【作者】 陈复生；

【导师】 吴玉澄； 杨剑波；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2003， 博士

【摘要】 家蚕多倍体研究和应用，是家蚕遗传分析、育种素材开发和品种选育以及生物防治的一个重要的研究领域，涉及的研究范围十分广泛，先驱们经过长期不懈的努力，取得了许多令人鼓舞的成就。但目前无论在理论上和实用化方面都还存在许多悬而未决的问题。本文在对前人的有关研究成果总结分析的基础上，进行相关的研究，主要取得以下结果： 1．利用微波方法处理二倍体雌雄交配产下的蚕卵，通过对细胞染色体数的观察和次代蚕产下卵的大小、产附等形态观察，确认已经诱发出多种多倍体，开发了家蚕多倍体诱发新手段。 2．用过冷却方法处理家蚕产下卵诱发四倍体，对影响诱发率的产下卵发育阶段、过冷却处理温度、过冷却处理时间、过冷却处理后的保护温度等主要因素进行不同水平分析，筛选出诱发效率高的卵产下140～160min、处理温度-9～-11℃、处理时间24h、处理后保护温度17—19℃的优选组合。 3．探讨了不同卵龄的过冷却处理对四倍体诱发率和后代三倍体性比发生的影响。四倍体雌蚕的诱发率高低在于不同杂交组合材料的卵龄处于的不同时期，处于细胞第一分割期处理的卵数越多则诱发率越高。解释了过冷却处理诱发的四倍体后代三倍体蚕发生性比从3：1向5：1变化的现象。通过分析四倍体减数分裂中性染色体的行为分析，后代三倍体蚕性比开始的3：1发生，是仅仅Z与Z、W与W之间的联会，其后在这种联会增加的同时，ZW之间的联会也逐渐开始，从而接近5：1。 4．通过雌蛾SB(Sex-chromatin body)数检查和产下卵的性状表现以及通浙江大学博士学位论文过标一记基因、后代的性别、蚁蚕体色等分离比观察;在人工诱发的ZZWW4n后代中获得3A+ZZWw个体。根据ZZWW3n产下大小不规则卵的表现，否定了W染色体上存在决定卵大小的数量基因ESd(Egg determining gene)的推测。5.利用人工诱发的ZZwW4n与ZZZn交配，后代有3A+22、ZZw、ZwW和3A+ZZWW三倍体的发生，证明四倍体雌蚕减数分裂中自由联会、选择联会分离方式同时存在。解释了ZWW发生的概率极少是因为生殖过程中受精和转座W干扰等多种原因;222雄蚕、ZZWW雌蚕发生的概率也比ZZW雌蚕低，是因为Z~Z一ZWW~ZWW联会分离样式在所有的联会分离样式中所占比率低的缘故。6.用对雌雄二倍体交配产下卵进行，根据得到的大卵个体雌蛾与22二倍体雄蛾交配的后代表现，确认有ZZZZWW六倍体个体发生。解析了由于温汤诱发处理刺激在家蚕胚胎期发生染色体非减数分裂和加倍的ZZZZWW六倍体发生机理。7.初步调查了现行家蚕品种过冷却处理的四倍体诱发率，检索了不同组合的诱发率高低并分析了不同组合产生的诱发率高低不同的原因。为今后家蚕品种及其组合的多倍体诱发的筛选和多倍体育种提供了依据。更多还原

【Abstract】 Study and application of polyploid in the silkworm is one of leading important research fields in genetic analysis, exploitation of breeding material, breeding and biological control, which can be related to many other research areas. Although many delighting achievements had been acquired by the continuous hard working of former researchers, there still exists a lot of problems that needs further studing. Based on this, we carried out this studing, and the results were described as follows:1 Polyploid can be induced by microwave treatment in the eggs of hybrid silkworm, and the induced ones were identified by their difference in the size of the egg, shape of the egg, chromosome and Sex-chromatin body(SB) numbers of the cell. Here, I introduce a new method to induce polypoid in silkworm , Bombyx mori, which was established by myself.2 Tetraploid can be induced by the method of super-cooling in silkworm . Different, factors such as stage of embryo after egg laying, temperature, super-cooling times and preservation temperature after super-cooling treatment determine the rate of polyploid induction. In our study, we had designed different levels for each factor, and found that the combination of 150 minutes after egg laying, treated at -10癈, for 24 hours and preservation at 17-19癈 is the optimizedcondition for polyploid induction.3 In this paper, we discussed the effect of the super-cooling treatment at different embryo stages based on their rate of tetraploid induction and the sex ratio of triploid in the next generation. In all hybrids, the tetraploid ratio of the female silkworm was determined by the number of embryo which are at their first cleavage period. If the number of embryo at the first cleavage period is more, the rate of tetraploid induction also be higher. In this paper we discussed the reasons for the change in sex ratio from 3 female : 1 male to 5 female : 1 male from the triploid female silkworm which is generated from a tetraploid female silkworm. This may be due to the active mechanism involved during meiosis of tetreploid parent chromosomes. In triloids, the selective synapsis is found to occur within Z and Z chromosomes, W and W chromosomes firstly, and hence the sex ratio here is 3 female : 1 male. With the increase in selective synapsis, the frequency of free synapsis between Z and W chromosome also will increase, resulting in the sex ratio of 5 female: 1 male.4 Based on the SB numbers of the female adult, the characters of eggs laid, the separate ratio of marker gene, sex, and body color of newly hatched larvae, we obtained silkworms of 3A + ZZWW type from the next generation of the induced ZZWW tetraploids. Depending on the characters of abnormal eggs laid by ZZWW triploids , we overrule the hypothesis laid by previouse researcher that the quantitive Egg determining gene (Esd) determining the egg size on the W chromosome. 5 From our finding that the triploids types like 3A+ZZ, ZZW, ZWW and 3A+ZZWW are generated from the cross between induced ZZWW tetraploid type and ZZ diploid type, we can conclude that there are free synapsis andselect synapsis during raeiosis in the female silkworm simultaneously. The results of our experiments is supplementary with the previous studies already reported. Due to the interference of W chromosome transposition, impregnation and other factors during the procreation, the ratio of ZWW type is found to be very low in our exprements. The ratio of Z-Z-ZWW-ZWW synapsis separate model is lower than other synapsis separate models in our study and this may be the reason for having a lower rate of ZZZ type in male and ZZWW type in female than the rate of ZZW type in male silkworm .6 The eggs of female formed from the cross betweem ZW and ZZ, when subjected to hot water treatment seems to enlarge some eggs’ size, these big eggs when crossed with ZZ , they produced a progeny with some ZZZZWW hexaploid. These above facts proved that stimulation at embryo stage could cause the chromosome to double or no-meiosis.7 We had also inves更多还原