• <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 ?

    Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity

    2022-10-25 06:26:48JunLiuJinhuiChenLinSunJiaweiSuQinLiShihuiYangJianhuaZhangWenqing
    Rice Science 2022年6期

    Lü Jun, Liu Jinhui, Chen Lin, Sun Jiawei, Su Qin, Li Shihui, Yang Jianhua, Zhang Wenqing

    Research Paper

    Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity

    Lü Jun#, Liu Jinhui#, Chen Lin, Sun Jiawei, Su Qin, Li Shihui, Yang Jianhua, Zhang Wenqing

    (State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; These authors contributed equally to this work)

    MicroRNAs (miRNAs) can participate in plant-insect interactions, which regulate plant defense networks. In this study, we analyzed the miRNA expression profiles of six rice varieties before and after brown planthopper (BPH)-feeding. We identified 45 differentially expressed miRNAs between BPH- susceptible and BPH-resistant rice varieties and 144 miRNAs that responded to BPH-feeding. Thus, miRNAs may be involved in multiple pathways regulating rice defense response against BPH. In addition, we found that the genetic history of rice varieties determined the regulation mode of the miRNA and affected the amounts, types, changing trends and response periods of miRNAs in response to BPH- feeding. To conclude, we scanned seven potential cross-kingdom miRNAs, of which miR5795 may target thegene in BPH, causing a 16.07% reduction in BPH oviposition. The results provide new miRNA information of rice-BPH interactions and BPH-resistant rice variety breeding.

    ; insect-resistant rice; miR5795; fecundity

    Brown planthopper (BPH) is one of the most devastating pests, severely reducing rice yield (Jena and Kim, 2010). Currently, chemical pesticides are often used to control BPH (Wuet al, 2018). However, insecticides have caused several ecological and environmental problems in recent years (Baoet al, 2012). Therefore, the cultivation of resistant rice varieties has ecological benefits and is the most economical and environ- mentally friendly measure for BPH control (Yang and Zhang, 2016).

    After long-term adaptation, rice has gradually developed mechanisms to defend BPH attacks (Chenet al, 2012). It is generally believed that rice resistance to BPH is controlled by resistance genes (Chenget al, 2013; Fujitaet al, 2013). Currently, around 40 major BPH-resistance genes have been identified in rice (Akankshaet al, 2019). Nine genes,,/,,,,and,have been characterized by map-based cloning approaches. Different resistance genes confer different levels of insect resistance in rice, and their mechanisms of action and resistant spectrum are also varied (Chenget al, 2013).encodes a nucleotide- binding and leucine-rich repeat protein that activates the salicylic acid signaling pathway and induces callose deposition in phloem cells and trypsin inhibitor production after planthopper infestation, thus reducing BPH fitness (Duet al, 2009).encodes three plasma membrane-localized lectin receptor kinases (OsLecRK1?OsLecRK3), which combine to confer broad-spectrum and permanent insect resistance in rice (Liuet al, 2015).encodes a protein in the exocyst, interacts with, and participates in cell wall maintenance and reinforcement (Guoet al, 2018). Since less than 18% of the genome encodes protein- coding genes, most genomic landscapes are comprise of non-coding elements. Studies of only coding genes are not sufficient to reveal the molecular mechanism underlying rice resistance to BPH infestation.

    MicroRNAs (miRNAs) are a class of 21–24-nucleotide- long endogenous non-protein-coding small RNAs present in eukaryotes (Bartel, 2004). As essential post- transcriptional regulators, miRNAs play significant roles in plant-insect interactions. For example, miR396 and miR156 negatively regulate rice resistance against BPH through regulation of flavonoid and jasmonic acid biosynthesis (Geet al, 2018; Daiet al, 2019). In recent years, evidence of cross-kingdom miRNAs has been reported in several studies (Zhanget al, 2012; Zhouet al, 2015; Chinet al, 2016; Zhuet al, 2017). In particularly, plant miR162a can target the TOR (target of rapamycin) gene of honeybee () (Zhuet al, 2017), which motivates investigations on the interactions between rice and BPH.

    This study focused on analyzing the miRNA expression profiles of six rice varieties (including two BPH-susceptible and four BPH-resistant varieties) before and after BPH-feeding. We predicted and analyzed the functions of miRNAs to clarify the modulatory defense roles of miRNAs in the interactions between rice and BPH. In addition, we selected a group of potential cross-kingdom miRNAs from differentially expressed miRNAs, and predicted their targets in BPH to screen out rice miRNAs that can regulate BPH genes. Subsequently, rice miR5795 was observed to possibly target thegene in BPH.

    RESULTS

    Differentially expressed miRNAs between BPH-susceptible and BPH-resistant rice varieties

    We performed deep sequencing and characterization of miRNAs in two BPH-susceptible rice varieties (TN1 and Nipponbare) and four BPH-resistant rice varieties [R476, Mudgo, IR36 and Rathu Heenati (RH)] before and after BPH-feeding. Total reads of the 47 libraries were filtered to remove low-quality reads, incorrect adaptors, poly-A and those shorter than 18 nt, and then the clean reads were obtained (Table S1). The length distribution of the small RNAs was mostly concentrated in the range of 19–24 nt, as previously reported (Fig. S1-A) (Bartel, 2004). Subsequently, the total small RNAs were aligned with the miRNA database in miRBase (release 22) to determine the known miRNAs. In total, 575 known miRNAs were identified, which were dominatly 21 nt- long with 5′-U as the first base (Fig. S1-B), consistent with the characteristics of Argonaute (Ago) 1 protein in plants (Miet al, 2008).

    To determine whether there was a difference in the expression of miRNAs between BPH-susceptible and BPH-resistant rice varieties, principal component analysis (PCA) was performed on the miRNA expression of samples before BPH-feeding. The result of PCA showed that the four BPH-resistant rice varieties were concentrated in the area above PC2. In comparison, two BPH-susceptible rice varieties were located in the area below PC2, indicating differences in the miRNAs expression between BPH-susceptible and BPH-resistant rice varieties (Fig. 1-A).

    In addition, a total of 45 differential miRNAs were identified through differential expression analysis (Fig. 1-B). Among them, 25 miRNAs were substantially expressed in BPH-susceptible rice varieties, while 20 miRNAs were highly expressed in BPH-resistant rice varieties (Fig. 1-B and -C). Furthermore, target prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichments of the predicted targets were carried out to understand the possible defense modulatory roles of the differentially expressed miRNAs. The KEGG pathway enrichments revealed that some of the targets were involved in common rice defense responses such as plant hormone signal transduction, MAPK signaling pathways and secondary metabolite biosynthesis (Fig. 1-D).

