【作者】 刘洪展；

【导师】 唐学玺； 孙修勤；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2013， 博士

【摘要】 为了完善刺参病害发生的理论和探讨生态防治的方法,本文利用常规技术从患病仿刺参中分离了致病菌,并初步分析了其致病性；此外,模拟刺参养殖环境,测定了感染病菌后刺参相关非特异性免疫指标的变化；利用病菌感染刺参构建的cDNA文库进行EST分析,并鉴定了若干免疫相关基因；筛选了病原菌的拮抗菌,并对其拮抗特性进行了研究。试验结果如下：采用微生物学方法鉴定了假交替单胞菌属2株细菌和1株病毒,其中病菌Pseudoalteromonas sp和病毒粒子对刺参有较强的感染性和致病性。实验表明,随着病原菌感染浓度升高,刺参体腔液溶菌酶活性和AKP活性、SOD和CAT及PO的诱导活性、补体C3含量和呼吸爆发活性均显著下降,而体腔液ACP活性和补体C4含量稍微上升；刺参体表粘液的SOD活性和溶菌酶活性对病菌感染较敏感,含量明显受到抑制；而体表粘液中的CAT活性、ACP活性和补体C4含量在病原菌感染浓度较低时增加、在感染浓度较高时减少。病毒感染条件下,刺参体液中的溶菌酶、酸性磷酸酶、SOD和PO活性降低,而CAT活性增强,体腔液中的免疫酶活性比体表粘液中的免疫酶活性变化更明显。当水体中氨氮浓度较高时,刺参体腔液中的超氧物歧化酶(SOD)和碱性磷酸酶(ALP)活性降低、溶菌酶(LSZ)活性升高；而在感染病菌的情况下,体腔液SOD、ALP和LSZ活性下降,而GPx活性持续上升；在中等氨氮浓度时,体腔液酶活性减少幅度最小或增加幅度最大。硒可诱导动物的抗病毒和抗病菌活性,亚硒酸钠浓度较低时,随着亚硒酸钠浓度增加,刺参体液超氧物岐化酶(SOD)、谷胱甘肽过氧化物酶(GP、)、溶菌酶(LSZ)、酚氧化酶(PO)和碱性磷酸酶(ALP)活性升高；而在感染病原菌的情况下,体液SOD、GPX和LYZ的活性表现为先升高后下降的趋势,PO和ALP的活性则持续上升。在高浓度NO2-条件下,刺参体腔液SOD、CAT、AKP和P0的诱导活性下降,LSZ和GPx活性显著被抑制；病原菌感染时加剧刺参体腔液CAT、GPx、LSZ和P0活性被抑制,但体腔液SOD活性被诱导升高；缺氧处理刺参时,体腔液ACP诱导活性被抑制,而AKP活性被促进；分析表明,病原菌感染可促进体腔液ACP诱导活性,明显拮抗高浓度NO2-对AKP活性的抑制。在较低的适宜盐度条件下,病原菌感染可促进刺参体液SOD、GPx和AKP活性的诱导水平,而盐度过高时,病原菌感染加剧使刺参体液SOD、GPx和AKP活性水平降低；此外,适宜盐度处理下,刺参体液LSZ活性均可被诱导,而病原菌感染后,体液LSZ的诱导活性被抑制。水温低于23℃时,升温和病原菌感染均可促进刺参体液SOD、GPx、LSZ和P0的活性；高温胁迫条件下,病原菌感染可明显抑制体液上述酶的活性；此外,水温上升可抑制刺参体液AKP活性,而病原菌感染诱导AKP活性。缓慢升高温度可以增强刺参体液的SOD和PO活性,但体液GPx和LSZ活性变化不大,而高温预处理后刺参体液SOD和LSZ活性明显下降；因此,适当的温度刺激可减少刺参的发病。构建的cDNA文库库容为3.24×105cfu/mL,插入片段大小为0.8-2.5kb,文库质量较高。对文库进行测序和初步的生物信息学处理,得到了高质量的表达序列标签(ESTs)1106条。经过软件拼接,共得到533条单基因簇,包含165个序列重叠群和368条单一序列。同源序列基因分析表明,仿刺参cDNA文库中有25种免疫相关的基因。采用real-time PCR技术对免疫相关基因进行分析,证明了体腔液中血清凝集素、补体C3和补体3类似蛋白基因的表达水平显著升高,提示其在刺参免疫防御中发挥作用。为了开发更多有效的病原拮抗菌用于刺参疾病防治,本文利用点种法和滤纸片法筛选到了5株拮抗菌,利用牛津杯法进一步确认YAAJ6和YASM12拮抗菌株的活性最高。活体试验表明,该2株拮抗菌是通过促进体腔液SOD、ALP、LSZ活性和细胞吞噬活性而提高刺参对病原菌的抗性。深入的研究表明,拮抗菌胞外产物在28℃或pH8.0时抑菌活性最高,但对蛋白酶K和链霉素蛋白酶敏感。本研究证实,病原细菌和病毒病原均可导致刺参非特异性免疫力的下降；水环境的变化会加剧刺参受病原菌感染的可能性,但适宜的环境条件可诱导刺参免疫反应；水温驯化处理可一定程度提高刺参的免疫能力；筛选有效拮抗菌并加以应用,是刺参病害防治的有效手段之一。更多还原

