WO2023077376A1 - 高级脂肪醇在提高豆科植物固氮能力以及抗旱能力方面的应用 - Google Patents

高级脂肪醇在提高豆科植物固氮能力以及抗旱能力方面的应用 Download PDF

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WO2023077376A1
WO2023077376A1 PCT/CN2021/128777 CN2021128777W WO2023077376A1 WO 2023077376 A1 WO2023077376 A1 WO 2023077376A1 CN 2021128777 W CN2021128777 W CN 2021128777W WO 2023077376 A1 WO2023077376 A1 WO 2023077376A1
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legumes
higher fatty
content
increasing
application
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PCT/CN2021/128777
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English (en)
French (fr)
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蔡典贤
孙昀浩
彭小明
喻国辉
秦迪
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珠海市润农科技有限公司
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Priority to CN202180003269.3A priority Critical patent/CN116744783A/zh
Priority to PCT/CN2021/128777 priority patent/WO2023077376A1/zh
Publication of WO2023077376A1 publication Critical patent/WO2023077376A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/20—Cereals
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity

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  • the invention relates to the application of higher fatty alcohols in improving the nitrogen fixation ability and drought resistance ability of leguminous plants.
  • legumes are an important source of starch, protein, oil and vegetables in human food, and are also important feeding materials for animal husbandry and aquaculture.
  • peanut oil and soybean oil are important vegetable source edible oils
  • soybean meal is also an important feed protein source.
  • my country's edible oil and feed protein have long been dependent on imports. According to data from the General Administration of Customs, in 2016, my country's edible oil dependence on foreign countries reached 67.7%. In 2020, my country's soybean imports will reach 100.33 million tons, a year-on-year increase of 13.3%. The import dependence of edible oil and soybean is too high, and it is very easy to be restrained by foreign market conditions, which poses a threat to my country's grain and oil security.
  • the object of the present invention is to provide an application of a higher fatty alcohol in improving the nitrogen fixation ability and drought resistance ability of leguminous plants.
  • the invention provides an application of a higher fatty alcohol in preparing a preparation for increasing the content of lysophosphatidylcholine in leguminous plants.
  • the application of the higher fatty alcohol in increasing the number of root nodules is achieved by increasing the content of lysophosphatidylcholine in legumes.
  • higher fatty alcohols can be used to improve the nitrogen fixation capacity of legumes by increasing the number of root nodules of legumes.
  • the present invention provides an application of a higher fatty alcohol in the preparation of a preparation for increasing the content of isoflavones in leguminous plants, and the isoflavones are 3,9-dihydroxy pterostilbene.
  • the present invention provides an application of a higher fatty alcohol in the preparation of a preparation for increasing the content of isoflavones in leguminous plants, and the isoflavones are calycosin and daidzein.
  • the present invention provides the application of a higher fatty alcohol in the preparation of preparations for improving the germination rate and germination potential of peanuts and shortening the germination time.
  • the invention provides the application of a higher fatty alcohol in the preparation of a preparation for improving the drought resistance of leguminous plants.
  • the preparation containing higher fatty alcohol can improve the transcription level of genes related to phenylpropane metabolic pathway and increase the transcription level of genes related to isoflavone biosynthesis pathway. As well as increasing the content of isoflavones in leguminous plants, so as to realize the application of improving the drought resistance ability of legumes.
  • the higher fatty alcohol is one of lauryl alcohol and cetyl alcohol or a mixture of both.
  • the preparation is a water emulsion, which includes higher fatty alcohol, emulsifier, thickener, and water.
  • the preparation containing higher fatty alcohol of the present invention is not limited to emulsion in water, as long as it adopts all dosage forms of the inventive concept of the present invention, it is applicable, such as wettable powder, emulsion, sprayable solution, concentrated emulsion, aerosol, seed coating .
  • the present invention uses advanced transcriptome and metabolome sequencing and analysis techniques to screen and determine that emulsifiers containing higher fatty alcohols can affect the content of signal substances lysophosphatidylcholine and lysophosphatidylethanolamine in legumes through external application , phenylpropane metabolic pathway and isoflavone biosynthetic pathway-related gene transcription levels, and accumulate isoflavones, thereby improving the nitrogen fixation and drought resistance of legumes.
  • Figure 1 is a schematic diagram of the biosynthetic pathway of isoflavones in peanut leaves.
  • This embodiment provides a water emulsion containing higher fatty alcohol, which is composed of the following components by weight percentage: 24% lauryl alcohol, 3% cetyl alcohol, 3% emulsifier, 5% thickener, and the balance of water.
  • the emulsifier is polyoxyethylene fatty acid ester
  • the thickener is methyl cellulose
  • the present embodiment also provides the preparation method of above-mentioned emulsion in water, comprises the following steps:
  • This embodiment provides a water emulsion containing higher fatty alcohol, which is composed of the following components by weight percentage: 24% lauryl alcohol, 3% emulsifier, 5% thickener, and the balance of water.
  • the emulsifier is polyoxyethylene fatty acid ester
  • the thickener is methyl cellulose
  • the present embodiment also provides the preparation method of above-mentioned emulsion in water, comprises the following steps:
  • This embodiment provides an aqueous emulsion containing higher fatty alcohol, which consists of the following components by weight percentage: 3% cetyl alcohol, 3% emulsifier, 5% thickener, and the balance of water.
