CN114605564B - 杂聚寡糖及其在提高植物抗病性中的应用 - Google Patents
杂聚寡糖及其在提高植物抗病性中的应用 Download PDFInfo
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Abstract
本发明公开了一种杂聚寡糖及其在提高植物抗病性中的应用。所述的杂聚寡糖含有7个D‑葡萄糖残基和1个D‑半乳糖残基，每个杂聚寡糖分子含有或不含有一分子的丙酮酰基团，和一分子的琥珀酰基团，其结构为
本发明的杂聚寡糖能够诱导植物叶片产生H2O2，增强植物抗性酶的活性，激发植物的植保素的产生，从而提高植物对病原菌感染的抗性，适用于防治病原菌引起的多种农作物和植物的病害。
Description
技术领域
本发明属于植物抗病诱导剂领域，涉及一种杂聚寡糖及其在提高植物抗病性中的应 用。
背景技术
植物在长期的进化发展过程中，为应对病原菌的侵害，形成了有效的自身免疫***。 植物抗性诱导剂可以激发植物的免疫***，起到抗病、防病、治病的目的。在植物与病原相互作用中，一方面植物分泌β-1,3葡聚糖酶、壳聚糖酶和几丁质酶直接水解病原菌 的细胞壁，抑制其生长；另一方面病原菌又分泌多聚半乳糖醛酸酶和果胶酶降解植物的 细胞壁。相互水解产生的寡糖片段，可以激发植物产生病程相关蛋白、植保素等，增强 植物的抗病性。这是寡糖作为植物抗性诱导剂的基本原理。
目前在寡糖作用机理方面的研究，已由一般的观察发展到分子、细胞水平。大量研究表明寡糖在发挥诱导子作用时，大致通过以下方式：寡糖与细胞膜上的受体相互识别，引起受体构象改变产生跨膜信号，经过一系列的信号传导、放大和整合，调控防卫基因 的表达，积累次生代谢产物，诱导植物自生免疫抗性，抵抗病原物质的侵染。
寡糖诱导子来源于天然产物，目前研究显示其具有如下作用特点：(1)生态友好；(2)对常规方法防治无效的病原菌有效，特别是对化学农药产生抗性的病菌；(3)作 用于植物自身，而不是直接作用于病原生物，避免了对非致病生物的不良作用；(4)提 供植物对微生物、昆虫、线虫等多种生物胁迫的保护；(5)可与其他作用方式不同的防 治方法组合使用以扩大防治范围；(6)***抗性中激活了多种基因，其中一些可能在植 物抵抗热、冷、干旱及紫外线胁迫时起到保护作用。
近年来，研究较多的寡糖诱导子主要有以下种类：(1)壳寡糖：是将壳聚糖经特殊的生物酶技术降解得到的一种聚合度在2～20之间的寡糖产品，壳寡糖可诱导植物的抗 病性，对多种真菌、细菌和病毒产生免疫和杀灭作用，对棉花黄萎病、水稻稻瘟病、番 茄晚疫病等病害具有良好的防治作用，可以开发为生物农药、生长调节剂和肥料等；(2) 几丁寡糖：是几丁质的降解产物，是2～10个N-乙酰氨基葡萄糖以糖苷键连接而成的 糖类总称，对多种植物病原菌具有很好的抑制作用，如小麦纹枯病、烟草黑胫病等；(3) 葡寡糖：是人们最早开始研究、认识较***的一类寡糖诱导子，能有效地诱导植保素的 合成与积累，并作为一种早期的信息分子对植物的抗病侵染、分子信号调控、生长发育、 形态建成以及对环境的适应等有着重要意义，对小麦白粉病、马铃薯晚疫病等有良好的 防治作用；(4)寡聚半乳糖醛酸：是由2～20个半乳糖醛酸通过α-1,4-糖苷键连接而成 的寡糖，来源于果胶多糖，广泛分布于高等植物的根、茎、叶及果实的细胞初生壁和细 胞间层中，对细胞组织起着软化和粘合作用，对辣椒病毒病、苹果花叶病毒病有良好的 防治作用。
由昆布多糖制备的低聚β-1,3-葡寡糖在多种植物上证实具有良好的防病效果，并且 已经有相关产品上市。但是昆布多糖生产寡糖受到原料来源和水解工艺的限制，所得的寡糖聚合度偏低且不均一，难以充分发挥作用。因此，亟需寻找一种理想的替代原料， 高效制备高聚合度、结构均一的寡糖，探索新型寡糖对植物诱导作用机制。
