The Key Technology Of Standardization And Transcriptome Analysis In Laboratory Red Crucian Carp

Objective:1.According to the standardization requirements of laboratory red crucian carp,further screen and establish the microsatellite DNA molecular marker of laboratory red crucian carp C1HD strain,and use these markers to analyze its population genetic homozygosity.2.According to the standardization requirements of laboratory red crucian carp,establish the standard method of breeding and reproduction,and optimize the current method of gynogenetic reproduction for the conservation of its inbred strain.3.To promote the application of laboratory red crucian carp,explore the research of the biological effects of nuclear radiation and screen the miRNA biomarkers sensitive to nuclear radiation.Method:1.By the whole genome sequence information of red crucian carp,using bioinformatics analysis technology to extract microsatellite DNA sequences with repeated 1 to 6 base motifs,then design specific primers for PCR amplification and method distinguish the PCR amplification products by polyacrylamide gel electrophoresis.Comparing laboratory red crucian carp with its related species?goldfish,dongtinglake carp,hybrid red carp,koi carp?,to screen and verify the C1HD line's specific microsatellite marker.At last,use these markers to analyze the genetic homozygosity of the laboratory red crucian carp C1HD strain's gynogenetic test population.2.Setting up different environmental conditions for the indoor aquarium and outdoor cement tank,analyze the effects of comprehensive environmental factors such as lighting,landscaping,and water flow on the growth and reproduction of the laboratory red crucian carp,and comprehensively compare its aquaculture effects.Consist of 5 indoor experimental combinations and 1 outdoor experimental group,which are group 1?lighting,imitating ecological landscaping,water flow?,group 2?lighting,imitating ecological landscaping,no water flow?,group 3?lighting,no landscaping,water flow?,group 4?no light,imitating ecological landscaping,water flow?,group 5?no light,no landscaping,no water flow?,group 6?outdoor group?,each group is fed about 1 year;during the feeding process,the growth indexes and classification morphological indexes were dynamically measured and analyzed,and the histological characteristics of the development of the gonadal and other major organs were observed by tissue paraffin section.3.Based on the technical methods established in the laboratory's previous research on laboratory red crucian carp's gynogenesis,the key index parameters which was broodstock spawning,sperm DNA inactivation,and fertilized egg chromosome doubling were optimized.Carry out oxytocin effect experiments on three commonly used oxytocins such as LRH-A2,HCG,DOM and screen the best;adopt gradient test for ultraviolet inactivation time of sperm,cold shock time and temperature of fertilized egg,while emergence rate and fry survival rate indicators were counted,select the best time and temperature parameters.4.The laboratory red crucian carp was irradiated with 0.3 Gy at a total dose of¹³⁷Cs for one-time whole body irradiation by bioirradiator.The liver tissues of the irradiated group and the control group were taken for histological paraffin section observation and miRNA transcriptome sequencing.The sequencing data were screened for differential expression using bioinformatics analysis Significant miRNAs,predict their target genes and perform GO function annotation analysis and KEGG function enrichment analysis,and then verify the expression level of differentially expressed miRNAs by q-PCR,and finally determine the miRNA biomarkers sensitive to nuclear radiation.Result:1.The results of microsatellite DNA molecular markers indicate that:?In the whole genome of red crucian carp,the distribution type of microsatellite is mainly simple type,accounting for 81.6%;the repeating units in simplex type are mainly single base and two bases,accounting for 54.8%and 33.5%,respectively.?Randomly select 1000 microsatellite loci from the whole genome sequence information of red crucian carp,PCR amplified and repeated to obtain 19 loci with clear and relatively stable bands.Among them,9 microsatellite sites numbered SSR-2,SSR-4,SSR-20,SSR-21,SSR-86,SSR-142,SSR-434,SSR-763,and SSR-788 are laboratory red crucian carp's specific site.?The population genetic analysis of 9 specific sites showed that the genetic distances of laboratory red crucian carp and glodfish,hybrid red crucian carp,dongting lake crucian carp and koi carp were 0.6760,0.4842,0.7344,2.1358,and Nei's genetic similarity are 0.5082,0.6162,0.4798,0.1181,which can be distinguished accordingly.?According to the analysis of population genetic homozygosity