• <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 ?

    Transcriptomic Analysis of Deinococcus radiodurans During The Early Recovery Stage From Ultraviolet Irradiation*

    2023-07-19 02:52:30ZHANGCaiYunQIUQinTianMABinGuang

    ZHANG Cai-Yun, QIU Qin-Tian, MA Bin-Guang

    (Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan 430070, China)

    Abstract Objective Deinococcus radiodurans (D. radiodurans) is an extremophile with strong resistance to ultraviolet (UV),ionization, desiccation and chemical reagents. However, the molecular responses of this bacterium in the early recovery stage after UV irradiation are not fully understood. The aim of this work is to reveal the transcriptomic responses of D. radiodurans at this stage.Methods In this study, the transcriptomes of D. radiodurans under normal and UV irradiation culture conditions were determined by using RNA-seq technique. To identify the key genes and their regulatory relationships among the differentially expressed genes(DEGs), functional enrichment analysis was performed. Some key DEGs were selected and validated by real-time quantitative PCR.The transcriptome data from previous studies were adopted to find DEGs common to UV irradiation, ionizing radiation and desiccation stresses. The protein-protein interaction (PPI) network of DEGs was constructed; the hub genes and major modules in the PPI network were identified; functional enrichment analysis was performed for these hubs and modules. Results The results showed that the number of up-regulated genes was more than twice that of down-regulated genes in the early recovery stage after UV irradiation, and most of them were related to stress response and DNA repair. The main repair pathways in the early stage of recovery include single-strand annealing (SSA) pathway (involving genes ddrA-D), nonhomologous end joining (NHEJ) pathway (involving genes ligB and pprA) and nucleotide excision repair (NER) pathway (involving genes uvrA-C), the first two of which are for homologous recombination (HR), while the NER pathway removes pyrimidine dimers caused by UV irradiation. By comparing the transcriptome data under UV irradiation, ionizing radiation and desiccation stresses, it was found that the common responsive DEGs mainly involve Deinococcus-specific genes and the genes related to DNA/RNA metabolism. Several important hub genes and interaction modules were identified from the PPI network of DEGs, whose functions are concentrated in double-strand break repair,DNA topological change and replication. Conclusion These results indicate that in the early recovery stage after UV irradiation, a variety of genes in D. radiodurans undergo responses at transcriptome level, several repair pathways are initiated to cope with this stress, and some repair pathways are common to other stress conditions.

    Key words Deinococcus radiodurans, RNA-seq, ultraviolet irradiation, DNA damage repair, protein-protein interaction (PPI)network

    Deinococcus radiodurans(D. radiodurans) is an extremophile, whose cells are spherical and usually live in dimer or tetramer form, and the colonies are red[1].D. radioduranshas strong adaptability to various stress conditions, such as ionizing radiation,desiccation, high temperature and chemical reagents[2], which is due to its strong ability to repair DNA damage (such as chromosome double-strand breaks (DSB)). Therefore, this species has become an ideal model organism for studying tolerance and repair mechanisms under extreme stress conditions.The tolerance ofD. radioduransto damage by ionizing radiation, ultraviolet (UV) irradiation and desiccation may be the result of three mechanisms:prevention, tolerance and repair. Studies have found that ultraviolet light can damage DNA of organisms,producing cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs)[3],and such a damage can interfere with the replication and transcription mechanisms of DNA.D. radioduransshows strong resistance to ultraviolet irradiation damage, and its resistance to ultraviolet irradiation is about 20 times that ofEscherichia coliwhen measured from the perspective of survival rate[2].D. radioduranshas a complex damage repair pathway. For example, deletion ofuvsE,uvrA1, anduvrA2in nucleotide excision repair pathways was found to reduce but not completely eliminate the ability of this bacterium to remove CPDs and 6-4PPs from genomic DNA[3], indicating thatD. radioduranshas multiple repair pathways to resist UV damage.What pathways are involved in the repair of UV damage remains to be further explored.

    Transcriptomics studies the response of organisms to environmental stress from the aspect of gene expression, and its main techniques are microarray and mRNA sequencing (RNA-seq). Up to now, many works have studied various stress responses ofD. radioduransfrom the perspective of transcriptomes, including ionizing radiation[4],desiccation stress[5], heat stress[6], salt stress[7],cadmium stress[8], H2O2stress[9], mitomycin C (MMC)stress,etc.D. radioduranshas strong tolerance to UV irradiation, but there is still a lack of systematic transcriptome study on its response to UV irradiation stress. In this work, we used RNA-seq technology to detect the transcriptome changes ofD. radioduransin response to UV irradiation, and compared and analyzed the differentially expressed genes (DEGs) ofD. radioduransunder normal culture and UV irradiation conditions. Through functional analysis of the DEGs, we found key genes related to UV irradiation stress response, among which many classic damage-repair genes play a role in the early recovery stage from UV irradiation. We analyzed the similarities and differences of DEGs in response to UV irradiation, ionizing radiation and desiccation stresses, and identified common responsive genes.Moreover, the protein-protein interaction (PPI)network of DEGs was constructed and the hub nodes and major modules were identified, which enhanced our understanding on gene functions in UV damage repair. This study is the first one using RNA-seq technology to measure the transcriptomic response ofD. radioduransto UV irradiation, providing new data and insights for further understanding the molecular mechanism of radiation tolerance in this species.

    1 Materials and methods

    1.1 Strain,growth conditions and treatment

    The R1 strain ofD. radioduranswas used in this study, including two culture conditions. (1) Normal culture condition: 30℃, TGY medium, culture to the middle logarithmic stage; (2) UV irradiation condition: the bacterial solution under normal culture condition was irradiated with 2.5 J·m-2·s-1UV (254 nm)for 5 min (namely, irradiation dose of 750 J/m2) and sampled 10 min later (namely, in the early stage of recovery according to the definition in a previous work[4]). For each culture condition, three biological replicate samples were taken for RNA sequencing.

    1.2 Transcriptome data analysis

    1.2.1 RNA-seq data acquisition and preprocessing

    The bacterial samples were sent to Tianjin Nuohe Source Biology Co., Ltd. for RNA extraction,subsequent cDNA library construction and onmachine sequencing. The sequencing standard was double-ended 150 bp, and the sequencing platform was NovA-PE150. Clean RNA-seq data from the company was used for subsequent analysis.

    Clean data were checked for sequencing quality before biological analysis. The FastQC program was used for quality check, and Trimmomatic[10]was used for quality control of the data. The genome and annotation information ofD. radioduransR1 strain was downloaded from the NCBI genome database(Genome_Assembly_ID 300483) and used as the reference genome. The sequencing data were aligned to the reference genome by using TopHat[11](which calls Bowtie2[12]). StringTie[13]was used for transcript assembly; the preDE.py script in StringTie was used to extract quantitative results and generate the count matrix.

