• <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 ?

    米根霉脂肪酶基因pro-ROL和m-ROL在畢赤酵母中的密碼子優(yōu)化、表達和酶學性質(zhì)的比較分析

    2011-02-26 13:21:04楊江科嚴翔翔黃日波張搏
    生物工程學報 2011年12期

    楊江科,嚴翔翔,黃日波,張搏

    1 武漢工業(yè)學院生物與制藥工程學院,武漢 430023

    2 廣西科學院國家非糧生物質(zhì)能源工程技術(shù)研究中心,南寧 530070

    Introduction

    Rhizopus orgzae lipases (EC 3.1.1.3) have been widely utilized in hydrolysis of triglycerides, synthesis of aromatic esters and biofuel, and kinetic resolutions of prochiral compounds[1-3]. Typically, Rhizopus orgzae lipase (ROL) consists of three parts that includes a signaling peptide responsible for the translocation and secretion of ROL, pre-sequence, and mature lipase (m-ROL) domain[4]. The pre-sequence is an intramolecular chaperone involved in the correct folding and efficient secretion of m-ROL. Although intramolecular chaperones have been found in several protein families[5-6], to our knowledge, Rhizopus lipase is the only member of the lipase family to possess intramolecular chaperone-like pre-sequences. It remains unknown how the pre-sequence contributes to folding, post-translational processing and maturation of Rhizopus lipase.

    Currently, Rhizopus lipase has been successfully expressed in Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. In E. coli, pro-ROL and m-ROL exists as insoluble inclusion bodies. To obtain their active forms, a complex refolding process was needed[4,7]. m-ROL exhibited no enzymatic activity when expressed in S. cerevisiae, suggesting the pre-sequence is necessary for correct protein folding of m-ROL[8-9]. However, m-ROL displayed enzymatic activity when expressed in Pichia[10-11]. It means that without the help of pre-sequence the mature domain of ROL can form the active conformation in P. pastoris by itself. But questions such as whether pro-ROL would actively express in P. pastoris and whether pro-ROL and m-ROL were enzymatically identical characteristics still need to be clarified.

    P. pastoris expression system is an easy and efficient system suitable for high-density fermentation, and now broadly used to produce recombinant heterologous proteins[12-14]. Like most organisms, Pichia displays a non-random pattern of synonymous codon usage and showed general bias towards a subset of codons. This bias can affect the heterogenous expression efficiency of genes in Pichia. Codon optimization by substituting rare codons with frequently used ones has been established as an efficient measure to enhance the expression level[15-17].

    In this study, we cloned and expressed R. orzyae lipase genes, pro-ROL and m-ROL, in P. pastoris. To improve its expression levels, we conducted overlap extension PCR to optimize the codons of m-ROL gene. We further compared the enzymatic activity of pro-ROL and m-ROL to assess the function of the pre-sequence on the maturation of ROL, and to acquire parameters for potential biotechnological applications.

    1 Materials and methods

    1.1 Reagents, media and strains

    All molecular biological reagents were purchased from TaKaRa, Dalian. p-Nitrophenyl acetate (pNPA, C2), p-Nitrophenyl butyrate (pNPB, C4), p-Nitrophenyl caprate (pNPC, C10), p-Nitrophenyl laurate (pNPL, C12), and p-Nitrophenyl palmitate (pNPP, C16) were purchased from Sigma.

    R. oryzae strains HU3005 was obtained from the China Centre of Industrial Culture Collection (CICC) with deposition number CICC3005. E. coli strain DH10B was cultured at 37 °C in Luria-Bertani (LB) medium unless otherwise noted. Ampicillin (100 mg/L) and kanamycin (50 mg/L) were added when necessary.

    1.2 Lipase genes cloning

    R. oryzae pre-mature lipase gene pro-ROL was amplified using the primer pairs ROL1 (5¢-CTGAATTCGTTCCTGTTTCTGGTAAATC-3¢, EcoR I site) and ROLA1 (5¢-CTGCGGCCGCTTA CAAACAGCTTCCTCGT-3¢, Not I site), and mature lipase gene m-ROL was amplified with the primer pairs MROL2 (5¢-CTGAATTCTCTGAT GGTGGTAAGGTTG-3¢, EcoR I site) and MROLA2 (5¢-CTGCGGCCGCTTACAAACAGC TTCCTTCGT-3¢, Not I site) (Fig. 1).

    1.3 RNA extraction

    Total RNA from R. oryzae was extracted by Trizol reagent (Gibcol) according to the manufacturer’s protocol. The first strand cDNA was synthesis by using the RevertAid First Strand cDNA Synthesis Kit (Fermentas). PCR was carried out in a 50 μL reaction containing 200 mmol/L dNTPs, 0.1 mmol/L primers, 1.5 mmol/L MgCl2, and 1 U of pfu DNA polymerase (TaKaRa). The PCR conditions is as followed: denaturation at 94 °C for 4 min, 28 cycles of 94 °C for 30 s, 56 °C for 50 s and 72 °C for 1 min, and final elongation at 72 °C for 6 min. The PCR product was cloned into the pMD18-T simple vector (TaKaRa), and then sequenced by Sangon Ltd., Shanghai. The sequence of R. oryzae lipase gene was deposited into GenBank with the accession number GQ502721.

