CN114958906B

CN114958906B - Gene and promoter related to low potassium stress of tobacco and application of gene and promoter

Info

Publication number: CN114958906B
Application number: CN202210710520.6A
Authority: CN
Inventors: 喻奇伟; 何轶; 罗贞宝; 范龙; 康俊; 戴彬; 李彩斌; 蔡何青; 曹廷茂; 张小全; 张松涛; 贾宏昉
Original assignee: Henan Agricultural University; Guizhou Tobacco Co Ltd Bijie Branch
Current assignee: Henan Agricultural University; Guizhou Tobacco Co Ltd Bijie Branch
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2023-08-08
Anticipated expiration: 2042-06-22
Also published as: CN114958906A; LU504522B1

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates toNtIAA13Genes and promoters, in particular to genes and promoters related to low potassium stress of tobacco and application thereof.NtIAA13Application of gene in regulating and controlling growth and development of tobacco under low potassium stress tolerance, and application of gene in regulating and controlling growth and development of tobacco under low potassium stress toleranceNtIAA13The sequence of the gene is shown as SEQ ID No. 1. A promoter for use in the initiation of a gene associated with tobacco growth under low potassium stress. By constructing a low potassium inducible specific promoter proNtIAA13NtIAA13Expression vector of gene for realizingNtIAA13The gene is specifically and highly expressed under the condition of low potassium stress, so that the potassium content of tobacco leaves is improved on one hand, and the low potassium resistance of tobacco is improved on the other hand. The invention can be widely applied to tobaccoIAA13Related theory of gene expression inhibition, and application researches on tobacco for improving potassium content, resisting low-potassium stress and the like.

Description

Gene and promoter related to low potassium stress of tobacco and application of gene and promoter

Technical Field

The invention belongs to the field of plant genetic engineering, and relates toNtIAA13Genes, in particular to genes and promoters related to low potassium stress of tobacco and application thereof.

Background

Potassium is a mineral nutrient element necessary for plant growth and development, participates in various vital activities such as carbon and nitrogen metabolism in plants, is closely related to disease resistance and stress resistance of the plants, and affects the yield and quality of the plants. In order to improve the yield and quality of crops, a large amount of potash fertilizer is applied in agricultural production, and on one hand, the use of the potash fertilizer increases the production cost due to the shortage of potassium ore resources and higher imported potash fertilizer cost in China; on the other hand, the use of a large amount of potash fertilizer brings about environmental pollution, brings about adverse effects on ecological environment, and does not accord with the 'double reduction' policy of agricultural development in China. Therefore, how to improve the utilization efficiency of potassium in crops under the condition of reducing the use of potassium fertilizer is an important research direction of modern agricultural development at present. The patent CN 103031331A discloses a protein for improving the low-potassium resistance of rice, which is used for cultivating a new variety of rice with low-potassium stress resistance, and the patent CN103965308A discloses a gene StWRKY6 with low-potassium stress resistance in potatoes; the genes of the low potassium stress resistant pathways in different plants are different.

Tobacco is an important economic crop in China and is also an important model plant for researching the molecular mechanism of crops. At present, bioinformatic analysis of tobacco genome data shows that at least 77 Aux/IAA members exist in tobacco, but the functions of the members are still unclear. In the early stage, we found a gene from tobaccoUnknown auxin repressor geneNtIAA13，The gene is subjected to low potassium stress to enhance expression, so that the tobacco under low potassium stress is studied intensivelyNtIAA13The regulation and control mechanism of the plant potassium is important to improve the potassium absorption and utilization efficiency of the plant, and can provide theoretical basis for breeding tobacco potassium high-efficiency utilization varieties.

In addition, because the over-expression of the 35S strong promoter is easy to cause the influence on certain physiological development of tobacco, the use of the inducible promoter is more beneficial to the growth and development of plants. Tobacco is a potassium-like crop, potassium can obviously improve the combustibility and safety of tobacco leaves, enhance chromaticity and filling property, improve the quality of the tobacco leaves, and the potassium content of the tobacco leaves is always used as an important index for evaluating the quality of flue-cured tobacco at home and abroad. The potassium content of the tobacco leaves in China is generally low and has a large difference from the international high-quality tobacco leaves, which becomes one of the main factors limiting the production of the high-quality tobacco leaves in China. Therefore, it is an urgent and significant object to search for a gene and a promoter that can increase the potassium content and the potassium utilization efficiency of flue-cured tobacco, and to conduct intensive studies on the gene.