    1.2.2 Screening of differentially expressed genes(DEGs)

    The R package DESeq2[14]was used for DEGs analysis. The gene expression levels in FPKM(fragments per kilobase of transcript per million mapped reads) obtained above were merged for the three biological replicates, and the transcriptomes under normal culture and UV irradiation conditions were compared to obtain differentially expressed genes (DEGs). The criteria for DEG identification were:Fold change>2 andFalse Discovery Rate(FDR)<0.05.

    1.2.3 GO and KEGG enrichment analysis for DEGs

    GO database (http://geneontology.org/) was used for mapping and enrichment analysis of the obtained DEGs before and after UV irradiation. Based on the KEGG database, the R package clusterProfiler[15]was used for KEGG pathway enrichment analysis. In this process, theD. radioduransR1 genome was used as background with threshold ofFDR<0.05.

    1.3 Validating RNA-seq results by real-time quantitative PCR (RT-qPCR)

    For each of the two above-mentioned culture conditions (normal and UV irradiation), three biological replicate samples were taken for RT-qPCR analysis to validate the RNA sequencing results.Using housekeeping gene DR_1343 (glyceraldehyde 3-phosphate dehydrogenase) as internal reference,three key DEGs (DR_B0100, DR_1262, DR_2275)were selected for RT-qPCR experiments and the designed primer sequences are listed in Table S1. The 2-ΔΔCtmethod was used to calculate the relative expression of DEGs.T-test was used to evaluate the statistical significance (P-value) in data comparison.

    1.4 Construction of the protein-protein interaction (PPI) network for DEGs

    The PPI network ofD. radioduranswas downloaded from STRING database (https://cn.stringdb.org/)[16], and the subnetworks related to DEGs were screened out and imported into Cytoscape V3.9.1[17]for visualization. Unconnected nodes were deleted from the network. In the visualized PPI network,nodes represent proteins, and edges between nodes represent interactions. The color of node shows the regulation direction of a DEG (red for up-regulation,green for down-regulation, and yellow for insignificant change). The node size reflects the connectivity degree of a node.

    1.5 Identification of hubs and modules in the PPI network of DEGs

    The Centiscape V2.2 plug-in[18]in Cytoscape was used to analyze the PPI network topology. For each node in the network, the values of three network centrality indexes (degree, betweenness, eigenvector)were calculated. For each centrality index, the average value over the whole PPI network was calculated, and the nodes with higher values than the average were selected as high-centrality subset. Therefore, three high-centrality subsets were obtained, corresponding to the above three centrality indexes. The VENNY 2.1 software (https://bioinfogp.cnb.csic.es/tools/venny/) was used to draw the Venn diagram; the genes in the intersection of three high-centrality subsets were selected as the main gene set. The main gene set was uploaded to STRING database again to construct PPI network, and the PPIs with weighted score≥0.4(namely, medium confidence) were screened out as the PPI network of main genes. CytoHubba plug-in[19]was used to calculate the connectivity of each main gene, and the top 15 genes with the highest connectivity were considered as hub nodes in the PPI network. The MCODE plug-in[20]was used to identify the major modules (K-Score>5) from the PPI network of main genes.

    2 Results and discussion

    2.1 Growth status and viability of D. radiodurans cells under UV irradiation stress

    After UV stress, the recovery ofD. radioduransusually goes through three stages: early recovery,middle recovery and late recovery[4]. To investigate the adaptation mechanism ofD. radioduransto UV stress, we measured the survival rates for the Normal and UV irradiation culture conditions (Table 1). The survival rate (UV/Normal) ofD. radioduranscells was about 76.6% after UV irradiation of dose 750 J/m2.

    Table 1 The viable counts per 100 μl of broth before and after UV irradiation

    2.2 Key functional genes related to UV irradiation stress

    Through transcriptome data analysis, we found that a total of 750 genes had significant changes (Fold change>2 andFDR<0.05) in expression level after UV irradiation. Table 2 lists the top 30 most significantly up-and down-regulated DEGs.Interestingly, the fold changes of the top 30 upregulated genes were all more than 8 times(log2FC>3), while the fold changes of the top 30 down-regulated genes were all less than 8 times(log2FC>-3), indicating that positive responses are more likely taken byD. radioduransunder UV irradiation. In addition, many previously reported genes that may be involved in damage repair inD. radioduranswere also identified in our study(Table 3), suggesting that cells utilize many repair processes in response to UV stress. We hope to further investigate the roles of these genes in the early repair of UV damage.

    Table 2 The 30 genes with most significant differences in expression after UV irradiation

    Table 3 The DEGs involved in the known damage repair pathways

    2.2.1 Response of the four replicons to UV irradiation stress

    The percentage of responsive genes in each of the four genomic components (replicons) of Chr1,Chr2, pMP1 and pSP1 was compared, and the percentage of responsive genes in pSP1 was the highest (40%). Among the 40 genes of pSP1, the expression levels of 16 genes were significantly changed and all of them were up-regulated, indicating that the small plasmid played an important role in the damage repair after UV irradiation. This point was also found in the repair after ionizing radiation, where most genes on pSP1 were significantly up-regulated in the middle and late stages of post-radiation recovery, and almost all genes were activated in the late stage of recovery[4].

    2.2.2 Non-homologous end joining (NHEJ) pathway participates in early UV damage repair

    DR_A0346 (pprA), DR_B0100 (ligB), DR_B0098 and DR_B0099 were significantly induced after UV irradiation. It was found thatpprAandligBwere involved in the non-homologous end joining(NHEJ) pathway inD. radiodurans.As observed by atomic force microscopy (AFM), PprA protein preferentially binds to DNA double-stranded ends,promotes the ligation of DNA ends catalyzed by ATP-dependent DNA ligase (DR_B0100), and protects it from degradation by exonuclease[21]. DR_B0098-DR_B0100 are three genes encoded in an operon,which may be directly involved in DNA repair[4].DR_B0098 contains a phosphatase domain of the HD hydrolase family and a polynucleotide kinase domain,and resembles a human protein with similar structure that plays an important role in DSB repair[22]. Studies have shown that in the presence of DR_B0098, PprA can stimulate LigB DNA ligase activity several times,and PprA and DR_B0098 proteins are essential for LigB function[23]. At 1.5 to 5 h of ionizing radiation,the expression level of each of these genes was induced 5 to 10 times[4]. Therefore, this pathway plays an important role in both UV damage repair and ionization damage repair.

