【作者】 李艳和；

【导师】 王卫民；

【作者基本信息】 华中农业大学 ， 水产养殖， 2013， 博士

【摘要】 外来物种克氏原螯虾作为我国一个重要的淡水养殖对象,为我国的经济作出了一定的贡献。但是,作为外来入侵物种的克氏原螯虾给入侵地带来的危害也相当严重。弄清外来生物扩散模式、入侵路径以及外来物种在入侵过程中各种影响因素的相互作用等,对合理预防入侵物种的危害,制定科学合理的控制策略具有重要意义。为了摸清克氏原螯虾种群扩散模式和入侵路线及其入侵过程中的相关影响因素,合理控制其危害,本论文从外来物种克氏原螯虾在我国的种群遗传多样性、种群遗传结构等方面进行分析以推测克氏原螯虾的首个入侵地,推断其入侵路径,研究其扩散模式；同时测定了克氏原螯虾线粒体全基因组,并进行了基因结构和适应性进化分析,从分子水平上研究入侵种克氏原螯虾和甲壳动物线粒体基因在适应不同的环境压力下和进化中发生怎样的变化；并试图分析克氏原螯虾免疫相关基因proPO在不同因子刺激后的表达情况,从免疫基因入手研究其与克氏原螯虾成功入侵是否具有相关性。主要研究结果如下：(1)建立了一种从克氏原螯虾的虾壳中提取DNA的方法,用虾壳作为DNA来源,其样本取样时对实验动物伤害小,样本易长期保存,且提取的DNA降解程度比从肌肉中提取的DNA降解程度低,可适用于克氏原螯虾种群遗传多样性分析及相关分子生物学应用研究。(2)利用12对微卫星引物和线粒体COI以及16S rRNA序列对37个克氏原螯虾种群(35个种群采自我国,1个种群采自美国,1个种群采自日本)的种群遗传多样性和种群遗传结构进行了分析,并进一步分析克氏原螯虾群体的扩散模式,探明种群的来源地以及是来自单个还是多个引进事件。结果表明,我国的克氏原螯虾种群都具有较高的遗传多样性；克氏原螯虾可能来自日本；人为介导的扩散是我国克氏原螯虾入侵的重要途径之一；不排除多次引入事件或隐性入侵的存在。(3)基于利用微卫星标记和线粒体COI以及16S rRNA序列的克氏原螯虾的种群遗传结构分析及种群遗传多样性的研究,进一步利用克氏原螯虾线粒体控制区序列和核基因proPOx内含子序列及一些历史数据推测克氏原螯虾入侵路径。结果表明,克氏原螯虾系从日本引入我国,然后主要依靠自然扩散和人工介导的扩散沿着长江中下游进行扩散。此外,我们只发现克氏原螯虾从长江中下游扩散到长江中上游,而没发现从长江上游往长江中下游扩散的。结果亦表明我国的克氏原螯虾的扩散路线比我们预想的要复杂。(4)克隆克氏原螯虾线粒体序列,分析其碱基组成和排列等,同时对37个克氏原螯虾种群的线粒体COI基因和92种甲壳动物线粒体的13个基因进行了适应性进化分析,结果发现,线粒体基因是相对保守的,在进化过程中以净化选择为主,其变异基本以同义突变为主。(5)利用荧光定量PCR方法分析克氏原螯虾proPO基因的mRNA在9种组织(血淋巴、肝胰腺、卵巢、肌肉、精巢、皮肤、腮、肠、胃)中的分布情况以及在CPG ODN (CpG oligodeoxynucleotide,含CpG的寡脱氧核苷酸)、嗜水气单胞菌(Aeromonas hydrophila)、对虾白斑病病毒(White spot syndrome virus, WSS V)等因子刺激后不同时间段的表达情况。结果显示,克氏原螯虾proPO基因在血淋巴中转录水平最高,其次是肝胰腺、卵巢、肌肉和精巢中转录水平较高,在表皮和鳃中有较弱表达,而在肠和胃中几乎无表达；克氏原螯虾proPO在螯虾的免疫系统中发挥着重要的作用,其表达情况会因不同的刺激源或刺激压力(如受CpG ODN、 A. hydrophila和WSSV的刺激)以及不同的组织而有所差别。更多还原

