CN111909252A - Ginseng PgbHLH149 transcription factor and application thereof - Google Patents

Abstract

The invention provides a ginseng PgbHLH149 transcription factor and application thereof, relating to the field of plant transcription factors. According to the application, through effective exogenous substance screening tests for relieving ginseng iron toxicity stress, a proper amount of silicon element is added under iron toxicity stress to effectively relieve ginseng iron toxicity symptoms, and by combining a transcriptome and an element analysis result, a new transcription factor PgbHLH149 transcription factor in a bHLH transcription factor family is discovered, and the expression level of the PgbHLH149 gene and the iron accumulation level in ginseng body are in a trend of obvious negative correlation, namely the higher the expression level of the PgbHLH149 gene is, the lower the iron accumulation level in ginseng body is. Experiments of PgbHLH149 transgenic Arabidopsis show that the overexpression of the PgbHLH149 gene in Arabidopsis can improve the resistance of transgenic plants to iron toxicity stress.

Description

Ginseng PgbHLH149 transcription factor and application thereof

Technical Field

The invention relates to the field of plant transcription factors, in particular to a PgbHLH149 transcription factor and application thereof.

Background

Ginseng is a perennial herb of the family Araliaceae, belonging to the genus Panax, is a rare Chinese medicinal material with high medicinal value, and is known as the king of all herbs. Jilin province is the main ginseng producing area in China, the yield accounts for 80% of the whole country and 60-70% of the world, and the ginseng cultivation industry plays a significant role in Jilin province agricultural economy. The red skin disease is a common physiological disease of ginseng roots, mainly causes red brown patches on the periderm of the ginseng roots, and the lesions gradually expand along with the increase of the age of ginseng, thereby causing the reduction of the grade of ginseng commodity. In areas such as the Jilin province and the soil loosening, the incidence of some plots is sometimes as high as more than 80%, which causes huge economic loss to ginseng farmers, seriously affects the economic benefit of ginseng planting, and restricts the health and sustainable development of the ginseng industry in China.

The occurrence of erythroderma is a complex process and is the result of the combined action of various factors (soil water content, soil organic matter, iron and aluminum elements, phenolic substances and the like). Wherein the iron content in the soil is closely related to erythroderma, and when the iron content in the soil or the nutrient solution is excessive, the periderm of the root of the ginseng appears reddish brown plaques, namely the erythroderma of the ginseng. Therefore, iron toxicity (excessive iron content) is an important factor for inducing the red skin disease of ginseng. Therefore, the iron-toxicity resistance of the ginseng is improved, and the excavation of iron-toxicity resistance related genes can provide theoretical and technical support for preventing and relieving red skin disease of the ginseng and also provide molecular basis for breeding red skin resistant patients.

Disclosure of Invention

The first purpose of the invention is to provide a ginseng PgbHLH149 transcription factor which is related to ginseng iron toxin resistance.

The second purpose of the invention is to provide a DNA molecule carrying the genetic information of the ginseng PgbHLH149 transcription factor.

The third purpose of the invention is to provide a recombinant vector of the DNA molecule carrying the genetic information of the ginseng PgbHLH149 transcription factor.

The fourth object of the present invention is to provide a recombinant cell containing the above recombinant vector.

The fifth purpose of the invention is to provide the application of the DNA molecule in improving the resistance of plants to iron toxicity stress.

The sixth purpose of the invention is to provide an RNA molecule carrying genetic information of the ginseng PgbHLH149 transcription factor.

In order to achieve the above object, the present invention firstly provides a ginseng PgbHLH149 transcription factor, which is a protein having an amino acid sequence shown in SEQ ID No.3, or a protein having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No. 3.

Specifically, substitution and/or deletion and/or addition of one or several amino acid residues means substitution and/or deletion and/or addition of not more than ten amino acid residues.

The invention also provides a DNA molecule for coding the ginseng PgbHLH149 transcription factor.

In some embodiments of the invention, the DNA molecule comprises the DNA sequence shown in SEQ ID NO.2 or a DNA sequence which has more than 70% homology with the DNA sequence shown in SEQ ID NO.2 and encodes the same functional protein.

Specifically, the homology of 70% or more may be 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

The present invention also provides a recombinant vector comprising the DNA molecule as described above.

In some embodiments of the invention, the vector is a cloning vector or an expression vector.

The invention also provides a recombinant cell comprising the recombinant vector.

In some embodiments of the invention, the recombinant cell is a plant cell.

The invention also provides the application of the DNA molecule in improving the resistance of plants to iron toxicity stress.

In some embodiments of the invention, the plant is arabidopsis thaliana.

In some embodiments of the invention, the application is a method of growing an anti-iron virus plant, the method comprising: the expression vector comprising the DNA molecule described above is transformed into a plant cell, and the transformed plant cell is cultured into an anti-iron virus plant.

