刺参Apostichopus japonicus耐高温品系选育的基础研究
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摘要
刺参是目前支撑我国海水增养殖业高效、稳定、持续发展的重要物种之一，但该产业面临着种质匮乏、病害严重、养殖环境局部恶化和产品品质难以保证等诸多问题。本文围绕刺参增养殖过程中的种质苗种问题，以培育耐高温品系为研究目标，在评价我国沿海不同地理群体刺参及其子一代在热刺激条件下HSP70基因mRNA表达差异的基础上，以低纬度地理群体为亲本，经温度刺激后比较了幼体及幼参的生长及HSP70 mRNA表达差异，获得了性状优良的耐高温群体。主要研究结果如下：
1、研究了我国沿海刺参不同地理群体在热激后HSP70基因mRNA的表达差异，比较了在不同温度下不同地理群体幼参的生长特性。青岛、日照和养殖等低纬度群体成参HSP70基因mRNA表达变化较大连、长岛等高纬度群体明显；热激后低纬度群体幼参HSP70基因mRNA表达量较高纬度群体高；高温条件下，高纬度群体子一代成活率低生长。温度升高对高纬度群体生长的影响较大。
2、利用荧光实时定量PCR检测了HSP70基因在不同组织、不同温度、不同时间mRNA水平的表达差异。刺参的各组织都有HSP70的mRNA表达，且对热刺激都有应答。自然水温12℃时，在＋12℃温度刺激时刺参的呼吸树中的HSP70 mRNA水平较高；刺参成参、幼参呼吸树中HSP70基因的mRNA水平在热激后6-12h达到最高；低温刺激后HSP70 mRNA下调表达。
3、分别从受精卵和初耳幼体开始在不同温度下进行幼体培育，27℃以上温度培育的刺参幼体，其生长和成活率显著低于24℃以下温度培育的幼体；不同起始刺激阶段和不同温度都是显著影响因子。从初耳幼体进行高温培育，幼体的变态受到明显影响，26℃以上温度培育幼体的变态率显著低于24℃以下培育温度。
4、以日照刺参为亲本，在26℃培育温度下获得了耐高温群体幼参，并从生长、HSP70mRNA水平的表达差异与常温培育的群体（普通群体）进行了比较。耐高温群体幼参在较高温度下成活率和HSP70mRNA表达水平明显高于普通群体，而二者在特定生长率随温度变化上没有差异。
5、在浙江苍南进行了选择群体耐高温能力和生长验证实验。耐温群体的温度耐受能力提高了0.7℃，进入夏眠时间延后15d左右。耐温刺参群体的特定生长率和成活率在3月温度回升后远高于对照组。
Sea cucumber Apostichopus japonicus (Selenka) aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in north China. However, there are many problems in this industry, such as deteriorations of germ plasm, environment and product quality. especially lacking of good artificial seed.
In this paper, comparative study on growth and mRNA expression level of HSP70 between different populations collected from different coastal area of China. Temperature stimulation was carried out in order to obtain a population which can tolerate higher temperature. The main results are as follows:
1. Studies on difference expression level of HSP70 mRNA in sea cucumber collected from different coastal area and their F1 were carried out. In addition, comparative study on growth of F1 at different temperature was executed. Results showed that the mRNA expression level of HSP70 of sea cucumber collected from Qingdao, Rizhao and Fujian were higher than those of Dalian and Changdao.
2. Fluorescent real-time quantitative RT-PCR was used to examine the mRNA expression level of HSP70 in different tissues of sea cucumber challenged by different temperature. When the temperature was above 12 (temperature of natural sea water is 12 ), the mRNA expression level of HSP70 was higher than those of the natural temperature in the respiratory tree. In the respiratory tree of adult and juvenile sea cucumber, HSP70 mRNA level increased and reached maximal level at 9h after heat stimulation.
3. The temperature stimulation was initially started at fertilized eggs and early auricularia stage. The viability rate of larvae varied with different temperature. Both the initial stimulation stage and temperature had significant effect on the larval viability rate. However, there was no the correlation between maximal body length and temperature or initial stimulation time. Moreover, temperature influenced larval metamorphosis and settlement significantly. In conclusion, the larvae, stimulated by the temperature above 26 , which viability rate and metamorphosis rate were significantly lower than those stimulated by lower temperature.
