WO2022082864A1 - 智能应答胁迫信号的高效抗逆模块SyDcw及其在作物育种中的应用 - Google Patents

智能应答胁迫信号的高效抗逆模块SyDcw及其在作物育种中的应用 Download PDF

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WO2022082864A1
WO2022082864A1 PCT/CN2020/126327 CN2020126327W WO2022082864A1 WO 2022082864 A1 WO2022082864 A1 WO 2022082864A1 CN 2020126327 W CN2020126327 W CN 2020126327W WO 2022082864 A1 WO2022082864 A1 WO 2022082864A1
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stress
sydcw
functional module
drought
resistance
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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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    • 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 an artificial intelligence high-efficiency stress-resistant module for responding to stress signals in cultivating varieties with improved biological resistance to drought and high-salt stress.
  • the purpose of the present invention is to create an anti-stress functional module 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.
  • tissue-specificity of promoters and the design of stress response a functional module with specific response to stress signals and tissue-specific and high-efficiency stress resistance was artificially constructed, named SyDcw.
  • the stress-resistant functional system SyDcw has the ability to improve the drought and salt tolerance of model plants, and can be used for the cultivation of new varieties of new-generation stress-resistant crops.
  • the specific research work is as follows:
  • SyDcw Using synthetic biology to design stress response functional modules, through artificial optimization of protein functional elements, tissue specificity of promoters and stress response design, artificial construction of functional modules with specific responses to stress signals and tissue-specific and efficient stress resistance, Named SyDcw.
  • the full-length nucleic acid sequence of the anti-stress functional module SyDcw was obtained by artificial chemical synthesis.
  • the anti-stress module SyDcw was connected to the pBI-121 vector to construct a plant expression vector pBI-SyDcw, and the expression vector was transformed into Agrobacterium tumefaciens EHA105 (see Example 1 for details);
  • the stress-resistant functional system SyDcw is integrated and recombined with the model plant rape, and by the method of resistance screening and PCR verification, the positive transgenic plants with stable inheritance are obtained by culturing (see Example 2 for details).
  • 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 5-6 true leaves were grown for adversity treatment. Plants were irrigated with the same amount of stress solution every day, and samples were taken at 0, 1, 3, 7, and 14 days of stress treatment, and the growth state was observed to determine physiological indicators.
  • the experimental results show that: under normal conditions, the stress-resistant functional system SyDcw has no effect on the growth and development of host plants, and has the function of significantly improving the drought resistance and salt tolerance of model plants under stress conditions, which can be used for the cultivation of a new generation of stress-resistant crops.
  • SEQ ID NO.1 Nucleotide sequence of the anti-stress functional module SyDcw.
  • SEQ ID NO. 2 Amino acid sequence of functional module coding element.
  • FIG. 1 Construction diagram of Bn-SyDcw module carrier
  • the plasmids, strains, and model plants listed in the following examples are only used to further illustrate the present invention in detail, 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;
  • Brassica napus material Rapeseed 84100-18 is preserved in this laboratory.
  • Example 1 Design of anti-stress functional module SyDcw and construction of recombinant Agrobacterium tumefaciens
  • Cloning vector pJET a commercially available product from ThermoFisher;
  • Agrobacterium tumefaciens EHA105 preserved in this laboratory.
  • SyDcw The full-length nucleic acid sequence of the anti-stress functional system SyDcw was obtained by artificial chemical synthesis. Its size is 2828bp. It was cloned into the vector pJET, and the recombinant cloned plasmid pJET-SyDcw containing the complete anti-reverse functional module SyDcw was constructed and verified by sequencing; then the anti-reverse module containing sticky ends was obtained by double digestion with EcoRI and HindIII.
  • SyDcw fragment and shuttle vector pBI-121 vector fragment the anti-stress module SyDcw was connected to the pBI-121 vector, and the plant expression vector pBI-SyDcw was constructed.
  • the expression vector was transformed into Agrobacterium tumefaciens EHA105, and kanamycin antibiotic resistance was used. Positive recombinant strains were screened and verified by colony PCR sequencing.
  • the full-length nucleic acid sequence of the anti-stress functional module SyDcw was obtained by artificial chemical synthesis, and the plant expression vector pBI-SyDcw containing the functional module SyDcw was successfully constructed and transformed into Agrobacterium tumefaciens EHA105. After PCR, enzyme digestion, and sequencing, the inserted sequence was verified to be correct, and the strain was named EHA-SyDcw.
  • 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 module SyDcw was transformed into rapeseed by Agrobacterium-mediated co-cultivation of explants. 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-SyDcw expressing the anti-stress functional module is obtained, which can be used for the subsequent research on the anti-stress performance.
  • Example 3 Stress resistance analysis of rapeseed by transferring the anti-stress functional module SyDcw
  • 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, and 14 days of stress treatment, and the growth state was observed to determine physiological indicators.
  • transgenic rape Bn-SyDcw and wild-type rape was not different, and the agronomic characters were not affected.
  • the reverse functional module SyDcw 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

