CN115851812B - Dragon fruit HuC3H35 gene and encoding protein and application thereof - Google Patents
Dragon fruit HuC3H35 gene and encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses a dragon fruit HuC3H35 gene and a coding protein and application thereof, wherein the nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO. 1; or a nucleotide sequence with a coding amino acid sequence shown as SEQ ID NO. 2. The pitaya HuC3H35 gene provided by the invention participates in salt stress and high temperature stress response by expressing CCCH type zinc finger protein HuC3H35 of plants, can be applied to genetic breeding of plants aiming at salt stress and high temperature stress, improves tolerance of the plants to salt and high temperature, cultivates salt-resistant and high temperature-resistant crop varieties, and has important reference significance in reducing safety hazard brought by saline land and high temperature to grain crops.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a dragon fruit HuC3H35 gene and a coding protein thereof in improving salt stress resistance and high temperature stress resistance of plants.
Background
Pitaya is native to Mexico and south America, etc., and is a perennial climbing tropical fruit of the Cactaceae family. Dragon fruits are widely planted in southeast Asian countries such as Vietnam, thailand, and the south of China. The dragon fruits are divided into wampee white meat, red skin red meat and the like, and the fruit pulp is sweet and delicious, contains a large amount of vegetable proteins, dietary fibers and betalains, and has rich nutritional value and health care value. The dragon fruit can also be used for brewing wine, making various foods such as fruit juice, desserts and the like, and has great economic value and commercial value.
Salt is one of main abiotic stress for limiting the worldwide agricultural development, and at present, nearly one third of the world area is affected by salinization, and the large-area salinized soil seriously affects the growth and development of crops, so that the yield and quality of the crops are affected. Meanwhile, high temperature stress caused by global warming has severely threatened the yield and quality of crops.
Zinc finger proteins are a class of transcription factors containing zinc finger domains, which are widely found in animals, plants, yeasts and viruses. Zinc finger proteins regulate gene expression at the transcriptional and posttranscriptional levels through interactions with DNA, RNA and other proteins, and play an important role in biological functions such as plant growth and development, stress response, and plant hormone response. The zinc finger domain consists of Cys (cysteine), cys and His (histidine) and is formed by binding zinc ions. Zinc finger proteins can be divided into 9 general classes, based on the number and arrangement of Cys and His residues: C2H2, C2HC5, CCCH, C3HC4, C4HC3, C6, C8. CCCH-type zinc finger proteins are a class of zinc finger proteins with a low number of structural elements, typically comprising 1 to 6C-X4-15-C-X4-6-C-X3-4-H (X is another amino acid) CCCH-type motifs. CCCH-type zinc finger proteins regulate gene expression at the transcriptional and posttranscriptional levels, primarily by binding to DNA or RNA, thereby affecting plant growth and stress response. The research shows that the Arabidopsis AtC H23/AtTZF1 (tandem zinc finger) has binding activity to DNA and RNA, and can move between cell nucleus and cytoplasm, which shows that AtC3H23/AtTZF1 can regulate gene expression by binding DNA and RNA. Subsequent studies have found that AtC H23/AtTZF1 is able to bind and cause degradation of AREs (AU-rich elements) -containing mRNA. The recombinant proteins AtC H49/AtTZF3 and AtC H20/AtTZF2 showed RNase activity in vitro, indicating that they may be involved in the metabolic process of mRNA. OsTZF1 in rice is induced to express by drought, high salt and hydrogen peroxide, and over-expression of OsTZF1 improves drought and salt resistance of rice, improves salt stress tolerance of plants by reducing the level of Reactive Oxygen Species (ROS), and can be combined with mRNA containing U-rich motif and AREs motif in cytoplasm, so that OsTZF1 possibly improves stress resistance of various plants by influencing RNA metabolism of stress response genes.
Under the pressure of continuously accelerating the modern process of human beings, increasing population, reducing cultivated land area and insufficient fresh water resources, the mechanism of salt resistance and high temperature resistance of plants is researched, genetic resources of salt resistance and high temperature resistance are excavated, and the cultivation of new salt resistance and high temperature resistance materials and new varieties has great significance for global crop production. The dragon fruit has the characteristics of stronger salt resistance, drought resistance, heat resistance, barren resistance and the like, and can be used as a plant stress resistance genetic resource library for deep research.