on 9 specific sites of laboratory red crucian carp,the average observed homozygosity of its experimental gynogenetic population is 0.53,and the average Nei's expected heterozygosity is 0.33.2.The results of laboratory red crucian carp's breeding show:?The laboratory red crucian carp can be kept alive and grow in theenvironment of indoor aquarium and outdoor pond,and can be developed and matured.?The growth speed of the laboratory red crucian carp in the indoor aquarium is faster than that in the outdoor fish pond,and there is a significant difference?p<0.05?;among them,The growth rate of each group is from fast to slow:group 4>group 1>group 3>group 5>group 2>group 6?outdoor group?.?The body weight and body length of the laboratory red crucian carp were positively and highly correlated.The correlation coefficients of body weight and length of each group were:0.9860 for group 1,0.9850 for group 2,0.9792 for group 3,0.9792for group 3,0.9742 for group 4,0.9724 for group 5 and 0.9907 for group 6.?The development of gonads in the laboratory red crucian carp is:the sexual maturity of the fish in the indoor aquarium is slightly slower than that in the outdoor fish pond,but all can reach sexual maturity within 1 year.Among them,the first group's fish in the indoor aquarium developed gonads relatively quickly among the indoor.From the observation result of paraffin section at 7 months old,the first group develops fastest,and the development of gonadal phase is in phase III;from the observation result of paraffin section of laboratory red crucian carp at 9 months old,The gonads are all mature stage IV.?There was no difference in the morphology and other major organ development of the laboratory red crucian carp among indoor group and outdoor group.3.The results of the optimization test for gynogenesis show that:?The combined usage of LRH-A2+HCG+DOM has the best efficiency,and the effect time is about 8h.?Mirror sperms with two 15 W ultraviolet lamps at a distance of 12 cm inactivated for 45 min is better,the fertilization rate is 49.23%.?The survival rate of the fry of the fertilized eggs at 30 min and 35 min was0.07%and 3.09%,and the difference was respectively significant;the cold shock temperature was 3.0?,and the fertilization rate and emergence rate were better.4.Transcriptome sequencing analysis results of laboratory red crucian carp after irradiation showed that:?The paraffin section of radiation tissue showed that the liver cells of the experimental group occured nuclear deflation,but there was no visible damage on the body surface.?Analysis of sequencing data of liver miRNA transcriptome showed that the genome comparison rate of the 6 samples from the experimental group and the control group was 96.58%-97.87%,and its comparison rate of known miRNA was 62.07%-70.52%,which included 789 known miRNAs and 995 new predicted miRNAs.?34 differentially expressed miRNAs were detected in differential expression analysis?p<0.05?,16 of them were up-regulated and 18 were down-regulated.Among them,the numbers are abu-miR-152,abu-miR-19b,ssa-miR-125b-2-3p,dre-miR-18c,dre-miR-126a-3p,gmo-miR-27d-3p,dre-miR-9-7-3p and gmo-miR-140-5p were the most significant?p<0.01?.Conclusion:1.The 9 microsatellite sites numbered SSR-2,SSR-4,SSR-20,SSR-21,SSR-86,SSR-142,SSR-434,SSR-763,SSR-788 have laboratory red crucian carp's specificity of the C1HD strain,which can distinguish the laboratory red crucian carp from its relative species?goldfish,hybrid red crucian carp,dongtinglake crucian carp,koi?.2.The rearing environment and method for setting up laboratory aquariums are:rearing laboratory red crucian carp in aquariums larger than 120 cm×45 cm×70 cm,with a density of 180 fish/m³?50 mm length?,and using aeration for chlorine exposure water,light?150 LX,12 h/12 h?,ecological setting and water flow?12 h/12 h?and other environmental conditions,maintain water temperature 18-24?,laboratory red crucian carp can grow normally and reproduce normally.3.Induced labor with LRH-A2+HCG+DOM?3 ug/kg+800 U/kg+2 mg/kg?,and used two 15 W ultraviolet lamps at a distance of 12 cm to inactivate sperm of mirror carps for 45 min,and fertilized eggs cold shock at 3?for 35 min,a great gynogenetic effect of laboratory red crucian carp can be obtained.4.After 0.3 Gy ¹³⁷Cs radiation treatment,the laboratory red crucian carp showed no damage to the body surface but nuclear shrinkage of the hepatocyte nucleus;screening in the liver transcriptome obtained 34 miRNAs with obvious differential expression,among which the numbers were abu-miR-152,abu-miR-19b,ssa-miR-125b-2-3p,dre-miR-18c,dre-miR-126a-3p,gmo-miR-27d-3p,dre-miR-9-7-3p,gmo-Eight miRNAs,miR-140-5p,may be used as biomarkers sensitive to radiation injury in laboratory red crucian carp.