【作者】 孙守慧；

【导师】 刘宽余； 高其康； 高长启； 马玉堃；

【作者基本信息】 东北林业大学 ， 森林保护， 2003， 博士

【摘要】 本文以野蚕黑卵蜂(Telenomus theophilae)寄主识别利它素为对象，以野蚕黑卵蜂及其寄主野桑蚕(Theophila mandarina)和替代寄主家蚕(Bombyx mori)为材料，对野蚕黑卵蜂寄主识别利它素的合成、分泌、信息传递途径和活性肽以及家蚕雌蛾生殖系统中具有利它素免疫活性和生物活性的组分进行了的研究。主要结果如下： 1、利用透射电镜免疫金定位技术的研究结果表明，在家蚕和野桑蚕雌蛾性附腺分泌部分泌细胞中的内质网上、高尔基体上、分泌颗粒中、微绒毛上、贮液囊中和腺腔分枝、分泌部腺腔中、贮存部腺腔中以及产出卵表层上都有胶体金特异性标记的现象，再结合家蚕和野桑蚕卵壳的ELISA标记结果，总结归纳了野蚕黑卵蜂寄主识别利它素的合成、分泌及信息传递的途径：野蚕黑卵蜂寄主识别利它素蛋白在寄主和替代寄主雌蛾性附腺分泌部分泌细胞中的内质网上合成、高尔基体上再修饰后，经过分泌颗粒的浓缩与传递，被贮液囊周围的微绒毛所吸收，通过分泌细胞腺腔分枝到分泌部腺腔中，再由分泌部腺腔逐渐汇聚到管状的贮存部腺腔中。在雌蛾产卵时，分泌到中输卵管，覆盖在寄主卵上，随寄主卵一起排出体外。 2、野蚕黑卵蜂寄主识别利它素经凝胰乳蛋白酶酶切后主要分为两大部分，一部分为结晶区域(沉淀)，另一部分为无定形区域(上清)。家蚕利它素结晶区域电泳的条带随着酶解时间的延长逐渐从高分子量向低分子量移动，低分子量区的条带越来越显著，酶解70小时后有4条带，分子量分别是8.8kD、9.5kD、12.8kD和14.4kD。野桑蚕利它素结晶区域电泳的条带同家蚕很相似，酶解70小时后也是4条带，分子量分别是9.4kD、10.3kD、12.6kD和14.2kD。 3、野蚕黑卵蜂寄主识别利它素酶解产物的ELISA、DIGFA和生物测定结果一致，结晶区域和无定形区域都有免疫反应和生物活性，进一步用Western blotting检测，在结晶区域和无定形区域都有免疫活性组分存在。这些组分是候选的野蚕黑卵蜂寄主识别利它素的活性肽。家蚕和野桑蚕的结晶区域氨基酸组成非常相似，它们都至少由6种氨基酸组成，百分比排列顺序依次都是酪氨酸、赖氨酸、谷氨酸、甲硫氨酸、甘氨酸和丝氨酸，家蚕和野桑蚕结晶区域3种最主要氨基酸的摩尔百分含量分别是：酪氨酸52.4％和53.3％、赖氨酸13.3％和22.1％、谷氨酸12.3％和9.5％。 4、为了进一步研究野蚕黑卵蜂寄主识别利它素，我们对其主要来源----家蚕和野桑蚕的雌蛾性附腺的粗提物用双向电泳进行了分离，结果分析表明，二者蛋白组分非常相似，均显示5个主要斑点，分布在分离胶的上、中、下部，蛋白含量最高的组分位于分离胶的上部，pH值在3.5-6.0之间。为了深入研究野蚕黑卵蜂寄主识别利它素的组分构成及其分子结构，建立了反相色谱的最佳分离条件，分别对家蚕和野桑蚕的雌蛾性附腺粗提物进行分离、纯化。两者都分离出一组亲水性的馏分和3个疏水性的馏分。对家蚕性附腺粗提物色谱分离的馏分用时间窗口进行收集，经ELISA检测各馏分都有免疫反应，但在强度上有显著差别，其中保留时间6.172分钟(10分钟分离程序)的色谱峰馏分免疫强度最高，疏水性馏分的免疫反应强度普遍高于亲水性的馏分。将性附腺粗提物与低温物理法分离的上清液的色谱图对比，确定了免疫强度最高的色谱峰对应于野蚕黑卵蜂寄主识别利它素，收集这个馏分后经SDS卫AGE进一步分离成两个组分，制备成质谱分析样品。 5、为了快速和直接地检测野蚕黑卵蜂寄主识别利它素的免疫活性，我们建立了野蚕黑卵蜂寄主识别利它素的快速斑点金免疫渗滤试验(Dotk切munogofd Filtrationassay，DIGEA)检测方法。并应用此方法成功地检测了野蚕黑卵蜂寄主识别利它素的结晶区域和无定形区域的免疫活性。 6、利用ELISA、生物测定和SDS.RAG毛的方法研究和探讨了家蚕卵巢管和解剖卵壳等非性附腺源的野蚕黑卵蜂寄主识别利它素，结果分析表明，这些物质来源于家蚕和野桑蚕的生殖系统，在含有野蚕黑卵蜂寄主识别利它素多克隆抗体上的抗原决定簇和有较野蚕黑卵蜂寄主识别利它素低的生物测定反应级数方面具有相似性。更多还原

