【作者】 颜海燕；

【导师】 钟伯雄；

【作者基本信息】 浙江大学 ， 特种经济动物饲养学， 2002， 硕士

【摘要】 家蚕伴性赤蚁（sch）品种在胚胎发育后期具有高温干燥敏感性，在高温干燥的催青条件下，伴性赤蚁的孵化率极低，但是赤蚁的不孵化并不是由于胚胎死亡，而是一种孵化不可能。其中对高温最敏感的时期是气管显现期（己₃），对干燥最敏感的时期是转青期（己₅），这两个时期分别称为高温敏感期和干燥敏感期，高温干燥敏感性基因定义为伴性温敏基因（t），t与Z染色体上21.5位置的sch基因紧密连锁，呈隐性伴性遗传。研究发现，伴性温敏现象在其它品种中也存在。温敏致死（孵化不可能）的分子机制仍不很清楚，本研究采用蛋白质组研究技术，通过不同催青条件下具温敏性状品种的胚胎蛋白质分析，探索温敏不孵化的分子机理。 本研究首先综合比较了磷酸缓冲液和Tris-HCl缓冲液两种蛋白质抽提缓冲液、以及由不同的离液剂、表面活性剂和还原剂所组成的六种蛋白质溶解缓冲液，发现用磷酸缓冲液抽提蚕卵蛋白质、用TCA沉淀其中的易溶性蛋白质、再用含有8M 尿素，2M硫尿，4％W／V CHAPS, 20mM Trisbase, 30mM DTE, 2％carrier amphotytes （pH 3.5-10）的溶解缓冲液溶解蛋白质是抽提家蚕胚胎蛋白质样品的较好方法，抽提的蛋白质样品经双向电泳获得的图谱比较完整、稳定。 以具伴性温敏基因的品种和正常基因品种为亲本的杂交一代，在高温干燥条件下，经质谱分析和同源性检索发现，主要差异蛋白与卵黄蛋白具有同源性，其中具温敏基因的正交组合，蛋白斑点6、蛋白斑点8与卵黄蛋白同源，而不具温敏基因的反交组合，蛋白斑点6’与cDNA文库中编号为wdV30370 T3“$”的五龄幼虫第三天的翅原基基因克隆推导的蛋白序列同源，蛋白斑点8’与卵黄蛋白同源。综合蛋白斑点6和蛋白斑点8的变化，推测是由于高温干燥的不利环境，胚胎需要提前并且快速地消耗卵黄蛋白以合成功能蛋白质，但是又由于高温干燥的影响，使得正常消耗卵黄蛋白的某些酶的合成以及其活性受到影响，卵黄蛋白在体内的吸收通道改变或被断截，氨基酸供应的正常途径受影响，其供应保证不了在高温干燥条件下合成功能蛋白质的需要，即使最终卵黄蛋白被消耗掉大部分，但是由于氨基酸供应时间的滞后造成合成功能蛋白质时间的延迟，起不到抵抗不良环境的作用，以及最终在胚胎体内的积留造成胚胎合成功能蛋白的氨基酸供应的不足以及合成的功能蛋白质的不足，引起了温敏基因品种的生理机理不完全从而导致了不孵化。蛋白斑点8的蓄积以及最终的残 留推测是在以温敏基因品种作父本的杂交一代的雌个体中存在，是引起不孵化的原 因。 为了进一步分析胚胎在高温干燥催青条件下的蛋白质变化，揭示高温干燥催青的 机理，实验将蚕卵的胚胎从卵黄、卵壳及浆膜中分离出来，单一提取胚胎蛋白质，进 行蛋白质的双向电泳。研究发现在常温常湿、高温干燥、高温多湿三种不同的温湿度 条件下，温敏基因品种的蛋白质电泳图谱各有不同，从戊3胚子到己4胚子期间，明 显差异的蛋白共有30个。推测高温干燥催青条件使温敏基因品种相应地产生了协同 机制，其中的一部分基因被抑制，而另一部分基因被激活，由此造成了家蚕胚胎发育 过程中酶的合成和活性受到影响，进而使胚胎正常的生理生化反应被改变，合成功能 蛋白质的时间和途径也受影响，从而导致了温敏基因品种的不孵化。胚胎30个差异 蛋白质可能助证了这一问题。 卵黄蛋白的降解途径和降解卵黄蛋白的酶以及胚胎的正常生理代谢发生变化后 是如何造成代谢途径的改变，使胚胎在孵化时缺乏所需的营养而导致不孵化，有待于 进一步的研究。更多还原

