低钙环境下青海湖裸鲤幼鱼能量代谢及钙调节相关基因表达规律研究

发布时间：2018-04-21 18:30

本文选题：低钙 + 青海湖裸鲤　；参考：《上海海洋大学》2016年硕士论文

【摘要】：青海湖裸鲤(Gymnocypris przewalskii)俗称湟鱼,属鲤形目(Cypriniformes),鲤科(Cyprinidae),裂腹鱼亚科(Schizothoracinae),裸鲤属(Gymnocypris),是青海湖重要的经济鱼类,处于青海湖整个生态系统的核心地位。青海湖裸鲤生活于低钙、低氧、高pH和高海拔的青海湖中,对低钙环境具有较高的耐受性,为高原低温盐碱性水域经济鱼类。本实验室前期研究发现低钙胁迫下裸鲤生长缓慢。本论文在前期研究的基础上,进一步研究裸鲤幼鱼能量代谢、组织钙分配,并在此基础上采用半定量和定量PCR法研究鳃、肾钙离子变化相关的CaBPs(Ca2+-binding proteins)、PMCA(plasma membrane calcium ATPase)和NCX(sodium calcium exchanger)基因的表达情况,以期揭示青海湖裸鲤适应低钙环境的相关钙基因调节机制。本论文采用室内生长试验方法,依据裸鲤自然条件下生存环境,在盐度为10的基础上设置对照组(Ca2+浓度113.46 mg·L-1)和低Ca2+浓度组(Ca2+浓度依次是11.35 mg·L-1、3.78 mg·L-1、0.38 mg·L-1,为同盐度正常海水值的1/10、1/30、1/300)四个试验组。结果如下:1.为探讨青海湖裸鲤幼鱼在低钙水环境中生长及能量代谢规律,通过持续60d生长试验,对青海湖裸鲤日增重、特定生长率和能值的5个指标进行了测定。结果表明:(1)青海湖裸鲤幼鱼在低钙水环境中,日增重和特定生长率(SGR)随着水中钙离子浓度的降低而减小,其中(1/300)低钙组日增重和SGR均与对照组有显著性差异(P0.05)。(2)随着水中钙离子浓度的降低,生长能和粪能相应的降低,其中生长能、粪能(1/300)低钙组均与对照组之间差异性显著(P0.05);代谢能低钙组之间差异性显著(P0.05),但除了(1/300)低钙组与对照组差异性显著外(P0.05),其他各组与对照组均无显著性差异。说明水环境中低钙浓度是造成青海湖裸鲤生长缓慢的原因之一。2.为观察青海湖裸鲤在低钙水环境中钙组织分配情况,通过持续60d低钙胁迫试验,对青海湖裸鲤幼鱼血液中离子变化、组织和残饵、粪便中钙含量的测定。结果如下:(1)血液中除了na+、k+外,ca2+、mg2+、cl-均随着水环境钙离子浓度降低而显著性降低(p0.05)。随着水环境中钙离子浓度的降低,血液中ca2+、mg2+(1/300)低钙组与对照组差异性显著(p0.05),且近似等于1/2。另外,血液中cl-(1/300)低钙组显著低于对照组(p0.05);血液中na+(1/300)显著高于对照组(p0.05);血液中k+各试验组之间均无差异。(2)青海湖裸鲤幼鱼在低钙水环境中,脊柱(1/300)低钙组显著低于对照组(p0.05),且降低了1.4倍;耳石、鳃盖骨和肌肉组织中的钙离子浓度各处理组之间没有显著性差异。(3)青海湖裸鲤幼鱼在低钙水环境中养殖60d,饵料中钙离子的摄入量和粪便排出量在低钙组与对照组之间均有显著性差异(p0.05),且(1/300)低钙组较对照组降低了1.7倍。说明青海湖裸鲤组织钙离子分布受水环境中钙离子含量的影响。3.为探索青海湖裸鲤低钙水环境中钙相关基因调节机制,通过荧光定量pcr法,分别观察青海湖裸鲤鳃、肾中cabps、pmca和ncx基因的表达情况。试验结果表明:(1)鳃中cabps表达量随钙离子浓度的降低而升高,并在试验结束时出现最大值,显著高于对照组5倍(p0.05);肾中cabps表达量出现了随着钙离子浓度降低而下降的趋势,在试验结束时,肾中cabps(1/300)低钙浓度组表达量较对照组显著降低了3.7倍(p0.05)。(2)青海湖裸鲤鳃中pmca表达量出现了随着钙离子浓度降低先升高后下降的趋势,(1/300)低钙组在养殖30d时,鳃中pmca表达量达到最大值,较对照组显著升高了2.6倍(p0.05),试验结束时,鳃中pmca表达量较对照组显著降低了1.9倍(p0.05);肾中pmca表达量没有显著性变化。(3)青海湖裸鲤鳃中ncx表达量出现了随着钙离子浓度降低而下降的趋势,试验结束时,(1/300)低钙组鳃中ncx表达量较对照组显著降低了3倍(p0.05);肾中ncx表达量没有显著性变化。说明青海湖裸鲤cabps、pmca和ncx在低钙水环境中起到了重要的调节作用,裸鲤通过调节鳃和肾cabps、pmca和ncx的表达量,来稳定机体自身需要的钙离子含量。4.为进一步研究pmca在青海湖裸鲤鳃、肾中的作用机理,采用ca2+-atp酶试剂盒的方法,对青海湖裸鲤各试验组ca2+-atp酶进行测定。结果表明:(1)经过60d低钙水环境胁迫后,青海湖裸鲤幼鱼鳃ca2+-atp酶活力随着水环境中ca2+浓度的降低而逐渐降低。其中,(1/300)低钙浓度组较为明显,约是对照组的64.6%。(2)经过60d低钙水环境胁迫后,青海湖裸鲤幼鱼肾ca2+-atp酶活力随着水环境中ca2+浓度的降低呈先上升后下降的趋势。其中,(1/300)低钙浓度组较为明显,是对照组的58.6%。说明外界水环境中钙离子含量较低时,进入其体内ca2+含量也随之降低,机体通过调节鳃、肾pmca的表达量来改变ca2+-atpase酶活力,进而调节机体Ca2+的摄入量和减少体内细胞Ca2+的外排,维持细胞内钙稳定。因此,Ca2+-ATPase活力变化对血液中钙离子平衡有重要意义。
[Abstract]:The Qinghai Lake naked carp (Gymnocypris przewalskii), commonly known as the huangfish, belongs to the cyprinid (Cypriniformes), the cyprinid (Cyprinidae), the Schizothoracinae, and the Nudy carp (Gymnocypris). It is an important economic fish of the Qinghai Lake and is at the core of the whole ecological system of Qinghai Lake. The naked carp lives in low calcium, hypoxia, high pH and high altitude. In Qinghai lake, it has high tolerance to low calcium environment and is the economic fish of low temperature saline alkali water in the plateau. In this laboratory, we found that the growth of bare carp was slow under low calcium stress. On the basis of previous studies, this paper further studied the energy metabolism and tissue calcium distribution of young carp, and on this basis, the semi quantitative and quantitative PCR were adopted. The expression of CaBPs (Ca2+-binding proteins), PMCA (plasma membrane calcium ATPase) and NCX (sodium calcium exchanger) gene expression in the gill and renal calcium ions was studied in order to reveal the regulation mechanism