CN111925427B - 水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用 - Google Patents
水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用 Download PDFInfo
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Abstract
本发明公开了水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,所述水稻基因OsT5H的核苷酸序列如SEQ ID NO.1所示;所述应用的途径为通过缺失或沉默OsT5H基因促进水稻突变体栓质化。本发明首次发现在OsT5H基因缺失的情况下水稻栓质沉积,且耐盐性提高,可为保水抗旱型水稻种植资源的培育奠定基础。
Description
技术领域
本发明涉及植物基因工程技术领域,主要涉及水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用。
背景技术
5羟色胺,是一种在动物中常见的神经递质,主要有调节情绪、影响睡眠和免疫系统等作用。至今已经被报道存在超过90种植物中,分布在各种组织中,种属之间的含量差异较大。各种研究表明,5羟色胺在植物的整个生命周期中都有很重要的作用,从种子萌发到生长开花,果实成熟到衰老,都发挥着重要的作用。例如,植物受外界环境胁迫时,5羟色胺可以发挥去除自由基的作用。
前期研究,申请人证实了5羟色胺与水杨酸之间有着拮抗性,在水稻-褐飞虱互作这件起到了负向调控作用(Lu H.P.,Luo T.,Fu H.W.,Wang L.,Tan Y.Y.,Huang J.Z.,Wang Q.,Ye G.Y.,Gatehouse A.M.R.,Lou Y.G.,Shu Q.Y.,2018.Resistance of rice toinsect pests mediated by suppression of serotonin biosynthesis.Nature Plants,4(6):338.)。目前,研究者们对5羟色胺在植物中的作用主要围绕病害防御,作物生长发育,衰老等方面,根部发挥怎么样的功能,至今并不清楚。
发明内容
本发明提供了水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,首次发现在OsT5H基因缺失的情况下水稻栓质沉积,且耐盐性提高,可为保水抗旱型水稻种植资源的培育奠定基础。
具体技术方案如下:
本发明提供了水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,所述水稻基因OsT5H的核苷酸序列如SEQ ID NO.1所示;所述应用的途径为通过缺失或沉默OsT5H基因促进水稻突变体栓质化。
本发明通过电镜观察和荧光染色的方法发现经缺失或沉默OsT5H基因的突变体植株的根部和种皮都有较厚的栓质沉积,说明OsT5H基因的缺失或沉默使得突变体植株发生进一步地栓质化。
进一步地,水稻栓质化的组织部位为根部或种皮。
进一步地,所述应用的途径为通过缺失或沉默OsT5H基因提高水稻根部栓质单体的含量,来促进水稻突变体栓质化。
进一步地,所述栓质单体为苯丙氨酸衍生物,所述苯丙氨酸衍生物为p-香豆酸、阿魏酸4-乙基苯甲酸、香兰素、香草酸、2,4,5-三甲基苯甲酸、1-羟基环己基苯基甲酮、6,7-二羟基-4-甲基香豆素、早熟素I、5,7-二甲氧基-3-羟基黄酮和/或2',4'-二甲氧基-3-羟基黄酮。
进一步地,所述栓质单体为脂肪族组分,所述脂肪族组分为甘油、脂肪酸、初级醇、ω-羟基酸和/或α,ω-二羧酸。
本发明通过气相色谱-质谱法(GC-MS)测定野生型水稻植株和突变体水稻植株的根部栓质单体,发现突变体水稻植株中苯丙氨酸衍生物和脂肪族组分的含量均显著提高,说明缺失或沉默OsT5H基因使得突变体植株根部栓质单体的含量提高,进一步促进水稻突变体栓质化。
此外,本发明还提供了5羟色胺在抑制水稻根部栓质化中的应用。
本发明将转基因品系OsT5Hpro::OsT5H-GUS-His置于不同浓度的NaCl溶液中进行水培,发现转基因品系OsT5Hpro::OsT5H-GUS-His所表达的OsT5H-GUS融合蛋白的表达量在较低浓度的NaCl溶液中表达较高。说明5羟色胺可以作为根中栓质生物合成的抑制剂。
与现有技术相比,本发明具有以下有益效果:
本发明首次发现在OsT5H基因缺失的情况下水稻栓质沉积,且耐盐性提高,可为保水抗旱型水稻种植资源的培育奠定基础。
附图说明
图1为野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE根部和种皮的栓质化程度示意图;
其中,a为透射电子显微镜(Electron microscope)显示的栓质厚度;b为荧光黄088(Fluorol yellow)染色结果;c为苏丹红7B(Sudan7B)染色结果;四氮唑盐(Tetrazolimred)染色结果。