Disclosure of Invention

The invention provides a gene and a promoter related to low-potassium stress resistance of tobacco and application thereof, creates a new flue-cured tobacco strain resistant to low-potassium stress, has important application value for reducing potassium fertilizer application and cost and environmental pollution, and provides ideas and methods for cultivating new high-efficiency potassium varieties.

The technical scheme of the invention is realized as follows:

NtIAA13application of gene in regulating and controlling growth and development of tobacco under low potassium stress tolerance, and application of gene in regulating and controlling growth and development of tobacco under low potassium stress toleranceNtIAA13The sequence of the gene is shown as SEQ ID No. 1.

A promoter for use in the initiation of a gene associated with tobacco growth under low potassium stress.

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

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

A recombinant vector comprising the aboveNtIAA13Genes and promoters as described above.

The recombinant vector is applied to regulating and controlling the growth and development of tobacco under low potassium stress resistance.

The recombinant vector is applied to the cultivation of new flue-cured tobacco varieties which efficiently utilize potassium.

Further, the method comprises the following steps: and (3) genetically transforming the recombinant vector into tobacco to be transformed, so that the dominant tobacco with high-efficiency utilization of potassium can be cultivated.

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

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

The invention has the following beneficial effects:

1. the invention utilizes agrobacterium transformation method to obtainNtIAA13Over-expressed plants, found to beNtIAA13Under low-potassium stress, the gene can improve the potassium content and potassium utilization efficiency of the tobacco plant by regulating and controlling the expression of the related genes of the high-affinity potassium transporter NtHAK gene family, further regulate and control the growth and development of the tobacco plant under low-potassium stress, improve the tolerance of the tobacco plant to low-potassium stress, and provide theoretical basis for cultivating new varieties of low-potassium resistant flue-cured tobacco.

2. The application finds thatNtIAA13The highest expression was in the root, second in the stem, and lowest in the leaf (fig. 4A). Further determining that the whole tobacco seedlings cultured for 21 days are under different stresses (N, P, K, ca and Mg),NtIAA13relative expression levels of genes. The results show that low potassium stress upregulates tobaccoNtIAA13Expression of the gene (FIG. 4B). These findings indicate that the number of the active cells,NtIAA13the gene participates in the growth and development of tobacco and can regulate and control the low potassium stressNtIAA13Gene expression; by constructing low-potassium induced specific promoterNtIAA13The gene creates a new flue-cured tobacco strain resistant to low potassium stress, and provides ideas and methods for cultivating new potassium efficient varieties. The application constructs an over-expression vector by utilizing a low-potassium induction specific promoter proNtIAA13, creates a new low-potassium stress resistant flue-cured tobacco strain, and has important application value for reducing potassium fertilizer application, cost and environmental pollution.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic view ofNtIAA13Electrophoretogram of gene clone.

FIG. 2 is a schematic view ofNtIAA13Subcellular localization maps in tobacco epidermal cells.

FIG. 3 shows an embodiment of the present inventionNtIAA13Expression patterns at different sites in tobacco and under different stress of lack of elements.

FIG. 4 shows the phenotypic characteristics of biomass, chlorophyll content and antioxidant capacity of wild type tobacco (WT) and proNtIAA13 transgenic material under different potassium supply conditions provided by the examples of the invention.

FIG. 5 shows the potassium content and potassium accumulation of wild-type tobacco (WT) and proNtIAA13 as provided by the examples of the present invention.

FIG. 6 shows the expression of the high affinity potassium transporter gene of wild type tobacco (WT) and proNtIAA 13:NtIAA 13 transgenic material provided by the examples of the invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.

