【作者】 罗育发；

【导师】 颜亨梅；

【作者基本信息】 湖南师范大学 ， 动物学， 2004， 硕士

【摘要】 本研究应用RAPD、DNA序列分析等分子标记技术，根据有关种群遗传学和动物系统学原理与方法，对我国狼蛛科主要类群的遗传多样性、分子系统发育关系及其在农药胁迫下所引起的遗传分化和分子适应性机理等方面进行了初步研究，获得了以下结果： 1 进行了RAPD技术在蜘蛛遗传多样性研究中的方法探讨。利用蜘蛛的附肢作为其基因组DNA抽提的材料，且该改进的方法所抽提的蜘蛛基因组DNA的产量与纯度足以满足RAPD分析的要求；通过优化RAPD-PCR反应条件，可以获得清晰度高，重复性好的RAPD图谱以用于蜘蛛（如拟水狼蛛Pirata subpraticus拟环玟豹蛛（Pardosa pseudoan nulata）武昌獾蛛Trochosa wuchangensis等）的遗传多样性研究；在不投食饲养武昌獾蛛一个月以上的前提下，用随机引物S₉₂（5′CAGCTCACGA 3′）比较分析了武昌獾蛛的头胸部与其附肢各自的RAPD图谱的差异，差异不显著（P＞0.05）。结果表明：蜘蛛的附肢和头胸部均可作为其遗传多样性研究材料，但以前者为材料可以有效防止异源DNA的干扰。因而，其基因组DNA在作为蜘蛛遗传多样性的RAPD分析模板时要优于头胸部。 2 根据蜘蛛外生殖器结构特征和RAPD分子标记分析的结果表明：武昌獾蛛Trochosa wuchangensis自然种群存在明显种群内遗传变异现象。即使同一地理种群的武昌獾蛛也有多种变异类型，且该种内变异并不完全是因分布地域不同而产生的。尹长民教授等（1997）所报道的武昌獾蛛外生殖器结构可明显分为三大类型：（ⅰ）两垂兜间合抱部分梨形，长≦宽；纳精囊远端分叶时内叶稍短于外叶。（ⅱ）两垂兜间合抱部分梨形，长＞宽；内叶明显短于外叶。（ⅲ）两垂兜间合抱部分窄长，长＞宽；外叶明显细。作者实验中所获得的武昌獾蛛雌性成体RAPD谱带特征与尹老师以外生殖器形态特征为依据所划分的三种变异类型基本一致。同时对ⅰ型和ⅱ型内个体间的遗传相似度进行方差分析表明，ⅱ型内个体间不存在显著差异，但ⅰ型内个体间存在显著差异（P＜0.05）。研究还表明性别不同的武昌獾蛛，其成体的RAPD谱带也有差异，即随机引物592扩增的结果在不同性别的武昌灌蛛相互间显示种的特异性，并发现所扩增的253 bp带为雄性武昌灌蛛的种的特异性谱带。引物552和引物52，对不同生长发育期的武昌灌蛛样本的RAPD一PCR扩增中都显示其相互间存在不一致的谱带特征。经统计分析发现成熟个体的谱带类型稳定，变化较小;未成熟个体的谱带类型不稳定，变异较大。 3应用1 ZSr洲A基因序列分析探讨了狼蛛优势种群对长期农药胁迫的分子响应机制。通过对12SrRNA基因第三结构域的序列比对分析，研究显示:（i）拟水狼蛛厂丫了刀tasu如了了刁tfcus在长期农药胁迫下的12SrRNA基因第三结构域序列发生了明显改变，即碱基组成由315 bp减少为300 bp，转换/颠换为17/5，核昔酸变异百分数（p一distance=序列差异/序列大小）达到0.095，同源性仅为85.8%;（ii）拟环纹豹蛛儿厂由sa pse“由a朋u1二ta在长期农药胁迫下的12SrRNA基因第三结构域序列也发生改变，即碱基组成由301 bp减少为299bp，转换/颠换为11/10，核昔酸变异百分数为0.083，同源性为91 .7%;（iii）比较拟水狼蛛和拟环纹豹蛛在长期农药胁迫下，其12SrRNA基因第三结构域的序列所发生的改变，以拟水狼蛛所受的影响最明显，可见连续丢失几段4一10以上bp的片段，说明拟水狼蛛对农药的胁迫更为敏感，（iv）供试两种狼蛛标本的基因组DNA的四个碱基对中以A和T的变异频率最高，属农药敏感型碱基对。（v）根据拟水狼蛛和拟环纹豹蛛12SrRNA基因片段的碱基共同突变位点的规律，发现192一228片段中有37个位点是长期农药胁迫下狼蛛的12SrRNA基因片段中共同突变位点频率最高的区域。作者认为这就是该2种狼蛛对农药胁迫的敏感区。同时，发现在无直接农药胁迫的环境中，这两个狼蛛种群中的12SrRNA在192一咒8该狭窄区域内的相似性明显增加，其原因有待进一步研究。 4将自测的我国蜘蛛目狼蛛科5属6个种和从互联网Genbank中检索到相关物种的线粒体基因组12SrDNA的序列进行同源性比较，计算核昔酸使用频率。然后据此进行分子分析，利用2个外群（漏斗蛛科的机敏漏斗A群了eI7adz’几P1’叮了z’s和缘漏斗刀gelena了2初右at）和2种建树方法（邻近法Neighbour Joining，NJ和最大简约法Maxi^Parsimony，MP）分析我国狼蛛科内的亲缘关系。获得平均为291.6bP的序列中，A+T平均含量为78.54%，而G+C含量只要21.50%，颠换取代（tranversion）的速度多数大于或接近转换取代（transition）的速度，其中161个核昔酸位点存在变异。研究结果表明:在蜘蛛目狼蛛科有差异的161 bp中，属内种间仅为1.08%，狼蛛科属间为6.85%一14.80%。所构建的分子系统树表明:科内的属和属内的种均优先聚在一起;狼蛛科现行分类系统中各亚科的演化关系顺序为:马蛛亚科一狼蛛亚科一豹蛛亚科;狼蛛科各属的演化关系顺序为:水狼蛛属一马蛛属（或水狼蛛属和马蛛属）一罐蛛属一狼蛛属一豹蛛属;水狼蛛属为最早分出的一支或者水狼蛛属和马蛛属最先聚为一族，二者关系较近，是较为原始的类群，同为马蛛亚科。这与传统的分类观点有所差别。所更多还原