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Abstract

提供了一种具有提高宿主细胞抵抗高盐、干旱胁迫能力的功能模块SyDcw。通过农杆菌介导侵染转化的方法将该抗逆功能模块的重组载体在模式植物油菜中整合重建。该功能模块在模式植物宿主细胞中表达后，能增强作物的耐高盐和抗干旱的能力，可用于农作物新品种抗逆性改良。

Description

智能应答胁迫信号的高效抗逆模块SyDcw及其在作物育种中的应用 技术领域
本发明属于合成生物学领域，涉及一种人工智能应答胁迫信号的高效抗逆模块在培育提高生物抵抗干旱和高盐胁迫能力的品种中的应用。
背景技术
据保守估计，我国主要农作物因干旱、盐碱等环境胁迫因素造成的减产每年高达总产的8-15％，严重的年份甚至可以导致作物绝收。由于作物耐盐抗旱性是一个复杂的性状，同时受到多个基因和因素的影响，传统单基因的基因工程育种策略并不理想，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。当前，以大数据、人工智能为代表的新兴科技交叉融合加速，以基因智能改造与定向表达为核心的新一代合成生物学工程技术进入一个日新月异、产业蓬勃发展的新阶段，在工农业生产、环境保护、健康保健等领域具有巨大应用前景。
发明内容
本发明的目的是创建一种抗逆功能模块能够提高生物抵抗干旱和高盐胁迫的能力。
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优化、启动子的组织特异性和逆境响应设计，人工构建具有特异性响应逆境胁迫信号并组织特异性高效抗逆的功能模块，命名为SyDcw。
通过如下研究，首次鉴定了抗逆功能体系SyDcw具有提高模式植物抗旱耐盐能力，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、人工设计抗逆功能体系SyDcw的构建
利用合成生物学设计逆境胁迫应答功能模块，通过蛋白质功能元件的人工优化、启动子的组织特异性和逆境响应设计，人工构建具有特异性响应逆境胁迫信号并组织特异性高效抗逆的功能模块，命名为SyDcw。利用人工化学合成的方法获得了抗逆功能模块SyDcw全长核酸序列。将抗逆模块SyDcw连接于pBI-121载体上，构建植物表达载体pBI-SyDcw，将该表达载体转化根癌农杆菌EHA105(详见实施例1)；
2、转抗逆功能模块SyDcw油菜的获得
通过农杆菌介导的转基因植物构建方法，将抗逆功能体系SyDcw与模式植物油菜整 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)。
3、转抗逆功能模块SyDcw油菜的耐盐性与抗旱性分析
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，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0,1,3,7,14d取样拍照，观测生长状态测定生理指标。
实验结果表明：正常条件下，抗逆功能体系SyDcw对宿主植株生长发育无影响，逆境条件下具有显著提高模式植物抗旱耐盐能力的功能，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：抗逆功能模块SyDcw的核苷酸序列。
SEQ ID NO.2：功能模块编码元件的氨基酸序列。
附图说明：
图1 Bn-SyDcw模块载体构建图；
图2转基因油菜Bn-SyDcw和非转基因油菜(WT)耐盐实验对比；
图3转基因油菜Bn-SyDcw和非转基因油菜(WT)抗旱实验对比。
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；
甘蓝型油菜材料：油菜种子84100-18为本实验室保存。
实施例1 抗逆功能模块SyDcw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
