• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Changes in Global DNA Methylation Intensity and DNMT1 Transcription During the Aging Process of Scallop Chlamys farreri

    2015-06-01 09:24:20LIANShanshanHEYanLIXueZHAOBosongHOURuiHUXiaoliZHANGLinglingandBAOZhenmin
    Journal of Ocean University of China 2015年4期

    LIAN Shanshan, HE Yan, LI Xue, ZHAO Bosong, HOU Rui, HU Xiaoli, ZHANG Lingling, and BAO Zhenmin

    Key Laboratory of Marine Genetics and Breeding (MGB) of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

    Changes in Global DNA Methylation Intensity and DNMT1 Transcription During the Aging Process of Scallop Chlamys farreri

    LIAN Shanshan, HE Yan, LI Xue, ZHAO Bosong, HOU Rui, HU Xiaoli, ZHANG Lingling, and BAO Zhenmin*

    Key Laboratory of Marine Genetics and Breeding (MGB) of Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao266003,P. R. China

    DNA methylation is an important epigenetic regulatory mechanism that influences genomic stability, gene activation, X-chromosome inactivation and other factors. A change in DNA methylation is usually associated with aging and cellular senescence. DNA methyltransferase 1 (DNMT1) is the most abundant DNA methyltransferase, and it plays an important role in maintaining the established methylation pattern during DNA replication in vertebrates. Although the effect of aging on DNA methylation has been well studied in vertebrates, little research has been conducted in invertebrates, especially in marine bivalves. In this study, we examined global DNA methylation levels in four groups of adult Zhikong scallopChlamys farreriat different ages. The results showed that both the age and tissue type had a strong effect on the DNA methylation. In addition, a significant decrease in DNA methylation with aging (1-4 years) can be detected in mantle, kidney and hepatopancreas. We further measured the change inDNMT1transcript abundance using quantitative reverse transcription PCR (qRT-PCR), which revealed thatDNMT1transcription significantly decreased with aging in mantle and hepatopancreas and strongly correlated with DNA methylation (R= 0.72). Our data provided greater insight into the aging-related decline of DNA methylation, which could aid in gaining a better understanding of the relationship between DNA methylation and the aging process in bivalve mollusks.

    aging; DNA methylation;DNMT1transcription;Chlamys farreri

    1 Introduction

    Epigenetic changes in DNA play an essential role in determining gene transcription. As an important epigenetic modification, DNA methylation is involved in the regulation of development, aging and carcinogenesis in mammals (Liuet al., 2003; Klose and Bird, 2006; Calvanese, 2009). Approximately 70%-80% of cytosines in CpG dinucleotides are methylated in vertebrates (Bird and Taggart, 1980). However, with the aging of animals, DNA demethylation increases (Wilsonet al., 1987; Mazin, 1993, 1994; Kresset al., 2001, 2006; Richardson, 2002; Rodriguezet al., 2008), and the total 5 mC loss could be as high as 91% in cows, 93% in mice, and even 99% in rats for old animals (Mazin, 1993). In humans, DNA methylation associates with chronological age over long time scales and is linked to complex aging-related diseases. The global loss in DNA methylation during aging and in tumor cells was also found in human beings, which could mainlybe the result of the progressive loss of DNMT1 efficacy (Fraga and Esteller, 2007). In a recent study, Hannumet al. (2013) measured more than 450000 CpG markers in hundreds of people from 19 to 101 years of age and found that 70387 (15%) of the markers had significant associations with the aging rate; the genome-wide methylation pattern represents a strong and reproducible biomarker of the biological aging rate. The genome-wide loss of DNA methylation during aging could be relevant to genome instability, the risk of carcinogenesis, aging disorders or other complex age-associated diseases, and the retardation of cell proliferation in tissues of aging organisms (Mazin, 1993; Barres and Zierath, 2011; Lao and Grady, 2011; Tappet al., 2013).

    Maintenance of the DNA methylation pattern in vertebrates is mediated by DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the 5-position of cytosines, principally in the CpG dinucleotides (Law and Jacobsen, 2010). As the most abundant DNMT, DNMT1 plays an important role in maintaining the established methylation pattern during DNA replication (Flores and Amdam, 2011). It has been reported that DNMT1 has acrucial effect on global genomic methylation. For example, the inactivation of DNMT1 causes DNA demethylation, and homozygous null deletions of DNMT1 result in an 80% genomic loss of DNA methylationin mouse (Leiet al., 1996; Takebayashiet al., 2007). In human cells,DNMT1transcription steadily declines throughout the aging process (Lopatinaet al., 2002). These findings suggest that in vertebrates, reduced genome-wide methylation during aging can be attributed to a decreased abundance ofDNMT1.

    Invertebrates display a wide diversity of DNA methylation patterns (Suzukiet al., 2007). For example, with the lack of essential DNMTs, 5-methylcytosine could not be detected in the nematode wormCaenorhabditis elegansat any time during development or aging (Simpsonet al., 1986). In comparison, the fruit flyDrosophila melanogasterlacks most of the classical DNMTs and displays limited cytosine methylation (Hunget al., 1999; Lykoet al., 2000). At the same time, the honey beeApis melliferabears a fully functional set of DNMTs, and DNA methylation is widespread across its genome (Elangoet al., 2009).

    Although bivalve organisms comprise more than 30000 species and constitute the second largest group of mollusks, only limited research on DNA methylation patterns has been conducted in this taxonomic group (Gavery and Roberts, 2010; Riviereet al., 2013). A recent study has revealed changes in the DNA methylation during the early life of oysters and the importance of DNA methylation for proper larval development (Riviereet al., 2013). Such observations might indicate time-dependent patterns of DNA methylation in mollusks and the evolution of 5 mC during the process of aging. In this study, we examined the changes in DNA methylation andDNMT1transcript abundance in the marine bivalveChlamys farreriat 1 to 4 years of age. Detailed information on the changing patterns in different tissues was also collected. This study will aid us in obtaining a better understanding of the role of DNA methylation in the aging process of bivalve mollusks.

