【摘要】 目的:研究苏铁珊瑚根共生念珠藻16SrDNA-23SrDNA基因间隔序列(ITS)拷贝数。方法:提取苏铁珊瑚根共生念珠藻全DNA,PCR扩增其16SrDNA-23SrDNA基因间隔序列(ITS)克隆并测序;采用Cluster X 1.83对所得序列进行比对,用DNAman软件对比对结果进行人工校正;采用割胶回收电泳检测、菌液PCR及混合模板PCR扩增三种方法验证ITS-L2。结果:ITS-S(420 bp)不含任何氨基酸编码序列,ITS-L1(676 bp)包含一个异亮氨酸和一个丙氨酸编码序列;ITS-L1和ITS-L2在琼脂糖凝胶电泳检测时长度分别为676 bp和1000 bp,而谱带经克隆测序后结果表明ITS-L1和ITS-L2碱基序列和长度几乎相同,仅有两个碱基发生变异。结论:ITS-L2为假阳性,可能是由ITS-S和ITS-L1形成的异源双链;苏铁珊瑚根共生念珠藻含有两种类型的ITS拷贝,一种是含有tRNAIle和tRNAAla编码序列的ITS,另一种是不含tRNAIle和tRNAAla编码序列的ITS。更多还原

【Abstract】 Objective: To investigate the multicopy number of ITS sequences of Nostoc in Cycads revolute. Methods: The total DNA of Nostoc in Cycads revolute was extracted. 16S rDNA-23SrDNA internal transcribed spacer(ITS) was amplified from Nostoc, which is symbiont in cycads revolute, then was TA-cloned and sequenced. The sequences were blasted by Cluster X 1.83, and DNA man was used to calibrate the results; Electrophoresis detection of PCR amplification with bacteria solution or mixture template was used to check the results. Results: The PCR amplification had three products [420 bp(ITS-S),676 bp(ITS-L1),1000 bp(ITS-L2)]. And the sequences indicated that none of the tRNA coding region was included in the ITS-S(420 bp), while ITS-L1(676 bp) included both tRNAIle and tRNAAla coding regions. ITS-L1 and ITS-L2 were at 676 bp and 1000 bp. But the sequences results showed they were almost of the same length and base sequences, and only two bases were different. Conclusion: It was inferred that ITS-L2 was false positive, and it maybe heteroduplexes composed of ITS-S and ITS-L1. There are two types of multicopy of ITS sequences of Nostoc in cycads revolute. One includes both tRNAIle and tRNAAla coding region, and the other includes none.更多还原