4. From the results that have been shown, the sea cucumber collected from Rizhao was selected as broodstock. Juvenile sea cucumbers were obtained by the stimulation at the temperature of 26 . Comparative studies on the growth and different expression of HSP70 of these populations and the natural population were also carried out. The larval viability rate, specific growth rate and the mRNA expression level of HSP70 of artificial seeding group were significantly higher than the control group. 5. Similar results also had been obtained from the comparative growth experiment in Cangnan, Zhejiang province. These results showed that the tolerance temperature of artificial breeding sea cucumbers enhanced 0.7 and the time to begin aestivation delayed 15d.
1、研究了我国沿海刺参不同地理群体在热激后HSP70基因mRNA的表达差异，比较了在不同温度下不同地理群体幼参的生长特性。青岛、日照和养殖等低纬度群体成参HSP70基因mRNA表达变化较大连、长岛等高纬度群体明显；热激后低纬度群体幼参HSP70基因mRNA表达量较高纬度群体高；高温条件下，高纬度群体子一代成活率低生长。温度升高对高纬度群体生长的影响较大。
2、利用荧光实时定量PCR检测了HSP70基因在不同组织、不同温度、不同时间mRNA水平的表达差异。刺参的各组织都有HSP70的mRNA表达，且对热刺激都有应答。自然水温12℃时，在＋12℃温度刺激时刺参的呼吸树中的HSP70 mRNA水平较高；刺参成参、幼参呼吸树中HSP70基因的mRNA水平在热激后6-12h达到最高；低温刺激后HSP70 mRNA下调表达。
3、分别从受精卵和初耳幼体开始在不同温度下进行幼体培育，27℃以上温度培育的刺参幼体，其生长和成活率显著低于24℃以下温度培育的幼体；不同起始刺激阶段和不同温度都是显著影响因子。从初耳幼体进行高温培育，幼体的变态受到明显影响，26℃以上温度培育幼体的变态率显著低于24℃以下培育温度。
4、以日照刺参为亲本，在26℃培育温度下获得了耐高温群体幼参，并从生长、HSP70mRNA水平的表达差异与常温培育的群体（普通群体）进行了比较。耐高温群体幼参在较高温度下成活率和HSP70mRNA表达水平明显高于普通群体，而二者在特定生长率随温度变化上没有差异。
5、在浙江苍南进行了选择群体耐高温能力和生长验证实验。耐温群体的温度耐受能力提高了0.7℃，进入夏眠时间延后15d左右。耐温刺参群体的特定生长率和成活率在3月温度回升后远高于对照组。
Sea cucumber Apostichopus japonicus (Selenka) aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in north China. However, there are many problems in this industry, such as deteriorations of germ plasm, environment and product quality. especially lacking of good artificial seed.
In this paper, comparative study on growth and mRNA expression level of HSP70 between different populations collected from different coastal area of China. Temperature stimulation was carried out in order to obtain a population which can tolerate higher temperature. The main results are as follows:
1. Studies on difference expression level of HSP70 mRNA in sea cucumber collected from different coastal area and their F1 were carried out. In addition, comparative study on growth of F1 at different temperature was executed. Results showed that the mRNA expression level of HSP70 of sea cucumber collected from Qingdao, Rizhao and Fujian were higher than those of Dalian and Changdao.
2. Fluorescent real-time quantitative RT-PCR was used to examine the mRNA expression level of HSP70 in different tissues of sea cucumber challenged by different temperature. When the temperature was above 12 (temperature of natural sea water is 12 ), the mRNA expression level of HSP70 was higher than those of the natural temperature in the respiratory tree. In the respiratory tree of adult and juvenile sea cucumber, HSP70 mRNA level increased and reached maximal level at 9h after heat stimulation.
3. The temperature stimulation was initially started at fertilized eggs and early auricularia stage. The viability rate of larvae varied with different temperature. Both the initial stimulation stage and temperature had significant effect on the larval viability rate. However, there was no the correlation between maximal body length and temperature or initial stimulation time. Moreover, temperature influenced larval metamorphosis and settlement significantly. In conclusion, the larvae, stimulated by the temperature above 26 , which viability rate and metamorphosis rate were significantly lower than those stimulated by lower temperature.
4. From the results that have been shown, the sea cucumber collected from Rizhao was selected as broodstock. Juvenile sea cucumbers were obtained by the stimulation at the temperature of 26 . Comparative studies on the growth and different expression of HSP70 of these populations and the natural population were also carried out. The larval viability rate, specific growth rate and the mRNA expression level of HSP70 of artificial seeding group were significantly higher than the control group. 5. Similar results also had been obtained from the comparative growth experiment in Cangnan, Zhejiang province. These results showed that the tolerance temperature of artificial breeding sea cucumbers enhanced 0.7 and the time to begin aestivation delayed 15d.
引文
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