发明内容
本发明的目的在于提供一种杂聚寡糖及其在提高植物抗病性中的应用。
本发明所述的杂聚寡糖，含有7个D-葡萄糖残基和1个D-半乳糖残基，每个杂聚 寡糖分子含有或不含有一分子的丙酮酰基团，和一分子的琥珀酰基团，其结构如下所示：
本发明所述的杂聚寡糖通过将保藏编号为CCTCC NO:M 2018797的土壤杆菌Agrobacterium sp.ZCC3656生产的胞外多糖Riclin经β-葡聚糖酶酶解后制得。具体为：
(1)多糖Riclin的酶解：
将保藏编号为CCTCC NO:M 2018797的土壤杆菌Agrobacterium sp.ZCC3656生产的 胞外多糖Riclin溶于水溶液中，加入β-葡聚糖酶混匀后，于60℃恒温水浴中反应，震荡混匀直至酶解完全；
(2)杂聚寡糖的纯化：
将步骤(1)酶解后的溶液离心去除不溶性杂质，取上清液加入体积比为4:1的氯仿-正丁醇混合溶剂，去除溶液中的蛋白，剧烈振荡，静置分层，取水相离心去除蛋白层， 保留上清液，重复数次上述步骤直至完全去除蛋白层，最后加入95％的乙醇析出杂聚寡 糖。
上述土壤杆菌Agrobacterium sp.ZCC3656已在中国专利申请201811493131.2中公 开。
本发明提供上述的杂聚寡糖在提高植物抗病性中的应用。
本发明中，所述的植物包括但不限于农作物及其它植物，例如烟草、小麦、番茄、马铃薯、苹果、草莓、水稻、大豆等。
本发明中，所述的杂聚寡糖在提高植物抗病性中的应用，所述的植物抗病性为对植 物病原菌感染的抗病性，包括但不限于对花叶病毒感染的抗病性、对禾谷镰孢菌感染的抗病性、对黄枝孢菌感染的抗病性、对致病疫霉菌感染的抗病性、对苹果盘二孢菌感染 的抗病性、对青枯假单胞杆菌感染的抗病性等。
本发明还提供一种植物抗病诱导剂，其有效活性成分包含上述杂聚寡糖。
本发明所述的植物抗病诱导剂中，所述的杂聚寡糖的浓度为5～5000mg/L，优选为50mg/L～200mg/L。
本发明的杂聚寡糖作为植物抗病诱导剂应用于农作物及其它多种植物，能够显著提 高植物的抗病性，具体为：1)作用于烟草叶片时，随着剂量增加，能明显提高烟草叶 片过氧化氢的含量；2)作用于小麦叶下表皮细胞时，荧光强度的增加，说明杂聚寡糖 能明显诱导小麦叶片细胞过氧化氢的释放；3)作用于番茄叶片时，杂聚寡糖能明显提 高叶片葡聚糖酶、几丁质酶、苯丙氨酸氨解酶的酶活，而且随着剂量的增加，诱导时间 的增加，酶活不断的增加；4)作用于马铃薯叶片时，采用致病疫霉菌片侵染马铃薯叶 片，可以看出，杂聚寡糖能明显降低病原菌对植物的侵害，减轻甚至消除致病菌的感染。
与现有技术相比，本发明具有以下优点：
(1)本发明的杂聚寡糖能够诱导植物产生对病原菌感染的抗病性，诱导效果显著，能明显降低病原菌对植物的感染；
(2)本发明采用外施杂聚寡糖的方法，使用方法简单，而且无生物毒性，不引起 环境污染；
(3)本发明的杂聚寡糖通过菌株发酵制备，方法简单，成本较低，可规模化生产，并且其具有均一化结构，作为新型生物农药具有广泛应用前景。
附图说明
图1为杂聚寡糖诱导烟草叶片H2O2含量变化图；
图2为H2DCF-DA荧光检测杂聚寡糖诱导小麦叶片细胞H2O2含量的变化图，CK 为喷水对照组，50ppm为杂聚寡糖浓度为50mg/L，500ppm为杂聚寡糖浓度为500mg/L；
图3杂聚寡糖诱导番茄叶片抗性相关酶酶活变化图，a为葡聚糖酶酶活；b为几丁质酶酶活，c为苯丙氨酸氨解酶酶活；
