CN114836432A - Arabidopsis thaliana PIF1 gene for improving germination rate of seeds under dark and salt stress conditions and application thereof - Google Patents

Abstract

The invention discloses an arabidopsis PIF1 gene for improving seed germination rate under dark and salt stress conditions and application thereof, wherein the sequence of the arabidopsis PIF1 gene is shown as SEQ ID NO. 1. Identifying that pif1 is a T-DNA inserted functional deletion mutant by a PCR amplification method, counting the germination rate of seeds under dark and salt stress conditions by using wild arabidopsis thaliana Col and pif1 as test materials, and detecting the expression level of a salt stress response marker gene by a real-time fluorescent quantitative PCR method, thereby further confirming that the sensitivity of the pif1 mutant seeds to salt stress is remarkably reduced in the germination stage.

Description

Arabidopsis thaliana PIF1 gene for improving germination rate of seeds under dark and salt stress conditions and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to an arabidopsis PIF1 gene for improving the germination rate of seeds under dark and salt stress conditions and application thereof.

Background

The adversity stress is one of the main factors influencing the normal growth and development of plants and limiting the crop yield, and the crop yield loss caused in the global range is very large, so that the average yield can be reduced by 65-87%. China has abundant land resources, but the proportion of saline-alkali soil is high. The reported saline-alkali soil has large area and many types in China, and the area of the saline-alkali soil is gradually enlarged. Seriously affecting the sustainable development of agriculture. Therefore, improving the salt tolerance of crops, reducing the inhibition of salt stress on the growth of crops and limiting the yield become one of the hot spots of the current research. Under salt stress, plants turn off some normally expressed genes, but start expression of some specific genes associated with salt stress, with changes in expression patterns that facilitate resistance to salt stress. Therefore, the key genes participating in the plant response to the salt stress are separated and identified, the biological functions of the key genes in the plant response to the salt stress regulation network are determined, the important theoretical significance is provided for deeply understanding the molecular mechanism of the plant adapting to the salt stress, and the important gene resources are provided for improving the salt tolerance of crops.

After the plants are subjected to salt stress, osmotic stress is firstly caused, ion imbalance is then caused, nutrient element deficiency is caused, and finally oxidative stress is caused to change the permeability of a biological membrane, disorder of physiological and biochemical metabolism and accumulation of toxic substances. In the process of long-term evolution and environmental adaptation, plants form a series of physiological and molecular mechanisms adapting to salt stress, which mainly comprise the steps of adjusting ion transport, regionalizing to maintain ion balance, improving hormone level, increasing accumulation of osmoregulation substances, improving activity of antioxidant enzyme and regulating expression of salt stress response genes to respond to the salt stress. Among the signaling pathways of plants in response to salt stress, the sos (salt excess sensitive) signal is one of the more clear pathways studied. Zhu et al (1998) identified SOS1, SOS2 and SOS3 as being involved in modulating cellular Na under salt stress in the SOS signaling pathway by screening multiple SOS mutants in Arabidopsis thaliana ⁺ /K ⁺ The important components of the balance. SOS3 is an upstream component in the signaling pathway when plants are exposed to high concentrations of Na ⁺ Second messenger Ca in cells under stress ²⁺ At increasing concentrations, SOS3 senses Ca ²⁺ The signal is combined with the FISL structural domain at the C terminal of the SOS2 protein to activate the protein kinase activity of SOS2, the SOS3-SOS2 protein kinase complex regulates the expression of SOS1, SOS1 plays a role in converting Na ⁺ Discharging and maintaining intracellular Na ⁺ /K ⁺ So that the plant can endure salt stress. With the development of modern molecular biology, researches on plant response to salt stress mechanisms are also advanced to the levels of genomics, transcriptomics, proteomics, metabolomics, ionomics and the like. The research of utilizing the omics technology can more comprehensively reveal the mechanism of plant response to salt stress and provide a powerful means for the identification of salt-tolerant genes and the excavation of important components in a salt stress signal pathway. For example, after salt stress, the transcriptomics of plants such as barley, cotton, soybean, alfalfa and the like are analyzed, a plurality of genes which code nitrogen absorption, hormone synthesis and transportation, active oxygen elimination, ion transport and exchange, biomembrane stabilization and signal transduction pathways are obviously changed, and the functions of the screened important genes are identified by utilizing a forward or reverse genetics method, so that the functions of the differentially expressed genes in response to the salt stress are determined.

