【作者】 吴韶辉；

【导师】 蔡妙珍；

【作者基本信息】 浙江师范大学 ， 植物学， 2009， 硕士

【摘要】 酸性土壤在全球分布广泛,由此引发的铝毒问题被认为是酸性土壤中限制作物生长最重要的因素,严重影响酸性土壤上作物的生产力。铝毒的原初效应是抑制植物的根系伸长,进而抑制植物对水分和养分的吸收。植物对铝的外部排斥机制是其耐铝的机制之一,通过外部排斥机制能使大量的铝被拒之于根表之外免遭其毒害。根尖粘液是主要由根冠细胞和边缘细胞分泌的高分子物质,其在植物外部排拆铝中所起的作用逐渐受到重视。本文采用两种不同铝耐型的大豆（Glycine max L.）浙春3号（铝敏感性品种）、浙秋2号（铝耐性品种）为材料,采用悬空培养法培养收集根冠粘液,分析粘液多糖的单糖组成和铝胁迫下大豆根尖粘液分泌量、主要成分等基本的生物学特性变化,研究根冠粘液结合铝的能力以及阻止铝进入根尖的贡献大小,以及粘液中结合铝的主要物质和根尖生理特性,揭示根冠粘液在大豆根际排斥铝的作用和机理,有望为植物铝毒及耐铝机制和遗传改良研究探索新途径。研究结果如下:（1）建立了采用三氟乙酸水解豌豆根尖粘液中的多糖,衍生化后用气相色谱分离测定6种单糖的分析方法。实验表明,根尖粘液用三氟乙酸水解并乙酰化,采用OV-17石英毛细管柱（35m×0.32 mm×0.33μm）为色谱柱,利用气相色谱法对6种单糖进行分离测定,6种糖全部达到基线分离,总分析时间为42 min。用保留时间定性,外标法定量。方法相线性范围为0.0025～2.5 mg/mL,各标准曲线相关系数r＞0.9991。检出限0.0216～0.1578μg/mL,加样回收率为96.7%～107.4%。实验结果表明本法是一种有效测定逆境胁迫下植物根尖粘液中糖类物质组成的方法。（2）通过悬空培养法对大豆进行培养,收集根尖粘液,研究铝胁迫下粘液的分泌特性。结果表明:铝胁迫诱导根尖粘液分泌,耐铝大豆浙秋2号粘液产生量高于铝敏感大豆浙春3号。粘液中含有较高含量的糖类、蛋白质、氨基酸、有机酸等物质,这些成分在浙秋2号中的含量普遍高于浙春3号。400μmol/L Al处理下,浙春3号和浙秋2号的蛋白质含量分别达到对照的4.7倍、3.9倍;氨基酸含量达对照的8.0倍、5.2倍,总有机酸含量是对照的7.0倍、3.9倍;粘液的pH呈弱碱性,在铝毒下变化较小。上述结果表明,铝毒胁迫下,糖类物质是粘液中响应最大的成分,可能是粘液中响应铝毒的主要物质。（3）对大豆根尖粘液糖类含量及组成成分进行测定,结果表明:多糖是粘液中的主要成分,占粘液成分的90%。铝胁迫引起总糖含量显著增加,浙春3号和浙秋2号经400μmol/L铝处理后总糖含量分别增加了218.2%、88.1%。粘液中的多糖主要由木糖、半乳糖、阿拉伯糖、葡萄糖、盐藻糖和甘露糖6种单糖组成。铝胁迫下其中葡萄糖和木糖的含量较高且对铝胁迫的响应程度大,可作为大豆根尖粘液多糖响应铝胁迫的标志性单糖。（4）采用悬空培养条件下去除和保留根根尖粘液法,分别测定根相对伸长率,根尖、根尖细胞壁和粘液中的铝含量以及PME活性。结果表明:保留根尖粘液时,浙春3号和浙秋2号的根相对伸长率平均比相同铝浓度处理下去除粘液处理组分别高15.8%和21.6%,表明粘液显著缓解铝毒对大豆根系生长的抑制作用。根尖保留粘液缓解铝胁迫引起的PME活性增加,浙秋2号的PME活性低于浙春3号。400μmol/L铝处理下浙秋2号粘液吸收了49.5%的铝,而浙春3号仅14.2%。尽管粘液结合了大量的铝,但是毒害形态的单核铝含量非常低,粘液单核铝仅占总铝含量的0.06%～1.16%。荧光染色结果表明根尖吸收的铝在细胞壁上的积累,保留粘液处理组的根尖荧光强度明显低于去除粘液处理组。细胞壁的铝含量测定结果也表明,去除根尖粘液后细胞壁上的铝含量显著增加。上述结果表明,根尖保留粘液能抑制铝进入根尖,减少铝与细胞壁的结合;推测粘液能够将毒害形态铝结合形成稳定的结合物或者非毒害形态,降低铝对根生长的抑制作用,缓解了铝毒害。（5）采用悬空培养条件下去除和保留根根尖粘液法,分别测定根尖细胞壁成分含量变化和根尖呼吸酶活性。去除根尖粘液引起细胞壁果胶、半纤维素1和半纤维素2增加;铝耐型的浙秋2号去除根尖粘液后各成分含量增长率较高,最高达239.4%（果胶）、194.8%（半纤维素1）和759.3%（半纤维素2）,表明粘液显著缓解由铝胁迫引起的大豆根尖细胞壁多糖组分的增加。粘液对呼吸酶活性影响结果显示,去除粘液后H⁺-ATPase和Ca²⁺-ATPase活性显著下降,浙春3号和浙秋2号的H⁺-ATPase最低分别仅为保留粘液组的84.1%、68.8%,Ca²⁺-ATPase活性最低分别仅为保留粘液组的65.2%、45.7%。上述结果暗示粘液能有效降低铝引起的细胞壁多糖含量,减少细胞壁上能与铝结合的结构,降低了细胞壁对铝的结合能力;同时稳定根尖呼吸酶活性,促进根尖细胞正常伸长。（6）采用悬空培养法培养大豆,收集根冠粘液测定其中的果胶和酚类含量。粘液中还含有能结合金属阳离子的物质如果胶、酚类。铝处理下粘液中的果胶、酚类显著增加,400μmol/L铝处理下浙春3号和浙秋2号的果胶分别比对照增加293.8%和195.7%,酚类分别比对照增长215.6%、214.0%。铝胁迫下粘液中酸性磷酸酶活性显著增加,200μmol/L时活性最高,此时浙春3号和浙秋2号分别达对照的8.9倍、10.3倍,铝浓度继续增加,酶活性下降。粘液中果胶、酚类的增加提高了粘液结合铝的能力,酸性磷酸酶活性增加促进根尖的磷代谢,代谢产生的磷酸盐具有结合铝的能力,表明果胶、酚类和酸性磷酸酶对粘液结合铝的能力起到重要作用。更多还原

【Abstract】 Aluminum（Al） toxicity is an important factor for crop production and quality in acid soil;it has become hot spots in plant stress physiology research.Al primarily affects root growth by interfering with processes critical for the regulation of growth in the root apex. The exclusion mechanism that excludes Al from entering root apex is considered one of the most important mechanisms of Al resistance.Mucilage layer is high molecular complex