Cloning And Evolutionary Analysis On The Bitter Taste Receptor Gene Of Apodemus Peninsulae And A.agrarius

Taste sensation is the basis of animal foraging behavior.Animals have five taste senses: sweet,salty,sour,bitter,and umami;and different taste sensation performs different function.And,bitter taste is an important basis for animals to identify toxic and harmful food,so it is one of the ways for animals to carry out the defense to harmful substances.It can help animals to reduce the intake of toxic substances,and has an important impact on animal survival,reproduction and evolution.The taste function of animals is realized by taste buds,which are composed of taste receptor cells,and the taste receptors are the result of the expression of bitter taste receptor(Tas2rs)genes.Therefore,the study on animal Tas2 r genes has important ecological significance.So far,there many studies on Tas2 r genes are conducted in vertebrates,especially on the adaptive evolution of Tas2 rs genes among different species and the function of Tas2 rs genes.Studies on the Tas2 rs genes in rodents are mainly about mice and rats,while studies on other rodents,especially the affinis in the same region,are relatively rare.Therefore,this research will take Apodemus peninsulae and A.agrarius as research object,and the Tas2 rs genes of the two species were cloned by first generation sequencing technology,and the Tas2 rs genes of other rodents were obtained by TBlast N.Using some biology softwares such as Oligo7.0,Blastn,Clustal W,Mega7.0,PAML4.0,DAMBE,Fig Treev1.4.3,Mrbayes3.2.1,PAUP,TMHMM server 2.0,Bio Edit,Ex PASy Proteomics Server,Timetree and so on,the base composition,phylogeny,selection pressure,hydrophobicity and transmembrane structure of Tas2 r genes were analyzed.The main results are as follows:1.In the study,the sequences of Tas2 r genes family in A.peninsulae and A.agrarius were obtained by the first generation sequencing technology.There are totally 25 Tas2 r genes in both mice,but their composition is different.A.peninsulae has 23 functional Tas2 r genes(Tas2r2,Tas2r3,Tas2r4,Tas2r5,Tas2r6,Tas2r7,Tas2r10,Tas2r13,Tas2r15,Tas2r16,Tas2r17,Tas2r19,Tas2r20,Tas2r25,Tas2r29,Tas2r34,Tas2r35,Tas2r36,Tas2r38,Tas2r39,Tas2r41,Tas2r43,Tas2r44),One partial gene(Tas2r40)and one pseudogene(Tas2r14);A.agrarius has 21 functional Tas2 r genes(Tas2r3,Tas2r4,Tas2r5,Tas2r6,Tas2r7,Tas2r10,Tas2r13,Tas2r15,Tas2r16,Tas2r17,Tas2r19,Tas2r25,Tas2r29,Tas2r34,Tas2r35,Tas2r29,Tas2r34,Tas2r35,Tas2r36,Tas2r38,Tas2r39,Tas2r41,Tas2r43,Tas2r44),two partial genes(Tas2r20,Tas2r40)and two pseudogenes(Tas2r2,Tas2r14).The length of CDS sequence of Tas2 r gene is about 930 bp,and the amino acid of the functional Tas2 r genes has 7 transmembrane structure.2.TBLASTN analysis showed that the composition of the Tas2 rs gene was different in different species.3.The results of phylogenetic analysis indicated that the squirrel and the blacklined squirrel clustered in one group,but the Tas2 r genes with the same name in different species were not clustered together;the topological structure of system phylogenetic tree in Tas2 r genes has the same topology as the phylogenetic tree of cytochrome b genes.4.The results of select pressure analysis: the results of the shoot model indicated that the Tas2 r genes of the mouse was strongly purified;the results of the shoot site model indicated that the Tas2 r genes of the mouse had a positive selection mutation,and the Apodemus peninsulae had positive selection sites Tas2r20(46P,54L)and Tas2r34(28I,88 S,111C,225V);the positive selection site of Tas2r36 gene of Apodemus agrarius was at 83M;the positive selection site of Tas2r16 gene of Heterocephalus glaber was at 255 M and 281T;the positive selection site of Mus musculus Tas2r36 gene was 148C;the site model testified positive selection sites for all testing positive selection genes.5.Comparison results of bitter taste receptor genes in different species: the bitter taste receptor genes were different in different diets,and there were more functional bitter taste receptor genes in phytophagous and omnivorous animals.Conclusions:The results of phylogenetic analysis of murine bitter receptor genes are more in line with the traditional taxa of the species,and have little relationship with the main feeding preferences of rodents.Evolutionary analysis indicates that the bitter receptor genes of Apodemus peninsulae and Apodemus agrarius adapt to their diet and living environment.There are positive selection mutation sites in Apodemus peninsulae,Apodemus agrarius Tas2r20,Tas2r34 and Tas2r36 genes,and the positive selection sites locate in the extracellular and transmembrane domains of the amino acid structure of the bitter receptor gene,which have good recognition ability for some bitter substances.By comparing with different feeding species,we find that the number of functional bitterness receptor genes in phytophagous and omnivorous animals is relatively large,which are related to the differentiation and change of their feeding habits.