Light not only acts as energy to affect photosynthesis, but also acts as a signal to regulate the growth and development of plants. The phytochrome is a light receptor of red light and far-red light, and phytochrome action factors PIFs are positioned at the downstream of the phytochrome, and have important functions in the aspects of regulating and controlling seed germination, photomorphogenesis of seedlings, participation in shade-avoiding reaction, response to adversity stress and the like. For example, PIF1 participates in the regulation and control of the germination process of the light-mediated seeds, PIF3 and PIF4 participate in the response of low temperature, high temperature and drought stress, PIF4 and PIF5 participate in the regulation and control of shade-avoiding reaction, and the like. PIFs belong to a subfamily of bHLH transcription factor families, under the condition of illumination, a phytochrome is converted from a Pr physiological inactivation type into a physiological activation type Pfr, and is transferred to cell nucleus from cytoplasm to cause the ubiquitination and degradation of the PIFs, and the expression of the bHLH specific recognition target gene promoter is regulated by a G-box element of the PIFs sequence conserved domain bHLH, so that a plant generates a specific physiological and biochemical reaction. Currently, a total of 7 members of the PIFs family are found in the model plant arabidopsis thaliana, which are: PIF1, PIF3, PIF4, PIF5, PIF6, PIF7, and PIF 8. Wherein PIF1 is involved in GA and ABA signal transduction pathways, and can bind to promoter of DELLA protein (RGA1 and GAI) which is important component of GA signal pathway, so that mRNA level of RGA1 and GAI is increased, and seed germination is inhibited under dark condition. In recent years, scientists identified two important target genes of SOM and DAG1 at the downstream of PIF1, light-promoted degradation of PIF1 leads the expression level of SOM and DAG to be increased, and SOM and DAG further regulate and control the expression of GA synthesis key genes GA3ox1 and GA3ox2, and promote GA biosynthesis and seed germination. Although PIF1 has an important function in participating in the regulation of seed germination under light stress conditions, whether PIF1 is involved in regulating seed germination under salt stress conditions has not been reported so far.

The salt stress inhibits the processes of plant seed germination, seedling growth and the like, and the germination rate of the seeds is obviously reduced under the condition of the salt stress. Although many studies have now used forward and reverse genetics to identify a series of important components that regulate seed germination under salt stress conditions, the experimental systems of these studies have been performed under light conditions. Under natural conditions, when seeds are sown in soil, the seeds are exposed to dark environmental conditions in the germination process, and if components which are identified in a light test system and improve the germination rate of the seeds under the condition of salt stress do not have the regulation and control effect in the dark environment, the components are difficult to further utilize in agricultural production. According to the invention, the model plant Arabidopsis thaliana PIF1 function-deficient mutant PIF1 is used as a test material, and the fact that the tolerance of PIF1 seeds to salt stress in the germination process is obviously enhanced under a dark condition is found, and the germination rate of the PIF1 seed is greatly improved compared with that of a wild control. By utilizing the important discovery of the invention, the function of the endogenous PIF1 of the plant is lost through gene editing, RNA interference or antisense RNA technology, so that a new plant variety with improved seed germination rate under the conditions of darkness and salt stress can be cultivated, and the invention has wide application value.

Disclosure of Invention

The invention aims to solve the problems that: the method comprises the steps of identifying PIF1 as a function-deletion mutant with T-DNA insertion by a PCR amplification method, counting the germination rate of seeds under the conditions of darkness and salt stress by using wild type Arabidopsis Col and PIF1 as test materials, detecting the expression level of a salt stress response marker gene by a real-time fluorescent quantitative PCR method, and further confirming that the sensitivity of the seeds of the PIF1 mutant to the salt stress is remarkably reduced in the germination stage.

