实验海洋生物学重点实验室知识库

皱纹盘鲍（Haliotis discus hannai）是鲍科动物中最具经济价值的物种，同时也是我国北方最重要的传统养殖贝类之一。皱纹盘鲍属典型的狭盐性贝类，对低盐尤为敏感，但迄今有关皱纹盘鲍对盐度胁迫的响应尚缺乏系统深入的研究。本研究从水和离子的跨膜运输两个方面对皱纹盘鲍渗透压调控相关基因进行了基础性的研究。
水通道蛋白是水分子跨膜运输进出细胞的重要通道，在渗透压调控过程中发挥了至关重要的作用。本论文中，根据生物信息学方法，同时结合分子生物学技术，在皱纹盘鲍基因组中分析鉴定出18种水通道蛋白基因，并通过Sanger测序验证获得其完整序列信息。对已鉴定出的皱纹盘鲍水通道蛋白进行聚类分析表明这18种水通道蛋白可分为C-AQP、AQP-8、AQGP和S-AQP共4大亚家族，根据每个成员所属的亚家族名称，分别将其命名HdAQPc1-HdAQPc9、HdAQP8.1-HdAQP8.4、HdAQPg1-HdAQPg3、HdAQPs1-HdAQPs2。对这18种水通道蛋白氨基酸序列的结构分析表明所有水通道蛋白均含有高度保守的NPA结构域。对其Ar/R结构域氨基酸残基构成进行分析可以看出在C-AQP亚家族的Ar/R结构域的前两个氨基酸大都为苯丙氨酸（Phe）和组氨酸（His），这两种氨基酸残基的侧链基团为大的环状结构，这使得其所形成的孔径相对较小，对其通道孔径进行分析表明C-AQP亚家族的通道孔径大约1.6 Å左右；但是在C-AQP亚家族中HdAQPc3的Ar/R结构域中，前两个氨基酸位点被两个丙氨酸（Ala）所取代，HdAQPc8和HdAQPc9的Ar/R结构域中第二个氨基酸残基被异亮氨酸（Ile）所取代；对该亚家族水通道蛋白的功能验证结果表明，在C-AQP亚家族中虽然HdAQPc3和HdAQPc8的孔径相对较大，但并不具备甘油运输能力，但HdAQPc9表现出很强的甘油运输能力；另外由于Ar/R结构域中第四个氨基酸位点精氨酸（Arg）被色氨酸（Trp）取代，可能使得HdAQPc4能够运输其他渗透压效应物，并起到渗透压调控的作用。在AQP-8亚家族中，其Ar/R结构域中第一个位点通常为His，这与C-AQP亚家族明显不同；功能分析表明表达HdAQP8.2的菌株在高渗条件下仍能正常存活，说明其对甘油并不具有渗透性，而在低渗条件下同样能正常存活，说明除甘油外，酵母仍存在其他渗透压效应物，HdAQP8.2介导了该物质的跨膜运输；而该亚家族中的其他三种水通道蛋白均未表现出该特性，说明其他三种水通道蛋白均不具有甘油运输能力。在AQGP亚家族中，Ar/R结构域的前两个氨基酸残基大都以侧链基团最小的甘氨酸（Gly）为主，这使得AQGP成员的孔径相对较大，功能验证分析表明只有HdAQPg1表现出很强的甘油运输能力，其余两种水通道蛋白虽然孔径相对更大，但均未表现出该特性。对经不同盐度处理后皱纹盘鲍不同组织中各水通道蛋白的表达进行检测，结果表明在不同组织中，对同样的盐度胁迫处理各水通道蛋白的响应模式不尽相同，另外在同一种组织中，各水通道蛋白对不同的盐度胁迫处理的响应模式也不尽相同。这说明渗透压平衡的维持是多种水通道蛋白参与，彼此相互协调共同维持的结果，同时也体现出皱纹盘鲍机体内渗透压效应物的多样性。
Na+/K+-ATPase（NKA）属于离子载体蛋白，通过消耗1分子ATP将胞内3个Na+逆浓度梯度转运至胞外，同时将2个K+逆浓度梯度转运至胞内，体内产生的ATP约1/4至3/4被NKA所消耗来维持细胞内外Na+和K+的浓度差以及渗透压平衡。在本研究中，通过分子生物学技术，结合生物信息学方法，在皱纹盘鲍基因组中克隆得到1种NKA的α亚基和1种β亚基cDNA序列。通过氨基酸序列多重比对，可以看出NKA的α亚基氨基酸序列高度保守（73.55%-89.24%），并且氨基酸序列中的保守区域与其跨膜结构域高度吻合，相反β亚基的氨基酸序列相似度较低（23.96%-47.46%）。分别用高盐度海水或低盐度海水对皱纹盘鲍处理不同时间后，发现高盐度处理导致血淋巴中各种离子浓度均升高，与之相反，低盐度处理导致血淋巴中的各种离子浓度均降低，但是在组织中，离子浓度在盐度骤然变化处理后，均维持在一个相对稳定的水平，并没有出现大幅度的波动。对经盐度骤然变化处理不同时间后组织中NKA的α亚基和β亚基mRNA表达量进行检测，结果显示在盐度变化初期，高盐度处理导致两种亚基均出现小幅度的降低，之后均出现大幅度的升高；相反，低盐度处理后初期，两种亚基均出现小幅度的上升，但是随后出现大幅度的下跌。通过Western blotting对NKA的α亚基的蛋白表达量进行检测，结果显示高盐度处理导致其表达量升高，而低盐度处理导致其表达量降低；同时对NKA的酶活性进行检测，结果与Western blotting结果相一致。最后检测了盐度骤然变化处理后各组织中cAMP的浓度变化趋势，结果显示高盐度处理导致cAMP浓度升高，而低盐度处理导致cAMP浓度降低。通过分析组织中cAMP浓度与NKA两种亚基mRNA的表达量以及NKA的活性之间的相关性，结果显示cAMP浓度与NKA两种亚基mRNA的表达量以及NKA的酶活性均呈正相关。该研究表明NKA在皱纹盘鲍渗透压调节中起重要作用，并且NKA的表达受cAMP调控。
 