    Identification of putative miRNAs that respond to BPH-feeding

    During the interactions between rice and BPH, BPH- feeding may induce the specific expression of miRNAsin rice, which will help to regulate the defense responseof rice against BPH. To investigate the effects of BPH- feeding on miRNA expression in rice, the miRNA expression profiles of the six rice varieties before and after BPH-feeding at 8 and 32 h were analyzed, respectively. A total of 144 miRNAs (TN1, 13; Nipponbare, 5; IR36, 28; Mudgo, 32; RH, 55; and R476, 53) that responded to BPH-feeding were identified with some of them being identified repeatedly (Table 1). In terms of quantity, the miRNAs of BPH-resistant rice varieties in response to BPH-feeding were more than those of BPH-susceptible rice varieties, indicating that BPH-resistant rice varieties were more sensitive to BPH-feeding and had a stronger response. Regarding the changing trend of miRNAs, the number of up- regulated miRNAs after BPH-feeding was higher than that of down-regulated miRNAs in BPH-susceptible rice varieties, while the miRNAs of RH and Mudgo were predominantly down-regulated. Comparing the two time points, R476 and Mudgo showed no or slight response at the early stage (8 h) of BPH-feeding but a strong response at the late stage (32 h). In contrast, RH and IR36 had an intense response at the early stage and a mild response at the late stage.

    Fig. 1. miRNAs differentially expressed in brown planthopper (BPH)-susceptible and BPH-resistant rice varieties before BPH-feeding.

    A, Principal component analysis (PCA) of miRNAs in rice samples before BPH-feeding.

    B, Volcano plot of differential miRNA expression in BPH-resistant and BPH-susceptible rice varieties. ‘down’ and ‘up’ mean miRNA expression was down-regulated and up-regulated in BPH-resistant rice varieties compared with BPH-susceptible rice varieties, respectively, and ‘non’ means the expression of miRNA was not significantly different between the BPH-resistant and BPH-susceptible rice varieties.

    C, Heatmap of differential miRNA expression profile.

    D, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of target genes of differential miRNAs.

    RH, Rathu Heenati.

    Furthermore, we found only a small number of miRNAs (32, 22.22%) involved in the response of two or more rice varieties to BPH-feeding, while most miRNAs (112, 77.78%) were involved in response of only one rice variety (Fig. 2-A). The prediction results of target genes indicated that a single miRNA can regulate the expression of many genes, and the different miRNAs regulated different target genes (Fig. 2-B).However, the target genes of miRNAs that responded to BPH-feeding in four BPH-resistant rice varieties shared 12 identical KEGG pathways (Fig. 2-C). The results indicated that different miRNAs were involved in the rice-BPH interactions, and the genetic back- ground of rice determined its miRNA regulation mode after BPH-feeding. There were differences in the amounts, types, changing trends and response periods of miRNAs in response to BPH-feeding in rice varieties with different BPH-resistance genes. However, these miRNAs responded to BPH-feeding by regulating common signaling pathways, such as the synthesis and metabolism of secondary metabolites, flavonoids and amino acids, as well as the regulation of plant hormones.

    Table 1. Summary of rice miRNAs in response to brown planthopper (BPH)-feeding.

    The backgrounds with orange and blue colors indicate miRNA expression was up-regulated and down-regulated, respectively.

    RH, Rathu Heenati.

    Fig. 2. Different miRNAs participate in rice-brown planthopper (BPH) interactions after BPH-feeding.

    A, UpSet diagram of miRNAs in rice response to BPH-feeding. ‘●’ indicates that the miRNAs appeared in only one rice variety, and ‘●?●’ indicates that the miRNAs appeared in two or more rice varieties.

    B, UpSet diagram of miRNA target genes in rice response to BPH- feeding.‘●’ indicates that the miRNA target genes appeared in only one rice variety, and ‘●?●’ indicates that the miRNA target genes appeared in two or more rice varieties.

    C, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway is shared by four BPH-resistant rice varieties.

    NB, Nipponbare; RH, Rathu Heenati.

    Screening of potential cross-kingdom miRNAs in rice

    Fisher’s exact test results showed that the miRNAs differentially expressed in BPH-susceptible and BPH- resistant rice varieties and the miRNAs that responded to BPH-feeding were significantly correlated, meaning that 25 miRNAs shared in 2 groups were more likely to be directly involved in the rice-BPH interactions (Fig. 3-A). Among them, seven miRNAs (miR168b, miR1428g-5p, miR1432-5p, miR5795, miR3980b-5p, miR1882h and miR2876-5p) terminated in a 5′-A residue. These types of miRNAs tended to bind to the Ago2 protein rather than to the Ago1 protein that plays an important role in plants (Miet al, 2008). In addition, Ago2 protein can promote the secretion of miRNAs outside the cell (Lvet al, 2014).

    We speculated that miRNAs terminating with 5′-A residue can be secreted extracellularly and ingested by BPH. Therefore, we predicted the targets of these seven miRNAs in BPH. A total of 3 861 miRNA- target pairs were predicted using three bioinformatic algorithms (miRanda, TargetScan and RNA22) (Fig. 3-B). It was noted that these targets were involved in multiple pathways related to the fecundity of BPH, including the pathways related to the vitellogenin biosynthesis such as PI3K-Akt, AMPK and insulin signaling pathways, and the pathways related to oocytedevelopment such as oocyte meiosis and progesterone-mediated oocyte maturation. In contrast, other pathways such as pancreatic secretion, bile secretion, protein digestion and absorption may be related to feeding, digestion and detoxification (Fig. 3-C).

    Rice miR5795 mediates fecundity of BPH

    To verify whether the miRNAs in rice have the potential to mediate the fecundity of BPH, we scanned 15 representative genes related to the fecundity of BPH for potential binding sites in 7 miRNAs (Table S2). The results showed that miR5795 and miR1428g-5p had potential binding sites in the CDS (coding sequence) and 3′-UTR (untranslated region) of multiple genes, respectively (Table 2). Subsequently, the fecundity of BPH was tested in three seasons (winter, spring and summer) after injection of miRNA mimics. In order to exclude the effects of seasonal differences, a two-way analysis of variance (ANOVA) was used to analyze the fecundity data. The results showed that miR5795 reduced the number of eggs laid by 16.07% (< 0.01) and the hatching rate by 16.45% (< 0.01) of BPH (Fig. 4-A to -C), while miR1428g-5p did not affect the fecundity of BPH (Fig. S2-A to -C). Additionally, ovarian development was inhibited significantly at 72 h after injection of miR5795 mimics (Fig. S3).

    Fig. 3. Screening of potential cross-kingdom miRNAs in rice.

    A, Venn diagram of two sets of miRNAs.

    B, Venn diagram of target prediction of rice miRNAs using three bioinformatic algorithms (miRanda, TargetScan and RNA22) based on the total transcripts of

    C, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of target genes inof miRNAs.

    Table 2. Number of targets of rice miRNAs on fecundity of Nilaparvatalugens.

    The target prediction of miR5795 showed that it had a potential binding site on the()gene in BPH, which is usually used as a molecular marker for insect reproduction (Fig. 4-D). Therefore, we fused the potential binding site oninto a reporter plasmid. Thereafter, the fused plasmid was co-transfected into a cell line in combination with miR5795 mimics. Results indicated that miR5795 resulted in a 24.30% decrease in luciferase activity (Fig. 4-E). However, when the predicted ‘seed site’ was mutated, the fused luciferase reporters were unaffected by miR5795 (Fig. 4-E). Thus, miR5795 can specifically recognize the potential binding site of NlVg in BPH. Furthermore, to determine the potential effects of miR5795 onexpression, we detected the mRNA and protein levels ofin BPH after injection of miR5795 mimics. Although the mRNA levels ofat 48 and 72 h after miR5795 mimics injection were significantly higher than those in the control (Fig. 4-F), the protein translation ofwas inhibited (Fig. 4-G and -H). Given the incomplete base pairing of miR5795 with(Fig. 4-D), miR5795 mediated the fecundity of BPH possibly by inhibiting NlVg protein translation.

