Analyzation Of Single Nucleotide Polymorphisms In CAT Gene And SOD Gene And Associated With Resistant Hypoxia Traits In Litopenaeus Vannamei

Pacific white shrimp(Litopenaeus vannamei),owing to rapid growth,low nutrient need,strong resistance to disease,wide adaptability and suitable for high-density farming,has become the Chinese highest yield and largest size of marine farming shrimp species in recent years.In this study,the catalase gene and superoxide dismutase gene of Litopenaeus vannamei were used as the object of study,using direct sequencing technique,which will be use to analyze the Single Nucleotide Polymorphisms(SNP)of SOD gene,and CAT gene,which will be used to analyze the association between the polymorphisms and hypoxia tolerance traits.Aimed to find the SNP sites and provided the molecular basis for the genetic breeding of L.vannamei.Finally,real-time quantitative RT-PCR is used to detect the difference between SOD gene's and CAT gene's expression in under different pleasure level and recovery conditions.The aim was to provide molecular aspects for the study of low dissolved oxygen tolerance of L.vannamei theoretical basis.The results are as follows:1.Single nucleotide polymorphisms in CAT gene and SOD gene and their association with resistant hypoxia traits in Litopenaeus vannameiUsing direct sequencing technique to study the Single Nucleotide Polymorphisms(SNP)in CAT gene between hypoxia resistant group and susceptible group of Litopenaeus vannamei,a SNP site was found in 155 bp of catalase gene,which is A/G transversion and synonymous with mutation.Two SNPs site were identified in the SOD gene.One A/G mutation is located at 1575 bp and the other C/T mutation at 1602 bp in the SOD gene(g.1602C>T).Both of three loci belong to synonymous mutations.The result showed that: CAT g.155A>G,SODg.1575A>G and SODg.1602C>T,the expected heterozygosity and observed heterozygosity of this locus were 0.244~0.393 and 0.281~0.344 respectively,which fitted to the Hardy-Weinberg equilibrium(P>0.05)in the three SNPs sites.The polymorphism information content of CATg.155A>G mutation site and the SODg.1575A>G mutation site was less than 0.25 which in a low degree of polymorphism(PIC<0.25).The polymorphism information content of SODg.1602C> T position was he PIC value of the point is 0.314,which is in the median degree of polymorphism(0.25<PIC<0.5).The correlation between different genotypes and hypoxia tolerance traits of L.vannamei was analyzed by chi-square test.The results indicated that the genotypes of intron 1 of CAT gene were significantly associated with the hypoxia tolerance traits(X2 = 9.277,P<0.01).Among them,the AG genotype was dominant in the hypoxia tolerance(52.17%).There was no significant correlation between the genotypes of SODg.1575A>G(P>0.05).While the genotypes of SODg.1602C>T were significantly associated with the hypoxia tolerance traits(X2 = 6.515,P = 0.038).And the allele genotypes of SODg.1602C>T were very significantly associated with the hypoxia tolerance traits(X2 = 6.874,P = 0.009).There results could provide a basis for a further research on SOD gene and CAT gene in association with hypoxia tolerance traits in L.vannamei.2.Effect of hypoxia stress and reoxygenation condition on the transcription of SOD gene and CAT gene expression of L.vannamei.Tested the change in SOD gene and CAT gene expression in L.vannamei's liver and pancreas in hypoxia resistance and sensitive family with RT-PCR technology under the hypoxia for 48 hours and reoxygenation for 24 hours.The result showed that: the SOD gene and CAT gene expression trend to fall first and rise,then fall.In hypoxia sensitive family,they both showed first decline and then declinein the hypoxia resistance family.The expression of SOD and CAT gene in hypoxia sensitive family was significantly decreased under the stress of hypoxia for 6 hours,then it reached the highest level after the under stress for 12 hours.The expression of SOD and CAT gene decreased gradually at 24 hours to 48 hours.The expression of SOD gene and CAT gene in hypoxia resistance family reached the highest level under the stress of hypoxia for 6 hours,then both declined after 12 hours.The expression of SOD in the hypoxia resistance family and hypoxia sensitive family increased first,then descreased,but it occured contrary trend in expression of CAT under the reoxygenation condition.3.Changes in SOD gene and CAT gene expression of L.vannamei under the high-ammonia exposure and the recovery condition.Tested the change in SOD gene and CAT gene expression in Litopenaeus vannamei's Liver and pancreas in hypoxia resistance and sensitive family with RT-PCR technology under high-ammonia exposure for 48 hours and recovery condition for 24 hours.The result showed that: the SOD gene expression trend to fall first and rise,then fall in ammonia sensitive family and resistance ammonia family,then it reached the highest level after exposured to high-ammonia for 6hours,the lowest level occured at 48 hours under the high-ammonia exposure condition.The expression of SOD gene in the ammonia sensitive family and resistance ammonia family trend to continuous decrease,it reached the lowest point after 48 hours.The expression of SOD gene and CAT gene gradually rise with 24 hours under the recovery condition.