WO2022082866A1

WO2022082866A1 - Stress-resistant gene line acdwem and use thereof in improvement of salt tolerance, drought resistance and high temperature resistance of crops

Info

Publication number: WO2022082866A1
Application number: PCT/CN2020/126331
Authority: WO
Inventors: 谷晓峰; 林敏�; 王劲; 周正富; 燕永亮; 左开井
Original assignee: 隆平生物技术(海南)有限公司
Priority date: 2020-10-20
Filing date: 2020-11-04
Publication date: 2022-04-28
Also published as: CN113151293B; CN113151293A

Abstract

Provided is a stress-resistant functional line AcDwEm that has the ability to improve the resistance of host cells to high salt, drought and high temperature stress, with the nucleotide sequence thereof as shown in SEQ ID NO: 1. A recombinant vector containing the stress-resistant functional line AcDwEm is constructed, and is integrated and reconstructed in model plants of oilseed rape and rice by means of an agrobacterium-mediated infestation transformation method. After the stress-resistant functional line AcDwEm is expressed in host cells of the model plants, the high-salt resistance, drought resistance and high-temperature resistance of crops can be significantly enhanced, and the stress-resistant functional line AcDwEm can be used for improving the stress resistance of new varieties of crops.

Description

Stress resistance gene line AcDwEm and its application in improving crop salt tolerance, drought tolerance and high temperature tolerance

technical field

The invention belongs to the field of synthetic biology, and relates to the application of a multi-module anti-stress functional circuit with the ability to improve biological resistance to drought and high salt stress.

Background technique

Soil salinization, frequent droughts and prolonged high temperatures are the most damaging abiotic stresses in global agriculture, significantly reducing agricultural productivity through adverse effects on seed germination, plant growth and development, plant vigor and crop yield.

At present, genetic engineering strategies are more and more widely used in the world to breed stress-resistant varieties. However, since crop salt tolerance, drought tolerance, high temperature tolerance is a complex trait, and is affected by multiple genes and factors at the same time, single-gene transformation operations are not ideal, and cultivated plants with improved stress tolerance do not perform well under stress-free conditions.

Since the beginning of the new century, the original innovation and integrated application of the new generation of synthetic biology have accelerated breakthroughs, and the whole genome design breeding technology has promoted the upgrading of traditional agricultural varieties, nurturing a new round of agricultural scientific and technological revolution and industrial transformation. Therefore, using modern synthetic biology design methods, artificially designing protein functional elements, promoters, and combining multiple genes to artificially construct response functional modules that specifically respond to high-salt stress signals, drought signals and high temperature stress signals, or It is expected to create an anti-stress functional system that improves the ability of organisms to resist drought and high salt stress.

SUMMARY OF THE INVENTION

The purpose of the present invention is to create an anti-stress functional circuit that can improve the ability of organisms to resist drought and high salt stress.

The invention utilizes the modern synthetic biology design method to optimize and transform the anti-stress element. Through the artificial design of protein functional elements, the tissue-specific and stress-responsive design of promoters, the response functional modules, anti-stress functional stabilizer modules and tissue-specific high-efficiency anti-stress functional modules that specifically respond to high temperature stress signals are artificially constructed. A new anti-stress functional circuit of intelligent response-directed expression, named AcDwEm.

Through the following studies, it was identified for the first time that the stress resistance function line AcDwEm has the ability to improve the drought resistance, salt tolerance, high temperature and high temperature resistance of model plants, and can be used for the cultivation of a new generation of stress-resistant crop varieties. The specific research work is as follows:

1. Construction of artificially designed anti-stress circuit AcDwEm

Design stress response functional modules through synthetic biology, design and construct response functional modules, anti-stress function stabilizer modules and tissue-specific high-efficiency anti-stress functional modules that specifically respond to high temperature stress signals, and assemble to form a new anti-stress module with intelligent response and directional expression. Function line, named AcDwEm. The full-length nucleic acid sequence of the anti-stress functional circuit AcDwEm was obtained by artificial chemical synthesis. The anti-stress circuit AcDwEm was connected to the pBI-121 vector to construct a plant expression vector pBI-AcDwEm, and the expression vector was transformed into Agrobacterium tumefaciens EHA105 (see Example 1 for details);

2. The acquisition of AcDwEm in rapeseed and rice with the anti-stress function line

Through the method of constructing transgenic plants mediated by Agrobacterium, the stress resistance functional circuit AcDwEm was integrated and recombined with the model plants rape and rice, and the positive transgenic plants with stable inheritance were obtained by the methods of resistance screening and PCR verification (see Example 2 for details). , 4).