    DISCUSSION

    Plants and insects interact in complex ways (Lucas- Barbosa, 2016). To resist the invasion of insects, plants have evolved constitutive and inducible defense mechanisms to reduce tissue damage (Liet al, 2018). miRNAs are involved in rice-BPH interactions (Wuet al, 2017; Nandaet al, 2020; Tanet al, 2020). In contrast with previous studies, we analyzed the miRNA expression of two BPH-susceptible rice varieties, TN1 and Nipponbare, as well as four rice varieties carrying different BPH-resistance genes, Mudgo (), IR36 (), RH () and R476 (). To the best of our knowledge, this is the first report to analyze the miRNA expression of multiple BPH-susceptible and BPH-resistant rice varieties.

    Fig. 4. miR5795 in rice may targetgene in brown planthopper (BPH) and subsequently decrease its fecundity.

    A?C, The number of eggs per pair (A), hatching rate (B) and the number of hatched eggs per pair (C) ofafter injection of miR5795 mimics.

    D, Predicted binding sites between miR5795 andwith energy of -87.65 kJ/mol.

    E, Dual-luciferase assay between miR5795 and NlVg. psiCHECK-2, Vector without insertion sites; WT, Wild type vector; MT, Mutation type vector.

    F, mRNA expression level ofin whole bodies of BPH after injection of miR5795 mimics at 24, 48 and 72 h.

    G, Western blot for NlVg in whole bodies of BPH after injection of miR5795 mimics at 24, 48 and 72 h. β-actin was used as an internal reference.

    H, Protein expression levels of NlVg in whole bodies, ovaries and fat bodies of BPH after injection of miR5795 at 72 h.

    Data are Mean ± SE.= 3 in A?C, F and H, and= 9 in E. *,< 0.05; **,< 0.01.

    In this study, we found that the miRNA expression profiles of BPH-susceptible rice varieties differed from those of BPH-resistant rice varieties, and these differences may be the reason for their different resistances (Fig. 1-A to -C). KEGG enrichment results indicated that the differential expression of miRNA might lead to differences in the synthesis of secondary metabolites, plant hormones and MAPK signaling pathways (Fig. 1-D). Dai et al (2019) reportedthat miR396 can regulate the biosynthesis of flavonoids, thereby negatively regulating BPH resistance in rice. Our analysis showed that flavonoid biosynthesis might participate in the defense response of rice to BPH. Moreover, we found that the expression levels of miR396 in RH and Mudgo were down-regulated, implying that BPH resistance in rice increased, which was consistent with Dai et al (2019).

    It was noted that different rice varieties had significant differences in the amounts, types, changing trends and response periods of miRNAs in response to BPH-feeding (Table 1), which was consistent with previous studies. For example, P15 rice variety (carrying with) responds to BPH-feeding with far more miRNAs than susceptible rice variety PC, and only a few miRNAs between the two varieties are identical (Wuet al, 2017). In terms of changing trends and response periods, most of the differentially expressed miRNAs in IR36 are up-regulated, and the early stage response of BPH-feeding is stronger than that at late stage, which is similar to P15 (Wuet al, 2017). In addition, Nanda et al (2020) found that the genetic history of BPH affects the rice-BPH interactions. In general, the rice-BPH interactions mediated by miRNAs involve complex regulatory networks, which are affected by the genetic history of rice and BPH. However, we also found that the four BPH-resistant rice varieties shared multiple defense pathways, revealing the similarity in defense strategies mediated by BPH-resistance genes (Fig. 2-C).

    Plant miRNAs are passed into animals feeding on respective plants, which can regulate the gene expression of the animal (Zhouet al, 2017). Therefore, rice miRNAs could have been transferred to BPH during feeding, which may regulate gene expression of BPH. In this study, we investigated the potential of cross- kingdom miRNAs in rice to expand the understanding of miRNA transfer between species. In cross-kingdom miRNA screening, miRNAs terminating with 5′-U residue were not selected because they tend to bind to the Ago1 protein that is important in plants (Miet al, 2008). In addition, BPHs are phloem-feeders,so they can ingest miRNAs that are secreted extracellularly (Liet al, 2018). However, the miRNAs bind to the Ago1 protein are unlikely to be secreted extracellularly (Dunoyeret al, 2010). In contrast, the miRNAs terminating with 5′-A residue bind to the Ago2 protein, which play an important role in plant biological and abiotic stress responses. The plant Ago2 protein has functions similar to animal Ago2 protein, which promotes miRNAs to load into vesicles, secretes miRNAs out of the cell, and functions outside the cell (Lvet al, 2014). As a result, miRNAs terminating with 5′-A residue are more likely to be secreted into the ligament and ingested by BPH, allowing for cross-border regulation of the BPH genes. The KEGG pathway enrichment results showed that the targets of the seven miRNAs were mainly involved in fecundity, feeding, digestion and detoxification (Fig. 3-C).

    The target sites of miR1428g-5p and miR5795 were mainly located in the CDS region of the gene, which was consistent with most plant miRNA target sites (Llaveet al, 2002; Rhoadeset al, 2002). Our results showed that miR5795 might target thegene in BPH and subsequently decreased BPH fecundity (Fig. 4). In a related study, plant miR162a was reported to regulate the honeybeegene and inhibit the ovarian and overall development of juveniles in a transboundary manner (Zhuet al, 2017). However, in this study,mRNA expression level was increased after miR5795 injection (Fig. 4-F), while miR162a resulted in the reduction of honeybeemRNA expression level. Therefore, the mechanism of gene regulation by miR5795 and miR162a may be distinct and required further investigation. In addition, the degrees of base complementarity of miRNA and mRNA determine regulation (Zhanget al, 2007). miR162a is more complementary to its target gene, which may regulateexpression through mRNA degradation, and miR5795 was complementary only in the seed region and may be regulated in translation inhibition (Fig. 4).

    Studies of cross-kingdom miRNAs have progressed in recent years. However, it is still unknown whether BPH can ingest miR5795 under natural conditions, and further research is needed. Overall, our study provides a better understanding of the regulatory mechanisms of rice-BPH interactions and gives data references and identifies new genetic materials for breeding of rice varieties containing insect-resistance genes.

