大麦小孢子DNA提取方法的研究  

STUDIED ON THE METHOD OF DNA EXTRACTION FROM BARLEY MICROSPORES

作　　者：李文泽[1] 宋子红[1] 徐丽娟[1] 郭宝太[1] 于常春[1] 

机构地区：[1]莱阳农学院农学系

出　　处：《莱阳农学院学报》1998年第4期237-241,共5页Journal of Laiyang Agricultural College

基　　金：国家自然科学基金

摘　　要：以冬性大麦品种Ｉｇｒｉ为材料，研究了小孢子游离、破壁及ＤＮＡ提取方法。结果发现：①提取时间和旋切刀具的转速，对小孢子的得率均有直接影响。时间越长，得率越高。旋切刀具的转速越高，其游离小孢子效果越好，最高为２２０００ｒｐｍ，达到９４．０。但是当转速在１２０００ｒｐｍ以上时，各处理差异不明显；②超声波处理不同时间对小孢子破壁率影响明显，以１０ｓ／１０ｓ／１０ｓ／１０ｓ／１０ｓ／１０ｓ（间隔１０ｓ）处理的小孢子破壁率最高达到６８．８５％；最低的处理为２０ｓ／２０ｓ／２０ｓ（间隔２０ｓ），仅为１２．７７％。前者是后者的５．４倍。此外，超声波的输出功率对小孢子破壁率的作用也十分明显，功率越高，效果越好。超声波法的破壁率明显地优于液氮研磨法。前者是后者的１１．６倍；③ＣＴＡＢ法和微量快速提取法２种ＤＮＡ提取方法的ＤＮＡ得率和质量无明显差异，均适合于大麦小孢子ＤＮＡ的提取。The barley cultivar of winter type “Igri”, which was grown in the field, was used as donor plant. The spikes at the mid-uninucleate stage were excised, and then pretreated at 4℃ for 28 days .They were used to study the methods of microspore isolation, dissolution of pollen wall and DNA extraction. The results showed that the yield of isolated microspores was significantly affected by time and speed of microblending. The longer the time of microblending was, the higher the yield of isolated microspores was. The effect of speed of microblending on the yield of isolated microspores was the same as the time. On the other hand, the rate of dissolution of pollen wall was also significantly affected by the different time treatments of ultrasonic appliance. It for 10sec, repeated 6 times and with 10sec interval between repeated treatments was highest in all treatments, which reached 68.85%. However, it for 20sec, repeated 3 times and with 20sec interval between repeated treatments was lowest in all, which reached only 12.77%. Meanwhile, the rate of dissolution of pollen wall was affected by the output control of ultrasonic appliance. The higher the output was, the higher the rate was. We found that the ultrasonic appliance was much better than grinding with liquid N on the rate of dissolution of pollen wall, which was by 11.6 times higher than that of liquid N. Finally, there were not significantly differences on the yield and the quality of DNA extraction between methods of DNA extraction used in this research. They were all very suitable for microspore DNA extraction in barley.

关 键 词：大麦 小孢子 游离 破壁 DNA提取 超声波 

分 类 号：S512.302[农业科学—作物学]