| Sinonovacula constricta,commonly known as razor razor,is widely distributed coastal mudflats in China.It is cultivated along the coasts of Zhejiang,Fujian,Shandong,Jiangsu,Liaoning,Guangdong,Guangxi and other provinces.It has a long history of breeding and is one of the four largest cultivated shellfish in China.After five successive generations of selection and breeding,the research team has obtained a new high-quality and high-yielding razor clam variety "Shenzhe No.1",which is genetically stable and has obvious growth advantages.In order to adapt to global warming,the deterioration of the mudflat environment and to ensure the sustainable development of the razor clam industry,a new strain of resistant razor clams was selected on the basis of fast growth.This thesis is based on the establishment and early screening of the "Shenzhe No.1" population for high temperature tolerance;the determination and analysis of relevant antioxidant enzymes,antioxidants and antioxidant pathways;and the cloning,sequence analysis and expression of the antioxidant gene GPx(Glutathione Peroxidase).The specific findings are as follows.1.Continue to select the new high-temperature tolerance varieties s of S.constricta "Shenzhe No.1",16 full-sib families were established in the early stage of this experiment,and the growth of each family was recorded on the 2nd,8th,22 nd and 36 th days.Three families(family 1,2,9)with low survival rate during the larval stage were eliminated,and 13 families with better growth were kept for further cultivation.In the spat stage,three families of longer shell lengths(family 14,15,16)and three families of shorter shell lengths(family 3,4,5)were selected,and a total of6 families were selected to the semi-lethal temperature experiment.The results showed that the shell length of families 3,4,and 5 was significantly lower than other families at the end of spat stage.The shell length and shell height of families 8,14,15,and 16 were significantly larger than other families(P<0.05).The survival rate of families 6 and 14 in spat stage was significantly higher than other families,and family14 had the highest survival rate,reaching 83.17±0.85%.In the semi-lethal temperature experiment,the highest semi-lethal temperature of family 16 reached36.64℃,and there was a significant difference in semi-lethal temperature between families with longer shell length and families with shorter shell length(P<0.05),which identified lines 14,15 and 16 as heat-tolerant families..After comprehensively evaluating the shell length,survival rate and heat tolerance data of each family,it is finally determined that family 14 is the best family suitable for production,and families 15,16 can be used as candidate families.2.The trend of peroxidase(POD)and total antioxidant capacity(T-AOC)increased with increasing temperature in all lines,reaching the highest level at 34 ℃and showing an inflection point;the content of Glutathione(GSH)increased with increasing temperature;meanwhile,the three antioxidant capacities of the longer shell length lines were significantly higher than those of the shorter shell length lines at high temperature(P<0.05).The expression level of tumor protein 53(p53)increased with increasing temperature until 34 ℃,but decreased afterwards,and the expression level at 37 ℃ was lower than that at room temperature;the average expression level of Sestrin 1(SESN1)in longer shell length lines was significantly higher than that in shorter shell length lines(P<0.05).The selection of heat-tolerant progeny with resistance to high temperature was carried out by strain selection and high-temperature experiments,and the need for strain selection was demonstrated from a physiological and biochemical point of view by the determination of antioxidant capacity.3.Cloning of the glutathione peroxidase GPx gene showed that the c DNA of the razor clam GPx gene was 1338 bp in length.Phylogenetic tree analysis showed that the razor clam GPx gene was most closely related to the Ruditapes philippinarum GPx gene,and the results of the phylogenetic analysis were consistent with the traditional morphological classification.The results of the phylogenetic analysis were also consistent with the traditional morphological classification.Quantitative Real-time PCR(QRT-PCR)showed the highest relative expression in the coat membrane,followed by the hepatopancreas and gills,and lower relative expression in the axoneme and gonad tissue(P<0.05).QRT-PCR showed that the expression of GPx gene differed in the mantle,gill andhepatopancreas under acute temperature stimulation,indicating that glutathione peroxidase plays an important role in all tissues of S.constricta at high temperatures.Summary: taking the new variety of S.constricta "Shenzhe No.1" as the research object,several families have been established in this population,with antioxidant capacity(antioxidant enzyme activity,reducing substance content,expression of key genes in antioxidant pathway)as the research object,a new strain of high temperature resistant S.constricta was screened.The study cloned the full-length sequence of the GPx gene of S.constricta,and performed its temporal and spatial expression analysis and high temperature stress expression analysis,confirming that GPx may play an important role in the high temperature stress of S.constricta.In the context of current global warming,this research screened new strains of resistant to high temperature of S.constricta,which will help improve the success rate and efficiency of cultivation of S.constricta in production. |
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