CN117187294B - Application of BnaC5.ACBP4 gene in improving flooding resistance of plants - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to the application of BnaC5.ACBP4 gene in improving plant waterlogging tolerance. The present invention provides the application of BnaC5.ACBP4 gene in improving plant waterlogging tolerance. In the present invention, wild type and BnaC5.ACBP4 overexpression lines of rapeseed are subjected to flooding treatment. The experimental results show that compared with the wild type, the BnaC5.ACBP4 overexpression line confers stronger flooding resistance to rapeseed, indicating that the BnaC5.ACBP4 gene has a positive regulatory effect on the crop's response to flooding stress. This is achieved by constructing Transgenic plants to improve plant flooding resistance provide theoretical support based on the principles of molecular biology, that is, by improving the flooding resistance of rapeseed, flood-tolerant rapeseed varieties can be cultivated.

Description

Application of BnaC5.ACBP4 gene in improving plant waterlogging tolerance

Technical field

The present invention relates to the field of biotechnology, and in particular to the application of BnaC5.ACBP4 gene in improving plant waterlogging tolerance.

Background technique

Rapeseed (Brassica napus) belongs to the genus Brassica of the Brassicaceae family and is one of the four major oil crops in the world. In addition to being used to extract edible oil and feed, rapeseed can also be used to make margarine and artificial protein in the food industry. It is also widely used in metallurgy, machinery, rubber and medicine, and has important economic value.

A large amount of continuous rainfall in a short period of time will cause plants to be partially or completely flooded. Since the diffusion rate of oxygen in water is slower than that in air, coupled with the consumption of oxygen by soil microorganisms, the damage caused by waterlogging disasters to plants is indirect, that is, When plants are flooded for a long time, a large number of mineral elements and major intermediate metabolites in plant roots are dissolved and lost; secondly, anaerobic respiration will produce toxic secondary metabolites that are detrimental to plant growth, such as ethanol and acetaldehyde. In addition, when there is too much water in the soil, the gas phase replaces the liquid phase, and the ethylene released by soil anaerobic microorganisms is also absorbed by plants, eventually leading to hypoxia in plant roots and even entire plant cells, and in severe cases, plant death.

Waterlogging caused by flooding has many adverse effects on the growth and development of plants, restricting plant growth and reducing productivity and quality. Through extensive research in rice and the model plant Arabidopsis thaliana, people have revealed the molecular regulatory mechanism of plant response to waterlogging and hypoxia stress, and have made good research progress. However, in actual production, it has rarely been successfully applied to crops such as rapeseed to improve their waterlogging tolerance.

The escape strategies that plants have evolved in response to flooding stress are also driven by ethylene, but with different downstream signals in different plants. Transcription factors (TFs) integrate signals and activate hypoxia-related genes. The transcription factors in rice are the group VII ethylene response factor (VII-ERFs) family. The stability of the oxygen sensing mechanism of Arabidopsis depends on the oxidation reaction of the N-terminal cysteine (Cys) of VII-ERFs. This oxidation reaction requires the mediation of plant cysteine oxidase (PCO), which requires molecular oxygen. as a co-substrate. Oxidized Cys can convert RAP2.12 into a target for proteolytic enzymes for proteolysis via the ubiquitin-mediated N-end rule pathway. Under aerobic conditions, RAP2.12 is localized on the plasma membrane by interacting with plasma membrane-anchored acyl-CoA-binding protein 1 (ACBP1) and ACBP2. During hypoxia, RAP2.12 is released and transported to the nucleus. Activate the expression of downstream hypoxia-responsive genes (such as ADH1, SUS4). During aerobic restoration, RAP2.12 is rapidly degraded through the N-terminal pathway and protease-mediated proteolysis, terminating the hypoxic response.

Transgenic technology is a method used to change the genome of an organism. Through transgenic technology, genes of one species can be cut out from its native genome and inserted into the genome of another species to achieve cross-species transfer of genes. Doing so can result in the target species acquiring or modifying a specific trait or function.

