超声波法提取木材中的阿拉伯半乳聚糖的研究

应用超声波技术浸提兴安落叶松(Larix gmelinii Rupr．)木材加工剩余物中的阿拉伯半乳聚糖。研究了影响阿拉伯半乳聚糖得率的提取条件,包括超声波功率、料液比、原料粒度和浸提时间。将超声波提取与传统提取和微波提取进行比较,并利用红外光谱和紫外吸收光谱分析浸提物。超声波法提取的粗产品在280nm处的紫外吸光度最小;产品质量最接近标准品。在单因素试验中超声波提取的粗阿拉伯半乳聚糖最高提取率为16．61％,比传统浸提法高。     

Arabinogalactan proteins may facilitate the movement of pollen tubes from the stigma to the ovules in Actinidia deliciosa and Amaranthus hypochondriacus

Sexual plant reproduction is a complex process that involves a series of interactions between the male gametophyte and the different cell types of the pistil. These interactions are believed to direct the pollen tube growth until its final target, the embryo sac. Arabinogalactan proteins are complex proteoglycans that are believed to be involved in these processes. The pistil is enriched in these highly glycosylated proteins and we provide results that show the selective presence of different AGP epitopes at the surface of the cells or in the ECM of the tissues that correspond exactly to the pollen tube growth pathway in Amaranthus hypochondriacus and Actinidia deliciosa. We also show that in Actinidia deliciosa, which is a dioecious plant with the male flowers having rudimentary ovaries where fertilization does not occur, there is no presence at all of the epitopes recognised by the monoclonal antibodies utilized in this study. This revised version was published online in July 2006 with corrections to the Cover Date.     

柱层析法纯化落叶松木材中的阿拉伯半乳聚糖

分别采用大孔吸附树脂柱层析和聚酰胺柱层析技术纯化兴安落叶松中的阿拉伯半乳聚糖，研究了两种层析柱洗脱过程中的杂质含量、糖含量随时间变化的情况，对比分析了它们对阿拉伯半乳聚糖的纯化效果。结果表明，经大孔树脂层析柱纯化后的阿拉伯半乳聚糖产品得率为68．28％，糖含量为95．02％；经聚酰胺层析柱纯化后的产品得率为75．67％，糖含量为98．30％。综合产品得率、糖含量以及成本等多方面因素，选择直接用聚酰胺层析柱来纯化阿拉伯半乳聚糖较为合适。     

落叶松阿拉伯半乳聚糖十六烷基醚的合成

以氯代十六烷和兴安落叶松提取的阿拉伯半乳聚糖（AG）为反应物，二甲亚砜为溶剂，氢氧化钠为催化剂，通过威廉森合成法制得阿拉伯半乳聚糖十六烷基醚（HEAG），探讨了时间、温度和氯代十六烷与AG的反应物配比对合成反应的影响。实验结果表明，合成时间30min、温度25℃和反应物配比为0．3：1（mL：g）时，HEAG平均取代度为0．212mmol／g，产率为65．56％。经表面张力和脂溶性的测试，表明HEAG的脂溶性比AG高，在非极性溶剂正己烷和石油醚中的溶解度分别增加了30．66％和21．95％；在极性较大的溶剂氯仿和水中溶解度分别降低了60．90％和44．24％。在水中HEAG溶解度为6．70g。     

脯氨酸羟化酶GhP4H2在棉花纤维发育中的功能研究

阿拉伯半乳聚糖蛋白(arabinogalactan proteins, AGP)在棉花纤维发育过程中发挥重要作用。AGP由富含羟脯氨酸的主链蛋白和大量Ⅱ型阿拉伯半乳聚糖(arabinogalactan,AG)侧链组成,其合成过程要经历2次翻译后修饰,首先是氨基酸主链上的脯氨酸被脯氨酸羟化酶(prolyl-4-hydroxylases,P4H)羟基化,随后在糖基转移酶(glycosyltransferases, GT)催化作用下将阿拉伯半乳聚糖或寡糖添加到羟脯氨酸残基上。我们前期利用P4Hs的抑制剂处理棉花离体胚发现,棉纤维伸长受到抑制,暗示P4H参与棉纤维生长发育过程。为深入研究P4H在棉纤维发育中的功能,我们从棉花中分离鉴定了1个在棉纤维发育过程中高量表达的脯氨酸羟化酶基因GhP4H2。本研究分别构建了GhP4H2过表达和RNA interference (RNAi)载体,通过农杆菌介导法转化棉花,获得转基因植株,发现过表达转基因棉花株系T₁～T₃代成熟棉纤维变短,AGP含量增加,AG多糖侧链也发生变化。对过表达转基因植株和野生型植株棉纤维...     

Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins (AGPs) are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.  In this study, we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.  Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes, 38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.  The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.  We further tested their expression patterns using RT-PCR and RT-qPCR.  Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads (RPKM) values during pollen tube growth, implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.  We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.  Therefore, 19 PbrAGPs (PbrAGP1 to PbrAGP19) were selected to test their influences on pollen tube growth.  Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen, and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.  Additionally, pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.  PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.  In summary, this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.     