    1.4 Codon optimization

    Overlap extension PCR amplifications were conducted to substitute eight less-frequently used codons with the frequently used codons (Table 1). Three fragments which overlap each other were amplified with the primers containing the mutant nucleotides (Table 2), and then assembled into the full-length m-ROL gene by a second PCR using 5¢- and 3¢-end primers (Fig. 2).

    Fig. 1 Primary structure of R. oryzae lipase ROL.

    Table 1 The optimized codons of R. oryzae lipase gene m-ROL and their usage in Pichia

    Table 2 The primers used for codon optimization of ROL and the size of PCR products.

    Fig. 2 Phylogeny tree of R. oryzae lipase (ROL) and the related species. Bracketed are the GenBank accession numbers. P. nodorum: Phaeosphaeria nodorum; P. tritici: Pyrenophora tritici.

    1.5 Plasmid construction, transformation, and recombinants selection

    PCR product of m-ROL and pro-ROL were digested with EcoR I and Not I, and then inserted into vector pPIC9K (Invitrogen) to obtain plasmids pPIC9K-m-ROL and pPIC-pro-ROL, with a fusion expression of α-factor. Sac I was used to linearize the plasmid pPIC9K-m-ROL and pPIC-pro-ROL for the single crossover with P. pastoris genome to generate the methanol-utilized phenotype (Mut+). About 6 μg of linearized DNA was mixed with 80 μL of yeast competent cells, and transformed into P. pastoris GS115 cells by electroporation conducted on Gene Pulser (Bio-rad) according to the manufacturer’s suggestion for S. cerevisiae. Positive clones were initially selected by MD medium (1.34% yeast nitrogen base, 4×10?5% biotin, 2% dextrose) plates and then checked by colony PCR.

    The lipase activity of the recombinants was initially checked by BMMY agar plate containing olive oil and rhodamine B. Positive clones displayed orange fluorescent halos visible upon UV irradiation[18].

    1.6 Fermentation

    A single colony of recombinant bacteria was selected and inoculated into 50 mL BMGY (1% yeast extract, 2% peptone, 100 mmol/L potassium phosphate buffer with pH 6.0, 1.34% yeast nitrogen base, 4×10?5% biotin, 1% glycerol) medium, and grown at 28 °C in a shaking incubator (250 r/min) until the culture reached an OD600of 3.0. The cells were harvested, and a portion was transferred into 50 mL BMMY (1% yeast extract, 2% peptone, 100 mmol/L potassium phosphate buffer pH 6.0, 1.34% yeast nitrogen base, 4×10?5% biotin, and 0.5% methanol) medium to obtain a cell suspension with OD600=1.0. The cells were grown for another 5 d and expression of lipase was induced by methanol at a final concentration of 0.5%. Lipase activity of the supernatant of fermentation broth was checked at intervals. For every type of recombinant, three colonies were randomly selected for fermentation.

    1.7 Lipase activity and characterization

    Lipase activity was quantified at pH 7.5 by free fatty acid titration with 50 mmol/L NaOH after incubation in a thermostated vessel for 10 min. The assay mixture consisted of 5 mL 50 mmol/L Tris-HCl buffer, 50 mmol/L NaCl, 4 mL emulsified olive oil and 1 mL enzyme solution. One unit (U) of the activity was defined as the amount of enzyme liberating 1 micromole of fatty acid per min at 40 °C. Properties of lipase such as its thermal stability and optimal pH were determined as previously reported[13-14]. All experiments were carried out in triplicate. Protein content of the fermentation broth was determined by the Bradford method[25].

    1.8 Phylogenic analysis

    Phylogenetic analysis was performed by molecular evolutionary genetics analysis (MEGA3.1) software, which was also used to produce a phylogenetic dendrogram reflecting the evolutionary relationship between the cloned genes and other reference strains by the neighbor-joining method according to the Kimura 2-parameter model[19].

    2 Results

    2.1 Cloning of R. oryzae lipase genes

    R. oryzae premature lipase gene pro-ROL and mature lipase gene m-ROL were amplified by RT-PCR. The ORFs of pro-ROL and m-ROL genes were 1 101 bp and 810 bp, respectively. Phylogenetic analysis based on the similarity of amino acid sequences between R. oryzae and the related reference lipases revealed three distinct clades (I to III). Clade I consisted of lipases from Rhizopus and Rhozomucor. Cloned R. oryzae lipase exhibited a 95% amino acid similarity with R. stolonifer (Fig. 2).

    2.2 Codon optimization and expression in P. pastoris

    Bias codon usages between different microorganisms are one of the main factors that restrict gene expression in heterogeneous host. To achieve a high level expression of m-ROL in Pichia, the less frequently used codons consisting of eight amino acids were substituted with the frequently used ones by overlap extension PCR (Fig. 3). After transformation, yeast recombinants carrying codon-optimized m-ROL, pro-ROL and original m-ROL were acquired. Both m-ROL and pro-ROL showed lipase activity on plates. However, the enzymatic activity of the recombinants carrying codon-optimized m-ROL (optimized-ROL) is significantly higher than the non-optimized m-ROL and pro-ROL (Fig. 4A). After methanol-inducible expression in Pichia, the molecular weight of the enzymes m-ROL and pro-ROL determined by PAGE were 30 kDa and 35 kDa, respectively (Fig. 4B). The expression level of codon-optimized m-ROL is higher than the original m-ROL and pro-ROL. After 72 h of fermentation, the enzymatic activity (Fig. 5A) and protein content (Fig. 5B) of the codon-optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.