of calcium gene regulation for the low calcium environment of Qinghai Lake naked carp. This paper adopts the indoor growth test method, according to the natural conditions of the naked carp. Under the subsistence environment, on the basis of the salinity of 10, the control group (Ca2+ concentration 113.46 mg. L-1) and low Ca2+ concentration group (Ca2+ concentration was 11.35 mg L-1,3.78 mg L-1,0.38 mg. L-1, for the same salinity normal sea water value 1/10,1/30,1/300) four test groups. The results are as follows: 1. the growth of the young carp in the low calcium water environment of Qinghai Lake and the results are as follows. 5 indexes of daily weight gain, specific growth rate and energy value of Qinghai Lake naked carp were measured by continuous 60d growth test. The results showed that: (1) the daily weight gain and specific growth rate (SGR) of young Qinghai Lake naked carp decreased with the decrease of calcium concentration in water, and the daily gain of (1/300) low calcium group was increased. SGR was significantly different from the control group (P0.05). (2) with the decrease of calcium concentration in the water, the growth energy and fecal energy decreased correspondingly, and the growth energy and the fecal energy (1/300) low calcium group were significantly different from the control group (P0.05), and the difference of the metabolic energy low calcium group was significant (P0.05), but the difference was significant except (1/300) low calcium group and the control group. P0.05, the low calcium concentration in the water environment was one of the reasons for the slow growth of the naked carp in the Qinghai Lake..2. was used to observe the distribution of calcium tissue in the low calcium water environment of the Qinghai Lake naked carp, and through the continuous 60d low calcium stress test, the changes in the blood ions in the blood of the young carp of Qinghai Lake were changed, and the tissues and the tissues were observed. The results were as follows: (1) in the blood, except na+, k+, ca2+, mg2+, cl- decreased significantly (P0.05) with the decrease of calcium concentration in water environment (P0.05). With the decrease of calcium concentration in water environment, the difference of ca2+, mg2+ (1/300) low calcium group in blood was significant (P0.05), and approximately equal to 1/2.. The cl- (1/300) low calcium group was significantly lower than the control group (P0.05), and na+ (1/300) in the blood was significantly higher than that of the control group (P0.05), and there was no difference between the k+ test groups in the blood. (2) in the low calcium water environment of the Qinghai Lake naked carp, the lower calcium group of the spine (1/300) was significantly lower than that of the opposite group (P0.05), and decreased by 1.4 times; the calcium in the otolith, the gill cover bone and the muscle tissue was in the lower calcium water environment. There was no significant difference between the treatment groups of the ion concentration. (3) the young fish of the Qinghai Lake carp were cultured in the low calcium water environment. The intake of calcium ion and the excretion amount in the bait were significantly different between the low calcium group and the control group (P0.05), and (1/300) the low calcium group was 1.7 times lower than that of the control group, indicating the calcium ion of the naked carp tissue in the Qinghai Lake. The distribution of calcium content in the water environment.3. is