WO2016004688A1 - 基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法 - Google Patents

基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法 Download PDF

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WO2016004688A1
WO2016004688A1 PCT/CN2014/088253 CN2014088253W WO2016004688A1 WO 2016004688 A1 WO2016004688 A1 WO 2016004688A1 CN 2014088253 W CN2014088253 W CN 2014088253W WO 2016004688 A1 WO2016004688 A1 WO 2016004688A1
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spirulina
solution
adjuvant
preparation
transpiration
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PCT/CN2014/088253
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王根轩
李岩
王智威
何令超
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浙江大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom

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  • the invention belongs to the field of biology, and particularly relates to a preparation method of a transpiration inhibitor based on spirulina-induced stomatal immune closure.
  • plant transpiration inhibitors on the market can be divided into four categories: one is Film-forming antitranspirant, and the common film-forming agents are Wilt-Pruf and Vapor Guard.
  • Film-forming antitranspirant and the common film-forming agents are Wilt-Pruf and Vapor Guard.
  • Mobileaf, Folicote, Plant guard, etc. are mostly expensive patented products abroad, which are difficult to be naturally degraded and have long-lasting pollution.
  • 2Reflecting antitranspirant commonly used are: Kaoline and Kaolinite. These preparations are used in large amounts, have poor effects, and have side effects of directly reducing photosynthetic rate. Less practical applications.
  • 3 Metabolic antitransferants commonly used are PMA (phenylmercuric acetate), ABA, NaHSO 3 , CaCl 2 , CCC, 2,4-D, atrazine, alachlor, triadimefon, Fulvic acid (FA), which has serious environmental toxicity (such as phenylmercuric acetate) and physiological toxicity except for fulvic acid, which is harmful to the environment and human health, and growth regulators such as ABA are not only environmentally toxic but also expensive. It is difficult to promote use.
  • biological induction anti-transpiration agent biological induction antitranspirant
  • biological induction antitranspirant is a bio-type plant anti-transpiration agent based on stomatal-induced microorganisms invented by Wang Genxuan et al., but since microorganisms may produce substances that have side effects on plants, Further improvement can be promoted by practical application. Therefore, under the conditions of increasingly serious water crisis and frequent droughts, it is urgent to further research and develop new high-efficiency and non-toxic bio-sensing anti-transpiration preparations and water-saving technologies.
  • the object of the present invention is to provide a preparation method of a transpiration inhibitor based on spirulina-induced stomatal immune closure in view of the deficiencies of the prior art.
  • the method of the invention comprises the following steps:
  • Spirulina platensis was inoculated into the sterilized liquid spirulina culture medium and cultured in a light incubator.
  • the culture temperature was controlled at 25-30 ° C, and 8-10 photoperiods were cultured for one photoperiod.
  • the light intensity of 2500 ⁇ 3500 lx was firstly used for 12h, and then placed in the dark for 10h to obtain the cultured spirulina solution; then the cultured spirulina solution was centrifuged in a centrifuge at a speed of 2000.
  • the freshwater spirulina (Spirulina platensis) was purchased from the Freshwater Algae Species Bank of the Chinese Academy of Sciences, and the deposit number is FACHB-439;
  • the liquid spirulina medium is a mixture of solution I, solution II and vitamin B 12 ; wherein the volume ratio of solution I to solution II is 1:1, and the concentration of vitamin B 12 is 5 ⁇ 10 -6 g/L
  • Solution I was prepared from sodium bicarbonate 13.61g/500ml, sodium carbonate 4.03g/500ml, dipotassium hydrogen phosphate 0.5g/500ml
  • solution II was prepared from sodium nitrate 2.50g/500ml, sodium chloride 1.00g/500ml, potassium sulfate 1.00g/500ml, magnesium sulfate 0.20g/500ml, calcium chloride 0.04g/500ml, ferrous sulfate 0.01g/500ml, ethylenediaminetetraacetic acid 0.08g/500ml;
  • Step (1) spirulina A is added to step (2) adjuvant B, and stirred and mixed; wherein the volume ratio of step (1) spirulina A to step (2) adjuvant B is 0.03 to 0.06 :1.