图2为野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE在盐溶液中的耐受性表现及转基因品系OsT5Hpro::OsT5H-GUS-His在盐溶液中OsT5H-GUS融合蛋白的表达量结果;
其中,a为野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE在水培过程中的照片;b为野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE水培时取出某一单株幼苗的照片;c为转基因品系OsT5Hpro::OsT5H-GUS-His在盐溶液中OsT5H-GUS融合蛋白的表达量结果;OsT5H-GUS–His表示OsT5H基因与报告基因GUS-His的融合蛋白;OsHSP90表示内参基因的表达量。
图3为萘乙酸处理对转基因品系OsT5Hpro::OsT5H-GUS-His的OsT5H-GUS融合蛋白表达的影响结果;
其中,a和b为萘乙酸处理前(a)和后(b)转基因品系OsT5Hpro::OsT5H-GUS-His植株根部GUS染色结果的图片;c为不同浓度的萘乙酸处理后转基因品系OsT5Hpro::OsT5H-GUS-His的OsT5H-GUS融合蛋白的表达量,OsT5H-GUS–His表示OsT5H基因与报告基因GUS-His的融合蛋白;OsHSP90表示内参基因的表达量;d为不同浓度的萘乙酸处理后转基因品系OsT5Hpro::OsT5H-GUS-His的OsT5H-GUS融合蛋白的活性。
图4为野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE根中栓质成分的分析结果;
其中,a为野生型嘉浙B在溶剂浸提根解聚释放的栓质单体的气相色谱分析,经质谱鉴定的脂肪类化合物名称已经标注在图上;b为a图中17-33分钟的气相图的放大图,主要是栓质单体的苯丙氨酸衍生物。其鉴定的化合物如下:①:4-Ethylbenzoic acid(4-乙基苯甲酸)、②:Vanillin(香兰素)、③:Vanillic acid(香草酸)、④:Benzoic acid,2,4,5-trimethoxy(2,4,5-三甲基苯甲酸)、⑤:Methanone(1-hydroxycyclohexyl)phenyl(1-羟基环己基苯基甲酮)、⑥:6,7-Dihydroxy-4-methylcoumarin(6,7-二羟基-4-甲基香豆素)、⑦:Precocene I(早熟素I)、⑧:p-Coumaric acid(p-香豆酸)、⑨:Ferulic acid(阿魏酸)、⑩:5,7-Dimethoxy-3-Hydroxyflavone(5,7-二甲氧基-3-羟基黄酮)、2′,4′-Dimethoxy-3-Hydroxyflavone(2',4'-二甲氧基-3-羟基黄酮),c为上述苯丙氨酸衍生物在野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和突变体OsT5H-OE中的含量;d为⑨Ferulic acid(阿魏酸)的含量,发现其含量相比别的苯丙氨酸衍生物的含量更高。
图5为野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE中栓质单体中各种脂肪酸含量的分析结果;
其中,a为甘油,b为初级醇,c-d为不同碳链长度的脂肪酸,e为α、ω-二羧酸,f为ω-羟基酸。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
实施例中所涉及的分子生物学和生物化学方法均为已知的技术,主要参考由Ausubel编写的Current Protocols in Molecular Biology,以及Green MR和Sambrook J编写的Molecular Cloning:ALaboratory Mannual,4th ED.。实施例中所用的实验材料如无特殊说明均为市售产品。
下列实施例中涉及的western印迹法,具体方法为:将研磨均匀的幼苗根部组织加入至蛋白质提取液中(50mM Tris–HCl,pH 7.4;2mM EDTA;2%Triton X-100;1mM PMSF;5%mercaptoethanol;20mM NaF).取上清,进行SDS-PAGE,进行蛋白分离,湿转,将蛋白转移至PVDF膜,经一抗,二抗孵育之后进行曝光。
其他未详细说明的实验方法均为现有的常规实验方法。
实施例1
1、实验材料
本实例中采用的野生型水稻材料是嘉浙B(简称WT),并通过辐射诱变获得5羟色胺缺失的突变体嘉浙LM,该突变体在5羟色胺合成基因OsT5H中缺少一个碱基“G”,导致移码突变;另外,在野生型粳稻品种锡稻一号Xi1(简称Xi1)的基础上,通过CRISPR获得5羟色胺合成基因OsT5H的沉默品系OsT5H-KO,过表达品系OsT5H-OE,以及转基因品系OsT5Hpro::OsT5H-GUS-His;该转基因品系OsT5Hpro::OsT5H-GUS-His的产物是OsT5H-GUS的融合蛋白,可以通过测定GUS酶活的方法,来表征OsT5H基因的表达水平和对外界环境处理的响应程度。
