| Temperature stress can cause free radical metabolism disorder,damage normal physiological function and immune defense ability of body cells and tissues,affect animal metabolism,growth,development,immunity and reproduction,and seriously lead to animal death.Litopenaeus vannamei is one of the major shrimp species in the world.Temperature is one of the important factors for influencing the culture of L.vannamei.In this study,the transcriptome sequencing data of hepatopantras tissues of L.vannamei under high and low temperature stress were analyzed,and candidate genes related to temperature stress were obtained.Quantitative real-time PCR was used to investigate the expression levels of these genes in temperature stressed L.vannamei,and fluorescent in situ hybridization was used to analyze the function and expression mapping of these genes.The main research contents are as follows:1.Expression characteristics of Luc7 L,PUF and ddit4 l genes in L.vannamei under high temperature stressThree candidate genes,Luc7 L,PUF and ddit4 l,were screened from hightemperature gradual and high-temperature abrupt transcriptome data,based on reference sequences from the whole genome data(NCBI,ASM378908v1)of L.vannamei,and partial c DNA sequences(complete ORF and partial UTR)were obtained by direct PCR amplification.Lv-Luc7 L gene contains a 1200 bp ORF encoding 399 amino acids and contains a conserved domain of LUC7 supergene family at 22～264 position.The Lv-PUF gene contains 1890 bp ORF encoding 629 amino acids and contains a conserved structural domain of the half-pint supergene family at positions 40～629,and three functional structural domains of the RRM at positions 131～204,28～301,and 533～615.The homology of the amino acid sequences encoded by the above three genes was compared.The Lv-ddit4 l sequence contains a495 bp ORF encoding 164 amino acids,and contains a functional domain of RTP801 C supergene family between 62 and 160 amino acids,which is highly conserved among species.The homology of Lv-ddit4 l and Chionoecetes opilio-ddit4 l was 59.76%.Lv-Luc7 L had the highest homology with Penaeus monodon and Penaeus japonicus(95.00%).Lv-PUF had the highest homology with Penaeus monodon(80.78%).Phylogenetic analysis of the above three genes showed that they were closely related to crustaceans.High temperature stress experiment can be divided into two kinds: gradual change of high temperature and acute change of high temperature.The gradual stress process of high temperature was as follows: After constant temperature culture at 26℃(control),L.vannamei was heated from 26℃ to 38℃ at a rate of 2℃ every 12 h.After 38℃ was maintained for 12 h,the temperature returned to 26℃ at a rate of 2℃every 12 h.The sampling times were as follows: A-26℃ group: normal temperature26℃ culture;B-32℃ group: 12 h after heating from 26℃ to 32℃;C-38℃ group: 12 h after heating from 32℃ to 38℃;D-32℃ group: 12 h after reheating from 38℃ to32℃;E-26℃ group: from 32℃ to 26℃ for 12 h.The process of sudden temperature stress was as follows: After constant temperature at 26℃(control),L.vannamei was directly moved from 26℃ seawater to 36℃ seawater for 24 h,and then directly moved from 36℃ seawater to 26℃ seawater for 48 h.The sampling times were as follows: F-36℃(10 min)group: 10 min after moving from 26℃ seawater to 36℃seawater;G-36℃(24 h)group: 24 h after moving from 26℃ seawater to 36℃seawater;H-26℃ group: 48 h after moving from 36℃ seawater to 26℃ seawater.The expression of Luc7 L,PUF and ddit4 l genes in five different tissues(eye stalk,gill,hepatopancreas,nerves and muscle)during two experiments of gradual change of high temperature and acute change of high temperature were analyzed by q RT-PCR.The results showed that Lv-Luc7 L,Lv-PUF and Lv-ddit4 l were expressed in five tissues of L.vannamei at 26℃ water temperature,and the expression levels were basically the same without significant difference(p > 0.05).The expression levels of Lv-ddit4 l were significantly up-regulated in eye stalk,gill and hepatopancreas during the whole temperature gradient from 32℃ to 38℃(p < 0.05).The expression of Lv-ddit4 l was significantly upregulated in gill,hepatopancreas,nerve and muscle during the whole temperature sudden change at 36℃(p < 0.05),and the expression of Lv-ddit4 l was the highest in hepatopancreas at the early stage of sudden change.When water temperature slowly increased to 38℃(C-38℃),the expression level of Lv-Luc7 L in eye stalk,gill,hepatopancreas,nerve and muscle was significantly up-regulated(p < 0.05).The expression in hepatopancreas,nerve and muscle increased firstly and then decreased with temperature.The expression levels of Lv-Luc7 L were significantly up-regulated in gill,hepatopancreas,nerve and muscle during the whole acute change of high temperature within 36℃(p < 0.05).The expression level of Lv-PUF in gills,hepatopancreas,nerves and muscles were significantly upregulated when high temperature gradually changed to the highest temperature(C-38℃)(p < 0.05).The expression in hepatopancreas,nerve and muscle increased firstly and then decreased with temperature.The expression of this gene in