    METHODS

    Plant, insect and cell lines

    The seeds of six rice varieties, i.e., TN1, Nipponbare, IR36, R476, Mudgo and RH, were sown in pots (12?cm in diameter and 12?cm in height), and rice seedlings were grown in a greenhouse under standard growth conditions. The BPH population was obtained from rice fields in Guangdong Province, China. All BPHs were reared in the same walk-in chamber at 26 oC ± 1 oC under a photoperiod of 16 h light and 8 h dark with a relative humidity of 80% ± 10% for a susceptible rice variety Huanghuazhan. The 293T cells were cultured in 1× Dulbecco’s Modification of Eagle’s Medium (Corning Inc., NY, USA) supplemented with 10% fetal bovine serum premium (PAN Biotech GmbH, Aidenbach, Germany) at 37 oC under 5% CO2.

    miRNA sequencing analysis

    Three-week-old individual rice plants were infested with twenty 3rd, 4th and 5th instar BPH nymphs that had been starved for 2 h. The stems were collected at 0, 8 and 32 h in triplicate after BPH-feeding. RNA was extracted and purified, and adaptors were added to the 5′- and 3′-ends using T4 ligase. RNA was then amplified for library construction and sequence using BGISEQ-500 (Beijing Genomics Institute, Shenzhen, China). Raw reads were filtered to remove low quality reads, incorrect adaptors, poly-A and those shorter than 18 nt. The clean reads were used to search against the miRBase database (release 22, http://www.mirbase.org/) for known rice miRNA identification. The frequency of miRNA counts was normalized as transcripts per million (TPM). PCA analysis was conducted using TPM of all miRNAs by the R package (http://www.r- project.org/).

    Differential expression analysis of miRNAs

    The-value of differential expression was calculated using the R DESeq2 package (Loveet al, 2014). We used the absolute value of log2(Fold change)? ≥ 1 and

    Prediction and functional annotation of miRNA target genes

    We used the psRNATarget (v2) software (Daiet al, 2018) to predict miRNA target genes on rice, while used miRanda (v3.3a) (Johnet al, 2004), TargetScan (v7.0) (Agarwalet al, 2015) and RNA22 (v2) (Loher and Rigoutsos, 2012) softwares to predict miRNA target genes on BPH. The known fecundity- related genes in BPH are listed in Table S2. KEGG (https:// www.kegg.jp/) was used to identify the functions of target genes.

    Fecundity bioassay

    Each one-day-old female BPH injected with a drop of 20? μmol/L miRNA mimics (50?nL) was paired with two untreated male BPHs and then transferred to fresh rice plants. The pairs of adults were removed after 7 d, and then the number of hatched nymphs was counted daily. When the nymphs no longer hatched for two consecutive days, the number of unhatched eggs was counted. The experiment was replicated three times during three seasons (winter, spring and summer). Ten ovaries of female BPHs were dissected in PBS solution (0.02? mol/L, pH 7.4) at 72 h after injection. The ovaries were viewed and photographed with an Olympus photomicroscope (Olympus Corporation Company, Kyoto, Japan). The ovaries of brachypterous BPH were divided into five stages according to ovariole development (Dong et al, 2011).

    Luciferase reporter assay

    The binding site was fused into the downstream position of the firefly luciferase gene in the psiCHECK-2 reporter plasmid. Cultured cells were prepared for transfection by seeding 1 × 106cells/mL in a 96-well plate. After culturing the cells for 12–18 h, transfection was performed using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). The transfection mixture per well contained 0.3 μL FuGENE reagent, 100 ng fused plasmid and 0.5 μL miR5795 mimics. The cells were collected at 48 h after transfection and lysed using 50 μL passive lysis buffer in a linear shaker at 1 500 r/min for 15 min. The luciferaseactivity detection was performed using a multifunctional microplate reader (TriStar LB941; Berthold Technologies, Bad Wildbad, Germany) and a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) following the manufacturer’s instructions.

    qRT-PCR analysis

    Total RNA was extracted from samples using MagZolTM Reagent (Angen Biotech, Guangzhou, China) following the manufacturer’s instructions. One microgram of RNA was used for the first-strand complementary DNA (cDNA) synthesis using Color Reverse Transcription Kit (EZB Bioscience, Roseville, MN, USA). qRT-PCR was performed using a Light Cycler 480 (Roche Diagnostics, Indianapolis, IN, USA) with 2× Color SYBR Green qPCR Master Mix (EZB Bioscience, Roseville, MN, USA) following the manufacturer’s instructions. Each reaction mixture included 1 μL of cDNA template equivalent to 1 ng of total RNA, 0.3 μL of each primer (10 μmol/L) and 5 μL of SYBR Mix in a total volume of 10 μL. Reactions were performed in triplicate for each sample and three reactions for each biological replicate (= 3) were performed. The gene expression levels were normalized to the expression level ofgene (Chenet al, 2013). The specific primers used for qRT-PCR are listed in Table S3. The amplification conditions were as follows: 95 oC for 30 s, followed by 40 cycles of 95 oC for 5 s, 60 oC for 30 s and 72 oC for 5 s.

    Western blot analysis

    Total protein from whole bodies ofwas extracted from three females at 24, 48 and 72 h after miRNA mimics injection. Fat bodies and ovaries were extracted from 20 females in each of the three replicates performed. Whole or fat bodies or ovaries were lysed in RIPA Lysis Buffer (Yeasen Biotech, Shanghai, China). The homogenate was centrifuged at 12 000 ×at 4 oCfor 15 min, and protein content in the supernatant was measured using the Bradford method. The western blot technique was modified according to Mitsumasuet al (2008). Total protein (10 μg) were separated using 10% SDS-PAGE and transferred to poly (vinylidene difluoride) membranes (0.45 μm, Millipore-Sigma, Burlington, MA, USA), and the membranes were immunoblotted with anti-NlVg (vitellogenin, 1:500; Abmart, Berkeley Heights, NJ, USA) and anti-β-actin (1:4 000; Abcam, Cambridge, UK). The secondary antibody was immunoglobulin G (IgG) goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) (1:20 000; TransGen Biotech, Beijing, China). The membranes were visualized using electrochemi-luminescence (MilliporeSigma, Burlington, MA, USA) and Image Lab (Bio-Rad Laboratories, Hercules, CA, USA). The protein bands were quantified by importing the images into the ImageJ analysis software (v1.52a).

    Statistical analysis

    For the statistical analysis of the fecundity bioassay of BPH, the number of eggs laid was transformed by square root, the hatching rate values were transformed by arcsine square root, and the differences between the two groups were analyzed using two-way ANOVA to exclude the effects of seasonal differences. All results are expressed as Mean ± SE, and statistical differences were considered significant at< 0.05 and< 0.01.

    ACKNOWLEDGEMENTS

    This study was supported by the Key Realm Research and Development Program of Guangdong Province, China (Grant No. 2020B0202090001) and the Foundation of Guangzhou Science and Technology Key Project, China (Grant No. 201904020041).

    SUPPLEMENTAL DATA

    The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

    Fig. S1. Length and 5′-terminal nucleotide distribution of miRNAs.

    Fig. S2. miR1428g-5p in rice did not affect fecundity of brown planthopper.

    Fig. S3. Developmental stages of ovaries at 72 h after injection of miR5795 mimics.

    Table S1. Sequencing data for rice samples (quoted BGI-tech).

    Table S2. Known fecundity-related genes in.

    Table S3. Primer sequences used in this study.

    Agarwal V, Bell G W, Nam J W, Bartel D P. 2015. Predicting effective microRNA target sites in mammalian mRNAs., 4: e05005.