secreted from root cap and root border cells（BC）,which is considered to play a major role to suffer the stress of Al.In this paper,two significantly different Al genotypes of soybean （Glycine max L.） were selected as material,such as Zhechun No.3（Al sensitive genotype） and Zheqiu No.2（Al tolerant genotype） which were respectively Al-tolerant genotype and Al-sensitivity genotype.Mucilage was collected from root tips of soybean which was grown in aeroponic culture for 24 h with Al supply.Monosaccharide content of polysaccharide,the mucilage secretion quality and its primary components were examined to evaluat its capacity in binding Al.Besides,the main effective compound and characteristic of root physiological were also analysed,in order to reveal the function and mechanism of mucilage on Al resistance.Explore some new pathways for Al toxicity, Al-resistant mechanism and genetic improvement.The main results were as follows:（1）A novel gas chromatography method was developed to determine six kinds of monosaccharide units hydrolyzed by trifluoroacetic acid from polysaccharide in pea root cap mucilage.The baseline separation of the monosaccharide units were achieved with a medium polar capillary column OV-17（35 m×0.32 mm×0.33μm）.The total analysis time was 42 min.Retention times were used to identify the individual sugar in the chromatogram and external standard method was used for quantitative analysis.The linear range of the method was 0.0025～2.5 mg/mL and the correlation coefficient were over 0.9991.Minimum detectable limit at a signal-to-noise of 2:1 was 0.0216～0.1578μg/mL. The recovery ration of the six kinds of standard mono sugars was 96.7%～107.4%.The results indicated that this method was effective and quick in analyzing the components of sugars in the mucilage collected from plant root tips under adversity stress.（2） Mucilage was collected from soybean through areoponic culture to study its. characteristics.Results shown as follows,Al obviously enhanced mucilage secretion, especially in Zheqiu No.2.Abundant of sugar,protein,amino acid and organic acid composed main components of mucilage.All these were much higher in Zheqiu No.2 than in Zhechun No.3.The protein,amino acid and organic acid contents of Zhechun No.3 and Zheqiu No.2 were 4.7 and 3.9,8.0 and 5.2,7.0 and 3.9 times of control,respectively. Mucilage presented weak alkali and didn’t show much varity under Al stress.These results revealed that sugar shown significant change under Al toxicity,which might be the main Al-response compound in mucilage.