【作者】 赵爱春；

【导师】 鲁成； 梶浦善太；

【作者基本信息】 西南农业大学 ， 特种经济动物饲养， 2002， 硕士

【摘要】 家蚕是遗传学上具有重要地位的模式生物，是生物工程研究的理想材料，也是一种重要的经济昆虫，在我国的国民经济中占有极为重要的地位。家蚕分子连锁图谱的构建对促进家蚕分子生物学的研究和分子辅助育种具有重要的意义。利用分子标记技术寻找绿茧基因紧密连锁的分子标记，对绿茧基因进行定位，加速优质绿茧蚕品种的育成是十分迫切而必要的。 天蚕蛾科的昆虫是目前所记载的鳞翅目昆虫中产卵最大的种类，这些昆虫卵的形成中，卵黄的形成和卵巢的发育相当活跃，其卵巢是生理学、生物化学及分子生物学上研究发育过程所期望的模式系统，对其卵的形成的研究具有重要意义。本实验利用天蚕基因组文库等，克隆了卵黄蛋白主要组分的卵黄原蛋白的全基因。主要结果如下： 1 家蚕AFLP图谱的构建 扩增片段长度多态性(Amplified Fragment Length Polymorphism，简称AFLP)技术是一种具有稳定性好和多态率高的优点的DNA指纹分析技术。目前已广泛应用于多种生物的连锁图图谱的构建、基因和QTL定位等，具有广泛的应用前景，近几年开始用于蚕。 本研究中通过AFLP技术构建了一张家蚕的分子连锁图谱并定位独立绿茧基因在该连锁图谱上。具体结果如下： ’①使用了28对AFLP引物在家蚕品系C。和大造的回交子代中共扩增出3956个位点，其中 1018个为多态性位点，每组引物扩增出36．4个多态位点，693个位点符合盂德尔1：1分 离；虐（9个位点（包括693个AFLP标记和CC形态标记）中的402个构成了33个连锁群，占总构 图位点数的y，占总多态性的39．5％，占呈1：互分离标记的68．l％；另外还有122个标记 不连锁，28个标记构成8个伪连锁群，伪连锁群是几个间距为OCM的不同的标记构成的连 锁群；94个标记构成了47个二联体，48个标记构成11个类似二联体的连锁群即几个间 距为0。回的不同的标记和另一个标记构成的连锁群：③33个连锁群中，包括25个主连锁群和8个次连锁群，主连锁群包含7～32个标记，次连 锁群包含3～6个标记。标记间距离为0．0～！19．ga，平均间距为9．豆C回；标记数最多的 为第14连锁群，含有32个标记，最少的为3个标记；最长的连锁群0。U昨的跨距为 496．二训 最短的p。u昨9为粤．7。饲：④构成33个AFLP分子标记连锁群的标记数33个连锁群的总图距为3676 7CM，每条连锁群 的平均图距为111．4CM，家蚕有28个经典的连锁群，而本实验所构建的连锁群数大于28， 说明本实验中构建的连锁群有些应是相互连锁的，只是本实验数据中缺乏特定的标记将其 整合在一起；⑤位于家蚕经典的第15号连锁群的形态标记阶被定位在该图谱中的第22连锁群的 L－P4T6－107与L－P6T4－84之间，这表明该分子连锁群可能与传统的家蚕形态标记图谱中的 第15号连锁群对应。⑤本研究所构建的分子连锁图谱中的标记有较高的成族的现象，这可能是由于两个方面造成 的，一方面是这些位点之间本身较为紧密连锁：另一方面是本研究中作图群体个体数的限 制，不能有效分辨图距较小的两个标记。 本研究中构建的家蚕图谱和绿茧基因的定位为进一步克隆家蚕绿茧基因和育成优质家蚕绿茧品种奠定了一定的基础。在育种中，借助与QTL和绿茧基因紧密连锁的分子标记并结合 7 V它们控制的数量和形态性状进行有目的的选择，加速优质绿茧品种育成的进程。2天蚕卵黄原蛋白的克隆 天蚕卵黄原蛋白（Vg）是卵黄蛋白主要前体，幼虫·蛹蜕皮（变态蜕皮）到蛹早期阶段时在雌的脂肪体中合成。天蚕天然的卵黄原蛋白和卵黄磷蛋白（Vt）有若干不同种类的分子量，主要是由两个分子量分别为185kDa和44kDa的大小亚单位形成的四体蛋白构成。 本实验中首先利用入 DASH 11／fbmHI载体构建了一个天蚕基因组 wA文库，然后根据天蚕的卵黄原蛋白的。wA序列设计不同的引物，筛选能在天蚕基因组DNA中特异扩增出适合大小的DNA片段的引物组合，并利用此引物组合成探针对构建的天蚕基因组文库进行筛选，最后对阳性克隆测序获得Vg基因核酸序列。具体结果如下：①利用 A DASHD／Bhmrrl载体构建了一个天蚕基因组 IhA文库。文库扩增共用了总扩增量为 能产生 10e个噬菌斑的包装终产物。获得的扩增文库的滴度为 1．ZXI吓fU／．1；故本实验 获得的扩增文库己经达到了筛选基因组中任一基因的统计要求的规模，滴度也适合长期稳 定保存和利用；②本实验根据天蚕卵黄原蛋白基因CDNA序列设计了ZI个前弓！物和18个反向引物。筛选获 得一对能在天蚕基因组DNA中特异扩增产生大?更多还原