图4为杂聚寡糖诱导马铃薯叶片抗致病疫霉侵染效果图，CK为喷水对照组，50ppm为杂聚寡糖浓度为50mg/L，500ppm为杂聚寡糖浓度为500mg/L。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详述。
下述实施例中采用的土壤杆菌Agrobacterium sp.ZCC3656已在中国专利申请201811493131.2中公开。
实施例1
杂聚寡糖的筛选纯化
1.多糖的制备及分离
接种土壤杆菌Agrobacterium sp.ZCC3656至Htm(磷酸二氢钠1g，无水氯化钙0.07g，氯化镁0.2g，硫酸亚铁0.0125g，硝酸钾3g，硫酸锰0.003g，氯化锌0.0075g，水 1000mL，蔗糖20g，pH 7.0～7.2)液体培养基中，28℃、250rpm摇床培养48h；向发 酵液中加入2倍体积的工业乙醇(95％乙醇)，发现有白色丝状多糖析出；6000×g离心 收集沉淀，置于60℃真空干燥箱，干燥8h的固体多糖，用粉碎机粉碎，即为多糖的粗 品。
2.杂聚寡糖的制备
称取多糖溶于水溶液中，加入β-葡聚糖酶混匀后，于60℃恒温水浴中反应，间隔1h震荡混匀，直至多糖溶液不黏。
3.杂聚寡糖的纯化
将酶解后的溶液，9000rpm离心30min去除不溶性杂质。离心后的上清液采用Sevage法去除溶液中的蛋白，即加入1/4体积的氯仿-正丁醇(4:1)混合剂，剧烈振荡30min，静置分层，取水相7000rpm离心10min去除蛋白层，将上清液移至干净容器 中，重复数次上述操作至无蛋白层。最后加入3倍体积的95％的乙醇析出杂聚寡糖，置 于抽真空干燥箱内，50℃烘干。
实施例2
杂聚寡糖对提高烟草花叶病抗病性的应用
1.烟草试验模型的建立：
试验烟草品种为红花烟草。实验室培养条件为：12h光照-12h黑暗，恒温30℃，并保持土壤湿润。试验分为六组，分别为：喷水对照组、喷不同浓度杂聚寡糖组(分别为 5mg/L、25mg/L、50mg/L、100mg/L、250mg/L)，喷施后15min内收集叶片测定相关信 号分子。
2.烟草叶片H2O2含量测定：
称取100～200mg新鲜植物叶片，迅速在液氮中研磨成极细的粉末；加入1mL丙酮，旋涡震荡萃取，10000rpm离心10min，将上层清液移入干净离心管中；取0.8mL萃取 液于2mL离心管中，加入0.1mL四氯化钛试剂(5mL四氯化钛试剂含有浓盐酸2.175mL、 四氯化钛1mL、去离子水1.825mL)，再加入0.2mL浓氨水，5000rpm离心10min，收 集沉淀；沉淀用丙酮洗涤3～5次至沉淀呈白色；向洗涤后的沉淀加入1mL2MH2SO4， 震荡使沉淀溶解；移取200μL样品与96-well比色板中，读取415nm吸光值，结果如图 2所示。
3.烟草花叶病模型的建立：
试验烟草品种为红花烟草。室外培养，保持土壤湿润。试验分为五组，分别为喷水对照组、喷不同浓度杂聚寡糖组(分别为50mg/L、100mg/L、200mg/L、500mg/L)，喷 施杂聚寡糖后两天，将烟草花叶病毒均匀喷洒于烟草叶片表面，观察叶片感染情况。从 开始喷洒烟草花叶病毒后开始，每隔一周，记录烟草叶片感染情况，连续记录三周。结 果如表1所示。
表1杂聚寡糖对提高烟草花叶病抗病性的应用
4.数据分析
多组之间的比较用ANOVA统计学方法进行统计分析。P＜0.05被认为有显著性区别。
结合图1和表1可以看出，当喷施杂聚寡糖后，烟草叶片内的过氧化氢含量明显上升，而且随着寡糖浓度的增加，过氧化氢含量呈线性依赖上升。烟草对花叶病毒的感染 情况可以看出，杂聚寡糖能明显提高烟草对花叶病毒的抵抗能力，提高烟草对病毒的防 治效果，降低烟草叶片的感染比率。由此可见，杂聚寡糖可以成为提高植物抗病性的潜 在生物农药。
实施例3
杂聚寡糖对小麦提高赤霉病抵抗力的应用
