【作者】 卢忠燕；

【导师】 夏庆友； 鲁成；

【作者基本信息】 西南农业大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 潘庆中(1992)发现伴性赤蚁(sch)胚胎期具有温敏致死性：在常规催青环境中能正常孵化的赤蚁，若用高温干燥环境催青，则孵化率很低。为探讨sch致死(孵化不能)的机理，本文首先分析了催青期温湿度，尤其是湿度对sch的影响和作用机制；并利用双向电泳技术和指纹质谱法对与sch有关的蛋白质进行了分析。 1 催青期温湿度对伴性赤蚁sch系统致死性的影响 不同催青条件对不同家蚕系统处理结果表明，温度、湿度因子对赤蚁sch孵化抑制均有较大的独立作用，分别表现出一定的“温度敏感性”和“湿度敏感性”。温湿度的联合作用(高温干燥)对sch的孵化抑制更强，sch具有温湿度敏感致死性。 高温干燥或干燥单独作用均引起胚子的水分异常散失。“温度+干燥”对胚子水分影响最大，失水达到了16％-18％；常温干燥处理，失水率约为5-9％，与常规催青下的失水率也有较大差异。高温干燥催青下赤蚁sch孵化率最低，常温干燥处理其孵化也受到较大影响。高温干燥造成蚕卵水分的变化，水分(干燥)和孵化率之间有一定的关系。“湿度敏感性” 的基础应是水分。 常规催青下，家蚕己2期前其水分散失有相同的趋势，失水率曲线都呈直线上升；催青至己3、己4期，失水率呈突然下降的趋势，下降了3％-5％，说明胚胎发育中己3至己4期保持低水分散失可能是胚胎发育正常的生理要求。己3至己4期是家蚕对湿度(水分)最敏感的时期。 不同家蚕系统催青后失水率有一定差异。高温干燥和常温干燥处理后夏sch的失水率比较高，N100在高温干燥和常温干燥处理后失水率最低，表明系统不同(赤蚁、黑蚁)，抵抗高温、干燥的能力有差异。 伴性赤蚁sch受温度、湿度的影响其孵化抑制为不可逆过程。高温、干燥处理后，常温常湿保护至孵化，并不能使蚕卵在异常条件下所散失的水分得到恢复，并使蚁蚕孵化，表现出“性状固定”的现象。 不同家蚕品系在不同催青条件下，其孵化前的含水率有一致的趋势；高温干燥处理对蚕卵体内水分影响最大，蚕卵含水率最低，常温干燥含水率较低，高温高湿含水率比较高。不同家蚕系统，高温干燥和常温常湿催青其孵化前的含水率变化有较大不同，高 bW＄kXW2002 RWf＃gr＆J 温于燥和常温常湿催青其sob的含水率差异最大，说明高温干燥对Sob孵化前的水分影 响最大，水分降低最甚。 夏SCh的水分散失临界温度大致为43’C，秋白为48’C，秋白X夏Sob为53，C左右。 表明夏sCb对温度最敏感，其水分的散失受温度的影响明显大于秋白和秋白X夏sCh。 关于 Sob的致死机理推论如下：SCh在己 3至己 4期对温湿度敏感，受到异常条件（高 温、干燥）的冲击，将获得“孵化不能”性状并固定。温度、湿度可以速过影响胚胎体 内水分平衡而引起这种孵化不能，表现出一定的“温湿度敏感性＼ 2 Sob蚕胚胎朋致死性的差异蛋白分析及质谱鉴定 利用双向电泳技术分析了Sob、la和夏芳在常温常湿、高温干燥催青条件下的蚕 卵蛋白质差异，赤蚁SCh和la中分子量大约50kD左右的两个蛋白点P3干、P34有明 显差异，与常温常湿条件下的相比，高温于燥下这两个蛋白点浓度较弱，表达量较低。 夏芳在不同催青条件下，P3亿和 P3叫蛋白点的大小、表达量基本没有差异，其表达量 较高。说明P3刁、P3叶与sCh的孵化抑制有关。 对P3一进行胰蛋白酶消化并测定其肽质量指纹谱，其主要多肽片段为：923．687、 1397．654、1505．669、1791．797、1828刀15、1896．938 2023刀15、2065．1899、2212．155、 2384．009、2466．165、3256．448Dao 通过对建立的家蚕质谱数据库（Digest Database）进行查询，发现序列号为 6906263 （Genebank）基因所编码的蛋白质的胰蛋白酶消化位点与P3A质谱结果有车个位点相 同，且成直线分布，说明P3＊可能与6906263基因所编码的蛋白质为同一蛋白。经同 源性比较，序列号为6906263的氨基酸序列与果蝇La蛋白的序列相似性为50％，其蛋 白质分子量为44．884kD，等电点为7．07；与实验测出的P34分子量so kD比较接近。 因此P3＊蛋白有可能为La蛋自或与La蛋白结构相似的一种蛋白质。更多还原