Disclosure of Invention
Based on the above, one of the purposes of the invention is to provide an application of a dragon fruit HuC3H35 gene and a coding protein thereof in improving plant stress resistance, wherein the gene and the coding protein thereof can improve salt stress resistance and high temperature stress resistance of plants.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
the application of the dragon fruit HuC3H35 gene in improving the stress resistance of plants, wherein the nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO. 1; or a nucleotide sequence with the coding amino acid sequence shown as SEQ ID NO. 2; the stress resistance is salt stress resistance or high temperature stress resistance.
The application of the protein coded by the dragon fruit HuC3H35 gene in improving the stress resistance of plants is disclosed, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
In some embodiments, the plant is a dragon fruit or an arabidopsis thaliana.
Another object of the present invention is to provide an application of the dragon fruit HuC3H35 gene and the encoded protein thereof in genetic breeding for improving salt and high temperature tolerance of plants, and the dragon fruit HuC3H35 gene and the encoded protein thereof can be used for cultivating salt and high temperature resistant plant varieties.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
the application of a dragon fruit HuC3H35 gene in genetic breeding for improving the tolerance of plants to salt and high temperature is disclosed, wherein the nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO. 1; or a nucleotide sequence with the coded amino acid sequence shown as SEQ ID NO. 2.
The application of the protein coded by the dragon fruit HuC3H35 gene in improving the stress resistance of plants is disclosed, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
In some embodiments, the plant is a dragon fruit or an arabidopsis thaliana.
It is another object of the present invention to provide an over-expression vector for improving salt stress resistance and high temperature stress resistance of plants, and improving tolerance of plants to salt and high temperature.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
the over-expression vector is inserted with a dragon fruit HuC3H35 gene, and the nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO. 1; or a nucleotide sequence with the coded amino acid sequence shown as SEQ ID NO. 2.
In some of these embodiments, the over-expression vector is pCAMBIA1302-HuC3H35.
The invention also provides application of the overexpression vector inserted with the dragon fruit HuC3H35 gene in improving stress resistance of plants, wherein the stress resistance is salt stress resistance or high temperature stress resistance.
The invention also provides application of the overexpression vector inserted with the dragon fruit HuC3H35 gene in genetic breeding for improving salt tolerance or high-temperature tolerance of plants.
In some of these embodiments, the plant is arabidopsis thaliana or pitaya.
The invention also provides a biological agent for improving the salt stress resistance or the high temperature stress resistance of plants.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a biological agent for improving stress resistance of plants, wherein the active ingredient of the biological agent contains the over-expression vector, and the stress resistance is salt stress resistance or high temperature stress resistance.
In some of these embodiments, the plant is arabidopsis thaliana or pitaya.
The invention also provides a method for improving the stress resistance of the plants.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a method for improving stress resistance of plants comprises improving expression of a dragon fruit HuC3H35 gene, wherein a nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO. 1; or a nucleotide sequence with the coding amino acid sequence shown as SEQ ID NO. 2; the stress resistance is salt stress resistance or high temperature stress resistance.
In some of these embodiments, the plant is arabidopsis thaliana or pitaya.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, the inventor screens out a dragon fruit HuC3H35 gene, and through the transgenic method, the HuC3H35 gene is over-expressed, and the following is found: along with the improvement of the expression quantity of the HuC3H35 gene, the resistance of arabidopsis thaliana to salt and high temperature is correspondingly improved, which shows that the HuC3H35 gene participates in salt stress and high temperature stress response by expressing CCCH type zinc finger protein HuC3H35 of plants, therefore, the invention has important significance for comprehensively understanding the biological functions of the CCCH type zinc finger protein HuC3H35 in plants, provides more abundant genetic resources for plant salt resistance or high temperature resistance molecular breeding, can be applied to the genetic breeding of plants aiming at salt stress and high temperature stress, improves the tolerance of plants to salt and high temperature, cultivates salt and high temperature resistance crop varieties, and has important reference significance for reducing the safety hazard and the like brought by salt land and high temperature to grain crops.
Drawings
FIG. 1 is a map of pCAMBIA1302 vector used to construct the expression vector of example 1 of the present invention.
FIG. 2 shows the detection of positive transgenic plants overexpressing the HuC3H35 gene in example 1 of the present invention, wherein WT is wild type Arabidopsis and 7, 16, 17 are positive transgenic Arabidopsis plants.