3. Analysis of salt tolerance and drought resistance performance of AcDwEm rapeseed with anti-stress function line

NaCl and polyethylene glycol PEG-6000 were used as additives to simulate salt stress and drought stress, respectively, and the stress treatment was carried out by watering. The obtained positive transgenic seeds and wild-type seeds were cultured to emerge, and subjected to stress treatment. Plants were irrigated with the same amount of stress solution every day, and samples were taken at 0, 1, 3, 7, 14, and 21 days of stress treatment, and the growth status was observed to determine physiological indicators.

4. Analysis of high temperature resistance performance of AcDwEm rice

Seeds of wild-type rice and positive transgenic rice were germinated and treated with high temperature. The culture environment was set up with light at 45°C for 14 hours and dark conditions at 45°C for 10 hours, and the treatment was carried out for 7 days, and the growth state of the plants was observed.

The experimental results show that: under normal conditions, the stress resistance function line AcDwEm has no effect on the growth and development of host plants, and under stress conditions, it has the function of significantly improving the salt tolerance, drought tolerance, drought tolerance and high temperature tolerance of rapeseed and rice, which can be used for the cultivation of a new generation of stress resistant crops.

Sequence Listing Information

SEQ ID NO. 1: Nucleotide sequence of the anti-reverse functional circuit AcDwEm.

SEQ ID NO. 2: Nucleotide sequence of functional module 1.

SEQ ID NO.3: The amino acid sequence of the encoded protein of functional module 1.

SEQ ID NO. 4: Nucleotide sequence of functional module 2.

SEQ ID NO.5: The amino acid sequence of the encoded protein of functional module 2.

SEQ ID NO. 6: Nucleotide sequence of functional module 3.

SEQ ID NO. 7: The amino acid sequence of the encoded protein of functional module 3.

Description of drawings:

Fig. 1 Construction diagram of AcDwEm vector of anti-reverse circuit;

Fig. 2 Comparison of the results of salt tolerance and drought resistance experiments of transgenic rape Bn-AcDwEm and non-transgenic rape (WT);

Figure 3. Comparison of high temperature resistance test results between transgenic rice Os-AcDwEm and non-transgenic wild-type rice.

Detailed ways

The plasmids, strains and model plants cited in the following examples are only used to further illustrate the present invention, and do not limit the essential content of the present invention. Where the specific experimental conditions are not indicated, all are in accordance with the conventional conditions well known to those skilled in the art or in accordance with the conditions suggested by the manufacturer. The plasmids, bacterial strains, and plant sources cited in the examples are as follows:

Cloning vector pJET: a commercially available product from ThermoFisher;

Shuttle vector: pBI-121: preserved in this laboratory;

Agrobacterium tumefaciens EHA105: preserved in this laboratory;

Rice material: Rice seeds ZH11 are preserved in this laboratory.

Brassica napus material: Rapeseed 84100-18 is preserved in this laboratory.

Example 1 Design of the anti-stress circuit AcDwEm and construction of recombinant Agrobacterium tumefaciens

1. Experimental materials

Cloning vector pJET: a commercially available product from ThermoFisher;

Shuttle vector: pBI-121: preserved in this laboratory;

Agrobacterium tumefaciens EHA105: preserved in this laboratory.

2. Experimental method

1. Design stress response functional modules through synthetic biology, design and construct response functional modules, anti-stress functional stabilizer modules and tissue-specific high-efficiency anti-stress functional modules that specifically respond to high temperature stress signals, and assemble to form a new intelligent response directional expression Anti-reverse function circuit, named as AcDwEm. The full-length nucleic acid sequence of the anti-reverse functional circuit AcDwEm was obtained by artificial chemical synthesis. Its size is 3737bp. It was cloned into the vector pJET, and the recombinant cloned plasmid pJET-AcDwEm containing the complete anti-reverse functional circuit was constructed and verified by sequencing. Then, the anti-reverse circuit AcDwEm with sticky ends was obtained by double digestion with EcoRI and HindIII. Fragment and shuttle vector pBI-121 vector fragment, connect the anti-reverse circuit AcDwEm to the pBI-121 vector, construct the plant expression vector pBI-AcDwEm, transform the expression vector into Agrobacterium tumefaciens EHA105, use kanamycin antibiotic resistance Positive recombinant strains were screened and verified by colony PCR sequencing.