The invention also provides an RNA molecule for coding the ginseng PgbHLH149 transcription factor.

A "plant" is any plant at any developmental stage.

A "plant cell" is the structural and physiological unit of a plant, comprising protoplasts and a cell wall. Plant cells may be in the form of isolated individual cells or cultured cells, or as a higher organized unit, such as a plant tissue, plant organ, or part of a whole plant.

The invention has the beneficial effects that:

according to the application, through effective exogenous substance screening tests for relieving ginseng iron toxicity stress, a proper amount of silicon element is added under iron toxicity stress to effectively relieve ginseng iron toxicity symptoms, and a transcriptome and element analysis result is combined, so that a new transcription factor PgbHLH149 transcription factor in a bHLH transcription factor family is discovered, the expression level of the PgbHLH149 gene and the iron accumulation level in ginseng body are in a negative correlation trend, namely the higher the expression level of the PgbHLH149 gene is, the lower the iron accumulation level in ginseng body is. Experiments of transforming PgbHLH149 into Arabidopsis show that the over-expression of the PgbHLH149 gene in Arabidopsis can improve the resistance of Arabidopsis to iron toxicity stress.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.

FIG. 1 shows the phenotypic differences of ginseng leaves under different iron treatment conditions;

FIG. 2 shows the difference in iron content in ginseng under different iron treatment conditions;

FIG. 3 shows the difference in the expression level of the PgbHLH149 gene under different iron-treated conditions;

FIG. 4 shows the result of electrophoresis of the PCR product in example 2;

FIG. 5 is a map of an overexpression vector of the PgbHLH149 gene;

FIG. 6 shows the Arabidopsis floral dip transformation and screening steps;

FIG. 7 shows photographs of phenotypes of Arabidopsis thaliana of the T1 generation after treatment with different iron concentrations than that of wild type Arabidopsis thaliana;

FIG. 8 is the statistics of plant stress rate.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

Beneficial elements such as calcium, silicon, potassium and the like are nutrient elements beneficial to the growth and development of plants, and can improve the resistance of the plants to abiotic stress such as iron stress. In the rice cultivation, exogenous elements such as calcium, silicon, potassium and the like are applied to effectively relieve iron toxicity. Researches find that the beneficial elements can regulate and control the iron stability in the rice body by reducing the accumulation of excessive iron, and simultaneously increase the activity of rice antioxidant enzymes (APX, POD and the like), thereby improving the iron toxicity resistance of the rice and relieving the iron toxicity. Therefore, the beneficial elements have important application values in the aspects of improving the iron toxicity resistance of plants and relieving the damage of iron toxicity to the plants.

The bHLH transcription factor contains basic/helix-loop-helix structures (bHLH), is widely present in plants, participates in signal transduction of various biotic and abiotic stresses, and plays an important role in the growth and development process of eukaryotes. A large number of researches prove that the bHLH family protein participates in an iron deficiency stress response signal regulation and control pathway and plays an important role in improving the iron deficiency stress resistance of plants. However, the current research focuses on the aspect that the bHLH transcription factor responds to the iron deficiency stress of plants, and the regulation and control research of the bHLH transcription factor in the iron toxicity stress is almost not available, and the regulation and control of the bHLH transcription factor in the iron toxicity stress response is not reported.

The inventor discovers that the addition of a proper amount of beneficial elements can enable the ginseng root and the ginseng leaf to show resistance under the iron toxicity stress and the red skin symptom of the ginseng root to disappear through effective exogenous material screening tests for relieving the iron toxicity stress of the ginseng. Analyzing and comparing ginseng under different treatment conditions by a high-throughput sequencing technology (RNA-Seq), discovering a bHLH family gene-PgbHLH 149 gene (named by the inventor), and simultaneously combining with RACE technology to obtain a full-length cDNA sequence (SEQ ID NO.1) of the PgbHLH149 gene, wherein the full-length cDNA size of the PgbHLH149 gene is 1160bp, the coding region of the PgbHLH149 gene is a DNA sequence shown in the 247-th 885 bit in the SEQ ID NO.1, and the PgbHLH149 gene has an Open Reading Frame (ORF) of 639bp, a 5 'untranslated region of 246bp and a 3' untranslated region of 275 bp. The expression of the PgbHLH149 gene is induced by elements such as silicon under iron toxicity stress, the ginseng shows resistance to iron toxicity stress when the iron toxicity plus silicon elements are treated, the expression level of the PgbHLH149 gene in the ginseng is obviously increased, and meanwhile, the iron accumulation level in the ginseng is obviously reduced. Therefore, it is presumed that the PgbHLH149 gene is involved in the resistance of ginseng to iron toxicity stress, and the resistance of ginseng to iron toxicity stress can be improved by inducing the overexpression of the gene.