to explore the regulation mechanism of calcium related genes in the low calcium water environment of Qinghai Lake, and to observe the expression of cabps, PMCA and NCX genes in the gills of Qinghai Lake naked carp and the kidneys by fluorescence quantitative PCR. The results showed that: (1) the expression of cabps in the gills increased with the decrease of calcium ion concentration. At the end of the test, the maximum value was 5 times higher than that of the control group (P0.05). The expression of cabps in the kidney decreased with the decrease of calcium ion concentration. At the end of the experiment, the expression of cabps (1/300) low calcium concentration in the kidney was significantly reduced by 3.7 times than that of the control group (P0.05). (2) the expression of PMCA in the gills of the Qinghai Lake naked carp appeared with it. The tendency of calcium ion concentration to decrease first and then decrease, (1/300) the expression of PMCA in the gills reached the maximum when the low calcium group was cultured for 30d, which was 2.6 times higher than the control group (P0.05). At the end of the experiment, the expression of PMCA in the gill was 1.9 times higher than that of the control group (P0.05), and there was no significant change in the PMCA expression in the kidney. (3) n in the gills of the Qinghai Lake naked carp. The expression of Cx decreased with the decrease of calcium ion concentration. At the end of the experiment, the expression of NCX in the gill of (1/300) low calcium group was significantly reduced by 3 times than that of the control group (P0.05), and there was no significant change in the expression of NCX in the kidney. It indicated that the cabps of Qinghai Lake naked carp, PMCA and NCX played an important role in the low calcium water environment. The expression of the gill and kidney cabps, PMCA and NCX, to stabilize the calcium content of the body itself needed to further study the mechanism of PMCA in the gills of the Qinghai Lake naked carp and the kidney, and the method of ca2+-atp enzyme kit to determine the ca2+-atp enzyme in the test groups of the naked carp of Qinghai Lake. The results showed: (1) after the environmental stress of 60d low calcium water, green carp was stressed. The ca2+-atp enzyme activity of the gill of the young carp of the sea lake gradually decreased with the decrease of the concentration of ca2+ in the water environment. Among them, the group of (1/300) low calcium concentration was more obvious. It was about the 64.6%. (2) of the control group. After 60d low calcium water environment stress, the ca2+-atp enzyme activity of the kidney of the young carp of Qinghai Lake rose first and then decreased with the decrease of the concentration of ca2+ in the water environment. Among them, (1/300) low calcium concentration group is more obvious, the control group 58.6%. shows that when the content of calcium ion in the outside water environment is low, the content of ca2+ in its body also decreases. The body changes the activity of ca2+-atpase enzyme by regulating the gills and the expression of renal PMCA, and then regulating the intake of Ca2+ in the body and reducing the excretion of Ca2+ in the body of the body. Maintaining intracellular calcium homeostasis, therefore, changes in Ca2+-ATPase activity are important for calcium homeostasis in the blood.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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