| The main environmental stress factors of trepang growth mainly include temperature, salinity, DO, infection, dry and so on. The strong stimulus will cause trepang fall in the body’s immune ability and feeding ability, slow growth speed, outbreaks of disease, etc. But at this present relatively straightforward way of outdoor pond farming, summer heat, estuary area of fresh water injection, water pollution caused by bacteria infection, and stimulate the disturbance of dew trepang transportation and in the process of production is inevitable. This experiment carries on the comparison to three trepang different strains, each strain is divided into large and small two kinds of specifications. Small specification trepang weight was 49.72±6.53 g, large size trepang weight at about 97.58±18.12 g. In the laboratory simulation of these four causes of trepang stress, through the experiment of stress method to evaluate three trepang strains of different specifications of experimental art ability. Combining trepang growth rate, mortality, and also determine the ways to evaluate the immune enzyme activity trepang new strains, the selection trepang and three other farms trepang strains of economic characters. Experimental results for, in the high temperature stress experiment and low salt stress experiment, trepang new strain has better performance and more highly survival rate, specific growth rate is bigger, nonspecific immune enzyme activity is higher.Especially in the case of high temperature stress, trepang new strain has good resistance to high temperature properties.For the anniversary of the water temperature change on trepang new strains trepang active status and the influence of the physiological and biochemical, six trepang aquaculture ponds for one-year tracking, trajectory observation record its feeding, defecation, activity.When water temperatures rise to trepang aestivation node, samling sample.measure digestive enzymes and fixed sample for paraffin section in the gut. Results show that the new strain has more significant resistance to high temperature properties: summer dormancy shorten about one month. When water temperature rose to 28 ℃, protease and amylase activity in the digestive tract is higher and the significant difference; Lipase vitality is very low under different temperature, there was no significant difference.This study according to previous person research on trepang response genes of high temperature environment, from the perspective of genomi NS2-S, adopting real-time fluorescent quantitative PCR technology, to improve the process temperature trepang gene transcription to determine the quantity of mRNA. 4 kinds of associated with heat stress expression genes, such as Hsp70, l(2)ef Myp and Psck9. Inside with β-actin, using relative quantitative 2-ΔΔCt method, for the four genes in the relative expression quantity under different temperature stress were studied. Trepang were used in this experiment to study after high temperature stress, Hsp70 and l(2)efl relatively increased expression and once again proved that Hsp70 and l(2)efl are raising genes, when the water temperature is 27℃, Hsp70 and l(2)efl increased with the increase of temperature the expression, trepang heat resistance by the expression of Hsp70. When the water temperature is 28 ℃,trepan were under high temperature stress, Hsp70 and l(2)efl express clearly. After high temperature stress Myp relative expression and Psck9 overall declining, Myp and Psck9 genes for downgrade. When the water temperature is 27 ℃, The relative expression of Mpy and Psck9 decreased sppgnificantly. In various high temperature stress, four kinds of heat stress related gene expression level of trepang new strains is different with other strains trepang.Microsatellite marker of DNA as a template is extracted by experiment, and laboratory existing 64 pairs of primers for PCR amplification, after amplification products with 1% agarose gel electrophoresis for testing, according to the stripe in the presence of selected eligible microsatellite DNA primers. PopGene32.0 statistical software to analyze each dot on the number of alleles(Na) and effective number of alleles(Ne) and observed heterozygosity(Ho), expected heterozygosity(He), gene flow(Nm), shannon- nasdaq for(I), Hardy Weinberg equilibrium test, population genetic differentiation between index(Fst) and Nei’s standard genetic distance(Ds); Linkage disequilibrium inspection GenePop software; PIC Calc 0.6 calculated each locus polymorphism information content(PIC); Using the MEGA 6.0 clustering analysis diagram. Three trepang strain sample average number of alleles, average effective number of genes in the group of GK trepang, highest 10 and 4.7070, respectively; NS2 and the allele for 9 NS1 group, NS1 and NS2 trepang strains the average effective number of alleles were 4.5260 and 4.1618 respectively. GK group of individual highest trepang expected heterozygosity and observed heterozygosity, 0.7197 and 0.4714 respectively; NS2 group trepang observed heterozygosity, lowest 0.4555, expected heterozygosity was 0.6789. Three trepang strains from generation to generation of polymorphism information content(PIC) were 0.6828, 0.6386 and 0.6691; The GK group PIC is the largest, quantitative value is 0.6828, trepang times NS1 group, is 0.6691, NS2 group minimum, 0.6386. Three groups of inbreeding coefficient Fis value are 0.3760, 0.2698 and 0.3760, respectively, are greater than zero. Fis value indicates that inbreeding coefficient, when the Fis <0 indicates that heterozygote excess, when Fis > 0 indicates that group heterozygote is missing, when the Fis=1 NS1 that group observation heterozygote completely missing. The experimental results show that three trepang strains have a certain degree of heterozygote missing phenomenon; 13 bits point 3 strains heterozygote missing points respectively: 12, 9 and 11. Three trepang strains genetic differentiation between two index is between 0.0141~0.0593, the genetic differentiation between the groups of the GK and NS2 index minimum(0.0141), the genetic differentiation between F and W index(0.0593), the largest genetic differentiation between GK and NS1 group generation index was 0.0432. Three trepang strain between the two values between 3.9629~17.5212 Nm, the gene flow between NS2 and GK group largest(17.5212), NS1 and NS2 gene flow between the minimum group(3.9629), GK and NS1 group gene flow between generation of 5.539. Three trepang strain between the genetic distance was 0.0628 ~ 0.3166, the genetic similarity index was 0.7286 ~ 0.9391. The genetic distance between NS2 and GK group recently, is 0.0628, the highest genetic similarity index was 0.9391; The genetic distance between the groups of NS2 and NS1, as far as 0.3166, the genetic similarity index, lowest 0.7286. NS2 and GK group recently, generation of genetic distance, get together for a first, because the NS1 group and the other two groups the genetic distance is relatively far, NS2 and GK generation gathered for a group of a generation finally and NS1. |
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