FIG. 3 shows the results of quantitative PCR assays for wild-type and HuC3H35 transgenic Arabidopsis thaliana in example 1 of the present invention, wherein WT is the wild-type Arabidopsis plant; huC3H35-OE7 is HuC3H35 over-expressed strain 7; huC3H35-OE16 is HuC3H35 over-expression strain 16; huC3H35-OE17 is HuC3H35 over-expression strain 17.
FIG. 4 is a phenotypic comparison of 200mM NaCl treated overexpressing Arabidopsis thaliana and wild type Arabidopsis thaliana in example 2 of the present invention, wherein WT is the wild type Arabidopsis plant; huC3H35-OE7 is HuC3H35 over-expressed strain 7; huC3H35-OE16 is HuC3H35 over-expression strain 16; huC3H35-OE17 is HuC3H35 over-expression strain 17.
FIG. 5 shows the expression levels of HuC3H35 gene at various time points of dragon fruit treated with high salt 450mM NaCl in example 3 of the present invention; 0 is the sample before treatment, 3,6, 24, 72 is the different time points (hours) after treatment.
FIG. 6 is a phenotypic comparison of overexpressed Arabidopsis thaliana and wild type Arabidopsis thaliana treated at a high temperature of 42℃in example 4 according to the present invention, wherein WT is the wild type Arabidopsis plant; huC3H35-OE7 is HuC3H35 over-expressed strain 7; huC3H35-OE16 is HuC3H35 over-expression strain 16; huC3H35-OE17 is HuC3H35 over-expression strain 17; survivin rates are Survival rates.
FIG. 7 shows the expression levels of HuC3H35 gene at various time points of dragon fruit treated at a high temperature of 45℃in example 5 of the present invention; 0 is the sample before treatment, 3,6, 24, 72 is the different time points (hours) after treatment.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
The nucleotide sequence of the dragon fruit HuC3H35 gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The nucleotide sequence of the vector pCAMBIA1302-HuC3H35 for over-expressing the HuC3H35 gene is shown as SEQ ID NO. 3.
SEQ ID NO.1
ATGATGATCGGAGAAACACACCGTCACCTCAACCTTCAGGTCCCACCATGGCAGGATCAGCACATCAATCATCCAACGGCCCAGATCTCATCATCTTCCTTACTCACCCCTTCATCTCCACCGTCCAATCTGCCCACTACCCCTCAATCCCCCTCATATGCTGACGTTTTGGCCTACCTGCTCGCCACAGGCGCCGGCGATGGTGACTCGTCGGCTGAATCTGACTTTCCCGCCGGTGACGACGACGAGTTCTACATGTACGAGTTCAAGGTCCGAAAGTGCACACGGGCCCGGGCTCATGATTGGACTGAGTGCCCGTTTGCCCACCCGGGCGAAAAAGCCCGGCGCCGTGACCCGAGAAAGTACTCCTATTCGGGTACGGCCTGCTCCGATTTCCGAAAGGGCAGTTGCAAGAAGGGGGACAGTTGTGAGTTTGCTCATGGGGTTTTCGAATGCTGGCTTCACCCTTCTAGGTATCGGACCCAGGCTTGCAAGGACGGCCCCGGCTGCAAGCGCCGGGTCTGTTTCTTCGCCCACTCGCCCGATCAGCTACGGGTCGGGTCGGGCTTGGGTAGTCCCGTGAGCCAGAAGTCTTCGGGGTCGGAGTTCTTCGATGACGGGTTATTCGGGTCGGGCTCGGTTTCGTCCATTGGGGACCTGGTCGCCTCGTTGAGGAATCTGCAGCTGAGTAAGGTGAAATCCATGCCGACTAGCGGGTCGAATTGGAGTGTCGGTATTGGGTCGCCGTTATTCGGTTCTCCAAGAGGGACCGGGTCACCTGTGGCGCGGGCCGGGTTCTTCAGTCTCCCCACCACCCCTACCCGGCCCGGGATCAGGTACTTGGATGCATGGGATAATAATAGGAGGGATCAGTTTGGAGATTATCAAGAGGAGGAGCCCGTTTTGGAACGGGTCGAGTCGGGCCGCGAATTACGGGTCCGTATGTTTGAGAAGCTAAGCAAGGAGAACTCGCTGCACGGGCTGGGTTTGGTTGAGGCATCCGGTTCGGGTCCTGATTTCGGGTGGGTGTCCGATCTCGTCAAGTGA