As shown in SEQ ID NO.2, the DNA coding sequence of the PgbHLH149 gene is shown, namely the DNA sequence of the open reading frame (position 247-885) in SEQ ID NO. 1.

The PgbHLH149 gene encodes a 212 amino acid protein that contains a helix loop helix domain, mediates protein dimer formation, and is associated with protein binding to DNA. The protein is a bHLH transcription factor (named as PgbHLH149 transcription factor by the inventor) in ginseng, and the amino acid sequence of the protein is shown as SEQ ID NO. 3.

tttcattccctctttgaggtatctttcccaccctgtcaccctctctattctttcttcgttccttcaccgttatttcttctccctcatcgttctttcctgaaatttatgttcagagccgcctgcatcattcagaaggtatttctgatttttaagcttgttctccaattttcttctgtttttacacgagaggaatttaggttgagaaactgaacattttgctaaagaatttcatcgatctcaaaatcgatggtatcattatttatctcgaatcccgaagcagcgtcaaattccaacagctcacgcgaatctaagcgcaaaaagagaagaaaaatcgaaacgagcgataaaattgaaaatcaccagcagctcaaaatcgatcgtagtagatggaaatcaagcgcagaacaccagatctactcaacgaaactcgtcgaagctctctgtaaccggaattcttctcaatcggcgacgtcaactgctggcggccgcgccgtccgtgaaactgccgaccgagtactcgccgtcgaggctaagggaaggacgcggtggagcagagctattctatctaacaggctcaagcttcaattgaatatgatcaagcataagaagagtaagaaagcgaaggtgaccggcgatgtccggtcgaagaaactggcggcgaagaagaaattgtccacgttgaaccggaaagtgcgtgttcttggccggctagttcccggttgccggaagctatcgtttccgaatcttctagaagaaactacggattatattgcggctttggaaatgcaagtccgagccatgactgctctcaccgggcttctcgccggctccggcccttcttcggatcggctcggctcaaatttatcccaatcatcgactttataaagtttttattgtcctaaaaaggaagtcattgttaattactgagtttttttttttaatcctacatcatttggttctttcatctttcttggtattttgattttgggtttcttaacattattgttatttatttattcattcattactacttaatgggttggttggaattttgattaaacatgacaactaattgtttcgttttttatactactaagcatagcatttgttaaacatgacaactaattgtttcgttttttatactactaagcatagcattt(SEQ ID NO.1)。

atggtatcattatttatctcgaatcccgaagcagcgtcaaattccaacagctcacgcgaatctaagcgcaaaaagagaagaaaaatcgaaacgagcgataaaattgaaaatcaccagcagctcaaaatcgatcgtagtagatggaaatcaagcgcagaacaccagatctactcaacgaaactcgtcgaagctctctgtaaccggaattcttctcaatcggcgacgtcaactgctggcggccgcgccgtccgtgaaactgccgaccgagtactcgccgtcgaggctaagggaaggacgcggtggagcagagctattctatctaacaggctcaagcttcaattgaatatgatcaagcataagaagagtaagaaagcgaaggtgaccggcgatgtccggtcgaagaaactggcggcgaagaagaaattgtccacgttgaaccggaaagtgcgtgttcttggccggctagttcccggttgccggaagctatcgtttccgaatcttctagaagaaactacggattatattgcggctttggaaatgcaagtccgagccatgactgctctcaccgggcttctcgccggctccggcccttcttcggatcggctcggctcaaatttatcccaatcatcgactttataa(SEQ ID NO.2)。

MVSLFISNPEAASNSNSSRESKRKKRRKIETSDKIENHQQLKIDRSRWKSSAEHQIYSTKLVEALCNRNSSQSATSTAGGRAVRETADRVLAVEAKGRTRWSRAILSNRLKLQLNMIKHKKSKKAKVTGDVRSKKLAAKKKLSTLNRKVRVLGRLVPGCRKLSFPNLLEETTDYIAALEMQVRAMTALTGLLAGSGPSSDRLGSNLSQSSTL(SEQ ID NO.3)。

Example 1 screening of Ginseng radix iron toxin-resistant related transcription factor Gene PgbHLH149

Ginseng seedlings are respectively treated by 0.05mM Fe (normal iron concentration), 0.4mM Fe (iron toxicity stress) and 0.4mM Fe + Si (0.5mM, 1.5mM and 2.0mM), effective exogenous substances capable of effectively relieving the iron toxicity stress of ginseng and proper concentrations thereof are screened, and the fact that the symptoms of the iron toxicity of ginseng can be effectively relieved by adding a proper amount of silicon element (0.5mM) under the iron toxicity stress is found (see the figure 1 and the figure 2).