    Akanksha S, Lakshmi V J, Singh A K, Deepthi Y, Chirutkar P M, Ramdeen, Balakrishnan D, Sarla N, Mangrauthia S K, Ram T. 2019. Genetics of novel brown planthopper(St?l) resistance genes in derived introgression lines from the interspecific crossvar. Swarna ×., 98: 113.

    Bao Y Y, Wang Y, Wu W J, Zhao D, Xue J, Zhang B Q, Shen Z C, Zhang C X. 2012.intestine-specific transcriptome of the brown planthopperrevealed potential functions in digestion, detoxification and immune response., 99(4): 256–264.

    Bartel D P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function., 116(2): 281–297.

    Chen H, Stout M J, Qian Q, Chen F. 2012. Genetic, molecular and genomic basis of rice defense against insects., 31(1): 74–91.

    Chen J, Liang Z K, Liang Y K, Pang R, Zhang W Q. 2013. Conserved microRNAs miR-8-5p and miR-2a-3p modulate chitin biosynthesis in response to 20-hydroxyecdysone signaling in the brown planthopper,., 43(9): 839–848.

    Cheng X Y, Zhu L L, He G C. 2013. Towards understanding of molecular interactions between rice and the brown planthopper., 6(3): 621–634.

    Chin A R, Fong M Y, Somlo G, Wu J, Swiderski P, Wu X W, Wang S E. 2016. Cross-kingdom inhibition of breast cancer growth by plant miR159., 26(2): 217–228.

    Dai X B, Zhuang Z H, Zhao P X. 2018. psRNATarget: A plant small RNA target analysis server (2017 release)., 46(W1): W49–W54.

    Dai Z Y, Tan J, Zhou C, Yang X F, Yang F, Zhang S J, Sun S C, Miao X X, Shi Z Y. 2019. The OsmiR396-OsGRF8-OsF3H- flavonoid pathway mediates resistance to the brown planthopper in rice ()., 17(8): 1657–1669.

    Dong S Z, Ma Y, Hou Y, Yu X P, Ye G Y. 2011. Development of an ELISA for evaluating the reproductive status of female brown planthopper,, by measuring vitellogenin and vitellin levels., 139(2): 103–110.

    Du B, Zhang W L, Liu B F, Hu J, Wei Z, Shi Z Y, He R F, Zhu L L,Chen R Z, Han B, He G C. 2009. Identification and characterization of, a gene conferring resistance to brown planthopper in rice., 106(52): 22163–22168.

    Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington J C, Voinnet O. 2010. Small RNA duplexes function as mobile silencing signals between plant cells., 328: 912–916.

    Fujita D, Kohli A, Horgan F G. 2013. Rice resistance to planthoppers and leafhoppers., 32(3): 162–191.

    Ge Y F, Han J Y, Zhou G X, Xu Y M, Ding Y, Shi M, Guo C K, Wu G. 2018. Silencing of miR156 confers enhanced resistance to brown planthopper in rice., 248(4): 813–826.

    Guo J P, Xu C X, Wu D, Zhao Y, Qiu Y F, Wang X X, Ouyang Y D, Cai B D, Liu X, Jing S L, Shangguan X X, Wang H Y, Ma Y H, Hu L, Wu Y, Shi S J, Wang W L, Zhu L L, Xu X, Chen R Z, Feng Y Q, Du B, He G C. 2018.encodes an exocyst- localized protein and confers broad resistance to planthoppers in rice., 50(2): 297–306.

    Jena K K, Kim S M. 2010. Current status of brown planthopper (BPH) resistance and genetics., 3(2/3): 161–171.

    John B, Enright A J, Aravin A, Tuschl T, Sander C, Marks D S. 2004. Human microRNA targets., 2(11): e363.

    Li C D, Wong A Y P, Wang S, Jia Q, Chuang W P, Bendena W G, Tobe S S, Yang S H, Chung G, Chan T F, Lam H M, Bede J C, Hui J H L. 2018. miRNA-mediated interactions in and between plants and insects., 19(10): 3239.

    Liu Y Q, Wu H, Chen H, Liu Y L, He J, Kang H Y, Sun Z G, Pan G, Wang Q, Hu J L, Zhou F, Zhou K N, Zheng X M, Ren Y L, Chen L M, Wang Y H, Zhao Z G, Lin Q B, Wu F Q, Zhang X, Guo X P, Cheng X N, Jiang L, Wu C Y, Wang H Y, Wan J M. 2015. A gene cluster encoding lectin receptor kinases confers broad-spectrum and durable insect resistance in rice., 33(3): 301–305.

    Llave C, Xie Z X, Kasschau K D, Carrington J C. 2002. Cleavage ofmRNA targets directed by a class ofmiRNA., 297: 2053–2056.

    Loher P, Rigoutsos I. 2012. Interactive exploration of RNA22 microRNA target predictions., 28(24): 3322–3323.

    Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2., 15(12): 550.

    Lucas-Barbosa D. 2016. Integrating studies on plant-pollinator and plant-herbivore interactions., 21(2): 125–133.

    Lv Z Y, Wei Y, Wang D, Zhang C Y, Zen K, Li L M. 2014. Argonaute 2 in cell-secreted microvesicles guides the function of secreted miRNAs in recipient cells., 9(7): e103599.

    Mi S J, Cai T, Hu Y G, Chen Y M, Hodges E, Ni F R, Wu L, Li S, Zhou H Y, Long C Z, Chen S, Hannon G J, Qi Y J. 2008. Sorting of small RNAs intoArgonaute complexes is directed by the 5′ terminal nucleotide., 133(1): 116–127.

    Mitsumasu K, Azuma M, Niimi T, Yamashita O, Yaginuma T. 2008. Changes in the expression of soluble and integral-membrane trehalases in the midgut during metamorphosis in., 25(7): 693–698.

    Nanda S, Yuan S Y, Lai F X, Wang W X, Fu Q, Wan P J. 2020. Identification and analysis of miRNAs in IR56 rice in response toBPH infestations of different virulence levels., 10(1): 19093.

    Rhoades M W, Reinhart B J, Lim L P, Burge C B, Bartel B, Bartel D P. 2002. Prediction of plant microRNA targets., 110(4): 513–520.

    Tan J Y, Wu Y, Guo J P, Li H M, Zhu L L, Chen R Z, He G C, Du B. 2020. A combined microRNA and transcriptome analyses illuminates the resistance response of rice against brown planthopper., 21(1): 144.

    Wu S F, Zeng B, Zheng C, Mu X C, Zhang Y, Hu J, Zhang S, Gao C F, Shen J L. 2018. The evolution of insecticide resistance in the brown planthopper (St?l) of China in the period 2012?2016., 8(1): 4586.

    Wu Y, Lv W T, Hu L, Rao W W, Zeng Y, Zhu L L, He Y Q, He G C. 2017. Identification and analysis of brown planthopper- responsive microRNAs in resistant and susceptible rice plants., 7(1): 8712.

    Yang L, Zhang W L. 2016. Genetic and biochemical mechanisms of rice resistance to planthopper., 35(8): 1559–1572.

    Zhang B H, Wang Q L, Pan X P. 2007. MicroRNAs and their regulatory roles in animals and plants., 210(2): 279–289.

