| Gymnocypris przewalskii, otherwise known as scale-less carp, is an important economic fish in Lake Qinghai. This lake is the largest inland water body in China, has a low concentration of dissolved oxygen, strong alkalinity and a high salinity level. The saline-alkali tolerance of G. przewalskii is very high. The major objective of this study is to construct the suppression subtractive cDNA library of G. przewalskii in response to saline-alkali stress, thus, to better understand how saline-alkali stress influences global gene expression in G. przewalskii and to provide theoretical basis in functional genes study. We then obtain full length cDNA of two acid-base related genes, Na+, K+-ATPase β1b subunit and carbonic anhydraseⅡ. Real-time PCR was used to analyse the acclimation of G. przewalskii to saline-alkali stress on the expression level.1. The suppression subtractive hybridization (SSH) method was adapted to construt the suppression subtractive cDNA library of G. przewalskii in response to saline-alkali stress.450positive clones from libraries were randomly selected to validate with PCR, and318clones showed a single channel result. The results showed that the quality of libraries were perfect. Consequently,195positive clones were selected randomly to sequence,166were successed. Sequences were qualified, clustered, assembled and compared through Blast. Altogether91genes were identified from the forward and reverse libraries,69of them were well annotated. These differentially expressed genes can be divided into a number of biological gene ontology groups related to catalytic activity, binding, cell, immune system process, transporter activity, structural molecule activity, cellular process, development, reproduction, biological regulation. Catalytic activity, binding and cell genes were up-regulated, while, binding, immune system process genes were down-regulated. Quantitative real-time PCR showed that the expression of Na+, K+-ATPase β1b (NKA-β1b) and glutamine synthase (GS) increased significantly at72h after exposure. The increasing expression of NKA-β1b and GS were matched the expression trend of the library, suggested its high quality. High saline-alkali water could change the osmotic pressure and acid-base balance in fish, injure surface mucous membrane of gill and kidney, and inhibit the immune system in a certain extent. Therefore, G. przewalskii may activate the metabolism, transporter and tolerance genes expressing through activate and enhance partial signal path. G. przewalskii improve the expression and activity of catalyticase to increase the ion exchange with the environment and maintain osmotic equilibrium; increase the expression of glutamine synthase to increase the content of glutamine and serum urea nitrogen, thus rebuild the acid-base balance; synthetize heat shock protein and annexin to increase the defense ability, reduce the response to stimulus.2. The ubiquitous membrane-spanning enzyme Na+, K+-ATPase is responsible for the active transport of Na+out of and K+into animal cells. It is important for maintaining intracellular homeostasis as well as for providing a driving force for acid-base regulation. The cDNA sequence of G.przewalskii NKA-β1b subunit gene was attained by rapid amplification of cDNA ends and found to have a total length of1258bp, encoded a peptide chain of260amino acids. One transmembrane regain was predicted using the TMHMM program analysis. Two putative N-glycosylation sites were found. Phylogenetic analysis showed that NKA-β1b of G. przewalskii is most closely linked to Danio rerio NKA β gene, following by Oncorhynchus mykiss, Xenopus laevis and Homo sapiens. Tissues distribution analysis revealed that the mRNA of NKA-β1b is expressed in all tissues that were examined, primarily in the gill. Significant changes in the expression of NKA-β1b mRNA were observed when exposed to salinity, alkalinity and saline-alkali stress in G. przewalskii, especially in the saline-alkali stress and high carbonate alkalinity stress. These result shows that NKA β play an important role in acute salinity, high carbonate alkalinity acclimation in G. przewalskii. The expressions of NKA-β1b mRNA were then returned to the control level or even lower, suggested the high saline-alkali regulated ability of G. przewalskii. In the high carbonate alkalinity stress group, the expressions of NKA-β1b mRNA in kidney incresed earlier and larger than in gill, inducated the important function of kidney in acid-base regulation.3. The full-length cDNA of carbonic anhydrase Ⅱ of G. przewalskii (GpCA Ⅱ) was amplified and sequenced. Tissues distribution and expression of GpCA II were examined to study the function of CAⅡ in G. przewalskii. GpCAⅡ has a total length of1400base pairs and open reading frame of783bp. The deduced protein encoded by GpCA Ⅱ consists of260amino acids, and has an approximate unmodified molecular weight of28.87kDa. A comparison of the deduced amino acid showed that GpCAⅡ is85%and77%identical to Tribolodon hakonensis CAⅡ and Danio rerio CAⅡ, respectively. Phylogenetic analysis showed that GpCAⅡ is most closely linked to the cytoplasmic CAⅡ of T. hakonensis, and grouped with teleost cytoplasmic CA isoform. The active sites in GpCAⅡ are highly conserved within the animal CA family, which suggests that GpCAⅡ has a catalytic function. Tissues distribution analysis revealed that the mRNA of GpCAⅡ is expressed in all tissues that were examined, primarily in the gill, pituitary gland and heart tissues. In the high alkaline stress group, the expression of GpCAⅡ in gill decreased significantly at12h after transferring, and the expression in kidney increased significantly at the same time. At96h after transferring, both gill and kidney decreased to lower level than the control group. These results suggested the important role GpCAⅡ played in acute high carbonate alkalinity acclimation. The regulation pattern of GpCAⅡ showed tissues depend. |
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