  • the present invention utilizes the principle that microorganisms induce immune closure. Specifically, the phenomenon that the plant stomatal sensing microorganisms can be actively closed so that they cannot enter the plant through the pores and cause disease is called stomatal immunity.
  • the mechanism is that specific receptors on plant stomatal guard cells can recognize microbes or related molecular patterns derived from microorganisms, triggering a series of signal cascades that mediate stomatal closure, including kinase activation, second messenger production, calcium Ion oscillation and improvement, egg White modification, cytoskeletal reorganization, elevation of cytoplasmic matrix pH, regulation of ions and water channels.
  • plant stomata play a vital role in controlling plant transpiration and photosynthesis.
  • the transpiration rate usually decreases faster than the photosynthetic rate, so the proper opening of the stomata is appropriately reduced.
  • Degree can improve the instantaneous water use efficiency of plants. This provides a theoretical basis for the development of novel stomatal-inducing microorganisms that immuno-closed transpiration inhibiting water-saving agents.
  • the transpiration and water-saving preparation based on the stomatal induction microbial immune closure mechanism has the advantages of high efficiency, water saving, safety, environmental protection, low cost and large-scale production compared with various existing transpiration inhibitors.
  • Spirulina is a photosynthetic autotrophic microalgae, which not only has no carbon emissions, but also forms a new carbon sink, and it has high nutritional value and pharmacological effects.
  • Step (1) Preparation of spirulina A by:
  • Spirulina platensis was inoculated into the sterilized liquid spirulina medium and cultured in a light incubator.
  • the light intensity was set to 3500 lx
  • the photoperiod was 12 h light/12 h dark
  • the culture temperature was controlled at 25. °C, culture for 8 days, to obtain spirulina solution after cultivation;
  • the cultured spirulina solution was then centrifuged in a centrifuge at 2000 rpm, centrifuged, washed and dried, followed by preparation of a pure spirulina solution having a concentration of 15 g/L.
  • the prepared pure spirulina solution is inactivated at 121 ° C, 0.15 MPa pressure in an autoclave for 70 minutes, after cooling, frozen at -20 ° C, to make spirulina A;
  • Step (3) Prepare a transpiration inhibitor by adding 0.03 L of step (1) spirulina A to 1 L of step (2) adjuvant B, and stirring and mixing.
  • Step (1) Preparation of spirulina A by:
  • Spirulina platensis was inoculated into the sterilized liquid spirulina culture medium and cultured in a light incubator.
  • the light intensity was set to 3000 lx
  • the photoperiod was 12 h light/12 h dark
  • the culture temperature was controlled at 28. °C, culture for 9 days;
  • the cultured spirulina solution was then centrifuged in a centrifuge at a speed of 2200 rpm, centrifuged, washed and dried, followed by preparation of a 13 g/L pure spirulina solution.
  • the prepared pure spirulina solution is inactivated at 121 ° C, 0.13 MPa pressure in an autoclave for 80 minutes, after cooling, frozen at -20 ° C, to prepare spirulina A;
  • Step (3) Prepare a transpiration inhibitor by adding 0.04 L of step (1) spirulina A to 1 L of step (2) adjuvant B, and stirring and mixing.
  • Step (1) Preparation of spirulina A by:
  • Spirulina platensis was inoculated into the sterilized liquid spirulina culture medium and cultured in a light incubator.
  • the light intensity was set to 2500 lx
  • the photoperiod was 12 h light/12 h dark
  • the culture temperature was controlled at 30. °C, culture for 10 days;
  • the cultured spirulina solution was then centrifuged in a centrifuge at a speed of 2500 rpm, centrifuged, washed and dried, followed by preparation of a pure spirulina solution having a concentration of 10 g/L.
  • the prepared pure spirulina solution is inactivated at 121 ° C, 0.12 MPa pressure in an autoclave for 90 minutes, after cooling, frozen at -20 ° C, to prepare spirulina A;
  • Step (3) Prepare a transpiration inhibitor by adding 0.06 L of step (1) spirulina A to 1 L of step (2) adjuvant B, and stirring and mixing.
