【作者】 朱金金；

【导师】 王志跃； 刘桂琼；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 禽类品种间和个体间的性状差异本质上都是由于基因种类的差异和基因表达量的差异所致。对同一个体不同组织或不同个体相同组织的基因表达差异进行比较研究,可获得与生命过程相关的信息,揭示不同性状的发生发展机理。禽类输卵管的子宫阴道交接部存在一种特殊的管状腺,该腺体由单层柱状上皮细胞构成,腺管细胞顶部无纤毛,细胞核排列整齐,位于细胞的基部,因精子在此贮存而被称为贮精腺。贮精腺的存在使禽类精子在输卵管内可以长期存活,并能长时间保持受精能力。自贮精腺被发现以来,人们从贮精腺的组织学和生殖生理学等方面进行了广泛的研究,但这些研究还不足以解释贮精腺的贮精机理。本实验利用mRNA差异显示技术,以29bp的10条锚定引物和25bp的10条随机引物进行DDRT-PCR分析不同处理及不同持续受精能力的母鸡的贮精腺组织。通过对100个引物组合的筛选后发现有63对引物组合的扩增效果较好,其中有差异条带的引物组合有23对,共获得差异条带30条,二次扩增重复出现的差异条带有13条,通过克隆、测序及序列分析,获得6个ESTs序列。对获得的6个ESTs序列进行基因信息学分析发现:SST01部分序列与鸡第2号染色体上的Neurexophilin基因的一部分相似,其同源性达97.8%,推测SST01与贮精腺生理功能的神经调控有关;SST02部分序列与鸡第3号染色体上的丝裂素活化蛋白激酶基因的一部分相似,其同源性达98.7%,推测SST02与贮精腺上皮细胞的增殖、分化有关;SST03部分序列与鸡第8号染色体上磷脂酰肌醇蛋白聚糖基因的一部分相似,其同源性达98.5%,推测SST03与细胞的生长分化有关;其他3个ESTs序列与鸡己有核酸数据库中的cDNA克隆或EST具有较高相似性,可认为是鸡的新EST序列,但其功能未知。利用半定量RT-PCR检测SST01、SST04和SST05的EST序列的,发现这些ESTs表达的上调与贮精腺贮精能力增强有关,但这些ESTs的全序列信息及其功能以及作用方式还不清楚,这些都有待今后进一步的研究。通过本实验的实施,可以从基因水平上对贮精腺贮精能力的遗传基础进行研究,为进一步研究确定贮精腺的贮精机理提供重要的信息,也为新扬州鸡品质特性的研究探索了一种新的方法和思路。对于各表达序列标签及其代表的基因在染色体上的精确定位、基因的结构和功能等方面,尚需通过大量分析和实验进行进一步研究。更多还原

【Abstract】 The essential differences between breeds and individuals of poultry are due to the diffrernt gene expressing or the different expressing amount of the same gene. Comparative research on differential expression of genes will obtain the information of life process and uncover the mechanisms of occurrence and development of different traits.A kind of special glandula tubulare named sperm storage tubule (SST) exist in fowl’s uterovaginal junction (UVJ), the glandular organ is comprised primarily of tall columnar, non-ciliated, cell nucleus which located at basement of cell line up in order. For the existence of SST, fowl’s spermatozoa can survive for long-term, and maintain fertilizability for longer time. Since the discovery of SST, many researches have been done to reveal the histology and reproductive physiology of SST, but the mechanism of sperm storage in SST is still unknow.By using differential display reverse transcription PCR(DDRT-PCR) that piloted by combinations of ten anchor primers and ten random primers, 30 different expressed fragments were obtained, which were cut from polyacrylamide gel electrophoresis products of SST glandular tissue cDNA. With Re-PCR and electrophoresis, 13 different expressed fragments were obtained and 6 ESTs were obtained after cloning and sequencing.By bioinformatics analysis of the 6 ESTs: SST01 is semilar to part of Neurexophilin gene, the homology is 97.8%, presume that SST01 is concerned with the neural regulation of the physiological function; SST02 is semilar to part of MAPKs gene, the homology is 98.7%, presume that SST02 is concerned with epithelial cell proliferation of SST; SST03 is semilar to part of glypican gene, the homology is 98.5, %presume that SST03 is concerned with cell proliferation, differentiation of SST; other 3 ESTs are highly homological to cDNA and dbEST comparing with red jungle fowl in the database of NCBI and dbEST, but function is unknow.Using semiquantitative RT-PCT to test the EST of SST01, SST02 and SST03, the up-regulation expression of these ESTs are concerned with enhancing capability of SST for sperm storage, but the entire sequence function and action mode were unknown, thus further research were needed.This study on the gene level of the hereditary basis of SST provided important information for researching the mechanism of SST for storing sperm and also provided a new method to study on the quality characteristic of New Yangzhou chicken. Further research were needed in the study of the accurate localization of SST gene on chromosome and the structure and functional studies of EST gene.更多还原