As one of the most important commercially fishery industry animal, Pacific abalone Haliotis discus hannai was widely cultured in northern China. At present, the response to osmotic change in Pacific abalone has not been studied systematically. In this study, the related genes involved in osmotic pressure regulation in Pacific abalone were studied. It has important theoretical guiding significance to production and practice.

Aquaporin (AQP) mediated the transmembrane transport of water molecule. In addition, it plays a crucial role in the regulation of osmotic pressure. In this study, we identified 18 AQPs from Pacific abalone genome using bioinformatics combined with molecular biology. The accurate sequences were confirmed by sanger sequencing. Cluster analysis indicated that the 18 AQPs could be clearly divided into 4 clusters include all the 4 subfamilies named C-AQP, AQP-8, AQGP and S-AQP. According to their subfamilies, they were respectively named HdAQPc1-c9, HdAQP8.1-8.4, HdAQPg1-g3 and HdAQPs1-s2. Multiple alignment analysis shown that the 18 amino acid sequences were highly conserved in the two NPA motifs. Comparison of the amino acids in the Ar/R region shown that in the C-AQP subfamily, the first two sites were Phe and His in general. Hole analysis shown that the diameter of the AQPs in this subfamily was about 1.6 Å. But the Ar/R amino acids component in HdAQPc3, HdAQPc8 and HdAQPc9 were very special. In HdAQPc3, both of the first two amino acids were replaced by Ala. In HdAQPc8 and HdAQPc9, the second amino acid was replaced by Ile. Hole analysis shown that the replacements result in a wider diameter than the others in this subfamily. While the functional analysis shown that only HdAQPc9 possess the ability for glycerin transport. HdAQPc3 and HdAQPc8 may possess the ability for the other compounds transport. In addition, the fourth site of the Ar/R region in HdAQPc4 replaced by Trp. This replacement may enable HdAQPc4 to transport other osmotic effectors and play the role of osmotic pressure regulation. In AQP-8 subfamily, His usually appeared at the first site of the Ar/R region. In general, this region of the AQP-8 subfamily does not contain hydrophobic amino acids. Functional analysis show that the stain expressing HdAQP8.2 could survived at the hypertonic condition. This pattern indicated that the AQP inability to transport glycerin. Moreover, this stain also grew well at the hypotonic condition. This pattern indicated that glycerol is not the only osmotic effector in yeast. Additionally, HdAQP8.2 mediated the transport of these solutes. In AQGP subfamily, the first two site of the Ar/R region are usually Gly, the smallest α amino acid. This lead the hole diameter of the AQPs in this subfamily relatively wider. While functional analysis indicated that only HdAQPg1 could mediated the transport of glycerin. These data suggested that the other two AQPs in this subfamily may mediate the transport of other macromolecular substances. Detection of the 18 AQPs expression in different tissues of Pacific abalone after high or low salinity treatments shown that the response patterns of each AQPs to the same salinity change were diverse in different tissues, and the response patterns of each AQPs to different salinity change were also diverse in the same tissue. These data indicate that the maintenance of osmotic pressure balance is the result of multiple AQPs participating and coordinating with each other. Moreover, it also reflects the diversity of osmotic pressure effectors in the Pacific abalone.

As an ion transporter, Na⁺/K⁺-ATPase (NKA) could driving 3 Na⁺ export and 2 K⁺ import across the plasma membrane against their electrochemical gradients using the energy derived from ATP hydrolysis. In the present study, we have cloned and characterized the full-length cDNAs of NKA α subunit and β subunit from Pacific abalone. Multiple alignment shown that the predicted protein sequence of the NKA α subunit, as the catalytic subunit, was well conserved (73.55%-89.24%). Moreover, the highly conserved regions were consistent with the transmembrane regions. In contrast, the protein sequence of the β subunit had low similarity with those of other species (23.96%-47.46%). When treated the Pacific abalones with sudden salinity change (high or low salinity), the concentration of ions in hemolymph increased or decreased rapidly. While in tissues, the concentration of ions was maintained within the normal range. These data suggested that Pacific abalone has a strong osmotic regulation ability when faced with salinity change. The mRNA expression levels of NKA α and β subunits in tissues were detected after the sudden salinity change treatment. From these data we could conclude that at the initial stage, high salinity treatment led to a small decrease in both subunits and then a large increase. On the contrary, both subunits showed a small increase, but then a large decrease after the low salinity treatment. Meanwhile, the protein expression trend of NKA α subunit and its activity in tissues were measured, which was consistent with the mRNA expression pattern. Finally, the concentration of cAMP in tissue after sudden salinity change were measured. These data indicated that the high salinity treatment leads the increase of cAMP concentration. In contrast, low salinity treatment leads the decrease of cAMP concentration. Correlation analysis shown that the concentration of cAMP was positive correlated with the NKA mRNA expression and its activity. Therefore, NKA played an important role in the process of osmotic regulation in Pacific abalone, and its expression was regulated by cAMP.