本文選題：低鈣 + 青海湖裸鯉��； 參考：《上海海洋大學》2016年碩士論文

【摘要】：青海湖裸鯉(Gymnocypris przewalskii)俗稱湟魚,屬鯉形目(Cypriniformes),鯉科(Cyprinidae),裂腹魚亞科(Schizothoracinae),裸鯉屬(Gymnocypris),是青海湖重要的經(jīng)濟魚類,處于青海湖整個生態(tài)系統(tǒng)的核心地位。青海湖裸鯉生活于低鈣、低氧、高pH和高海拔的青海湖中,對低鈣環(huán)境具有較高的耐受性,為高原低溫鹽堿性水域經(jīng)濟魚類。本實驗室前期研究發(fā)現(xiàn)低鈣脅迫下裸鯉生長緩慢。本論文在前期研究的基礎上,進一步研究裸鯉幼魚能量代謝、組織鈣分配,并在此基礎上采用半定量和定量PCR法研究鰓、腎鈣離子變化相關的CaBPs(Ca2+-binding proteins)、PMCA(plasma membrane calcium ATPase)和NCX(sodium calcium exchanger)基因的表達情況,以期揭示青海湖裸鯉適應低鈣環(huán)境的相關鈣基因調(diào)節(jié)機制。本論文采用室內(nèi)生長試驗方法,依據(jù)裸鯉自然條件下生存環(huán)境,在鹽度為10的基礎上設置對照組(Ca2+濃度113.46 mg·L-1)和低Ca2+濃度組(Ca2+濃度依次是11.35 mg·L-1、3.78 mg·L-1、0.38 mg·L-1,為同鹽度正常海水值的1/10、1/30、1/300)四個試驗組。結(jié)果如下:1.為探討青海湖裸鯉幼魚在低鈣水環(huán)境中生長及能量代謝規(guī)律,通過持續(xù)60d生長試驗,對青海湖裸鯉日增重、特定生長率和能值的5個指標進行了測定。結(jié)果表明:(1)青海湖裸鯉幼魚在低鈣水環(huán)境中,日增重和特定生長率(SGR)隨著水中鈣離子濃度的降低而減小,其中(1/300)低鈣組日增重和SGR均與對照組有顯著性差異(P0.05)。(2)隨著水中鈣離子濃度的降低,生長能和糞能相應的降低,其中生長能、糞能(1/300)低鈣組均與對照組之間差異性顯著(P0.05);代謝能低鈣組之間差異性顯著(P0.05),但除了(1/300)低鈣組與對照組差異性顯著外(P0.05),其他各組與對照組均無顯著性差異。說明水環(huán)境中低鈣濃度是造成青海湖裸鯉生長緩慢的原因之一。2.為觀察青海湖裸鯉在低鈣水環(huán)境中鈣組織分配情況,通過持續(xù)60d低鈣脅迫試驗,對青海湖裸鯉幼魚血液中離子變化、組織和殘餌、糞便中鈣含量的測定。結(jié)果如下:(1)血液中除了na+、k+外,ca2+、mg2+、cl-均隨著水環(huán)境鈣離子濃度降低而顯著性降低(p0.05)。隨著水環(huán)境中鈣離子濃度的降低,血液中ca2+、mg2+(1/300)低鈣組與對照組差異性顯著(p0.05),且近似等于1/2。另外,血液中cl-(1/300)低鈣組顯著低于對照組(p0.05);血液中na+(1/300)顯著高于對照組(p0.05);血液中k+各試驗組之間均無差異。(2)青海湖裸鯉幼魚在低鈣水環(huán)境中,脊柱(1/300)低鈣組顯著低于對照組(p0.05),且降低了1.4倍;耳石、鰓蓋骨和肌肉組織中的鈣離子濃度各處理組之間沒有顯著性差異。(3)青海湖裸鯉幼魚在低鈣水環(huán)境中養(yǎng)殖60d,餌料中鈣離子的攝入量和糞便排出量在低鈣組與對照組之間均有顯著性差異(p0.05),且(1/300)低鈣組較對照組降低了1.7倍。說明青海湖裸鯉組織鈣離子分布受水環(huán)境中鈣離子含量的影響。3.為探索青海湖裸鯉低鈣水環(huán)境中鈣相關基因調(diào)節(jié)機制,通過熒光定量pcr法,分別觀察青海湖裸鯉鰓、腎中cabps、pmca和ncx基因的表達情況。試驗結(jié)果表明:(1)鰓中cabps表達量隨鈣離子濃度的降低而升高,并在試驗結(jié)束時出現(xiàn)最大值,顯著高于對照組5倍(p0.05);腎中cabps表達量出現(xiàn)了隨著鈣離子濃度降低而下降的趨勢,在試驗結(jié)束時,腎中cabps(1/300)低鈣濃度組表達量較對照組顯著降低了3.7倍(p0.05)。(2)青海湖裸鯉鰓中pmca表達量出現(xiàn)了隨著鈣離子濃度降低先升高后下降的趨勢,(1/300)低鈣組在養(yǎng)殖30d時,鰓中pmca表達量達到最大值,較對照組顯著升高了2.6倍(p0.05),試驗結(jié)束時,鰓中pmca表達量較對照組顯著降低了1.9倍(p0.05);腎中pmca表達量沒有顯著性變化。(3)青海湖裸鯉鰓中ncx表達量出現(xiàn)了隨著鈣離子濃度降低而下降的趨勢,試驗結(jié)束時,(1/300)低鈣組鰓中ncx表達量較對照組顯著降低了3倍(p0.05);腎中ncx表達量沒有顯著性變化。說明青海湖裸鯉cabps、pmca和ncx在低鈣水環(huán)境中起到了重要的調(diào)節(jié)作用,裸鯉通過調(diào)節(jié)鰓和腎cabps、pmca和ncx的表達量,來穩(wěn)定機體自身需要的鈣離子含量。4.為進一步研究pmca在青海湖裸鯉鰓、腎中的作用機理,采用ca2+-atp酶試劑盒的方法,對青海湖裸鯉各試驗組ca2+-atp酶進行測定。結(jié)果表明:(1)經(jīng)過60d低鈣水環(huán)境脅迫后,青海湖裸鯉幼魚鰓ca2+-atp酶活力隨著水環(huán)境中ca2+濃度的降低而逐漸降低。其中,(1/300)低鈣濃度組較為明顯,約是對照組的64.6%。(2)經(jīng)過60d低鈣水環(huán)境脅迫后,青海湖裸鯉幼魚腎ca2+-atp酶活力隨著水環(huán)境中ca2+濃度的降低呈先上升后下降的趨勢。其中,(1/300)低鈣濃度組較為明顯,是對照組的58.6%。說明外界水環(huán)境中鈣離子含量較低時,進入其體內(nèi)ca2+含量也隨之降低,機體通過調(diào)節(jié)鰓、腎pmca的表達量來改變ca2+-atpase酶活力,進而調(diào)節(jié)機體Ca2+的攝入量和減少體內(nèi)細胞Ca2+的外排,維持細胞內(nèi)鈣穩(wěn)定。因此,Ca2+-ATPase活力變化對血液中鈣離子平衡有重要意義。
[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.

【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4

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