  • sterile water (control) and anti-transpiration agents formulated according to the three example methods were sprayed onto the surface of the leaves of the plants.
  • the anti-transpiration agent was prepared according to the above three examples, and then the anti-transpiration agent prepared by the sterile water and the three examples was sprayed onto the living broad bean respectively. After 24 hours, the width of the 50 pores was randomly measured under the microscope. The length, while using the Li-6400 photosynthetic apparatus to measure the stomatal conductance, photosynthetic rate, transpiration rate, and the ratio of photosynthetic rate and transpiration rate to calculate the water use efficiency, the results are expressed by the mean and standard error.
  • the transpiration inhibitors prepared by the three examples can significantly reduce the stomatal opening (porosity/stomach length) of the broad bean, the stomatal conductance and improve the water use efficiency of the broad bean leaves, and achieve a good inhibition of plant transpiration. Effect.
  • the freshwater spirulina (Spirulina platensis) used in the above examples was purchased from the Freshwater Algae Species Bank of the Chinese Academy of Sciences' Typical Culture Collection Committee under the accession number FACHB-439;
  • the liquid spirulina medium used is a mixture of solution I, solution II, vitamin B 12 ; wherein the volume ratio of solution I to solution II is 1:1, and the concentration of vitamin B 12 is 5 ⁇ 10 -6 g/L;
  • Solution I was prepared from sodium bicarbonate 13.61g/500ml, sodium carbonate 4.03g/500ml, dipotassium hydrogen phosphate 0.5g/500ml;
  • solution II was prepared from sodium nitrate 2.50g/500ml, sodium chloride 1.00g/500ml, potassium sulfate 1.00 g/500 ml, magnesium sulfate 0.20 g/500 ml, calcium chloride 0.04 g/500 ml, ferrous sulfate 0.01 g/500 ml, and ethylenediaminetetraacetic acid 0.08 g/500 ml were prepared.

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Abstract

本发明公开基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法。首先将淡水螺旋藻接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，然后置于离心机中离心，稀释得到纯螺旋藻溶液；置于121℃、0.12～0.15Mpa压力的高压灭菌锅中灭活制成螺旋藻剂A；将CaCl2·2H2O和蔗糖溶解于蒸馏水中，搅拌均匀，得到辅剂B；将螺旋藻剂A加入到辅剂B中，搅拌混匀即可。本发明具有可再生性、低成本性（螺旋藻为光合自养生产的微生物）、环境友好性和高效性等优点，属于新一代微生物诱导性型蒸腾抑制节水剂，具有强大的应用前景和潜在市场竞争力。

Description