  • the emulsifier is polyoxyethylene fatty acid ester
  • the thickener is methyl cellulose
  • the present embodiment also provides the preparation method of above-mentioned emulsion in water, comprises the following steps:
  • cetyl alcohol in a container and heat it to 60 degrees to melt, then add an emulsifier into the container, process it through a high-shear homogenizer at a speed of 5000 rpm, rotate for 10 minutes, and then add water at 60 degrees to the container , processed by a high-shear homogenizer at a speed of 10,000 rpm, rotated for 10 minutes, cooled to 40 degrees, and then added a thickener to the container, processed by a high-shear homogenizer, and rotated at 10,000 rpm After 30 minutes, it's ready.
  • This embodiment provides an emulsion in water, which consists of the following components by weight percentage: 3% of emulsifier, 5% of thickener, and the balance of water.
  • the emulsifier is polyoxyethylene fatty acid ester
  • the thickener is methyl cellulose
  • the emulsifier into the container, process it through a high-shear homogenizer at a speed of 5,000 rpm, and rotate for 10 minutes, then add water at 60 degrees to the container, and process it through a high-shear homogenizer at a speed of 10,000 rpm. Minute, rotate for 10 minutes, cool down to 40 degrees, then add a thickener to the container, process it through a high-shear homogenizer, and rotate it at 10,000 rpm for 30 minutes to get it.
  • Embodiment 5 The water emulsion that contains higher fatty alcohol is to the impact of peanut lysophosphatidylcholine and lysophosphatidylethanolamine content
  • Lysophosphatidylcholine and lysophosphatidylethanolamine are important signal substances in plant cells. Normal physiology comes from the formation of phospholipids after being hydrolyzed by phospholipase to remove a long carbon chain, which can be induced by various pressures. Lysophosphatidylcholine plays an important role in root nodule formation. Lysophosphatidylethanolamine can activate the activity of phenylalanine ammonia-lyase, a key enzyme related to immunity and resistance to abiotic stress, and inhibit premature senescence in plants.
  • Peanut samples were treated with the aqueous emulsion containing higher fatty alcohols of the present invention, metabolite determination and transcriptome sequencing were performed, and the influence of the aqueous emulsion of the present invention on the content of peanut lysophosphatidylcholine and lysophosphatidylethanolamine was analyzed.
  • the signal substance lysophosphatidylcholine Alkali (12:0 (12 carbons without double bonds)) and lysophosphatidylethanolamine (16:3 (16 carbons with three double bonds)) content detection results are shown in Table 2, the results of detecting the average number of three biological repetitions It shows that compared with the water control, the content of lysophosphatidylcholine in the mixed sample is increased by 4070 times, and the content of lysophosphatidylcholine in the mixed sample is also increased by 4070 times compared with the emulsifier treatment, indicating that the increase of the signal substance is not caused by the emulsifier.
  • the content of signal substances in the emulsifier treatment was consistent with that in the clear water treatment.
  • the content of another signal substance, lysophosphatidylethanolamine, in the mixed sample increased by 960 times, which was consistent with the increase in the emulsifier treatment, indicating that the increase in the content of the signal substance was caused by the treatment of higher fatty alcohols.
  • Dodecyl alcohol treatment alone has the same increase fold compared with the control and emulsifier treatment, and is lower than the mixed treatment of dodecanol and cetyl alcohol, which is consistent with the situation of lysophosphatidylcholine.
  • Table 2 Ratio of lysophosphatidylcholine and lysophosphatidylethanolamine content in peanut plant leaves of each treatment group (up-regulation factor)
  • the type of lysophospholipid is marked: 12:0 means 12 carbons without double bonds, and 16:3 means 16 carbons with three double bonds
  • Embodiment 6 The effect of water emulsion containing higher fatty alcohol on gene transcription related to peanut phenylpropane metabolic pathway
  • Lysophospholipids participate in the regulation of phenylalanine ammonia-lyase activity, which is a specific metabolic pathway for signaling substances and one of the most important plant secondary metabolic pathways, which play an important role in plant growth and development and plant-environment interactions.
  • This pathway includes multiple branched pathways leading to isoflavone metabolites. Isoflavone metabolites are involved in physiological processes such as helping plant cells reduce UV damage, resist disease occurrence, and tolerate uncomfortable temperature and high-salt drought conditions.
  • Several higher fatty alcohol preparations involved in the present invention can significantly increase the content of lysophospholipid signal substances in peanut leaves, indicating that they may further affect downstream metabolic pathways. Therefore, the transcription levels of genes encoding key enzymes in the phenylpropane metabolic pathway were further detected on peanut leaves treated with S samples, A samples, B samples, and water.
  • Table 3 Difference ratios of transcription levels of genes related to phenylpropane metabolic pathway in peanut plant leaves
  • Example 7 Effects of Water Emulsion Containing Higher Fatty Alcohol on Gene Transcription and Metabolite Content of Peanut Isoflavone Biosynthetic Pathway
  • the phenylpropane metabolism pathway involves many physiological activities of plants.
  • the isoflavone biosynthesis pathway was selected as the object, and 72-hour S samples and Peanut leaves were treated with clean water, and the transcription levels of pathway-related genes and corresponding metabolite omics assays were performed, and joint analysis was performed. The results are shown in Figure 1 and Tables 4 and 5.
  • the up-regulation level in the average number of three biological repetitions of the relevant genes is shown in Table 4, in which the transcription level of the 2-hydroxyisoflavone synthase gene was up-regulated by 5.80 times, and the transcription level of the isoflavone 4'-O-methyltransferase gene was up-regulated by 4.53 times, isoflavone/4'-methoxyisoflavone 2'-hydroxylase gene transcription level was up-regulated 2.25 times, glutamine reductase gene transcription level was up-regulated 4.21 times.