SEQ ID NO.2
MMIGETHRHLNLQVPPWQDQHINHPTAQISSSSLLTPSSPPSNLPTTPQSPSYADVLAYLLATGAGDGDSSAESDFPAGDDDEFYMYEFKVRKCTRARAHDWTECPFAHPGEKARRRDPRKYSYSGTACSDFRKGSCKKGDSCEFAHGVFECWLHPSRYRTQACKDGPGCKRRVCFFAHSPDQLRVGSGLGSPVSQKSSGSEFFDDGLFGSGSVSSIGDLVASLRNLQLSKVKSMPTSGSNWSVGIGSPLFGSPRGTGSPVARAGFFSLPTTPTRPGIRYLDAWDNNRRDQFGDYQEEEPVLERVESGRELRVRMFEKLSKENSLHGLGLVEASGSGPDFGWVSDLVK
SEQ ID NO.3
ATGATGATCGGAGAAACACACCGTCACCTCAACCTTCAGGTCCCACCATGGCAGGATCAGCACATCAATCATCCAACGGCCCAGATCTCATCATCTTCCTTACTCACCCCTTCATCTCCACCGTCCAATCTGCCCACTACCCCTCAATCCCCCTCATATGCTGACGTTTTGGCCTACCTGCTCGCCACAGGCGCCGGCGATGGTGACTCGTCGGCTGAATCTGACTTTCCCGCCGGTGACGACGACGAGTTCTACATGTACGAGTTCAAGGTCCGAAAGTGCACACGGGCCCGGGCTCATGATTGGACTGAGTGCCCGTTTGCCCACCCGGGCGAAAAAGCCCGGCGCCGTGACCCGAGAAAGTACTCCTATTCGGGTACGGCCTGCTCCGATTTCCGAAAGGGCAGTTGCAAGAAGGGGGACAGTTGTGAGTTTGCTCATGGGGTTTTCGAATGCTGGCTTCACCCTTCTAGGTATCGGACCCAGGCTTGCAAGGACGGCCCCGGCTGCAAGCGCCGGGTCTGTTTCTTCGCCCACTCGCCCGATCAGCTACGGGTCGGGTCGGGCTTGGGTAGTCCCGTGAGCCAGAAGTCTTCGGGGTCGGAGTTCTTCGATGACGGGTTATTCGGGTCGGGCTCGGTTTCGTCCATTGGGGACCTGGTCGCCTCGTTGAGGAATCTGCAGCTGAGTAAGGTGAAATCCATGCCGACTAGCGGGTCGAATTGGAGTGTCGGTATTGGGTCGCCGTTATTCGGTTCTCCAAGAGGGACCGGGTCACCTGTGGCGCGGGCCGGGTTCTTCAGTCTCCCCACCACCCCTACCCGGCCCGGGATCAGGTACTTGGATGCATGGGATAATAATAGGAGGGATCAGTTTGGAGATTATCAAGAGGAGGAGCCCGTTTTGGAACGGGTCGAGTCGGGCCGCGAATTACGGGTCCGTATGTTTGAGAAGCTAAGCAAGGAGAACTCGCTGCACGGGCTGGGTTTGGTTGAGGCATCCGGTTCGGGTCCTGATTTCGGGTGGGTGTCCGATCTCGTCAAG
It is to be understood that modifications of the base sequences referred to in the following examples without changing the amino acid sequence, taking into account the degeneracy of the codons, are also within the scope of the invention.
The invention is described in further detail below with reference to specific embodiments and figures.
EXAMPLE 1 construction of overexpression vector of Dragon fruit HuC3H35 Gene and acquisition of transgenic Material
In this example, the overexpression vector pCAMBIA1302-HuC3H35 is constructed by using the pCAMBIA1302 vector (the map of which is shown in FIG. 1), and the transgenic material is obtained by the following steps:
(1) Amplifying the target gene
Using cDNA of the dragon fruit HuC3H35 gene as a template and using an upstream primer: 5'-TGACCATGGTAGATCTGATGATGATCGGAGAAACACAC-3' (SEQ ID NO. 4) and a downstream primer 5'-CTTCTCCTTTACTAGTCTTGACGAGATCGGACACC-3' (SEQ ID NO. 5) are used as primers, and a 1044bp target fragment is obtained by PCR amplification.