1.小麦试验模型的建立：
试验小麦品种为棉农6号。实验室培养条件为：12h光照-12h黑暗，恒温25℃，并 保持土壤湿润。
2.小麦叶片过氧化氢荧光检测
试验分为三组，分别为空白对照组(即以水为对照)、低浓度杂聚寡糖组(寡糖终浓度为50mg/L)、高浓度杂聚寡糖组(寡糖终浓度为500mg/L)。H2O2荧光检测方法为： H2DCF-DA用DMSO配置成1mmol/L的贮存液，小心撕取植物叶片下表皮，将下表皮 放入pH为5.6的柠檬酸缓冲液中，孵育4h，更换柠檬酸缓冲液，并加入H2DCF-DA。 使其终浓度为10μmol/L，水平摇晃孵育30min；孵育结束后，用柠檬酸缓冲液冲洗3遍， 洗去多余的荧光染料；将叶片下表皮放置于载玻片上，采用BP460-490的激光滤色镜， 光照波长范围为460-490nm，观测荧光强度，结果如图2所示。
3.小麦赤霉病模型建立：
试验小麦品种为棉农6号。室外培养，保持土壤湿润。试验分为五组，分别为喷水对照组、喷不同浓度杂聚寡糖组(分别为50mg/L、100mg/L、200mg/L、500mg/L)，喷 施杂聚寡糖后两天，将禾谷镰孢菌孢子悬浮液均匀喷洒于小麦叶片表面，观察叶片感染 情况。从开始喷洒禾谷镰孢菌孢子悬浮液后开始，记录两周后小麦叶片感染情况，结果 如表2所示。
发病率＝发病株数/调查株数×100％，
病情指数＝Σ(各级病叶数×相对级数值)/(调查总叶数×最高级代表值)×100，
防效效果＝(对照病情指数-处理病情指数)/对照病情指数×100％。
表2杂聚寡糖对小麦提高赤霉病抵抗力的应用
4.数据分析
多组之间的比较用ANOVA统计学方法进行统计分析。P＜0.05被认为有显著性区别。
结合图2和表2可以看出，当小麦叶下表皮细胞没有杂聚寡糖诱导时，气孔细胞产生过氧化氢的荧光强度很弱，而终浓度为500mg/L的杂聚寡糖能明显诱导气孔细胞长生 大量过氧化氢。小麦对赤霉病的感染情况可以看出，杂聚寡糖的喷施，能提高小麦对禾 谷镰孢菌孢子的抵抗能力，喷施杂聚寡糖后，防效显著上升，发病率明显降低。由此可 见，杂聚寡糖可以成为提高植物抗病性的潜在生物农药。
实施例4
杂聚寡糖对番茄提高叶霉病抵抗力的应用
1.番茄试验模型的建立
试验番茄品种为惠砧一号。实验室培养条件为：12h光照-12h黑暗，恒温25℃，并保持土壤湿润。
2.番茄叶片抗性相关酶的酶活测定
试验分为三组，分别为空白对照组(即以水为对照)、低浓度杂聚寡糖组(寡糖终浓度为50mg/L)、高浓度杂聚寡糖组(寡糖终浓度为150mg/L)。喷施杂聚寡糖后，分 别于4h、24h、72h取样，测定相关蛋白酶酶活。
(1)粗酶液提取：25mg叶片迅速在液氮中研磨成极细的粉末，加入500μL蛋白提 取液(包含20mM PBS buffer、5mM EDTA·2Na、2.5mM DTT、2.5mM Na2S2O3·5H2O、 0.5％pvp)。
(2)葡聚糖酶活性测定：反应液中含200μL1％昆布多糖，100μL粗酶提取液， 200μL50mM、pH5.0乙酸钠缓冲液，混匀后37℃反应2h，沸水浴10min终止反应；取 100μL酶解液，加入100μLDNS，沸水浴5min，然后冰水浴冷却，读取540nm吸光值。
(3)几丁质酶活性测定：反应液中含200μL1％几丁质底物，100μL粗酶提取液， 200μL50mM、pH5.0乙酸钠缓冲液，混匀后37℃反应2h，沸水浴10min终止反应；取 100μL酶解液,加入100μLDNS，沸水浴5min，然后冰水浴冷却，读取540nm吸光值。