【Abstract】 It is discovered that the silkworm embryo of sex-linked chocolate (sch) possesses temperature sensitivity in its later development stage. Sex-linked chocolate (sch) hatched scarely when incubated under high temperature and dry condition, whereas the embryo is still alive. The stage which is most sensitive to high temperature is trachea colouring embryo(ji-3), while the stage sensitive to dry condition is body pigmentation embryo(ji-5), so these two development stages have been called high temperature sensitivity stage and dry condition sensitivity stage respectively. The gene of high temperature and dry condition sensitivity is defined as sex-linked temperature sensitivity gene(t), which exists in 21.5 location of Z chromosome linking closely to sch gene, and presents concealed heredity. This phenomenon exists in other races, but the molecular mechanism of temperature sensitivity is unknown. This research was carried out to explore the molecular mechanism of not-hatching embryo by analyzing the protein of embryo with temperature sensitivity under different incubating condition with proteome technology.By comparing two protein extraction buffers and six protein solubilization buffers consisting of different chaotrope, sulfobetaine and reducing agents, it is apparent that the best method to extract embryo protein is by using phosphate buffer and the solubilization buffer containing 8M urea,2M triourea, 4%W/V CHAPS, 20mM Trisbase, 30mM DTE, 2% carrier amphotytes (pH 3.5-10) to solve the protein of embryo.By MALDI-TOF-MS analysis and homology searching, it was found that the main specific protein in positive and negative crossing generation when crossing the race with sex-linked temperature sensitive gene and normal gene races as parents under high temperature and dry condition are homologous to Bombyx mori 6G1 gene for 3OK major plasma protein. In the positive generation, protein spot 6 and 8 are homologous to the 6G1 gene, while in the negative crossing generation, protein spot 6’ is homologous to the protein sequence which is conferred by the Bombyx mori cDNA clone wdv30370 T3 " $ " named Bombyx. mori wing disk C108 5th-instar day-3 larvae., and protein spot 8’ is also homologous to the 6G1 gene. Synthesizing the variety of protein spot 6 and 8, thus it is considered that embryo need to consume the major protein to synthesize the functional protein ahead and quickly. But at the same time ,the synthesizing activity of enzyme which is used to consume the major plasma protein normally is effected by the incubatingenvironment of high temperature and dry condition. Thus, the absorbing route of the major plasma protein is changed or cut, and the regular route of the amino acid application is also changed. It can not satisfy the need to synthesize the functional protein under high temperature and dry condition. Even if the major plasma protein is mostly consumed finally, but the embryo still can not resist the unfourable environment because of the lag time of the functional protein synthesized. The accumulation of major plasma protein in the embryo results in insufficient amino acid to synthesize the functional protein, and makes the mechanism of embryo development incomplete. It is also considered that the accumulation of protein 8 at last exists in the female pasent of the crossing generation with the temperature sensitivity gene race as male parent, which is the reason of not hatching..For further analyzing the protein variety and to understand the mechanism of high temperature and dry condition, the embryo is separated from yolk, egg shell and serosa to extract protein. Also 2D-PAGE patterns of embryo protein were achieved. There are 30 specific proteins between three different condition: normal temperature and humidity, high temperature and dry condition, high temperature and wet condition from the shortening stage to body pigmentation stage. It can be considered that embryo come into being cooperation mechanism accordingly for resisting the bad incubating condition under high temperature and dry更多还原