【Abstract】 Silkworm is an important model Lepidopteran insect with a long history of significant agriculture value. It is very important for researchers to construct the linkage map of silkworm to make great progress of molecular biology’ and molecular-marker-assisted breeding of silkworm, and also necessary to obtain the molecular markers which are closely linked to the green cocoon gene, Gc. to improve the green cocoon line of silkworm for a relative short period.Until now the insects of Saturniidae are a kind of Lepidoptera insect with the biggest eggs. When the eggs of these insects is developing, the formation of yolk and the development of ovary are very much dynamic, which is the expected model system of development in physiology, biochemistry and molecular biology. It is very important to study about the formation eggs. Vitellogenin gene from Antheraea yamamai. which is mainly compose of yolk protein, was cloned by utilizing to the constructed genomic DNA library of Antheraea yamamai. The mail results were represented as the following: 1 Construction of the AFLP linkage map of silkworm, Bombyx moriAmplified Fragment Length PoKinorphism (AFLP). a novel DNA fingerprinting technique, is10a reliable and powerful approach in detecting polymorphism loci. It has a good applied prospect considering the fact that It has been widely used to construct the linkage map and to locate the gene and the quantitative trait loci (QTL) and so on. At last years it had been applied to the study of silkworm, Bombyx mori.AFLP was applied to construct a linkage map, and Gc was mapped on the linkage map in this research. The following are the detail results:First, 3956 loci were amplified using 28 primer combinations in 44 progenies from a backcross population of C100 and Dazao, but 1018 of loci were polymorphic and 693 of loci were the segregation ratio of 1:1 conforming to Mendel’s segregation rules. On average, 36.4 polymorphic loci were amplified by one primer pair.Second, 402 of 694 loci, which include 693 AFLP marker and Gc specific marker, consist of 33 linkage groups. The number of the loci of constructing map is 39.5 percent of that of polymorphic loci and 68 percent of that of the 1:1 segregation ratio. The other included unlinked markers each other an those linkage groups on which contains two markers.Third, 33 linkage groups compose of 25 major linkage groups with 7 to 32 markers and 8 relatively small linkage groups with 3 to 6 markers. The length of the longest linkage group. group2, is 496.1 cent Morgan (cM), while that of the shortest linkage group, group29, is 4.7 cM. The number of the markers on each linkage group varied from 3 to 32, in which group 14 and group33 contains 32 and 3 markers respectively. On average, die distance between two markers is 9.1 cM.Fourth, the total length of 33 linkage groups is 3676.7 cM, and the length of one linkage group is 111.4 cM on average. In this research 33 linkage groups wrere obtained, however silkworm has only 28 chromosomes. It means that some of the linkage groups should be linked together, but we not link them together for the absence of specific markers.Fifth, the marker about Gc, which was located on group 15 of classic linkage map. was mapped between die markers L-P4T6-107 and L-P6T4-84 on group22 in the research.Sixth, there are relatively high clustering markers. The reason could be the markers are too close to have recombination and result from relatively small scale of individuals of BC1 population in the research.This work provides an essential basic for cloning green cocoon gene and improving quickly11green cocoon lines of silkworm. With the molecular markers of linking closely to QTL and green cocoon gene, we can screen die breeding progenies by following the molecular markers and then select the phenotype to improve quickly the green cocoon Line of silkworm. 2 Clone for the Vitellogenin gene from Antheraea yamamaiVitellogenin of Anther aea yamamai is a precursor of major yolk protein synthesized in the female fat body at lar更多还原