  • Table 4 Ratio of difference in transcription levels of genes related to isoflavone biosynthesis pathway in leaves of peanut plants (S/control)
  • Embodiment 8 The water emulsion containing higher fatty alcohol affects the content of soybean lysophosphatidylethanolamine
  • Example 5 Based on the research of Example 5, 6 and 7, the water emulsion containing higher fatty alcohols of the present invention and clear water are used to process soybean plant samples, and the determination of transcriptome and metabolite group is carried out to analyze the higher fatty alcohols of the present invention. Effects of water emulsions on soybean plant physiology.
  • lysophospholipid 24:0 means 24 carbons without double bonds
  • Example 9 Effect of water emulsion containing higher fatty alcohol on gene transcription related to soybean phenylpropane metabolic pathway
  • Phenylpropane metabolism is one of the most important plant secondary metabolic pathways, which plays an important role in plant growth and development and plant-environment interaction. This pathway includes multiple branched pathways leading to metabolites such as lignin, isoflavones, etc.
  • isoflavones attract rhizobia and induce nod gene expression, thereby inducing root nodule formation.
  • Lignin is mainly accumulated in the secondary cell wall and participates in the process of providing mechanical support, hydrotrophic transport, resistance to diseases and insect pests, and resistance to non-physiological stress.
  • Isoflavone metabolites are involved in physiological processes such as helping plant cells reduce UV damage, scavenge active oxygen, resist disease occurrence, and tolerate uncomfortable temperature and high-salt drought conditions. Isoflavone substances are also important factors of soybean quality.
  • transcriptome differential analysis was performed, and the results of three biological repeats were detected, as shown in Tables 7 and 8, the transcription levels of key enzyme-encoding genes in the phenylpropane metabolic pathway and isoflavone biosynthetic pathway There was a highly significant change and was significantly upregulated in S-treated samples.
  • Table 7 Difference ratio (fold up-regulation) of transcription levels of genes related to phenylpropane metabolic pathway in leaves of soybean plants in each treatment group
  • Table 8 Difference ratio (fold up-regulation) of transcription levels of genes related to isoflavone biosynthesis pathway in leaves of soybean plants in each treatment group
  • Embodiment 10 The water emulsion containing higher fatty alcohol affects the physiological phenotype of peanut
  • the water emulsion containing higher fatty alcohols of the present invention can significantly affect the signal substances lysophosphatidylcholine and lysophosphatidylethanolamine of peanuts, and further affect the bioactivity of phenylpropane metabolic pathways and isoflavones.
  • the specific physiological phenotypes of these physiological pathways include disease resistance, production increase, anti-ultraviolet radiation, etc., in order to verify that the physiological metabolic pathways caused by higher fatty alcohols in the present invention do have the above-mentioned physiological activities, environmental variables can be controlled in the laboratory. Validation of conditions.
  • MDA malondialdehyde
  • POD peroxidase
  • the measurement results of the average number of three biological repetitions are shown in Tables 9 and 10.
  • the results show that the mixture of lauryl alcohol and cetyl alcohol can significantly improve the germination potential of peanuts, which is higher than that of lauryl alcohol or cetyl alcohol alone and the control, and that of lauryl alcohol and cetyl alcohol alone is also significantly higher than that of the control. But there was no difference between the two fatty alcohols alone. Germination rate, germination index and vigor index all showed the same rule.
  • the average germination days can be significantly shortened by mixed treatment and lauryl alcohol alone treatment, and the germination days can also be significantly shortened by cetyl alcohol treatment compared with the control.
  • a, b and c represent the significant difference relationship of data in statistics, group b is significantly higher than group c, and group a is significantly higher than group b.
  • Table 9 Physiological indicators of peanut seed germination after treatment
  • the phenylpropane metabolic pathway and isoflavone metabolites can increase the resistance of plants to stress.
  • the single or mixed treatment of dodecyl alcohol and cetyl alcohol can significantly improve the drought resistance of peanuts, and the treatments significantly increased
  • the plant height of peanuts, the proline content of leaves treated with lauryl alcohol and cetyl alcohol was significantly higher than that of lauryl alcohol and cetyl alcohol alone and the control, and the proline content of leaves treated with the two alcohols was also significantly higher than that of the control .
  • the content of malondialdehyde was consistent with that of proline.
  • the POD content of the mixed treatment was significantly higher than that of other treatments and the control, and the content of dodecanol and cetyl alcohol alone was higher than that of the control, but the difference was not significant. Data for dry weight are consistent with POD.
  • Table 10 Physiological indicators of peanut plants after simulated drought treatment
  • Embodiment 11 The water emulsion containing higher fatty alcohol is used in the production and use effect of peanut field
  • Sample S is the emulsion in water prepared in Example 1
  • the germination rate, growth status, number of flower buds, and root nodules of the test group peanuts treated with the aqueous emulsion containing higher fatty alcohols of the present invention are significantly better than those of the comparison group, and no damage by diseases and insect pests occurs.
  • the rotten fruit rate of the test group was less than 1%, and the peanut weight per plant was 16.36% higher than that of the control group.
  • Embodiment 12 The effect of water emulsion containing higher fatty alcohol in soybean field production
  • Sample S is the emulsion in water prepared in Example 1
  • test group peanuts processed by the water emulsion containing higher fatty alcohol of the present invention its growth status, pod number, root nodule number are all significantly better than contrast group.
  • the soybeans in the experimental group and the control group were subjected to high temperature and dry weather for more than 40 days, and the control group was significantly affected.