上述制备突变体嘉浙LM的方法来自γ射线诱变;制备5羟色胺合成基因OsT5H的沉默品系OsT5H-KO,过表达品系OsT5H-OE,以及转基因品系OsT5Hpro::OsT5H-GUS-His是通过将相应的DNA片段连接进载体pCAMBIA1301之中,并通过农杆菌介导转化水稻愈伤组织获得(制备过程均采用现有技术手段)。
2、5羟色胺合成基因OsT5H与水稻栓质化的关系
2.1突变体水稻根部和种皮的栓质化程度
探究野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE根部和种皮的栓质化程度,具体方法如下:
(1)获取野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE的根部切片,直接用电子显微镜进行观察(图1a)。
(2)在野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE的根部切片上滴上荧光黄溶液(0.01%(w/v),用高温处理1h(70℃),清洗,并用带有GFP过滤器的外荧光显微镜观察(图1b)。
(3)取野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE的成熟种子,在室温下用含有0.01%(w/v)Triton X-100和10%(v/v)商业漂白剂的水中24小时,使种子褪色。用蒸馏水和100%乙醇依次漂洗后,用氯仿:甲醇(2:1,v/v)孵育30min,用100%乙醇漂洗,风干。种子最终在室温下用聚乙二醇400中的苏丹红7B溶液中染色1至4小时,用水冲洗(图1c)。
(4)取野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE的干种子,在1%(w/v)四氮唑红(2,3,5-三苯基四氮唑)的水溶液中在黑暗中培养4至48小时(图1d)
结果如图1所示:图1a中,与野生型嘉浙B和野生型粳稻Xi1相比,突变体嘉浙LM和突变体OsT5H-KO根部切片的栓质化程度明显增强;图1b中,突变体嘉浙LM和突变体OsT5H-KO根部切片荧光黄的信号显著高于野生型嘉浙B和野生型粳稻Xi1。图1c中,苏丹7B可以结合显示重红色(附图中根据颜色深浅来体现)的脂肪族结构域,说明突变体嘉浙LM和突变体OsT5H-KO的种皮含有较厚的栓质。图1d中,较高含量的栓质可以抵抗突变体对四氮唑盐的渗透性,呈现出颜色降低;所以突变体嘉浙LM和突变体OsT5H-KO的种皮颜色明显浅于于野生型嘉浙B和野生型粳稻Xi1。
2.2突变体水稻的耐盐性以及盐溶液中OsT5H-GUS-His融合蛋白的表达量规律
探究突变体OsT5H-KO幼苗和过表达植株OsT5H-OE幼苗在盐溶液中的耐受性,具体方法如下:
(1)将野生型粳稻Xi1、突变体OsT5H-KO幼苗和过表达植株OsT5H-OE幼苗置于浓度为500mmol/L的NaCl溶液中进行水培,观察幼苗形态(图2a和图2b)。
(2)在不同浓度(0、1、10、50、100、500、1000mmol/L)的NaCl溶液中培养转基因品系OsT5Hpro::OsT5H-GUS-His,并采用western印迹法检测转基因品系OsT5Hpro::OsT5H-GUS-His根部OsT5H-GUS融合蛋白的表达量(图2c);
结果如2所示:从图2a和图2b可以看出,过表达植株OsT5H-OE幼苗的枯萎程度明显高于突变体OsT5H-KO幼苗,突变体OsT5H-KO幼苗表现出更强的抗性,而过表达植株OsT5H-OE表现出更高的敏感性;图2c中可以看出,转基因品系OsT5Hpro::OsT5H-GUS-His的OsT5H-GUS融合蛋白的表达量在较低浓度的NaCl溶液中表达较高。
由此说明,5羟色胺可以作为根中栓质生物合成的抑制剂。
2.3萘乙酸处理对水稻根系OsT5H-GUS融合蛋白表达量的影响
探究萘乙酸处理对转基因品系OsT5Hpro::OsT5H-GUS-His的OsT5H-GUS融合蛋白表达量的影响,具体方法如下:
(1)将转基因品系OsT5Hpro::OsT5H-GUS–His的植株幼苗种植在含有萘乙酸的水溶液中水培12小时,取出后,对转基因品系OsT5Hpro::OsT5H-GUS-His的根系进行GUS染色,GUS染色缓冲液如下:50mM NaH2PO4·2H2O,50mM Na2HPO4·12H2O,Na2-EDTA 10mM,372mg,TritonX-100(0.1%V/V),K3[Fe(CN)6]铁氰化钾0.5mM,K4[Fe(CN)6]亚铁氰化钾0.5mM。X-Gluc母液(20mM):染色缓冲液=1:9。37温度下,反应12小时,即可观察到蓝色染色。
(2)采用western印迹法检测不同萘乙酸处理浓度下转基因品系OsT5Hpro::OsT5H-GUS–His植株幼苗根部的OsT5H-GUS融合蛋白的表达量和活力。
结果如图3所示,经萘乙酸(NAA)处理后,转基因品系OsT5Hpro::OsT5H-GUS–His的侧根增多,并且OsT5H-GUS融合蛋白在根尖或各侧根尖处的表达量减少,栓质合成沿着根轴从顶端到基部增加,根尖几乎没有栓质成分(图3a)。经NAA处理后,侧根出现位置靠前,意味着栓质区域也向前,OsT5H基因或5羟色胺可作为栓质化抑制剂,栓质的形成意味着OsT5H基因的降低表达。这种表型也可以在每个侧根上表现出来,NAA处理促进OsT5H向根尖集中。NAA处理之后,无论是从蛋白水平还是从酶活角度都表现出了抑制OsT5H的趋势。