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

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

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

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

Example 1

Is not aligned with the prior artNtIAA13Cloning and expression of the invention from tobacco K326NtIAA13Gene and promoter sequences, constructionNtIAA13Promoter initiationNtIAA13Over-expression vector of gene and genetic transformation are carried out to obtainNtIAA13Over-expressing transgenic plants using wild type tobacco K326 andNtIAA13the over-expression transgenic plant analyzes the biomass, antioxidant capacity, potassium content and potassium accumulation of the plant under low potassium stress and the expression level of related genes of high affinity potassium transporter, and the over-expression transgenic plant is found by analysisNtIAA13The gene overexpression can regulate the growth and development of the tobacco strain under low-potassium stress, improve the tolerance of the tobacco strain to the low-potassium stress,

tobacco auxin inhibitor geneNtIAA13The sequence of (2) is shown as SEQ ID No.1, and the sequence of (2) is shown as SEQ ID No. 2.

Tobacco auxin response factorNtIAA13The cloning method of the gene and the promoter specifically comprises the following steps:

the first step: extracting total RNA of tobacco leaves; using K326 root system as material, using Eastep Super Total RNA Extraction Kit (Shanghai Probeg Mei Gray biological products Co., ltd.) kit to extract total RNA, measuring OD260/280 value of the total RNA, and detecting its integrity by 1.2% agarose gel electrophoresis;

and a second step of: obtaining total cDNA of tobacco root system and total DNA of genome; reverse transcription was performed using 1ug of K326 young leaf total RNA sample as a template and HiScript type III 1st Strand cDNA Synthesis Kit (+gDNA wind) kit (Nanjinouzan Biotechnology Co., ltd.) to obtain cDNA; extracting total DNA of tobacco genome by adopting EasyPure Plant Genomic DNA Kit of Beijing full gold biotechnology Co., ltd and referring to the instruction book;

in a preferred embodiment of the invention, the reverse transcription conditions are: 42 ℃ 40min,50 ℃ 30min,99 ℃ 5min,5 ℃ 5min.

And a third step of: according to predictions on NCBINtIAA13Design of Gene and promoter sequencesNtIAA13Cloning primers of genes and promoters, and constructing primer sequences of eukaryotic expression vectors are shown in table 1:

TABLE 1 primer sequences

In a preferred embodiment of the invention, the eukaryotic expression vector is proNtIAA13CAMBIA1305, which replaces the 35S constitutive promoter, was started to modify the vector.

Fourth step: tobacco leafNtIAA13PCR amplification of genes and promoters. Respectively using 1 mu L of tobacco root system cDNA and genome DNA as templates, adopting a standard 50 mu L PCR reaction system, and amplifying by using Phusion super-fidelity DNA polymeraseNtIAA13A full-length CDS sequence and a promoter sequence of the gene;

in a preferred embodiment of the invention, the parameters for PCR amplification of the fragment of interest are as follows: ntIAA13 CDS sequence (pre-denatured at 94℃for 4min,35 cycles, incubated at 72℃for 10min. The course of the cycles is 94℃denaturation for 1min,58℃annealing for 1min, and 72℃extension for 1 min);NtIAA13about 1500bp sequence of gene promoter (94 ℃ C. Pre-denaturation for 4min,35 cycles, 72 ℃ C. Heat preservation for 10min. The cycle process is 94 ℃ C. Denaturation for 1min,58 ℃ C. Annealing)1min, extension at 72℃for 1.5 min);

the method for constructing the over-expression vector provided by the embodiment of the invention specifically comprises the following steps:

and (5) constructing and checking an over-expression vector. To construct the overexpression vector proNtIAA13pCAMBIA1305, first, tobacco genomic DNA is used as a template for amplification using primersNtIAA13Construction of the entire promoter sequence of (A)NtIAA13Promoter-driven CAMBIA1305 intermediate vector, then clonedNtIAA13Full length CDS sequence (FIG. 1), construction by cleavageNtIAA13Promoter initiationNtIAA13Is a vector of (a) an expression vector of (b); selecting positive clones for bacterial liquid PCR identification, sequencing positive clones, verifying correct sequences and successfully constructing a vector;

subcellular localization of NtIAA13 in tobacco epidermal cells

To analyze subcellular localization, a cell without terminal codons was usedNtIAA13The cDNA was fused to the N-terminus of the Green Fluorescent Protein (GFP) gene in the pBWA (V) HS vector. Subcellular localization was transiently expressed in leaf epidermal cells of Nicotiana benthamiana and the same transformation was performed with 35S:: GFP on control cells. The transformed tobacco skin samples were then stored at 25 ℃ for 16 hours in the dark. Transient expression of GFP was monitored by confocal laser scanning microscopy (Nikon C2-ER). As a result, as shown in FIG. 2, ntIAA13 was localized in the nucleus and was a regulatory factor.

