【作者】 谢凯；

【导师】 洪一江；

【作者基本信息】 南昌大学 ， 动物学， 2010， 硕士

【摘要】 池蝶蚌(Hyriopsis Schlegeli)原产于日本滋贺县的琵琶湖,属软体动物门、瓣鳃纲、蚌科、帆蚌属,与我国三角帆蚌为同属不同种。池蝶蚌无论形态、生长速度、产量、成活率、育珠能力皆优于其它淡水育珠蚌,更具有经济优势,因而国内已大力发展池蝶蚌作为我国的重要育珠对象之一。本研究构建了经过嗜水气单胞菌诱导的池蝶蚌血细胞cDNA全长文库并对部分序列进行了序列分析,并重点对免疫相关基因亲环蛋白(Cyp A)进行了较详细的序列分析。主要结果如下：1.利用灭活的嗜水气单胞菌(Aeromonas Hydrophila)诱导处于旺盛生长期(四龄)的池蝶蚌14h,采用Trizol试剂提取诱导后的池蝶蚌血细胞的总RNA。根据Creator SMART试剂盒用户手册,逆转录后,用LD-PCR合成双链cDNA,并逐步构建成池蝶蚌血细胞的全长cDNA文库。本文库为首次成功构建的淡水贝类池蝶蚌血细胞全长cDNA文库。原始文库的滴度为4×106cfu/cm3,重组率为90%,扩增后文库的滴度为3.55×109pfu/ml。2.从文库共随机测序102个样品,所得双向测序结果去除载体,拼接,并多序列比对去除重复序列后,余59条已知功能序列,其余为未知功能序列。序列中最小长度270 bp,最大长度为2153bp,平均大小608.6 bp,表明插入片段大小理想。已知基因根据其功能可分为以下几大类：基础代谢类基因,细胞凋亡,细胞分裂调节、蛋白质的装配、修饰与降解,DNA合成、转录、翻译及重组相关基因,核糖体基因和免疫相关基因。3.从文库中筛选获得免疫相关基因池蝶蚌亲环蛋白A(HsCyp A)全长基因并进行序列分析,池蝶蚌Cyp A基因全长1229个碱基,其在53-547位存在一个长495 bp的开放阅读框,编码164个氨基酸。其起始密码子是atg,终止密码子是taa。HsCypA序列还包括52bp的5’非编码区(untranslated region,UTR),682bp的3’UTR,和29 bp的poly(A)尾,没有明显的加尾信号。ORF编码区蛋白的分子量为17.26 kDa,理论等电点PI=8.9。对Cyp A氨基酸序列二级结构进行了较详细的分析并进行了三维建模,同时构建了其系统进化树,分析表明亲环蛋白家族是一个在进化上非常保守的蛋白家族。本研究为课题组基础性工作,其研究成果将为大规模筛选文库、发现新的功能基因提供有效的平台,为免疫相关特定基因的研究提供了较好的依据。更多还原

【Abstract】 Hyriopsis schlegelii originated in Biwa lake of Japan and was introduced to China in 1997. Compared with other freshwater pearl mussel, the Hyriopsis schlegelii has high rate of growth, better disease-resistance and pearling production. Considering economical interests, it is necessary to select breed with better disease-resistance phenotype.A full-length haemocytes cDNA library was constructed in order to clone genes involving in Aeromonas Hydrophila defenses from Hyriopsis Schlegeli.Inactivated Aeromonas Hydrophila were used to chalenge Hyriopsis Schlegeli (fourth instar). And then we extracted the total RNAs from haemocytes using TRIzol reagent. The"anchor first-strand cDNA"containing a symmetrical sfiI restriction enzyme sites (A&B) was synthesized by transcription of total RNA with the SMART technique.The SMART switching mechanism at 5’end of the RNA transcript technique was used to construct the cDNA library.The titer of the unamplified cDNA library was 4×106cfu/cm3 with a recombinantrate of 90% and that of amplified one was 3.55×109cfu/cm3. A total of 102 clones was selected to be sequenced and 59 were functional sequences.The shortest sequence was 270bp and longest one was 2153 bp.The average length is 608.6 bp. The results indicated that a high quality library was meet the needs for the consequence gene clone and expressed sequence tag analysis.Cyclophylin A(Cyp A,) was isolated from cDNA library by screening.The full length of the Hyriopsis Schlegeli Cyp A (HsCyp A)was 1229 bp, Sequence analysis showed that HsCyp A cDNA contained 52bp 5’untranslated region(UTR),682bp 3’ UTR and 29 bp poly(A) tail.Which also includes an open reading frame (ORF) of 495 bp and encoded 164 amino acid residues,which doesn’t exist signal peptides.It’s molecular weight is 17.26 kDa and pI 8.9.We also analysised it’s secondary structure and homology-modeled the HsCyp A. A phylogenetic tree showed that they might have similarities in biological functions evolving from the same ancient gene.更多还原