The technical scheme provided by the invention for solving the problems is as follows: an Arabidopsis thaliana PIF1 gene for improving seed germination rate under dark and salt stress conditions, wherein the gene sequence of the Arabidopsis thaliana PIF1 gene is shown as SEQ ID NO. 1.

The invention also discloses application of the arabidopsis PIF1 gene, which is characterized in that PIF1 is a functional deletion type mutant with T-DNA insertion through a PCR amplification method, wild arabidopsis Col and PIF1 are used as test materials, the germination rate of seeds under dark and salt stress conditions is counted, the expression level of a salt stress response marker gene is detected through a real-time fluorescent quantitative PCR method, and the obvious reduction of the sensitivity of the seeds of the PIF1 mutant to salt stress in the germination stage is further verified.

Preferably, the T-DNA insertion loss-function mutant is identified by the following steps,

(1) extracting DNA of seeds of which the wild type arabidopsis Col and pif1 mutants germinate for 48 hours;

(2) designing specific primer sequences of PIF1 on two sides of a T-DNA insertion site of the PIF1 mutant by taking DNA as a template;

the designed PCR amplification primer sequence is as follows:

an upstream primer: 5'-GACAGTTCCGCCGTAGCTGG-3'

A downstream primer: 5'-AGCAGCACGAGATCTCTTGG-3', respectively;

(3) PCR amplification is carried out by using the extracted DNA as a template, and then agarose gel electrophoresis is carried out on the product.

Compared with the prior art, the invention has the advantages that: test results show that the seed germination rate of the functional deletion type mutant PIF1 of the model plant Arabidopsis PIF1 is remarkably and greatly improved compared with that of a wild type Arabidopsis Col control under the conditions of darkness and salt stress. And the salt stress induced degree of the expression of the marker genes COR15A, DREB2A, RAB18 and ADH1 responding to the salt stress is detected to be obviously reduced in the pif1 mutant by a real-time fluorescent quantitative PCR method, so that the sensitivity of the germination of the seeds of the pif1 mutant to the salt stress is reduced under the dark condition. By utilizing the important discovery of the invention, the function of the endogenous PIF1 of the plant is lost through biological technical means such as T-DNA insertion, chemical mutagenesis, gene editing, RNA interference or antisense RNA technology and the like, and a new plant variety with the seed germination rate improved under the conditions of darkness and salt stress can be cultivated. The loss of function of the PIF1 gene may be directed to any trait that increases the germination rate of ornamental, commercial or other plant seeds under conditions of darkness and salt stress.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a PCR method for detecting that the model plant Arabidopsis thaliana pif1 is a loss-of-function mutant due to the insertion of foreign T-DNA, M: DL2000, Col and pif1 represent PCR amplification electrophoretograms using DNA of wild type Arabidopsis thaliana Col and pif1 mutants as templates, respectively.

FIG. 2 is a phenotype (A) and statistical germination rate (B) of seeds of wild type Arabidopsis thaliana Col and pif1 mutants sown in 1/2MS medium without (control group) or with 150mM NaCl (salt stress treated group) for 5 days of germination growth under dark conditions < P < 0.05.

FIG. 3 is a graph of seeds of wild type Arabidopsis thaliana Col and pif1 mutants seeded on 1/2MS medium without (control group) or with 150mM NaCl (salt stress treated group) for germination and growth for 2 days under dark conditions and the expression levels of the salt stress responsive genes COR15A, DREB2A, RAB18 and ADH1 were detected by real-time fluorescent quantitative PCR method with P < 0.05.

Detailed Description

The embodiments of the present invention will be described in detail with reference to the accompanying drawings and examples, so that how to implement the technical means for solving the technical problems and achieving the technical effects of the present invention can be fully understood and implemented.

Example 1: PCR identification of Arabidopsis thaliana T-DNA insertion mutant pif1

(1) DNA of seeds germinated for 48 hours by the wild type Arabidopsis thaliana Col-0 and pif1 mutants was extracted.