    2.3 Characterization of pro-ROL and m-ROL

    We determined the optimal substrate, pH and temperature of pro-ROL and m-ROL (Fig. 6). pNP esters with different carbon chain lengths were selected as substrates. pro-ROL and m-ROL showed differential preferences to these substrates. pro-ROL preferred short- and middle-chain substrates (C4and C10), while m-ROL preferred middle-chain substrates with optimal activity observed using C10substrates (Fig. 6A).

    Fig. 3 Flow chart of codon optimization of m-ROL by overlap extension PCR (A), and the gel picture of the three fragments (B), F1 (500 bp), F2 (270 bp ) and F3 (71 bp). M: DNA marker (2 000, 1 000, 750, 500, 250, 100 bp)

    Fig. 4 Expression of codon-optimized ROL (optimized ROL), pro-ROL and original m-ROL in P. pastoris. (A) The morphology of different recombinants on the plate under the UV light. (B) SDS-PAGE of supernatant product of recombinant lipase.

    Fig. 5 Time course of the production of lipases by recombinants carrying pro-ROL, m-ROL and the codon-optimized m-ROL (optimized-ROL). (A) and (B) directed out lipase activity and protein content of the fermentation broth, respectively.

    Fig. 6 Enzymatic characterization of pro-ROL and m-ROL. (A) Substrate specificity of pro-ROL and m-ROL towards ester of nitrophenol. (B) and (C) Effects of temperature and pH on lipase activity.

    The optimal pH was determined by incubating lipases in reaction buffer with different pH values. As shown in Fig. 6B, both pro-ROL and m-ROL preferred alkaline environments. Unlike pro-ROL, which has an optimal pH value 8.0, the optimal pH value of m-ROL was 9.0. The suitable pH range, determined as having>60% remaining activity, of m-ROL is 8.5?9.5, while the suitable pH range of pro-ROL is from 7.5 to 8.5. Enzymatic activity significantly decreased when the lipases were not maintained at their optimal pH.

    To determine the optimal reaction temperature and thermal stability, lipase activities were assessed under different temperatures (20 °C?60 °C) and calculated as percentages of the maximal activity. As shown in Fig. 6C, the optimal temperatures for both pro-ROL and m-ROL were 30 °C. At 50 °C, enzymatic activity was not detected for m-ROL, whereas pro-ROL had 51% activity. Additionally, pro-ROL was more thermally stable than m-ROL.

    3 Discussion

    Pre-sequence plays an important role on the folding, post-translational processing and secretion of R. oryzae lipase[4,7,9]. Previous reports showed lack of enzymatic activity when m-ROL was expressed in S. cerevisiae[8]. This suggested that the lack of the pre-sequence in m-ROL may have inhibited formation of its active conformation. In our study, we actively expressed pro-ROL and m-ROL in P. pastoris. The enzymatic characteristics of pro-ROL in Pichia are different than those in S. cerevisiae. In Pichia, pro-ROL prefers short and middle-length carbon chains (C4-C10), while in S. cerevisiae, pro-ROL favors middle-chain substrates[8]. It is possible that in P. pastoris, post-translational modification may modulate the function of the pre-sequence and facilitate correct protein folding of m-ROL. Moreover, intramolecular chaperones may also affect enzymatic properties by mediating the folding and conformational changes of the mature protein domain[20]. In this study, pro-ROL was more thermally stable than m-ROL. Similar phenomenon was observed by Beer and his colleague. In their study, they found that in contrast to m-ROL, pro-ROL and prepro-ROL had considerably higher thermostability[7]. This suggests that the pre-sequence domain may also protect the m-ROL region from dramatic conformational change when exposed to high temperatures.

    A special characteristic of lipase is the interfacial activation. Generally, the active site (catalytic triad) is completely buried beneath a short amphiphilic helical segment, which prevents substrate access. When the enzyme contact with the hydrophobic substrates, it will go through a series structural rearrangement, and the enzyme changes from a closed to a open conformation that make the substrates entre into the activity site[21]. Although there were no report on the effect of pre-sequence on the substrate selectivity of lipase before, but it is possible that this pre-sequence of pro-ROL can spatially retard the conformational change of mature ROL domain. When the long-chain substrates contacted with enzymes, although the enzymes have conformational change, the open size of enzyme is still not enough to permit the long-chain substrates entre into the active site. On the contrary, this half-open conformation will be more accessible to the shorter chain substrate (C4). As observed in this study, the favorable substrate of pro-ROL and m-ROL are C4, while for m-ROL is C10 (Fig. 6A).

    Codon-usage frequency is a major impediment to the efficient expression of foreign gene in heterologous host. In ROL gene, four codons 155Leu (CTC), 171Ser (AGC), 254Leu (CTC) and 267Ser (AGC), which have the lowest usage frequency, are the bottleneck of gene expression in P. pastoris. Our study shows that codon optimization by overlap extension PCR to substitute less frequently used codons with frequently used ones could significantly improve the expression level of ROL. Compared to other methods such as chemical gene synthesis, overlap extension PCR is simple and effective. The initial enzymatic activity achieved based on the flask fermentation method could reach 132.7 U/mL. Although our product yield is not as high as a previous study[22]using a large-scale bioreactor (644.0 U/mL), it is still significantly better than other reported results[23-24]. We believe that lipase production and enzymatic activity from recombinants carrying optimized ROL gene may be improved under a batch-induced mode with further optimization in pH, methanol concentration and aeration during the fermentation process.

