摘要
通过试验寻求得到一种快速、简便抽提飞蝗 (Locustasp .)总DNA的方法 ,使每头雄性和雌性成虫分别可以得到 5 0和 10 0 μg的总DNA。所得到的总DNAOD2 60 /OD2 80 为 1 5～ 2 2 ,分子量 45kb。为了获得高分子量的DNA产品 ,使RPAD结果具重复性 ,酚氯仿抽提后的DNA沉淀用灭菌Tip头挑出 ,而不用离心收集。对各种分析条件如模板、Taq酶、dNTP及引物的浓度、不同的PCR仪、反应管进行了比较试验 ,发现在一定的范围内 ,它们对RAPD结果影响不大。用优化的试验条件对我国 3个飞蝗亚种 5个地理种群进行RAPD分析 ,结果在 3个亚种UPGMA聚类图中 ,东亚飞蝗和西藏飞蝗的 2个种群以 10 0 %Bootstrap分别聚类在一起 ,亚洲飞蝗与东亚飞蝗的 2个种群以 6 6 %的Bootstrap聚类在一起。在 3个亚种所有个体的UPGMA聚类图中 ,亚种内的所有个体都聚类在一起 ,各自形成独立分支 ,说明 3个飞蝗亚种有明显的区别。西藏飞蝗的 2个种群之间 ,群居型与散居型东亚飞蝗之间在聚类图中混合聚类 。
In this study,we had developed a fast and easy method to extract total DNA from migratory locusts.We could extract about 50?μg and 100?μg DNA from single male and female individual respectively.The extracted total DNA was about 45?kb in length with OD 260 /OD 280 between 1 5 and 2 2,which could satisfy the requirements of RAPD and PCR on DNA template.In order to obtain high molecular weight DNA from migratory locusts and repeatable RAPD results,we used autoclaved tips to collect precipitated DNA after being extracted with phenol/chloroform instead of collecting by centrifugation.We tested experimental conditions including different PCR amplifiers,tubes and concentrations of template DNA,Taq DNA polymerase,dNTP and primer that might affect RAPD results.Within some limits,the concentrations of template DNA,Taq DNA polymerase,dNTP and primer did not greatly affect RAPD profiles.The optimized reaction conditions of RAPD for three Chinese locust subspecies in 25?μL reaction volume were as follows:20?ng template DNA,0 1?mol/L dNTP,0 2?μmol/L primer,1?U Taq polymerase.Some advice was also given,especially all individuals of all populations should be amplified with every primer at the same reaction conditions.With the optimized reaction conditions for RAPD,we analyzed the genetic relationship among three locust subspecies in China.The genetic distances generated by RAPDDIST between populations belonging to the same subspecies were less than those to different subspecies.In the UPGMA Phenogram of three locust subspecies,the two populations of L m manilensis and L m tibetensis were clustered together with a bootstrap of 100%,respectively. The Bameng population of L m migratoria was clustered with two populations of L m manilensis with a bootstrap of 66%,meaning that the Bameng population of L m migratoria could also clustered with L m tibetensis .In the UPGMA Phenogram of all individuals of three subspecies,all individuals of one subspecies were clustered together to form a branch,so three subspecies were significantly different in DNA polymorphism.Thus,RAPD method could be used to separate the three subspecies.There was a gene flow between the populations of L m tibetensis and L m manilensis .In order to get the true relationships among the three subspecies,more samples and primers should be used in the analysis on the three locust subspecies with RAPD,and other molecular techniques should be applied to test the results of RAPD.
基金
国家基金委重点项目! (39430 0 30 )
关键词
飞蝗
DNA提取
RAPD优化
种群遗传
蝗科
Migratory locust
Extraction of total DNA
RAPD optimization
Population genetic
