WO2022082866A1 - 抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用 - Google Patents

抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用 Download PDF

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WO2022082866A1
WO2022082866A1 PCT/CN2020/126331 CN2020126331W WO2022082866A1 WO 2022082866 A1 WO2022082866 A1 WO 2022082866A1 CN 2020126331 W CN2020126331 W CN 2020126331W WO 2022082866 A1 WO2022082866 A1 WO 2022082866A1
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stress
acdwem
resistance
high temperature
rice
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谷晓峰
林敏�
王劲
周正富
燕永亮
左开井
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隆平生物技术(海南)有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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);
  • 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).
  • 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.
  • 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 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.
  • 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.
  • 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);
  • 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;
  • 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
  • Cloning vector pJET a commercially available product from ThermoFisher;
  • Agrobacterium tumefaciens EHA105 preserved in this laboratory.
  • 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.
  • 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.
  • 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.
  • Brassica napus material Rapeseed 84100-18 is preserved in this laboratory.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • transgenic rape Bn-AcDwEm was no different from that of wild type rape, and the agronomic characters were not affected.
  • 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.
  • 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
  • Rice material Rice seeds ZH11 are preserved in this laboratory.
  • 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 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.
  • 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.
  • 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.
  • transgenic rice Os-AcDwEm were not different from those of wild type rice.
  • the inverse functional circuit AcDwEm significantly improves the high temperature resistance of the host rice, and has great potential for breeding applications.

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Abstract

提供了一种具有提高宿主细胞抵抗高盐、干旱、高温胁迫能力的抗逆功能线路AcDwEm，其核苷酸序列如SEQ ID NO:1所示。构建了包含该抗逆功能线路AcDwEm的重组载体，并通过农杆菌介导侵染转化的方法将其在模式植物油菜和水稻中整合重建，所述抗逆功能线路AcDwEm在模式植物宿主细胞中表达后，能显著增强作物的耐高盐、抗干旱和抗高温的能力，可用于农作物新品种抗逆性改良。

