The Construction Of Fujian Goat Ovary CDNA Library And Reproduction Traits Related Genes Polymorphism Analysis

This study choose Fujian Mindong goat, Daiyun goat and Fuqing goat for experimental samples,extract Fuqing goat’s ovarian tissue’s total RNA and three kinds of goat’s DNA, designed two experiments:Experiment one, using SMART technology and DSN homogenization technology, establish Fuqing goat’sovarian tissue normalized full-length cDNA library. Analysis the sequence of selected monoclonal samplesby EST method to understand their biological function, in order to further provide evidence for screeningfunctional genes related to goat fertility; Experiment two, using PCR-SSCP, PCR-RFLP molecularmarkers to SNPs polymorphism detection of five reproductive candidate genes (BMP15, GDF9, LH-β,GnRHR, BMPR-IB). Using the least squares variance analysis method to carry on the significance testsand providing theoretical reference basis for the correlation between different kinds of gene polymorphismsand reproductive traits. The results are as follows:1.Established the Fuqing goat’s ovarian tissue normalized full-length cDNA library. Fuqing goat’sovarian original library’s drop is1.0x10~6pfu/mL, restructuring at a rate of99%. After verified by colonyPCR, cloning fragment size distribution between0.5kb to2kb. Picked up200positive clones randomlly toEST sequencing,removing carrier sequence, pollution and small fragments sequence by Phred software, got183effective length sequences greater than500bp.164sequences are significant homology and sequencesof unknown function are19. Using Phrap software to sequence splicing, get156Unigenes, including crossfold group (contigs)7,(singleton)149, library redundancy rate is14.8%.Blast the homologous sequencefunction classification, divided into three categories, including cells related sequences, molecular functionrelated sequences, and biological processes related sequences.2.Genetic polymorphism analysis in BMP15, GDF9, LH-β, GnRHR, BMPR-IB genes by PCR-SSCPand PCR-RFLP methods and analysis the relationship between different genotypes and the lambing numberby least squares test, the results show that:In this experiment, there are two mutations in BMP15gene affecting the lambing performance. C873Gmutation: heterozygous mutation CG type with high lambing number of1.74headage, which is more thanCC type0.3headage, the difference was not significant with0.6more headage than homozygous mutantGG type. The lambing number is obviously increased with mutant A963G, GG type’s average lambingnumber is1.85, which is0.2more than type AG and0.5more than AA type with significant differences.Preliminary estimate, BMP15gene can be treated as a candidate gene realated to Fujian local goat’s lambing number trait.The effect of GDF9gene in lambing performance was not significant, mutant A1673C, though threekinds of mutation genotype frequency are in difference, the distribution is normal, each genotypeindividual’s lambing mean have no significant difference.LH-β gene exon2has a C1128T mutation, CC type’s individual number is fewer than other types. Seefrom the least-squares analysis results, the effect of mutation to goat’s lambing trait is significant. Theheterozygote mutant CT and homozygous TT type’s individual lambing number were higher than the wildtype CC, and extremely significant difference. Preliminary estimate, LH-β gene can be treated as fertilitycandidate gene in Fujian goats.GnRHR gene’s mutation G757A, homozygous mutant AA type’s lambing number obviously fewerthan that of AG and GG type individuals,with significant differences.It is initially determined that mutationG757A has certain influence in Fujian goats lambing number.BMPR-IB gene’s mutation A1766G has little effect on goat’s lambing number properties, theaverage lambing number between each mutation genotype individual has not significant difference, but allof them can reach more than1.6headage, and heterozygote mutant AG type’s average lambing number isrelatively higher.