高原鼢鼠肝脏组织细胞周期相关基因的进化和表达

高原鼢鼠Myospalax baileyi是一种世居青藏高原的地下鼠,对严重的低氧环境有很强的适应性。低氧诱导细胞周期G1、G2期阻滞。为了探讨高原鼢鼠适应低氧环境的分子机制,应用生物信息学方法对p53下游细胞周期基因p21、CyclinD1、CyclinE、CDK6、CDK2、14-3-3-σ、Gadd45α、B99和CyclinB1的序列和编码的氨基酸序列进行了进化分析,并以SD大鼠Rattus norvegicus为对照,研究了这些基因在不同海拔(3300 m、2260 m)条件下的表达模式。结果表明:(1)高原鼢鼠细胞周期相关基因的序列与以色列鼹鼠Nannospalax galili同源性最高,达到90%以上;p21、CyclinD1、CyclinE和CyclinB1编码蛋白与以色列鼹鼠存在明显的趋同进化位点;SIFT评估发现,p21和CyclinB1氨基酸序列分别在第27号位点和第105号位点的变异对细胞周期调控功能有显著影响;(2)与低海拔条件相比,在高海拔条件下,高原鼢鼠肝脏组织中与G1期相关的基因p21表达水平显著上升,p21下游基因CyclinD1、CyclinE、CDK6和CDK2表达水平显著下降,而在SD大鼠中没有显著变化;与G2期相关的基因Gadd45α、B99、14-3-3-δ和CyclinB1在高原鼢鼠和SD大鼠中随海拔变化不发生明显变化。在不同海拔条件下,高原鼢鼠肝脏组织中的上述细胞周期相关基因的表达水平均极显著高于SD大鼠(P<0.01)。以上结果提示,高原鼢鼠经过长期的低氧适应,通过上调p21基因的表达抑制下游CyclinD1、CyclinE、CDK6和CDK2基因的表达,导致细胞周期G1期阻滞,从而提供充足的时间进行DNA修复,保证了DNA复制的准确性;同时高原鼢鼠肝脏组织中细胞周期的调控不仅与细胞周期基因的表达水平有关,而且可能与细胞周期因子p21的第27号位点和CyclinB1的第105号位点的变异有关。

Evolution and Expression of Liver Cell Cycle-Related Genes in Myospalax baileyi

The plateau zokor(Myospalax baileyi) is a specialized subterranean rodent living on the Qinghai-Tibet Plateau which has strong ability in adaptation to hypoxic environments. Hypoxia induces arrest of cell cycles G1 and G2. In order to understand the molecular mechanism of the adaptation of M. baileyi to hypoxic environments, the homologies, positive selection sites and the convergent evolution sites of p21, CyclinD1, CDK6, CyclinE, CDK2, 14-3-3-σ, Gadd45α, B99 and CyclinB1 genes were analyzed by MEGA 7.0, PAML 4.8 program and Ancestor program, respectively. In addition, the expression levels of these genes in the liver tissues under different altitudes(3 300 m and 2 260 m) were determined by real-time PCR and compared with those of SD rat(Rattus norvegicus). The results showed that,(1) the nucleotide and amino acid sequences of the cell cycle-related genes in M. baileyi had high homologies(>90%) with Nannospalax galili;p21, CyclinD1, CyclinE and CyclinB1 of M. baileyi and N. galili occurred convergent sites. SIFT test showed that 27 and 105 variation sites might influence the regulation of p21 and CyclinB1, respectively.(2) Compared to low altitude of 2 260 m, the expression level of p21(cell cycle G1-related gene) in M. baileyi liver tissues under high altitude of 3 300 m was significant increased, while its downstream genes CyclinD1, CyclinE, CDK6 and CDK2 were significant decreased. By contrast, the expression levels of these genes were not significantly different in R. norvegicus. Moreover, no significant differences of the expression levels of cell cycle G2-related genes such as Gadd45α, B99, 14-3-3-δ and CyclinB1 were detected in M. baileyi and R. norvegicus under different altitudes. Compared to R. norvegicus, the expression levels of all the tested cell cycle-related genes were significantly higher in M. baileyi. These results suggested that, the up-regulated p21 and down-regulated CyclinD1, CyclinE, CDK6 and CDK2 genes, which can induce the arrest of cell cycle G1 and thus provide sufficient time for DNA repair and ensure the accuracy of DNA replication, contribute to the long-time adaptation of M. baileyi to hypoxic environments. The regulation of cell cycle in the liver of M. baileyi was not only related to the expression levels of cell cycle-related genes, but may also be related to the variation sites at residue 27 and 105 of p21 and CyclinB1, respectively.