【作者】 王慧超；

【导师】 朱勇； 夏庆友；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2003， 硕士

【摘要】 家蚕是一种重要的经济昆虫，是研究昆虫性别调控的理想模式生物。在家蚕中很容易获得多倍体和人工雌雄嵌合体。家蚕的性别决定是无脊椎动物中的一个上位体系模式，因此，家蚕的性别调控机制研究将有助于理解无脊椎动物复杂的性别调控机制。家蚕的性别调控机理也相当复杂，目前，其分子生物学研究还处于起步阶段。 本研究的一部分内容是：通过RAPD-PCR方法来获得雌特异性分子标记，然后通过克隆、测序及序列分析结果，来初步推测该分子标记是否来自于家蚕的W染色体，或是否与家蚕的性别调控相关。最后，进一步通过PCR验证来确证该片段的雌特异性。 dsx基因是位于果蝇性别决定级联末端，控制其性别分化的主基因。Bmdsx是与果蝇dsx基因同源的家蚕基因，最初是从家蚕的EST数据库中得到的。另外，迄今为止，该基因也是唯一的、得到实验研究证明了的家蚕性别调控基因，现有的研究表明，Bmdsx基因不仅在cDNA序列上与dsx基因同源，而且在功能上也具有相同性，即Bmdsx基因是调控家蚕性别分化的主基因。而且，已有的实验研究表明，该基因是一个单拷贝基因。 本研究的另一部分内容是：首先根据Bmdsx的cDNA序列来设计一对特异引物，运用TaKaRa RNA LA^TM PCR（AMV）1.1试剂盒进行该基因在家蚕组织中的mRNA表达分析，从而，为进行该基因在特定组织中的定量表达分析和Bmdsx基因在家蚕中的RNAi检测实验奠定基础。 1．雌特异性RAPD分子标记的筛选、克隆、测序、序列分析及其雌特异性的验证 以家蚕Y010、C100的雌雄蚕为材料，分别提取基因组DNA。然后运用随机引物来对同一品种家蚕的雌雄基因组DNA进行筛选，通过大量的引物筛选，从中得到一条355hp的雌持异性分子标记。然后对该标记进行了成功的克隆，并进行了序列测定，以及运用blastr和blasin软件进行了序列分析。序列分析结果表明，所得雌特异性分子标记的功能目前还不清楚。最后，又根据所测得的序列进行设计引物，通过Y010、C100和另外的4个品种，共6个家蚕品种进行了对该分子标记的雌特异性验证。从而初步推断该分子标记来自于家蚕的W染色体。当然，是否真正来源于家蚕的W染色体还有待于进一步的实验研究。2．Bm批基因在家蚕不同组织中的表达分析 运用日本东京大学Shimada等人检测Bmdri基因时所设计的引物，采用TakaRa RNA LAuLAuPC…Verl＊试剂盒说明书进行实验操作，最终分析被检测组织中有无 Bmfor基因的InRNA表达。实验结果表明，在家蚕C108（旱，占）5龄6、7天的中肠组 织中均得到了预期的实验结果：在雌蚕的中肠组织中得到了475brt约)特异e带；在雄蚕 中肠组织中得到了226hp特异性条带（而且226hp的主带又得到了克隆、测序及序列分析 鉴定的印证）。即，BIndix基因在雌蚕组织中是以雌特异性方式进行表达的；在雄蚕组织中 是以雄特异性方式进行表达的，这与Shimada等人的实验结果是相吻合的。同时，本部分 研究也有一个新的发现，即在四岛嵌合体化蛹后第8天和第10天的卵巢中，同时得到了 雌（约475hp）．雄(226bpwh两条主带。这说明B，ndri基因mANA在田岛嵌合体中同时以雌 雄两种方式进行表达。结合本实验结果与前人的实验结果可知：①由到目前为止，ffedsx 在所有己被检测过的家蚕组织中均有表达，可初步推断激dsx可能在家蚕的大部分或全部 组织中有表达，是否确实是这样，还有待于进一步进行研究。②家蚕的性别分化在胚胎早 期就形成了，但BmdSX基因从幼虫期到成虫期均有表达，这说明该基因可能和dsx基因对 于果蝇拥有某种性别表型中是必不可少的这一点相类似，即，BzdSI基因可能对于家蚕拥 有某种性别表型是必不可少的。更多还原