3. Experimental results

The full-length nucleic acid sequence of the anti-stress functional circuit AcDwEm was obtained by artificial chemical synthesis, and the plant expression vector pBI-AcDwEm containing the functional circuit SyAcDwEm was successfully constructed and transformed into Agrobacterium tumefaciens EHA105. After PCR, enzyme digestion, and sequencing, it was verified that the inserted sequence was correct, and the strain was named EHA-AcDwEm.

Fourth, the experimental conclusion

The construction of recombinant Agrobacterium tumefaciens EHA-AcDwEm expressing the anti-stress circuit AcDwEm was completed.

Example 2 Acquisition of Agrobacterium-mediated anti-stress function line AcDwEm rape

1. Experimental materials

Recombinant strain EHA-AcDwEm: obtained in Example 1

Brassica napus material: Rapeseed 84100-18 is preserved in this laboratory.

2. Experimental method

Rapeseeds were removed, immersed in 75% ethanol and 0.1% HgCl2 for sterilization, placed evenly in plant tissue culture medium, and cultured in a tissue culture room at 24°C for one week. The hypocotyls of rapeseed seedlings were cut by sterile surgery, placed on pre-medium, and cultured in the light for 2-3 days, and the explants were pre-cultured.

The recombinant Agrobacterium strain EHA-AcDwEm expressing the anti-stress circuit was transferred and activated, and the strain was collected by centrifugation and resuspended to OD600=1.0. The pre-cultured explants were soaked in Agrobacterium solution for 90s, and then transferred to the co-culture medium after drying, and cultured in the dark for 2-3d. Well-grown explants were then transferred to induction medium for culture.

The explants with good callus growth were selected and transferred to the screening medium supplemented with antibiotics, cultivated in the light for 45-50 d, and then differentiated into buds. Transfer the differentiated and budded callus to the rooting medium, cultivate in the light for 2 weeks, when the roots appear and the stem grows 4-5cm, transfer to the culture soil for seedling training, and transplant to the greenhouse after acclimation, PCR detection of positive rapeseed Seedling.

3. Experimental results

Using the Agrobacterium-mediated co-cultivation method of explants, the anti-stress functional circuit AcDwEm was transformed into rapeseed. After infecting the rapeseed explants, the steps of induction culture, screening culture, rooting culture, and seedling transplantation were verified by PCR. The transgenic rape Bn-AcDwEm expressing the anti-stress functional circuit was obtained, which can be used for the subsequent research on the anti-stress performance.

Fourth, the experimental conclusion

Through Agrobacterium-mediated transformation, the anti-stress circuit AcDwEm canola Bn-AcDwEm was finally obtained

Example 3 Stress resistance analysis of AcDwEm rapeseed by transferring the anti-stress function line

1. Experimental materials

Genetically modified rape: Bn-AcDwEm

Control: non-transgenic wild-type canola

2. Experimental method

NaCl and polyethylene glycol PEG-6000 were used as additives to simulate salt stress and drought stress, respectively, and the stress treatment was carried out by watering.

The obtained transgenic rapeseed seeds and wild-type seeds that have been identified as positive were cultured in MS solid state, and after the seedlings grew true leaves, they were transplanted into plastic pots equipped with substrates, and MS nutrient solution was irrigated until the seedlings grew for 5-6 months. True leaves were subjected to adversity treatment.

Plants were irrigated with the same amount of stress solution every day, and samples were taken at 0, 1, 3, 7, 14, and 21 days of stress treatment, and the growth status was observed to determine physiological indicators.

3. Experimental results

The growth state observation results show:

Before the treatments of salt stress and drought stress, the growth state of transgenic rape Bn-AcDwEm was no different from that of wild type rape, and the agronomic characters were not affected.

Under 15% severe drought stress for 7 days, the wild type rapeseed began to wilt and yellow leaves, and the growth rate of transgenic rapeseed Bn-AcDwEm slowed down, but the growth of leaves and stems were not significantly affected;

After 14 days of drought treatment, the wild-type rapeseed was completely dead, and the transgenic rapeseed began to wilt.

