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摘要: 采用十六烷基三甲基溴化铵(CTAB)-硅珠法提取光皮桦Betula luminifera嫩叶DNA,利用近缘种日本白桦B. platyphylla var. japonica和欧洲白桦B. pendula的引物,建立光皮桦简单重复序列标记(SSR)反应体系。在体系建立过程中,采用单因素法分别对影响聚合酶链式反应(PCR)反应的因素进行分析。结果表明:在20 L反应体系中,模板DNA用量、Mg2+浓度、脱氧核糖核苷酸(dNTPs)浓度、引物浓度、Taq DNA聚合酶用量分别为60 ng,1.500 mmolL-1,0.175 mmolL-1,2.500 mmolL-1,1.0 16.67 nkat时结果最好;引物退火温度比日本白桦扩增时提高1 ℃时效果佳。PCR扩增产物经聚丙烯酰胺凝胶电泳后随机挑取其中3对引物扩增的多态性片段进行克隆测序,对引物的通用性做进一步检验,序列经比对后得到的与原物种序列相似度(max ident值)均在95.0%以上。可见,近缘种日本白桦和欧洲白桦的SSR引物可以在光皮桦上应用。图7表2参17
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关键词:
- 林木育种学 /
- 光皮桦 /
- 简单重复序列(SSR)分子标记
Abstract: To find the optimal simple sequence repeats(SSR) system for Betula luminifera,the cetyltrimethyl-ammonium bromide (CTAB) method was used to extract DNA from tender leaves,using the known primers from related species:B. platyphylla var. japonica and B. pendula. Then,one factor was changed at a time to optimize the polymerase chain reaction(PCR). Next,Polyacrylamide Gel Electrophoresis(PAGE) was used to detect the amplified products with three primers segments randomly selected from thirty-six pairs of primers and sequenced to further check the universality of the primer. Results showed that in a volume of 20 L,the optimal reaction system was 60 ng template DNA,1.500 mmolL-1 Mg2+,0.175 mmolL-1 deoxyribonucleotide triphosphate(dNTP)s,2.500 mmolL-1 primer,and 1.0 16.67 nkat Taq polymerase. Compared with B. platyphylla var. japonica and B. pendula,the optimal annealing temperature of the primer was 1C higher. The blast results from primer AF310851,AB084479,and AB084480 showed that the values of max ident were all above 95.0%. This indicated that the SSR primers of birch could be used with B. luminifera.[Ch,7 fig. 2 tab. 17 ref.] -
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链接本文:
https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2010.03.023

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