《植物生理学报》 2014, 50(6): 880-884

一种高效提取普通小球藻叶绿体DNA的新方法

许夏冰^1,2, 谭德冠², 孙雪飘², 弓淑芬², 张家明^2,*

¹海南大学农学院, 海口570228; ²中国热带农业科学院热带生物技术研究所, 农业部热带作物生物学与遗传资源利用重点实验室, 海南省热带生物质能源工程技术研究中心, 海口571101

通信作者：张家明；E-mail: zhangjiaming@itbb.org.cn；Tel: 0898-66984866

摘　要：

本文采用尿素-月桂酰肌氨酸钠(urea-sarkosyl)法, 用于分离带有坚硬细胞壁小球藻的高纯度叶绿体DNA (cpDNA)。将对数生长期的小球藻收集后置于冰上研磨, percoll密度梯度离心收集叶绿体层, 显微观察表明叶绿体经梯度离心后形态完整。采用尿素-月桂酰肌氨酸钠法、蛋白酶K消化及酚/氯仿/异戊醇抽提, 获得了高纯度的cpDNA。检测结果显示, cpDNA分子长度为22 kb, A₂₆₀:A₂₈₀值为1.87±0.01, 产率达(2.52±0.01) μg•g^-1 (DW); cpDNA编码的16S rDNA扩增呈阳性, 而由细胞核编码的18S rDNA扩增呈阴性。表明cpDNA纯度高, 没有受到核基因组DNA的污染, 符合小球藻cpDNA高通量测序的要求。同时, 该方法也适合提取具有相似细胞壁成分的其他微藻的基因组DNA和cpDNA。

关键词：普通小球藻; 叶绿体DNA; 尿素; 月桂酰肌氨酸钠; percoll分离液

收稿：2014-01-07   修定：2014-04-09

资助：海南省重点实验室和工程技术研究中心建设专项(gczx-2013001)和海南省自然科学基金项目(310068)。

A New Method for Efficient Extraction of Chlorella vulgaris Chloroplast DNA

XU Xia-Bing^1,2, TAN De-Guan², SUN Xue-Piao², GONG Shu-Fen², ZHANG Jia-Ming^2,*

¹College of Agronomy, Hainan University, Haikou 570228, China; ²Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Key Laboratory of Tropical Crops Biology and Genetic Resources Utilization, Ministry of Agriculture, Hainan Provincial Tropical Bioenergy Engineering Research Center, Haikou 571101, China

Corresponding author: ZHANG Jia-Ming; E-mail: zhangjiaming@itbb.org.cn; Tel: 0898-66984866

Abstract:

The purpose of this study was to establish a method for isolation of high quality chloroplast DNA (cpDNA) from a green microalgae Chlorella vulgaris with rigid cell wall. C. vulgaris cells at logarithmic phase were collected and ground on the ice. Intact chloroplasts were isolated from the abrasives by using percoll density gradient centrifugation, and were verified by microscope observation. High quality cpDNA was then isolated from the chloroplasts with urea-sarkosyl method, digested with protease K and purified by phenol/ chloroform/isoamylol extraction. The purified cpDNA had a molecular length of 22 kb, an A₂₆₀:A₂₈₀ value of 1.87±0.01, and a yield of 2.52 μg•g^-1 (DW). The nuclear-encoded 18S rDNA could not be amplified from the purified cpDNA, while the chloroplast-encoded 16S rDNA was amplified, indicating that the cpDNA was not contaminated by nuclear DNA. These results showed that the cpDNA isolated by urea-sarkosyl method meets the requirements of high-throughput sequencing. This method may also be used to isolate total DNA and cpDNA from other microalgae with similar cell wall components.

Key words: Chlorella vulgaris; cpDNA; urea; sarkosyl; percoll separation medium