    In this study, we actively expressed pre-mature (pro-ROL) and mature lipase gene (m-ROL) of R. oryzae in P. pastoris. The differences in enzymatic characteristics between these two forms of lipase may be caused by the intramolecular chaperone-like pre-sequence in pro-ROL, which could affect the conformation of m-ROL domain. Codon optimization by overlap extension PCR has effectively improved the expression level by 4.6-fold. This newly codonoptimized ROL gene may be useful for future large-scale production of R. oryzae lipase. Further characterization of pro-ROL and m-ROL may offer a valuable tool in industrial applications.

    [1] Gotor-Fernández V, Brieva R, Gotor V. Lipases: useful biocatalysts for the preparation of pharmaceuticals. J Mol Catal B: Enzym, 2006, 40(3/4): 111?120.

    [2] Fukuda H, Hama S, Tamalampudi S, et al. Whole-cell biocatalysts for biodiesel fuel production. Trends Biotechnol, 2008, 26(12): 668?673.

    [3] Tamalampudi S, Talukder MR, Hama S, et al. Enzymatic production of biodiesel from Jatropha oil: a comparative study of immobilized-whole cell and commercial lipases as a biocatalyst. Biochem Eng J, 2008, 39(1): 185?189.

    [4] Beer HD, Wohlfahrt G, Schmid RD, et al. The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence. Biochem J, 1996, 319(Pt 2): 351?359.

    [5] Shinde U, Inouye M. Folding mediated by an intramolecular chaperone: autoprocessing pathway of the precursor resolved via a substrate assisted catalysis mechanism. J Mol Biol, 1995, 247(3): 390?395.

    [6] Chen YJ, Inouye M. The intramolecular chaperonemediated protein folding. Curr Opin Stru Biol, 2008, 18(6): 765?770.

    [7] Beer HD, McCarthy JEG, Bornscheuer UT, et al. Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae. Gene Struct Expr, 1998, 1399(2/3): 173?180.

    [8] Takahashi S, Ueda M, Tanaka A. Function of the prosequence for the in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae. Appl Microbiol Biotechnol, 2001, 55(4): 454?462.

    [9] Ueda M, Takahashi S, Washida M, et al. Expression of Rhizopus oryzae lipase gene in Saccharomyces cerevisiae. J Mol Catal B: Enzym, 2002, 17(3/5): 113?124.

    [10] Minning S, Schmidt-Dannert C, Schmid RD. Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties. J Biotechnol, 1998, 66(2/3): 147?156.

    [11] Cos O, Resina D, Ferrer P, et al. Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures. Biochem Engin J, 2005, 26(2/3): 86?94.

    [12] Fernández L, Pérez-Victoria I, Zafra A, et al. High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris. Protein Expr Purif, 2006, 49(2): 256?264.

    [13] Shu ZY, Duan MJ, Yang JK, et al. Aspergillus niger lipase: heterologous expression in Pichia pastoris, molecular modeling prediction and the importance of the hinge domains at both sides of the lid domain to interfacial activation. Biotechnol Prog, 2009, 25(2): 409?416.

    [14] Yang JK, Zhang B, Yan YJ. Cloning and expression of Pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification. Appl Biochem Biotechnol, 2009, 159(2): 355?365.

    [15] Müller M. Codon optimization of papillomavirus genes. Methods Mol Med, 2005, 119: 433?444.

    [16] Tokuoka M, Tanaka M, Ono K, et al. Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl Environ Microbiol, 2008, 74(21): 6538?6546.

    [17] Daniell H, Ruiz G, Denes B, et al. Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function. BMC Biotechnol, 2009, 9(1): 33.

    [18] Kouker G, Jaeger KE. Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol, 1987, 53(1): 211?213.

    [19] Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform, 2004, 5(2): 150?163.

    [20] Ma BY, Tsai CJ, Nussinov R. Binding and folding: in search of intramolecular chaperone-like building block fragments. Protein Eng Des Sel, 2000, 13(9): 617?627.

    [21] Chapus C, Semeriva M, Bovier-Lapierre C, et al. Mechanism of pancreatic lipase action. 1. Interfacial activation of pancreatic lipase. Biochemistry, 1976, 15(23): 4980?4987.

    [22] Surribas A, Stahn R, Montesinos JL, et al. Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies. J Biotechnol, 2007, 130(3): 291?299.

    [23] Ramchuran SO, Vargas VA, Hatti-Kaul R, et al. Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris. Appl Microbial Biotechnol, 2006, 71(4): 463?472.

    [24] Yao HY, Yu SW, Zhang LD, et al. Isolation of a novel lipase gene from Serratia liquefaciens S33 DB-1, functional expression in Pichia pastoris and its properties. Mol Biotechnol, 2008, 38(2): 99?107.

    [25] Zor T, Selinger Z. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem, 1996, 236: 302?308.