    2.2.3 Single-strand annealing (SSA) pathway participates in early UV damage repair

    The genes involved in the single-stranded annealing (SSA) pathway were found to be: DR_0423(ddrA), DR_0070 (ddrB), DR_0003 (ddrC), DR_0326(ddrD), DR_A0346 (pprA) and DR_0100 (ssb). These genes were significantly induced in the early stage of UV irradiation. Some studies have found that the reaction products consistent with annealing mainly appear in the early stage after irradiation (i.e., 1.5 h)[4],which was also proved in this study, namely, genes related to SSA were significantly up-regulated in the early stage after UV irradiation, indicating that the SSA pathway was initiated for repair. DdrB protein can bind to single-stranded DNA and promotes annealing, and single-stranded binding protein (SSB)plays a promoting role in this pathway[24].Alternatively, DdrB may facilitate the accurate assembly of countless small fragments generated by extreme radiation exposureviaSSA, thereby generating suitable substrates for subsequent genomic reconstruction by the ESDSA pathway[25].

    2.2.4 Nucleotide excision repair (NER) pathway participates in early UV damage repair

    D. radioduranshas a strong tolerance to UV irradiation. Pyrimidine dimers produced by UV damage can interfere with the processes of DNA replication and transcription, while nucleotide excision repair (NER) can remove pyrimidine dimers to ensure normal cell life activities.Mfd,uvrA,uvrB,uvrC,uvrD,polA,ligA,uveE,yejH/rad25andsms/radAare genes involved in the NER pathway, among which DR_1771 (uvrA1), DR_2275 (uvrB) and DR_1354 (uvrC) are significantly different in expression level after UV irradiation. UvrABC complex plays an important role in nucleotide excision repair inD. radiodurans, and the NER pathway has a unique ability to clear DNA damage[26]. This complex recognizes and binds to the damage site and then cleaves, with about 4 nucleotides cleaved at the 3' end and 8 nucleotides cleaved at the 5' end. Finally, DNA is synthesized and the gap is completed under the actions of DNA polymerase and DNA ligase to complete DNA damage repair[27]. Compared with the previous results on the response to ionizing radiation[4], UvrA1 in the NER pathway was expressed at the early stage of UV irradiation and at 1.5 h after ionizing radiation; UvrA2 (DR_A0188)was not expressed at the early stage of UV irradiation,but expressed at 5 h after ionizing radiation; UvrB and UvrC in NER pathway were expressed after UV irradiation and at 3 h after ionizing radiation; UvrD was involved in NER, methylation-dependent mismatch repair (mMM) and SOS repair, and was not expressed after UV irradiation, but was expressed at 1.5 h after ionizing radiation. Two possible pathways for pyrimidine dimer clearance inD. radioduransare ultraviolet damage endonuclease UvsE-dependent excision repair (UVER) and nucleotide excision repair(NER). These two pathways make recovery from UV damage redundant. Some studies have shown that UvrA2 plays a minor role in UV resistance, and NER is more related to UV resistance than UVER[3], which is consistent with our transcriptomic findings, that is,no significant difference was found for UvrA2 and UvsE expression after UV irradiation. One possible explanation is that the contribution of NER and UVER pathways to UV resistance is related to the growth stage[28], which needs to be further investigated.

    2.2.5 Superhelix related genes play a role in the repair of early UV damage

    GyrA and GyrB are two DNA-gyrase subunits inD. radiodurans, both of which are significantly induced after UV irradiation, indicating that the regulation of DNA supercoiling is important for DNA repair, and GyrA and GyrB may be nucleoid associated proteins (NAPs) ofD. radiodurans[29].Both subunits were also found to be induced after ionizing radiation[4], in which GyrA protein is essential for DNA replication and genome reconstruction after severe ionizing radiation exposure[30].

    2.2.6 Mismatch repair (MMR) system participates in the repair of early UV damage

    MutS2 and MutS1 are involved in the base mismatch repair system and are significantly induced after UV irradiation. MutS recognizes and binds to base mismatch[31]. MutS1 is a key protein involved in mismatch repair system, which can ensure the replication and recombination fidelity ofD. radiodurans[32], while MutS2 is involved in RecA-independent repair mechanism inD. radiodurans,which enhances the resistance of cells to oxidative stress-induced DNA damage, so as to make organisms exhibit remarkable DNA repair capabilities[33].

    2.2.7 Homologous recombination (HR) repair is involved in DNA repair

    RecA protein is a recombinant enzyme with unwinding ability, which plays an important role in homologous recombination (HR) and extended synthesis-dependent strand annealing (ESDSA). RecA is essential for genome recovery after irradiation, and its induction is considered to be a major marker for the initiation of homologous recombination[34]. In the present study, RecA protein was only induced more than two-fold at the early stage of UV irradiation(Table 3). Previous studies have found that RecA-dependent homologous recombination occurs 5 h or more after irradiation[4], so it is possible that this protein is not sufficiently induced in the early stage of UV irradiation, and other genes related to homologous recombination are also rarely induced. In addition,there are RuvA/B/C proteins inD. radiodurans,whose functions are similar to the DNA branch transferase and dissociase involved in homologous recombination of DNA in human cells, and among them two proteins (RuvB, RuvC) were significantly induced (Table 3).

    2.2.8 Other key functional genes related to UV irradiation stress

    In addition, our study also found that DR_1262(rsr), an important gene against UV stress, was significantly up-regulated. Rsr protein is an RNA-binding protein. Studies have shown that Rsr protein can bind some small RNA produced in cells after UV irradiation and improve the resistance of cells to UV irradiation[35].

    DR_0928 in the base excision repair (BER)pathway, which is expressed in the early and middle stages of ionizing radiation[4], is also induced after UV irradiation (Table 3). RecA and LexA proteins are involved in SOS repair[22], which are induced at 1.5 h after ionizing radiation[4], are also induced after UV irradiation (Table 3). However,D. radioduransdoes not possess a functional SOS response system, so this remains to be investigated.

    ABC transporters (DR_1356-DR_1359) are highly induced and may be involved in the transshipment of damaged products, and these transporters are also highly induced after ionizing radiation[4]. Up-regulated kinases of unknown characteristics (DR_2467, DR_0394) may be involved in a variety of cellular processes, and when expressed inEscherichia coli, these uncharacterized kinases affect DNA topology[22]. Several genes encoding proteins from the extended family of stress response(DinB family) were also induced in early UV irradiation, including DR_1263 and two members of this family (DR_0053, DR_0841), which were strongly induced in a RecA-like manner, supporting their important role in UV irradiation response.