    2 Materials and Methods

    2.1 Sample Collection

    Zhikong scallop individuals (1, 2, 3 and 4 years of age) were provided by a shellfish farm in Rongcheng (Shandong Province, China). A total of 24 individuals (6 of each age) were randomly collected and then acclimated at 15℃in filtered seawater for one week. Then, six tissues, including mantle, gill, gonad, kidney, adductor muscle and hepatopancreas, were dissected, immediately frozen in liquid nitrogen and kept at -80℃.

    2.2 Global DNA Methylation Analysis

    Genomic DNA was extracted using a standard phenol-chloroform protocol. RNase A was added to avoid RNA contamination. DNA concentration and purity was determined by the NanoVue Plus UV spectrophotometer (GE Healthcare). DNA methylation was quantified using the Methylamp? Global DNA Methylation Quantification Kit from Epigentek (Brooklyn, NY) following the manufacturer’s instructions. The methylated fraction of the DNA is recognized by 5-methylcytosine antibody and quantified through an ELISA-like reaction. For each sample, methylation analysis was performed in triplicate. The methylation percentage of each sample was calculated according to the slope of the standard curve. The standard curve was generated by plotting the OD values of a dilution series made from a 100% methylated DNA standard that was supplied in the kit.

    2.3 Total RNA Extraction

    The total RNA was extracted using the method described by Huet al. (2006). Genomic DNA contamination in RNA samples was removed by DNase I treatment. The RNA concentration and purity was determined using the NanoVue Plus UV spectrophotometer (GE Healthcare), and the RNA integrity was verified by agarose gel electrophoresis.

    2.4 Analysis ofDNMT1Transcript Abundance

    The transcript abundance ofDNMT1was detected using quantitative reverse transcription PCR (qRT-PCR). Firststrand cDNA was synthesized from 500 ng total RNA using oligo (dT)18and MMLV reverse transcriptase (Promega, Madison, WI, USA). A control reaction without reverse transcriptase was performed to preclude the DNA contamination. The amplification mixture contained 2 μL of diluted cDNA (1:50), 4 μL of primers (2 μmol L-1each) and 10 μL of SYBR Green Real-time PCR Master Mix (TOYOBO, Osaka, Japan). All of the PCR reactions were performed in duplicate and run on a 7500 Real-Time PCR System (Applied Biosystems, CA, USA), using the following program: initial denaturation at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min. Here, β-actin (ACTB), elongation factor 1 beta (EF1β) andribosomal protein L16 (RPL16) were chosen as internal reference genes (IRGs) (Table 1). PCR efficiencies and optimal Ct values were estimated using the online software real-time PCR Miner (Zhao and Fernald, 2005). TheDNMT1transcripts were quantified relative to the three IRGs using the algorithm proposed by Hellemanset al. (2007).

    Table 1 List of primers used for qRT-PCR

    2.5 Statistical Analysis

    All of the data were subjected to one-way ANOVA using SPSS 16.0 (Norusis, 2008). Fisher’s least significant difference (LSD) test was applied when the ANOVA indicated a significant (P< 0.05) difference. The relationship between the data from different assays was determined using the Pearson correlation coefficient (R).

    3 Results and Discussion

    3.1 Age Effect on DNA Methylation

    To roughly estimate the change in the DNA methylation during scallop aging, the entire soft tissue was subjected to global DNA methylation analysis. Based on the results (Fig.1a), the DNA methylation ratio ranged from 0.1% to 0.5% across all of the samples, which was much lower than the values obtained in the vertebrates, such as human, mice and zebrafish (Vuceticet al., 2010; Liuet al., 2011; Fanget al., 2013). One-way ANOVA showed that age was significantly associated with the DNA methylation fraction (P< 0.001). It appeared that with the increase in the age of the scallops, DNA methylation declined, which is similar to the findings in most of the vertebrates and in thein vitromodels (Kresset al., 2001, 2006; Rodriguezet al., 2008; Mazin, 2009; Bollatiet al., 2009). We also noticed that the 1-year-old scallops had a significantly higher DNA methylation fraction than the other three age groups, and a dramatic drop in DNA methylation was detected when the scallops entered their second year. Afterward, the DNA methylation declined gradually, and a significant decrease was found only between the 2- and 4-year-old individuals (P< 0.01). Although the above results were based on all of the soft tissue, they indicated an age effect on the DNA methylation in scallop and provided some clues on the age-dependent changes of DNA methylation in marine bivalves.

    3.2 Effect of the Tissue Type on the DNA Methylation

    DNA methylation could be different among the tissues, which leads to an inaccurate estimation of the age effect based on the results from all of the soft tissue. Therefore, we further tested the DNA methylation fraction in six tissues (mantle, gill, gonad, kidney, adductor muscle and hepatopancreas) of 2-year-old scallop. According to oneway ANOVA, the DNA methylation fractions were significantly different among the tissue types (P< 0.001). As shown in Fig.1b, the kidney and adductor muscle had the highest (approximately 0.3%) DNA methylation ratio, followed by gonad and hepatopancreas (approximately 0.2%), and the lowest DNA methylation ratio was found in mantle and gill (approximately 0.1%). The observed tissue difference in the DNA methylation is similar to the finding in mammals (Romanov and Vanyushin, 1981; Gama-Sosaet al., 1983; Maegawaet al., 2010), which suggests that the tissue type should be considered when examining the effect of age on DNA methylation.

    Fig.1 Effects of age (a) and tissue type (b) on DNA methylation. One-way ANOVA followed by Fisher’s LSD test was used for the comparisons. The vertical bars represent the mean ± S.E. (n= 6). The values marked with different letters differed significantly from one another (P< 0.05).

    3.3 Tissue-Specific Effect of Age on DNA Methylation

    Consistent with the observation on the entire soft tissue, a decline in the DNA methylation fraction with age was also observed across all of the six tissues (Fig.2a). However, a significant difference among the ages was detected only in mantle, kidney and hepatopancreas. Based on the previous studies, all of these three tissues participate in excreting and depurating metals and other toxic materials in marine bivalves (Carmichael and Fowler, 1981; Cembellaet al., 1994; Arévaloet al., 1998; Blancoet al., 2002; Suzukiet al., 2005). Considering the depuration function, the damage from toxic residue and the burden of oxidative metabolism in these three tissues, their aging rate could be higher than that of the others, which would result in a significant decrease in the methylation ratio.