The application of genetically modified technology in agriculture, such as genetically modified crops, refers to inserting foreign genes into the genome of crops to give them some new traits or improve existing traits. These foreign genes can come from other individuals of the same species or from different species. Through transgenic technology, traits such as disease resistance, insect resistance, drought tolerance, and salt-alkali tolerance can be added to crops to improve crop yield and quality. The laboratory uses genetically modified technology to improve the hypoxia-tolerant traits of rapeseed to increase its yield and quality.

Specific steps include the following aspects:

Gene selection: Determining the specific genes that need to be transferred. Select appropriate genes for transfer based on target traits

Gene cloning: Using molecular biology methods, the target gene is amplified and purified from DNA to obtain a sufficient amount of DNA.

Vector construction: Select a suitable vector (such as plasmid or virus) and insert the target gene into the appropriate position of the vector for stable expression in the target cells.

Gene delivery: Introduce the constructed vector into target cells. This can be achieved by different methods, such as biolistics, Agrobacterium-mediated transformation, electroporation, etc.

Gene integration: After obtaining transgenic cells, the target gene will be successfully integrated into the genome of the target cell and become a permanent genetic characteristic.

Selectable markers and screening: To determine which cells have successfully transferred the target gene, a selectable marker or screening method is often included in the transgenic process. These methods can vary depending on the purpose of the transgene.

Identification and verification: The genetically modified cells or tissues are identified and verified at multiple levels such as molecular biology and physiology to ensure that the target genes are correctly expressed in the target cells and have no adverse effects on the cells or tissues.

A comprehensive safety assessment must be conducted before plant transgenic operations are carried out. This includes analysis and evaluation of aspects such as genomic structure, function, expression and delivery of transgenic plants to ensure that transgenic plants will not cause negative impacts on the environment and human health. In addition, the toxicity, sensitization, allergy and other aspects of genetically modified plants need to be evaluated to ensure the food safety of genetically modified plants.

Dark submergence treatment is a method commonly used in laboratories to simulate the low-oxygen environment caused by flood disasters. It is often used in model plants such as Arabidopsis thaliana and aims to simulate it by establishing a dark, low-oxygen environment with controllable conditions. and monitoring a series of responses of plants after they are exposed to hypoxia during flooding stress.

Therefore, how to discover more types of flooding tolerance-related genes to provide a theoretical basis for cultivating and improving flooding-tolerant rapeseed and other crop varieties is an urgent problem that those skilled in the art need to solve.

Contents of the invention

The purpose of the present invention is to provide the application of BnaC5.ACBP4 gene in improving plant waterlogging tolerance.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides the use of the BnaC5.ACBP4 gene in improving plant flooding tolerance; the nucleotide sequence of the BnaC5.ACBP4 gene is shown in SEQ ID NO: 1.

The present invention also provides biological materials containing the BnaC5.ACBP4 gene; the biological materials are expression vectors or strains.

The present invention also provides the application of the biological material in cultivating waterlogging-tolerant plants.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention shows that the BnaC5.ACBP4 overexpression strain gives rapeseed a stronger ability to tolerate flooding compared with the wild type, indicating that the BnaC5.ACBP4 gene has a positive regulatory effect on improving the ability of crops to tolerate flooding stress. , which provides molecular biology support for improving the plant's ability to tolerate flooding stress by constructing transgenic plants, that is, by improving the flooding tolerance of rapeseed, cultivating flooded rapeseed varieties.

Description of drawings

In order to explain this embodiment or the technical solution in the prior art more clearly, the accompanying drawings required to be used will be briefly introduced below. The drawings in the following description are only examples of this embodiment. Other workers can repeat experiments based on existing results and methods to verify the results of the drawings. By using existing results to carry out further research, other workers may make new discoveries.

Figure 1 is a map of the pBWA(V)BS-BnACBP4-OSGFP vector (exogenous gene BnaC5.ACBP4 referred to by Gname (Gene name)) in Example 1; the vector sequence is shown in SEQ ID NO: 9.

Figure 2 shows that overexpression of BnaC5.ACBP4 in rapeseed in Example 1 can significantly improve flooding resistance.

Figure 3 shows that compared with wild-type rapeseed in BnaC5.ACBP4 overexpression in Example 1, the expression of hypoxia response gene ADH1 and cysteine oxidase PCO1 is significantly up-regulated.

