感染与未感染桃拉病毒凡纳宾对虾基因差异表达文库的构建

Construction of Differential Expression Library for Survival Shrimps Infected with TSV and Uninfected Shrimps

为了寻找对虾抗桃拉病毒(TSV)相关基因信息,研究对虾抗TSV分子机理。应用抑制性差减杂交(SSH)技术,构建TSV感染后存活凡纳宾对虾与未感染凡纳宾对虾的基因差异表达文库,并通过相对定量PCR以及斑点杂交分别对SSH的效率以及文库的可靠性进行验证。结果表明,SPR凡纳宾对虾和SPF凡纳宾对虾攻毒后的死亡率分别为12%和90%;在SSH系统中,相对于未进行杂交的cDNA样品,杂交后的cDNA样品的β-肌动蛋白(β-actin)几乎没有被检测出,说明SSH操作系统是高效率的。通过文库构建则获得正负2个文库,其中正库含阳性克隆1051个,负库含阳性克隆580个;而经斑点杂交验证,共获差异表达克隆321个,其中攻毒组高表达克隆270个,未攻毒组高表达克隆51个。可见,SPR凡纳宾对虾具有较强的抗TSV能力,以之为基础构建的对虾基因差异表达文库具有高度的可靠性,由文库得到的基因信息对获取对虾抗(TSV)相关基因具有潜在的应用价值。

In order to find out the antivirus genes in shrimp, and research the antivirus mechanism. SPR and SPF Penaeus vannamei were both infected with TSV for comparing their antivirus capability. Suppressive subtractive hybridization（SSH） was performed to screen genes that were differentially expressed in the hemocytes between survival shrimps infected with TSV and the uninfected shrimps , forward and reverse subtractive eDNA library were constructed. Relative quantitative PCR and dot-blot analysis were performed to validate the efficiency of SSH and the quality of subtraetive eDNA library. The mortality of SPR Penaeus vannamei infected was 12 % and SPF Penaeus vannamei was 90 %. In SSH system , β-aetin hardly can be examined in subtraetive eDNA compared with unsubtraetive eDNA. Forward subtracted eDNA library and reverse subtracted eDNA library were constructed , including 1051 positive clones and 580 positive danes respectively. Validated by dot-blot analysis, total of 321 differential express clones were attained,270 clones from infected group and 51 clones from uninfected group. SPR Penaeus vannamei possess of strong antivirus capability, and the subtractive eDNA library were credible and useful for further study in the antivirus genes in shrimp.