Determination ofNtIAA13Is regulated by low potassium signals

Wild (WT) tobacco seeds were sterilized, uniformly spread in a sponge seedling tray, and placed in a constant temperature and humidity culture chamber (28 ℃ in the daytime, 14 ℃ in the evening, 22 ℃ in the evening, 10 hours, every 24 hours of circulation; humidity set at 60%). To detect different tobacco tissuesNtIAA13Tissue-specific expression of the gene, sampling the roots, stems and leaves of tobacco cultured for 10 weeks in sections, and determining by qRT-PCR method (the adopted primers are shown in Table 1), the result shows that,NtIAA13the highest expression was in the root, second in the stem, and lowest in the leaf (fig. 3A). Further determining that the whole tobacco seedlings cultured for 21 days are under different stresses (N, P, K, ca and Mg),NtIAA13gene relativityExpression level. The results show that low potassium stress upregulates tobaccoNtIAA13Expression of the gene (FIG. 3B). These findings indicate that the number of the active cells,NtIAA13the gene participates in the growth and development of plants and can regulate and control the low potassium stressNtIAA13Gene expression.

ObtainingNtIAA13Overexpression transgenic tobacco plants

Amplification from tobacco leaves with the specific primers in Table 1NtIAA13CDS sequences of (2) forNtIAA13Is over-expressed by (a). The PCR product was ligated into pCAMBIA1305 vector driven by the proNtIAA13 promoter and the nopaline synthase terminator. The construct was transferred by electrophoresis to the Agrobacterium tumefaciens strain EHA105 and transformed into tobacco (Nicotiana tabacum cv, K326). By introduction ofNtIAA13Over-expressing the construct, 16 transgenic tobacco lines were obtained. Analysis identified two transgenic lines (designated OX1 and OX 2) with better overexpression was confirmed and used in this study.

VerificationNtIAA13Tolerance of overexpressed transgenic tobacco to low potassium stress. By introduction ofNtIAA13Overexpression constructs, wild-type (WT) tobacco seeds andNtIAA13-OXthe transgenic tobacco seeds (T2 generation) are sterilized, uniformly spread in a sponge seedling tray, and placed in a constant temperature and humidity culture room (28 ℃ in the daytime, 14 hours at 22 ℃ in the evening, 10 hours in each 24 hours of circulation, and the humidity is set to be 60%). When the seeds germinated, two leaves were grown, 1/4 Hoagland nutrient solution was initially added for 4 days, then 1/2 Hoagland nutrient solution was added for 4 days, then Hoagland nutrient solution was added for 14 days. Tobacco seedlings for about 30 days were subjected to normal potassium (2 mmol/L) low potassium (0.1 mmol/L) stress treatment for 7 days, harvested, and leaf and root rinsed with deionized water.

Example of embodiment effect analysis

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

1. study the biomass of tobacco (fig. 4A); indicating that under low potassium stress conditions,NtIAA13-OXtransgenic plants are grown relatively well at higher biomass than wild-type (WT) tobacco.

2. Determination of chlorophyll content (figure)4B) The method comprises the steps of carrying out a first treatment on the surface of the Indicating that under low potassium stress conditions,NtIAA13-OXthe chlorophyll content of the transgenic plants was significantly higher than that of the control WT.

3. The activity of antioxidant enzyme SOD and MDA content were determined (FIGS. 4C, D); under the condition of low potassium stress, the method comprises the following steps,NtIAA13-OXthe MDA content of the transgenic tobacco is obviously lower than that of the wild type, and the activity of SOD is obviously higher than that of the wild type. This indicatesNtIAA13The over-expression of (2) can enhance the antioxidant capacity of tobacco.

4. Determining potassium content and potassium accumulation (FIG. 5); at 7 days of low potassium treatment, compared to control WT,NtIAA13the potassium content and potassium accumulation of the over-expressed transgenic lines OX1, OX2 were significantly higher than that of the wild type. The low-potassium treatment reduces the absorption and utilization of potassium by tobacco plants,NtIAA13over-expression can enhance the absorption and accumulation of potassium by tobacco.