    Zhang L, Hou D X, Chen X, Li D H, Zhu L Y, Zhang Y J, Li J, Bian Z, Liang X Y, Cai X, Yin Y, Wang C, Zhang T F, Zhu D H, Zhang D M, Xu J, Chen Q, Ba Y, Liu J, Wang Q, Chen J Q, Wang J, Wang M, Zhang Q P, Zhang J F, Zen K, Zhang C Y. 2012. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: Evidence of cross-kingdom regulation by microRNA., 22(1): 107–126.

    Zhou G Y, Zhou Y, Chen X. 2017. New insight into inter-kingdom communication: Horizontal transfer of mobile small RNAs., 8: 768.

    Zhou Z, Li X H, Liu J X, Dong L, Chen Q, Liu J L, Kong H H, Zhang Q Y, Qi X, Hou D X, Zhang L, Zhang G Q, Liu Y C, Zhang Y J, Li J, Wang J, Chen X, Wang H, Zhang J F, Chen H L, Zen K, Zhang C Y. 2015. Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses., 25(1): 39–49.

    Zhu K G, Liu M H, Fu Z, Zhou Z, Kong Y, Liang H W, Lin Z G, Luo J, Zheng H Q, Wan P, Zhang J F, Zen K, Chen J, Hu F L, Zhang C Y, Ren J, Chen X. 2017. Plant microRNAs in larval food regulate honeybee caste development., 13(8): e1006946.

    24 February 2022;

    11 May2022

    Copyright ? 2022, China National Rice Research Institute. Hosting by Elsevier B V

    This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

    Peer review under responsibility of China National Rice Research Institute

    http://dx.doi.org/10.1016/j.rsci.2022.05.003

    Zhang Wenqing (lsszwq@mail.sysu.edu.cn)

    (Managing Editor: Wu Yawen)