Wherein, the PCR reaction system is as follows:
max DNA Polymerase 10. Mu.L, 10. Mu.M upstream/downstream primers each 0.5. Mu.L, cDNA template 1. Mu.L, ddH 2 O8. Mu.L. The components are evenly mixed and then placed on a PCR instrument for reaction.
The PCR reaction procedure was as follows: 98 ℃ for 5min;98℃10s,55℃ 15s,72℃20s,38 cycles; 72 ℃ for 5min; electrophoresis detects and recovers the products with correct band sizes.
(2) Linearization vector pCAMBIA1302
Vector plasmid pCAMBIA1302 was digested simultaneously with BglII and SpeI. The 20 mu L double enzyme digestion reaction system is as follows: plasmid template 5. Mu.L, 10 XFastDiest Buffer 2. Mu.L, bglII 1. Mu.L, speI 1. Mu.L, ddH 2 O11. Mu.L. Cleavage reaction conditions: and enzyme cutting at 37 ℃ for 60min. After the reaction, the digested product was recovered by agarose gel DNA kit.
(3) Carrier connection
The gene fragment of interest was ligated into the linearized pCAMBIA1302 vector after cleavage using the In-Fusion cloning (homologous recombination) method. The 5. Mu.L ligation system was: 5 XIn-Fusion HD Enzyme Premix, 0.5. Mu.L, pCAMBIA1302 Vector, 3.5. Mu.L, 1. Mu.L of the desired fragment. After being evenly mixed, the mixture is placed at 50 ℃ for 45min and is connected, and the overexpression vector pCAMBIA1302-HuC3H35 is obtained, and the nucleotide sequence of the overexpression vector pCAMBIA1302-HuC3H35 is shown as SEQ ID No. 3.
(4) Coli transformed with ligation product
2 μl of the ligation product obtained in the step (3) (namely, the overexpression vector pCAMBIA1302-HuC3H 35) is transformed into escherichia coli DH5 alpha, and the mixture is uniformly mixed and then subjected to ice bath for 30min; heat shock in 42 deg.c water bath for 90s; placing on ice, and ice-bathing for 2min; 700ml of liquid LB medium was added, resuscitated at 37℃for 40min and plated on LB plates containing Kan (kanamycin) overnight at 37 ℃. Selecting monoclonal, amplifying and culturing in liquid LB culture medium containing Kan, sequencing and identifying, and extracting plasmid.
(5) Agrobacterium transformation by positive cloning
And (3) transforming agrobacterium with the positive clone extraction plasmid obtained in the step (4), and selecting positive agrobacterium.
(6) Infecting wild type Arabidopsis inflorescences
And (3) adopting a genetic transformation method mediated by agrobacterium tumefaciens, and impregnating the positive agrobacterium obtained in the step (5) with a wild type arabidopsis inflorescence, and taking the wild type arabidopsis not transformed with the HuC3H35 gene as a control.
(7) Screening and detection of transgenic T0 generation positive plants
T0 generation seeds were surface sterilized and plated on MS solid medium plates containing kan resistance. And (3) transferring the germinated seedlings to nutrient soil for cultivation after the germinated seedlings grow to 2 leaves. Taking out its leaves when it flowers quicklyDNA was extracted and PCR was performed using hpt-F/R primers (Guangzhou division of Beijing qing department of biological Co., ltd.) using the following PCR reaction system: 2 XTaq Mix 5. Mu.L, 10. Mu.M upstream/downstream primers each 0.25. Mu.L, DNA template 0.5. Mu.L, ddH 2 O4. Mu.L. The components are evenly mixed and then placed on a PCR instrument for reaction. The PCR reaction procedure was as follows: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 30s,36 cycles, 72℃for 5min; electrophoresis detects and recovers the products with correct band sizes.
As a result, as shown in FIG. 2, lanes 2 to 4 are transgenic Arabidopsis plants, all have a band of about 500bp, lane 1 is wild type Arabidopsis, and no band.
(8) Expression level detection of transgenic plants overexpressing HuC3H35
The expression level of the HuC3H35 gene in transgenic arabidopsis was detected by qRT-PCR technique. Use of fluorescent quantitation kit Hieff TM qPCR
qRT-PCR was performed by Green Master mix (No Rox), and the qRT-PCR reaction system was: 2X SYBR GreenMasterMix. Mu.L, 10. Mu.M forward/reverse primer each 0.2. Mu.L, diluted cDNA 1. Mu.L, ddH 2 O3.6. Mu.L. qRT-PCR was performed using 384 wells, the instrument was Light Cycler480 from RoChe, and the following procedure was used: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 30s and extension for 45 cycles; making a dissolution curve by 15s at 95 ℃,60 s at 60 ℃, 15s at 95 ℃ and 1 cycle; finally, the temperature is reduced by 30s at 50 ℃. And selecting Arabidopsis thaliana action as an internal reference gene for quantitative analysis.