  • the control group suffered from more than 40 days of high temperature and drought weather, and the growth status was weak. From the perspective of the number of pods per plant and the total weight of fruit per plant, it was already in a state of loss of harvest. The effect produced after the emulsion treatment maintains a normal growth condition, and the final fruit harvest situation shows that the soybean weight per plant increases by 199.0% compared with the control group.
  • the application of the water emulsion containing higher fatty alcohols of the present invention on leguminous plants can affect the signal substance lysophosphatidylcholine and lysophosphatidylethanolamine in legumes by external application. content to increase the root nodule content of legumes to promote nitrogen fixation and increase yield.
  • the gene transcription levels related to the phenylpropanoid metabolism pathway and the isoflavone biosynthesis pathway are up-regulated, and the accumulation of isoflavones promotes drought resistance and improves quality.

Abstract

高级脂肪醇在制备提高豆科植物的溶血磷脂酰胆碱含量的制剂的应用，高级脂肪醇通过提高豆科植物的溶血磷脂酰胆碱含量来增加根瘤数量，从而提高豆科植物固氮能力；高级脂肪醇在制备提高豆科植物的异黄酮类化合物含量的制剂的应用；高级脂肪醇在制备提高豆科植物抗旱能力的制剂的应用，含有高级脂肪醇的制剂通过提高苯丙烷代谢途径相关基因的转录水平、提高异黄酮生物合成途径相关基因的转录水平以及提高豆科植物的异黄酮类化合物含量，来提高豆科植物抗旱能力；高级脂肪醇在制备提高花生的发芽率和发芽势、缩短发芽时间方面的制剂的应用。

Description

高级脂肪醇在提高豆科植物固氮能力以及抗旱能力方面的应用 技术领域
本发明涉及高级脂肪醇在提高豆科植物固氮能力以及抗旱能力方面的应用。
背景技术
豆科植物作为重要的经济作物，是人类食品中淀粉、蛋白质、油和蔬菜的重要来源，也是畜牧及水产养殖业的重要饲养原料。其中，花生油和大豆油是重要的植物源食用油，豆粕也是重要的饲料蛋白源。但是我国食用油和饲料蛋白长期依赖进口，根据海关总署数据显示，2016年，我国食用油对外依存度就达到67.7％。2020年我国大豆的进口数量未10033万吨，同比增长13.3％。食用油和大豆的进口依存度过高，极容易受到国外市场行情的牵制，对我国的粮油安全造成威胁。
通过抗逆、高产的新品种选育以及病虫害绿色防控技术的应用，一定程度上推进了我国花生大豆等豆科作物农业生产水平，但大豆和花生的总体产量低下，病虫害发生频繁，不良气候多发，连作障碍严重，过度使用化学农药及肥料及收获后保质不佳等关键问题，依然严重制约相关产业的健康快速发展。
发明内容
为了克服现有技术的不足，本发明的目的在于提供一种高级脂肪醇在提高豆科植物固氮能力以及抗旱能力方面的应用。
为解决上述问题，本发明所采用的技术方案如下：
本发明提供一种高级脂肪醇在制备提高豆科植物的溶血磷脂酰胆碱含量的制剂的应用，高级脂肪醇通过提高豆科植物的溶血磷脂酰胆碱含量来实现增加根瘤数量方面的应用。
进一步地，高级脂肪醇通过增加豆科植物根瘤的数量来实现提高豆科植物 固氮能力方面的应用。
在另一方面，本发明提供一种高级脂肪醇在制备提高豆科植物的异黄酮类化合物含量的制剂的应用，所述异黄酮类化合物为3,9-二羟基紫檀碱。
进一步地，高级脂肪醇通过提高豆科植物的3,9-二羟基紫檀碱的含量来实现提高豆科植物植保素含量方面的应用。
在另一方面，本发明提供一种高级脂肪醇在制备提高豆科植物的异黄酮类化合物含量的制剂的应用，所述异黄酮类化合物为毛蕊异黄酮和大豆黄素。
进一步地，高级脂肪醇通过提高豆科植物的毛蕊异黄酮和大豆黄素的含量来实现提高豆科植物抗逆境活性方面的应用。