(4)苯丙氨酸氨解酶活性测定：反应液中含100μL粗酶提取液，500μL20mM L- 苯丙氨酸溶液，400μL50mM pH8.5 Tris-HCl缓冲液，37℃反应2h；加入0.2mL 6M HCl 终止反应，加入2mL***萃取肉桂酸，3000rpm离心1min；将萃取剂移入干净的5mL 离心管中，重复萃取一次，合并萃取液，氮气吹干；加入0.5mL甲醇复溶，读取290nm 吸光值。结果如图3所示。
3.番茄叶霉病模型建立
试验番茄品种为惠砧一号。室外培养，保持土壤湿润。试验分为五组，分别为喷水对照组、喷不同浓度杂聚寡糖组(分别为50mg/L、100mg/L、200mg/L、500mg/L)，喷 施杂聚寡糖后两天，将黄枝孢菌孢子悬浮液均匀喷洒于番茄叶片表面，观察叶片感染情 况。从开始喷洒黄枝孢菌孢子悬浮液后开始，记录两周后番茄叶片感染情况。结果如表 3所示。
发病率＝发病株数/调查株数×100％，
病情指数＝Σ(各级病叶数×相对级数值)/(调查总叶数×最高级代表值)×100，
防效＝(对照病情指数-处理病情指数)/对照病情指数×100％。
表3杂聚寡糖对番茄提高叶霉病抵抗力的应用
4.数据分析
多组之间的比较用ANOVA统计学方法进行统计分析。P＜0.05被认为有显著性区别。
结合图3和表3可以看出，当番茄叶片喷施杂聚寡糖后，几丁质酶在72h时的酶活有显著性提高，而葡聚糖酶和苯丙氨酸氨解酶在24h时酶活已经有显著提高。对番茄喷 施杂聚寡糖，能提高番茄对黄枝孢菌孢子的抵抗能力，喷施杂聚寡糖后，防效显著上升， 发病率明显降低。由此可见，杂聚寡糖可以成为提高植物抗病性的潜在生物农药。
实施例5
杂聚寡糖对马铃薯提高疫霉病抵抗力的应用
1.马铃薯试验模型的建立
试验马铃薯品种为费乌瑞它。实验室培养条件为：12h光照-12h黑暗，恒温25℃，并保持土壤湿润。
2.马铃薯致病疫霉侵染试验
试验分为三组，分别为空白对照组(即以水为对照)、低浓度杂聚寡糖组(寡糖终浓度为50mg/L)、高浓度杂聚寡糖组(寡糖终浓度为500mg/L)。试验前，将马铃薯叶 片放置在平板上，保持叶柄处湿润，防止叶片枯萎。在叶片上喷施不同浓度的杂聚寡糖， 放置于20℃保湿培养，36h后将致病疫霉菌片贴在马铃薯叶片上，避光保持24h，之后 12h光照-12h黑暗培养，两天后观察叶片贴菌片处感染情况，结果如图4所示。
3.马铃薯致病疫霉侵染大田试验
试验分为四组，分别为空白对照组(即以水为对照)、低浓度杂聚寡糖组(寡糖终浓度为50mg/L)、中浓度杂聚寡糖组(寡糖终浓度为250mg/L)、高浓度杂聚寡糖组(寡 糖终浓度为500mg/L)。从开始喷洒致病疫霉孢子悬浮液后开始，每隔一周，记录马铃 薯叶片感染情况，连续记录三周。
4.数据分析
结合图4和表4可以看出，当马铃薯叶片喷施杂聚寡糖后，在用治病菌片侵染时，没有用杂聚寡糖的对照组可以看到叶片感染严重，而喷施杂聚寡糖的叶片感染轻微甚至没有感染。杂聚寡糖喷施马铃薯后，能提高马铃薯对致病疫霉的侵害，喷施杂聚寡糖后， 防效显著上升，发病率显著下降。由此可见，寡糖能很好的诱导马铃薯叶片产生抗性， 提高抗病能力，可作为一种潜在的生物农药。
表4杂聚寡糖对马铃薯提高晚疫病抵抗力的应用
实施例6
杂聚寡糖对苹果提高褐斑病抵抗力的应用
1.苹果试验模型的建立
试验苹果品种为红富士。室外培养，保持土壤湿润，试验时期为叶子繁茂、结苹果之前。
2.苹果盘二孢侵染试验
试验分为三组，分别为空白对照组(即以水为对照)、低浓度杂聚寡糖组(寡糖终浓度为50mg/L)、高浓度杂聚寡糖组(寡糖终浓度为500mg/L)。喷施杂聚寡糖后两天， 将苹果盘二孢孢子悬浮液均匀喷洒于苹果树叶片表面，观察叶片感染情况。从开始喷洒 苹果盘二孢孢子悬浮液后开始，每隔一周，记录苹果树叶片感染情况，连续记录三周。 结果如表5所示。
表5杂聚寡糖对苹果提高褐斑病抵抗力的应用
