海洋无脊椎动物组织总RNA提取方法的探讨  

Extraction of total RNA from marine invertebrate tissue

作　　者：冯政夫[1] 王琳[1] 李文侠[1] 部凡[1] 胡彦江[1] 朱伟[1] 

机构地区：[1]青岛农业大学生命科学学院,山东青岛266109

出　　处：《海洋科学》2014年第11期24-28,共5页Marine Sciences

基　　金：国家自然科学基金项目(31272678);山东省现代农业产业技术体系项目(SDAIT-15-011-10);青岛农业大学实验技术研究项目(SYJK 12-14)

摘　　要：RNA提取是分子生物学研究的基础实验,由于海洋无脊椎动物组织结构以及体成分的特殊性,导致现有的RNA提取方法不能完全胜任此工作。作者以刺参（Apostichopus japonicus）、栉孔扇贝（Chlamys farreri）和凡纳滨对虾（Litopenaeus vannamei）为研究对象,对异硫氰酸胍RNA提取方法进行了改进。栉孔扇贝和凡纳滨对虾的肝胰腺富含RNase,刺参体壁结构致密、富含胶原蛋白,而成熟期精巢富含多糖,因此在这些动物组织中提取RNA时,在原有方法基础上增加了瞬时匀浆、增加氯仿及酸性酚的抽提次数,或添加高盐溶液等方法。实验结果显示,改进方法后获得的RNA具有完整的28S、18S和5S r RNA条带,A260nm/A280nm和A260nm/A230nm比值分别为1.89~2.0和1.98~2.11,β-actin基因扩增条带清晰,所获得的组织RNA的纯度和质量符合c DNA合成和基因功能研究的要求。RNA extraction is the basic experiment of the molecular biology study. The particularity of marine in- vertebrate in tissue structure and component results in some disadvantage to obtaining pure RNA using general RNA extraction methods. In this study, sea cucumber, Apostichopus japonicus, scallop, Chlamys farreri and the white shrimp, Litopenaeus vannamei were used as the experimental materials. The method was modified from the method of Guandine Throcyanate Reagent. Hepatopancreas of scallop and white shrimp were rich in endogenous RNase. The sea cucumber body wall was compact and rich in collagen and its testis was rich in polysaccharides. During the process of RNA extraction from these tissues, extraction time of chloroform and phenol-chloroform was increased, and brief homogenate and high concentrated solution of NaC1 were used. The results show that A26onm/A28onm and A26onrn/A23onm ratio of RNA were 1.89-2.0 and 1.98-2.11, respectively. The bands of 28S, 18S and 5S were integrity, and the amplified band of fl-actin gene was clear. The data demonstrate that the RNA obtained was suitable for cDNA synthesis and future functional genomic studies.

关 键 词：无脊椎动物 RNA提取 刺参(Apostichopus japonicus) 栉孔扇贝(Chlamys farreri) 凡纳滨对虾(Litopenaeus vannamei) 

分 类 号：S488[农业科学—农药学]