    一级片'在线观看视频| 久久久精品国产亚洲av高清涩受| 久久婷婷青草| 乱人伦中国视频| 欧美成人午夜精品| 日韩一区二区三区影片| 又黄又粗又硬又大视频| 又大又黄又爽视频免费| 国产免费现黄频在线看| 亚洲国产精品999| 秋霞在线观看毛片| 丰满少妇做爰视频| 天堂8中文在线网| 有码 亚洲区| 日本色播在线视频| 久久鲁丝午夜福利片| 亚洲国产av影院在线观看| 亚洲精品一二三| 国产成人精品一,二区| 国产精品蜜桃在线观看| av又黄又爽大尺度在线免费看| 亚洲精品久久久久久婷婷小说| 纵有疾风起免费观看全集完整版| 欧美激情高清一区二区三区 | 一本—道久久a久久精品蜜桃钙片| 九草在线视频观看| 午夜福利一区二区在线看| 亚洲伊人色综图| 午夜激情av网站| 国产片内射在线| 极品人妻少妇av视频| 91aial.com中文字幕在线观看| 亚洲精品美女久久av网站| 午夜免费男女啪啪视频观看| 两个人免费观看高清视频| 三级国产精品片| av国产精品久久久久影院| 十分钟在线观看高清视频www| 青草久久国产| 国产精品成人在线| 欧美精品一区二区免费开放| 亚洲精品视频女| 18+在线观看网站| 青春草国产在线视频| 亚洲四区av| 午夜91福利影院| 久久韩国三级中文字幕| 久久久国产一区二区| 亚洲av.av天堂| 精品久久久久久电影网| 汤姆久久久久久久影院中文字幕| 午夜福利在线免费观看网站| kizo精华| 国产成人精品一,二区| 九九爱精品视频在线观看| 国产精品国产三级国产专区5o| 国产av国产精品国产| 欧美精品国产亚洲| 精品国产乱码久久久久久小说| 国产有黄有色有爽视频| 欧美成人午夜精品| 精品一区二区三区四区五区乱码 | 精品第一国产精品| 国产白丝娇喘喷水9色精品| 高清欧美精品videossex| av网站免费在线观看视频| 最近手机中文字幕大全| 一级毛片黄色毛片免费观看视频| 亚洲一区中文字幕在线| 一边亲一边摸免费视频| 满18在线观看网站| 啦啦啦啦在线视频资源| 69精品国产乱码久久久| 丝袜喷水一区| 又黄又粗又硬又大视频| 国产男人的电影天堂91| 午夜福利网站1000一区二区三区| 久久久精品免费免费高清| 婷婷色av中文字幕| 日韩中文字幕欧美一区二区 | 亚洲成人一二三区av| 国产1区2区3区精品| 久久午夜综合久久蜜桃| 男女高潮啪啪啪动态图| 久久午夜综合久久蜜桃| 国产精品一区二区在线不卡| 如日韩欧美国产精品一区二区三区| av.在线天堂| 大陆偷拍与自拍| 人人妻人人爽人人添夜夜欢视频| 国产午夜精品一二区理论片| 久久亚洲国产成人精品v| 99re6热这里在线精品视频| 亚洲欧美色中文字幕在线| 18禁动态无遮挡网站| 国产亚洲一区二区精品| 日本欧美国产在线视频| 丰满乱子伦码专区| 国产黄频视频在线观看| 欧美人与性动交α欧美精品济南到 | 超碰成人久久| 精品少妇久久久久久888优播| 成人18禁高潮啪啪吃奶动态图| 美女福利国产在线| 高清不卡的av网站| 成人国语在线视频| 亚洲色图 男人天堂 中文字幕| 一级片免费观看大全| 免费女性裸体啪啪无遮挡网站| 亚洲经典国产精华液单| 日韩在线高清观看一区二区三区| 国产深夜福利视频在线观看| 免费黄网站久久成人精品| 在线观看www视频免费| 国产精品久久久久久精品古装| 亚洲国产精品成人久久小说| 免费观看性生交大片5| 夫妻午夜视频| 精品久久久久久电影网| 青草久久国产| 欧美精品一区二区大全| 亚洲第一区二区三区不卡| 国产在线视频一区二区| 80岁老熟妇乱子伦牲交| 亚洲人成网站在线观看播放| 亚洲男人天堂网一区| 丁香六月天网| 成人二区视频| 国产精品99久久99久久久不卡 | 久久午夜福利片| 青春草国产在线视频| 高清在线视频一区二区三区| 丁香六月天网| 晚上一个人看的免费电影| 亚洲成人一二三区av| 亚洲少妇的诱惑av| 国产亚洲av片在线观看秒播厂| 青春草视频在线免费观看| 五月开心婷婷网| 黄色配什么色好看| 国产熟女欧美一区二区| 男女啪啪激烈高潮av片| 成人国产麻豆网| 亚洲国产av新网站| 成年女人毛片免费观看观看9 | 婷婷色av中文字幕| 黄色 视频免费看| 丝袜在线中文字幕| 国产亚洲av片在线观看秒播厂| 日韩一区二区视频免费看| 久久久久久久国产电影| 天天躁夜夜躁狠狠躁躁| 国产欧美日韩一区二区三区在线| 免费黄网站久久成人精品| 丝袜在线中文字幕| 