5. Determination of nitrate transporter genesNtHAK1、NtHAK5、NtHAK6、NtNKT1Expression level of (1) (fig. 6); under the condition of low potassium stress, the method comprises the following steps,NtIAA13-OXtransgenic tobaccoNtHAK1、NtHAK5The gene expression level is obviously higher than that of the wild typeNtHAK6、NtNKT1The gene expression amount is not greatly different from the wild type.

The above results indicate thatNtIAA13The over-expression of the gene can regulate the growth and development of the tobacco plant under low-potassium stress, improve the tolerance of the tobacco plant to the low-potassium stress, and provide a basis for the salt-tolerant tobacco cultivation technology and the comprehensive utilization of tobacco leaves.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

<110> Pijie Co, a tobacco company of Guizhou province

University of agriculture in Henan province

<120> Gene related to tobacco Low Potassium stress, promoter and use thereof

<130> sequence Listing

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 879

<212> DNA

<213> Nacobbus aberrans

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atggaaccaa cacttggttt acttgacact ggtgaaaaag gttgttcaaa tatggccaaa 60

gtggaacaag attacaatat ggacatgtgt tctgaggaag aaacagagct tgagttgggg 120

ttaggactta gtctaaacag tggtggtggg gtggtggcaa aagctaagaa atcagcatgg 180

ggtgattatg gtagaatttt aactgctaaa gattttccta atggattttc agctgcaggg 240

agatctatta ttaataatag tggtgtttct tctggtacta aaagagctgc tgattttgtt 300

ggttctaata ctgatgttgg atctcctcct actggttcca gtcaggttgt gggatggcca 360

cccataaggg catacagaat gaacagcttg gttaatcaat caaaggttct aaatgctgaa 420

gaagacaagg gagttggcgg gaacgataag aaggagcatt caaagaagaa aatcaatcat 480

ggaaatgcca aggacgacgc gacttctatc aaagaaaaag ggcatcttgg ttttgtaaag 540

gtgaatatgg atggtttgcc tattggaaga aaggtggatt tgaatgctca cacttgctat 600

gaatccttag cagaaacctt agaggatatg ttctgcaaat caactaaaag tggtgaaaag 660

gaacaaacaa caaagtcttt taagctcttg gatggatcat ctgaatttgt gctcacatat 720

gaggataaag aaggagactg gatgcttgtt ggagatgttc catgggagat gtttgtcaac 780

agtgtgaaaa ggctaagaat tatgaggact tctgaggcta atggacttgg tccaagaatc 840

cctcagaagc aggagagaca aaaagggaaa ccaatctaa 879

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tgttgtcatt taatgtataa taatttttac attattgttc atccgtatat caatcaatgc 180

attaaagtta cattactagt ccatgaccta aatacacaac aaggtggagt aaaacgagaa 240

aaattacttc atactccata caataacaaa taacttttgt aaaaatgcct cttttcattt 300

catctaatta gctgtcctac tttgcatgca tgagactgag aatgatatgc acgagaatta 360

ttagttgaaa ggacaatcat tttaatgttg taaaacaaat ttctattttt attgaattac 420

acatatttat attgtttctg attttttaca acaagattga tttaaatgat aaatcctgtg 480

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ctattatttc ttattgacta tttggtatgt tgtattaatt ctacgattgt caattttatt 840

actttatctt aggataactt atcttgtgat tactattcca atctttgaca gataaaaatt 900

atccttatac tatttttaat ctggattagt aaccaaataa aaattaattt tttctaaatt 960

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ccaaaaacac tttcaagtag taatatcaaa agcatggcaa aatacaagcc aaaccaaaaa 1260

ccaaaaatca tgagctcgag aaattgaaat gaatacaata aaagaaactt gaggaagaag 1320

aattttgaca gtgcttgttg cactagcaga catatgggtc ccataatcat cactagctcc 1380

accatacatc cccaccccca cccccacccc ccaccccata ataaaccact atacattctc 1440

ttcattattc tctcaaatcc attttcttga aatcttactc acaaaatctt gaaacccctt 1500

ttgaatg 1507

Claims

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.