【作者】 梁君荣；

【导师】 苏永全；

【作者基本信息】 厦门大学 ， 海洋生物学， 2002， 博士

【摘要】 花尾胡椒鲷（Plectorhynchus cinctus（Temminck et Schlegel））是我国东南沿海重要的优良海水网箱养殖新品种。鉴于当前海水养殖鱼类均为小群体，有效亲鱼数量少，常处于被迫高度近交状态，遗传资源极易衰退的现状，为了花尾胡椒鲷资源的可持续利用，十分有必要对其遗传背景进行研究。本文率先应用同工酶电泳技术，染色体分析技术和DNA分子标记技术从不同层次对花尾胡椒鲷养殖群体的种质资源进行了深入研究。首次报道了花尾胡椒鲷体色变异种的发现，并首次应用生物学方法和多种分子标记技术对花尾胡椒鲷变异种与正常种的生物学性状差异和DNA水平的遗传差异进行了综合性检测，筛选出了花尾胡椒鲷与体色相关的DNA分子标记。主要结果分述如下： 1．应用聚丙烯酰胺凝胶电泳法（PAGE）对花尾胡椒鲷广东柘林湾海水养殖群体48尾进行了11种同工酶分析，共检测到26个基因位点，其中sSOD-1、sSOD-2、sMDH和IDDH-2等4个座位表现出多态性。该群体的多态位点比例P为15.4％，平均每个位点的杂合度H_e为0.047。同时对该群体进行了RAPD检测，34个随机引物共扩增出197条DNA片段，并获得45个多态性DNA片段。其中多态位点比例为22.84％，平均遗传差异度为7.7％。从同工酶和RAPD检测结果一致说明，与一般脊椎动物的遗传变异水平相比，该养殖群体的遗传多样性处于中等水平，仍保留了一定的遗传变异水平。 2．比较花尾胡椒鲷不同养殖群体遗传多样性的同工酶检测结果，发现不同养殖群体的遗传多样性水平存在差异，表现为广东柘林湾养殖群体较福建厦门养殖群体保持较多的遗传变异水平，厦门养殖群体在mSOD—1₁₁₀、mSOD—2₁₁₀、sMDH₁₂₀和IDDH—2₁₃₈等基因上发生了稀有基因的散失，而广东柘林湾养殖群体也发生了GCDH₈₀等位基因的散失，表明花尾胡椒鲷不同养殖群体的种质资源已发生不同程度的衰退。 3．采用秋水仙素腹腔注射，肾细胞直接制片法对养殖花尾胡椒鲷进行染色体核型分析，确定花尾胡椒鲷具有48条A型染色体，核型公式为2n=48t，NF=48。此结果表明花尾胡椒鳃的染色体核型与其它石妒科（Pomadasyidae）鱼类的染色体核型都不问，表现出石妒科鱼类染色体核型的多态性。 4．从花尾胡椒碉总基因组中扩增出6gobp左右的16s rRNA的基因片段，对该基因片段分别进行EcoR、Mscl、Sac、Hindlll、Pst五种内切酶的酶切图谱分析。其中只有 Mscl酶切了花尾胡椒绸的 165 rRNA目的基因片段，其余内切酶未将 165 rRNA 切开。该内切图谱与同科不同属的斜带置绸 （Hopalogenys miens）的同源性 165 rRNA基因片段的相同内切酶的酶切图谱比较，体现出酶切图谱的多态性，即花尾胡椒鳃和斜带置鳃的 165 rRNA目的基因片段的内切酶图谱在 Mscl、Sac两种酶切图谱上表现出差异。该多态性片段体现出两种鱼在酶切位点上的差异。 5．对花尾胡椒绸165 rRNA扩增片段进行测序，并选取上游421hp大小片段与石妒科其它 3种鱼类用镜绸属的o附alogenys miens、仿石妒属的 Haemulonsciurus和石妒属的PomadasyS maCulatus)的同源性序列进行比较发现，花尾胡椒鳃的 165 rRNA基回序列与其它 3种石妒科鱼类的遗传差异度均在 11．72～14．35％之问。4种鱼共出现105个多态位点，变异座位gO个，占总序列的21．38％。在所排列的 165 rRNA基因同源序列中，第 252～274位碱基之间的mA存在高频率的多态性核昔酸位点，这段约20hP大小的核昔酸区可以作为鉴别花尾胡椒绸及其它3种石妒科鱼类的特异性标记。 6．基于 165 rRNA基因序列构建的 4种石妒科鱼类 UPGAE和 MJ系统进化树一致表明花尾胡椒鳃和斜带豢鳃的亲缘关系较近，而石鲸属的P maculatus和仿石莎属的H．sciurus亲缘关系较近。而依据染色体核型推断的儿种石妒科鱼类的进化关系为：花尾胡椒鳃为最原始的种类，H SCICruS为较特化的种类，而斜带蔑鳃和横纹置鳃介于两者之问。这些研究都为石鲸科鱼类的系统进化关系提供了细胞学水平和基因DNA分子水平的依据。 7．1999年在海口拓林湾养殖网箱首次发现花尾胡椒鳃变异种，表现为体白黑眼。2002年8月在海南养殖网箱中发现同样的变异种，对其生物学性状检测发现，除体色外，花尾胡椒绸变异种较正常种在生长速度、运动活力、肌肉氨基酸组成和含量卜存在差异，变异种表现为摄食活跃、游动积极、生长速度较快R肌肉中鲜味氨基酸含量较高。 －8．采用 165 rRNA基因序列分析、PCR－RFLP、RAJ，D、fLP、ISSR和 BSA等多种分子标记方法并结合BSA法首次对花尾胡椒绸及其变异种的遗传差异进行多角度、多方面的检测，结果表明花尾胡椒绸及其变异种在总基因组DNA。微卫星DNA、mtDNA、DNA序列碱基组成上均表现出很高的同源性和一定的遗传差异。各种方法检测所得的遗传相似性为96石1～98．57％，遗传差异度为1．43～3．39％。在体色变异种和正常种之间，共扩增出17个特异性DNA片段（13条RAPD差异带，3条AFLP差异带和 1条ISSR差异带）。 9．应用 RAPD、ISSR分于标记和 BSA方法相结合，首次进行了花?更多还原