精品人妻一区二区三区麻豆| 一区福利在线观看| 黄色一级大片看看| 国产熟女欧美一区二区| 免费观看在线日韩| 日韩一本色道免费dvd| 欧美日韩成人在线一区二区| 久久免费观看电影| 18在线观看网站| 日韩不卡一区二区三区视频在线| 亚洲内射少妇av| 91在线精品国自产拍蜜月| 成人国语在线视频| 精品久久久久久电影网| 国产精品人妻久久久影院| 亚洲情色 制服丝袜| 99久久综合免费| 99久久精品国产国产毛片| 美国免费a级毛片| 午夜老司机福利剧场| 亚洲av免费高清在线观看| 曰老女人黄片| 久久久久国产精品人妻一区二区| 一级a爱视频在线免费观看| 亚洲欧美日韩另类电影网站| 久久久久精品性色| 婷婷色av中文字幕| 国产深夜福利视频在线观看| 亚洲美女搞黄在线观看| 亚洲伊人色综图| 丝袜在线中文字幕| 在现免费观看毛片| 人人妻人人添人人爽欧美一区卜| 欧美国产精品一级二级三级| 亚洲av电影在线观看一区二区三区| 成人漫画全彩无遮挡| 国产日韩一区二区三区精品不卡| 久久99一区二区三区| 青春草视频在线免费观看| 国产精品免费视频内射| 超碰成人久久| 亚洲精品国产色婷婷电影| 成人黄色视频免费在线看| 天堂中文最新版在线下载| 女人久久www免费人成看片| 99国产精品免费福利视频| 亚洲人成网站在线观看播放| 亚洲视频免费观看视频| 日韩,欧美,国产一区二区三区| 狠狠婷婷综合久久久久久88av| 成人黄色视频免费在线看| 有码 亚洲区| 欧美日韩国产mv在线观看视频| 侵犯人妻中文字幕一二三四区| 亚洲三区欧美一区| 涩涩av久久男人的天堂| 91精品三级在线观看| 成人毛片60女人毛片免费| 91国产中文字幕| 观看av在线不卡| 欧美亚洲 丝袜 人妻 在线| 中文欧美无线码| 久久久久久伊人网av| 久久热在线av| 一级黄片播放器| 在线观看人妻少妇| 久久毛片免费看一区二区三区| 免费看不卡的av| 国产白丝娇喘喷水9色精品| 2022亚洲国产成人精品| 26uuu在线亚洲综合色| 一边亲一边摸免费视频| 春色校园在线视频观看| 黑人巨大精品欧美一区二区蜜桃| 少妇被粗大的猛进出69影院| 亚洲精品一二三| 寂寞人妻少妇视频99o| 亚洲美女视频黄频| 国产精品免费大片| 成年美女黄网站色视频大全免费| videossex国产| 国产精品久久久久久精品古装| 成人午夜精彩视频在线观看| 岛国毛片在线播放| 在线看a的网站| 狠狠婷婷综合久久久久久88av| 国产高清不卡午夜福利| 欧美黄色片欧美黄色片| 国产成人精品一,二区| 韩国精品一区二区三区| 国产无遮挡羞羞视频在线观看| 婷婷色综合www| 国精品久久久久久国模美| 国产男女内射视频| 亚洲国产av新网站| 在线观看免费日韩欧美大片| 免费观看av网站的网址| 九草在线视频观看| 成年女人在线观看亚洲视频| 婷婷成人精品国产| 美女视频免费永久观看网站| 亚洲精品国产av成人精品| 国产亚洲午夜精品一区二区久久| av在线观看视频网站免费| 国产白丝娇喘喷水9色精品| 两个人免费观看高清视频| 伊人久久大香线蕉亚洲五| www.自偷自拍.com| 久久人人97超碰香蕉20202| 九九爱精品视频在线观看| 大香蕉久久网| freevideosex欧美| 亚洲伊人久久精品综合| 欧美少妇被猛烈插入视频| 少妇猛男粗大的猛烈进出视频| a级毛片黄视频| 国产精品久久久久久久久免| 精品一区二区三卡| 看免费成人av毛片| 一级片免费观看大全| 波多野结衣一区麻豆| 日本猛色少妇xxxxx猛交久久| 99久国产av精品国产电影| 免费黄频网站在线观看国产| 国产成人精品福利久久| 大香蕉久久成人网| 免费观看a级毛片全部| 爱豆传媒免费全集在线观看| 一级毛片电影观看| 9热在线视频观看99| 欧美在线黄色| 欧美+日韩+精品| 久久久亚洲精品成人影院| 久久久精品国产亚洲av高清涩受| 人人妻人人添人人爽欧美一区卜| 亚洲国产欧美日韩在线播放| 久久久久精品人妻al黑| 亚洲av中文av极速乱| www.