摘要
本研究为解决暗纹东方鲀养殖过程中病虫害的防治方法,从提高鱼体自身免疫出发,探讨存在哪些与哺乳类、鸟类BlyS基因相关性的DNA序列以及这些序列的性质。人B淋巴细胞刺激因子(又称为BAFF、BlyS、TALL-1、zTNF4、THANK)是1999年发现的肿瘤坏死因子超家族的成员之一,它与某些免疫疾病的致病机制密切相关,低量或过量表达均能引起机体的免疫失衡。根据四种已知高度保守的BlyS同源片段设计引物,同时以草鱼、青鱼模板做外对照,人模板做阳性对照,经PCR扩增、割胶回收、克隆测序。结果表明:以鱼基因组为模板经PCR扩增获得了542bp、349bp、221bp三条片段;以人基因组为模板获得5条片段,将最亮一条测序,发现是位于14号染色体上的片段,与人BlyS没有相关性。542bp序列在暗纹东方鲀和草鱼中是非常保守的,A含量最低占18.8%,判断此段序列属于红鳍东方鲀基因组文库中“N(任意核苷酸)”区,填补了红鳍东方鲀基因组文库(2007)数据的空白。349bp和221bp在暗纹东方鲀、红鳍东方鲀及草鱼中都是保守的。因此542bp、349bp、221bp三条片段属于引物特异性扩增,但对于BlyS来说是非特异性片段。暗纹东方鲀和红鳍东方鲀序列比对分析显示:349bp序列中,转换颠换各发生3次、1次,插入/缺失12次。221bp序列中,碱基替换各1次,插入/缺失共6次,以上结果符合大多数生物DNA序列进化中转换大于颠换的现象,但插入或缺失出现的频率要远远高于碱基转换和颠换。在暗纹东方鲀和红鳍东方鲀中碱基突变的主要方式为插入或缺失。同时用生物学软件将此三段DNA序列按照三种读码框自动翻译成氨基酸序列,能够折叠成类似BlyS蛋白空间结构的可能性很小,在蛋白质数据库中做Blast,同样没有发现与BlyS蛋白相关性序列。结论:此三条序列与BlyS蛋白的相关性很小,并且也不属于其他性质的基因或蛋白,推测可能是新发现的三段序列,有待进一步研究。
The research aimed to explore whether there was BlyS gene in the genome of Fugu obscurus,which exists in mammals and birds.Human B lymphocyte stimulator(also known as BAFF,BlyS,TALL-1,zTNF4,THANK)was found and considered to be one member of tumor necrosis factor(TNF)family.It was closely related to pathogenic mechanisms of immue system diseases.Low level or excessive expression of it all can induce immune imbalance.Specific primers were designed based on the reported conserved BlyS gene regions,which were from four species:Homo sapiens, Mus musculus,Sus scrofa and Gallus gallus.Ctenopharyngodon idellus and Mylopharyngodon piceus were taked as external control group.Homo sapiens were positive control.By PCR amplification,purifycation and withdraw,the products were cloned into pUCm-T vector and transformed into E.coli and sequenced.Three specific bands were obtained through PCR amplification,with the size 542,349 and 221bp. 542bp-sequence was highly conserved in Fugu obscurus and Ctenopharyngodon idellus.The A content of 542bp-sequence was 18.8%and the lowest among four kinds of bases.It belonged to "N(arbitrary nucleotide)",which weren't sequenced in Fugu rubripes genomic library(2007).Sequences with the size of 349bp and 221bp were conservative in Fugu obscurus,Fugu rubripes and Ctenopharyngodon idellus. 542,349 and 221bp were specific amplification.The comparison results between Fugu obscurus and Fugu rubripes showed that the main type of base mutation was insertion or deletion in the process of molecular evolution.Meanwhile,we translated the DNA-sequences by biological sol,are,such as DNAstar or DNA club.They were translated to three amino acids sequences respectively.These amino acids were impossible to shape the same three-diamension as BlyS;then they were blasted in protein data bank,it was not found any similarity with B lymphocyte stimulator.So, the possibility that amplified bands were the related sequences to BlyS gene was less. The actions of three sequences need futhur study,which were neither other known genes nor proteins.
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