【作者】 王兵；

【导师】 李思发；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 1、慢性盐度胁迫下“吉丽”罗非鱼NKCC1a mRNA表达的盐度-组织特异性应用实时荧光定量PCR技术(qRT-PCR),以“吉丽”罗非鱼[尼罗罗非鱼(Oreochromis niloticus,♀)×萨罗罗非鱼(Sarotherodon melanotheron,♂)]为材料,研究NKCC1a基因表达的盐度-组织特异性。研究结果表明: (1)NKCC1a基因mRNA表达量存在显著的组织特异性,在低于25盐度环境中,NKCC1a mRNA在鳃、肝、肾及肠中均有表达;当盐度从0提高到48时,表达量在鳃中与盐度变化呈高度正相关(R>0.9,P<0.01),在肠和肾中与盐度变化呈负相关(R≈-0.7,P<0.05),但在肝中则不受盐度变化的影响。(2)当盐度提高到64,表达量在鳃和肠3 h后达最高值,5 h后下降,前后变化差异显著(P<0.05);表达量在肝中则是在5h后达最高值,变化差异也显著(P<0.05);表达量在肾中持续下降,但差异不显著。以上结果揭示,在盐度高于25的环境中,“吉丽”罗非鱼主要由鳃组织的NKCC1a排出多余的离子以维持鱼体的水盐平衡,由此认为鳃组织在“吉丽”罗非鱼高渗透压调节中起最主要作用。2、急性盐度胁迫下“吉丽”罗非鱼NKCC1a mRNA表达变化研究“吉丽”罗非鱼[体质量(21.4±5.4)g]从淡水直接转移至盐度为20、25水体中进行急性盐度胁迫实验。结果显示:(1) 20水体中,24h内死亡率仅为12%,之后死亡率几乎没有变化;25水体中,5.35h时死亡率达到100%。(2) 20盐度胁迫下,NKCC1a mRNA相对表达量在鳃中先升高,在6h达到峰值,随后呈下降趋势,6h表达量是淡水对照的37.49倍,12h和24h表达量与淡水对照间差异显著(P<0.05);肠和肾组织中表达量总体呈先下降然后恢复的趋势。在肠中,转移后8h降低至最小值,仅为淡水对照的28.4%, 12h和24h表达水平与淡水对照间差异不显著;在肾中,转移后6h表达量降至最小值,仅为淡水对照的20.86%,24h表达量与淡水对照间差异不显著(P>0.05)。(3) 25盐度胁迫下,NKCC1a mRNA相对表达量在鳃中呈上升趋势,4h表达量是淡水对照的4.31倍;但5h表达量与4h相比呈下降趋势,且二者差异显著(P<0.05)。肾中表达量在5h内无显著变化(P>0.05)。肠中表达量与20盐度下相似,呈显著下降趋势(P<0.05)。结果提示:鳃和肾NKCC1a表达的有限调节能力与“吉丽”罗非鱼不能适应25盐度有关;急性盐度胁迫下可能主要由鳃和肾来完成过多单价离子的分泌。3、“吉丽”罗非鱼及其亲本“新吉富”罗非鱼、萨罗罗非鱼耐寒力评估在上海地区室外自然降温条件下,连续3年观察测定了“新吉富”罗非鱼、“吉丽”罗非鱼(“新吉富”罗非鱼×萨罗罗非鱼)及萨罗罗非鱼对低温的耐受力,计算和分析了平均半致死水温。2008年,“新吉富”罗非鱼死亡水温范围是7.5～5.8℃,平均半致死水温是6.8℃,对水温降低过程中罗非鱼行为活动的变化进行了观察;2009年,“新吉富”罗非鱼和“吉丽”罗非鱼的死亡水温范围分别是10.8～7.6℃、12.4～9.2℃,平均半致死水温是:9.2℃和11.4℃;2010年,3种实验鱼的死亡水温范围分别是11.0～7.0℃、12.8～11.4℃和13.8～12.4℃,平均半致死水温分别是8.3℃、12.5℃、13.1℃,且3者间差异显著(P<0.05)。综合3年实验结果,开始死亡和100%死亡的水温是:“新吉富”罗非鱼分别为—11℃、5.8℃,“吉丽”罗非鱼分别为—12℃、9.2℃,萨罗罗非鱼分别为—13.8℃、12.4℃。3种遗传型罗非鱼耐寒力之间存在显著或极显著的种间差异,杂交能一定程度地改良耐寒能力。更多还原

【Abstract】 1、Tissue-specific changes of NKCC1a mRNA under gradual stress of salinity in“GILI”tilapiaQuantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to study the tissue-specific changes of NKCC1a expression by salinity in case of the hybrid (named“GILI”tilapia) by O.niloticus(♀)×S.melanotheron(♂). Being a new variety,“GILI”tilapia is characterized by high salt tolerance and fast growth both and is suitable for marine culture. The present results showed that: (1) The NKCC1a mRNA expression level has a significant tissue specialty: the NKCC1a gene expressed in all the tested tissues of gill, liver, kidney and intestine when salinity below 25. When the salinity increased from 0 to 48, the NKCC1a mRNA expression level showed a positive correlation in gill (R>0.9, P<0.01), but negative correlation in intestine and kidney (R≈-0.7, P<0.05) and no correlation in liver. (1)When the salinity raised to 64, the mRNA expression level reached the highest peak after 3h in gill and intestine, and decreased after 5h, and there were significant differences from 1h to 3h and 3h to 5h (P<0.05). In liver, the maximum level appealed in 5h and significantly higher than those in 1h and 3h(P<0.05). In kidney, the levels descended continually with the increase of time, but no statisticaly significant differences were detected among them. (2)Above results revealed that, in the“GILI”fish which acclimates to environmental salinity over 25, it is primarily the gill chloride cell to secrete the cation ions and to maintain water and salt balance. So, it is considered that gill plays a leading role in hyper-osmoregulatory of“GILI”tilapia. Meanwhile, we are expected to provide a guidline