(2) Specific primers are designed on both sides of the T-DNA insertion site of pif1, and the primer sequences are as follows:

an upstream primer: 5'-GACAGTTCCGCCGTAGCTGG-3'

A downstream primer: 5'-AGCAGCACGAGATCTCTTGG-3'

(3) The extracted DNA is used as a template, and whether the pif1 mutant has the insertion of the exogenous T-DNA fragment is identified by a PCR method.

PCR reaction procedure: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 1min at 72 ℃ and 32 cycles; 10min at 72 ℃; keeping the temperature at 4 ℃.

The PCR reaction system is as follows:

as shown in FIG. 1, the PCR detection result shows that the specific amplified fragment of the endogenous PIF1 gene can be detected due to no insertion of the exogenous T-DNA fragment in the wild type Arabidopsis (Col), the length is 390bp, but the specific amplified fragment of the PIF1 gene is not detected in the PIF1 mutant, and the target amplified band cannot be detected by using the DNA of the PIF1 mutant as a template due to the insertion of the exogenous T-DNA fragment in the PIF1 mutant, so that the PIF1 is a mutant with the function of the endogenous PIF1 deleted due to the insertion of the T-DNA.

Example 2: PIF1 functional deletion remarkably improves germination rate of arabidopsis seeds under dark and salt stress conditions

(1) Seeds of wild type Arabidopsis thaliana Col and pif1 loss-of-function mutant were sterilized with 1% sodium hypochlorite for 10min, and then rinsed 10 times with sterile water.

(2) 1/2MS medium was prepared and autoclaved (sucrose concentration of medium 1%). The sterilized arabidopsis seeds were sown in 1/2MS medium containing no (control group) or 150mM NaCl (salt stress treated group) to germinate and grow for 5 days under dark conditions: the temperature was 22 ℃ and dark.

(3) Wild-type arabidopsis thaliana Col and pif1 loss-of-function mutants grown for 5 days at germination in 1/2MS medium were scanned for phenotype (fig. 2A) and statistical seed germination (fig. 2B). Statistical results show that in a control group under the dark condition, the germination rates of the pif1 function-deficient mutant and the wild-type Col are not obviously different, but in a salt stress treatment group under the dark condition, the germination rates of the pif1 function-deficient mutant and the wild-type Col are obviously and greatly improved, the germination rate of the wild-type seed is 61.67%, and the germination rate of the pif1 mutant is 92.77%. The test results show that the PIF1 functional deletion can obviously improve the germination rate of the arabidopsis seeds under the conditions of darkness and salt stress.

Example 3: the salt stress response marker gene is obviously reduced in the induction degree of the pif1 mutant

(1) RNA of seeds germinated for 48 hours by wild arabidopsis Col and pif1 mutants is extracted by using a guanidine-phenol iso-sulfate method.

(2) Specific primer sequences are designed according to known CDS sequences of arabidopsis salt stress induction marker genes COR15A, DREB2A, RAB18 and ADH1, and are specifically as follows:

primer sequence for COR 15A:

an upstream primer: 5'-GCCAGAAAACTCAGTTCGTCG-3'

A downstream primer: 5'-ATACGCCGCAGCTTTCTCAGC-3'

Primer sequence of DREB 2A:

an upstream primer: 5'-CCGGTGGAGTGGAGCCGATG-3'

A downstream primer: 5'-CGCTCAGCCAATGCTTATCCGC-3'

Primer sequence of RAB 18:

an upstream primer: 5'-CTTGGGAGGAATGCTTCAC-3'

A downstream primer: 5'-CTTCTTCTCGTGGTGCTCAC-3'

Primer sequence for ADH 1:

an upstream primer: 5'-ATGAAGCTGGAGGGATTGTTGAG-3'

A downstream primer: 5'-AGAGGAGCATCCGGATTGATCTTA-3'

(3) Taking 1. mu.g of RNA as a template for reverse transcription, and taking P as a template ₂₈₅₃ Reverse transcriptase ImProm-II from Promega was used as primer ^TM Carrying out reverse transcription of P ₂₈₅₃ The primer sequence, reverse transcription procedure and reverse transcription system are as follows.