【Abstract】 The genetic resources of Plectorhynchus cinctus were investigated usingisoenzyme electrophoresis, karyotype analysis and molecular marker. The mutant of Pcinctus was firstly reported, and the genetic and biological character differencbsbetween P cinctus and its mutant were studied, DNA markers related to the bodycolor of P. cinctus were aslo abstained. The results are as bellowi1. The genetic diversity of P cinctus in the cultured stock from Zhe-lin bay inGuangdong was analyzed. 48 samples were examined by polyacrylamid gelelectrophoresis (PAGE). Ainong the 26 loci in ll enzymes, four polymorphic lociwere found. The meart level of polymophism was 15.4% and heterozygosity was0.047. With 34 random primers fOr RAPD-PCR, 197 infOrmative fragments wereobtained, including 45 variation fragments. The polymorphism and the meandtherence within the same stock were 22.84% and 0.077 respectively. The resultsindicated that the genetic diversity of P cinctus from Zhe-lin bay wereintermediate, compared with the other vertebrates.2. The genetic variations of two different cultured stock of P cinctus assessed byisoenzymes.The result indicated the stock of P cinctus from Zhe-lin bay showedhigher genetic diversity than that from Xiamen. The former lost one rare allele ofGCDHso. and the latter the four rare alleles of mSOD-11lo, mSOD--2,,.,, sMDHl2oand lDDH-2l38. All the above results implied the genetic resource had beendecline in both cultured stocks.3. The karyotype of the renal cell of Pcinclus was examined by PHA injection--airdrying method. The resu1t showed that the number of chromosome was 48, and4the karyotype formula was 2n=48=48t, NF=48, which was not in accordance withthe karyotype of the other three members of Pomadasyidae.4. 680 bp fragments of 16S rRNA gene in mtDNA in P cinctus and HaPatogenysnitens were amplified and digested with 5 restriction enzymes of Ecor I, Mse I 9Sac I, Pst’ I and HindI1I. The results showed the fragment of 16SrRNA gene in Pcinctus was only digested by Mse I and the fragment in H nitens by Mse I andSac I. But the number of cleavage site of Mse I was dtherent betWeenP cinctusand H nitens. These differences could be used as the genetic marker to distigushPcinctus from H nitens.5. The fragment of l6S rRNA gene of P cinctus was sequenced, the region of 421bpwere compared with the same regions of the other three members ofPomadasyidae. The results proved that the dtherences of polymorphrism betweenP cinctus and the other three members were from 11.72% to 14.35%. There were105 polymorphic sites, 90 variation loci within the fOur species. Onehypervariable domain from 250bp to 274bp of 16S rRNA was fOund, and thehypervariable domain could be regarded as the DNA marker for the fOur species.6. UPGMA and MP molecular phylogenetic trees of the above four species inPomadasyidae were established by Phylip 3.0. Two molecular phylogenetic treesconsistent with each other. Two trees supported that both H nitens and P cinctusoriginated from one ancestor, but both Haemulon Sciurus and PomadaSySmaculates from another ancestor.7. Mutant of P cinctus was firstly found in Zhe-lin bay. It lost the body color of wildtype and was white in skin but black in eyes. The difference of biologicalcharacter between P cinctus and its mutant was analyzed. The results showed thatthere were differences in grow rate, movement, the compose and content of aminoacid in the muscle.8. 16S rRNA gene sequence analysis. PCR-RFLP. RAPD. MLP. ISSR and BSAtechniques were applied to study on the genetic difference between the P cinctusand its mutant. The results showed that P cinctus and its mutant shared high5genetic homology in mtDNA. Microsatellite DNA and genome DNA. The geneticsimilarity abtained from the different techniques were 96.61--98.57%, the geneticdtherence were 1.43--3.39%. 17 special DNA markers including 13 RAnDmarkers, 3 AFLP markers and 1 ISSR marker were更多还原