for revealing the expression mode of NKCC1a gene in euryhaline teleosts in fresh, saline and hypersaline water.2、The expression changes of NKCC1a mRNA under sharp stress of salinity in“GILI”tilapiaThe stress response of“GILI”tilapia, juveniles[body mass(21.4±5.4)g],following abrupt transfer to salinities 20 and 25 were studied. The present results showed that: (1) the mortality was only 12% at salinity 20 during the first 24h, and almost unchanged after that; a mortality of 100% was observed at salinity 25 within 5.35h. (2) At salinity 20, The NKCC1a mRNA expression in the gill increased rapidly, and reached the peak at 6h, being 17.49-fold of the control, then gradually decreased; and the level of 12h and 24h were significantly higher than the fresh water (P<0.05); The expression of the intestine and kidney appeared to have the initial level after a decline trend. In the intestine, it decreased to the minimum at 8h, being 28.4% of the freshwater, but there were no significant difference among 12h、24h and freshwater(P>0.05); In the kidney, the level of 6h was 20.86% of the control, and there were no significant difference between 24h and freshwater. (3) At salinity 25, The mRNA expression of the gill content a decline trend after a rise of 4hs, the level of 4h were 4.31 times higher than the control, and there were significant difference between 4h and 5h (P<0.05); in the kidney, there were no significant changes during the 5hs; the expression of the intestine were similarly with that at 20 ppt, and decreased significantly (P<0.05). Collectively, above results indicated that:“GILI”tilapia could not directly adaption to salinity 25 may be related to the limit capacity of gill and kidney to up/down-regulate NKCC1a expression, and gill and kidney plays a leading role in the process of secrete the monovalent ions after abrupt salinity transfer.3、Cold tolerance of GILI Tilapia, NEW GIFT Nile tilapia and Black-chin tilapiaCold tolerances of three tilapias(NEW GIFT Nile tilapia、GILI Tilapia and Black-chin tilapia)to low temperatures naturally decreased in the Shanghai area were studied and the average lethal temperature of 50% individuals were calculated continuously for 3 years. It is found that Low lethal water temperatures ranged from 7.5℃to 5.8℃,and the average lethal temperatures of 50% individuals was 6.8℃for NEW GIFT Nile tilapia in 2008. Meanwhile, behaviors of tilapias under decreasing water temperatures were observed. The range of Low lethal water temperatures were 10.8～7.6℃、12.4～9.2℃for NEW GIFT Nile tilapia and GILI Tilapia in 2009, and the average lethal temperatures of 50% individuals were 9.2℃and 11.4℃. But they were 10.8～7.6℃、12.4～9.2℃and 13.8～12.4℃for NEW GIFT Nile tilapia、GILI Tilapia and Black-chin tilapia in 2010. The average lethal temperatures of 50% individuals were 8.3℃, 12.5℃and13.1℃, separately , also there were significant differences among them. The comprehensive results showed that:water temperatures that beginning die and100% die were 11℃、5.8℃respectively for NEW GIFT Nile tilapia, 12.4℃、9.2℃respectively for GILI Tilapia,13.8℃、12.4℃respectively for Black-chin tilapia. There were significant or highly significant species/strain-specific differences among these three genotypic tilapias in cold tolerance. The hybridization could improve cold tolerance to a certain extent.更多还原