在另一方面，本发明提供一种高级脂肪醇在制备提高花生的发芽率和发芽势、缩短发芽时间方面的制剂的应用。
本发明提供一种高级脂肪醇在制备提高豆科植物抗旱能力的制剂的应用，含有高级脂肪醇的制剂通过提高苯丙烷代谢途径相关基因的转录水平、提高异黄酮生物合成途径相关基因的转录水平以及提高豆科植物的异黄酮类化合物含量，来实现提高豆科植物抗旱能力方面的应用。
在本发明中，高级脂肪醇为十二醇、十六醇其中一种或者二者的混合。
进一步地，所述制剂为水乳剂，其包括高级脂肪醇、乳化剂、增稠剂、水。
本发明含有高级脂肪醇的制剂不限于水乳剂，只要是采用本发明的发明构思的所有剂型，均适用，比如可湿性粉剂、乳剂、可喷的溶液、浓乳剂、气雾剂、种衣剂。
相比现有技术，本发明的有益效果在于：
本发明利用先进的转录组、代谢组测序和分析技术，筛选并确定了含有高级脂肪醇的乳化剂可以通过外部施用来影响豆科植物中信号物质溶血磷脂酰胆碱及溶血磷脂酰乙醇胺的含量、苯丙烷代谢途径及异黄酮生物合成途径相关基 因转录水平，累积异黄酮类物质，从而提高豆科植物的固氮能力和抗旱能力。
下面结合附图和具体实施方式对本发明作进一步详细说明。
附图说明
图1为花生叶片异黄酮生物合成途径的示意图。
具体实施方式
实施例1
本实施例提供一种含有高级脂肪醇的水乳剂，按重量百分比由以下组分组成：十二醇24％、十六醇3％、乳化剂3％、增稠剂5％、水余量。
在本实施例中，所述乳化剂是脂肪酸聚氧乙烯酯，所述增稠剂是甲基纤维素。
本实施例还提供上述水乳剂的制备方法，包括以下步骤：
将十二醇、十六醇放入容器中加热至60度熔化，再于容器中加入乳化剂，通过高剪切均质机处理，转速5000转/分，转动10分钟，再于容器中加入60度的水，通过高剪切均质机处理，转速10000转/分，转动10分钟，降温至40度，然后再于容器中加入增稠剂，通过高剪切均质机处理，以10000转/分转动30分钟后，即得。
实施例2
本实施例提供一种含有高级脂肪醇的水乳剂，按重量百分比由以下组分组成：十二醇24％、乳化剂3％、增稠剂5％、水余量。
在本实施例中，所述乳化剂是脂肪酸聚氧乙烯酯，所述增稠剂是甲基纤维素。
本实施例还提供上述水乳剂的制备方法，包括以下步骤：
将十二醇放入容器中加热至60度熔化，再于容器中加入乳化剂，通过高剪 切均质机处理，转速5000转/分，转动10分钟，再于容器中加入60度的水，通过高剪切均质机处理，转速10000转/分，转动10分钟，降温至40度，然后再于容器中加入增稠剂，通过高剪切均质机处理，以10000转/分转动30分钟后，即得。
实施例3
本实施例提供一种含有高级脂肪醇的水乳剂，按重量百分比由以下组分组成：十六醇3％、乳化剂3％、增稠剂5％、水余量。
在本实施例中，所述乳化剂是脂肪酸聚氧乙烯酯，所述增稠剂是甲基纤维素。
本实施例还提供上述水乳剂的制备方法，包括以下步骤：
将十六醇放入容器中加热至60度熔化，再于容器中加入乳化剂，通过高剪切均质机处理，转速5000转/分，转动10分钟，再于容器中加入60度的水，通过高剪切均质机处理，转速10000转/分，转动10分钟，降温至40度，然后再于容器中加入增稠剂，通过高剪切均质机处理，以10000转/分转动30分钟后，即得。
实施例4
本实施例提供一种水乳剂，按重量百分比由以下组分组成：乳化剂3％、增稠剂5％、水余量。
在本实施例中，所述乳化剂是脂肪酸聚氧乙烯酯，所述增稠剂是甲基纤维素。
将乳化剂放入容器中，通过高剪切均质机处理，转速5000转/分，转动10分钟，再于容器中加入60度的水，通过高剪切均质机处理，转速10000转/分，转动10分钟，降温至40度，然后再于容器中加入增稠剂，通过高剪切均质机 处理，以10000转/分转动30分钟后，即得。
实施例5：含有高级脂肪醇的水乳剂对花生溶血磷脂酰胆碱和溶血磷脂酰乙醇胺含量的影响
溶血磷脂酰胆碱和溶血磷脂酰乙醇胺为植物细胞中重要的信号物质，正常生理来源于磷脂被磷脂酶水解去一条长碳链后形成，可以被多种压力诱导产生。溶血磷脂酰胆碱对根瘤形成具有重要作用。溶血磷脂酰乙醇胺能够激活免疫和抗非生物逆境相关关键酶苯丙氨酸解氨酶活性、抑制植物早衰。用本发明所述含有高级脂肪醇的水乳剂处理花生样品，进行代谢物测定和转录组测序，分析本发明所述水乳剂对花生溶血磷脂酰胆碱和溶血磷脂酰乙醇胺含量的影响。
(1)脂肪醇样品：以表1所列本发明制作成的水乳剂
表1：样品编号
Figure PCTCN2021128777-appb-000001
(2)供试植物：花生(栽培种汕油35号)
培养条件：70/0μmol m -2s -1(光照/黑暗周期光照强度)，14小时/10小时(光照/黑暗周期的时间)，27℃/24℃，70％相对湿度，四周龄。
(3)样品处理和数据收集
选取健康且组内生长状态相近的植株，用上述样品的稀释液(900倍兑水稀释)对叶片进行喷施，直至叶面完全覆盖液膜。对照组喷施等量的用于稀释原液的无菌水。于48小时后进行第二次喷施处理。每组设置3个生物学重复。
于第一次处理后72小时，收取各组4克叶片样品。液氮速冻3分钟后用干冰保温送武汉迈特维尔生物科技有限公司实验室进行代谢产物组学测序(下述所有代谢组及转录组测序数据均由武汉迈特维尔生物科技有限公司提供)。
