5种笛鲷mtDNA及Cyt b基因片段的RFLP比较

采用线粒体DNA限制性片段长度多态性分析(mtDNA-RFLP)和线粒体DNA的细胞色素b基因(Cyt b)的部分序列扩增限制性片段长度多态性分析(mtDNA PCR-RFLP)两种方法，对笛鲷属5种鱼类进行种间系统发育的比较研究，结果显示：(1)画眉笛鲷依照其形态学上体侧条带颜色差异，可分为褐带画眉笛鲷和黄带画眉笛鲷两个种；(2)千年笛鲷和星点笛鲷、褐带画眉笛鲷和黄带画眉笛鲷、金带笛鲷和金焰笛鲷之间遗传距离相对较近；(3)两种分析方法都可以为种的区分提供方便有效的分子标记；(4)两种分析方法在构建发育系统树上不完全一致。本文从mtDNA角度深入研究了南海海域笛鲷的系统发生，表明了mtDNA作为一种广泛应用的分子标记的实用性，同时也有一些潜在问题需要进一步研究。

Comparison on RFLP of mitochondrial DNA and Cyt b gene segment for 5 species in Lutjanus

The phylogenetic relationships of 5 species of snappers ( Lutjanus stellatus , L . sebae, L. vitta, L . f ulvus and L . fulvif lamma) were studied comparatively, which were based on 2 methods: restriction fragment length polymorphism of mitchondrial DNA ( mtDNA-RFLP) , RFLP of cytochrome b ( Cyt b ) gene segment ( > 900bp) amplified by polymerase chain reaction( PCR-RFLP) . The sample numbers of Lutjanus stellatus , L . sebae, L . vitta, L . fulvus and L . fulviflamma were 17, 17, 35, 17 and 17, respectively, which are collected in the South China Sea from Sep. 2002 to Jun. 2003. The mtDNA being isolated and purified from fresh liver s of samples range from 17. 05? 0. 46 to 17. 77? 0. 91 kb. Having been digested by 19 restriction endonucleases recognized 6 base pair s ( EcoR. , Hind ? , Bgl . , Bgl ? , Sty . , Xba . , Dra . , Pst . , Pvu ? , Xho . , BamH . , Sca . , EcoR . , Sma . , Mlu . , Sal . , Hinf . , Kpn . , Hp a . ) , the mtDNA were analysed. The result of mtDNA-RFLP show s that L . vitta could be divided into HE and HD, based on the co lor of stripe on the middle of the side. The physical maps of mtDNA from L. stllatus and L . sebae have been constructed based on the endonucleases that are chosen from 19 restriction endonucleases. By comparison of the sequence of cyto chrome b gene PCR-RFLP using different amplification templates ( one from purified mtDNA and the other from nDNA) , the possible interferences of nuclear mitochondrial pseudogenes ( Numts) were excluded and the possible pseudogenes from nDNA were preliminarily discussed. Having been digested by 6 restriction endonucleases recognized 4 base pairs( Sty . , Hinf . , Hpa ? , Msp . , Sau96. , Sin . , Taq . , Hae ? ) , the sequences were analy sed. The results of mtDNA PCR-RFLP support that L . vitta could be divided into two species: HE and HD by the 0. 0751 of divergency index. The results reveal that all of the 2 analysis methods are useful for seeking o ut the genetic markers among species effectively. And the results from RFLP indicate that the L . vitta could be divided into two species based on the color of stripe on the middle of the side. At the same time, the constructions of phylo genic trees by neighbor- joining( NJ) method show that there are some differences along different methods on the specific level when they are used for phy logenesis.