白斑狗鱼早期发育阶段的高温耐受性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1335

摘要/Abstract

摘要：

对白斑狗鱼早期发育阶段的高温耐受性进行分析,旨为白斑狗鱼的养殖和生态适应性研究奠定理论基础。采用高温胁迫法研究白斑狗鱼受精卵及仔鱼的高温耐受性,用24hUILT₅₀和CT_max进行评估;采用实时荧光定量PCR（qRT-PCR）技术和相对定量2^-ΔΔCt法,分别比较了不同温度和不同时间高温胁迫下HSP70基因在白斑狗鱼幼鱼脑、鳃、心和肝各组织中的表达情况。结果显示,白斑狗鱼受精卵破膜时间随着孵化温度的上升逐渐缩短,鱼苗上浮率呈现先缓慢上升后急剧下降的趋势。随着驯化水温的提高,白斑狗鱼仔鱼在高温胁迫下的24hUILT₅₀值升高,且CT_max出现的时间推迟。在不同温度高温胁迫下,白斑狗鱼幼鱼脑、鳃和心脏组织HSP70基因的表达量呈现先上升后下降的趋势,肝脏组织呈现持续上升的趋势。白斑狗鱼幼鱼脑、鳃和心脏组织在30℃高温胁迫0-24 h时,HSP70基因的表达量呈现先上升后下降的趋势,肝脏组织呈现先下降后上升再下降的趋势。白斑狗鱼幼鱼的脑、鳃、心和肝4种组织在不同温度和不同时间的高温胁迫下,HSP70基因的表达量发生不同程度的变化,其变化具有时间和组织特异性,但应答幅度不尽相同。经过高温驯化后,白斑狗鱼提高了温度耐受性,可能是通过HSP70参与重要调控作用。

关键词: 白斑狗鱼, 高温耐受性, 临界温度, 热休克蛋白70

Abstract:

Analyzing the high temperature tolerance in early development stage of Esox lucius is aimed to lay a theoretical foundation for the breeding and ecological adaptation of E. lucius. High temperature stress method was used to study the high temperature tolerance of the fertilized eggs and juvenile E. lucius,which was evaluated by 24hUILT₅₀and CT_max. Real-time fluorescence quantitative PCR（qRT-PCR）and relative quantitative 2^-ΔΔCt method were used to compare the expressions of HSP70 gene in the brain,gill,heart and liver of juvenile E. lucius under different temperatures and high temperature stressed times. The results showed that the breaking time of the membranes of the fertilized eggs in E. lucius was shortened gradually as the incubation temperature increased. However,the floating rate of the fry showed the tendency of rising slowly then falling sharply. With the increase of domestication temperature,the 24hUILT₅₀ value of the juveniles increased and the occurrence time of CT_max delayed under high temperature stress. Under the stress of different high temperature,the expressions of HSP70 gene in the brain,gill and heart tissues of juvenile E. lucius increased first and then decreased,while that in the liver tissue continued to increase. When juvenile E. lucius were subjected to 30℃ high temperature stress for 0-24 h,the HSP70 gene expressions in their brains gill and heart tissues increased first and then decreased,while that in the liver tissue tended to be decreasing first and then increasing and then decreasing. The expressions of HSP70 gene in the brain,gill,heart and liver of juvenile E. lucius varied at different degrees under different temperatures and different high temperature stressed times,but the variations were time-specific and tissue-specific,with different response ranges. After high temp-erature domestication,the temperature tolerance of E. lucius increased,which may be involved in important regulation through HSP70.

Key words: Esox lucius, high temperature tolerance, critical temperature, heat shock protein 70

张钰, 海萨·艾也力汗, 热比古丽·沙吾提, 时春明, 张人铭. 白斑狗鱼早期发育阶段的高温耐受性分析[J]. 生物技术通报, 2021, 37(5): 76-83.

ZHANG Yu, HAYSA· Ayelhan, RABIGUL· Sawut, SHI Chun-ming, ZHANG Ren-ming. Analysis of High Temperature Tolerance in Early Development of Esox lucius[J]. Biotechnology Bulletin, 2021, 37(5): 76-83.

图/表 8

表1 荧光定量PCR引物

Table 1 Fluorescent quantitative PCR primers

基因 Gene	引物序列 Primer sequence（5'-3'）	片段长度Frag-ment length/bp
HSP70	F：ATCAGCCAGGGGTGCTCATT	200
HSP70	R：CTTGTTCTCCTTCCCAGTGC	200
管家基因β-actin Housekeeping genes	F：AAGATGAAATCGCCGCACTG	215
管家基因β-actin Housekeeping genes	R：TGCCAGATCTTCTCCATG	215

表2 不同孵化水温对白斑狗鱼仔鱼上浮率的影响

Table 2 Effects of different incubation water temperature on floatation rate of E. lucius fry

温度 Temperature/℃	受精卵数量/粒 The number of fertilized eggs/Grain	50%破膜时间 50% film breaking time/h	正常上浮鱼苗数量/尾 Number of floating fry/Tail	上浮率 Floating ratio/%
15±0.5	959	188	526	73.47
17±0.5	1096	139	602	73.58
19±0.5	1254	123	653	69.76
21±0.5	950	102	372	52.46

图1 白斑狗鱼在不同驯化温度下的24hUILT50

Fig.1 24hUILT50 of E. lucius at different acclimation temperatures

表3 白斑狗鱼仔鱼在不同驯化温度下的CTmax

Table 3 CTmax of E. lucius fry at different acclimation temperatures

驯化温度Acclimation temperature/℃	升温速率Heating rate/（℃/d）	CT_max/℃	CT_max出现时间CT_max appearance time/min
18±0.5	1±0.5	32.90	1152
21±0.5	1±0.5	33.06	1259
24±0.5	1±0.5	33.36	1418
27±0.5	1±0.5	33.27	1735

图2 不同温度高温胁迫下白斑狗鱼HSP70基因在各组织中的表达图中不同字母表示同一组织在不同温度高温胁迫下HSP70基因的相对表达量存在显著差异（P<0.05）

Fig.2 Expression of HSP70 gene in E. lucius different tissues under different high temperature stress Different letters refer to there is significant difference within the relative expression of the HSP70 gene in the same tissue under different high temperature stress(P<0.05)

图3 相同温度高温胁迫下白斑狗鱼HSP70基因在不同组织中的表达图中不同字母表示不同组织在相同温度高温胁迫下HSP70基因的相对表达量存在显著差异（P<0.05）

Fig.3 Expression of HSP70 gene in E. lucius different tissues under the same high temperature stress Different letters indicate there is significant difference within the relative expression of the HSP70 gene in different tissues under the same high temperature stress(P<0.05)

图4 不同时间高温胁迫下白斑狗鱼HSP70基因在各组织中的表达图中不同字母表示同一组织在不同时间高温胁迫下HSP70基因的相对表达量存在显著差异（P<0.05）

Fig.4 Expression of HSP70 gene in E. lucius different tis-sues under different times of high temperature stress Different letters indicate there is significant difference within the relative expression of the HSP70 gene in the same tissue under different times of high temperature stress(P<0.05)

图5 相同时间高温胁迫下白斑狗鱼HSP70基因在不同组织中的表达图中不同字母表示不同组织在相同时间高温胁迫下HSP70基因的相对表达量存在显著差异（P<0.05）

Fig.5 Expression of HSP70 gene in E. lucius different tissues under the same time of high temperature stress Different letters indicate there was significant difference within the relative expression of the HSP70 gene in the different tissues under the same time of high temperature stress(P<0.05)

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