    In comparison with the change in the DNA methylation fraction among the different age-related groups in the mantle (<0.1%), a higher drop (up to 0.3%) was found in the hepatopancreas and kidney. The higher drop in DNA methylation rate in the two tissues was possibly caused by the relatively low toxin effect (Cembellaet al., 1994; Bauderet al., 2001; Blancoet al., 2002) and faster cell self-renewal rate in the mantle compared with kidney and hepatopancreas, which could help to slow down the decline in the methylation rate. In addition, research showed that the promoter methylation of some stress-responding genes, which are involved in cellular responses to environmental stresses, are mediated by DNMT1 and DNMT3B together (Yinget al., 2005). DNMT3 can also contribute to the methylation pattern change in the mantle and should be verified in the future.

    3.4 Tissue-Specific Effect of Age onDNMT1Transcription

    Inhibition ofDNMT1could lead to reduced methylation levels in various animals, such as frog, mouse, and human (Stancheva and Meehan, 2000; Sadoet al., 2000; Rheeet al., 2002; Gaudetet al., 2003), which would indicate that DNMT1 plays a critical role in maintaining the global DNA methylation level. In this study,DNMT1transcription also declined with age in all six tissues (Fig.2b), which is similar to the findings in various mammals, including mouse and human (Vertinoet al., 1994; Hamataniet al., 2004; Kimet al., 2009; Liuet al., 2009). The 1-year-old scallops contained moreDNMT1transcripts than the other age groups across all of the tissues, which is consistent with the trend in the DNA methylation levels. A significant difference among the ages was detected in the mantle and hepatopancreas, with the 4-year-old scallops containing remarkably lower abundance ofDNMT1mRNA than the 1-year-old scallops. There was a similar reduction tendency in the global DNA methylation and inDNMT1transcription of gill, gonad and muscle of 4-year-old scallops, although the reduction was not significant. In addition, based on the data from different tissues of all four groups, the DNA methylation ratio was significantly and positively (R= 0.72;P< 0.001) correlated with the relative abundance ofDNMT1transcripts (Fig.3), which is similar to related results (R= 0.746) in human cells (Leiet al., 2009). Thus, DNMT1 is likely to participate in maintaining DNA methylation in scallops in a similar way as in other organisms. In addition, the agedependent decrease in DNA methylation can be attributed to the down-regulation ofDNMT1during the aging process of scallop.

    Fig.2 Changes in the DNA methylation ratio (a) and relative abundance ofDNMT1transcripts in six tissues of scallop at 1-4 years of age (b). One-way ANOVA followed by Fisher’s LSD test was used for the comparisons. The vertical bars represent the mean ± S.E. (n= 6). The values marked with different letters differed significantly from one another (P< 0.05).

    Fig.3 Correlation between the relative percentages (%) of DNA methylation andDNMT1mRNA abundance (n= 24).

    In summary, we found that both the age and tissue type had strong effects on DNA methylation inChlamys farreri. A significant decrease in DNA methylation with age was observed in the mantle, kidney and hepatopancreas. Agreeing with the results of DNA methylation,DNMT1transcription in mantle and hepatopancreas also declined with age. In addition, theDNMT1transcript abundance was significantly correlated with the DNA methylation ratio, which suggests the important role of DNMT1 in maintaining DNA methylation in scallop. This study can contribute to a better understanding of aging-related DNA methylation changes in bivalve mollusks.

    Acknowledgements

    This study was supported by the National Natural Science Foundation of China (31130054), the National HighTechnology Research and Development Program of China (2012AA10A401), and Doctoral Fund of Ministry of Education of China (20120132130002).

    Arévalo, F., Bermúdez, M., and Salgado, C., 1998. ASP toxicity in scallops: Individual variability and tissue distribution. In:Harmful Algae. Reguera, B.,et al., eds., Xunta de Galicia and the IOC of UNESCO, Paris,499-502.

    Barres, R., and Zierath, J. R., 2011. DNA methylation in metabolic disorders.The American Journal of Clinical Nutrition, 93: 897S-900S.

    Bauder, A. G., Cembella, A. D., Bricelj, V. M., and Quilliam, M. A., 2001. Uptake and fate of diarrhetic shellfish poisoning toxins from the dinoflagellateProrocentrum limain the bay scallopArgopecten irradians.Marine Ecology Prog Researchs Series, 213: 39-52.

    Berletch, J. B., Andrews, L. G., and Tollefsbol, T. O., 2007. A method to detect DNA methyltransferase I gene transcriptionin vitroin aging systems.Biological Aging, 372: 73-80.

    Bird, A. P., and Taggart, M. H., 1980. Variable patterns of total DNA and rDNA methylation in animals.Nucleic Acids Research, 8: 1485-1497.

    Blanco, J., Acosta, C., Bermúdez, M., and Salqado, C., 2002. Depuration and anatomical distribution of the amnesic shellfish poisoning (ASP) toxin domoic acid in the king scallopPectenmaximus.Aquat Toxicol, 60: 111-121.

    Bollati, V., Schwartz, J., Wright, R., Litonjua, A., Tarantini, L., Suh, H., Sparrow, D., Vokonas, P., and Baccarelli, A., 2009. Decline in genomic DNA methylation through aging in a cohort of elderly subjects.Mechanisms of Aging and Development, 130: 234-239.

    Calvanese, V., Lara, E., Kahn, A., and Fraga, M. F., 2009. The role of epigenetics in aging and age-related diseases.Aging Research Reviews, 8: 268-276.

    Carmichael, N., and Fowler, B., 1981. Cadmium accumulation and toxicity in the kidney of the bay scallopArgopecten irradians.Marine Biology, 65: 35-43.

    Cembella, A. D., Shumway, S. E., and Larocque, R., 1994. Sequestering and putative biotransformation of paralytic shellfish toxins by the sea scallopPlacopecten magellanicus:Seasonal and spatial scales in natural populations.Journal of Experimental Marine Biology and Ecology, 180: 1-22.

    Elango, N., Hunt, B. G., Goodisman, M. A., and Yi, S. V., 2009. DNA methylation is widespread and associated with differential gene expression in castes of the honeybee,Apis mellifera.Proceedings of the National Academy of Sciences, 106: 11206-11211.