【Abstract】 The red swamp crayfish, Procambarus clarkii, is an important freshwater aquaculture species in China. The crayfish has caused serious damages for invasion areas as an invasive species although it has attributed greatly to our economy of China. Information on dispersal pattern and various impacts for invasion success would be useful for the establishment of possible methodologies for the prevention and control of their invasions. In order to identify the spread pattern, invasion route, and related impacts in the course of invasion for the prevention and control of the crayfish, the genetic diversity and population genetic structure etc. were investigated in this study to infer initial invasion place, retrace the dispersal routes, and study dispersal pattern of P. clarkii. The mitochondrial genome of P. clarkii were cloned, and its gene structure and adaptive evolution for mitochondrial gene were analyzed, to investigate what variations the mitochondrial gene of P. clarkii and crustacean would happen while enduring different environment pressure and evolution in molecular level. And the expression of the prophenoloxidase (proPO) gene was studied after challenges by varied stimuli to infer whether there is a correlation between immunogene and invasion success of P. clarkii. The results are summarized as follows:(1) An efficient protocol for DNA extraction from red swamp crayfish exoskeleton was described. If the samples are collected from crayfish exoskeleton, the crayfish exoskeleton samples are less invasive and can be easily preserved long. And crayfish exoskeleton samples showed less apparent DNA degeneration than muscle DNA samples. The crayfish exoskeleton samples could be used for the population genetic research and related applications in molecular biology.(2) The genetic structure and diversity of P. clarkii populations at37sampling sites (35from China, one from the USA and one from Japan) were analyzed using both mitochondrial gene sequences (COI and16S rRNA) and12nuclear microsatellites. And based on those analyses, the dispersal pattern of P. clarkii, and whether the introduction of P. clarkii in China was derived from a single or multiple event(s) were further analyzed. The results showed that:(ⅰ) relatively high genetic diversity was detected in Chinese populations,(ⅱ) the population from Japan and those collected from China, particularly from NanJing (BGt and XG) and its some neighboring sites (CJr, NT and NB), have similar genetic composition,(iii) human-mediated dispersal was one important dispersal route for P. clarkii in China, and (iv) multiple introductions and/or other unintentional introductions in China might exist.(3) Based on the study of genetic structure and diversity of P. clarkii populations using both mitochondrial gene sequences (COI and16S rRNA) and nuclear microsatellites, the sequences of mitochondrial control region and nuclear proPOx intron were used to infer the invasion history of P. clarkii in China in combination with historical records. The results revealed P. clarkii is most possible from Japan, which originated via introductions from an unsampled source, then mainly expanded and transferred in the middle and lower reaches of Changjiang River in China by natural expansion and human-mediated jump dispersal, and less spread from the upper reaches of Changjiang River to the middle and lower reaches of Changjiang River. Additionally, the results also show that the dispersal pathways for P. clarkii might be much more complex than previously assumed.(4) The mitochondrial genome of P. clarkii was obtained. And the base composition and gene arrangement of the mitochondrial genome were analyzed. We investigated adaptive evolution for mitochondrial COI gene of37P. clarkii populations, and13mitochondrial genes concatenated of92crustacean mitochondrial genomes. The results showed that mitochondrial genes were conserved, given priority to purifying selection in the course of evolution, and given priority to synonymous mutations for variation.(5) The expression of the prophenoloxidase (proPO) gene was investigated in nine tissues, including haemocytes, hepatopancreas, ovary, muscles, spermary, cuticular epidermis, branchia, intestines, and stomach of red swamp crayfish Procambarus clarkii by real-time PCR before and after challenges by CpG oligodeoxynucleotide (ODN), Aeromonas hydrophila and white spot syndrome virus (WSSV). The results showed that the highest level of mRNA was observed in haemocytes, followed by those in the hepatopancreas, ovary, muscles, spermary, cuticular epidermis, branchia, intestines, and stomach sequentially. The proPO gene may play an important role in the immune system of the crayfish, and proPO expression might be activated in different tissues by different mechanisms in response to various stresses, such as CpG ODN, A. hydrophila and WSSV challenges.更多还原