3、栓质单体含量的测定以及栓质成分分析
(1)利用气相色谱-质谱法(GC-MS)测定野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE根部的栓质单体的含量。
(2)对野生型嘉浙B、突变体嘉浙LM、野生型粳稻Xi1、突变体OsT5H-KO和过表达植株OsT5H-OE根部的栓质成分分析。
具体方法为:取4周大的水稻的根,用水彻底清洗,然后用纸巾擦干。新鲜采集的根组织在80℃下用热异丙醇浸泡10min。冷却后,用CHCl3:CH3OH(2:1,v/v)、CHCl3:CH3OH(1:1,v/v)、CHCl3:CH3OH(1:2,v/v)和CH3OH连续提取可溶性脂质24小时,在转速为33rpm的转轮上进行。样品在通风橱中在室温下干燥2d,然后在干燥器中干燥2d。称量干燥后的残留物,用含2%(v/v)2的甲醇中用1M硫酸在85℃下进行3h的转化甲基化解聚,2-二甲氧基丙烷,内标物为10μg十七烷酸(C17:0)和10μg十五醇(C15:0-OH)。冷却后,加入3ml的NaCl(2.5%,w/v),在正己烷中提取两次释放的脂肪酰链,以提取根部的残余脂质。用3mL盐水溶液(200mMNaCl和200mM Tris,pH 8.0)洗涤一次提取物,在温和的氮气流下干燥,并溶解在150μLBSTFA-TMC(N,O-双(三甲基硅烷基)三氟醚中乙酰胺:三甲基氯硅烷(BSTFA-TMCS,99:1)。游离羟基在110℃下衍生30分钟,剩余的BSTFA-TMCS在氮气下蒸发,样品溶解在己烷中,使用配有HP-5MS(30m×0.32mm×0.25μm)和火焰离子化检测器的安捷伦气相色谱仪进行分析。初始温度50℃保温4min,以7℃/min升至300℃,保温5min。
结果如图4所示,栓质解聚后,可以成功地检测出每种成分;几乎所有的脂肪酸单体在突变体嘉浙LM中都较高,突变体OsT5H-KO和过表达植株OsT5H-OE中也显示了相同的结果。
结果如图5所示,突变体嘉浙LM和OsT5H-KO植株中苯丙氨酸衍生的p-香豆酸和阿魏酸含量较高。此外,还发现了从苯丙氨酸代谢介质产物衍生的其他化合物,虽然这些类似物化合物尚未在栓质中得到证实,也许是水稻栓质特有的化合物,均表现出突变体嘉浙LM和OsT5H-KO的含量较高。脂肪族组分也表现出相同的结果。不仅甘油和脂肪酸(图5a,图5c-d),而且初级醇(图5b)、ω-羟基酸(ω-OH酸)(图5f)、α,ω-二羧酸(α,ω-二酸)(图5e)也显示出较高的水平。
序列表
<110> 浙江大学
<120> 水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2026
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 1
gttcatacca acacatctcc attgctgctt aagtttcttg gaccaaacgt gcaccccaag 60
tgttcgacga tatggagctc accatggcgt cgacgatgtc gctcgcgctg ctcgtgctct 120
ccgcggcgta cgtgttggtc gcgttgagga ggagccggtc gtcgtcgtca aagccacggc 180
ggctgccgcc gtcgccgccg gggtggccgg tgatcgggca cctccacctc atgtccggca 240
tgccgcacca cgcgctggcc gagctggcgc gcaccatgcg cgcgccgctg ttccggatgc 300
ggctggggag cgtgccggcg gtggtgatct ccaagccgga cctcgcccgc gccgcgctca 360
ccaccaacga cgccgcgctg gcgtcgcggc cgcacctgct ctccggccag ttcctgtcgt 420
tcggctgctc cgacgtgacg ttcgcgccgg cggggccgta ccaccggatg gcgcgccgcg 480
tggtggtgtc ggagctcctg tcggcgcgtc gcgtcgccac gtacggcgcc gtcagggtca 540
aggagctccg ccgcctgctc gcgcacctca ccaagaacac ctcgccggcg aagcccgtcg 600
acctcagcga gtgcttcctc aacctcgcca acgacgtgct ctgccgcgtc gcgttcggcc 660
gccggttccc gcacggcgag ggcgacaagc tcggcgcggt gctcgccgag gcgcaggacc 720