基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法 技术领域
本发明属于生物领域，具体涉及一种基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法。
背景技术
随着全球气候变化和人口剧增，淡水资源的缺乏已成为全球面临的重大挑战。农业用水所占比重一直是比较大的。因此，开发新型植物蒸腾抑制节水剂，有效控制气孔的开度，降低叶片的过度蒸腾，提高水分利用效率，是解决中国水资源危机和发展节水农业的有效途径。根据其不同的作用方式和特点，目前市场上的植物蒸腾抑制剂可分为4类：①成膜型抗蒸腾剂(Film-forming antitranspirant)，常见的成膜型药剂有Wilt-Pruf、Vapor Guard、Mobileaf、Folicote、Plant guard等，大都是国外昂贵的专利产品，很难自然降解，具有持久污染。②反射型抗蒸腾剂(Reflecting antitranspirant)，常用的有:高岭土(Kaoline)和高岭石(Kaolinite)，此类制剂的使用量大、效果差并且具有直接降低光合速率的副作用等，目前也较少实际应用。③代谢型抗蒸腾剂(Metabolic antitranspirant)，常用的有PMA(醋酸苯汞)、ABA、NaHSO3、CaCl2、CCC、2,4-D、阿特拉津、甲草胺、三唑酮、黄腐酸(FA)，其中除黄腐酸外大都具有严重的环境毒性(如醋酸苯汞)和生理毒性，危害环境和人类健康，同时ABA等生长调节剂不仅具有环境毒性而且价格昂贵，因此很难推广使用。④生物感应型抗蒸腾剂(biological induction antitranspirant)，它是王根轩等人发明的一种基于气孔感应微生物的生物型植物抗蒸腾剂，但由于微生物可能产生一些对植物有副作用的物质，那还需要进一步的完善才能被实际应用推广。因此在水资源危机日益严重和旱灾频发的条件下，迫切需要进一步地研究开发新型高效无毒的生物感应型抗蒸腾制剂和节水技术。
发明内容
本发明的目的是针对现有技术的不足，提供了一种基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法。
本发明方法包括以下步骤：
步骤(1).制备螺旋藻剂A：
首先将淡水螺旋藻(Spirulina platensis)接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，培养温度控制在25～30℃，培养8～10个光照周期，一个光照周期为24小时，首先采用光强为2500～3500lx的光光照12h，再置于黑暗中10h，得到培养后的螺旋藻液；然后将培养后的螺旋藻液置于离心机中离心，转速为2000～2500转/分，离心后取沉淀洗涤并干燥，然后用蒸馏水进行稀释，得到浓度为10～15g/L的纯螺旋藻溶液；将配制好的纯螺旋藻液置于121℃、0.12～0.15Mpa压力的高压灭菌锅中灭活70～90分钟，待冷却后，于-20℃冻存，制成螺旋藻剂A；
所述的淡水螺旋藻(Spirulina platensis)购买于中国科学院典型培养物保藏委员会淡水藻种库，保藏编号为FACHB-439；
所述的液体螺旋藻培养基为溶液Ⅰ、溶液Ⅱ、维生素B12的混合液；其中溶液Ⅰ与溶液Ⅱ的体积比为1:1，维生素B12的浓度为5×10-6g/L；溶液Ⅰ由碳酸氢钠13.61g/500ml、碳酸钠4.03g/500ml、磷酸氢二钾0.5g/500ml配制得到；溶液Ⅱ由硝酸钠2.50g/500ml、氯化钠1.00g/500ml、硫酸钾1.00g/500ml、硫酸镁0.20g/500ml、氯化钙0.04g/500ml、硫酸亚铁0.01g/500ml、乙二胺四乙酸0.08g/500ml配制得到；
步骤(2).制备辅剂B：
将CaCl2·2H2O和蔗糖溶解于蒸馏水中，搅拌均匀，得到辅剂B；辅剂B中CaCl2·2H2O的浓度为2g/L，蔗糖的浓度为0.4g/L；
步骤(3).制备蒸腾抑制剂：
将步骤(1)螺旋藻剂A加入到步骤(2)辅剂B中，搅拌混匀即可；其中步骤(1)螺旋藻剂A与步骤(2)辅剂B的体积比为0.03～0.06：1。
本发明利用了微生物诱导免疫闭合的原理。具体而言，植物气孔感应微生物可主动的关闭，使其不能通过气孔进入植物体内而致病的现象称为气孔免疫。其机制是植物气孔保卫细胞上的特异性受体能够识别微生物或源于微生物的相关分子模式，引发一系列介导气孔关闭的信号级联反应，包括激酶的激活，第二信使的产生，钙离子的振荡和提高，蛋 白的修饰，细胞骨架的重组，细胞质基质PH的升高，离子和水通道的调节。在自然界中，植物气孔在控制植物的蒸腾和光合作用起着至关重要的作用，随着气孔开度在一定阈值内减少，蒸腾速率通常比光合速率减少的快，所以适当的减少气孔的开度，可以提高植物的瞬时水分利用效率。这为开发新型的气孔感应微生物而免疫闭合的蒸腾抑制节水剂提供理论基础。通过研究发现，喷施一定浓度的螺旋藻到植物的叶片上可以引起植物气孔开度(气孔开度/气孔长度)和气孔导度的大幅度下降，进而增大了气孔阻力，减少了水分的蒸腾散失，且在一定时期内提高了植物的水分利用效率。
本发明具有的有益效果是：
1.基于气孔感应微生物免疫闭合机理的抑制蒸腾节水制剂，比各类已有的蒸腾抑制剂更具有高效节水、安全环保、低成本和适应大规模生产的优势。
2.螺旋藻为光合自养微藻，不仅没有碳排放，还可形成新的碳汇，同时它具有很高的营养价值和药理效应。
3.节水效果显著，适用范围广，是干旱和半干旱地区理想的植物蒸腾抑制节水剂。
具体实施方式
下面结合具体实施例对本发明做进一步的分析。
实施例1.