As shown in FIG. 3, the expression level of the HuC3H35 gene in transgenic Arabidopsis plants (HuC 3H35-OE7, huC3H35-OE16, huC3H35-OE 17) was far higher than that in wild type Arabidopsis plants, indicating that the HuC3H35 gene was successfully overexpressed in transgenic Arabidopsis.
EXAMPLE 2 phenotype of Arabidopsis plants overexpressing the HuC3H35 Gene under salt stress
This example examined the phenotype of Arabidopsis plants overexpressing the HuC3H35 gene under salt stress.
Wild type Arabidopsis seeds and HuC3H35 over-expressed plants were sown on MS medium, cultured at room temperature for 7 days, transplanted into a pot containing vermiculite, grown at room temperature for 20 days, high salt treatment was performed by pouring 200mM NaCl, and the performance was observed after two weeks of treatment.
As shown in FIG. 4, the HuC3H35 overexpressing plants (HuC 3H35-OE7, huC3H35-OE16, huC3H35-OE 17) grew significantly better than the wild-type, indicating that the overexpressing plants were more salt tolerant.
EXAMPLE 3 HuC3H35 Gene expression Pattern in Dragon fruit under high salt treatment
In the embodiment, the expression level of the HuC3H35 gene of the dragon fruit at different time points under salt stress (450 mM NaCl) is detected by qRT-PCR technology, so that the mode of the HuC3H35 gene response to the salt stress in the dragon fruit is studied.
Use of fluorescent quantitation kit Hieff TM qPCR
qRT-PCR was performed by Green Master Mix (No Rox), and the qRT-PCR reaction system was: 2X SYBR Green MasterMix. Mu.L, 10. Mu.M forward/reverse primer each 0.2. Mu.L, diluted cDNA 1. Mu.L, ddH 2 O 3.6μL。
qRT-PCR was performed using 384 wells, the instrument was Light Cycler480 from RoChe, and the following procedure was used: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 30s and extension for 45 cycles; making a dissolution curve by 15s at 95 ℃,60 s at 60 ℃, 15s at 95 ℃ and 1 cycle; finally, the temperature is reduced by 30s at 50 ℃. The quantitative analysis is carried out by taking HueEF in the dragon fruit as an internal reference gene, and the result is shown in figure 5.
As can be seen from fig. 5, when the dragon fruit is subjected to salt stress, huC3H35 gene expression changes are not obvious at 3H and 6H, huC3H35 gene expression rises at 24H, and expression still rises at 72H, indicating that HuC3H35 gene is induced to be expressed under salt stress.
EXAMPLE 4 phenotype of Arabidopsis plants overexpressing the HuC3H35 Gene under high temperature stress
This example examined the phenotype of Arabidopsis plants overexpressing the HuC3H35 gene under high temperature stress.
Wild type arabidopsis seeds and HuC3H35 over-expressed plants were sown on MS medium, after 7 days of seed germination, treated at 42 ℃ for 2 hours, then grown for 2 days at room temperature, and after observation of phenotype and statistics of survival rate.
As shown in FIG. 6, the survival rate of the HuC3H35 over-expressed plants (HuC 3H35-OE7, huC3H35-OE16 and HuC3H35-OE 17) is obviously higher than that of the wild-type plants, which indicates that the over-expressed plants are more resistant to high temperature.
Example 5 expression pattern of HuC3H35 Gene in Dragon fruit at high temperature treatment
In the embodiment, the expression levels of the HuC3H35 gene of the dragon fruit at different time points under high temperature stress (45 ℃) are detected by qRT-PCR technology, so that the mode of the HuC3H35 gene response to the high temperature stress in the dragon fruit is studied.