Detailed ways

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention.

Example 1

1. Selection and positioning of major flooding genes

Previous studies in the laboratory have shown that the Arabidopsis chromosome 3 gene ACBP4 (AT3G05420) can interact with WRKY70 and ERFVII to directly or indirectly regulate the downstream hypoxia-responsive gene ADH1 through proteolysis mediated by cystine oxidase (PCO1) , enhance plant flooding resistance. In order to further study its waterlogging resistance in rapeseed, the present invention obtained its homologous gene BnaC5.ACBP4 and overexpressed it in rapeseed. The homologous gene of ACBP4, BnaC5.ACBP4, can be queried from the website https://bioinformatics.psb.ugent.be/. The nucleotide sequence of the BnaC5.ACBP4 gene is shown in SEQ ID NO: 1.

2.Construction of BnaC5.ACBP4 transgenic vector

(1) Obtain the target fragment

Perform PCR amplification of the target fragment of the gene BnaC5.ACBP4, and recover the product to obtain the target fragment; specifically, the following primers are synthesized, in order to add a FLAG tag (to facilitate other experiments unrelated to this patent, the presence or absence of the FLAG tag has no direct impact on this experiment ), the present invention performs segmented amplification. The first pair of primers is a FLAG tag-added primer (48766_0 primer), and the second pair of primers is a target gene sequencing primer.

48766_0 primer

48766_0(+):aacacgggggactttgcaacatggctatggctagagcaacatctgg

48766_0(-):tcctcgcccttcacgatacatggcgaatcatctttctcctgaggg

Target gene sequencing primers

M48766(500C):caccaaggtaacggccatt

M48766(1000C):caagagttgacatgtttagtacaacac

M48766(1500C):gcgttcattgcggcttgtt

As shown in SEQ ID NO: 2~6

(2) Make a 50 μL system and perform the amplification reaction according to the following procedures:

Table 1 PCR system

Element volume Ultra-pure water 20μL Boyuan high-fidelity enzyme Pfu 25μL Sense primer (100μM) 2μL Antisense primer (100μM) 2μL template 1μL total capacity 50μL

Table 2PCR procedures

(3) After amplification, use 1% agarose gel electrophoresis at 5v/cm voltage for 20 minutes. Cut out the electrophoresis fragment of Gname (Gene name, refers to gene BnaC5.ACBP4, 1998bp) under UV light and place it in a The sol is recovered in the system, and the DNA is dissolved and recovered with a total volume of 40 μL of water (the recovery product is labeled: rDNAG1). After the detection is correct, it is recombined with the vector.

2. Enzyme digestion vector

BsaI/Eco31I double enzyme digestion plasmid pBWA(V)BS-ccdB was used to recover the double enzyme digestion empty vector; Tables 3 and 4 show the enzyme digestion reaction system and conditions.

Table 3 Enzyme digestion reaction system

Element volume Ultra-pure water 13μL 10*buffer 2μL Restriction endonuclease BsaI or Eco31I 1μL Vector pBWA(V)BS--ccdB-OSGFP 4μL total capacity 20μL

Table 4 Enzyme digestion reaction conditions

temperature time 37℃ 1 hour

Use the PCR purification kit to purify the vector digest (the purified product is labeled pBWA(V)BS--ccdB-OSGFP(D)) for the next step of in vitro or in vivo recombination reaction.

3. Carrier construction

The pBWA(V)BS-Bn ACBP4-OSGFP vector was constructed using homologous recombination and golden gate seamless cloning methods (Figure 1). The vector sequence is shown in SEQ ID NO: 9; Tables 5 and 6 show pBWA(V)BS- Bn ACBP4-OSGFP vector recombination system and conditions;

Table 5 Recombination reaction system

Table 6 Recombination reaction conditions

temperature time 37℃ 30hours

Transform 8 μL of the ligation product into competent Escherichia coli. After transformation and recovery, apply (cananamycin) resistant medium and culture at 37°C for 12 hours. Perform plaque PCR to identify single clones.

4. Plaque PCR identification

Pick 10 bacterial plaques and conduct bacterial inoculation and PCR identification with 1.5ml EP tube at the same time.