In the high-salt stress experiment, 300mM NaCl stress for 7 days, the wild-type rapeseed was severely dehydrated and withered, and some leaves of the transgenic Bn-AcDwEm turned yellow, and the growth condition was significantly better than that of the wild-type;

After 14 days of high-salt treatment, the wild-type rape had basically dried up and died, and the transgenic rape Bn-AcDwEm was still alive, only the leaves were curled, the stems were wilted, and the growth was slowed down.

Fourth, the experimental conclusion

The reverse functional circuit AcDwEm is expressed in the model plant rape, which significantly improves the salt tolerance and drought resistance of the host plant, and has great potential for breeding applications

Example 4 The acquisition of Agrobacterium-mediated anti-stress function line AcDwEm rice

1. Experimental materials

Recombinant strain EHA-AcDwEm: obtained in Example 1

Rice material: Rice seeds ZH11 are preserved in this laboratory.

2. Experimental method

The rice seeds were peeled, sterilized by soaking in 75% ethanol and 0.1% HgCl2, evenly placed in plant tissue culture medium, and cultured in a tissue culture room at 24°C for 2 weeks. The rice callus was excised with sterile surgery, placed on pre-medium, and cultivated in the dark for 2 weeks.

The recombinant Agrobacterium strain EHA-AcDwEm expressing the anti-stress circuit was transferred and activated, and the strain was collected by centrifugation and resuspended to OD600=1.0. The pre-cultured explants were soaked in Agrobacterium solution for 30 minutes, and then transferred to the co-culture medium after drying, and cultured in the dark for 2-3 days. It was then transferred to induction medium for cultivation.

The selected callus was transferred to the screening medium supplemented with antibiotics, cultured in the dark for 2 weeks, and re-screened once and cultivated in the dark for 2 weeks. , in the differentiation culture for 1 week. Transfer the differentiated and budded callus to rooting medium, and transfer to the greenhouse when the roots appear and the stem grows 4-5 cm, and the positive rice seedlings are detected by PCR.

3. Experimental results

The anti-stress function line AcDwEm was transformed into rice by Agrobacterium-mediated callus co-culture. After infecting the rice callus, induction culture, resistance screening culture, rooting culture and seedling transplantation were verified. Finally, the transgenic rice Os-AcDwEm expressing the stress-resistant functional circuit was obtained, which could be used for subsequent studies on stress-resistant performance.

Fourth, the experimental conclusion

Through the Agrobacterium-mediated transformation method, we finally obtained the anti-stress function line AcDwEm rice Os-AcDwEm

Example 5 Stress resistance analysis of AcDwEm rice with a functional line of resistance to stress

1. Experimental materials

Transgenic rice: Os-AcDwEm

Control: Non-GMO Rice

2. Experimental method

Analysis of high temperature resistance of rice Os-AcDwEm of AcDwEm rice with anti-stress function

Seeds of wild-type rice and positive transgenic rice were germinated and treated with high temperature.

Rice seeds were grown in MS medium to emerge. When the rice seedlings grow to 2 leaves and 1 heart, stress treatment is carried out for about 12 days. The stress culture environment is set, 45 °C light for 14 hours, 45 °C dark conditions for 10 hours, and the treatment is carried out for 7 days, and the growth state of the plants is observed.

3. Experimental results

The observation results of the growth state show that,

In the absence of high temperature stress, the emergence and growth of transgenic rice Os-AcDwEm were not different from those of wild type rice.

Under high temperature stress treatment for 7 days, the leaves of wild-type rice were yellow and curled, and the stems were wilted and withered. The growth of transgenic rice Os-AcDwEm plants was hardly affected.

Fourth, the experimental conclusion

The inverse functional circuit AcDwEm significantly improves the high temperature resistance of the host rice, and has great potential for breeding applications.

Claims

The application of the gene of the nucleotide sequence shown in SEQ ID NO: 1 in improving the biological stress resistance function.
The application according to claim 1 is the application in improving the stress resistance of cells during the selection and breeding of crop varieties.
The application according to claim 1 or 2, wherein the stress resistance is to improve the cell's ability to resist drought, high salt and high temperature.
The application of the plasmid containing the anti-stress functional system of the sequence shown in SEQ ID NO: 1 in enhancing the biological anti-stress function.