精华液| 女性被躁到高潮视频| 99久久精品国产国产毛片| 一级a爱视频在线免费观看| 91成人精品电影| 亚洲av电影在线进入| 视频区图区小说| 黄色毛片三级朝国网站| 亚洲精品国产av蜜桃| 欧美人与善性xxx| 久久精品国产亚洲av高清一级| 99热国产这里只有精品6| av天堂久久9| 狂野欧美激情性bbbbbb| 赤兔流量卡办理| 美女高潮到喷水免费观看| av在线观看视频网站免费| 9色porny在线观看| 另类精品久久| 欧美人与善性xxx| 高清黄色对白视频在线免费看| 日日撸夜夜添| 午夜老司机福利剧场| 久久久a久久爽久久v久久| 久久女婷五月综合色啪小说| 国产一区二区三区av在线| 黄片播放在线免费| 国产一级毛片在线| 在线天堂中文资源库| 亚洲欧美成人综合另类久久久| 久久 成人 亚洲| 亚洲欧美精品综合一区二区三区 | 亚洲av免费高清在线观看| 18禁动态无遮挡网站| 91精品三级在线观看| 免费播放大片免费观看视频在线观看| 搡老乐熟女国产| 美女国产高潮福利片在线看| av在线老鸭窝| 久久久久久久久久人人人人人人| 两性夫妻黄色片| 中文字幕最新亚洲高清| 九色亚洲精品在线播放| 午夜影院在线不卡| 中文字幕另类日韩欧美亚洲嫩草| 久久久精品国产亚洲av高清涩受| 午夜福利影视在线免费观看| 亚洲第一av免费看| 免费观看av网站的网址| 亚洲成国产人片在线观看| 精品国产国语对白av| 亚洲欧美日韩另类电影网站| 国产爽快片一区二区三区| 欧美人与善性xxx| 汤姆久久久久久久影院中文字幕| 国产精品免费大片| 亚洲精品日本国产第一区| 天天躁狠狠躁夜夜躁狠狠躁| 国产av精品麻豆| 日韩制服骚丝袜av| 毛片一级片免费看久久久久| 成人二区视频| 免费在线观看完整版高清| 国产精品国产三级专区第一集| 一级毛片电影观看| 欧美精品亚洲一区二区| 国产又爽黄色视频| 男女免费视频国产| 18禁裸乳无遮挡动漫免费视频| 午夜91福利影院| 国产野战对白在线观看| 国产在线免费精品| 欧美日韩精品成人综合77777| 欧美日韩国产mv在线观看视频| 亚洲第一av免费看| 亚洲精品久久久久久婷婷小说| 亚洲精品av麻豆狂野| 欧美日韩亚洲高清精品| 黄色配什么色好看| 国产精品秋霞免费鲁丝片| 美女福利国产在线| 精品一区二区三卡| 91国产中文字幕| 好男人视频免费观看在线| 男人添女人高潮全过程视频| 国产成人精品久久二区二区91 | 国产有黄有色有爽视频| 国产亚洲午夜精品一区二区久久| 另类精品久久| 亚洲第一区二区三区不卡| 一级片'在线观看视频| 777久久人妻少妇嫩草av网站| 日本91视频免费播放| 亚洲av电影在线进入| 国产一区亚洲一区在线观看| 97精品久久久久久久久久精品| 午夜福利,免费看| 日韩制服骚丝袜av| 亚洲欧美精品综合一区二区三区 | 大话2 男鬼变身卡| 可以免费在线观看a视频的电影网站 | 看非洲黑人一级黄片| 亚洲欧美一区二区三区久久| 精品亚洲成a人片在线观看| 欧美精品人与动牲交sv欧美| 午夜免费鲁丝| 国产亚洲av片在线观看秒播厂| 亚洲精品日韩在线中文字幕| 啦啦啦在线免费观看视频4| 久久久久久人人人人人| 熟女电影av网| 久久ye,这里只有精品| 18禁观看日本| 色网站视频免费| √禁漫天堂资源中文www| 超碰97精品在线观看| 天堂中文最新版在线下载| 国产日韩一区二区三区精品不卡| 精品99又大又爽又粗少妇毛片| 色网站视频免费| 国产精品三级大全| 国产 精品1| 国产又爽黄色视频| av在线观看视频网站免费| 亚洲国产欧美网| 久久毛片免费看一区二区三区| 国产欧美日韩综合在线一区二区| 9热在线视频观看99| 黄片小视频在线播放| 亚洲内射少妇av| 亚洲成人一二三区av| 久久久久久久久久人人人人人人| av网站在线播放免费| 热re99久久精品国产66热6| 国产成人精品在线电影| av网站在线播放免费| 热re99久久精品国产66热6| 免费高清在线观看日韩| 一边亲一边摸免费视频| 欧美亚洲日本最大视频资源| 国产成人精品久久久久久| www.精华液| 老熟女久久久| 日韩一本色道免费dvd| 看免费av毛片| 极品少妇高潮喷水抽搐| 午夜福利网站1000一区二区三区| 亚洲av电影在线观看一区二区三区| 亚洲国产成人一精品久久久| 国产熟女欧美一区二区| 男女边吃奶边做爰视频| 激情视频va一区二区三区| 看十八女毛片水多多多| 中文精品一卡2卡3卡4更新| 国产日韩欧美视频二区| 日韩熟女老妇一区二区性免费视频| 久久久久视频综合| 三级国产精品片| 国产精品.