步骤(1).螺旋藻剂A的制备，方法是：
首先将淡水螺旋藻(Spirulina platensis)接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，光强设置为3500lx，光照周期为12h光照/12h黑暗，培养温度控制在25℃，培养8天，得到培养后的螺旋藻液；
然后将培养后的螺旋藻液置于离心机中离心，转速2000转/分，离心后，洗涤并干燥，紧接着配制浓度为15g/L纯的螺旋藻溶液。将配制好的纯螺旋藻液放在121℃，0.15兆帕压力的高压灭菌锅中灭活70分钟，待冷却后，于-20℃冻存，制成螺旋藻剂A；
步骤(2).制备辅剂B，方法是：
将2gCaCl2·2H2O和0.4g蔗糖溶解于1L蒸馏水中，搅拌均匀， 得到辅剂B；
步骤(3).制备蒸腾抑制剂，方法是：将0.03L步骤(1)螺旋藻剂A加入到1L步骤(2)辅剂B中，搅拌混匀即可。
实施例2
步骤(1).螺旋藻剂A的制备，方法是：
首先将淡水螺旋藻(Spirulina platensis)接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，光强设置为3000lx，光照周期为12h光照/12h黑暗，培养温度控制在28℃，培养9天；
然后将培养好的螺旋藻液置于离心机中离心，转速2200转/分，离心后，洗涤并干燥，紧接着配制浓度为13g/L纯的螺旋藻溶液。将配制好的纯螺旋藻液放在121℃，0.13兆帕压力的高压灭菌锅中灭活80分钟，待冷却后，于-20℃冻存，制成螺旋藻剂A；
步骤(2).制备辅剂B，方法是：
将2gCaCl2·2H2O和0.4g蔗糖溶解于1L蒸馏水中，搅拌均匀，得到辅剂B；
步骤(3).制备蒸腾抑制剂，方法是：将0.04L步骤(1)螺旋藻剂A加入到1L步骤(2)辅剂B中，搅拌混匀即可。
实施例3
步骤(1).螺旋藻剂A的制备，方法是：
首先将淡水螺旋藻(Spirulina platensis)接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，光强设置为2500lx，光照周期为12h光照/12h黑暗，培养温度控制在30℃，培养10天；
然后将培养好的螺旋藻液置于离心机中离心，转速2500转/分，离心后，洗涤并干燥，紧接着配制浓度为10g/L纯的螺旋藻溶液。将配制好的纯螺旋藻液放在121℃，0.12兆帕压力的高压灭菌锅中灭活90分钟，待冷却后，于-20℃冻存，制成螺旋藻剂A；
步骤(2).制备辅剂B，方法是：
将2gCaCl2·2H2O和0.4g蔗糖溶解于1L蒸馏水中，搅拌均匀，得到辅剂B；
步骤(3).制备蒸腾抑制剂，方法是：将0.06L步骤(1)螺旋藻剂A加入到1L步骤(2)辅剂B中，搅拌混匀即可。
使用时，将无菌水(对照)和根据三种实施例方法配制的抗蒸腾剂喷施到植物的叶片表面。首先按上述3种实施例配制抗蒸腾剂，然后分别喷施无菌水和3种实施例配制的抗蒸腾剂到活体蚕豆上，24小时后，在显微镜下活体随机测量50个气孔的宽度和长度，同时用Li-6400光合仪测定叶片的气孔导度，光合速率，蒸腾速率，并用光合速率和蒸腾速率的比值来计算水分利用效率，结果用平均值和标准误来表示。如下表：
Figure PCTCN2014088253-appb-000001
由上表可知三个实施例方法配制的蒸腾抑制剂能够明显的降低蚕豆的气孔开度(气孔开度/气孔长度)，气孔导度和提高蚕豆叶片的水分利用效率，达到良好的抑制植物蒸腾的效果。