The primers are as follows:

HS)35seq:tTCATTTGGAGAGAACACGGGggac(2303bp)

M48766(500C):caccaaggtaacggccatt(2945bp);

As shown in SEQ ID NO: 7～8

Do 10 PCR reactions of 25 μL system. The PCR reaction system and procedures are shown in Table 7 and Table 8.

Table 7 PCR reaction system

Element volume Ultra-pure water 9.5μL Boyuan PCRMix 12.5μL Upstream primer (100μM) 1μL Downstream primer (100μM) 1μL template 1μL total capacity 25μL

Table 8 PCR reaction procedure

The target band is a fragment of approximately 662bp. Take the bacterial liquid corresponding to the 2 positive bands, take 100 μL and send the sample for sequencing, and inoculate the remaining 400 μL bacterial liquid into 8 ml (cananamycin)-resistant LB, shake the bacteria in the test tube, and wait for the sequencing results to come out, corresponding to the correct sequence Take a tube to extract the plasmid.

5. Agrobacterium transformation

Transform 1 μg of pBWA(V)BS-BnACBP4-OSGFP vector into Agrobacterium competent cells, and apply (cananamycin, rifampicin) resistant medium, culture at 28°C for 48 hours, and perform colony PCR identification. The PCR reaction system and The procedure is the same as Table 7 and Table 8.

6. Agrobacterium-mediated genetic transformation of rapeseed

(1) Preparation work before sowing

Clean the covered square box for sowing, prepare 1000ml blue pipette tip, 50ml centrifuge tube, prepare M0 medium, prepare deionized water, send it to high temperature and high pressure sterilization, and pour the sterile M0 medium into the covered square box for use.

(2) Sowing

Soak the rapeseed seeds (variety Zhongshuang 11) with an appropriate amount of 75% alcohol for 1 minute, pour out the alcohol; wash it once with sterile water, pour out the water; then sterilize it with 50% 84 disinfectant for 10 minutes, and use an appropriate amount of sterile water. Wash the seeds 5 times with water; use sterile tweezers to sow the treated seeds into the M0 medium, 25 seeds per dish, then place the petri dish into a sterile culture box and culture it in the dark at 24°C for 6 days.

(3) Preparation work before dip dyeing

Clean the large square dish used for dip dyeing, glass dish, 50ml centrifuge tube, 200ml pipette tip, prepare culture medium, send it to high temperature and high pressure sterilization, pour the culture medium into the glass dish, seal it and set aside.

(4) Activation and preparation of Agrobacterium

The day before dip-staining, add antibiotics to the sterilized 100 mL liquid LB culture medium (add the corresponding antibiotics according to the resistance of the Agrobacterium strain), insert the Agrobacterium strain, and culture with shaking at 28°C and 200 rpm for 16 hours.

(5) Infection and co-culture of explants

When the OD value of the Agrobacterium cultured in step (4) is 0.8, pour the bacterial solution equally into two 50mL sterile centrifuge tubes (operated on a ultra-clean workbench), balance and centrifuge at 3000 rpm for 20 minutes, and discard the supernatant. , gently wash the cells with 1ml of suspension (DM) (AS acetosyringone added), then pour it out, add 1ml of DM and pipette to suspend, shake well, prepare and place on ice for later use;

At the same time, use sterile tweezers and a scalpel to vertically cut the hypocotyl of the above-mentioned seedlings cultured in the dark, and cut it out in DM liquid. The optimal length of the explant is 1.0cm; put the cut explant into the above-mentioned Agrobacterium-containing bacteria Dip in the liquid dish for 15 minutes. The number of explants in each dish is 150. Shake once every 10 minutes for a total of 5 times;

After dip-staining, use sterile tweezers to gently pinch out the explant, place it on sterile filter paper to remove excess bacterial fluid on the surface, then use sterile tweezers to place the explant on M1 culture medium, and co-culture at 24°C in the dark. 48h.

(6) Selective training

After co-culture, the explants were transferred to M2 medium for selective culture for 18 days. The culture conditions were: light culture at 24°C, 16 hours during the day/8 hours at night. The conditions for differentiation culture and rooting culture are the same as at this stage.