化学杀雄剂SQ-1和阿拉伯葡聚糖蛋白对小麦品种间杂交及远缘杂交成胚率的影响

【目的】探究化学杀雄剂SQ-1对小麦品种间、小麦与近缘植物间、小麦与远缘植物间杂交成胚率的影响,以及阿拉伯葡聚糖蛋白对小麦与玉米杂交成胚率和得苗率的影响。研究结果对于合理选用小麦杂交方式,提高小麦杂交结实率和利用玉米诱导小麦单倍体植株的效率具有重要意义。【方法】通过在小麦拔节期喷施化学杀雄剂SQ-1,开花期分别授以小麦花粉和远缘植物(黑麦、玉米)花粉,并在小麦授玉米花粉后的处理液中加入不同浓度的阿拉伯葡聚糖蛋白(arabinogalactan proteins,AGP),对小麦与玉米杂交后产生的幼胚进行离体拯救培养,统计授粉小花数、接种幼胚数、膨大颖果数、结实粒数、萌发单倍体幼胚数和单倍体植株数,计算结实率、颖果膨大率、成胚率、萌发率和成苗率并对所得数据进行差异显著性分析,结合细胞学观察结果,研究SQ-1对小麦品种间杂交及远缘杂交结实性的影响,以及AGP对小麦单倍体胚诱导率的影响。【结果】在不同小麦品种间杂交中SQ-1处理结实率19.8%—83.3%,人工去雄的结实率为69.4%—93.0%,SQ-1对不同品种的影响不同,Fielder对SQ-1的反应比较敏感;在中国春与兰州黑麦杂交中,SQ-1处理的结实率为65.5%,人工去雄处理的结实率为78.8%,两种处理方式产生的F1杂种的染色体数均为28条;在不同小麦品种与玉米品种郑单58杂交中,SQ-1处理小麦单倍体胚的成胚率为1.11%—1.41%,人工去雄小麦单倍体胚的成胚率为2.38%—14.29%;在小麦与玉米杂交后的处理液中添加0.5—2.0 g·L-1 AGP一定程度地提高了小麦单倍体胚获得率和成苗率。另外,在玉米花粉诱导的单倍体胚离体培养过程中,发现13.07%的胚发育出了2—6株苗;显微镜观察发现,玉米花粉诱导后18 d左右小麦单倍体胚上出现了多个突起,这些突起在离体培养条件下进一步发育为形态健全的小植株,其染色体数目均为21条。【结论】化学杀雄剂SQ-1减低了小麦品种间杂交及小麦与黑麦、玉米间杂交的成胚率,AGP提高了小麦与玉米间杂交单倍体成胚率和成苗率。     

Effects of inter-culture,arabinogalactan proteins,and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins(AGPs) and hydrogen peroxide(H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199(KN199) and Xinchun 9(XC9), together with low-frequency regeneration wheat line Chinese Spring(CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L–1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L–1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22(JM22), Jingdong 18(JD18) and Yangmai 18(YM18), respectively; whereas at 100 mg L–1 AGP level, CS(105.44%), Chuannong 16(CN16)(80.60%) and Ningchun 4(NC4)(62.87%) acted the best. Moreover CS(79.05%), JM22(7.55%), CN16(101.87%), YM18(365.56%), Yangmai 20(YM20)(10.48%), and CB301(187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4(35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP(or H2O2) application, can be further applied to wheat transgenic research.     

多糖存在下异丁香酚的脱氢聚合及LCC的形成

以异丁香酚为木质素生物合成的前驱物,采用混合法合成了木质素脱氢聚合物(DHP)和木质素-碳水化合物复合体(LCC),同时采用红外光谱、13C NMR波谱分析聚合产物的结构,探讨了漆酶催化脱氢聚合过程中形成的DHP与多糖(阿拉伯半乳聚糖)的聚合反应机理。结果表明:在漆酶的催化作用下,异丁香酚与阿拉伯半乳聚糖发生聚合形成了DHP与LCC,13C NMR波谱结果证实DHP中以β-O-4,β-β,β-5和β-1等结构为主,而LCC的13C NMR谱图中既有来自木质素的芳香族碳原子的信息,也有来自阿拉伯半乳聚糖的共振信号。     

桃FLA家族基因的生物信息学及表达分析

从桃基因组数据库筛选出了12个假定的成束状阿拉伯半乳糖蛋白(FLA)基因序列。对其蛋白结构进行分析,结果显示PpFLA7不具有特定的阿拉伯糖或半乳糖糖基化位点,不属于典型的FLA家族。PpFLA6的氨基酸组成和保守域的分布与其他成员相比差别较大。聚类分析结果表明PpFLAs家族可以分为CladeⅠ、CladeⅡ和CladeⅢ3类。利用荧光定量PCR(qPCR)对不同组织和成熟阶段果实中的FLA进行表达分析,发现各个成员在不同时期的组织中差异化表达,其中PpFLA4在果实成熟阶段显著上调表达,暗示其可能在桃果实的成熟阶段扮演重要角色。