    Fang, X., Thornton, C., Scheffler, B. E., and Willett, K. L., 2013. Benzo [a] pyrene decreases global and gene specific DNA methylation during zebrafish development.Environment Toxicol, 36: 40-50.

    Flores, K. B., and Amdam, G. V., 2011. Deciphering a methylome: What can we read into patterns of DNA methylation?The Journal of Experimental Biology, 214: 3155-3163.

    Fraga, M. F., and Esteller, M., 2007. Epigenetics and aging: The targets and the marks.Trends in Genetics, 23: 413-418.

    Gama-Sosa, M. A., Midgett, R. M., Slagel, V. A., Githens, S., Kuo, K. C., Gehrke, C. W., and Ehrlich, M., 1983. Tissuespecific differences in DNA methylation in various mammals.Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 740: 212-219.

    Gaudet, F., Hodgson, J., Eden, A., Jackson-Grusby, L., Dausman, J., Gray, J. W., Leonhardt, H., and Jaenisch, R., 2003. Induction of tumors in mice by genomic hypomethylation.Science, 300: 489-492.

    Gavery, M. R., and Roberts, S. B., 2010. DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas).BMC Genomics, 11: 483-491.

    Hamatani, T., Falco, G., Carter, M. G., Akutsu, H., Stagg, C. A., Sharov, A. A., Dudekula, D. B., Vanburen, V., and Ko, M. S., 2004. Age-associated alteration of gene expression patterns in mouse oocytes.Human Molecular Genetics, 13: 2263-2278.

    Hannum, G., Guinney, J., Zhao, L., Zhang, L., Hughes, G., Sadda, S., Klotzle, B., Bibikova, M., Fan, J. B., Gao, Y., Deconde, R., Chen, M., Rajapakse, I., Friend, S., Ideker, T., and Zhang, K., 2013. Genome-wide methylation profiles reveal quantitative views of human aging rates.Molecular Cell, 49: 359-367.

    Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., and Vandesompele, J., 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.Genome Biology, 8: R19.

    Hu, X., Bao, Z., Hu, J., Shao, M., Zhang, L., Bi, K., Zhan, A., and Huang, X., 2006. Cloning and characterization of tryptophan 2, 3-dioxygenase gene of Zhikong scallopChlamys farreri(Jones and PResearchton 1904).Aquaculture Research, 37: 1187-1194.

    Hung, M.-S., Karthikeyan, N., Huang, B., Koo, H. C., Kiger, J., and Shen, C.-K., 1999. Drosophila proteins related to vertebrate DNA (5-cytosine) methyltransferases.Proceedings of the National Academy of Sciences, 96: 11940-11945.

    Kim, K.-C., Friso, S., and Choi, S.-W., 2009. DNA methylation, an epigenetic mechanism connecting folate to healthy embryonic development and aging.The Journal of Nutritional Biochemistry, 20: 917-926.

    Klose, R. J., and Bird, A. P., 2006. Genomic DNA methylation:the mark and its mediators.Trends Biochemistry Science, 31:89-97.

    Kress, C., Thomassin, H., and Grange, T., 2001. Local DNA demethylation in vertebrates: How could it be performed and targeted?FEBS Letter, 494: 135-140.

    Kress, C., Thomassin, H., and Grange, T., 2006. Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks.Proceedings of the National Academy of Sciences, 103: 11112-11117.

    Lao, V. V., and Grady, W. M., 2011. Epigenetics and colorectal cancer.Nature Reviews Gastroenterology and Hepatology, 8:686-700.

    Law, J. A., and Jacobsen, S. E., 2010. Establishing, maintaining and modifying DNA methylation patterns in plants and animals.Nature Reviews Genetics, 11: 204-220.

    Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A., Jaenisch, R., and Li, E., 1996.De novoDNA cytosine methyltransferase activities in mouse embryonic stem cells.Development, 122: 3195-3205.

    Lei, W., Luo, Y., Yan, K., Zhao, S., Li, Y., Qiu, X., Zhou, Y., Long, H., Zhao, M., Liang, Y., Su, Y., and Lu, Q., 2009. Abnormal DNA methylation in CD4+T cells from patients with systemic lupus erythematosus, systemic sclerosis, and dermatomyositis.Scandinavian Journal of Rheumatology, 38:369-374.

    Liu, C., Ou, T., Wu, C., Li, R., Lin, Y., Lin, C., Tsai, W., Liu, H., and Yen, J., 2011. Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus.Lupus, 20: 131-136.

    Liu, L., Wylie, R. C., Andrews, L. G., and Tollefsbol, T. O., 2003.Aging, cancer and nutrition: The DNA methylation connection.Mechnismof Aging and Development, 124: 989-998.

    Liu, Y., Chen, Y., and Richardson, B., 2009. Decreased DNA methyltransferase levels contribute to abnormal gene expression in ‘senescent’ CD4+CD28-T cells.Clinical Immunology, 132: 257-265.

    Lopatina, N., Haskell, J. F., Andrews, L. G., Poole, J. C., Saldanha, S., and Tollefsbol, T., 2002. Differential maintenance andde novomethylating activity by three DNA methyltransferases in aging and immortalized fibroblasts.Journal of Cellular Biochemistry, 84: 324-334.

    Lyko, F., Ramsahoye, B. H., and Jaenisch, R., 2000. Development: DNA methylation inDrosophila melanogaster.Nature, 408: 538-540.

    Maegawa, S., Hinkal, G., Kim, H. S., Shen, L., Zhang, L., Zhang, J., Zhang, N., Liang, S., Donehower, L. A., and Issa, J. J., 2010. Widespread and tissue specific age-related DNA methylation changes in mice.Genome Research, 20: 332-340.

    Mazin, A. L., 2009. Suicidal function of DNA methylation in age-related genome disintegration.Aging Research Reviews, 8:314-327.

    Mazin, A. L., 1993. Genome loses all 5-methylcytosine a life span. How is this connected with accumulation of mutations during aging?Molecular Biology, 27: 160-173.

    Mazin, A. L., 1994. Enzymatic DNA methylation as an aging mechanism.Molecular Biology, 28: 21-51.

    Norusis, M., 2008. SPSS 16.0 guide to data analysis. Prentice

    Hall Press.