【Abstract】 Bombyx is one of the important economic insects. It is a suitable model organism to study the sex regulation of the insects. It is easy to generate polyploids and gynandromorphs artificially in this insect .Therefore , Bombyx should be a suitable material for studies on sex determination. Because the sex determination of Bombyx is a model of epistatic systems in inverteberates, the clarification of the sex-determining mechanism of this insect would be helpful for a comprehensive understanding of sex determination in invertebrates. The mechanism of sex regulation of Bombyx is very complex and the molecular studying of it is in initial stage.For the sake of generating female-specific fragment, RAPD-PCR will be used and if s product will be clarified by cloning , sequence analyzing , and further PCR verifying .Finally, on the basis of the results of sequencing, sequence comparing and PCR verifying, it can be inferred whether the female specific fragment will be from W chromosome of Bombyx or related with the sex regulation Bombyx.dsx gene lies in the terminal of Drosophila sex regulation cascades , which controls sex differentiation in Drosophila. Bmdsx, a homologue of dsx in Bombyx , is a sex gene of Bombyx and was initially obtained from the EST database of Bombyx. So far, Bmdsx, which had been confirmed by the experiments, is the only gene related with the sex regulation of Bombyx .The studies on hand show that Bmdsx is a homologue of dsx not only in the sequence but also in the function, that is, Bmdsx is a key gene of controlling sex differentiation of Bombyx. Moreover, Bmdsx is a single copy gene which have been experimented.Another part of this study is that a pair of specific primer is designed on the basis of the sequence of Bmdsx cDNA . In order to analyze the expression of this gene in the different tissue of Bombyx, TaKaRa RNA LA PCR kit is used .Then we can analyze the quantitative expression ofthis gene and have a test of Bmdsx RNAi in the different tissues ofBombyx.1. selecting, cloning, sequencing and verifying the female-specific RAPD molecular markerThe genomic DNA was extracted from the Bombyx Y010 and C100 , including male and female individuals. A 355bp female-specific molecular marker was obtained. On the basis of a number of random primers used to select the different fragment between male and female the molecular maker was successfully cloned, sequenced, and was compared with other DNA sequences with the software blastx and blastn. Finally, the specific primer was designed according to the results of the sequence and the specific fragment was tested by genomic DNA amplified of 6 kinds of Bombyx as the templates , including Y010 and C100 with the specific primer. Therefore it is inferred that the fragment was from the W chromosome of Bombyx. However, the further study will be carried out in order to verify whether the fragment was really from the W chromosome of Bombyx.2.The expression analysis of Bmdsx gene in different tissues of BombyxThe primer which was designed by Shimada el during testing the Bmdsx expression in a tissue of Bombyx. As is expected, the final results show that a 475bp band is amplified from the female tissue and 226bp band is amplified from the midgut of male Bombyx C108(o , O ) in 6 and 7days during the 5th instar, which is in harmony with the results of Shimada. And the 226bp band had been clarified by cloning and sequence analyzing. At the same time, we have a new result that 475bp band and a Bmdsx 226bp band coexisted in Tashima gynandromorphs, which shows Bmdsx gene is expressed in Tashima gynandromorphs in female and male specific ways. According to both the results of this study and the previous result, so far, Bmdsx was expressed in all the tissues detected of Bombyx, so it probably will be expressed in tissues of Bombyx in most of or all the tissues of Bombyx, which needs further study to identify. Sex differentiation of Bombyx had been determined during the early embryo developing. But Bmdsx was expressed from the stage of larvae to更多还原