Taking tissues such as roots, stems, leaves and the like of ginseng under different iron treatment conditions, quickly freezing a part of experimental samples by liquid nitrogen, and storing at a low temperature of-80 ℃ for RNA extraction. And (3) deactivating enzyme in the other part of the sample in an oven at 105 ℃ for 30min, and drying the sample to constant weight at 80 ℃ for element component analysis.

Freezing experimental sample stored at-80 deg.C with liquid nitrogen, grinding into powder, sequentially adding TRIzol reagent, chloroform and isopropanol to extract and precipitate total RNA of the sample, washing RNA precipitate with 75% ethanol by volume fraction, centrifuging, drying, adding appropriate amount of RNase-free ddH₂O dissolves the RNA. The concentration, purity and integrity of the extracted total RNA were checked by Nanodrop2000 and 1 wt% agarose gel electrophoresis, respectively, genomic DNA from RNA was digested by DNase I, and RIN values were determined using Agilent 2100. Crossing RNA to Shanghai MijishengThe company performs transcriptome (RNA-Seq) analysis. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

Pulverizing and grinding the dried experimental sample, sieving with 100 mesh sieve to obtain a sample to be tested, respectively and accurately weighing 0.25g of the sample to be tested in a 100mL triangular flask by adopting HNO₃-HClO₄After digestion, the Fe content of the solution was measured by inductively coupled plasma emission spectroscopy (ICP-OES).

FIG. 1 shows the phenotype differences of ginseng leaves under different iron treatment conditions, as shown in FIG. 1, the edges of ginseng leaves grown under iron toxicity (0.4mM Fe) condition are yellow, the area of the leaves is small, the growth condition is poor, all the areas of the ginseng leaves grown under iron toxicity + silicon element (0.4mM Fe +0.5mM Si) condition are green, the growth condition is good, and the phenotype of the leaves is basically consistent with that of the ginseng leaves grown under normal iron concentration (0.05mM Fe).

Fig. 2 shows the difference in iron content among the ginseng under different iron treatment conditions, as shown in fig. 2, the iron content among the ginseng under the iron toxicity (0.4mM Fe) condition was significantly increased compared to the normal iron concentration (0.05mM Fe) condition, whereas the iron content among the ginseng under the iron toxicity + silicon element (0.4mM Fe +0.5mM Si) condition was significantly decreased compared to the iron toxicity (0.4mM Fe) condition, which was close to the iron content among the ginseng under the normal iron concentration (0.05mM Fe) condition.

By combining the figure 1 and the figure 2, the resistance of the ginseng to the iron toxicity environment can be obviously improved by adding 0.5mM of silicon element in the iron toxicity environment, and the iron enrichment amount in the ginseng body is greatly reduced.

FIG. 3 shows the difference in the expression level of PgbHLH149 gene under different iron treatment conditions, as shown in FIG. 3, the expression level of PgbHLH149 gene under iron toxicity (0.4mM Fe) condition was significantly reduced compared to that under normal iron concentration condition (0.05mM Fe), while the expression level of PgbHLH149 gene under iron toxicity + elemental silicon (0.4mM Fe +0.5mM Si) condition was far higher than that under normal iron concentration condition (0.05mM Fe) and iron toxicity (0.4mM Fe).

By combining fig. 2 and fig. 3, it can be found that there is a tendency that the expression level of the PgbHLH149 gene and the iron accumulation level in the ginseng body are significantly negatively correlated, that is, the higher the expression level of the PgbHLH149 gene is, the lower the iron accumulation level in the ginseng body is, and particularly, the expression level of the PgbHLH149 gene in the ginseng body is significantly increased and the iron accumulation level in the ginseng body is significantly decreased in the iron toxicity + silicon element treatment.

Example 1 conclusion:

combining the transcriptome and the element analysis result, the application discovers a new transcription factor PgbHLH149 transcription factor in a bHLH transcription factor family, wherein the PgbHLH149 transcription factor is related to the iron toxicity resistance of the ginseng, and the excessive expression of the PgbHLH149 gene can be induced by adding a certain amount of silicon element in the iron toxicity stress environment, so that the iron toxicity stress resistance of the ginseng is improved.

Example 2 cloning and sequencing of PgbHLH149 Gene, a Gene of a transcription factor related to resistance to iron toxin in Ginseng

Since the sequence information of the PgbHLH149 gene provided by the transcriptome analysis result is not necessarily 100% accurate, the present inventors newly amplified and sequenced the PgbHLH149 gene: the cDNA of ginseng is used as a template, the PgbHLH149 gene sequence is divided into a plurality of small segments for amplification, the sequences of the small segments are designed to be crossed and overlapped from head to tail, the small segments are respectively sequenced, and the sequencing result is spliced to obtain the complete sequence of the PgbHLH149 gene.