3.数据分析
结合表5可以看出，当苹果树叶片喷施杂聚寡糖后，在用致病菌孢子悬浮液侵染时， 没有用杂聚寡糖的对照组可以看到叶片感染严重，而喷施杂聚寡糖的叶片感染轻微甚至 没有感染。对苹果树喷施杂聚寡糖，能提高苹果树对苹果盘二孢的抵抗能力，喷施杂聚寡糖后，发病率明显降低，由此可见，杂聚寡糖可以成为提高植物抗病性的潜在生物农 药。
实施例7
杂聚寡糖对草莓提高青枯病抵抗力的应用
1.草莓试验模型的建立
试验草莓品种为宁馨。室外培养，保持土壤湿润，试验时期为叶子繁茂、结草莓之前。
2.草莓青枯假单胞杆菌侵染试验
试验分为五组，分别为喷水对照组、喷不同浓度杂聚寡糖组(分别为50mg/L、100mg/L、200mg/L、500mg/L)，喷施杂聚寡糖后两天，将草莓青枯假单胞杆菌菌悬液 均匀喷洒于烟草叶片表面，观察叶片感染情况。从开始喷洒青枯假单胞杆菌菌悬液后开 始，记录两周后草莓叶片感染情况。结果如表6所示。
发病率＝发病株数/调查株数×100％，
病情指数＝Σ(各级病叶数×相对级数值)/(调查总叶数×最高级代表值)×100，
防效效果＝(对照病情指数-处理病情指数)/对照病情指数×100％。
表6杂聚寡糖对草莓提高青枯病抵抗力的应用
3.数据分析
结合表6可以看出，当草莓叶片喷施杂聚寡糖后，在用青枯假单胞杆菌菌悬液侵染时，没有用杂聚寡糖的对照组可以看到叶片感染严重，而喷施杂聚寡糖的叶片感染轻微 甚至没有感染。对草莓喷施杂聚寡糖，能提高草莓对青枯假单胞杆菌的抵抗能力，喷施 杂聚寡糖后，发病率明显降低，由此可见，杂聚寡糖可以成为提高植物抗病性的潜在生 物农药。
Claims (8)
1.杂聚寡糖，其特征在于，含有7个D-葡萄糖残基和1个D-半乳糖残基，每个杂聚寡糖分子含有或不含有一分子的丙酮酰基团，和一分子的琥珀酰基团，其结构如下所示：
所述的杂聚寡糖通过将保藏编号为CCTCC NO:M 2018797的土壤杆菌Agrobacteriumsp.ZCC3656生产的胞外多糖Riclin经β-葡聚糖酶酶解后制得。
2.根据权利要求1所述的杂聚寡糖的制备方法，其特征在于，所述的杂聚寡糖通过将保藏编号为CCTCC NO:M 2018797的土壤杆菌Agrobacterium sp.ZCC3656生产的胞外多糖Riclin经β-葡聚糖酶酶解后制得。
3.根据权利要求2所述的制备方法，其特征在于，具体步骤为：
（1）多糖Riclin的酶解：
将保藏编号为CCTCC NO:M 2018797的土壤杆菌Agrobacterium sp.ZCC3656生产的胞外多糖Riclin溶于水溶液中，加入β-葡聚糖酶混匀后，于60℃恒温水浴中反应，震荡混匀直至酶解完全；
（2）杂聚寡糖的纯化：
将步骤（1）酶解后的溶液离心去除不溶性杂质，取上清液加入体积比为4:1的氯仿-正丁醇混合溶剂，去除溶液中的蛋白，剧烈振荡，静置分层，取水相离心去除蛋白层，保留上清液，重复数次上述步骤直至完全去除蛋白层，最后加入95%的乙醇析出杂聚寡糖。
4.根据权利要求1所述的杂聚寡糖在提高植物抗病性中的应用。
5.根据权利要求4所述的应用，其特征在于，所述的植物为烟草、小麦、番茄、马铃薯、苹果、草莓、水稻或大豆。
6.根据权利要求4所述的应用，其特征在于，所述的植物抗病性为对花叶病毒感染的抗病性、对禾谷镰孢菌感染的抗病性、对黄枝孢菌感染的抗病性、对致病疫霉菌感染的抗病性、对苹果盘二孢菌感染的抗病性或对青枯假单胞杆菌感染的抗病性。
7.植物抗病诱导剂，其特征在于，有效活性成分包含权利要求1所述的杂聚寡糖，其浓度为5～5000mg/L。
8.根据权利要求7所述的植物抗病诱导剂，其特征在于，所述的杂聚寡糖的浓度为50mg/L～200mg/L。