(4)处理对2种信号物质含量的影响
使用十二醇和十六醇的混合制剂(S)、十二醇制剂(A)、乳化剂(C)以及CK(清水处理)处理花生叶片72小时后，生理代谢物中信号物质溶血磷脂酰胆碱(12:0(12个碳无双键))和溶血磷脂酰乙醇胺(16:3(16个碳有三个双键))含量检测结果如表2所示，检测三次生物学重复平均数的结果表明，混合样品相比清水对照，溶血磷脂酰胆碱含量上升4070倍，混合样品相比乳化剂处理，溶血磷脂酰胆碱含量也上升4070倍，说明该信号物质的上升非乳化剂引起。十二醇单独处理相比清水对照，溶血磷脂酰胆碱含量上升1850倍，十二醇单独处理相比乳化剂单独处理，溶血磷脂酰胆碱含量上升1850倍，说明十二醇处理引起的信号物质上升也不是由乳化剂引起。乳化剂处理与清水处理的信号物质含量一致。
同样，混合样品相比清水对照，另一信号物质溶血磷脂酰乙醇胺含量上升960倍，与相比乳化剂处理的上升倍数一致，说明信号物质含量上调是由高级脂肪醇处理引起。十二醇单独处理相比对照和乳化剂处理的上升倍数一致，并低于十二醇和十六醇混合处理，与溶血磷脂酰胆碱的情况一致。
从高级脂肪醇的单独处理和混合处理引起两种信号物质的含量变化结果来看，说明高级脂肪醇单独处理和混合处理都可以引起上述两种信号物质的含量上调。
表2：各处理组花生植株叶片中溶血磷脂酰胆碱和溶血磷脂酰乙醇胺含量比值(上调倍数)
Figure PCTCN2021128777-appb-000002
溶血磷脂的种类标注：12:0意思为12个碳无双键，16:3意思为16个碳三 个双键
实施例6：含有高级脂肪醇的水乳剂对花生苯丙烷代谢途径相关基因转录的影响
溶血磷脂参与苯丙氨酸解氨酶活性调节，是信号物质作用的具体代谢途径，也是最重要的植物次生代谢途径之一，对植物生长发育及植物环境互作具有重要作用。该途径包括多个分支途径，产生异黄酮类代谢物。异黄酮类代谢物参与帮助植物细胞减少紫外线损伤、抵抗病害发生、耐受不适温度和高盐干旱条件等生理过程。本发明涉及的几种高级脂肪醇制剂可以显著提高花生叶片中溶血磷脂类信号物质的含量，显示可能会进一步影响下游代谢途径。因此对使用S样品、A样品、B样品以及清水处理的花生叶片进一步开展了苯丙烷代谢途径关键酶编码基因的转录水平检测。
(1)脂肪醇样品：同实施例5
(2)供试植物：同实施例5
(3)样品处理和数据收集：同实施例5，转录组测序由武汉迈特维尔生物科技有限公司完成
(4)处理对苯丙烷代谢途径相关基因转录水平的影响
不同处理对花生叶片苯丙烷代谢途径的相关基因转录水平的影响如表3所示。转录组测定三次生物学重复平均数的结果显示，无论是十二醇单独处理，还是十六醇单独处理，或者2者的混合处理，都可以显著上调其中一些重要基因的转录水平。总体来看，十二醇和十六醇混合处理在调节基因转录水平方面优于单独处理，与代谢组测定信号物质的含量结果一致，显示十二醇和十六醇的单独或混合处理都引起花生叶片苯丙烷代谢途径相关基因的转录上调。
表3：花生植株叶片中苯丙烷代谢途径相关基因转录水平差异比值
Figure PCTCN2021128777-appb-000003
实施例7：含有高级脂肪醇的水乳剂对花生异黄酮生物合成途径相关基因转录和代谢产物含量的影响
苯丙烷代谢途径涉及到植物众多的生理活性，为进一步验证本发明制剂能够调节植物苯丙烷代谢相关途径基因转录水平和代谢物含量，选取异黄酮生物合成途径为对象，使用72小时的S样品及清水处理花生叶片，进行通路相关基因 转录水平和对应代谢产物组学测定，并进行联合分析。结果如图1和表4、5所示。
(1)脂肪醇样品：同实施例5
(2)供试植物：同实施例5
(3)样品处理和数据收集：同实施例5，转录组及代谢产物组测定由武汉迈特维尔生物科技有限公司完成
(4)处理对异黄酮生物合成途径相关基因转录和代谢产物含量
花生叶片异黄酮生物合成途径如图1所示，其中十二醇和十六醇的混合制剂处理后，引起多个基因的转录水平上调，灰色代表S制剂处理后对应的基因(矩形)转录水平或代谢产物(圆形)含量相比于对照组出现显著上调。相关基因三次生物学重复平均数中的上调水平如表4所示，其中2-羟基异黄酮合酶基因转录水平上调了5.80倍，异黄酮4'-O-甲基转移酶基因转录水平上调了4.53倍，异黄酮/4'-甲氧基异黄酮2'-羟化酶基因转录水平上调了2.25倍，谷氨酰胺还原酶基因转录水平上调了4.21倍。相应，几种重要的异黄酮类化合物的含量也大幅度上调，其中3,9-二羟基紫檀碱含量上调1223.67倍，毛蕊异黄酮上调2657.76倍，大豆黄素上调1982.35倍。研究表明，3,9-二羟基紫檀碱是重要的植保素前提，能够提高作物对病虫害的抵抗能力；毛蕊异黄酮和大豆黄素已被证明具有显著的抗逆境活性。
表4：花生植株叶片中异黄酮生物合成途径相关基因转录水平差异比值(S/对照)
Figure PCTCN2021128777-appb-000004
Figure PCTCN2021128777-appb-000005
实施例8：含有高级脂肪醇的水乳剂对大豆溶血磷脂酰乙醇胺含量的影响
基于实施例5，6和7的研究，用本发明所述含有高级脂肪醇的水乳剂和清水处理大豆植株样品，进行转录组和代谢产物组测定，分析本发明所述的含有高级脂肪醇的水乳剂对大豆植物生理的影响。