tcttcgccgg gttcaccatc ggcgacttct tccccgagct cgagcccgtc gccagcaccg 780
tcaccggact ccgccgccgc ctcaagaagt gcctcgccga cctccgcgag gcctgcgacg 840
tgatcgtgga cgaacacatc agcggcaacc gccagcgcat ccccggcgac cgcgacgagg 900
acttcgtcga cgtcctcctc cgcgtccaga aatcccccga cctcgaggtc cccctaaccg 960
acgacaatct caaggccctc gtcctggtac gccattaata cccaccatta aaactctcga 1020
attgactcgc cgccattgtt gctcctcctc tgatcttgac atgacgtgac aacaaccacc 1080
aggacatgtt cgtcgccggc acggacacca cgttcgcgac gctggagtgg gtgatgacgg 1140
agctagtccg ccacccacgg atcctcaaga aggcgcagga ggaggtccgg cgagtcgtcg 1200
gcgacagcgg ccgcgtcgag gagtcccacc tcggcgagct ccactacatg cgcgccatca 1260
tcaaggagac gttccggctg cacccggcgg tgccgttgct agtgccgcgc gagtccgtcg 1320
cgccgtgcac gctgggcggc tacgacatcc cggcgaggac gcgggtgttc atcaacacgt 1380
tcgccatggg gcgcgacccg gagatctggg acaacccgct ggagtactcg ccggagaggt 1440
tcgagagcgc cggcggcggc ggcgagatcg acctcaagga cccggactac aagctgctgc 1500
cgttcggcgg cgggcggcga gggtgccccg gctacacgtt cgcgctcgcc accgtgcagg 1560
tgtcgctcgc cagcttgctc taccacttcg agtgggcgct gcccgccggc gtgcgcgccg 1620
aggacgtcaa cctcgacgag acgttcggcc tcgccacgag gaagaaggag ccgctcttcg 1680
tcgccgtcag gaagagcgac gcgtacgagt ttaagggaga ggagcttagt gaggtttaag 1740
ttaaattaag taatgattag cgatagatag atttttatta tttttattat gttttgggat 1800
aataataagg gttaaagttt tgtgcttgct aagtaacctg gaggctggag ctggtaatat 1860
tgtgactaag cagagaagca agcctgtgtg atttcgatgt aaaagtacag gatatggtgc 1920
ctaatgaata aaagaagtca ataagtggcg tatttctctt gtaacttgta agctatatcg 1980
aattttaaac tttagagatt aatttatggt tctttttgtt atgatt 2026
Claims (5)
1.水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,其特征在于,所述水稻基因OsT5H的核苷酸序列如SEQ ID NO.1所示;所述应用的途径为通过缺失或沉默OsT5H基因促进水稻突变体栓质化。
2.如权利要求1所述的水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,其特征在于,水稻栓质化的组织部位为根部或种皮。
3.如权利要求1所述的水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,其特征在于,所述应用的途径为通过缺失或沉默OsT5H基因提高水稻根部栓质单体的含量,来促进水稻突变体栓质化。
4.如权利要求3所述的水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,其特征在于,所述栓质单体为苯丙氨酸衍生物,所述苯丙氨酸衍生物为p-香豆酸、阿魏酸4-乙基苯甲酸、香兰素、香草酸、2,4,5-三甲基苯甲酸、1-羟基环己基苯基甲酮、6,7-二羟基-4-甲基香豆素、早熟素I、5,7-二甲氧基-3-羟基黄酮和/或2',4'-二甲氧基-3-羟基黄酮。
5.如权利要求3所述的水稻OsT5H基因作为负调控因子在促进水稻栓质化中的应用,其特征在于,所述栓质单体为脂肪族组分,所述脂肪族组分为甘油、脂肪酸、初级醇、ω-羟基酸和/或α,ω-二羧酸。
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