEQ ID No.

1.

2. A promoter initiating NtPIN3 Application in genes, characterized by: The sequence of the promoter is shown in SEQ ID No. 2; NtPIN3 The gene sequence is shown in SEQ ID No.

1.

3. An overexpression vector, characterized in that: The overexpression vector contains the NtPIN3 The gene and the promoter sequence are shown in SEQ ID No.

2.

4. Use of the overexpression vector according to claim 3 in promoting tobacco growth and development under low iron stress.

5. Use of the overexpression vector according to claim 3 in cultivating flue-cured tobacco varieties that utilize iron.

6. The use according to claim 5, characterized in that: The steps are: genetically transforming the overexpression vector into the tobacco to be transformed, so as to cultivate the superior tobacco that can utilize iron.

7. The use according to claim 6, characterized in that: The tobacco to be transformed is the flue-cured tobacco variety Yunyan 87.

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