摘要
以湘林1号和湘林4号油茶优良无性系近成熟种子为材料,采用裂解法和改良的CTAB法提取总RAN;用磁珠法分离得到纯化的mRNA;在反转录酶和其他酶的作用下合成一链cDNA和二链cDNA;经与质料载体重组和转化大肠杆菌,成功地构建了世界上第一个油茶种子cDNA文库.经检测,文库存容量达106,重组率为88.21%.测序结果表明:克隆片段长度一般都大于600bp,说明构建的文库质量较高,能够满足ESTs文库构建及从文库中分离筛选重要基因等研究工作的需要,为进一步研究奠定了很好的物质和技术基础.
The total RNA was separated with splitting decomposition method and modified CTAB method from Camellia Oleifera seeds of XL-1 and XL-4; the mRNA was isolated from the total RNA by MagneSphereTechnique; and then activated by SuperscriptⅡenzyme, 1-strand and 2-strand cDNA were synthesized. Finally, by ligating the cDNA fragment with pBluescript vector and transforming the recombinants into E.coli, cDNA was reclaimed. This library reaches 10~6in capacity; the percentage of recombination is as high as 88.21%, and the length of fragments being cloned is over 600 bp. These results indicate that this is a high-quality cDNA library that is helpful to ESTs library construction and the gene isolation and will be beneficial to the splitting of genes in the future.
出处
《中南林学院学报》
CSCD
2004年第5期1-4,共4页
Journal of Central South Forestry University
基金
国家自然科学基金资助课题"油茶EST文库构建及重要基因的分离与鉴定"(编号:30371184)
教育部骨干教师资助课题"油茶cDNA文库构建及探针筛选"项目的部分内容.
关键词
林学
经济林
油茶
CDNA
文库构建
forestry
non-timber forest
Camelia oleifera seeds
cDNA
library construction
