牦牛HSP27基因的克隆及其在雌性生殖器官中的表达

doi:10.3864/j.issn.0578-1752.2015.20.017

摘要/Abstract

摘要： 【目的】克隆牦牛热休克蛋白27(heat shock proteins27，HSP27)基因，分析其生物学特性，研究正常生理条件下HSP27基因在母牦牛主要生殖器官中的表达差异。【方法】采取卵泡期后期、黄体期后期同侧的卵巢、输卵管和子宫，以及妊娠期早期（胚胎约长2.3cm）妊娠侧的卵巢、输卵管和子宫组织，以其cDNA为模板，利用RT-PCR扩增牦牛HSP27基因，并将纯化的PCR产物克隆到pMDTM18-T Vector 载体中进行测序；利用生物信息学软件对HSP27基因的特征进行生物信息学分析，预测功能结构域；采用实时荧光定量 RT-qPCR 测定繁殖周期母牦牛主要生殖器官中的相对表达量，用SPSS19.0软件对试验数据进行差异显著性分析。【结果】成功克隆出包含完整编码区的牦牛HSP27基因序列，其编码区长450bp（GenBank 登录号：KP716832），可编码149个氨基酸，其中含量最高的是亮氨酸Leu（10.7%），含量最低的是色氨酸Trp（0.7%）。HSP27编码区蛋白原子个数为2 366，分子式为C747H1189N205O220S5，分子量为16.722kD，理论等电点为5.33；HSP27编码蛋白为非跨膜可溶性蛋白。同源性分析显示，牦牛HSP27基因的核苷酸序列与家牛、印度水牛、绵羊、藏羚羊、虎鲸、野骆驼、野猪、马、灰狼、人和猩猩的核苷酸序列同源性分别为99.8%、98.4%、97.8%、97.6%、90.3%、89.7%、89.7%、86.7%、86.7%、85.5%和85.3%，牦牛HSP27基因的氨基酸序列与家牛、印度水牛、绵羊、藏羚羊、虎鲸、野骆驼、野猪、马、灰狼、人和猩猩的氨基酸序列同源性分别为100%、100%、100%、100%、96.3%、100%、96.8%、100%、100%、96.3%和96.3%；系统进化树表明，牦牛HSP27基因与家牛、印度水牛、绵羊和藏羚羊的进化水平较为相近，与灰狼、猩猩和人类的较远。RT-qPCR结果表明，牦牛HSP27基因在卵泡期、黄体期、妊娠期不同时期的卵巢、输卵管和子宫组织中均有表达，其中在卵泡期卵巢中的表达量最高，在黄体期子宫中的表达量最低；在不同繁殖周期，牦牛HSP27基因在卵巢中的表达均极显著大于在子宫中的表达，其在卵巢、输卵管和子宫的表达因妊娠而发生了显著变化。【结论】通过与其他物种HSP27基因的同源性对比分析，发现HSP27基因在进化中具有高度保守性，同时也存在种属特异性；通过对HSP27在繁殖周期母牦牛主要生殖器官中的表达情况分析，推测出HSP27可能参与了母牦牛妊娠的调控，为进一步探讨HSP27在牦牛生殖过程中发挥的作用提供了参考资料。

关键词: 牦牛, HSP27, 基因克隆, 生物信息学, 表达

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 pMD^TM18-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 of Bos 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%) and Gorilla gorilla (85.3%). The similarity of resulting amino acid sequence of HSP27 gene between yak and bos taurus, bubalus bubalis, ovis aries pantholops hodgsonii, orcinus orca, camelus ferus sus scrofa, equus caballus, canis lupus, homo sapiens and 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 gorilla and 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.

Key words: yak, HSP27, gene cloning, bioinformatics analysis, expression

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, 48(20): 4178-4187.

HE Hong-hong, CUI Yan, PAN Yang-yang, FAN Jiang-feng, HU Wei, ZHANG Yi-fu, LIU Peng-gang，LI Qin, YU Si-jiu. Cloning of Bos Grunniens HSP27 Gene and Its Expression in the Female Yak Reproductive Organs[J]. Scientia Agricultura Sinica, 2015, 48(20): 4178-4187.

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