一種西瓜葉片DNA快速提取方法

周賢達 孫輝 丁康芬 王鳳辰 王浩波

摘 要： 為了解決田間西瓜葉片提取DNA雜質較多及逐個樣品提取效率低的問題，建立了基于96孔PCR擴增板，每次提取96個樣，并調整提取液改善西瓜葉片DNA質量優化的方法，經超微量分光光度計檢測，提取的DNA濃度為287～573 ng·?L-1，OD260/OD280在1.96左右，OD260/OD230為1.6～1.9，表明蛋白質、酚類及小分子雜質很少，DNA質量較高；普通引物聚丙烯凝膠電泳檢測表明DNA質量穩定、擴增條帶清晰。此方法與目前常用的植物基因組DNA提取方法相比具有通量高、速度快、成本低的優點，是適宜于種子企業西瓜分子標記檢測相關工作的較好方法。

關鍵詞： 西瓜； DNA提取； PCR

Abstract： In order to solve the problem of extracting more DNA impurities and low efficiency of single sample extraction in field watermelon leaves， a method based on 96-well PCR amplification plate to extract 96 samples at a time was established and the extract was adjusted to improve the DNA quality of watermelon leaves in this study. The concentration of DNA that extracted by spectrophotometer was 287-573 ng·μL-1， OD260/OD280 was around 1.96， and OD260/OD230 was 1.6-1.9， which indicated that there were few impurities in protein， phenols and small molecules， and the DNA quality was high. Polyacrylamide gel electrophoresis detection of common primers showed that the quality was stable and the amplified bands were clear. Compared with the commonly used plant genomic DNA extraction methods， this method had the advantages of high flux， high speed and low cost， it was a better method which was suitable for seed acid related molecular markers in seed enterprises.

Key words： Watermelon； DNA extraction； PCR

使用分子標記手段可以大幅減輕田間育種和質量檢測的工作強度，提高工作效率。隨著種子產業和分子生物學的不斷發展，SSR等分子標記手段已越來越普遍地應用于種子室內純度測定、品種真實性鑒定和分子標記輔助育種等環節中[1-2]。SSR分子檢測的前提是高效提取符合質量要求的DNA，而不同來源的材料對提取的DNA質量有一定影響。正常的室內純度檢測所需DNA一般取材于發芽后的下胚軸或芽尖，DNA質量較高，但提取通量低、速度慢[3-6]。如果是對田間種植的西瓜品種進行純度鑒定抽檢，則只能用葉片提取；當開展分子標記輔助育種時，要保證有標記的單株正常在田間生長，由于室內發芽提取DNA損壞了單株，顯然不適合采用該方法，也需要以田間生長的葉片作為提取DNA的材料，而西瓜葉片提取的DNA往往含有大量的酚類物質等雜質[5-7]，達不到后續試驗的質量要求。……