| China is one of the world’s only breeding aquatic products is greater than the amount of fishing in the country,Freshwater fish occupy a large share in the aquatic products breeding。However, in recent years,Affected by the decrease of precipitation,river, lake water level dropped,and the mineralization degree is rising,Which can be used to reduce the resources of freshwater aquaculture;At the same time by the1800000000 mu of arable land red line constraints,Pond farming excessive occupation of cultivated land become history。These have become the bottleneck restricting the development of freshwater fish farming. Utilizing saline-alkaline water reasonably has opened a broad prospect for the development of Chinese fishery.Breeding new varieties of alkaline-resistant fish is the fundamental task, in which elucidation of fish alkaline-resistant mechanism and identifying candidate genes and exploring their functions play crucial roles.We use Leuciscus waleckii as the experimental material.Leuciscus waleckii(Dybowski) belongs to Osteichthyes, Cypriniformes,Cyprinidae and Leuciscus,it is of high salinity and alkalinity characteristics, and can survive in Dali Lake(Inner Mongolia) with alkalinity up to 53.57 mmol/L(p H 9.6),therefore, this fish has been regarded as a good object for evolutionary mechanism of salinity and alkalinity adaptation. L. waleckii in Dali Lake differs from their freshwater counterparts because they are characterized by breeding migration; they spawn in freshwater and grow in alkaline water. There is a marked difference in the acute phase alkaline tolerance between the alkaline population from Dali Lake and the fresh-water population from the Songhuajiang River, indicating the genetic heritability of alkaline tolerance. Scientists are paying more attention to L. waleckii with gradually recognized importance. However, the mechanism of its high tolerance on alkalinity is still a puzzle.Very limited physiological and genetic studies had been performed. Based on this point,our research team built hybrid F2 system(alkaline Dali Leuciscus X freshwater Songhua Leuciscus). Given the range of candidate genes associated with alkali resistance traits are large and microsatellite, SNPS and other means of molecularmarkers in fish population genetics applications already mature. This study intends to proceed from the angle of classical genetics, according to gene recombination principle,selecting the molecular markers that closely related to alkali resistance traits, then choose and locate functional genes by molecular markers. In this study, we examined 77 individuals performance of alkaline tolerance of hybrid F2, then we genotyped them using 39 SSRs makers including of 19 EST-SSRs. According to the performance of alkaline tolerance, individuals were divided into three groups, which composed of 43,20 and 14 individuals, respectively. Fisher exact probability test was conducted to examine the associations between maker and trait. The results showed that two markers were remarkably linked to alkaline tolerance(P<0.05), and one marker of EST-SSR.Sequence alignment of EST-SSR found that it has very high homology with Hypoxia inducible factor HIF-3( gene of Megalobrama amblycephala and Hypoxia inducible factor HIF-4( gene of grass carp(Ctenopharyngodon idllus), which mainly involved in hypoxia reaction process and strengthen hypoxia tolerance of fish. Combined with the research of Leuciscus waleckii digital differential expression analysis results,selected the HIF-3 gene and Rhag, Rhbg, Rhcga and Rhcg2 a genes together as the candidate gene or further analyses.The aim of this paper was to study the Alkali resistant mechanism of the fish at gene expression level, we selected L. waleckii in Dali Lake,which is alkali-resistant species and L. Waleckii in Songhua River( not Alkali-resistant species) as the experimental material,.According to the real-time fluorescence method.quantitative PCR was performed to analyze the relative expression levels in the gill of experimental fish.The results showed that the 5 candidate genes is different no matter in expression levels or the means of expressiong in two different kinds of fish.it showed that 5candidate genesi has a close relationship with the Alkali-resistant of Leuciscus waleckii.The five alkali-resistant genes identified in this study will be used as important elements for fish with alkali-resistante through transgenic technology in future.SMARTer? RACE c DNA Amplification Kit was successfully used for cloning the 5’ end and 3’-end of the five genes, combined with the DNAstar-Seq Man software to reconstruct the full-length c DNA with separate but overlapping 3’ and 5’ RACE PCR products following sequencing.These five full-length c DNA sequences were 2493bp、2026bp 、 2086 bp 、 2044 bp.The results set a foundation for further analyses on alkaline-responsive candidate genes, which would help us understand teleost adaptationunder extreme environmental stress and ultimately benefit future breeding for alkaline-tolerant fish strain. |
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