中国农业科学Issue(20)：4178-4187,10.DOI:10.3864/j.issn.0578-1752.2015.20.017
牦牛HSP27基因的克隆及其在雌性生殖器官中的表达
Cloning of Bos Grunniens HSP27 Gene and Its Expression in the Female Yak Reproductive Organs
摘要
Abstract
Objective]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 under normal physiological conditions by cloning the HSP27 gene and analyzing its biological characteristics.[Method] 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. The genetic characteristics of HSP27 gene were then analyzed utilizing bioinformation software to predict its product protein structure and potential functions. Next, RT-qPCR was employed to reveal the relative expression of HSP27 gene in the main reproductive organ during the reproductive cycle of the yak. Statistical analysis was performed using the software program SPSS (version 19.0, SPSS).[Result]HSP27 gene sequence containing a complete coding sequence, with the coding region length of 450bp (GenBank accession No: KP716832), This length could encode 149 amino acids of which Leucine (10.7%) was the most, and Tryptophan (0.7%) the least abundant. We determined that the atom number, molecular formula, calculated molecular weight and theoretical isoelectric point of the encoded protein were 2366, C747H1189N205O220S5, 16.722 kD, and 5.33 respectively. The HSP27 encoded protein of HSP27 was predicted to be a type of soluble and non-transmembrane protein. Nucleotide sequence analysis revealed that the HSP27 gene nucleotide sequence of yak was similar to those ofBos taurus(99.8%),Bubalus bubalis(98.4%),Ovis aries(97.8%),Pantholops hodgsonii(97.6%),Orcinus orca(90.3%),Camelus ferus(89.7%),Sus scrofa(89.7%),Equus caballus (86.7%),Canis lupus(86.7%),Homo sapiens(85.5%) andGorilla gorilla(85.3%). The similarity of resulting amino acid sequence of HSP27 gene between yak andbos taurus, bubalus bubalis, ovis aries pantholops hodgsonii, orcinus orca, camelus ferus sus scrofa, equus caballus, canis lupus, homo sapiensand gorilla gorilla were 100%、100%、100%、100%、96.3%、100%、96.8%、100%、100%、96.3% and 96.3%, respectively. The phylogenetic tree indicated that HSP27 gene of yak was close to bos taurus, bubalus bubalis, ovis aries and pantholops hodgsonii, but far from canis lupus, gorilla gorillaand homo sapiens. Moreover, the RT-qPCR indicated that HSP27 gene expressed universally in ovary, oviduct and uterus tissues during all reproductive periods, including follicular, luteal and pregnancy phases. The gene had its highest expression level in the follicle at follicle phase and its lowest expression in uterus in the luteal phase. 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 reproductive cycle. Its expression in ovaries, fallopian tubes and uterus changed dramatically because of pregnancy.[Conclusion]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, 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.关键词
牦牛/HSP27/基因克隆/生物信息学/表达Key words
yak/HSP27/gene cloning/bioinformatics analysis/expression引用本文复制引用
何翃闳,崔燕,潘阳阳,樊江峰,胡威,张译夫,刘鹏刚,李秦,余四九..牦牛HSP27基因的克隆及其在雌性生殖器官中的表达[J].中国农业科学,2015,(20):4178-4187,10.基金项目
国家自然科学基金（31472244，31360594）、甘肃省青年科技计划 （）