    Rhee, I., Bachman, K. E., Park, B. H., Jair, K., Yen, R. C., Schuebel, K. E., Cui, H., Feinberg, A. P., Lengauer, C., Kinzler, K. W., Baylin, S. B., and Vogelstein, B., 2002. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells.Nature, 416: 552-556.

    Richardson, B. C., 2002. Role of DNA methylation in the regulation of cell function: Autoimmunity, aging and cancer.The Journal of Nutrition, 132: 2401S-2405S.

    Rodriguez, J., Vives, L., Jordà, M., Morales, C., Mu?oz, M., Vendrell, E., and Peinado, M. A., 2008. Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells.Nucleic Acids Research, 36: 770-784.

    Romanov, G. A., and Vanyushin, B. F., 1981. Methylation of reiterated sequences in mammalian DNAs effects of the tissue type, age, malignancy and hormonal induction.Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 653: 204-218.

    Riviere, G., Wu, G., Fellous, A., Goux, D., Sourdaine, P., and Favrel, P., 2013. DNA methylation is crucial for the early development in the oysterC. gigas.Marine Biotechnology, 15:739-753.

    Sado, T., Fenner, M. H., Tan, S.-S., Tam, P., Shioda, T., and Li, E., 2000. X inactivation in the mouse embryo deficient forDnmt1distinct effect of hypomethylation on imprinted and random X inactivation.Development Biology, 225: 294-303.

    Simpson, V. J., Johnson, T. E., and Hammen, R. F., 1986. Caenorhabditis elegans DNA does not contain 5-methylcytosine at any time during development or aging.Nucleic Acids Research, 14: 6711-6719.

    Stancheva, I., and Meehan, R. R., 2000. Transient depletion of xDnmt1 leads to premature gene activation inXenopusembryos.Genes Development, 14: 313-327.

    Suzuki, M. M., and Bird, A., 2008. DNA methylation landscapes:Provocative insights from epigenomics.Nature Reviews Genetics, 9: 465-476.

    Suzuki, M. M., Kerr, A. R., De Sousa, D., and Bird, A., 2007. CpG methylation is targeted to transcription units in an invertebrate genome.Genome Research, 17: 625-631.

    Takebayashi, S.-I., Tamura, T., Matsuoka, C., and Okano, M., 2007. Major and essential role for the DNA methylation mark in mouse embryogenesis and stable association of DNMT1 with newly replicated regions.Molecular and Cellular Biology, 27: 8243-8258.

    Tapp, H. S., Commane, D. M., Bradburn, D. M., Arasaradnam, R., Mathers, J. C., Johnson, I. T., and Belshaw, N. J., 2013. Nutritional factors and gender influence age-related DNA methylation in the human rectal mucosa.Aging Cell, 12: 148-155.

    Vertino, P. M., Issa, J.-P., Pereira-Smith, O. M., and Baylin, S. B., 1994. Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts.Cell Growth and Differentiation: The Molecular Biology Journal of the American Association for Cancer Research, 5: 1395-1402.

    Vucetic, Z., Kimmel, J., Totoki, K., Hollenbeck, E., and Reyes, T. M., 2010. Maternal high-fat diet alters methylation and gene expression of dopamine and opioid-related genes.Endocrinology, 151: 4756-4764.

    Wilson, V. L., Smith, R., Ma, S., and Cutler, R. G., 1987. Genomic 5-methyldeoxycytidine decreases with age.Journal of Biological Chemistry, 262: 9948-9951.

    Ying, J., Srivastava, G., Hsieh, W. S., Gao, Z., Murray, P., Liao, S., Ambinder, R., and Tao, Q., 2005. The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.Clinical Cancer Research, 11:6442-6449.

    Zhao, S., and Fernald, R. D., 2005. Comprehensive algorithm for quantitative real-time polymerase chain reaction.Journal of Computational Biology, 12: 1047-1064.

    (Edited by Qiu Yantao)

    (Received October 10, 2013; revised January 19, 2014; accepted March 23, 2015)

    ? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

    * Corresponding author. Tel: 0086-532-82031960 E-mail: zmbao@ouc.edu.cn

    天天躁夜夜躁狠狠躁躁| 成人漫画全彩无遮挡| 新久久久久国产一级毛片| 一区二区日韩欧美中文字幕 | 91在线精品国自产拍蜜月| 日韩 亚洲 欧美在线| 最新的欧美精品一区二区| 国产极品粉嫩免费观看在线| 乱码一卡2卡4卡精品| 一级毛片电影观看| 高清欧美精品videossex| 边亲边吃奶的免费视频| 国产成人91sexporn| 亚洲少妇的诱惑av| 宅男免费午夜| 国产国语露脸激情在线看| 纯流量卡能插随身wifi吗| 国产色爽女视频免费观看| 一级片'在线观看视频| 久久狼人影院| videosex国产| 黄片无遮挡物在线观看| 亚洲精品国产av蜜桃| 久热久热在线精品观看| 亚洲色图 男人天堂 中文字幕 | 夫妻性生交免费视频一级片| 街头女战士在线观看网站| 建设人人有责人人尽责人人享有的| 国产极品天堂在线| 色婷婷av一区二区三区视频| 国产不卡av网站在线观看| 岛国毛片在线播放| 久久精品国产综合久久久 | 熟女电影av网| av女优亚洲男人天堂| 精品酒店卫生间| 国产不卡av网站在线观看| 天堂8中文在线网| 一本大道久久a久久精品| 精品国产露脸久久av麻豆| 一本久久精品| 熟女电影av网| 我要看黄色一级片免费的| 又黄又粗又硬又大视频| 永久网站在线| 热99国产精品久久久久久7| 亚洲av欧美aⅴ国产| 汤姆久久久久久久影院中文字幕| 久热久热在线精品观看| a级片在线免费高清观看视频| 免费大片18禁| a 毛片基地| 亚洲激情五月婷婷啪啪| 精品99又大又爽又粗少妇毛片| 最新中文字幕久久久久| 搡老乐熟女国产| 免费黄频网站在线观看国产| 久久人人爽av亚洲精品天堂| 色94色欧美一区二区| 国产精品成人在线| 精品一区二区三卡| 熟女电影av网| 纯流量卡能插随身wifi吗| 国产又色又爽无遮挡免| 蜜臀久久99精品久久宅男| 亚洲欧洲精品一区二区精品久久久 | 伦理电影大哥的女人| 成人黄色视频免费在线看| av天堂久久9| 另类精品久久| 国产午夜精品一二区理论片| av播播在线观看一区| 男女国产视频网站| 午夜激情av网站| 考比视频在线观看| 国产精品无大码| 久久青草综合色| 午夜精品国产一区二区电影| 插逼视频在线观看| 精品久久国产蜜桃| 国产精品一二三区在线看| 在线观看一区二区三区激情| 国产精品久久久久久久电影| 国产精品三级大全| 侵犯人妻中文字幕一二三四区| 青春草视频在线免费观看| 国产免费福利视频在线观看| 久久影院123| 免费看av在线观看网站| videosex国产| 九色成人免费人妻av| 九色亚洲精品在线播放| 国产精品不卡视频一区二区| 搡女人真爽免费视频火全软件| 国产成人免费无遮挡视频| 丰满饥渴人妻一区二区三| 亚洲精品一二三| 久久99蜜桃精品久久| 精品第一国产精品| 伊人久久国产一区二区| 免费观看在线日韩| 精品一区在线观看国产| 晚上一个人看的免费电影| 岛国毛片在线播放| 女人精品久久久久毛片| 嫩草影院入口| 成年动漫av网址| 国产在线视频一区二区| 肉色欧美久久久久久久蜜桃| 大话2 男鬼变身卡| 国产精品一区二区在线观看99| 九九在线视频观看精品| 欧美精品人与动牲交sv欧美| 国产麻豆69| 夫妻午夜视频| 热re99久久国产66热| 黄色视频在线播放观看不卡| 在线亚洲精品国产二区图片欧美| 日韩一区二区视频免费看| 人人妻人人澡人人爽人人夜夜| 亚洲一区二区三区欧美精品| av片东京热男人的天堂| 久久久久人妻精品一区果冻| 久久精品国产综合久久久 | 日本午夜av视频| 精品一区在线观看国产| 免费黄色在线免费观看| 少妇精品久久久久久久| 91精品三级在线观看| 成年美女黄网站色视频大全免费| 一级片免费观看大全| 免费在线观看黄色视频的| 夫妻午夜视频| 最后的刺客免费高清国语| 国产精品国产三级国产专区5o| 又黄又爽又刺激的免费视频.| 黄色配什么色好看| 男女边摸边吃奶| 久久这里只有精品19| 久久ye,这里只有精品| 日韩免费高清中文字幕av| 欧美亚洲日本最大视频资源| av视频免费观看在线观看| 久久久久久久久久人人人人人人| 制服诱惑二区| 丰满乱子伦码专区| 国产片特级美女逼逼视频| 两个人免费观看高清视频| av在线观看视频网站免费| 中文字幕av电影在线播放| 国产乱人偷精品视频| 成年美女黄网站色视频大全免费| 视频中文字幕在线观看| 日本欧美视频一区| 欧美精品一区二区免费开放| 日韩免费高清中文字幕av| 精品人妻在线不人妻| 91国产中文字幕| 丝袜美足系列| av视频免费观看在线观看| 男人操女人黄网站| 99久久精品国产国产毛片| 国产又色又爽无遮挡免| 色婷婷av一区二区三区视频| 建设人人有责人人尽责人人享有的| 女人久久www免费人成看片| 中国三级夫妇交换| 亚洲人与动物交配视频| 欧美日韩综合久久久久久| 久久久国产一区二区| 亚洲五月色婷婷综合| 免费看光身美女| 国产一区二区在线观看av| 9热在线视频观看99| 人妻一区二区av| 成人国产麻豆网| av不卡在线播放| 国产精品国产av在线观看| 五月伊人婷婷丁香| 国产黄色视频一区二区在线观看| 亚洲精品美女久久av网站| 一本—道久久a久久精品蜜桃钙片| 丝袜脚勾引网站| 久久久久久久久久成人| av免费在线看不卡| 国产精品一二三区在线看| 老司机影院毛片| 97在线视频观看| 亚洲国产欧美日韩在线播放| 永久免费av网站大全| 久久综合国产亚洲精品| 国产成人精品一,二区| 亚洲精品,欧美精品| av卡一久久| 一区二区日韩欧美中文字幕 | 久久久久国产精品人妻一区二区| 国产男女超爽视频在线观看| 久久热在线av| 国产精品麻豆人妻色哟哟久久| 