The cDNA is synthesized from the extracted total RNA of the ginseng by adopting an M-MuLV First Strand cDNA Synthesis Kit M-MuLV First chain cDNA Synthesis Kit, and is used as a template for PCR amplification. Designing a plurality of pairs of primers for amplifying the cDNA full-length sequence of the PgbHLH149 gene, wherein the plurality of pairs of primers are respectively as follows:

bHLH149-1F：TTTCATTCCCTCTTTGAGGTATCTT；

bHLH149-578R：CTGCGCTTGATTTCCATCTACT；

bHLH149-432F：TATTTATCTCGAATCCCGAAGCA；

bHLH149-806R：CGGTCACCTTCGCTTTCTTAC；

bHLH149-634F：TCAATCGGCGACGTCAACT；

bHLH149-955R：CAAAGCCGCAATATAATCCGTA；

bHLH149-781F：GAAGAGTAAGAAAGCGAAGGTGAC；

bHLH149-1282R：AAATGCTATGCTTAGTAGTATAAAAAACGA。

the primer pairs are respectively used for amplifying different regions in the cDNA full-length sequence of the PgbHLH149 gene, and the amplification product sequences of the primer pairs are crossed and overlapped.

And (3) PCR reaction conditions:

pre-denaturation at 95 ℃ for 3 min;

denaturation at 94 ℃ for 30 s;

annealing at 55 ℃ for 30 s;

extension at 72 ℃ for 30s, 33 cycles;

repairing and extending at 72 ℃ for 7 min.

PCR products amplified by the above primer pairs were separated by electrophoresis on a 1.0% agarose gel (see FIG. 4).

FIG. 4 shows the results of electrophoresis of the PCR products, and in FIG. 4, lane 1 shows the results of amplification of primers RT432-149-1F and bHLH 149-578R; 2 is the amplification result of primers RT432-149-432F and bHLH 149-806R; lane 3 shows the amplification results of primers RT432-149-634F and RT 432-149-955R; lane 4 shows the amplification results of primers RT432-149-781F and RT 432-149-1282R.

And recovering PCR products of the primer pairs from the gel, respectively connecting the PCR products to pGEM T-easy vectors, transforming escherichia coli competent cells, selecting monoclonal shake bacteria, carrying out PCR detection on bacteria liquid, and then sending the bacteria liquid to Shanghai bioengineering company for sequencing.

Because the DNA sequences of the PCR products amplified by the plurality of primer pairs are crossed and overlapped, the full-length cDNA sequence (SEQ ID NO.1) and the coding frame sequence (SEQ ID NO.2) of the PgbHLH149 gene are obtained after the DNA sequences obtained by sequencing the plurality of PCR products are spliced.

Example 3PgbHLH149 Gene transfer into Arabidopsis

Vector construction experiments:

1. designing a primer:

bHLH149-F:AGAACACGGGGGACGAGCTCATGGTATCATTATTTATCTCGAATCCCGA

bHLH149-R:CCATGGTGTCGACTCTAGAGGATCCTAAAGTCGATGATTGGGATAAATTTGAGC

2. and (3) a target gene amplification PCR reaction system:

reaction system	Amount of the composition used
		2×fast pfu master mix	10.0μl
Primer bHLH149-F	1μl
		Primer bHLH149-R	1μl
cDNA	1μl
		ddH2O	up to 20μl

Reaction procedure: 94 ℃ below zero: 3 min; 94 ℃ below zero: 30 sec; 58 ℃ C: 45 sec; 72 ℃ C: 1kb/30 s; 33 cycles; 72 ℃ C: 10 min; 16 ℃ C: forever.

The small fragment (PgbHLH149 gene fragment) was recovered.

3. Connecting the target gene with a target vector:

construction scheme of PgbHLH149 gene overexpression vector, and the map of the PgbHLH149 gene overexpression vector is shown in figure 5.

(1) The plasmid pCAMBIA2300-GFP was digested with SacI and BamHI, and the large fragment was recovered.

A double enzyme digestion reaction system:

reaction system	Amount of the composition used
		10×Tango buffer	2.0μl
PstI	1μl
		BamHI	1μl
Plasmids	1μg
		ddH2O	up to 20μl

Carrying out enzyme digestion at 37 ℃ for 2.5 h; inactivating at 65 deg.C for 20 min.

(2) And (3) recombining and connecting the large fragment obtained in the step (1) and the small fragment obtained by amplification (PgbHLH149 gene fragment) by using homologous recombination ligase, and transforming the product into DH5 alpha competent cells.

The connection method comprises the following steps:

adding the target DNA fragment and the linearized vector into an EP tube according to a certain molar ratio for recombination reaction, uniformly mixing, and standing at 37 ℃ for 30 min; the transformation is carried out immediately and the remaining ligation solution can be stored at 4 ℃ or-20 ℃ until use. Note that: a. the molar ratio of the target fragment to the carrier is 3: 1-10: 1, the molar ratio is lower than 3: 1 efficiency will decrease; b. the reaction time is 20-40 minutes, which is too long to be suitable for recombination.