    From Table 3, it can be found that among the 20 DEGs, there are 6 genes involved in the SSA pathway,3 genes involved in the NER pathway, 2 genes involved in the NHEJ pathway and 4 genes involved in the HR pathway. Among the top 30 up-regulated genes (Table 2), in addition to genes with known functions, some genes with unknown functions expressed, including DR_1143, DR_RS02180,DR_RS05900, DR_1977, DR_0206, and DR_RS05895, indicating that these genes also play an important role in the response to UV irradiation,which should be the focus of future research.

    2.2.9 PprI/DdrO mediated regulatory relationship

    A large number of proteins inD. radioduranswere significantly up-regulated in the early recovery from UV irradiation, among which PprI/DdrO mediated regulatory proteins were the most prominent. RDRM is a conserved palindromic sequence that exists upstream of many radiationinduced genes (e.g.,ddrO, ddrA, ddrB, ddrC, ddrD,ddrF, ddrR, recA, pprA, gyrA, gyrB, ssb, recQ, ruvB,uvrA, uvrB, uvrD). It has been found thatddrOacts as a repressor by binding to RDRM, the promoter that appears to be located at or very close to radiationinduced genes, includingddrOitself. However, PprI(IrrE) is a metalloproteinase required for the hydrolytic inactivation of DdrO protein to deactivate the suppressor gene, a process that is somehow activated after exposure ofD. radioduransto radiation[36]. IrrE-dependent cleavage of the DdrO,and hence up-regulation of RNA-dependent RNA polymerase (RDR), is essential for radiation tolerance.The genes that were significantly induced include theDeinococcus-specific genes (ddrO, ddrA, ddrB, ddrC,ddrD, ddrF, ddrR, pprA), DNA repair genes (recA,recQ, ruvB, uvrA, uvrB, uvrD, ssb), DNA superhelix genes (gyrA, gyrB) (Figure 1), among whichpprAis inhibited bypprMandlexA2[37-38], andrecAis inhibited byrecX[39].

    Fig. 1 PprI/DdrO mediated regulatory relationships in D. radiodurans

    2.3 Functional enrichment analysis of DEGs under UV irradiation

    For the selected criteria (log2(Fold change)>1 andFDR<0.05), there were 750 DEGs (Figure 2a),among which 544 genes were up-regulated and 206 genes were down-regulated, and the number of upregulated genes was more than twice that of downregulated genes. It seems that the tolerance ofD. radioduransto UV irradiation mainly depends on the enhancement of most stress response activities under irradiation stress to maintain cell survival and repair damage, rather than the slowing down or even stagnation of normal physiological activities. To determine the biological functions of DEGs under UV irradiation stress, we performed KEGG (Figure 2b)and GO (Figure 2c, d) functional enrichment analysis.To ensure the accuracy of the results, the hypothetical proteins in the protein functional annotation ofD. radioduranswere removed before enrichment analysis.

    Fig. 2 Differentially expressed genes and their enrichment

    According to the GO enrichment results, damage repair responses, including SOS response and DNA recombination repair, appeared inD. radioduranswhen exposed to UV irradiation stress. The translation process was enhanced, the translocation of translationrelated carboxylic acid and amino acid was enhanced,and the ribosome, rRNA, tRNA and other related genes were significantly up-regulated, presumably due to the massive synthesis of damage repair related proteins. ATP-binding and ATP-dependent activities were enhanced to maintain normal physiological status and to supply more energy for damage repair pathways. DNA binding is enhanced, due to the proteins in the damage repair pathway, such as DNA ligase LigB, DNA damage repair protein DdrB, singlestrand binding protein SSB, UvrABC component protein, and mismatch repair protein MutS, need to bind to DNA for damage repair. When exposed to UV irradiation, the number of down-regulated genes was significantly less than that of up-regulated genes, and mainly concentrated on the reduction of redox reactions: oxidoreductase activity in redox reactions decreased, cytochrome complex assembly decreased,and heme synthesis decreased. Cytochrome is involved in redox reactions with heme as a prosthetic group. Redox reactions provide energy for life activities, which is essentially the transfer of electrons and an important way of energy transfer. Therefore,UV irradiation has an impact on the energy metabolism ofD. radiodurans.

    KEGG pathway enrichment analysis was performed on DEGs to investigate the involved metabolic pathways (Figure 2b). Genes related to the ABC transporters pathway were significantly upregulated, while genes related to the biosynthesis of ubiquinone and other terpenoid-quinone and phenylalanine metabolism were significantly downregulated. Studies have found that ABC transporters inD. radioduransplay an important role in the supply of exogenous amino acids, and ABC transporters can be used for the uptake of amino acids and peptides[40].When adapting to irradiation pressure, bacterial cells need various ions and amino acids to synthesize corresponding proteins, which can explain the significant up-regulation of this metabolic pathway.On the other hand, it also indicates thatD. radioduranshas a certain ability to adapt to UV irradiation pressure. Ubiquitin, also known as coenzyme Q, is a hydrogen transporter in the respiratory chain and plays a role in electron transport. The decrease of ubiquitin synthesis corresponds to the decrease of redox reactions in the GO enrichment analysis, which jointly indicates that UV irradiation has an impact on the energy metabolism ofD. radiodurans.

    2.4 Validation of DEGs by RT-qPCR

    To further validate the expression level changes of DEGs in response to UV irradiation, 3 key functional genes inD. radiodurans, namely,DR_B0100 (RNA ligase family protein LigB),DR_1262 (TROVE domain-containing protein Rsr)and DR_2275 (excinuclease ABC subunit UvrB),were selected for RT-qPCR analysis. The results showed that the changes in expression level of the 3 genes after UV irradiation are consistent with the transcriptome sequencing results (Figure 3),indicating that the transcriptome data are reliable.

    Fig. 3 RT-qPCR results of 3 DEGs in response to UV irradiation

    2.5 Similarities and differences of DEGs under the three stress conditions of UV irradiation,ionizing radiation and desiccation

    The intersection (Figure 4) of DEGs (Fold change>3) under UV irradiation with ionizing radiation and desiccation stresses was taken to obtain the genes listed in Table S2. These genes can be broadly classified into the following categories: DNA metabolism, energy acquisition, putative regulatory proteins, RNA metabolism, protein synthesis,transport, specific genes of theDeinococcusgenus,and other genes[41]. We identified the common responsive genes (shown in bold fonts in Table S2)under UV irradiation, ionizing radiation and desiccation stresses. These genes are mainly concentrated in DNA metabolism, RNA metabolism andDeinococcus-specific genes. It seems that these are common molecular responses ofD. radioduransto UV, ionization and desiccation stresses. DNA metabolism includes some repair genes and superhelix related genes. Some genes were common response genes of UV irradiation and ionizing radiation but did not participate in the response to desiccation stress,while others were common response genes of UV irradiation and desiccation but did not participate in the response to ionizing radiation. These genes need to be further analyzed.