久久久| 亚洲精品国产一区二区精华液| 精品久久久久久电影网| 丰满少妇做爰视频| 日韩在线高清观看一区二区三区| 一级毛片 在线播放| 超碰97精品在线观看| 免费女性裸体啪啪无遮挡网站| 亚洲情色 制服丝袜| 国产免费又黄又爽又色| 国产极品粉嫩免费观看在线| 在线免费观看不下载黄p国产| 一级毛片黄色毛片免费观看视频| 亚洲 欧美一区二区三区| 秋霞伦理黄片| 精品国产一区二区久久| 少妇熟女欧美另类| 天天操日日干夜夜撸| 日韩免费高清中文字幕av| 亚洲精品美女久久av网站| 黑人欧美特级aaaaaa片| 人妻 亚洲 视频| 亚洲av.av天堂| 99热全是精品| 精品国产一区二区三区久久久樱花| 成人漫画全彩无遮挡| 午夜影院在线不卡| 日韩不卡一区二区三区视频在线| 一区二区三区激情视频| 最近最新中文字幕大全免费视频 | 热99久久久久精品小说推荐| 久久久精品国产亚洲av高清涩受| 侵犯人妻中文字幕一二三四区| 欧美激情高清一区二区三区 | 我的亚洲天堂| 精品视频人人做人人爽| 久久精品久久久久久噜噜老黄| 黄网站色视频无遮挡免费观看| 国产成人精品久久二区二区91 | 十分钟在线观看高清视频www| 国产成人欧美| 亚洲精品久久久久久婷婷小说| 婷婷色综合www| www.av在线官网国产| 国产精品女同一区二区软件| 男女无遮挡免费网站观看| 亚洲国产av新网站| 另类精品久久| 一级,二级,三级黄色视频| 老司机影院成人| 欧美激情高清一区二区三区 | 国产免费视频播放在线视频| 亚洲欧美成人精品一区二区| 日韩大片免费观看网站| 欧美 日韩 精品 国产| 精品福利永久在线观看| av国产精品久久久久影院| 久久毛片免费看一区二区三区| 国产男人的电影天堂91| 伊人久久国产一区二区| 日韩伦理黄色片| 国产精品久久久久久精品古装| 一级片免费观看大全| 精品国产国语对白av| 亚洲成av片中文字幕在线观看 | 美国免费a级毛片| 大香蕉久久网| 人人澡人人妻人| 秋霞伦理黄片| 中文字幕色久视频| 日韩制服丝袜自拍偷拍| 亚洲天堂av无毛| 国产精品国产三级国产专区5o| 大话2 男鬼变身卡| 国语对白做爰xxxⅹ性视频网站| 高清视频免费观看一区二区| 日韩av免费高清视频| 亚洲国产毛片av蜜桃av| 亚洲综合色惰| 免费黄网站久久成人精品| 视频在线观看一区二区三区| 婷婷色综合www| 久久久久久久久久久免费av| 青青草视频在线视频观看| 午夜福利乱码中文字幕| 18禁裸乳无遮挡动漫免费视频| 久久久久久久国产电影| 一区二区三区精品91| 国产 精品1| 制服丝袜香蕉在线| 亚洲欧美一区二区三区久久| 岛国毛片在线播放| 亚洲情色 制服丝袜| 久久精品熟女亚洲av麻豆精品| 菩萨蛮人人尽说江南好唐韦庄| 国产一级毛片在线| 2022亚洲国产成人精品| 久久久久精品性色| 天堂中文最新版在线下载| 国产免费一区二区三区四区乱码| 黄色配什么色好看| 天堂8中文在线网| 热99久久久久精品小说推荐| 色哟哟·www| 欧美在线黄色| 一级片'在线观看视频| 亚洲三级黄色毛片| 亚洲国产欧美在线一区| 涩涩av久久男人的天堂| 国产精品秋霞免费鲁丝片| 香蕉精品网在线| 在线观看免费日韩欧美大片| 久久人人爽人人片av| 男女边摸边吃奶| 777久久人妻少妇嫩草av网站| 在线看a的网站| 人妻少妇偷人精品九色| 一级片免费观看大全| 最近的中文字幕免费完整| 1024视频免费在线观看| 777米奇影视久久| 欧美 日韩 精品 国产| 亚洲美女黄色视频免费看| 成人二区视频| 波多野结衣一区麻豆| 久久久精品区二区三区| 在线观看免费日韩欧美大片| 热re99久久精品国产66热6| 国产精品一国产av| 国产一级毛片在线| 男人操女人黄网站| 国产成人欧美| 日本午夜av视频| 国产在线一区二区三区精| 美女国产视频在线观看| 国产成人精品一,二区| 精品午夜福利在线看| 精品视频人人做人人爽| 一级毛片电影观看| 美女高潮到喷水免费观看| 性色avwww在线观看| 最近最新中文字幕大全免费视频 | 国产熟女午夜一区二区三区| 飞空精品影院首页| 美女国产视频在线观看| 欧美变态另类bdsm刘玥| 高清不卡的av网站| 高清黄色对白视频在线免费看| 麻豆精品久久久久久蜜桃| 精品国产一区二区久久| 亚洲少妇的诱惑av| 熟女少妇亚洲综合色aaa.| 国产成人午夜福利电影在线观看| 久久久久久久精品精品| 精品酒店卫生间| 在线观看免费高清a一片| 色94色欧美一区二区| 亚洲国产欧美网| 免费女性裸体啪啪无遮挡网站| 国产精品嫩草影院av在线观看| 日韩人妻精品一区2区三区| 97精品久久久久久久久久精品| 久久99蜜桃精品久久| 免费黄网站久久成人精品|