Isolation And Analysis Of Hypoxia Stress Related Genes From Gynogenetic Hypophthalmichthys Molitrix

The rapid development of agriculture and industry has caused water deterioration, and the descending oxygen saturation which is caused by elevated temperatures lead to frequent fluctuations of the oxygen in the water, so the anoxic is more and more severe. Hypoxia of water has become a huge threat for survival of many fish, and would cause a direct impact on aquaculture production. Silver carp {Hypophthalmichthys molitrix) is one of the "Four Fish’", accounts for an important proportion in Chinese aquaculture. It is sensitive to hypoxia and will jump out of the water after stimulated by various stress. The mechanisms of response to hypoxia stress involved in metabolism, signal transduction, cellular defense, apoptosis, cell structure, blood vessel formation, oxygen transport and gene expression physiological processes or biochemical processes, so the hypoxia capacity of fish is controlled by multiple genes and other factors. It will be very important to clarifying the mechanisms of the hypoxia stress. The mechanism of the hypoxia will help us to breeding new varieties of sliver carp.In our research the gynogenesis silver carp’Changfenglian’with better consistent genetic background, resistance to hypoxia stress, and rapid growth rate was used as the experimental material. The electron microscopy structure of the liver, gill, kidney cells of gynogenetic clone silver carp under different dissolved oxygen levels was studied. Appropriate reaction system for silver carp was established by the exploration and optimization of the key steps of cDNA-amplified fragment length polymorphism technology. A number of hypoxia-related genes expressed sequence was acquired through screening the differences of the transcription between the hypoxia silver carp and the normal by the well-established cDNA-AFLP technique. The full-length cDNA of heat shock protein70gene was cloned from ’Changfenglian’and the expression profile was studied through RT-PCR. The conclusions of this thesis include:(1) The electron microscopy structure of the liver, gill, kidney cells of gynogenetic clone silver carp under low dissolved oxygen level did not show evident difference compared with that of cells under normal development. This result indicated that the cells of important orgas in ’Changfenglian’ had not physical damages during hypoxia stressa which suggested ’Changfenglian’ has high resistance to hypoxia stressa.(2) The kidney, liver and brain from silver carp ’Changfenglian’ stored at-80℃freezer were used for the material. Appropriate reaction system of silver carp was established with the exploration and optimization to the key steps of cDNA-amplified fragment length polymorphism technology. It was proved when RNA is extractted by TRIzol reagent, cDNA is digested with PstI and TaqI restriction enzyme, the digested time of Pstl is3h, TaqI is2h in the20μl system, the digested fragments are distributed mainly from250bp to750bp, and the concentration of the PstI is3.2pmol/μl and TaqI is0.32pmol/μl in the ligation reaction system good result could be achieved. Pre-amplification product was diluted10times as a template for selective-amplification reaction. The cDNA-AFLP technique in this study for the silver carp transcriptome will provide a platform the establishment of technical support.(3)206differential bands were found by cDNA-AFLP analysis between normal group silver carp and hypoxic group with108pairs of primers, recovery of90differentially expressed cDNA fragments, these bands showed increased expression. Using the BLAST for analysis of differentially expressed fragment and query-related information, finally32sequences were found with hypoxia stress-related functions. The32sequences were divided into eight categories based on the possible biological functions, including:signal transduction, oxidative stress, hypoxia response, body protection, damage repair, apoptosis, endoplasmic reticulum stress and energy metabolism.(4) Four genes detected for quantitative expression in liver. The syndecan binding protein gene expression levels reach a maximum after2h hypoxia, then reduces gradually; The expression level of Cu/Zn-of superoxide dismutase increases continuously and reachs a stable value after10h hypoxia; The expression of inducible nitric oxide synthase, firstly increases then falls below a certain value and increases again after8h hypoxia; The expression level of X-box binding protein1 gene doesn’t increases until4h hypoxia. The cytoskeletal proteins may play a certain positive role in hypoxic stress, and there are some intrinsic relationship between endoplasmic reticulum stress and hypoxic stress, as well as the apoptosis.(5) Based on the EST sequence encoded the heat shock protein70(Hsp70), gene-specific primers were designed and used to obtain the full-length cDNA of a novel Hsp70gene from silver carp’Changfenglian’ with the method of rapid amplification of cDNA ends (RACE). This novel gene was named as Hsp70. Hsp70is2339bp in length with an ORF of1950bp, a5’-untranslated region (UTR) of84bp, and a3’-UTR of305bp, and the ORF encodes a predicted polypeptide of649amino acids. Hsp70is abundantly expressed in heart, brain and gill of ’Changfenglian’ under normal development, lower expression level of Hsp70is detected in kidney and spleen, and little expression is detected in muscle.