    很黄的视频免费| 老司机深夜福利视频在线观看| 亚洲第一青青草原| 9191精品国产免费久久| 91麻豆av在线| 久久伊人香网站| 99国产精品一区二区三区| 神马国产精品三级电影在线观看 | 国产欧美日韩精品亚洲av| АⅤ资源中文在线天堂| 波多野结衣高清无吗| av免费在线观看网站| 国内精品久久久久精免费| 两个人看的免费小视频| 亚洲国产精品久久男人天堂| 中文在线观看免费www的网站 | 日日爽夜夜爽网站| 国产激情偷乱视频一区二区| netflix在线观看网站| 一本大道久久a久久精品| 国语自产精品视频在线第100页| 夜夜看夜夜爽夜夜摸| 神马国产精品三级电影在线观看 | 欧美三级亚洲精品| 老司机在亚洲福利影院| 可以在线观看毛片的网站| 久久 成人 亚洲| 欧美 亚洲 国产 日韩一| 久热爱精品视频在线9| 久久精品91蜜桃| 国产99白浆流出| 婷婷精品国产亚洲av在线| 在线观看一区二区三区| 男女床上黄色一级片免费看| 淫秽高清视频在线观看| 国产精品久久久久久人妻精品电影| 老鸭窝网址在线观看| 99在线人妻在线中文字幕| 国产精品九九99| 88av欧美| 99精品欧美一区二区三区四区| 成人亚洲精品一区在线观看| 亚洲黑人精品在线| 国产欧美日韩一区二区精品| 国产视频内射| 欧美国产日韩亚洲一区| 黄色毛片三级朝国网站| 99国产精品一区二区三区| 麻豆成人av在线观看| 午夜福利高清视频| 日韩精品免费视频一区二区三区| av有码第一页| 成人午夜高清在线视频 | 搡老岳熟女国产| 搡老妇女老女人老熟妇| 国产精品 欧美亚洲| 又黄又爽又免费观看的视频| 国产av一区二区精品久久| 亚洲黑人精品在线| 两个人看的免费小视频| 午夜福利高清视频| 少妇裸体淫交视频免费看高清 | 午夜久久久久精精品| 美女国产高潮福利片在线看| 午夜免费观看网址| 十八禁人妻一区二区| 天堂影院成人在线观看| 国内少妇人妻偷人精品xxx网站 | 国产一区二区在线av高清观看| 99在线视频只有这里精品首页| 一级毛片女人18水好多| 日韩免费av在线播放| videosex国产| 国产亚洲欧美98| 成人永久免费在线观看视频| 搡老岳熟女国产| cao死你这个sao货| 观看免费一级毛片| 伊人久久大香线蕉亚洲五| 成人亚洲精品一区在线观看| 色av中文字幕| av免费在线观看网站| 久久精品国产清高在天天线| 久99久视频精品免费| 99国产精品99久久久久| 天天躁狠狠躁夜夜躁狠狠躁| 久久天躁狠狠躁夜夜2o2o| 日韩欧美国产在线观看| 亚洲一区二区三区色噜噜| 免费高清视频大片| 国产麻豆成人av免费视频| 久久久久久久久中文| 亚洲精品在线观看二区| 一个人观看的视频www高清免费观看 | 久久久久久人人人人人| 中文字幕另类日韩欧美亚洲嫩草| 国产片内射在线| 一区二区三区激情视频| 欧美性猛交黑人性爽| 亚洲人成电影免费在线| 欧美国产精品va在线观看不卡| 巨乳人妻的诱惑在线观看| 国产视频内射| 搡老熟女国产l中国老女人| 老司机靠b影院| 亚洲无线在线观看| 久久久国产成人精品二区| 757午夜福利合集在线观看| 男女下面进入的视频免费午夜 | 欧美 亚洲 国产 日韩一| 男女下面进入的视频免费午夜 | 欧美日韩瑟瑟在线播放| 久久99热这里只有精品18| 午夜福利视频1000在线观看| 麻豆一二三区av精品| 国产av在哪里看| 18禁美女被吸乳视频| 国产激情久久老熟女| 女同久久另类99精品国产91| 亚洲精品粉嫩美女一区| 老司机午夜福利在线观看视频| 手机成人av网站| 久久99热这里只有精品18| 欧美乱妇无乱码| 麻豆一二三区av精品| 午夜福利18| 大型av网站在线播放| 天堂影院成人在线观看| 99国产极品粉嫩在线观看| 91av网站免费观看| 校园春色视频在线观看| 亚洲成人久久性| 亚洲人成77777在线视频| 婷婷丁香在线五月| 老汉色∧v一级毛片| 91成年电影在线观看| 精品午夜福利视频在线观看一区| 在线观看www视频免费| 国产一区二区在线av高清观看| 国产视频内射| 亚洲人成网站在线播放欧美日韩| 嫩草影院精品99| 精品久久久久久成人av| 久久久久久人人人人人| 精品免费久久久久久久清纯| 国内精品久久久久精免费| 波多野结衣高清无吗| 成人午夜高清在线视频 | 黑丝袜美女国产一区| 婷婷丁香在线五月| 夜夜看夜夜爽夜夜摸| www.999成人在线观看| 无人区码免费观看不卡| 久久国产精品影院| 亚洲熟妇熟女久久| 亚洲精品国产一区二区精华液| 一边摸一边做爽爽视频免费| 国产精品精品国产色婷婷| svipshipincom国产片| 色哟哟哟哟哟哟| 黄色视频,在线免费观看| 亚洲精品国产一区二区精华液| 精品国产超薄肉色丝袜足j| 亚洲av日韩精品久久久久久密| www国产在线视频色| 国产亚洲av高清不卡| 女性生殖器流出的白浆| 亚洲欧洲精品一区二区精品久久久| 国产爱豆传媒在线观看 | 国产v大片淫在线免费观看| 少妇 在线观看| 黄色视频不卡| 日本三级黄在线观看| 欧美亚洲日本最大视频资源| 免费人成视频x8x8入口观看| 久久久久久久久中文| 男女下面进入的视频免费午夜 | 在线观看免费视频日本深夜| 欧美激情高清一区二区三区| 精品久久久久久久久久久久久 | 国产成人影院久久av| 国产1区2区3区精品| 亚洲第一av免费看| 成人手机av| 亚洲av成人一区二区三| 亚洲精品色激情综合| 亚洲免费av在线视频| 精品不卡国产一区二区三区| 欧美激情高清一区二区三区| 一级毛片精品| 久久精品aⅴ一区二区三区四区| 午夜精品久久久久久毛片777| 美国免费a级毛片| 91麻豆精品激情在线观看国产| 国产成人一区二区三区免费视频网站| 精品福利观看| 天天添夜夜摸| 50天的宝宝边吃奶边哭怎么回事| www日本黄色视频网| 91国产中文字幕| 国产成+人综合+亚洲专区| 宅男免费午夜| www国产在线视频色| 国产99白浆流出| 黄频高清免费视频| 国产精品电影一区二区三区| 国产欧美日韩一区二区三| 一进一出抽搐动态| 老司机在亚洲福利影院| 国内毛片毛片毛片毛片毛片| 精品国内亚洲2022精品成人| 日韩欧美一区二区三区在线观看| 女人被狂操c到高潮| 熟女电影av网| av在线天堂中文字幕| 天堂影院成人在线观看| 国产又色又爽无遮挡免费看| 免费在线观看成人毛片| 99久久久亚洲精品蜜臀av| cao死你这个sao货| 美国免费a级毛片| 亚洲精品色激情综合| 99久久综合精品五月天人人| 欧美+亚洲+日韩+国产| 中亚洲国语对白在线视频| 精品久久久久久久人妻蜜臀av| 亚洲片人在线观看| 国产麻豆成人av免费视频| av免费在线观看网站| 久久久精品欧美日韩精品| 午夜久久久久精精品| 亚洲美女黄片视频| 99精品久久久久人妻精品| 国产精品乱码一区二三区的特点| 色综合站精品国产| 黄色a级毛片大全视频| 巨乳人妻的诱惑在线观看| 亚洲人成电影免费在线| 亚洲国产高清在线一区二区三 | 人人妻人人看人人澡| 国产蜜桃级精品一区二区三区| 亚洲人成伊人成综合网2020| 老熟妇仑乱视频hdxx| 老鸭窝网址在线观看| 99久久99久久久精品蜜桃| 久久中文看片网| 女人被狂操c到高潮| 少妇粗大呻吟视频| 欧美乱妇无乱码| 色老头精品视频在线观看| 国产精品日韩av在线免费观看| 欧美中文综合在线视频| 啦啦啦免费观看视频1| 无人区码免费观看不卡| 亚洲午夜精品一区,二区,三区| 久久久水蜜桃国产精品网| 成人亚洲精品av一区二区| 18禁观看日本| 香蕉国产在线看| 美女国产高潮福利片在线看| 一夜夜www| 一级毛片精品| 老汉色av国产亚洲站长工具| 观看免费一级毛片| 久久99热这里只有精品18| 成人国语在线视频| 此物有八面人人有两片| 久9热在线精品视频| 一二三四在线观看免费中文在| 国产精品av久久久久免费| 国产99久久九九免费精品| 在线永久观看黄色视频| 亚洲av电影不卡..