【Abstract】 Based on the previous researches, the synthesis, secretion, the approach of information transfer and activated peptide of the Telenomus theophilae ’s kairomone and the components that had immunity and biological activity of kairomone in the procreate system of Bombyx mori female moth were further performed by using the host Theophila mandarina and the replaceable host of T. theophilae, B. mori, as insect materials. Main findings of the study were as follows:1. Detailed studies on kairomone by using the immunolocalization with colloidal gold and transmission electron microscope indicated that the specific labels existed in the secretory cells of endoplasmic reticulum, Golgi complexes, secretory materials, mcrovilli, reservior, branched of reservior, lumen of secretory portion and lumen of reservoir. Combined with the results of ELISA labels in T. mandarina and B.mori, we summarized the synthesis, secrete and information transferring approaches of the T. theophilae-host-recognition kairomone. The kairomone proteins were synthesized in the endoplamic reticulum of the secretory cells that were in the accessory gland of the host and the replaceable host female mothes. After modified in Golgi complex and condensed and transferred by secretory materials, they were absorbed by the microvilli around the reservior. Finally, they got together in the Lumen of reservoir through the Lumen embranchment of the secretory cells and the lumen of the secretory portion. When the female moth laid eggs, they would be secreted to the mid-ovariole and were excreted out of the body with the host eggs by overlaying them.2. Telenomus theophilae host-recognition kairomone hydrolyzed by chymotrypsin contained two parts; they were crystalline (precipitation) and amorphous (supernatant) regions respectively. When hydrolyzed, the crystalline region from B.mori were cleaved into 4 bands of 8.8 kD,9.5 kD,12.8 kD and14.4kD respectively on SDS-PAGE gel. The case was 9.4 kD, 10.3 kD, 12.6 kD andl4.2kD for the crystalline region from T.mandarina .3. The products of kairomone hydrolyzed by chymotrypsin with ELISA and DIGFA analysis were consistent with those by bioassay. There appeared immunity and biological activity in the crystalline and amorphous regions. Western blotting showed that there existed immunity components in the crystalline and amorphous regions. The immunity components were the functional domain where Telenomus theophilae could found the hosts. The composition of amino acids in the crystalline region from B.mori and T.theophilae was very similar. Both of them were composed of at least six amino acids .They were tyrosine, lysine, glutamic acid, methionine, lysine and serine by scale-up. The molar percentage of tyrosine, lysine and glutamic acid was 52.4, 13.3% and 2.3% respectively for the crystalline region from B.mori, and 53.3%, 22.1%, 9.5% respectively for the crystalline region from T.theophilae.4. The kairomone was separated from the female accessory gland of B. mori and T.mandarina by using 2-D electrophoresis technology. The result showed that the proteins from the two kinds of accessory glands were highly similar. Both of the proteins had 5 spots and were distributed in the upper, mid and lower parts of theseparated gel. The materials with the highest contents of protein were in the upper separated gel with the pH value varied from 3.5 to 6.0. For the sake of the investigation of the constitution and the molecular structure of the kairomone, the perfect separation condition of reversed chromatogram technology was established, by which the raw materials of female accessory gland of B. mori and T. mandarina were separated and purred. A group of hydrophilic components and three groups of hydrophobic tic components were separated from both of the two species. The components which were separated by chromatogram from the accessory gland of B, mori was collected according to time window, and tested with ELISA technology. The result showed that each component had immunity. But the i更多还原