【Abstract】 Pan Qingzhong (1992) found that the embryo of sex-linked chocolate sch had temperature-sensitive lethality. When it is incubated under high temperature and low humidity, the hatching rate is very low, while under normal conditions, the hatching rate is as high as 95%. To explore lethal mechanism of sch , the paper analyzes effect of temperature and humidity, especially humidity on sch and analyzes proteins related to sch lethality by applying two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry. 1 The effects of high temperature and low humidity on the lethality oTschHatching rate under different incubations was investigated. It shows that temperature and humidity largely affect sch hatching, and the sch has temperature and humidity sensitivity. Combination effect of high temperature and low humidity on sch is larger than that of single.Water loss rate of embryo is abnormal under low humidity. Water loss rate of silkworm eggs under high temperature and low humidity is as high as 16%-18% and under normal temperature and low humidity is about 5%-9% while both were higher than that of normal condition. The hatching rate of sch is lowest under high temperature and low humidity and is also affected largely under normal temperature and low humidity. So water (humidity) relate to hatching rate. Water of sch may be the foundation of humidity sensitivity.Under normal condition water loss of silkworm eggs before the Ji2 stage has a similar trend. The loss rate increase gradually. Water loss rate of silkworm eggs decreases abruptly by 3%-5% in the Ji3 stage. It shows that low water lost may be the physiological demand of normal growth of the embryo, and the most sensitive stage of embryo development to humidity is between Ji3 and Ji4 stage.Water loss rate of different variety have difference. Water lost rate of Xia sch is higher under normal temperature and low humidity or high temperature and low humidity and it of N100 is lowest under low humidity or high temperature. It shows that ability of different variety resistant to high temperature and low humidity is different.The effects of high temperature and low humidity on the lethality of sch are an irreversible process. Although incubated under normal temperature and humidity after treated under high temperature and low humidity, silkworm eggs can’t restore water lost under abnormal conditions and thus can’t hatch.Water content of silkworm eggs before hatching is determined under four conditions. It shows that the treatment of high temperature and low humidity has largest influence on sch water of silkworm eggs andwater content rate of silkworm eggs is lowest. Water content rate of silkworm eggs is lower under normal temperature and low humidity and is higher under high temperature and high humidity. The difference of water content of sch between normal condition and under abnormal condition is large. So effect of water under high temperature and low humidity is largest.Critical temperature of water loss of silkworm egg was investigated. Critical temperature of water lost of Xia sch, Qiubai and Qiubai xXia sch is 43癈, 48癈, 53癈 respecially. Xia sch is most sensitive to temperature and its water loss is larger than that of Qiubai and Qiubai xXia sch.As mentioned above, the most sensitive stage of embryo to humidity is between Ji3 and Ji4 stage, Silkworm has "inability hatching" character which can be predetermined under abnormal conditions. The "inability hatching" is result of high temperature and low humidity through affecting water equilibrium. So sch display temperature sensitivity and humidity sensitivity.2 Search of differential proteins of chocolate sch embryo and it’s mass spectrometry identificationThe difference of proteins of silkworm egg of sch* la and Xiafang under normalcondition and under high temperature and high humidity are analyzed by two-dimensional gel electrophoresis. It shows that P3-3 and P3-4 protein of sch and la under different conditions are dif更多还原