(3) And (5) carrying out colony PCR detection.

(4) And (4) selecting positive colonies for sequencing verification.

Arabidopsis thaliana genetic transformation experiments:

1. 100. mu.l of Agrobacterium carrying the gene of interest was inoculated into 3ml of LB liquid medium (50. mu.g/ml kanamycin, 50. mu.g/ml gentamicin, 20. mu.g/ml rifampicin) and shake-cultured at 28 ℃ and 200rpm for 12-18 hours.

2. 200. mu.l of the shaken bacterial suspension was inoculated into 50ml of a resistant medium (50. mu.g/ml kanamycin, 50. mu.g/ml gentamicin, 20. mu.g/ml rifampicin) (2 bottles of 50ml medium per bacterial suspension), and shake-cultured at 28 ℃ and 200rpm for 12 to 18 hours.

3. Adding the shaken bacterial solution into a 50ml centrifuge tube, centrifuging for 5min at 5000g, and removing the supernatant.

4. The cells were resuspended in 30ml of 5% sucrose solution (100ml distilled water plus 5g sucrose), centrifuged at 5000g for 5min and the supernatant removed.

5. The cells were resuspended in 50ml of 5% (m/V) sucrose solution (100ml distilled water plus 5g sucrose), the OD600 values of the cells were measured spectrophotometrically, OD600 was made 0.8 by the formula C1VI ═ C2V2, Silwetl-77 (0.02% V/V) was added, covered with black cloth and left to stand for 1 h.

6. Cutting off the fruiting siliques of Arabidopsis, soaking the Arabidopsis inflorescence in the Agrobacterium suspension for 45s, continuously shaking the Arabidopsis during the process, and repeating the operation for two times.

7. Spraying black plastic bag on the water jacket after dipping flowers for moisture preservation, and removing the bag the next day.

8. And (3) repeating the steps (1) to (7) when the flowers are full, performing flower dipping transformation again, inserting a bamboo stick into the nutrition pot after transformation is completed without cutting the siliques, and bundling the arabidopsis on the bamboo stick by using a hemp rope.

9. Watering a little at the late stage of seed setting, collecting seeds after the seeds are mature, and drying the seeds in an incubator at 37 ℃ for 2 days, wherein the seeds which are set are T0 generation seeds.

10. And (4) subpackaging the seeds to be screened into 1.5ml centrifuge tubes, and marking.

11. Adding 500 μ l of 75% alcohol, sterilizing for 30s, and sucking out.

12. Add 10% NaClO, sterilize for 10min, reverse the course up and down, aspirate (NaClO configuration (200. mu.l + 1800. mu.l sterile water).

13. Adding 1ml of sterilized water, washing for 5 times, and sucking out.

14. 1ml of sterilized water was added, and the mixture was aspirated into MS medium containing Kan (kanamycin), and the water was aspirated and purified.

15. And (3) placing the MS culture medium containing the seeds in a culture room for 15 days until 2 true leaves grow out and transplanting the true leaves into soil for marking.

16. Repeating the steps 9-15 to obtain T1 generation Arabidopsis plants. The results are shown in FIG. 6.

FIG. 6 shows the Arabidopsis dip-flower transformation and screening steps, wherein A shows the planting of Arabidopsis wild type (Col-0) seeds into a seedling substrate; b shows arabidopsis grown for 4 weeks; c shows the growth of 7-8 weeks of Arabidopsis bolting, preparation of Agrobacterium dipping transformation; d, growing Arabidopsis thaliana for 10-11 weeks, and harvesting seeds (T0 generation seeds); e shows the case of T0 generation seeds screened on resistant medium; f shows the condition that positive plants of T1 generation obtained by screening are planted in a seedling raising substrate.

Transgenic T1 generation arabidopsis fluorescent quantitative PCR assay:

respectively irrigating the obtained T1 generation arabidopsis thaliana plants and wild type arabidopsis thaliana plants by nutrient solution containing ferrous sulfate with concentration of 0(Fe0), 0.1mmol/L (Fe1) and 1.6mmol/L (Fe3), setting 3 biological repetitions for each group of concentration, treating for 7 days, taking fresh arabidopsis thaliana leaves, respectively marking the wild type arabidopsis thaliana with CK-Fe0, CK-Fe1 and CK-Fe3, respectively marking the transgenic generation arabidopsis thaliana with T1-Fe0, T1-Fe1 and T1-Fe3, extracting total RNA of the leaves by a UNIQ-10 column Trizol total RNA extraction kit, using a randprimer as a Primer, and performing reverse transcription to form a first chain of cDNA as a downstream reaction template.