hepatopancreas was significantly changed in the hepatopancreas throughout the water temperature of 36°C during the abrupt change.The distribution of ddit4 l gene in hepatopancreas of L.vannamei was investigated by FISH under normal temperature(A-26℃)and early temperature shock(F-36℃ for 10 min)stress.The results showed that the ddit4 l gene was mainly localized in the nucleus of the cell.In the early stage of high temperature quenching(F-36℃ for 10 min),the fluorescence positive signal was significantly enhanced as well as the fluorescence range was expanded to the inner part of the hepatopancreas,and the expression was significantly increased.2.Fluorescence in situ hybridization of HSP70 gene in L.vannamei under heat stressAccording to the quantitative expression of HSP70 gene in our research group,the expression level of Lv-HSP70 gene was significantly up-regulated when the water temperature slowly rose to 38℃(C-38℃)in the gradient of high temperature.In the high temperature sudden change,the up-regulated gene expression was extremely significant at the early stage of sudden change at 36℃(F-36℃ for 10 min)(p < 0.01).Fluorescence in situ hybridization showed that HSP70 was localized in cytoplasm and nucleus.Under normal conditions,only a small amount of HSP70 exists in the cytoplasm,but under the temperature stress of gradual,the expression of HSP70 increases and shows a trend of increasing in the nucleus.HSP70 expression was increased in the cytoplasm when subjected to high temperature sudden change.3.Expression characteristics of MPPED2 genes in L.vannamei under low temperature stressThe MPPED2 gene was screened from gradual change of low-temperature and acute change of low-temperature transcriptome data,based on reference sequences from the whole genome data(NCBI,ASM378908v1)of L.vannamei.The partial c DNA(complete ORF and partial UTR)sequence of MPPED2 was obtained by using direct PCR amplification.The Lv-MPPED2 gene contains an 834 bp ORF encoding277 amino acids,which is predicted to contain three N-glycosylation sites at 134,165 and 244,and a conserved domain of the MPP＿239FB supergene family at 52～246.Homologous sequence comparison of the amino acid sequences encoded by MPPED2 gene showed that Lv-MPPED2 had the highest homology with Penaeus monodon(89.17%),followed by Penaeus japonicus(87.27%).Phylogenetic analysis of the above three genes revealed that L.vannamei first clustered closely with Penaeus monodon and then with Penaeus japonicus,and they were most closely related to each other,forming an independent branch.Low temperature stress experiment can be divided into low temperature gradient and low temperature sudden change.The gradual stress process of low temperature was as follows: After constant temperature culture at 26℃(control),L.vannamei was cooled from 26℃ to 10℃ at a rate of 2℃ every 12 h.After 10℃ was maintained for12 h,the temperature returned to 26℃ at a rate of 2℃ every 12 h.The sampling times were as follows: a-26℃ group: normal temperature 26℃ culture;b-18℃ group: 12 h after cooling from 26℃ to 18℃;c-10℃ group: 12 h after cooling from 18℃ to 10℃;d-18℃ group: 12 h after heating from 10℃ to 18℃;e-26 ℃ group: from 18℃ to 26℃for 12 h.The process of sudden low temperature stress was as follows: After the temperature was kept at 26℃(control),L.vannamei was directly moved from 26℃seawater to 14℃ seawater for 24 h,and then directly moved from 14℃ seawater to26℃ seawater for 48 h.The sampling times were as follows: f-14℃(10 min)group:10 min after moving from 26℃ seawater to 14℃ seawater;g-14℃(24 h)group: 24 h after moving from 26℃ seawater to 14℃ seawater;h-26℃ group: 48 h after moving from 14℃ seawater to 26℃ seawater.The expression of MPPED2 gene was analyzed in five different tissues(eye stalk,gill,hepatopancreas,nerves and muscle)during two experiments of low-temperature gradient and acute change of low-temperature.The results showed that Lv-MPPED2 was expressed in all five tissues of L.vannamei in the water temperature environment of 26℃,and the expression was basically the same without significant differences(p >0.05).The expression of Lv-MPPED2 in gill and hepatopancreas was significantly upregulated(p < 0.05)when the water temperature slowly decreased to the lowest temperature of 10°C(c-10°C),and the expression of Lv-MPPED2 in eye stalk,gill,hepatopancreas,nerve and muscle was significantly upregulated when the water temperature slowly returned to 18°C(d-18°C).Lv-MPPED2 expression in eye stalk,gill,hepatopancreas and muscle was significantly up-regulated(p < 0.05)when water temperature was maintained at 14°C for 24 h(g-14°C),with the most significant difference in hepatopancreas.The distribution of MPPED2 gene in hepatopancreas of L.vannamei was investigated by fluorescence in situ hybridization under normal temperature(a-26°C),low temperature gradient(c-10°C),and late low temperature sudden change(g-14°C24 h)stress.MPPED2 gene was mainly localized in the nucleus,and its expression level was enhanced under both gradual change of low-temperature and acute change of low temperature. |
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