LIU Hangkong,WANG Anzhu,ZHAO Caiping,et al.Study on Extracting DNA From Peach Leaves Efficiently[J].Northern Horticulture,2015,39(13):120-125.[doi:10.11937/bfyy.201513034]
高通量提取桃树叶片组织基因组DNA的研究
- Title:
- Study on Extracting DNA From Peach Leaves Efficiently
- 文章编号:
- 1001-0009（2015）13-0120-06
- Keywords:
- peach; leaf; high throughput; genome DNA
- 分类号:
- S 662.103.6
- 文献标志码:
- A
- 摘要:
- 以1个桃树杂交后代群体中不同成熟度的桃叶片组织为试材，以商品试剂盒为对照，采用高通量提取DNA的方法，研究了提取试剂、研磨仪使用钢珠大小和震动频率、时间等参数对提取DNA产物产率、质量的影响。结果表明：在常用的CTAB中添加0.3%的抗坏血酸，并提高PVP浓度至4%，NaCl浓度至12%，能有效解决试剂盒提取过程中出现色素及蛋白质、多糖等杂质问题。提取方法和研磨方法优化组合，筛选获得高通量提取不同成熟度桃叶片DNA的方法，表明改良的提取方法采用5 mm研磨珠、60s、30次/s或5 mm研磨珠、60s、25次/s的研磨预处理，不仅省时省工，适用不同成熟度的桃叶片组织，而且提取效果最好，产率最高，质量符合基因组测序要求。该研究为高效提取DNA提供了技术依据。
- Abstract:
- Taking different maturities of leaves from peach progenies population as material，commercial kit was experimented as control group.The effect of the parameters of retsch about the grinding bead sizes，the frequency and time of vibration on yield and quality of DNA were investigated by the method of high throughput extracting DNA.The results showed that adding 0.3% ascorbic acid，increasing the concentration of PVP to 4% and soaring the concentration of NaCl to 12% at the base of usual CTAB extraction buffer,the pigment and protein，polysaccharide were eliminated，and the problem appeared in the process of extracting DNA by the kits were effectively solved.Combining with the modified extraction method，the grinding pretreatment was screened.With the vibration frequency of 30 times/sec in 60 seconds by 5 mm grinding bead or the vibration frequency of 25 times/sec in 60 seconds by 5 mm grinding bead，a high throughput approach for DNA isolation from different maturity of leaves were acquired.It is not only considered to be a good ways to save time and dispense with much labor at extracting DNA，but also it was applied to extracting DNA from different maturity of peach leaves.More importantly，it could get high yield，reliable quality DNA for the genome sequencing.This study would provide guidance for technology of high performance DNA isolation.
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备注/Memo
第一作者简介：刘航空（1980-），男，陕西咸阳人，博士研究生，实验师，现主要从事果树栽培及遗传育种等研究工作。E-mail：lhk2205@163.com1.责任作者：韩明玉（1962-），男，陕西扶风人，博士，教授，研究方向为果树栽培及遗传育种。基金项目：国家“863”资助项目(2011AA1002060001)；国家园艺实验教学示范中心建设资助项目(I105021301)。