    Fig. 4 Venn diagram of DEGs in response to UV irradiation,ionizing radiation and desiccation stresses

    It has been reported that there is a strong correlation between desiccation resistance and γ-radiation resistance inD. radiodurans[5]; both radiation (3 kGy) and desiccation stresses induced 5 genes, and we found that UV irradiation induced high expression of the same 5 genes: DR_0423 (ddrA),DR_0070 (ddrB), DR_0003 (ddrC), DR_0326 (ddrD)and DR_A0346 (pprA). These 5 genes correspond to GO terms: “Response to Gamma radiation (GO:0010332)” and “Cellular response to Desiccation(GO:0071465)”. This indicates a strong correlation between UV irradiation and resistance to desiccation and γ-radiation, and since these five proteins are involved in the SSA pathway, it is speculated that the common properties of resistance to desiccation,γ-radiation and UV irradiation may be related to the SSA pathway.

    2.6 Hub and module analysis in the PPI network of DEGs

    The protein-protein interaction (PPI) network ofD. radioduranswas downloaded from the STRING database[16], and the DEGs related subnetwork was screened out. Unconnected nodes were deleted from the network. The initial PPI network consisted of 2 747 nodes and 100 965 edges (Figure 5). From the initial network, we can see that there are many genes,although they are not DEGs themselves, but they interact with DEGs, and these genes may still play an important role in the response to UV irradiation.

    To obtain the main PPI network from the initial network, we performed the following analysis. Firstly,nodes whose connectivity degree, betweenness and eigen vector values were higher than the average of the network were selected and their intersection was taken as the main gene set, resulting in a total of 336 nodes (Figure 6a). Next, these 336 main genes were submitted to the STRING database to construct a PPI network. Finally, unconnected nodes were removed from this network and nodes with interaction scores greater than 0.4 (medium confidence) were selected to obtain the main PPI network (Figure 6b), which consists of 314 nodes and 3 924 edges.

    Fig. 6 Hubs and modules in the main PPI network of DEGs

    To analyze the main PPI network, we used the CytoHubba plug-in[19]for Cytoscape[17]to identify the top 15 hub genes with high connectivity (Figure 6c)and rank them as follows:polA(degree=164),metG(degree=138),guaA(degree=136), DR_0603 (degree=134),dnaK(degree=134),guaB(degree=128),pheT(degree=126), DR_0183 (degree=122), DR_2168(degree=104),RecA(degree=98),fusA(degree=90),rpsG(degree=86),tsf(degree=82),rpoZ(degree=82),secD(degree=82). The names and functions of these hub genes are shown in Table 4. Subsequently, GO functional enrichment analysis was performed for these hub genes (Figure 7b), and according to the results, the most enriched GO biological processes(BP) include: GMP biosynthetic process, doublestrand break repair, translation; the most enriched GO molecular functions (MF) include: catalytic activity,acting on a nucleic acid, nucleic acid binding.

    Fig. 7 Function enrichment analysis

    Four major modules were discovered through the MCODE plug-in of Cytoscape (Figure 6d).Subsequently, GO function and KEGG pathway enrichment analysis were performed for genes in these four modules (Figure 7a), and according to the results, the most enriched GO biological processes include: translational initiation, DNA topological change, macromolecule biosynthetic process, cellular nitrogen compound biosynthetic process; the most enriched KEGG pathways include: RNA degradation,biosynthesis of cofactors, porphyrin metabolism,bacterial secretion system, DNA replication, protein export.

    3 Conclusion

    D. radioduransis the most radiation-resistant organism ever discovered on earth. Our study found thatD. radioduransnot only has the common DNA damage repair genes of general prokaryotes and eukaryotes, but also has some special multidomain gene families in the repair of UV irradiation damage.Through transcriptomic analysis, according to different DNA repair pathways, we explored genes or enzymes related to DNA repair in the early response to UV irradiation, especially the genes that may play a key role in DNA repair, such aspprA,ddrA-D,rsr,etc. Our results show pathways (SSA, NHEJ, NER,HR) that may be involved in early damage repair and genes that play important roles, and suggest that these genes may be regulated by PprI/DdrO. By comparing with ionizing radiation and desiccation stresses, the common molecular responses to the three stresses were found, which mainly involved DNA metabolism,RNA metabolism andDeinococcus-specific genes.The PPI network of DEGs was constructed, and hub genes includingpolA,metG,guaA, DR_0603,dnaK,guaB,pheT, DR_0183, DR_2168,recA,fusA,rpsG,tsf,rpoZ,secDwere identified. These genes also played important roles in the response to UV irradiation stress. At the same time, some highly expressed genes with unknown functions may also play a key role in UV irradiation resistance.Therefore, further elucidation of the functions and regulatory mechanisms of these genes is crucial for understanding the extreme resistance and damage repair mechanisms ofD. radiodurans.

    SupplementaryAvailable online (http://www.pibb.ac.cn or http://www.cnki.net):

    PIBB_20220471_Table S1.pdf

    PIBB_20220471_Table S2.pdf

    Data AccessibilityThe transcriptome data generated in this work were deposited in the National Genomics Data Center (NGDC) with accession number CRA009109. The data were also uploaded to the National Center for Biotechnology Information(NCBI) Sequence Read Archive (SRA) under the accession numbers SRR22460685 to SRR22460690.

