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用反义RNA与RNA干扰技术抗草鱼出血病的初步研究
Alternative TitleStudy of Disease Resistant on Grass Carp Reovirus by Antisense RNA and RNA Interference(RNAi)
廖莎
Subtype硕士
Thesis Advisor朱作言
2008-06-16
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordRna干扰 反义rna Shrna 草鱼出血病 表达抑制 转基因育种
Abstract  转基因技术是20世纪80年代发展起来的一项重要技术,通过向受精卵或早期胚胎中导入外源DNA,能有目的地对生物的遗传物质基础进行修饰,改造,培养出新的品系。鱼类转基因研究的一个重要方向就是定向育种,即通过转基因改良生产性状,如快速生长,抗病虫害,抗冻,抗盐碱等。随着RNA干扰现象的发现与反义技术的出现,人们已将RNAi与反义技术发展成为重要的基因治疗的新手段。   本研究利用RNAi与反义技术,以草鱼出血病为模型,稀有鮈鲫为模式动物,针对草鱼出血病病毒(GCRV)外壳蛋白VP7序列的cDNA片段构建shRNA表达载体pH1-siGCRV-pZero及反义载体pCAVP7c antisense,采用显微注射方法,将外源基因导入稀有鮈鲫受精卵,获得P0代转基因稀有鮈鲫。对培养至性成熟的P0代转基因稀有鮈鲫进行了攻毒实验,通过对攻毒后死亡时间的统计和转植基因表达的检测,得出以下结果:①转shRNA表达载体的稀有鮈鲫死亡率明显低于对照组,至攻毒后第20天死亡率仅有30%,而对照组在攻毒后第12天死亡率均达到100%;②反义组转基因稀有鮈鲫与对照组在死亡率上没有显著差异,但从死亡趋势线上可以看出反义组死亡峰值明显滞后于对照组,相差约18h,死亡峰值滞后与转植基因的表达相关。由此可以得出,不论是RNAi还是反义技术均能有效的抑制病毒的繁殖,具有一定的抗病效果。;   Gene-Modified technology was development in 1980s which targeted to modify the genetic elements by the foreign DNA trasfered to oosperms or earlyl embryoes. In fishery, the new gene-modified oragnism(GMO)had better characters such as growth faster, disease-resistant, antifreeze and tolerance of salt or alkali.Recently,while RNA interference(RNAi)and antisese technology appearanced,it became a significant new strategy for gene therapy.   This project aims to pineer new approaches developed in the last few years to tackle the viral diseases in fish. For such purpose, we use Gobiocypris rarus and GCRV as a model system to study RNAi and antisese RNA antiviral functions in fish and combine transgenic technology to potentially generate disease resistant fish strains. We produced the shRNA expression vector named pH1-siGCRV-pZero and the antisese expression vector named pCAVP7c antisense according to the VP7 cDNA sequences. Two gene-modified fish were geined by microinjected the two vector to Gobiocypris rarus embryoes. 3 monthes later,such fishes were infected with GCRV 991 strain. The fishes with antithesis gene or without forgein gene were 100% dead in 12dpi (day-post-infection), while the fishes with pH1-siGCRV-pZero were only 30% dead lasting to 20 dpi. Between the fishes with pCAVP7c antisense and without forgein gene there were no noticeable differences with the death rate. But the death peak of the fishes with antisense expression vector was obviously 18h later than the fish without foreign gene,according to the expression of the foreign gene.
Pages64
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12076
Collection学位论文
Recommended Citation
GB/T 7714
廖莎. 用反义RNA与RNA干扰技术抗草鱼出血病的初步研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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