91午夜精品亚洲一区二区三区| 两个人看的免费小视频| 巨乳人妻的诱惑在线观看| 只有这里有精品99| 亚洲av免费高清在线观看| 伊人久久国产一区二区| 久久99精品国语久久久| 最近中文字幕高清免费大全6| 亚洲成人一二三区av| 久久国产精品大桥未久av| 免费av中文字幕在线| 三级国产精品片| 男人添女人高潮全过程视频| av免费观看日本| 国产成人免费无遮挡视频| 亚洲成人一二三区av| 久久人妻熟女aⅴ| 丰满迷人的少妇在线观看| 丁香六月天网| 婷婷色综合大香蕉| 侵犯人妻中文字幕一二三四区| 国产成人精品在线电影| 天美传媒精品一区二区| 国产成人精品一,二区| 亚洲熟女精品中文字幕| 免费黄色在线免费观看| 久久99一区二区三区| 久久久国产一区二区| 国产精品熟女久久久久浪| 老熟女久久久| 中文字幕另类日韩欧美亚洲嫩草| 国产精品女同一区二区软件| 久久这里有精品视频免费| av福利片在线| 丰满饥渴人妻一区二区三| 中文字幕人妻丝袜制服| 久久精品久久精品一区二区三区| 日韩欧美一区视频在线观看| 亚洲av男天堂| 男人舔女人的私密视频| 日产精品乱码卡一卡2卡三| 国产在视频线精品| 精品酒店卫生间| 免费av中文字幕在线| 精品亚洲乱码少妇综合久久| 欧美日韩精品成人综合77777| 国产精品嫩草影院av在线观看| 国产探花极品一区二区| 欧美另类一区| 国产精品久久久av美女十八| 一区二区av电影网| 亚洲高清免费不卡视频| 国产在线一区二区三区精| 免费观看av网站的网址| 一二三四中文在线观看免费高清| 伦精品一区二区三区| 欧美精品国产亚洲| 91成人精品电影| 亚洲熟女精品中文字幕| a级毛色黄片| 国产免费福利视频在线观看| 一级毛片我不卡| 久久av网站| 国产欧美另类精品又又久久亚洲欧美| 亚洲美女搞黄在线观看| 国产一区二区三区av在线| 2021少妇久久久久久久久久久| 九草在线视频观看| 天美传媒精品一区二区| 免费在线观看完整版高清| 自拍欧美九色日韩亚洲蝌蚪91| 晚上一个人看的免费电影| a级毛片在线看网站| 性色avwww在线观看| 日韩,欧美,国产一区二区三区| 九色成人免费人妻av| 极品少妇高潮喷水抽搐| 国产欧美日韩综合在线一区二区| 高清欧美精品videossex| 九草在线视频观看| 最近中文字幕高清免费大全6| 午夜视频国产福利| 成人国语在线视频| 久久精品国产亚洲av天美| 超色免费av| 久久久久网色| 国产精品久久久久久久电影| 一二三四中文在线观看免费高清| 国产在线视频一区二区| 亚洲国产精品一区三区| 亚洲精品一二三| 啦啦啦在线观看免费高清www| 久久精品国产a三级三级三级| 如日韩欧美国产精品一区二区三区| 黄色视频在线播放观看不卡| 欧美精品一区二区大全| 久久99热这里只频精品6学生| 一区二区日韩欧美中文字幕 | 女人被躁到高潮嗷嗷叫费观| 如日韩欧美国产精品一区二区三区| 国产精品99久久99久久久不卡 | 亚洲成av片中文字幕在线观看 | 人人妻人人添人人爽欧美一区卜| 亚洲国产精品999| 一级a做视频免费观看| 免费黄频网站在线观看国产| 国产成人免费观看mmmm| 晚上一个人看的免费电影| 国产精品国产三级专区第一集| av女优亚洲男人天堂| 香蕉丝袜av| 亚洲成人手机| 女人久久www免费人成看片| 黄片播放在线免费| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 久久久久视频综合| 国产色爽女视频免费观看| 一区二区av电影网| 丝瓜视频免费看黄片| 五月伊人婷婷丁香| 国产精品蜜桃在线观看| 午夜av观看不卡| 韩国av在线不卡| 寂寞人妻少妇视频99o| 大片免费播放器 马上看| 久久久久国产网址| 亚洲色图综合在线观看| 免费大片黄手机在线观看| 久久影院123| 久热这里只有精品99| 一本色道久久久久久精品综合| 久久精品国产a三级三级三级| av福利片在线| 久久久久久人妻| 如日韩欧美国产精品一区二区三区| 亚洲精品第二区| 一级片免费观看大全| 在线 av 中文字幕| 亚洲av欧美aⅴ国产| 老司机亚洲免费影院| 欧美日韩一区二区视频在线观看视频在线| 男女国产视频网站| 亚洲欧美成人精品一区二区| 精品久久国产蜜桃| 涩涩av久久男人的天堂| 精品久久久久久电影网| 亚洲图色成人| 精品久久久久久电影网| 乱码一卡2卡4卡精品| 亚洲伊人久久精品综合| 久久精品国产a三级三级三级| 国产欧美日韩综合在线一区二区| 九九爱精品视频在线观看| 99热国产这里只有精品6| 超碰97精品在线观看| 91国产中文字幕| 人妻系列 视频| 久久久亚洲精品成人影院| 丝袜脚勾引网站| 交换朋友夫妻互换小说| 男人爽女人下面视频在线观看| 香蕉丝袜av| 一本久久精品| 国产成人精品久久久久久| 一本大道久久a久久精品| 插逼视频在线观看| 国产精品熟女久久久久浪| 久久人妻熟女aⅴ| 高清欧美精品videossex| 在线天堂最新版资源| 一级毛片我不卡| 99视频精品全部免费 在线| 午夜老司机福利剧场| 国产免费现黄频在线看| 性色avwww在线观看| 人体艺术视频欧美日本| 日本欧美国产在线视频| 久久久久久久亚洲中文字幕| 日韩熟女老妇一区二区性免费视频| 视频区图区小说| 国产片特级美女逼逼视频| 女性生殖器流出的白浆| 91久久精品国产一区二区三区| 十八禁高潮呻吟视频| 午夜日本视频在线| 中文字幕精品免费在线观看视频 | 久久久久久久精品精品| 亚洲国产毛片av蜜桃av| 看非洲黑人一级黄片| 最近的中文字幕免费完整| 老司机影院毛片| 日韩成人av中文字幕在线观看| 国产不卡av网站在线观看| 我的女老师完整版在线观看| 亚洲国产成人一精品久久久| 国产黄频视频在线观看| 大香蕉97超碰在线| 亚洲国产日韩一区二区| 丰满迷人的少妇在线观看| 一区二区日韩欧美中文字幕 | 人妻人人澡人人爽人人| 国产毛片在线视频| 女人精品久久久久毛片| 国产亚洲一区二区精品| 成人亚洲欧美一区二区av| 