Description

抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用 技术领域
本发明属于合成生物学领域，涉及一种多模块抗逆功能线路具有提高生物抵抗干旱和高盐胁迫能力的应用。
背景技术
土壤盐碱化、频繁干旱和长时期高温是全球农业最具破坏性的非生物胁迫，通过对种子萌发、植物生长发育、植物活力和作物产量的不利影响大幅降低农业生产力。
目前，在全世界范围内越来越广泛应用基因工程策略培育抗逆品种。但由于作物耐盐抗旱耐高温性是一个复杂的性状，同时受到多个基因和因素的影响，因此单基因转化操作并不理想，培育的耐逆性提高植物在无压力条件下表现不佳。
进入新世纪以来，新一代合成生物学的原始创新与集成应用加快突破，全基因组设计育种技术促进传统农业品种升级换代，孕育新一轮农业科技革命和产业变革。因此，运用现代合成生物学设计方法，通过人工设计蛋白质功能元件、启动子，并通过多个基因组合方式，人工构建特异性响应高盐胁迫信号、干旱信号和高温胁迫信号的应答功能模块，或有望能够创建出提高生物抵抗干旱和高盐胁迫的能力的抗逆功能体系。
发明内容
本发明的目的是创建一种能够提高生物抵抗干旱和高盐胁迫的能力的抗逆功能线路。
本发明利用现代合成生物学设计方法，优化改造抗逆元件。通过蛋白质功能元件的人工设计、启动子的组织特异性和逆境响应设计，人工构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。
通过如下研究，首次鉴定了抗逆功能线路AcDwEm具有提高模式植物抗旱耐盐耐高温能力，可用于新一代抗逆作物新品种的培育。具体研究工作如下：
1、人工设计抗逆功能线路AcDwEm的构建
通过合成生物学设计逆境胁迫应答功能模块，设计构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。利用人工化学合成的方法获得了抗逆功 能线路AcDwEm全长核酸序列。将抗逆线路AcDwEm连接于pBI-121载体上，构建植物表达载体pBI-AcDwEm，将该表达载体转化根癌农杆菌EHA105(详见实施例1)；
2、转抗逆功能线路AcDwEm油菜与水稻的获得
通过农杆菌介导的转基因植物构建方法，将抗逆功能线路AcDwEm与模式植物油菜和水稻整合重组，通过抗性筛选和PCR验证的方法，培养得到稳定遗传的阳性转基因植株(详见实施例2,4)。
3、转抗逆功能线路AcDwEm油菜的耐盐抗旱性能分析
分别以NaCl和聚乙二醇PEG-6000作为添加物质来模拟盐胁迫和干旱胁迫，采取浇灌的方式进行胁迫处理。将获得的已鉴定为阳性的转基因种子与野生型种子培养出苗，进行逆境处理。每天为植株浇灌等量的胁迫液，分别在胁迫处理的0,1,3,7,14,21d取样拍照，观测生长状态测定生理指标。
4、转抗逆功能线路AcDwEm水稻的耐高温性能分析
将野生型水稻与阳性转基因水稻种子萌发出苗，进行高温处理。培养环境设置，45℃光照14小时，45℃黑暗条件10小时，处理7天，观测植株生长状态。
实验结果表明：正常条件下，抗逆功能线路AcDwEm对宿主植株生长发育无影响，逆境条件下具有显著提高油菜与水稻耐盐抗旱耐高温能力的功能，可用于新一代抗逆作物新品种的培育
序列表信息
SEQ ID NO.1：抗逆功能线路AcDwEm的核苷酸序列。
SEQ ID NO.2：功能模块1的核苷酸序列。
SEQ ID NO.3：功能模块1的编码蛋白的氨基酸序列。
SEQ ID NO.4：功能模块2的核苷酸序列。
SEQ ID NO.5：功能模块2的编码蛋白的氨基酸序列。
SEQ ID NO.6：功能模块3的核苷酸序列。
SEQ ID NO.7：功能模块3的编码蛋白的氨基酸序列。
附图说明：
图1抗逆线路AcDwEm载体构建图；
图2转基因油菜Bn-AcDwEm和非转基因油菜(WT)耐盐抗旱实验结果比较；
图3转基因水稻Os-AcDwEm和非转基因野生型水稻耐高温实验结果比较。
具体实施方式
以下实施例中所举的质粒、菌株、模式植物只用于对本发明作进一步详细说明，并不 对本发明的实质内容加以限制。凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件或按照制造厂商所建议的条件。实施例中所举的质粒、菌株、植株来源如下：
克隆载体pJET：为ThermoFisher公司市售产品；
穿梭载体：pBI-121：本实验室保存；
根癌农杆菌EHA105：本实验室保存；
水稻材料：水稻种子ZH11为本实验室保存。
甘蓝型油菜材料：油菜种子84100-18为本实验室保存。
实施例1 抗逆功能线路AcDwEm的设计与重组根癌农杆菌的构建
一、实验材料
克隆载体pJET：为ThermoFisher公司市售产品；
穿梭载体：pBI-121：本实验室保存；
根癌农杆菌EHA105：本实验室保存。
二、实验方法
1.通过合成生物学设计逆境胁迫应答功能模块，设计构建特异性响应高温胁迫信号的应答功能模块、抗逆功能稳定器模块和组织特异性高效抗逆功能模块，组装形成智能响应定向表达的全新抗逆功能线路，命名为AcDwEm。利用人工化学合成的方法获得了抗逆功能线路AcDwEm全长核酸序列。其大小为3737bp，将其克隆于载体pJET上，构建了含有完整抗逆功能线路的重组克隆质粒pJET-AcDwEm，并测序验证；然后通过EcoRI和HindIII双酶切获得含有粘性末端的抗逆线路AcDwEm片段及穿梭载体pBI-121载体片段，将抗逆线路AcDwEm连接于pBI-121载体上，构建植物表达载体pBI-AcDwEm，将该表达载体转化根癌农杆菌EHA105，利用卡那霉素抗生素抗性筛选阳性重组菌株，并通过菌落PCR测序验证。
三、实验结果
利用人工化学合成的方法获得了抗逆功能线路AcDwEm全长核酸序列，成功构建将含有功能线路SyAcDwEm的植物表达载体pBI-AcDwEm，并转化根癌农杆菌EHA105。经PCR、酶切，测序验证***序列正确，将该菌株命名为EHA-AcDwEm。