(1)脂肪醇样品：同实施例5
(2)供试植物：大豆(华春一号)
各植株培养条件：80/0μmol m -2s -1(光照/黑暗周期光照强度)，14小时/10小时(光照/黑暗时间周期)，28℃25℃，70％相对湿度，四周龄。
(3)样品处理：
选取健康且组内生长状态相近的植株，用上述样品的稀释液(900倍兑水稀释)对叶片进行喷施，直至叶面完全覆盖液膜。对照组喷施等量的用于稀释原液的无菌水。于48小时后进行第二次喷施处理。每组设置3个生物学重复。于第一次处理后72小时，收取各组4克叶片样品。液氮速冻3分钟后于干冰中送武汉迈特维尔生物科技有限公司实验室进行转录组及代谢产物组测定。
(4)处理对大豆叶片溶血磷脂酰乙醇胺含量的影响
使用S样品、清水处理大豆叶片72小时后，测定三次生物学重复平均数后发现大豆叶片中信号物质溶血磷脂酰乙醇胺(24:0)(24个碳无双键)含量是清水处理的组织的2.44倍(表6)。
表6：各处理组大豆植株叶片中溶血磷脂酰乙醇胺含量比值(上调倍数)
Figure PCTCN2021128777-appb-000006
溶血磷脂的种类标注：24:0意思为24个碳无双键
实施例9：含有高级脂肪醇的水乳剂对大豆苯丙烷代谢途径相关基因转录的影响
苯丙烷代谢是最重要的植物次生代谢途径之一，对植物生长发育及植物环境互作具有重要作用。该途径包括多个分支途径，产生如木质素、异黄酮等代谢物。大豆中，异黄酮吸引根瘤菌并诱导nod基因的表达，从而诱导根瘤的形成。(东北农业大学学报2007年10月《异黄酮的生物合成途径及其调控》作者：马君兰、李成等)。
木质素主要积累于次生细胞壁中，参与提供机械支持、水养运输、抵抗病虫害、抵御非生理胁迫的过程。异黄酮代谢物参与帮助植物细胞减少紫外线损伤、清除活性氧、抵抗病害发生、耐受不适温度和高盐干旱条件等生理过程。异黄酮物质也是大豆品质的重要因素。
(1)脂肪醇样品：同实施例5
(2)供试植物：同实施例8
(3)样品处理和数据采集：同实施例8
(4)处理对大豆叶片苯丙烷代谢途径相关基因转录的影响
使用S样品、清水处理大豆叶片72小时后，进行转录组差异分析，检测三次生物学重复平均数的结果如表7、8，苯丙烷代谢途径及异黄酮生物合成通路关键酶编码基因的转录水平发生极显著变化，在S处理样品中显著上调。
表7：各处理组大豆植株叶片中苯丙烷代谢途径相关基因转录水平差异比值(上调倍数)
Figure PCTCN2021128777-appb-000007
Figure PCTCN2021128777-appb-000008
表8：各处理组大豆植株叶片中异黄酮生物合成途径相关基因转录水平差异比值(上调倍数)
Figure PCTCN2021128777-appb-000009
Figure PCTCN2021128777-appb-000010
实施例10：含有高级脂肪醇的水乳剂对花生生理表型的影响
通过实施例5，6和7发现，本发明的含有高级脂肪醇的水乳剂能够显著影响花生的信号物质溶血磷脂酰胆碱和溶血磷脂酰乙醇胺，并进一步影响苯丙烷代谢途径和异黄酮的生物合成，这些生理途径的具体生理表型包括抗病、增产、抗紫外线等，为验证本发明所述的高级脂肪醇引起的生理代谢途径确实具有上述生理活性，在实验室中进行环境变量可控条件的验证。
(1)供试品种：同实施例5；
(2)供试方法：
使用150倍稀释的S样品、A样品、B样品及清水分别对花生种子进行拌种晾干后播种，测定了发芽势、发芽率、发芽指数、平均发芽日、活力指数，以及喷叶后(900倍稀释)用5％聚乙二醇6000灌根处理模拟干旱条件下生长8天后株高、叶片组织脯氨酸含量、丙二醛(MDA)含量、过氧化物酶(POD)活性、及植株干重生理生化指标。
(3)处理对花生生理的影响
三次生物学重复平均数的测定结果如表9、10所示。结果表明十二醇和十六醇混合可以显著提高花生的发芽势，比十二醇或十六醇单独处理和对照的发芽势都高，十二醇和十六醇单独处理的也显著高于对照，但2种脂肪醇单独处理 之间没有差异。发芽率、发芽指数和活力指数都表现出相同的规律。混合处理和十二醇单独处理可以显著缩短平均发芽日，十六醇处理也可以显著缩短与对照的发芽日。说明十二醇和十六醇混合在提高花生发芽率和芽势，缩短发芽时间方面具有明显的表型。其中a、b和c代表统计学中数据的显著性差异关系，b组显著性高于c组，a组显著性高于b组。
表9：处理后花生种子萌发生理指标
Figure PCTCN2021128777-appb-000011
苯丙烷代谢途径和异黄酮类代谢物可以增加植物对逆境的抗性，通过模拟干旱实验，确定十二醇和十六醇单独或混合处理可以显著提高花生对干旱的抗性，其中处理显著提高了花生的株高，十二醇和十六醇的混合处理的叶片脯氨酸含量显著高于十二醇和十六醇单独处理和对照，2种醇的单独处理叶片脯氨酸含量也显著高于对照。丙二醛含量与脯氨酸含量规律一致。混合处理的POD含量显著高于其他处理和对照，十二醇和十六醇单独处理高于对照但差异不显著。干重的数据与POD一致。
生理测定的结果进一步验证，高级脂肪醇通过影响溶血磷脂酰胆碱和溶血磷脂乙醇胺信号物质含量后，的确可以提高花生对干旱的抗性。