    极品教师在线视频| 搡老熟女国产l中国老女人| 男插女下体视频免费在线播放| 国产91精品成人一区二区三区| 白带黄色成豆腐渣| 成人亚洲精品av一区二区| 精品久久久久久久末码| 91麻豆精品激情在线观看国产| 亚洲一区二区三区色噜噜| 国产色爽女视频免费观看| bbb黄色大片| 久久精品影院6| 免费在线观看影片大全网站| 少妇的逼水好多| 国产成人aa在线观看| 精品久久久久久久久av| 黄色日韩在线| 男女那种视频在线观看| 高清毛片免费观看视频网站| 久久99热这里只有精品18| 在线a可以看的网站| 久久久久精品国产欧美久久久| 亚洲欧美清纯卡通| 免费高清视频大片| 三级男女做爰猛烈吃奶摸视频| 两人在一起打扑克的视频| 国产成人福利小说| 亚洲欧美日韩无卡精品| 久久国产精品影院| 中文字幕免费在线视频6| 18禁黄网站禁片午夜丰满| 天堂动漫精品| 国产亚洲精品久久久久久毛片| 中文字幕人妻熟人妻熟丝袜美| 国模一区二区三区四区视频| 午夜影院日韩av| 中文字幕av在线有码专区| 国产视频内射| 亚洲最大成人av| 99国产极品粉嫩在线观看| 国产探花极品一区二区| 免费在线观看亚洲国产| 嫩草影院精品99| 午夜免费成人在线视频| 国产欧美日韩精品一区二区| 亚洲成人久久爱视频| 亚洲成av人片免费观看| or卡值多少钱| 男人舔奶头视频| 9191精品国产免费久久| 欧美日韩亚洲国产一区二区在线观看| 偷拍熟女少妇极品色| 欧美又色又爽又黄视频| 亚洲专区国产一区二区| 国产欧美日韩精品一区二区| 久久久久性生活片| 一边摸一边抽搐一进一小说| 变态另类丝袜制服| 欧美bdsm另类| 91久久精品电影网| 欧美日韩国产亚洲二区| 国产精品一区二区免费欧美| 最近在线观看免费完整版| 性色av乱码一区二区三区2| 色综合亚洲欧美另类图片| 好男人电影高清在线观看| 久久久久免费精品人妻一区二区| 久久伊人香网站| 桃色一区二区三区在线观看| 亚洲精华国产精华精| 我要看日韩黄色一级片| av国产免费在线观看| 国产精品亚洲一级av第二区| 国产伦一二天堂av在线观看| 国内精品久久久久精免费| 男人狂女人下面高潮的视频| 精品欧美国产一区二区三| 99久久无色码亚洲精品果冻| 少妇的逼好多水| 啪啪无遮挡十八禁网站| 人妻制服诱惑在线中文字幕| 午夜久久久久精精品| 国产日本99.免费观看| 欧美成人a在线观看| 国产精品日韩av在线免费观看| 3wmmmm亚洲av在线观看| 999久久久精品免费观看国产| 内射极品少妇av片p| 黄色丝袜av网址大全| 美女 人体艺术 gogo| 69av精品久久久久久| 九九热线精品视视频播放| 国产一区二区三区视频了| 午夜激情欧美在线| 久久久成人免费电影| 久久久久久久亚洲中文字幕 | 亚洲五月天丁香| 我的老师免费观看完整版| 久久精品国产99精品国产亚洲性色| 久久久久久大精品| 日本黄色视频三级网站网址| 精品99又大又爽又粗少妇毛片 | 欧美激情国产日韩精品一区| 国产精品嫩草影院av在线观看 | 午夜免费成人在线视频| 色哟哟哟哟哟哟| 一级黄色大片毛片| 在线十欧美十亚洲十日本专区| 9191精品国产免费久久| 99热精品在线国产| 日韩欧美国产在线观看| 国产久久久一区二区三区| 蜜桃亚洲精品一区二区三区| 久9热在线精品视频| 国产亚洲精品久久久com| 免费搜索国产男女视频| 乱人视频在线观看| 亚洲熟妇中文字幕五十中出| 日本 av在线| 国产野战对白在线观看| 国产精品影院久久| 麻豆久久精品国产亚洲av| 亚洲精品在线美女| 国内精品美女久久久久久| 精品午夜福利视频在线观看一区| 亚洲七黄色美女视频| 综合色av麻豆| 国产精品一区二区三区四区久久| 嫩草影院新地址| 精品国产亚洲在线| 亚洲成av人片在线播放无| 久久午夜亚洲精品久久| 精品免费久久久久久久清纯| 久久精品国产清高在天天线| 激情在线观看视频在线高清| 在线免费观看不下载黄p国产 | 美女免费视频网站| 国产精品综合久久久久久久免费| 黄色一级大片看看| 国产精品人妻久久久久久| 国产在视频线在精品| 成人鲁丝片一二三区免费| 1024手机看黄色片| 午夜福利在线观看吧| 18禁在线播放成人免费| 亚洲熟妇中文字幕五十中出| 18禁黄网站禁片免费观看直播| 波多野结衣高清无吗| 每晚都被弄得嗷嗷叫到高潮| h日本视频在线播放| 欧美日韩国产亚洲二区| 怎么达到女性高潮| 久久人妻av系列| 乱码一卡2卡4卡精品| 亚洲美女搞黄在线观看 | av专区在线播放| 内地一区二区视频在线| 久久99热6这里只有精品| 性色avwww在线观看| 久久久久久久精品吃奶| 嫩草影院精品99| 看黄色毛片网站| 99精品久久久久人妻精品| 午夜福利高清视频| 免费无遮挡裸体视频| 欧美不卡视频在线免费观看| 黄色一级大片看看| 亚洲av五月六月丁香网| 别揉我奶头 嗯啊视频| 亚洲七黄色美女视频| 美女xxoo啪啪120秒动态图 | 99久久精品国产亚洲精品| 久久午夜亚洲精品久久| 99riav亚洲国产免费| 嫁个100分男人电影在线观看| 日韩av在线大香蕉| 一区二区三区四区激情视频 | 色在线成人网| 神马国产精品三级电影在线观看| av天堂在线播放| av在线观看视频网站免费| 亚洲精品在线观看二区| 国产午夜精品久久久久久一区二区三区 | 欧美zozozo另类| 国产精品精品国产色婷婷| 免费电影在线观看免费观看| 九九在线视频观看精品| 乱码一卡2卡4卡精品| 国产精品美女特级片免费视频播放器| av在线观看视频网站免费| 国产熟女xx| 两人在一起打扑克的视频| 日韩有码中文字幕| 午夜福利免费观看在线| 日韩欧美国产一区二区入口| 91狼人影院| 亚洲久久久久久中文字幕| 99在线视频只有这里精品首页| 国产真实乱freesex| 非洲黑人性xxxx精品又粗又长| 一级黄片播放器| 国产成人欧美在线观看| 久久精品国产99精品国产亚洲性色| 国产淫片久久久久久久久 | 欧美成人a在线观看| 国模一区二区三区四区视频| 午夜精品久久久久久毛片777| 国产成人av教育| 成人av在线播放网站| 欧美日韩福利视频一区二区| x7x7x7水蜜桃| 精品久久久久久久久久久久久| 男女那种视频在线观看| 亚洲欧美日韩高清在线视频| 精品久久久久久久人妻蜜臀av| 国产在线男女| 9191精品国产免费久久| 欧美激情在线99| 亚洲国产欧洲综合997久久,| 久久久久久国产a免费观看| 亚洲国产欧美人成| 舔av片在线| 一级毛片久久久久久久久女| 舔av片在线| 精品久久久久久久久av| 不卡一级毛片| 1000部很黄的大片| 人人妻,人人澡人人爽秒播| 身体一侧抽搐| 日本免费一区二区三区高清不卡| 亚洲 国产 在线| 欧美又色又爽又黄视频| 在线观看一区二区三区| 免费电影在线观看免费观看| 国产一区二区激情短视频| 欧美性感艳星| 男女视频在线观看网站免费| 性插视频无遮挡在线免费观看| 久久久久国内视频| 亚洲欧美日韩高清专用| av女优亚洲男人天堂| 夜夜夜夜夜久久久久| 免费黄网站久久成人精品 | 国语自产精品视频在线第100页| 三级国产精品欧美在线观看| 亚洲真实伦在线观看| 3wmmmm亚洲av在线观看| 99久久精品热视频| 亚洲18禁久久av| 最近视频中文字幕2019在线8| 久久6这里有精品| 国产成人影院久久av| 99热6这里只有精品| 一卡2卡三卡四卡精品乱码亚洲| 老女人水多毛片| 免费av观看视频| 麻豆成人av在线观看| 好男人电影高清在线观看| 国产精品98久久久久久宅男小说| 国产精品一区二区免费欧美| 欧美最黄视频在线播放免费| 又黄又爽又免费观看的视频| 97碰自拍视频| 久久精品国产亚洲av香蕉五月| 国产欧美日韩精品亚洲av| 51午夜福利影视在线观看| 精品欧美国产一区二区三| 男人狂女人下面高潮的视频| 人妻丰满熟妇av一区二区三区| 少妇熟女aⅴ在线视频| 国产精品一区二区性色av| 丁香欧美五月| 床上黄色一级片| 国产精品久久久久久人妻精品电影| 国产淫片久久久久久久久 | 国产精品电影一区二区三区| 国产精品1区2区在线观看.| 午夜免费成人在线视频| 88av欧美| 亚洲av熟女| 中文字幕精品亚洲无线码一区| 深爱激情五月婷婷| 长腿黑丝高跟| 男女之事视频高清在线观看| 国产真实乱freesex| 丰满人妻熟妇乱又伦精品不卡| 美女被艹到高潮喷水动态| 国产精品综合久久久久久久免费| 国产精品,欧美在线| 制服丝袜大香蕉在线| 99在线视频只有这里精品首页| 成人亚洲精品av一区二区| 午夜日韩欧美国产| .国产精品久久| 亚洲自拍偷在线| 综合色av麻豆| 1024手机看黄色片| 精品久久久久久成人av| 韩国av一区二区三区四区| 日本黄大片高清| 国产高清激情床上av| 黄色视频,在线免费观看| 亚洲成av人片免费观看| 免费黄网站久久成人精品 | 精品国产三级普通话版| 噜噜噜噜噜久久久久久91| 一本一本综合久久| 内地一区二区视频在线| 一本综合久久免费| 精品人妻偷拍中文字幕| 宅男免费午夜| 18禁裸乳无遮挡免费网站照片| 亚洲av不卡在线观看| 熟妇人妻久久中文字幕3abv| 日韩人妻高清精品专区| 日本精品一区二区三区蜜桃| 亚洲精华国产精华精| 成熟少妇高潮喷水视频| 国产伦在线观看视频一区| 欧美+亚洲+日韩+国产| 亚洲18禁久久av| 午夜免费激情av| 亚洲不卡免费看| 欧美乱色亚洲激情| 国产三级中文精品| 国产高清视频在线观看网站| 欧美成人一区二区免费高清观看| 老司机福利观看| 国产探花极品一区二区| 久久国产乱子伦精品免费另类| 国产精品,欧美在线| 中国美女看黄片| 国产成人啪精品午夜网站| 亚洲va日本ⅴa欧美va伊人久久| 日韩人妻高清精品专区| 免费高清视频大片| 久久久久国内视频| 色尼玛亚洲综合影院| 国产白丝娇喘喷水9色精品| 日本与韩国留学比较| 90打野战视频偷拍视频| 麻豆国产av国片精品| 亚洲人成网站高清观看| 免费av不卡在线播放| avwww免费| 成人午夜高清在线视频| 两人在一起打扑克的视频| 在线国产一区二区在线| 成人av一区二区三区在线看| 91麻豆精品激情在线观看国产| 欧美成人a在线观看| 蜜桃亚洲精品一区二区三区| 熟妇人妻久久中文字幕3abv| 日本与韩国留学比较| 欧美一区二区国产精品久久精品| 久久久国产成人免费| 精品久久久久久久久久免费视频| 在线观看66精品国产| 九九久久精品国产亚洲av麻豆| 免费人成视频x8x8入口观看| 熟女人妻精品中文字幕| 欧美另类亚洲清纯唯美| 好看av亚洲va欧美ⅴa在| 精品一区二区三区人妻视频| 丰满人妻一区二区三区视频av| 久久午夜亚洲精品久久| 午夜精品一区二区三区免费看| 国产极品精品免费视频能看的| 国产欧美日韩一区二区三| 天天躁日日操中文字幕| 免费黄网站久久成人精品 | 国产国拍精品亚洲av在线观看| 国产精华一区二区三区| 九色国产91popny在线| 久久香蕉精品热| 国产精品久久久久久精品电影| 老鸭窝网址在线观看| 直男gayav资源| 伦理电影大哥的女人| 成人三级黄色视频| 免费观看人在逋| 最新在线观看一区二区三区| 中文资源天堂在线| 国产男靠女视频免费网站| 欧美性猛交黑人性爽| 99久久精品国产亚洲精品| 9191精品国产免费久久| 99国产极品粉嫩在线观看| 我要搜黄色片| 极品教师在线视频| 国产精品一区二区三区四区免费观看 | 午夜福利在线在线| 亚洲无线在线观看| 99热6这里只有精品| 成人精品一区二区免费| ponron亚洲| 亚洲精品色激情综合| 午夜福利视频1000在线观看| 1024手机看黄色片| 亚洲成av人片免费观看| 久久精品影院6| 欧美黄色片欧美黄色片| 国产乱人视频| 在线十欧美十亚洲十日本专区| 91字幕亚洲| 长腿黑丝高跟| 欧美xxxx性猛交bbbb| 搡老熟女国产l中国老女人| 少妇被粗大猛烈的视频| 久久久国产成人免费| 