大码成人一级视频| 超碰97精品在线观看| 欧美日韩综合久久久久久| 欧美性感艳星| 一级爰片在线观看| 美女主播在线视频| 嫩草影院入口| 亚洲国产精品999| 精品一区二区三区四区五区乱码 | av不卡在线播放| 国产在线视频一区二区| 精品久久国产蜜桃| 在线观看人妻少妇| 99热国产这里只有精品6| 久久精品夜色国产| 国产精品久久久久久久电影| 午夜福利视频精品| 中文字幕人妻丝袜制服| 97人妻天天添夜夜摸| 精品人妻偷拍中文字幕| 一边摸一边做爽爽视频免费| 亚洲色图 男人天堂 中文字幕 | 久久99精品国语久久久| 亚洲国产成人一精品久久久| 十分钟在线观看高清视频www| tube8黄色片| 国产精品免费大片| 人人妻人人爽人人添夜夜欢视频| 少妇猛男粗大的猛烈进出视频| 久久久国产精品麻豆| 亚洲综合精品二区| 国产精品 国内视频| 青春草国产在线视频| 日本午夜av视频| 国产深夜福利视频在线观看| 在线观看三级黄色| a级片在线免费高清观看视频| 日本爱情动作片www.在线观看| 看十八女毛片水多多多| 99久国产av精品国产电影| 日韩人妻精品一区2区三区| 国产精品一二三区在线看| av福利片在线| 亚洲国产毛片av蜜桃av| 国产亚洲最大av| 亚洲在久久综合| 99久久综合免费| 制服丝袜香蕉在线| 日韩制服丝袜自拍偷拍| 国产成人免费观看mmmm| h视频一区二区三区| 国产视频首页在线观看| 国产一区二区激情短视频 | 亚洲美女搞黄在线观看| av又黄又爽大尺度在线免费看| 亚洲第一av免费看| 青春草亚洲视频在线观看| 欧美人与善性xxx| 嫩草影院入口| 亚洲精品aⅴ在线观看| 国产乱来视频区| 成人黄色视频免费在线看| 亚洲精品一区蜜桃| 午夜久久久在线观看| 亚洲欧美成人精品一区二区| 男男h啪啪无遮挡| 中文字幕人妻丝袜制服| 99热6这里只有精品| 中文字幕人妻熟女乱码| 男女午夜视频在线观看 | 日韩,欧美,国产一区二区三区| 99九九在线精品视频| 纵有疾风起免费观看全集完整版| 黄网站色视频无遮挡免费观看| 国产极品粉嫩免费观看在线| 国产免费又黄又爽又色| 国产综合精华液| 夫妻性生交免费视频一级片| 午夜激情av网站| 波多野结衣一区麻豆| 男女午夜视频在线观看 | 黄色一级大片看看| 免费日韩欧美在线观看| 黄片无遮挡物在线观看| 精品亚洲成a人片在线观看| 欧美国产精品一级二级三级| 九草在线视频观看| 老司机亚洲免费影院| 免费人妻精品一区二区三区视频| 婷婷色av中文字幕| 18禁裸乳无遮挡动漫免费视频| 热99久久久久精品小说推荐| 男男h啪啪无遮挡| 国产成人精品无人区| 另类精品久久| 中国三级夫妇交换| 国产伦理片在线播放av一区| 丰满少妇做爰视频| 日韩av不卡免费在线播放| 女人被躁到高潮嗷嗷叫费观| 在线观看三级黄色| 91精品三级在线观看| 午夜福利影视在线免费观看| 最新的欧美精品一区二区| 亚洲美女搞黄在线观看| 夫妻午夜视频| 女性生殖器流出的白浆| 欧美日韩av久久| 午夜av观看不卡| 国产色爽女视频免费观看| 国产精品一区二区在线不卡| 久久久久久久久久人人人人人人| 久久久久国产精品人妻一区二区| 日韩不卡一区二区三区视频在线| 久久这里有精品视频免费| 一区二区三区四区激情视频| 满18在线观看网站| 亚洲国产欧美日韩在线播放| 大码成人一级视频| 亚洲精品美女久久久久99蜜臀 | 丝袜人妻中文字幕| 精品亚洲乱码少妇综合久久| 国产亚洲最大av| 亚洲av电影在线进入| a 毛片基地| 欧美bdsm另类| 国产色婷婷99| 日韩成人av中文字幕在线观看| 狠狠婷婷综合久久久久久88av| 秋霞在线观看毛片| 亚洲av男天堂| 中国美白少妇内射xxxbb| 久久精品国产综合久久久 | 黑人巨大精品欧美一区二区蜜桃 | 深夜精品福利| 欧美 亚洲 国产 日韩一| 91aial.com中文字幕在线观看| av在线老鸭窝| 人人妻人人添人人爽欧美一区卜| 制服丝袜香蕉在线| 国产精品蜜桃在线观看| 免费久久久久久久精品成人欧美视频 | 婷婷色麻豆天堂久久| 内地一区二区视频在线| 丰满乱子伦码专区| 久久久久久久久久人人人人人人| 人妻系列 视频| 99久久中文字幕三级久久日本| 国产伦理片在线播放av一区| 我要看黄色一级片免费的| 日本av免费视频播放| 在线观看一区二区三区激情| av国产精品久久久久影院| 久久人妻熟女aⅴ| 欧美亚洲日本最大视频资源| 2018国产大陆天天弄谢| 亚洲国产日韩一区二区| 国产精品一区www在线观看| 欧美bdsm另类| 少妇的逼水好多| 大片免费播放器 马上看| 18在线观看网站| 乱人伦中国视频| 精品一区在线观看国产| 国产在线一区二区三区精| 亚洲av男天堂| 久久狼人影院| 久久综合国产亚洲精品| 精品国产一区二区三区久久久樱花| 黄网站色视频无遮挡免费观看| 一本色道久久久久久精品综合| 久久精品国产亚洲av涩爱| 久久国内精品自在自线图片| 观看av在线不卡| 美女中出高潮动态图| 午夜福利视频精品| 2022亚洲国产成人精品| 亚洲国产毛片av蜜桃av| av在线播放精品| 日韩人妻精品一区2区三区| 亚洲精品一区蜜桃| 免费大片18禁| 边亲边吃奶的免费视频| 99久久人妻综合| 国产精品一区二区在线不卡| 亚洲第一区二区三区不卡| 欧美亚洲日本最大视频资源| 精品久久国产蜜桃| 亚洲成人av在线免费| 亚洲成人一二三区av| 国产男女内射视频| 亚洲成人av在线免费| 亚洲精品中文字幕在线视频| 欧美精品国产亚洲| 亚洲经典国产精华液单| 国产精品一二三区在线看| 中文精品一卡2卡3卡4更新| 久久鲁丝午夜福利片| 丝袜脚勾引网站| 99热网站在线观看| 不卡视频在线观看欧美| 亚洲国产精品成人久久小说| 菩萨蛮人人尽说江南好唐韦庄| 亚洲,欧美,日韩| 五月天丁香电影| 精品一区二区三区四区五区乱码 | 高清av免费在线| 久久久久久久久久人人人人人人| 妹子高潮喷水视频| 久久久国产一区二区| 黑丝袜美女国产一区|