苏铁珊瑚根共生念珠藻ITS序列多拷贝研究

The Study on Multicopy of ITS Sequences of Nostoc in Cycads Revolute

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。

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 tRNAAIa 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.