Cloning Of Bos Grunniens HSP27 Gene And Its Expression Both In The Female Yak Reproductive Organs And Parthenogenetic Embryos

The objective of this study was carried out to study differences of the expression of Heat Shock Protein 27(HSP27)gene in the main reproductive organs of the female yak and the different development period of embryos after parthenogenetic activation under normal physiological conditions by cloning the HSP27 gene and analyzing its biological characteristics.Samples from the tissues of ipsilateral Ovary,oviduct and uterus during follicular anaphase,luteal anaphase and early pregnancy phase were collected and cDNA were isolated from each of the collected tissues.RT-PCR was used to clone the HSP27 gene,and purified PCR products were cloned on pMDTM18-T Vector to detect the sequence.Next,RT-qPCR was employed to reveal the relative expression of HSP27 gene in the main reproductive organs during the reproductive cycle and the different development period of embryos after parthenogenetic activation of the yak.Western-blot were used in the present study to detect the expression of HSP27 protein in the main reproductive organs during the reproductive cycle of the yak.Immunohistochemistry SP method and immunofluorescence were used in the present study to detect the distribution of HSP27 protein in the main reproductive organs during the reproductive cycle and the different development period of embryos after parthenogenetic activation of the yak.(1)HSP27 gene sequence containing a complete coding sequence,with the coding region length of 450bp(GenBank accession No: KP716832).Nucleotide sequence analysis and the phylogenetic tree revealed that the HSP27 gene of yak was close to bos Taurus(99.8%)and bubalus bubalis(98.4%),far from homo sapiens(85.5%)and gorilla(85.3%).(2)The RT-qPCR indicated that HSP27 gene expressed universally in the main reproductive organs during the reproductive cycle and the different development period of embryos after parthenogenetic activation of the yak.In addition,the expression level of HSP27 gene in ovary was significantly higher than in the uterus during each of the different stages of the main reproductive organs during the reproductive cycle.In the different development period of embryos after parthenogenetic activation of the yak,the gene had its highest expression level in 2~4 cell stage embryos and its lowest expression in blastocysts.With the development of embryos,the expression of HSP27 decreased gradually.(3)Western-blot results demonstrated that HSP27 expressed universally in in the main reproductive organs during the reproductive cycle of the yak,of which the expression quantity in the follicle at follicle phase was the highest,while in uterus in the luteal phase was the lowest.Immunohistochemistry result indicated that HSP27 was have a positive expressed in yak theca folliculi interna,stratum granulosum,cumulus,oviduct epithelium,endometrium epithelium and uterine glands,respectively.(4)Indirect immune fluorescent displayed that HSP27 mainly existed in the nucleus and cytoplasm.It is concluded from the present study that HSP27 gene in the yak is highly conservative in mammalian evolution according to the comparative nucleotide sequence analysis.Analyzing HSP27 gene expression in central reproductive organs during the breeding cycle and the different development period of embryos after parthenogenetic activation of the yak,it was found that HSP27 was related to follicular development and maturation and other important physiological processes related to reproduction and specifically may play an important biological role in pregnancy.