表10：模拟干旱处理后花生植株生理指标
Figure PCTCN2021128777-appb-000012
Figure PCTCN2021128777-appb-000013
实施例11：含有高级脂肪醇的水乳剂在花生大田生产使用效果
取样：样品S为实施例1制备的水乳剂
作物：花生汕油35号
地点：广东省韶关市始兴县罗坝镇
方法：在生产基地选取10亩试验组、10亩对照组按表11进行生产
表11：花生试验方案
Figure PCTCN2021128777-appb-000014
使用效果对比如表12所示：
表12：花生试验效果
Figure PCTCN2021128777-appb-000015
Figure PCTCN2021128777-appb-000016
在整个花生生长周期中，使用本发明的含有高级脂肪醇的水乳剂处理的试验组花生，其发芽率、生长状况、花苞个数、根瘤数量都明显优于对比组，未发生病虫害危害，从最后收果情况看，试验组的烂果率小于1％，其单株花生重量比对照组增产16.36％。
实施例12：含有高级脂肪醇的水乳剂在大豆大田生产使用效果
取样：样品S为实施例1制备的水乳剂
作物：大豆春华1号
地点：广东省韶关市曲江区樟市镇
方法：在生产基地选取10亩试验组、10亩对照组按表13进行生产
表13：大豆试验方案
Figure PCTCN2021128777-appb-000017
在整个大豆生长周期中，使用本发明的含有高级脂肪醇的水乳剂处理的试验 组花生，其生长状况、豆荚数、根瘤数量都明显优于对比组。
试验期间，试验组和对照组大豆遭遇四十多天高温干旱天气，对照组受影响明显。
使用效果对比如表14所示：
表14：大豆试验效果
Figure PCTCN2021128777-appb-000018
对照组因遭遇四十多天高温干旱天气，生长状况疲弱，从单株豆荚数、单株果实总重看，已处于失收状态，而试验组因为使用本发明的含有高级脂肪醇的制剂水乳剂处理后产生的作用，保持正常的生长状况，最后收果情况看，其单株大豆重量比对照组增产199.0％。
由实施例5、6、7、8、9可见，采用本发明所述含有高级脂肪醇的水乳剂兑水稀释对豆科植物(花生、大豆)处理，可以显著影响豆科植物中信号物质溶血磷脂酰胆碱及溶血磷脂酰乙醇胺的含量、苯丙烷代谢途径及异黄酮生物合成途径相关基因转录水平，累积异黄酮类物质，从而提高豆科植物的固氮能力、抗旱能力、提质增产；从实施例10可见，实验室可控环境变量条件下生理表型试验证明使用本发明的含有高级脂肪醇的水乳剂可以显著影响豆科植物种 子萌发、苗期长势和抗逆指标；实施例11大田使用效果显示，使用本发明的含有高级脂肪醇的水乳剂可以增加花生根瘤数量、减低病害导致的坏果发生率、提升品质、提高产量；实施例12大田使用效果显示，使用本发明的含有高级脂肪醇的水乳剂可以增加大豆根瘤数量、提高大豆抗旱能力、提高产量。
综合实施例5到实施例12，在豆科植物上应用本发明所述含有高级脂肪醇的水乳剂，可以通过外部施用来影响豆科植物中信号物质溶血磷脂酰胆碱及溶血磷脂酰乙醇胺的含量从而提高豆科植物的根瘤含量以促进固氮能力增产。同时上调苯丙代谢烷途径及异黄酮生物合成途径相关基因转录水平，异黄酮类物质累积来促进抗旱能力并提质。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。

Claims (10)

  1. 高级脂肪醇在制备提高豆科植物的溶血磷脂酰胆碱含量的制剂的应用，其特征在于，高级脂肪醇通过提高豆科植物的溶血磷脂酰胆碱含量来实现增加根瘤数量方面的应用。
  2. 如权利要求1所述的应用，其特征在于，高级脂肪醇通过增加豆科植物根瘤的数量来实现提高豆科植物固氮能力方面的应用。
  3. 高级脂肪醇在制备提高豆科植物的异黄酮类化合物含量的制剂的应用，其特征在于，所述异黄酮类化合物为3,9-二羟基紫檀碱。
  4. 如权利要求3所述的应用，其特征在于，高级脂肪醇通过提高豆科植物的3,9-二羟基紫檀碱的含量来实现提高豆科植物植保素含量方面的应用。
  5. 高级脂肪醇在制备提高豆科植物的异黄酮类化合物含量的制剂的应用，其特征在于，所述异黄酮类化合物为毛蕊异黄酮和大豆黄素。
  6. 如权利要求5所述的应用，其特征在于，高级脂肪醇通过提高豆科植物的毛蕊异黄酮和大豆黄素的含量来实现提高豆科植物抗逆境活性方面的应用。
  7. 高级脂肪醇在制备提高花生的发芽率和发芽势、缩短发芽时间方面的制剂的应用。
  8. 高级脂肪醇在制备提高豆科植物抗旱能力的制剂的应用，其特征在于，含有高级脂肪醇的制剂通过提高苯丙烷代谢途径相关基因的转录水平、提高异黄酮生物合成途径相关基因的转录水平以及提高豆科植物的异黄酮类化合物含量，来实现提高豆科植物抗旱能力方面的应用。
  9. 如权利要求1-8任一项所述的应用，其特征在于，高级脂肪醇为十二醇、十六醇其中一种或者二者的混合。
  10. 如权利要求9所述的应用，其特征在于，所述制剂为水乳剂，其包括高级脂肪醇、乳化剂、增稠剂、水。
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