Internal reference gene primer sequence:

β-Actin-F：GCCGACAGAATGAGCAAAGAG；

β-Actin-R：AGGTACTGAGGGAGGCCAAGA。

the target gene primer sequence:

bhlh149-F：CAGAACACCAGATCTACTCAACGA；

bhlh149-R：TACTCGGTCGGCAGTTTCAC。

the reaction procedure was pre-denaturation at 95 ℃ for 30s, 40 cycles (denaturation at 95 ℃ for 5 s; annealing at 60 ℃ for 30 s; extension at 72 ℃ for 20 s). After real-time fluorescent quantitative PCR, the relative change of the expression of the PgbHLH149 gene was 2^-△△CtThe method carries out data processing analysis.

The results show (table 1): the PgbHLH149 gene can be stably inherited and over-expressed in arabidopsis, and the PgbHLH149 gene expression level of transgenic arabidopsis is obviously higher than that of wild arabidopsis after iron toxicity stress.

TABLE 1 PgbHLH149 Gene expression changes in wild-type and transgenic Arabidopsis thaliana after treatment with iron nutrient solutions of different concentrations

Treatment of	PgbHLH149 Gene expression level
		CK-Fe0	1
CK-Fe1	14.1±5.8b
		CK-Fe3	43.9±9.0b
T1-Fe0	26165.1±11386a
		T1-Fe1	13472.3±9066ba
T1-Fe3	22092.3±11722ba

Transgenic T1 generation Arabidopsis thaliana iron toxicity resistance identification experiment:

respectively watering T1 generation arabidopsis thaliana plants and wild type arabidopsis thaliana plants with nutrient solution containing ferrous sulfate with concentration of 0(Fe0), 0.1mmol/L (Fe1), 0.8mmol/L (Fe2) and 1.6mmol/L (Fe3), setting 3 biological repeats for each group of concentration, recording plant phenotype after 9 days of treatment, taking pictures and counting the stress rate of plants under the condition of 3 groups of iron concentration treatment such as Fe1, Fe2 and Fe3 (after arabidopsis thaliana is subjected to iron toxicity stress, yellow spots or dry withered leaves of part of the plants can appear, even some plants die, the ratio of the sum of the number of yellow spots or dry plants and the number of dead plants on the leaves to the total number of the treated parts is the plant stress rate), and the result is shown in figure 7 and figure 8. In FIG. 7, A is a photograph showing the phenotype of Arabidopsis thaliana treated with different iron concentrations at the T1 generation, and B is a photograph showing the phenotype of Arabidopsis thaliana treated with different iron concentrations at the wild type. FIG. 8 is the statistics of plant stress rate. The results in fig. 7 and fig. 8 show that overexpression of the PgbHLH149 gene in arabidopsis can improve the resistance of transgenic plants to iron-toxic stress.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Sequence listing

<110> institute of specialty products of Chinese academy of agricultural sciences

<120> ginseng PgbHLH149 transcription factor and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1160