上述实施例使用的淡水螺旋藻(Spirulina platensis)购买于中国科学院典型培养物保藏委员会淡水藻种库，保藏编号为FACHB-439；
使用的液体螺旋藻培养基为溶液Ⅰ、溶液Ⅱ、维生素B12的混合液；其中溶液Ⅰ与溶液Ⅱ的体积比为1:1，维生素B12的浓度为5×10-6g/L；溶液Ⅰ由碳酸氢钠13.61g/500ml、碳酸钠4.03g/500ml、磷酸氢二钾0.5g/500ml配制得到；溶液Ⅱ由硝酸钠2.50g/500ml、氯化钠1.00g/500ml、硫酸钾1.00g/500ml、硫酸镁0.20g/500ml、氯化钙0.04g/500ml、硫酸亚铁0.01g/500ml、乙二胺四乙酸0.08g/500ml配制得到。

Claims (2)

  1. 基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法，其特征在于该方法包括以下步骤：
    步骤(1).制备螺旋藻剂A：
    首先将淡水螺旋藻Spirulina platensis接种到灭菌后的液体螺旋藻培养基中，置于光照培养箱中培养，培养温度控制在25～30℃，培养8～10个光照周期，一个光照周期为24小时，首先采用光强为2500～3500lx的光光照12h，再置于黑暗中10h，得到培养后的螺旋藻液；然后将培养后的螺旋藻液置于离心机中离心，转速为2000～2500转/分，离心后取沉淀洗涤并干燥，然后用蒸馏水进行稀释，得到浓度为10～15g/L的纯螺旋藻溶液；将配制好的纯螺旋藻液置于121℃、0.12～0.15Mpa压力的高压灭菌锅中灭活70～90分钟，待冷却后，于-20℃冻存，制成螺旋藻剂A；
    所述的淡水螺旋藻Spirulina platensis购买于中国科学院典型培养物保藏委员会淡水藻种库，保藏编号为FACHB-439；
    步骤(2).制备辅剂B：
    将CaCl2·2H2O和蔗糖溶解于蒸馏水中，搅拌均匀，得到辅剂B；辅剂B中CaCl2·2H2O的浓度为2g/L，蔗糖的浓度为0.4g/L；
    步骤(3).制备蒸腾抑制剂：
    将步骤(1)螺旋藻剂A加入到步骤(2)辅剂B中，搅拌混匀即可；其中步骤(1)螺旋藻剂A与步骤(2)辅剂B的体积比为0.03～0.06：1。
  2. 如权利要求1所述的基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法，其特征在于所述的液体螺旋藻培养基为溶液Ⅰ、溶液Ⅱ、维生素B12的混合液；其中溶液Ⅰ与溶液Ⅱ的体积比为1:1，维生素B12的浓度为5×10-6g/L；溶液Ⅰ由碳酸氢钠13.61g/500ml、碳酸钠4.03g/500ml、磷酸氢二钾0.5g/500ml配制得到；溶液Ⅱ由硝酸钠2.50g/500ml、氯化钠1.00g/500ml、硫酸钾1.00g/500ml、硫酸镁0.20g/500ml、氯化钙0.04g/500ml、硫酸亚铁0.01g/500ml、乙二胺四乙酸0.08g/500ml配制得到。
PCT/CN2014/088253 2014-07-10 2014-10-10 基于螺旋藻诱导气孔免疫闭合的蒸腾抑制剂的制备方法 WO2016004688A1 (zh)

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