国产精品乱码一区二三区的特点| 97碰自拍视频| 最近中文字幕高清免费大全6 | 一区二区三区激情视频| 国产国拍精品亚洲av在线观看| 精品无人区乱码1区二区| 直男gayav资源| 国产午夜精品论理片| 免费看a级黄色片| 欧美黄色片欧美黄色片| 久久久色成人| 波多野结衣巨乳人妻| 亚洲精品一区av在线观看| 女人十人毛片免费观看3o分钟| 久久久久精品国产欧美久久久| 身体一侧抽搐| av在线天堂中文字幕| 亚洲人成网站在线播| 日韩中文字幕欧美一区二区| www.999成人在线观看| 一级黄片播放器| 亚洲欧美日韩东京热| 亚洲综合色惰| 久9热在线精品视频| 亚洲,欧美,日韩| 久久久久久久久大av| 别揉我奶头~嗯~啊~动态视频| 色5月婷婷丁香| 男女做爰动态图高潮gif福利片| 欧美成狂野欧美在线观看| 中文资源天堂在线| 国产一区二区在线观看日韩| 十八禁人妻一区二区| 成人精品一区二区免费| 男女下面进入的视频免费午夜| 男插女下体视频免费在线播放| 成人欧美大片| 亚洲欧美日韩无卡精品| 国产精品久久视频播放| 美女被艹到高潮喷水动态| 欧美又色又爽又黄视频| 亚洲aⅴ乱码一区二区在线播放| 直男gayav资源| 久久国产乱子免费精品| 国产欧美日韩精品亚洲av| 久久国产乱子免费精品| 久久精品综合一区二区三区| aaaaa片日本免费| 免费无遮挡裸体视频| 丰满人妻一区二区三区视频av| 成人性生交大片免费视频hd| 99热这里只有是精品50| 婷婷精品国产亚洲av在线| av视频在线观看入口| 十八禁国产超污无遮挡网站| 婷婷精品国产亚洲av在线| 老鸭窝网址在线观看| 免费在线观看日本一区| 97碰自拍视频| 色播亚洲综合网| 丁香六月欧美| 国产精品久久久久久亚洲av鲁大| 国内毛片毛片毛片毛片毛片| 蜜桃久久精品国产亚洲av| 国产高清视频在线播放一区| 精品免费久久久久久久清纯| 观看免费一级毛片| 免费看a级黄色片| 十八禁人妻一区二区| 色综合婷婷激情| 黄色女人牲交| 三级毛片av免费| 国产精品国产高清国产av| 波多野结衣高清作品| 久久精品国产99精品国产亚洲性色| 中文亚洲av片在线观看爽| 噜噜噜噜噜久久久久久91| 欧洲精品卡2卡3卡4卡5卡区| 午夜福利高清视频| 一个人观看的视频www高清免费观看| 夜夜躁狠狠躁天天躁| 日韩人妻高清精品专区| 嫩草影院入口| av专区在线播放| av欧美777| 熟女人妻精品中文字幕| 99久久九九国产精品国产免费| 长腿黑丝高跟| 国产v大片淫在线免费观看| av在线蜜桃| 欧美zozozo另类| 中文字幕久久专区| 美女大奶头视频| 99在线视频只有这里精品首页| 国产精品乱码一区二三区的特点| 男女床上黄色一级片免费看| 亚洲av不卡在线观看| 久久久久久九九精品二区国产| 老鸭窝网址在线观看| 国产精品人妻久久久久久| 香蕉av资源在线| 久久精品国产99精品国产亚洲性色| 国产一级毛片七仙女欲春2| 日本免费a在线| av专区在线播放| 免费av毛片视频| 国产精品亚洲av一区麻豆| 999久久久精品免费观看国产| а√天堂www在线а√下载| 99精品在免费线老司机午夜| 久久亚洲精品不卡| 99久久精品国产亚洲精品| 午夜福利在线观看免费完整高清在 | 亚洲专区国产一区二区| 国产精品自产拍在线观看55亚洲| 亚洲,欧美精品.| 午夜福利成人在线免费观看| 精品一区二区三区人妻视频| 搡老妇女老女人老熟妇| 国产精品野战在线观看| 99久久精品热视频| 男插女下体视频免费在线播放| 国产探花在线观看一区二区| 国产蜜桃级精品一区二区三区| 国产精品久久电影中文字幕| 久久国产乱子伦精品免费另类| 午夜激情福利司机影院| 精品福利观看| 最近视频中文字幕2019在线8| 免费观看精品视频网站| 国产精品久久久久久精品电影| 久久久国产成人精品二区| 老熟妇仑乱视频hdxx| 桃红色精品国产亚洲av| 欧美激情久久久久久爽电影| 国产精品爽爽va在线观看网站| av在线老鸭窝| 一a级毛片在线观看| 麻豆av噜噜一区二区三区| 日韩免费av在线播放| 免费观看的影片在线观看| 一个人看的www免费观看视频| 亚洲av第一区精品v没综合| 中文字幕av在线有码专区| www.999成人在线观看| 色尼玛亚洲综合影院| 久久亚洲精品不卡| 一级av片app| 久久欧美精品欧美久久欧美| 久9热在线精品视频| 中文亚洲av片在线观看爽| 91久久精品国产一区二区成人| 午夜视频国产福利| 亚洲av成人av| 国产色爽女视频免费观看| 日本一本二区三区精品| 少妇丰满av| 一级av片app| 亚洲 国产 在线| 麻豆成人午夜福利视频| 亚洲电影在线观看av| 一区二区三区免费毛片| 一个人看的www免费观看视频| 女人十人毛片免费观看3o分钟| 亚洲一区二区三区不卡视频| 亚洲最大成人av| 亚洲中文日韩欧美视频| 女生性感内裤真人,穿戴方法视频| 亚洲欧美日韩高清在线视频| 国产私拍福利视频在线观看| 欧美国产日韩亚洲一区| 午夜福利欧美成人| 免费在线观看亚洲国产| 久久热精品热| 简卡轻食公司| 我的老师免费观看完整版| a在线观看视频网站| 国产精品1区2区在线观看.| 狠狠狠狠99中文字幕| 少妇熟女aⅴ在线视频| 91在线精品国自产拍蜜月| 91九色精品人成在线观看| 噜噜噜噜噜久久久久久91| 真人做人爱边吃奶动态| 天堂√8在线中文| 亚洲三级黄色毛片| 欧美精品国产亚洲| av在线老鸭窝| 在线免费观看的www视频| 亚洲天堂国产精品一区在线| 一进一出好大好爽视频| av在线老鸭窝| 精品午夜福利在线看| 国内精品久久久久精免费| 亚洲精品色激情综合| 久久久久久久亚洲中文字幕 | 久久99热6这里只有精品| 国产精品影院久久| 桃色一区二区三区在线观看| 午夜福利18| 亚洲精品456在线播放app | 日韩免费av在线播放| 熟女人妻精品中文字幕| 日韩人妻高清精品专区| 精品无人区乱码1区二区|