Use of fluorescent quantitation kit Hieff TM qPCR
qRT-PCR was performed by Green Master Mix (No Rox), and the qRT-PCR reaction system was: 2X SYBR Green MasterMix. Mu.L, 10. Mu.M forward/reverse primer each 0.2. Mu.L, diluted cDNA 1. Mu.L, ddH 2 O 3.6μL。
qRT-PCR was performed using 384 wells, the instrument was Light Cycler480 from RoChe, and the following procedure was used: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing at 60℃for 30s and extension for 45 cycles; making a dissolution curve by 15s at 95 ℃,60 s at 60 ℃, 15s at 95 ℃ and 1 cycle; finally, the temperature is reduced by 30s at 50 ℃. The quantitative analysis is carried out by taking HueEF in the dragon fruit as an internal reference gene, and the result is shown in figure 7.
As can be seen from fig. 7, when the dragon fruit is subjected to salt stress, the expression amounts of the dragon fruit are significantly higher than those before high temperature stress from 3H to 6H,24H and 72H, and the result shows that the HuC3H35 gene is induced to be expressed under high temperature stress.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (13)
1. Dragon fruitHuC3H35The application of the gene in improving the stress resistance of plants is characterized in that the dragon fruitHuC3H35The expression mode of the gene is over-expression; the dragon fruitHuC3H35The nucleotide sequence coding sequence of the gene is shown as an amino acid shown as SEQ ID NO. 2; the stress resistance is salt stress resistance or high temperature stress resistance; the plant is Arabidopsis thaliana or dragon fruit.
2. The use according to claim 1, wherein the dragon fruit isHuC3H35The nucleotide sequence of the gene is shown as SEQ ID NO. 1.
3. Dragon fruitHuC3H35The application of the gene-encoded protein in improving the stress resistance of plants is characterized in that the dragon fruitHuC3H35The expression mode of the gene is over-expression; the amino acid sequence of the protein is shown as SEQ ID NO. 2; the stress resistance is salt stress resistance or high temperature stress resistance; the plant is Arabidopsis thaliana or dragon fruit.
4. Dragon fruitHuC3H35Use of a gene in genetic breeding for improving plant tolerance to salt or high temperature, characterized in that the dragon fruitHuC3H35The expression mode of the gene is over-expression; the dragon fruitHuC3H35The nucleotide sequence coding sequence of the gene is shown as an amino acid shown as SEQ ID NO. 2; the plant is Arabidopsis thaliana or dragon fruit.
5. The use according to claim 4, wherein the dragon fruit isHuC3H35The nucleotide sequence of the gene is shown as SEQ ID NO. 1.
6. Dragon fruitHuC3H35Use of a gene-encoded protein for genetic breeding to improve plant tolerance to salt or high temperature, characterized in that the dragon fruitHuC3H35The expression mode of the gene is over-expression; the amino acid sequence of the protein is shown as SEQ ID NO. 2; the plant is Arabidopsis thaliana or dragon fruit.
7. Inserted with dragon fruitHuC3H35An over-expression vector of a gene, characterized in that the dragon fruitHuC3H35The nucleotide sequence of the gene codes for the amino acid with the sequence shown as SEQ ID NO. 2.
8. The over-expression vector of claim 7, wherein the dragon fruit isHuC3H35The nucleotide sequence of the gene is shown as SEQ ID NO. 1.
9. The dragon fruit-inserted fruit of claim 7 or 8HuC3H35The application of the gene over-expression vector in improving the stress resistance of plants, wherein the stress resistance is salt stress resistance or high temperature stress resistance; the plant is Arabidopsis thaliana or dragon fruit.
10. The dragon fruit-inserted fruit of claim 7 or 8HuC3H35Use of an overexpression vector of a gene in genetic breeding to improve tolerance of a plant to salt or high temperature; the plant is Arabidopsis thaliana or dragon fruit.
11. A biological agent for improving stress resistance of plants, characterized in that the active ingredient of the biological agent contains the over-expression vector of claim 7 or 8, and the stress resistance is salt stress resistance or high temperature stress resistance; the plant is Arabidopsis thaliana or dragon fruit.
12. A method for improving stress resistance of plants is characterized by comprising the step of improving dragon fruitsHuC3H35Expression of the Gene, the Dragon fruitHuC3H35The nucleotide sequence coding sequence of the gene is shown as an amino acid shown as SEQ ID NO. 2; the stress resistance is salt stress resistance or high temperature stress resistance; the plant is Arabidopsis thaliana or dragon fruit.
13. The method of increasing stress resistance in plants of claim 12, wherein said dragon fruit isHuC3H35The nucleotide sequence of the gene is shown as SEQ ID NO. 1.
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