国家科技基础条件平台项目(No.2007DKA30470-003);江苏省自然科学基金项目(No.BK2006029)
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核糖体DNA内转录间隔区(internal transcribed spacers,ITS)是经常被用作种和种群水平系统研究的分子序列。本文分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)内转录间隔区,包括部分18S序列,ITS1、5.8S、ITS2全序列及部分28S序列。4尾奥利亚罗非鱼的10个克隆序列分析表明,其存在长度不同的a、b两种类型ITS1。a型长为536 bp,GC含量为69.96%;b型长为520 bp,GC含量为69.04%～69.42%。4尾尼罗罗非鱼的10个克隆序列分析表明,其只存在a型ITS1,长为536～540 bp,GC含量为69.42%～70.19%。与b型ITS1相比,a型ITS1在16～31 nt有16 bp片段(GGCCCGCCTGCGGCGC)的插入。奥利亚罗非鱼和尼罗罗非鱼共20条ITS序列中,5.8S长度均为157 bp,GC含量为56.69%～57.96%;ITS2为408 bp,GC含量为72.79%～74.26%。奥利亚罗非鱼和尼罗罗非鱼ITS区序列相似性高达98.2%,表明这两种罗非鱼亲缘关系很近。此外,本文对14尾奥利亚罗非鱼、15尾尼罗罗非鱼以及15尾奥尼罗非鱼[O.aureus(♂)×O.niloticus(♀)]ITS1的扩增结果显示,奥利亚罗非鱼均有a、b两种类型ITS1;15尾尼罗罗非鱼中1尾为a、b两类型ITS1,14尾为a型ITS1;15尾奥尼罗非鱼中则有6尾具有a、b两类型ITS1,9尾为单一的a型ITS1。分析表明,奥利亚罗非鱼在ITS1这个位点一致性高,但尼罗罗非鱼中有1尾混杂了奥利亚罗非鱼的基因,同时也说明分子生物学手段应用于种质鉴定比形态学手段更为精确。
Internal transcribed spacers in ribosomal DNA were often used as molecular sequence in phylogenetic study at the population and species level.PCR was used to isolate the complete sequence of internal transcribed spacer(ITS1-5.8S-ITS2) and partial 18S and 28S sequence from Oreochromis aureus and O.niloticus.The analysis of ten clones from four individuals indicated that O.aureus had two types of ITS1(ITS1a and ITS1b) with different lengths.ITS1a was 536 bp,GC content was 69.96%;ITS1b was 520 bp,GC content was 69.04%-69.42%.The analysis of ten clones from four O.niloticus individuals indicated that only ITS1a existed in O.niloticus which was 536-540 bp in length,and 69.42%-70.19% in GC content.Compared with ITS1b,ITS1a had 16 bp sequences(GGCCCGCCTGCGGCGC) which was inserted from 16 nt to 31 nt.5.8S of all the 20 sequences from O.aureus and O.niloticus was 157 bp,GC content was 56.69%-57.96%,and ITS2 was 408 bp,GC content was 72.79%-74.26%.The high identity of ITS between O.aureus and O.niloticus indicated that the genetic relationship of the two Oreochromis was very close.Furthermore,PAGE electrophoresis was used to investigate the ITS1 divergence of O.aureus,O.niloticus and O.aureus(♂) ×O.niloticus(♀) with distinguished morphological characters.The analysis showed that all of the 14 O.aureus individuals had two types of ITS1;1 individual of the 15 O.niloticus and 6 individuals of the 15 O.aureus(♂) ×O.niloticus(♀) had two types of ITS1.This paper showed the population of O.aureus had high identity while 1 individual of O.niloticus was genetic mixture with O.aureus in the locus of ITS1 and it also indicated that the means of molecular biology is more accurate than the means of morphology in germplasm identification.
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陈雪峰,李红霞,俞菊华,唐永凯,李建林.2009.奥利亚罗非鱼与尼罗罗非鱼rDNA内转录间隔区序列特征.动物学杂志,44(2):92-96.
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