【Abstract】 In order to supplement the theory that diseases occurred to sea cucumber, and to explore the technique of safe prevention and cure for its diseases, the pathogens are isolated from diseased sea cucumbers and primarily assayed its pathogenicity by general methods. Moreover, the change of some related non-specific immune indexes about sea cucumbers exposed to germs is detected under the simulated culture surroundings. Several genes related to the immune response are identified by analyzing constructed cDNA library and EST data in bacterial infected sea cucumbers. And then, the antagonistic bacterium is chosen from prepared various bacteria, but also its antagonistic characteristics are tested. The most results are as follows:The pathogens are identified as two psedoaltermonas bacteria and one undetailed virus respectively by microbiological assay. As virus particle, the bacterium Pseudoalteromonas sp shows more severe infectiosity and pathogenicity. The LSZ activity and AKP activity, induced SOD activity, CAT activity and PO activity, complement component C3contents and respiratory burst activity all significantly decrease, and that ACP activity and complement component C4contents show a little increase in the coelomic fluid of sea cucumbers suffering from bacterial infection with elevated concentration. However, the SOD activity and LSZ activity in the epimucus are sensitive to and obviously suppressed by bacterial infection. And furthermore, the epimucous CAT activity, ACP activity and complement component C4contents all rise when infecting bacteria is under low concentrations, but that decline during bacterial infecting with high concentrations. Under virus infecting conditions, the alteration of immune enzymes activity in the coelomic fluid is more evident than that in the epimucus, and the LSZ activity, ACP activity, SOD activity and PO activity are all reduced, and only CAT activity enhanced.The SOD activity and ALP activity are depressed, and but the LSZ activity raised in the coelomic fluid under high ammonia-N concentrations, and yet the above enzymes activity decline, the GPx activity continuously increase with additional bacterial infection, and as well the altering amplitude of coelomic enzymes activity decrease least or increase most under medium ammonia-N concentrations. The humoral SOD activity, GPx activity, LSZ activity, PO activity and ALP activity are raised with the increasing Na2SeO3concentrations under low Na2SeO3conditions, and that the humoral SOD activity, GPx activity and LSZ activity firstly ascend then decline at certain time, and yet PO activity and ALP activity keep rise during the additional bacterial infecting treatments. The induced SOD, CAT, AKP and PO activity decrease, and GPx activity and LSZ activity are markedly suppressed in the coelomic fluid under high nitrite-N concentrations, and additional hypoxia or bacterial infection aggravate depressing the CAT, GPx, LSZ and PO activity, but may induce SOD activity in the coelomic fluid. And besides, additional hypoxia treatments slow down induced ACP activity, but promote AKP activity, and that additional bacterial infection strengthen the induced ACP activity and obviously counteracts the suppression of AKP activity by high nitrite-N concentrations. The inducement of humoral SOD, GPx and AKP activity in the sea cucumbers is facilitated by additional bacterial infection under moderately low salinity. However, the reduction of above enzymes activity is quickened by additional bacterial infection under high salinity. Moreover, optimized saline treatments may induce humoral LSZ activity, but bacterial infection impairs the induced humoral LSZ activity. The induced humoral SOD, GPx, LSZ and PO activity all are mutually promoted by elevated temperature and bacterial infection during the seawater is below23℃, and that the above enzymes activity is apparently suppressed by bacterial infection under seawater high temperature stress. And then, the humoral AKP activity is repressed with risen seawater temperature and induced by bacterial infection respectively. The humoral SOD and PO activity may be reinforced, and the humoral GPx and LSZ activity alters insignificantly when the seawater temperature is slowly raised, but high temperature pretreatments lead to obvious decrease of humoral SOD and LSZ activity. And so, proper stimulus of temperature can alleviate disease severity of sea cucumbers despite the low stability of inducement on sea cucumbers.The cDNA library with an inserted fragment of0.8-2.5kb and also high quality is constructed, and its capacity is3.24×105cfu/mL.1106ESTs with high quality are obtained according to the results of library sequences assaying and elementarily bioinformatics processing.533single gene clusters including165overlapping sequence groups and368single sequences are confirmed by using related software to splice the EST.25immunity-related genes are found after analyzing the genes with homologous sequences. The results of analyzing immunity-related genes by real-time PCR technique indicate that genes encoding serum lectin isoform, complement component C3and complement component3-like protein in the coelomic fluid of infected A. japonicus showed most remarkable increase, which demonstrates their important effect in the immune defending of holothurian A. japonicus.Five antagonistic bacteria are selected by solid culture of dot-inoculating and filter paper piece-inoculating, and then further research by Niujin cup method validates the bacterium both YAAJ6and YASM12show highest antagonistic activity. Furthermore, the test in vivo demonstrates the above bacteria enhance disease resistance of A. japonicus to pathogenic infection by increasing SOD, ALP and LSZ activity in coelomic fluid and promoting the phagocytic activity of coelomocytes. Besides, the ECP of antagonistic bacteria shows the most resistant activity to pathogens at28℃or pH8.0, and yet the ECP is sensitive to protease K or streptomycin-protease.In conclusion, bacteria and virus can lead to the decrease of non-specific immunity in A. japonicus., and so cause the related symptoms about "skin ulceration" in holothurian, and unmoderate change of seawater environment advances the possibility of bacterial infecting on A. japonicus., but immune response is induced under reasonable environmental conditions, and also acclimatization treating may temporarily enhance the immune capacity. Otherwise, selecting and then applying the antagonistic bacteria on holothurian is feasible measures of disease prevention and cure.更多还原