四、实验结论
完成表达抗逆功能线路AcDwEm的重组根癌农杆菌EHA-AcDwEm的构建。
实施例2 农杆菌介导的转抗逆功能线路AcDwEm油菜的获得
一、实验材料
重组菌株EHA-AcDwEm：实施例1获得
甘蓝型油菜材料：油菜种子84100-18为本实验室保存。
二、实验方法
去油菜种子，分别用75％乙醇和0.1％的HgCl2浸泡消毒，均匀放置于植物组织培养基，24℃组织培养室培养一周。用消毒手术剪取油菜幼苗的下胚轴，置于预培养基上，光照培养2-3天，预培养外植体。
转接活化表达抗逆线路的重组农杆菌菌株EHA-AcDwEm，离心收集菌株重悬至OD600＝1.0。将预培养的外植体浸泡于农杆菌菌液中90s，晾干后转移至共培养基上，暗培养2-3d。随后将生长良好的外植体转移至诱导培养基上培养。
选取愈伤组织长势良好的外植体转移到添加抗生物的筛选培养基上，光照培养45-50d，在分化出芽。将分化出芽的愈伤组织转移到生根培养基，光照培养2周，待根系出现茎干长出4-5cm，转移至培养土中进行练苗，经驯化后移栽至温室，PCR检测阳性油菜苗。
三、实验结果
利用农杆菌介导的外植体共培养法，将抗逆功能线路AcDwEm转化油菜，经过侵染油菜外植体经过诱导培养、筛选培养、生根培养与练苗移植等步骤，经过PCR验证，最终得到表达抗逆功能线路的转基因油菜Bn-AcDwEm，可用于后续抗逆性能研究。
四、实验结论
通过农杆菌介导转化方法，最终获得转抗逆功能线路AcDwEm油菜Bn-AcDwEm
实施例3 转抗逆功能线路AcDwEm油菜的抗逆性分析
一、实验材料
转基因油菜：Bn-AcDwEm
对照：非转基因野生型油菜
二、实验方法
分别以NaCl和聚乙二醇PEG-6000作为添加物质来模拟盐胁迫和干旱胁迫，采取浇灌的方式进行胁迫处理。
将获得的已鉴定为阳性的转基因油菜种子与野生型种子在MS固体培养中，待苗长出真叶后移栽到装有基质的塑料盆中，浇灌MS营养液待幼苗长出5-6片真叶进行逆境处理。
每天为植株浇灌等量的胁迫液，分别在胁迫处理的0,1,3,7,14,21d取样拍照，观测生长状态测定生理指标。
三、实验结果
生长状态观测结果显示：
盐胁迫和干旱胁迫处理前，转基因油菜Bn-AcDwEm与野生型油菜生长状态无差异，农艺性状未受影响。
15％重度干旱胁迫下7天时，野生型油菜开始枯黄落叶萎蔫的表型，转基因油菜Bn-AcDwEm生长速率变慢，但叶片与茎干生长未受明显影响；
干旱处理14天时，野生型油菜已经完全枯死，转基因油菜开始出现萎蔫。
高盐胁迫实验中，300mM NaCl胁迫处理7天，野生型油菜出现严重失水干枯的情况，转基因油菜Bn-AcDwEm部分叶片泛黄，生长状况显著好于野生型；
高盐处理14天，野生型油菜已经基本干枯死亡，转基因油菜Bn-AcDwEm仍存活，仅叶片出现卷曲，茎干萎蔫，生长变缓。
四、实验结论
逆功能线路AcDwEm在模式植物油菜中表达，显著提高了宿主植物耐盐性能和抗旱性能，具有重大育种应用潜力
实施例4 农杆菌介导的转抗逆功能线路AcDwEm水稻的获得
一、实验材料
重组菌株EHA-AcDwEm：实施例1获得
水稻材料：水稻种子ZH11为本实验室保存。
二、实验方法
水稻种子去皮，用75％乙醇和0.1％的HgCl2浸泡消毒，均匀放置于植物组织培养基，24℃组织培养室培养2周。用消毒手术剪取水稻愈伤组织，置于预培养基上，黑暗培养2周。
转接活化表达抗逆线路的重组农杆菌菌株EHA-AcDwEm，离心收集菌株重悬至OD600＝1.0。将预培养的外植体浸泡于农杆菌菌液中30分钟，晾干后转移至共培养基上，暗培养2-3d。随后转移至诱导培养基上培养。
选取愈伤组织转移到添加抗生物的筛选培养基上，暗培养2周，复筛一次暗培养2周。，在分化培养1周。将分化出芽的愈伤组织转移到生根培养基，待根系出现茎干长出4-5cm，转移至至温室，PCR检测阳性水稻苗。
三、实验结果
通过农杆菌介导愈伤组织共培养法，将抗逆功能线路AcDwEm转化水稻，经过侵染水稻愈伤组织经过诱导培养、抗性筛选培养、生根培养与建苗移植等步骤，经过PCR验证，最终得到表达抗逆功能线路的转基因水稻Os-AcDwEm，可用于后续抗逆性能研究。
四、实验结论
通过农杆菌介导转化方法，最终获得转抗逆功能线路AcDwEm水稻Os-AcDwEm
实施例5 转抗逆功能线路AcDwEm水稻的抗逆性分析
一、实验材料
转基因水稻：Os-AcDwEm
对照：非转基因水稻
二、实验方法
转抗逆功能线路AcDwEm水稻Os-AcDwEm耐高温性能分析
将野生型水稻与阳性转基因水稻种子萌发出苗，进行高温处理。
将水稻种子在MS培养基中培养出苗。当水稻苗长到2叶1心时，大约12天左右进行胁迫处理，胁迫培养环境设置，45℃光照14小时，45℃黑暗条件10小时，处理7天，观测植株生长状态。
三、实验结果
生长状态观测结果显示，
无高温胁迫条件下，转基因水稻Os-AcDwEm出苗和生长与水稻野生型无差异。
高温胁迫处理7天，野生型水稻叶面枯黄卷曲，茎干萎蔫干枯，转基因水稻Os-AcDwEm植株，生长几乎未受到影响。
四、实验结论
逆功能线路AcDwEm显著提高了宿主水稻的耐高温性能，具有重大育种应用潜力。

Claims (4)

  1. SEQ ID NO:1所示核苷酸序列的基因在提高生物抗逆功能中的应用。
  2. 权利要求1所述的应用，是在农作物品种选育时，提高细胞抗逆能力中的应用。
  3. 权利要求1或2所述的应用，所述抗逆，是提高细胞抗干旱，耐高盐和耐高温能力。
  4. 含有SEQ ID NO:1所示序列的抗逆功能体系的质粒在增强生物抗逆功能上的应用。
PCT/CN2020/126331 2020-10-20 2020-11-04 抗逆基因线路AcDwEm及其提高作物耐盐抗旱耐高温的应用 WO2022082866A1 (zh)

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