几种饵料微藻基因组DNA的提取及其特异性引物的设计

Genomic DNA Extraction from Several Diet Microalgae and the Design of their Specific Primes

用改良CTAB法提取我国沿海东南部滩涂贝类常见的4种饵料微藻的基因组DNA,结果发现提取的DNA产率高、完整性好,认为是一种简便而高效的微藻基因组DNA提取方法.对其18S rRNA基因(18S rDNA)进行克隆与测序,并利用序列比对软件MEGA 5.0对各微藻的18SrDNA序列进行两两比对,设计出了能够用于快速区分此4对饵料微藻的特异性PCR引物(Cha.F/Cha.R、Iso.F/Iso.R、Pla.F/Pla.R及Nan.F/Nan.R).PCR扩增验证实验结果显示,4对引物均具有很强的特异性,无交叉扩增现象.扩增片段大小范围为100～200 bp,满足实时荧光定量PCR的实验要求,在检测滩涂贝类对此4种饵料微藻的摄食选择性研究方面具有重要意义.

A modified CTAB method is used to extract genomic DNA from four common diet microalgae of intertidal shellfish found in southeastern coast of China,The results show that this method is very suitable for extraction of high quality and productive genomic DNA from microalgae,By cloning and sequencing of 18S rRNA gene from the four diet microalgae,their sequences are compared with each other using the software MEGA5.0,Four pairs of specific primers which can be used to quickly distinguish the microalgae in samples are designed with the help of Primer 5.0.They are named Cha.F/Cha.R,Iso.F/Iso.R,Pla.F/Pla.R and Nan.F/Nan.R.Verification tests through the PCR amplification indicate that four pairs of the primers are highly distinctive and no cross amplification phenomenon is noted.The size of these amplified fragments ranges from 100 bp to 200 bp,which meets the experimental requirements of the Real-time fluorescent quantitative PCR（qPCR）.These specific primers will be of great help for the study on feeding selectivity for the diet microalgae of juvenile intertidal shellfish in China.