（3） Sugar composed almost 90%of soluble components in mucilage.Total sugar content in Zhechun No.3 and Zheqiu No.2 increased for 215.2%and 88.1%more than control under 400μmol/L Al treatments,respectively.The polysaccharides of sugar were mainly composed of six monosaccharides,which were the same as some other kinds of species.The monosaccharides variation of polysaccharide in mucilage might be characteristic reaction under Al stress.Xylose and glucose were the primary component and significantly response to Al toxicity which could use to be symbol monosaccharide to calculate Al tress grade.（4） Relative root elongation ratio,PME activities and Al content in root,mucilage and root cell wall of soybean were detected,which were cultred by earoponic culture.Relative root elongation ratio of Zhechun No.3 and Zheqiu No.2 increased for 15.8%and 21.6%on average when mucilage was kept on root.Mucilage also debased the increasing of PME activity which was much higher in Al-sensitive genotype.Most of Al was blocked outside the root by mucilage,49.5%of total Al was held back by mucilage of Zheqiu No.2,while its only 14.2%in Zhechun No.3.Monomeric Al was most toxicity form which shown only 0.06%～1.16%of total Al in mucilage,despite the large content of total Al in muciage. Morin fluorescence stain and cell wall Al content both shown that Al content siginificantly increased when mucilage was wipped off.Al binding capacity of mucilage could prevent Al from entering root tip and meristem of root cap,and then alleviated Al toxicity to root growth.（5） Root cell wall compositions and ATPase were also detected.Erase root mucilage significantly stimulate pectin and hemicelluloses production,especially in Zheqiu No.2. Pectin,hemicelluloses 1 and hemicelluloses 2 content reached 239.4%,194.8%and 759.3%that of control in Zheqiu No.2.If mucilage was wipped off,H⁺-ATPase viability in Zhechun No.3 and Zheqiu No.2 reduced for 15.9%and 31.1%,respectively,Ca²⁺-ATPase activity decreased 34.8%and 54.3%.Results indicated that Mucilage obviously alleviated the polysaccharide increasing of cell wall caused by Al stress,which decreased combination structure in cell wall,reduced Al content in cell wall.Meanwhile,mucilage stabilized ATPase activity which related to root antioxidant system.（6） Al bingding properties such as pectin and hydroxybenzene were also detected. Pectin content in Zhechun No.3 and Zheqiu No.2 were respectively 293.5%and 195.7% higher than control under 400μmol/L treatments,while hydroxybenzene increased 215.6% and 214.0%.Acid phosphatase activity（APA） also increased for 8.9 and 10.3 times more than control in Zhechun No.3 and Zheqiu No.2（200μmol/L）.APA decreased when Al concentration kept rising.The results above indicated that the increasing of pectin and hydroxybenzene enhanced Al binding ability.At the same time,APA increased root Pi metabolize.The production phosphate can also bind Al.The results indicated that pectin and hydroxybenzene may be the main component which enabled mucilage binding ability, and AP also played important role in Al binding.更多还原