P ₂₈₅₃ The primer sequence is as follows: 5'-GCGAATTCTTTTTTTTTTTTTTTTT-3'

Reverse transcription program:

5min at 72 ℃; 5min at 25 ℃; 60min at 42 ℃; 20min at 80 ℃; keeping the temperature at 4 ℃.

Reverse transcription system:

(4) and performing real-time fluorescent quantitative PCR amplification by using the primer by using the reverse transcribed cDNA as a template.

PCR reaction procedure: 3min at 94 ℃; 94 ℃ for 10s, 60 ℃ for 30s, 40 cycles.

And (3) PCR reaction system:

(5) by use of 2 ^-ΔΔCt The method analyzes the difference of the relative expression quantity of the target gene and performs significant difference analysis, wherein the relative expression quantity of the Delta CT wild-type is CT wild-type-CT internal control (wild-type), the Delta CT mutant is CT mutant-CT internal control (mutant), and the relative expression quantity of the salt stress response marker gene is 2 ^{(ΔCT wild-type-ΔCT mutant)} 。

After data statistics, salt stress is found to be capable of remarkably inducing the expression of COR15A, DREB2A, RAB18 and ADH1 in wild type Arabidopsis thaliana Col under dark conditions, and although salt stress can also remarkably induce the expression of COR15A, DREB2A and RAB18 in pif1 mutant, the induction degree is remarkably reduced compared with that of the wild type. In addition, the expression level of ADH1 in the PIF1 mutant is not significantly different between the control group and the salt stress treatment group, and the test results show that the sensitivity of the PIF1 mutant seeds to salt stress in the germination period is significantly reduced under the dark condition, which is consistent with the test result that the PIF1 function loss significantly improves the germination rate of the Arabidopsis seeds under the dark and salt stress conditions.

The results of the above examples show and confirm that the functional deletion of the model plant Arabidopsis endogenous PIF1 can significantly and greatly improve the germination rate of seeds under the conditions of darkness and salt stress. Therefore, by using the important discovery of the invention, the function of the plant endogenous PIF1 is deleted through biological technical means such as T-DNA insertion, chemical mutagenesis, gene editing, RNA interference or antisense RNA technology and the like, a new plant variety capable of improving the seed germination rate under the conditions of darkness and salt stress can be cultivated, and the invention has wide application value.

The foregoing is merely illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiments, and the specific structure thereof is allowed to vary. All changes which come within the scope of the invention as defined by the independent claims are intended to be embraced therein.

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Claims (3)

1. An arabidopsis PIF1 gene for improving seed germination rate under dark and salt stress conditions is characterized in that: the sequence of the Arabidopsis PIF1 gene is shown as SEQ ID NO. 1.

2. Use of the arabidopsis PIF1 gene of claim 1, wherein: the method is characterized in that the pif1 is a T-DNA inserted function-deficient mutant through a PCR amplification method, wild arabidopsis Col and pif1 are used as test materials, the germination rate of seeds under dark and salt stress conditions is counted, the expression level of a salt stress response marker gene is detected through a real-time fluorescent quantitative PCR method, and the fact that the sensitivity of the pif1 mutant seeds to salt stress in the germination stage is remarkably reduced is further verified.

3. The use of the Arabidopsis PIF1 gene according to claim 2, wherein: the method for identifying the mutant with the deletion of the insertion function of the T-DNA comprises the following specific operation steps,

(1) extracting DNA of seeds of which the wild type arabidopsis Col and pif1 mutants germinate for 48 hours;

(2) designing specific primer sequences of PIF1 on two sides of a T-DNA insertion site of the PIF1 mutant by taking DNA as a template;

the designed PCR amplification primer sequence is as follows:

an upstream primer: 5'-GACAGTTCCGCCGTAGCTGG-3'

A downstream primer: 5'-AGCAGCACGAGATCTCTTGG-3', respectively;

(3) PCR amplification is carried out by using the extracted DNA as a template, and then agarose gel electrophoresis is carried out on the product.

Images