摘要
利用mRNA差异显示技术研究中国孤雌生殖卤虫胚胎发育的相关基因,并对中国孤雌生殖卤虫差异显示中SYBR GreenⅠ、硝酸银和溴化乙锭三种染色方法进行了比较。以三种单碱基锚定引物对发育0 h,4 h,8 h,12 h,16 h和10天的中国孤雌生殖卤虫的 RNA进行逆转录和26种随机引物进行PCR反应,经非变性的6%聚丙稀酰胺凝胶电泳后,分别用 SYBRGreenⅠ、硝酸银和溴化乙锭染色,最后进行差异条带的回收和再扩增。结果表明:(1)6 种样本的DD PCR产物显示长200 3.0k不等的扩增片段,条带清晰,并呈现出有无和强弱的变化(2)SYBR GreenⅠ和硝酸银染色的灵敏度比溴化乙锭染色的灵敏度高(3)经 SYBR GreenⅠ和溴化乙锭染色的凝胶的回收和再扩增效率比硝酸银染色的高(4)水煮法和 NAP 25 过柱法对凝胶片段的回收效果几乎相当。利用mRNA差异显示技术研究中国孤雌生殖卤虫胚胎发育相关基因时,得到了较好的扩增片段;三种染色方法中 SYBR GreenⅠ染色灵敏度较高,并且SYBR GreenⅠ染色有利于凝胶片段的回收和再扩增。
Abstract: In this experiment the related genes of early embryonic development of Artemia Pathenogenatica from China have been studied by mRNA Differential Display, and three staining methods-SYBR GreenⅠ, Silver Nitrate and Ethidium Bromide have been compared. RNA samples in six development phases were reversed with three anchored primers and then amplified with twenty six random primers . After running on 6% non-denaturing PAGE gel, they were stained by SYBR GreenⅠ, Silver Nitrate and Ethidium Bromide. At last differentially expressed bands were excised and recovered. The results reveal: (1) Amplified bands of all six tested samples differ from 200bp to 3.0kb and the bands are bright and clear. (2)Sensitivity of SYBR GreenⅠand Silver Nitrate is higher than that of Ethidium Bromide.(3)The recovery and reamplification rates of PAGE gel stained by SYBR GreenⅠand Ethidium Bromide are higher than those stained by Silver Nitrate. (4)The gel recovery rate of boiling method is almost the same as that of NAP-25 Column method. In the study of the related genes of early embryonic development of Artemia Pathenogenatica from China excellent differentially expressed cDNA bands have been obtained; the staining sensitivity of SYBR GreenⅠis higher among the three methods, and it is easy to recover and reamplify the PAGE gel fragment stained by SYBR GreenⅠ.
出处
《海洋湖沼通报》
CSCD
北大核心
2005年第1期67-72,共6页
Transactions of Oceanology and Limnology
基金
国家自然科学基金项目(No.30271035)资助
