【作者】 李关荣；

【导师】 向仲怀； 鲁成；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养学， 2002， 博士

【摘要】 在蚕业生产上，由于雄蚕体质强健，虫蛹生命力强，茧层率高，茧丝长，解舒率高，雄蚕出丝率比雌蚕高20％左右，而且雄茧品质好，净度优，纤度细，雄茧缫丝等级高，雄茧单缫比雌雄茧混提高2个等级，达5A水平，且节约用桑15％左右(黄君霆，1980)，实现单养雄蚕是提高蚕茧品质、降低生产成本的关键措施之一。因此雄蚕品种的选育研究一直是国内外蚕业科学家关注的焦点之一。 为了实现单养雄蚕，国内外有很多研究者已经和正在寻找有效的途径如孤雌生殖(潘少西等，1996)、雄核发育(李木旺等，1996)、利用平衡致死系(夏建国等，1980)和限性品种(黄君霆等，1984)。雄核发育虽然在理论上讲—蛾所选卵可全部雄性化，但发育卵率低，发育卵的孵化率更低，技术上还未成熟，目前仅停留在理论研究阶段；利用平衡致死系的技术体系虽已成熟，且已育成平衡致死系统，能导入实用品种，操作也较简便，但品保繁琐复杂，成本高，导入实用品种亦较困难；利用限性普斑和限性虎斑虽人工鉴别雌雄简单，可用于种茧育，但均只能在五龄期以后才能鉴别雌雄，已不可避免地造成了浪费，利用限性茶斑系统可在三龄期鉴别雌雄，实现共育后雌雄分养，但茶斑基因对经济性状有一定的影响，利用限性茧色系统尚待进一步研究，利用限性卵色实现的黑白卵光电自动选卵效率高，但成本亦高。 1992年潘庆中等首次发现伴性赤蚁(sch)胚胎期具有温敏性，并证明此温敏性呈伴性遗传。这为雄蚕品种的选育研究开辟了新的途径。鲁成等(1996)研究了伴性赤蚁蚕催青期的温敏性，结果发现，sch基因与温敏致死性有密切关系。但该温敏致死性究竟是由于sch基因的多效性，还是与sch基因紧密连锁的另一个温敏致死基因在起作用，尚无结论。林健荣(1998)研究发现，sch的温敏性状是由位于Z染色体上的一个主基因控制，呈隐性的伴性遗传。朱勇等将sch的温敏性导入实用新品种夏芳和秋白，分别育成了秋白sch×夏芳和夏芳×秋白sch两个雄蚕品种。并已在生产上推广应用。可见，利用sch的温敏性实现单养雄蚕具有十分广阔的应用前景。从其发现至今的10年的时间里已育成了一些雄蚕品种，但迄今为止尚未完全了sch蚕温敏性与sch在分子水平上的确切关系，还未找到决定sch蚕温敏性的相关基因。sch蚕的温敏性是一复杂的生物学问题，其机理亟待阐明；温敏性状与伴性赤蚁基因(sch)之间的关系也不清楚，限制了其在研究及生产中的利用。从分子水平寻找与Sob蚕高温致死性有关的基因是阐明其分子机理的关键。 SCh蚕胚胎期表现的高温致死性是研究刺激与基因表达差异的理想系统。通过目前的基因表达比较研究技术，可望找出sCh蚕系统胚胎期高温致死的相关基因表达差异。伴性赤蚁胚胎期高温致死性是环境因子温度影响性别分化的典型例子，了解高温对sob蚕性别影响的分子生物学机理势在必行。自Lians和Pardee（1992）首次提出鉴别和研究不同细胞差别表达的一小部分基因的差别显示（Differential Displny）技术以来，此技术以其简便易行、灵敏度高、重复性好、具多面性和速度快的独特优点，在克隆差别表达基”因方面迅速超过了其它同类方法如减法杂交，m随机扩增和代表性差异分析，ow＆ uicroarra以及基因 冽表达的连续分析法（Serial Ananlysis ofGene EXpression，SAGE）等，迅速成了克隆差别表达基因最受青睐的技术手段。近年来正越来越多地用于其它高等真核生物的基因表达和基因克隆研究。在家蚕中运用差别显示技术研究的仅只数例。Masataka等（1999）运用差显技术发现了与家蚕滞育相关的BmEts基因；李斌等（2001）用荧光差异显示法（Fluorescent Differential Displap FDD）得到了家蚕肾形卵（hi）的5个特异克隆序列。为此，本研究采用纯种（近等位基因系）伴性赤蚁蚕卵为材料，分别采用常温常湿（25 C，RHSCh）和高温干燥（35oC，RH60％）催青，在催青过程中应用基因差别显示技术，分离、鉴定和克隆出sob蚕胚胎期的特异表达的基因片断，并对这些差示片段测序，然后用Northern杂交证实后，根据其特异的差示阳性片段设计基因特异引物，采用快速、简便的 5’－RACE（Ranid am刊ification ofcDNA ends）法找出其相应的CDNA基因，为进一步研究此温敏致死性的实质及其表达的分子机理奠定理论基础；为选育新型的转基因实用雄蚕品种提供有意义的易于筛选的目的性状基因，这在理论和实践中都具有重要的意义。 研究结果总结如下。l、基因差别差别显示的关键之一在于完整RNA的制备。本研究采用Chomcz卿更多还原