利用合成生物学设计逆境胁迫应答功能模块，通过蛋白质功能元件的人工优化、启动子的组织特异性和逆境响应设计，人工构建具有特异性响应逆境胁迫信号并组织特异性高效抗逆的功能模块，命名为SyDcw。利用人工化学合成的方法获得了抗逆功能体系SyDcw全长核酸序列。其大小为2828bp，将其克隆于载体pJET上，构建了含有完整抗逆功能模块SyDcw的重组克隆质粒pJET-SyDcw，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模块SyDcw片段及穿梭载体pBI-121载体片段，将抗逆模块SyDcw连接于pBI-121载体上，构建植物表达载体pBI-SyDcw，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模块SyDcw全长核酸序列，成功构建将含有功能模块SyDcw的植物表达载体pBI-SyDcw，并转化根癌农杆菌EHA105。经PCR、酶切，测序验证插入序列正确，将该菌株命名为EHA-SyDcw。
四、实验结论
完成表达抗逆功能模块SyDcw的重组根癌农杆菌EHA-SyDcw的构建。
实施例2 农杆菌介导的转抗逆功能模块SyDcw油菜的获得
一、实验材料
重组菌株EHA-SyDcw：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。
转接活化表达抗逆模块的重组农杆菌菌株EHA-SyDcw，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，将抗逆功能模块SyDcw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模块的转基因油菜Bn-SyDcw，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模块SyDcw的油菜Bn-SyDcw
实施例3 转抗逆功能模块SyDcw油菜的抗逆性分析
一、实验材料
转基因油菜：Bn-SyDcw
对照：非转基因油菜
二、实验方法
转抗逆功能模块油菜Bn-SyDcw的盐胁迫和干旱胁迫抗性实验
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0,1,3,7,14d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：
无胁迫的正常生长情况下，转基因油菜Bn-SyDcw与野生型油菜生长状态无差异，农艺性状未受影响。
1、高盐胁迫实验
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-SyDcw生长状况未受到影响，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-SyDcw叶片出现卷曲，茎干略有萎蔫生长变缓(图2)。
2、干旱胁迫实验
15％重度干旱胁迫下7天时，野生型油菜已经落叶，植株基本干枯萎蔫，转基因油菜Bn-SyDcw生长未受影响；
干旱处理14天时，野生型油菜已经干枯死亡，转基因油菜生长变缓，仅少量叶片出现卷曲(图3)。
四、实验结论
逆功能模块SyDcw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

Claims (6)

  1. SEQ ID NO:1所示核苷酸序列的功能模块提高细胞抗逆功能的应用。
  2. 含有SEQ ID NO:1所示功能模块的质粒提高细胞抗逆功能的应用。
  3. 权利要求1或2所述的应用，所述抗逆，是提高细胞对干旱、高盐胁迫的能力。
  4. 含有SEQ ID NO:1所示功能模块的重组工程菌株在提高生物抵抗干旱、高盐胁迫能力的应用。
  5. 权利要求1所述的功能模块编码的氨基酸序列，如SEQ ID NO:2所示。
  6. 核苷酸序列为SEQ ID NO:1所示的功能模块。
PCT/CN2020/126327 2020-10-20 2020-11-04 智能应答胁迫信号的高效抗逆模块SyDcw及其在作物育种中的应用 WO2022082864A1 (zh)

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