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| CA1173771A (en) * | 1980-05-21 | 1984-09-04 | Roger E. Cripps | Fluid displacement with heteropolysaccharide solutions, and the microbial production of heteropolysaccharides |
| GB8811365D0 (en) * | 1988-05-13 | 1988-06-15 | Shell Int Research | Process for preparing heteropolysaccharide & microorganisms for use in such process |
| JPH0689041B2 (ja) * | 1988-09-06 | 1994-11-09 | 理化学研究所 | オリゴガラクチュロン酸の製造法 |
| FR2934943B1 (fr) * | 2008-08-12 | 2011-06-17 | Algieplus | Utilisation d'apiogalacturonanes et de ses derives pour la stimulation des reactions de defense et de resistance des plantes contre le stress biotiques et abiotiques |
| CN102657169A (zh) * | 2012-05-07 | 2012-09-12 | 江苏瑞光生物科技有限公司 | 一种β-1,3葡聚寡糖植物抗病疫苗的使用方法 |
| CN105566510B (zh) * | 2015-12-18 | 2018-01-05 | 南京理工大学 | 一种葡寡糖及其制备方法和应用 |
| WO2019055717A1 (en) * | 2017-09-13 | 2019-03-21 | Evolve Biosystems, Inc. | OLIGOSACCHARIDE COMPOSITIONS AND THEIR USE DURING THE TRANSIENT PHASES OF INTESTINAL MICROBIOMA OF A MAMMALIAN |
| CN111286466B (zh) * | 2018-12-07 | 2023-05-05 | 南京理工大学 | 土壤杆菌及其所产抗炎症反应胞外多糖 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2665168A1 (fr) * | 1990-07-30 | 1992-01-31 | Rhone Poulenc Chimie | Composition comportant un succinoglycane. |
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| EP4206232A1 (en) | 2023-07-05 |
| US20230303724A1 (en) | 2023-09-28 |
| CN114605564A (zh) | 2022-06-10 |
| WO2022121962A1 (zh) | 2022-06-16 |
| EP4206232A4 (en) | 2024-03-06 |
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