【Abstract】 Based on the molecular markers, including random amplified polymorphic DNA (RAPD) and analysis of DNA sequences, the genetic diversity and molecular phylogeny of the main groups of wolf spiders from China were studied by using the principle and methods of group genetics and analysis of the DNA sequences. The results were summarized as follows:1. The method of applicability of RAPD in study of genetic diversity of spiders was discussed. The appendix of spiders can be employed to extract their genomic DNA; The production and purity of genomic DNA of spiders, on the basis of the method, could completely satisfy RAPD research. The conditions of polymerase chain reaction (PCR) were optimized for the highly clear and repeatable RAPD bands in study of genetic diversity of spiders. The RAPD bands of raised without food for above a month Trochosa wuchangensis’ cephaiothorax compared with its own appendix from random primers S92 amplified polymorphic DNA-PCR(P>0.05). The results showed:The appendix and cephaiothorax of spiders can be, as the stuff of extracted genomic DNA, both used to study their genetic diversity.But applicability of genomic DNA of appendix as RAPD-PCR models in study of genetic diversity of spiders is better than that of their own cephaiothorax, because of no interference from foreign DNA.2. The intraspecific variants of wolf spiders Trochosa wuchangensis were studied by structural features of genitalia combined with RAPD molecular mark technique. The results show that the intraspecific variants exist in the natural population of Trochosa wuchangensis. There are several kinds ofintraspecific variants in Trochosa wuchangensis, even in the same geographic population. The intraspecific variants do not totally result from the different geological zone. Three types that were found in the epigynum of Trochosa wuchangensis population by structural features of genitalia are nearly the same as the types detected by RAPD technique. According to the structural features of genitalia , 3 different types of the epigynum were found as follows: ( i )The two hoods pear-formed, the length<the width ;The inner lobe at the end of spermatheca almost equal the galea of that, (ii) The two hoods pear-formed partly, the length>the width ;The inner shorter than the galea of spermatheca . (iii) The two hoods like gall bladder, the lengthHhe width;the galea slightness clearly. Type ii , according to analysis on variance from the genetic similarity index both typeii and type iii of the individual differences, is unsiglificant buttype i is siglificant (P<0.05). 253 bp is the specific size of the DNA band of male spiders Trochosa wuchangensis in the RAPD patterns by the primer S92. The different growth periods of wolf spiders Trochosa wuchangensis show the intraspecific variants. The DNA bands of the different growth periods of Trochosa wuchangensis are different from the RAPD patterns by the primer S82 and S266. The statistic data showed that the types of RAPD patterns of the adults are stable (less variant), but the types of RAPD patterns of the young spiders are unstable (more variant).3. Molecular response mechanism of predominance populations of the wolf spiders under long-period pesticide force were studied based on the analysed mitochondrial 12S rRNA gene sequence data. Comparing the third domain mitochondrial 12S rRNA gene sequences, the results show that: i .The thirddomain mitochondrial 12S rRNA gene sequences of wolf siders Pirata subpiraticus were changed obviously under long-period pesticide force, For example, 315 bp of the base pair composition of 12S rRNA gene failed into 300 bp, transisions/transversions was 17/5, the precent of nucleotide variance (P-distance = sequence difference/sequence size) was 0. 095, homology was only 85.8%. ii.The third domain mitochondrial 12S rRNA gene sequences of the wolf siders Pardosa pseudoannulata were also changed under long-period pesticide force, For example, 301 bp of 12S rRNA gene dropped into 299 bp, transisions/transversions was 11/10,the precent of nucleotide varia更多还原