<212> DNA

<213> Panax ginseng

<400> 1

tttcattccc tctttgaggt atctttccca ccctgtcacc ctctctattc tttcttcgtt 60

ccttcaccgt tatttcttct ccctcatcgt tctttcctga aatttatgtt cagagccgcc 120

tgcatcattc agaaggtatt tctgattttt aagcttgttc tccaattttc ttctgttttt 180

acacgagagg aatttaggtt gagaaactga acattttgct aaagaatttc atcgatctca 240

aaatcgatgg tatcattatt tatctcgaat cccgaagcag cgtcaaattc caacagctca 300

cgcgaatcta agcgcaaaaa gagaagaaaa atcgaaacga gcgataaaat tgaaaatcac 360

cagcagctca aaatcgatcg tagtagatgg aaatcaagcg cagaacacca gatctactca 420

acgaaactcg tcgaagctct ctgtaaccgg aattcttctc aatcggcgac gtcaactgct 480

ggcggccgcg ccgtccgtga aactgccgac cgagtactcg ccgtcgaggc taagggaagg 540

acgcggtgga gcagagctat tctatctaac aggctcaagc ttcaattgaa tatgatcaag 600

cataagaaga gtaagaaagc gaaggtgacc ggcgatgtcc ggtcgaagaa actggcggcg 660

aagaagaaat tgtccacgtt gaaccggaaa gtgcgtgttc ttggccggct agttcccggt 720

tgccggaagc tatcgtttcc gaatcttcta gaagaaacta cggattatat tgcggctttg 780

gaaatgcaag tccgagccat gactgctctc accgggcttc tcgccggctc cggcccttct 840

tcggatcggc tcggctcaaa tttatcccaa tcatcgactt tataaagttt ttattgtcct 900

aaaaaggaag tcattgttaa ttactgagtt ttttttttta atcctacatc atttggttct 960

ttcatctttc ttggtatttt gattttgggt ttcttaacat tattgttatt tatttattca 1020

ttcattacta cttaatgggt tggttggaat tttgattaaa catgacaact aattgtttcg 1080

ttttttatac tactaagcat agcatttgtt aaacatgaca actaattgtt tcgtttttta 1140

tactactaag catagcattt 1160

<210> 2

<211> 639

<212> DNA

<213> Panax ginseng

<400> 2

atggtatcat tatttatctc gaatcccgaa gcagcgtcaa attccaacag ctcacgcgaa 60

tctaagcgca aaaagagaag aaaaatcgaa acgagcgata aaattgaaaa tcaccagcag 120

ctcaaaatcg atcgtagtag atggaaatca agcgcagaac accagatcta ctcaacgaaa 180

ctcgtcgaag ctctctgtaa ccggaattct tctcaatcgg cgacgtcaac tgctggcggc 240

cgcgccgtcc gtgaaactgc cgaccgagta ctcgccgtcg aggctaaggg aaggacgcgg 300

tggagcagag ctattctatc taacaggctc aagcttcaat tgaatatgat caagcataag 360

aagagtaaga aagcgaaggt gaccggcgat gtccggtcga agaaactggc ggcgaagaag 420

aaattgtcca cgttgaaccg gaaagtgcgt gttcttggcc ggctagttcc cggttgccgg 480

aagctatcgt ttccgaatct tctagaagaa actacggatt atattgcggc tttggaaatg 540

caagtccgag ccatgactgc tctcaccggg cttctcgccg gctccggccc ttcttcggat 600

cggctcggct caaatttatc ccaatcatcg actttataa 639

<210> 3

<211> 212

<212> PRT

<213> Panax ginseng

<400> 3

Met Val Ser Leu Phe Ile Ser Asn Pro Glu Ala Ala Ser Asn Ser Asn

1 5 10 15

Ser Ser Arg Glu Ser Lys Arg Lys Lys Arg Arg Lys Ile Glu Thr Ser

20 25 30

Asp Lys Ile Glu Asn His Gln Gln Leu Lys Ile Asp Arg Ser Arg Trp

35 40 45

Lys Ser Ser Ala Glu His Gln Ile Tyr Ser Thr Lys Leu Val Glu Ala

50 55 60

Leu Cys Asn Arg Asn Ser Ser Gln Ser Ala Thr Ser Thr Ala Gly Gly

65 70 75 80

Arg Ala Val Arg Glu Thr Ala Asp Arg Val Leu Ala Val Glu Ala Lys

85 90 95

Gly Arg Thr Arg Trp Ser Arg Ala Ile Leu Ser Asn Arg Leu Lys Leu

100 105 110

Gln Leu Asn Met Ile Lys His Lys Lys Ser Lys Lys Ala Lys Val Thr

115 120 125

Gly Asp Val Arg Ser Lys Lys Leu Ala Ala Lys Lys Lys Leu Ser Thr

130 135 140

Leu Asn Arg Lys Val Arg Val Leu Gly Arg Leu Val Pro Gly Cys Arg

145 150 155 160

Lys Leu Ser Phe Pro Asn Leu Leu Glu Glu Thr Thr Asp Tyr Ile Ala

165 170 175

Ala Leu Glu Met Gln Val Arg Ala Met Thr Ala Leu Thr Gly Leu Leu

180 185 190

Ala Gly Ser Gly Pro Ser Ser Asp Arg Leu Gly Ser Asn Leu Ser Gln

195 200 205

Ser Ser Thr Leu

210

Claims (9)

1. A ginseng PgbHLH149 transcription factor is characterized in that the transcription factor is a protein with an amino acid sequence shown in SEQ ID NO.3, or a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 3.

2. A DNA molecule encoding the ginseng PgbHLH149 transcription factor of claim 1.

3. The DNA molecule of claim 2, comprising the DNA sequence shown in SEQ ID No.2 or a DNA sequence having more than 70% homology with the DNA sequence shown in SEQ ID No.2 and encoding the same functional protein.

4. A recombinant vector comprising the DNA molecule of any one of claims 2 to 3.

5. A recombinant cell comprising the recombinant vector of claim 4.

6. The recombinant cell of claim 5, wherein the recombinant cell is a plant cell.

7. Use of a DNA molecule according to any one of claims 2 to 3 for increasing the resistance of a plant to iron-toxic stress.

8. The use of claim 7, wherein said use is a method of growing anti-ferritin plants, said method comprising: transforming a plant cell with an expression vector comprising the DNA molecule, and growing the transformed plant cell into an anti-iron-virus plant.

9. An RNA molecule encoding the ginseng PgbHLH149 transcription factor of claim 1.

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