Cloning And Sequence Analysis Of The Hamster Ssr Locus Screening And Barabensis The Mhc Genes

The landscapes of Tscherskia triton and Cricetulus barabensis in China are very extensive. The two species have strong reproductive ability,and can be considered as deleterious animals for agriculture.In these experiments the autuor progressed molecular study of them.Microsatellite sequence is an effective kind of genetic marker for molecular research.They are widely used in evolution study,structure analysis and genetic diversity in populations. Tscherskia triton,as one category of bandicoots,is extensive in the north of the Yangtse River. There were little outcome of microsatellite filtration,genetic diversity using in Tscherskia triton before.Therefore,the first experiment used the liver specimen of Tscherskia triton preserved in -70℃as materials to extract and purify genomic DNA with the classic phenol and chloroform extraction method.The materials were collected from Shunyi of Beijing and Gu'an,Raoyang of Hebei.According to the PCR amplication,using ten microsatellite locus primers from LOC-1 to ssrf11 of Tscherskia triton′related species-Cricetus cricetus,Mesocricetus auratus and Cricetulus barabensis to reclaim specific segments of DNA after polymerase chain reaction. Choose the white spots,make use of polymerase chain reaction to filtrate positive clones,get the 33 sequences and submit them to GenBank at last.There were no consistent sequences in GenBank using BLAST software.The submission number was from EF690673 to EF690693 and from EU140825 to EU140836.In the 33 microsatellites,perfect ones were 39.4%,imperfects were 24.2%,compounds were 36.4%.The motif number from 8 to 20 in most microsatellites was 60.6%,and the number exceed 21 was account for 39.4%.Most microsatellite sequences had the motif number exceeding 12.To this extent,they were feasible for primer designing and population polymorphic analysis.The major histocompatibility complex(MHC) is a specific chromosome region which has tight linkage and high polymorphic genes.It is extraodinary important in existance and maintenance of species.The prominent characters and fuctions of MHC have made it effective in immune genetics,colony genetics and breeding for disease resistance.MHC was used to study the structure as a molecular genetic marker in three populations of Cricetulus barabensis in the second experiment.Based on four reported MHC classⅠgene sequences of Cricetulus barabensis,sequences of human,rat and mouse,a pair of specific primers was designed,MHC classⅠα2 gene fragments were amplified from three populations in Yinan,Linqu and Wucun areas,Shandong province,China,then cloned and sequenced.The GenBank number was from FJ619112 to F J619183.The major results indicated that the polymorphism of the gene encoding MHC classⅠα2 domain in three populations of Cricetulus barabensis was rich,with 41 variable loci in 255 bp in length.The percentage of polymorphism was 16.08%.The percentage of polymorphism and Theta(per site) from S(θ_ω) of Wucun population were greater than Linqu population,while Yinan population was the least.The nucleotide diversity(Pi) and average number of nucleotide differences(K) of Linqu population were bigger than the others.The Haplotype diversity(Hd) in three populations was very high,indicating relatively high difference among nucleotide sequences.It was made up of conservative loci in amino acid sequences to combine antigen of three populations.There were alpha helixes and beta pucker in the protein structure,but Wucun population had a small difference.The Fixation Index(Fst) was negative and similar in these populations.Meanwhile,the haplotype was crossed.This result demonstrated that the differentiation of three populations was unconspicuous.The rich polymorphism of encoded MHC classⅠmolecularα2 chain gene is suitable as a molecular genetic marker for different populations of Cricetulus barabensis.In order to research the structure and fuction of MHCⅡgene in Cricetulus barabensis,the genomic DNA was also abstracted from the above three populations.The 249 bp fragment of exon 2 of DQA gene was amplified successfully by PCR and cloned into pMD18-T vector. Transfer the recombined carriers into E.coli DHSαafter recombination.The positive clones were further identified by PCR.The nucleotide sequence of this fragment was acquired and the peptide sequence was deduced.The GenBank number for the former sequence was FJ209306. Homologies of nucleotide sequence and amino acid sequence of exon 2 among Cricetulus barabensis,human,rat,mouse,pig,horse,cattle and rabbit were from 68.7%to 85%and from 56.8%to 83.5%,respectively.So Cricetulus barabensis is more similar to rat and mouse.This fragment is very polymorphic among species,and can be used as a molecular marker to study the genetic diversity of different species.