(7) Differentiation culture

The explants after selection and culture were transferred to M3 medium for differentiation and culture, and were subcultured every 20 days until budding.

(8) Rooting culture

After differentiation and sprouting, the sprouts were cut off from the callus using sterile tweezers and a scalpel, and then transferred to M4 medium for rooting. The vitrified sprouts will need to be cultivated for a period of time before they can return to normal and then take root.

(9)Transplanting and soil culture

The rooted rape plants were transplanted into sterilized soil (Pin's matrix soil), transferred to an incubator with 4°C and 2000lux light conditions for vernalization for 15 days, and then moved to 22°C, 13000lux (16 hours of light/8 hours of darkness). Circulation) light environment greenhouse for cultivation. A transgenic line overexpressing BnaC5.ACBP4 in rapeseed was obtained.

7. Dark flooding treatment

(1) Plant sample preparation:

Select plump fresh seeds (Zhongshuang 11), place them in a petri dish lined with two layers of sterile moist filter paper, and germinate at 25°C for 60 hours. Transplant the seedlings with cotyledons into sterile soil at 22°C, 13000lux (16-hour light/8-hour dark cycle) light environment greenhouse cultivation for 2 weeks without vernalization to obtain wild-type strains.

Select 20 appropriate samples each of the BnaC5.ACBP4 overexpression transgenic line and wild-type control line (Zhongshuang 11) of rapeseed with similar growth conditions, and place them in an insulated foam box with an inner size of 440*320*220cm. , use long bamboo sticks or steel balls to fix the flowerpot and soil;

(2) Flooding operation:

Completely soak the plant samples in the container in water for 48 hours, ensuring that all leaves of the sample are fully covered before the water level is 3cm higher. The flooding device can be set directly in the greenhouse for rapeseed cultivation to ensure that the water temperature matches the rapeseed growth conditions;

(3) Establish a dark environment:

Cover the foam box with a lid and use heavy objects to compress the gaps to simulate the low-oxygen conditions plants face during flooding;

(4)Monitoring and recording:

During the experiment, focus on physiological and morphological changes in plant samples. After the flooding is completed, the plants are moved to the original growth environment to recover for 4 days, and key indicators such as plant survival rate and plant dry weight are further evaluated.

(5) Analyze data:

Conduct data analysis on the experimental results and compare the differences between the treatment group and the control group to understand the adaptability of BnaC5.ACBP4 to the hypoxic environment.

As shown in Figure 2, after the flooding was completed and the plants were re-oxygenated in the original growth environment for 4 days (the culture conditions of the above step (1)), the results were similar to the BnaC5.ACBP4 overexpressing rapeseed line (BnaC5.ACBP4-OE). Compared with wild type (WT), the wilting of leaves was more serious. In addition, the survival rate and dry weight of the wild type and BnaC5.ACBP4 overexpressing rapeseed lines under waterlogging treatment were detected, and it was found that the survival rate and dry weight of the latter were significantly higher than those of the former.

As shown in Figure 3, after sampling the wild type and BnaC5.ACBP4 overexpression lines at 0h (Air) and 20h (Submergence) waterlogging, the rape homologous genes of the wildtype and BnaC5.ACBP4 overexpression lines after waterlogging treatment were detected. Expression of ADH1 and PCO1. As a result, in the BnaC5.ACBP4 overexpression line, the waterlogging response of ADH1 and PCO1 was stronger than that in the wild-type plant.

The rapeseed waterlogging treatment test showed that compared with the wild type, the BnaC5.ACBP4 overexpression line endowed the rapeseed with a stronger ability to tolerate flooding, indicating that its function is conserved.

The above are only the preferred embodiments of the present invention. It should be pointed out that those of ordinary skill in the art can also make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (3)

1. The application of the over-expressed BnaC5.ACBP4 gene in improving the flooding resistance of rape is characterized in that the nucleotide sequence of the BnaC5.ACBP4 gene is shown as SEQ ID NO: 1.

2. A biological material comprising the bnac5.Acbp4 gene of claim 1, wherein said biological material is an expression vector or strain.

3. Use of the biological material according to claim 2 for cultivating flooding-resistant rape.