【Abstract】 Due to the robustness of the male silkworm larvae, strong vitality of its pupae, high rate of cocoon layer they produced, long ,fine and evenly distributed silk fibers, the rate of silk produced by the male silkworm is about 20% more than that of the female; the grade of silk produced from silk fiber spu/7 by the male silkworm is raised to more than 2 grades than that of the silk fiber spun by mixed sexes, reaching to the level of 5A; what’s more, the male silkworm produce quality cocoon and need 15% less of the amount of mulberry than the mixed sexes (Huang Junting,1980). For all the above-mentioned advantages in rearing of the male silkworm, one of the key measures in sericultural practice is the attempt to realize the monoculture of male silkworm, which will both raise the quality of the cocoons and reduce the cost for silk production. For these purposes, the selective breeding of male silkworm races has always been one of the focuses in sericulture of many sericologists home and abroad.In order to realize the monoculture of male silkworm, many researchers across the world have attempted and are still attempting to look for effective means, e.g. parthenogenesis (Pan Shaoxi,1996), male nucleus development (Li Muwan et al., 1996), using balanced lethal systems(Xia Jianguo et al.,1980) and sex-limited races (Huang Junting et al.,1984). Theoretically speaking, all the eggs produced by a single silkworm moth can be masculinized in male nucleus development, but the rate of developed eggs is low and the rate of hatching is even lower. Therefore technically it is still not feasible but only used for theoretical studies; although it is technicallyfeasible to use the balanced lethal systems and the balanced lethal systems have been created with relatively simple manipulation,.it is much laborious in the maintainance of the races, its cost is high and it is difficult to introduce them into the pragmatic races; By using sex-limited normal marking and sex-limited zebra it is simple to identify male and female, and thus can be used for the breeding of seed cocoons, but the sex identification can only be done within the 5* instar when it has already caused some unavoidable loss; although by using the sex-limited mulrilunar system identification of sexes can be done within the 3rd instar, and thus male and female can be reared separately, it has certain negative effects on economic characters. As for the sex-limited cocoon color system, further research has yet to be done. The efficiency of the black and white egg automatic selection by photoelectric means is high but the cost is also high.Since the discovery of the high temperature lethality of the sex-linked chocolate (sch)during its embryonic stage and its sex-linked inheritance (Pan Qingzhong,1992), new horizon has been open for the selective breeding of male silkworm races. Many researches have been done on the silkworm sch.. Lu Cheng et al.(1996) studied the temperature sensitivity of silkworm sch during its incubating stages, and found that the sch gene was closed related with the temperature sensitive lethality; but the problem whether the temperature sensitive lethality was the result of the pleiotropism of sch gene or was determined by another so called lethal gene closely linked to sch, was unsolved. Lin Jianrong (1998) found that the temperature-sensitive trait of sch was determined by a major gene located on the Z chromosome and that it was in a form of sex-linked recessive inheritance. Zhu Yong et al have bred two male silkworm race combinations, namely Qiubai sch X Xiafang and Xiafang X Qiubai sch through introducing the temperature sensitivity of sch to practical race Xiafang and Qiubai; and these two combinations have been in practical tests. Within about 10 years since the discovery the temperature lethality of sch, some practical race combinations for the rear of male silkworm have already been created. Clearly, the temperature sensitivity of sch system has a broad potential for application in the monoculture of male silkworm.However, up更多还原