在线观看| 国产成人系列免费观看| 欧美黄色淫秽网站| 亚洲真实伦在线观看| 午夜福利在线观看吧| 熟妇人妻久久中文字幕3abv| 听说在线观看完整版免费高清| 午夜免费鲁丝| 国产精品,欧美在线| 亚洲第一欧美日韩一区二区三区| 欧美日本亚洲视频在线播放| 欧美色欧美亚洲另类二区| 变态另类成人亚洲欧美熟女| 亚洲黑人精品在线| 岛国在线观看网站| 成人亚洲精品一区在线观看| 免费一级毛片在线播放高清视频| 国产欧美日韩精品亚洲av| 国产人伦9x9x在线观看| 国产主播在线观看一区二区| 色av中文字幕| 日韩 欧美 亚洲 中文字幕| xxxwww97欧美| 此物有八面人人有两片| 热99re8久久精品国产| 妹子高潮喷水视频| 久久性视频一级片| 亚洲三区欧美一区| 亚洲精品久久国产高清桃花| 国产精品99久久99久久久不卡| 免费观看人在逋| 亚洲av中文字字幕乱码综合 | 久久精品国产综合久久久| 中文字幕人妻丝袜一区二区| 日本一本二区三区精品| 国产高清激情床上av| www国产在线视频色| 国产精品影院久久| 午夜激情福利司机影院| 18禁观看日本| 黄色片一级片一级黄色片| 老汉色∧v一级毛片| 高清毛片免费观看视频网站| 女同久久另类99精品国产91| av超薄肉色丝袜交足视频| 无限看片的www在线观看| 成人亚洲精品一区在线观看| 男男h啪啪无遮挡| 男女床上黄色一级片免费看| 欧美一级a爱片免费观看看 | 女警被强在线播放| 天天躁夜夜躁狠狠躁躁| 亚洲一卡2卡3卡4卡5卡精品中文| 色哟哟哟哟哟哟| 嫁个100分男人电影在线观看| 精品福利观看| 午夜福利在线观看吧| 成人国产综合亚洲| 欧美在线一区亚洲| 午夜激情av网站| 亚洲七黄色美女视频| 好看av亚洲va欧美ⅴa在| 久久久久久久午夜电影| 在线国产一区二区在线| 天天添夜夜摸| 无限看片的www在线观看| 天天添夜夜摸| 韩国av一区二区三区四区| 欧美日韩中文字幕国产精品一区二区三区| 精品久久久久久久人妻蜜臀av| 天堂√8在线中文| 国产在线精品亚洲第一网站| 免费在线观看完整版高清| 黄色丝袜av网址大全| 国产主播在线观看一区二区| 丰满人妻熟妇乱又伦精品不卡| av在线播放免费不卡| 精品午夜福利视频在线观看一区| 精品国产一区二区三区四区第35| 亚洲avbb在线观看| 男女做爰动态图高潮gif福利片| 欧美一级毛片孕妇| 国产不卡一卡二| 精品日产1卡2卡| 精品久久久久久,| 首页视频小说图片口味搜索| 亚洲自拍偷在线| 欧美黄色淫秽网站| 国产一区在线观看成人免费| 日韩欧美在线二视频| 中文字幕人成人乱码亚洲影| 午夜久久久在线观看| 日韩 欧美 亚洲 中文字幕| 婷婷丁香在线五月| 久久精品aⅴ一区二区三区四区| 亚洲国产中文字幕在线视频| 久久婷婷人人爽人人干人人爱| 亚洲国产精品久久男人天堂| 国产私拍福利视频在线观看| 欧美久久黑人一区二区| 日韩三级视频一区二区三区| 久久国产亚洲av麻豆专区| 欧美激情极品国产一区二区三区| 亚洲av成人一区二区三| 少妇裸体淫交视频免费看高清 | 黄网站色视频无遮挡免费观看| 嫁个100分男人电影在线观看| 最好的美女福利视频网| 中文字幕精品亚洲无线码一区 | 国产av又大| 变态另类成人亚洲欧美熟女| 大香蕉久久成人网| 老熟妇仑乱视频hdxx| 操出白浆在线播放| 婷婷精品国产亚洲av在线| 97超级碰碰碰精品色视频在线观看| 2021天堂中文幕一二区在线观 | 免费一级毛片在线播放高清视频| 亚洲无线在线观看| 国产成+人综合+亚洲专区| 午夜亚洲福利在线播放| 白带黄色成豆腐渣| 亚洲精品久久国产高清桃花| 亚洲精品中文字幕在线视频| 18禁黄网站禁片午夜丰满| 97碰自拍视频| 最新在线观看一区二区三区| 巨乳人妻的诱惑在线观看| 美女扒开内裤让男人捅视频| 亚洲成人国产一区在线观看| 夜夜躁狠狠躁天天躁| www.999成人在线观看| 亚洲精品av麻豆狂野| 视频区欧美日本亚洲| 91在线观看av| 亚洲一码二码三码区别大吗| 亚洲五月色婷婷综合| 青草久久国产| 成人国产一区最新在线观看| 国产精品亚洲av一区麻豆| 国产在线观看jvid| 国产精品久久电影中文字幕| 精品国产美女av久久久久小说| 午夜久久久久精精品| 亚洲成av片中文字幕在线观看| 啦啦啦韩国在线观看视频| 琪琪午夜伦伦电影理论片6080| 久久精品国产综合久久久| 制服丝袜大香蕉在线| 女性被躁到高潮视频| 色播亚洲综合网| 黑人巨大精品欧美一区二区mp4| 国产亚洲av嫩草精品影院| 少妇粗大呻吟视频| 国产区一区二久久| 草草在线视频免费看| 国产高清videossex| 国产精品二区激情视频| 热re99久久国产66热| 极品教师在线免费播放| 国产乱人伦免费视频| 久久久久九九精品影院| 久久久久久人人人人人| 亚洲一区高清亚洲精品| 曰老女人黄片| 一夜夜www| av免费在线观看网站| 午夜福利视频1000在线观看| 亚洲 国产 在线| 欧美丝袜亚洲另类 | www.自偷自拍.com| 首页视频小说图片口味搜索| 久久婷婷人人爽人人干人人爱| 久久热在线av| 久久久国产精品麻豆| 欧美绝顶高潮抽搐喷水| www.www免费av| 12—13女人毛片做爰片一| 国产日本99.免费观看| 天天躁狠狠躁夜夜躁狠狠躁| 国产又爽黄色视频| 成年女人毛片免费观看观看9| 久久精品夜夜夜夜夜久久蜜豆 | 91九色精品人成在线观看| 满18在线观看网站| 欧美三级亚洲精品| 老司机午夜十八禁免费视频| 巨乳人妻的诱惑在线观看| 亚洲欧洲精品一区二区精品久久久| 亚洲久久久国产精品| 亚洲国产欧美网| 狠狠狠狠99中文字幕| 麻豆成人av在线观看| 久久香蕉激情| 成人三级黄色视频| 久久久久精品国产欧美久久久| 亚洲国产欧洲综合997久久, | 久久精品91无色码中文字幕| 一区二区三区高清视频在线| 桃色一区二区三区在线观看| 国产av一区二区精品久久| 精品国产乱子伦一区二区三区| 亚洲无线在线观看| 白带黄色成豆腐渣| 最近在线观看免费完整版| 亚洲真实伦在线观看| 法律面前人人平等表现在哪些方面| 欧美黑人欧美精品刺激| 女人爽到高潮嗷嗷叫在线视频| 男女视频在线观看网站免费 | 亚洲精品国产精品久久久不卡| 国产成+人综合+亚洲专区| 色精品久久人妻99蜜桃| 亚洲成人久久性| 在线av久久热| 18禁国产床啪视频网站| 麻豆一二三区av精品| 亚洲七黄色美女视频| 成人特级黄色片久久久久久久| 美女 人体艺术 gogo| 精华霜和精华液先用哪个| 欧美久久黑人一区二区| 极品教师在线免费播放| 亚洲五月色婷婷综合| 黄色片一级片一级黄色片| 美女午夜性视频免费| 黑人欧美特级aaaaaa片| 亚洲狠狠婷婷综合久久图片| 国产精品1区2区在线观看.| 麻豆国产av国片精品| 亚洲成人精品中文字幕电影| 国产又黄又爽又无遮挡在线| 黑丝袜美女国产一区| 一二三四在线观看免费中文在| 欧美日韩福利视频一区二区| 国产一区在线观看成人免费| 中文字幕久久专区| 久久精品国产亚洲av高清一级| 精品国产亚洲在线| e午夜精品久久久久久久| 国产精品久久久久久精品电影 | 一个人观看的视频www高清免费观看 | 午夜老司机福利片| 日韩欧美一区视频在线观看| 亚洲av中文字字幕乱码综合 | 日韩欧美 国产精品| 老司机午夜福利在线观看视频| 欧美黄色片欧美黄色片| 国产亚洲精品一区二区www| www.www免费av| 午夜福利在线在线| 在线播放国产精品三级| 久久久精品欧美日韩精品| 一级黄色大片毛片| aaaaa片日本免费| 午夜福利一区二区在线看| 一级毛片精品| 欧美黑人巨大hd| 老司机靠b影院| 亚洲欧美精品综合久久99| www.999成人在线观看| 超碰成人久久| 成年人黄色毛片网站| 亚洲自拍偷在线| 黑人欧美特级aaaaaa片| 免费女性裸体啪啪无遮挡网站| 久久精品91蜜桃| 久久精品国产99精品国产亚洲性色| 久久久久久久久久黄片| 国产免费男女视频| 久久九九热精品免费| 欧美av亚洲av综合av国产av| 高潮久久久久久久久久久不卡| 18禁美女被吸乳视频| 丝袜在线中文字幕| 熟女少妇亚洲综合色aaa.| 久久精品影院6| 国产精品国产高清国产av| 久久香蕉激情| 国产高清激情床上av| 热re99久久国产66热| 欧美成人一区二区免费高清观看 | 亚洲成人久久性| 欧美另类亚洲清纯唯美| 视频区欧美日本亚洲| 日本成人三级电影网站| aaaaa片日本免费| 亚洲,欧美精品.| 国产伦一二天堂av在线观看| 久久午夜亚洲精品久久| 精华霜和精华液先用哪个| 精品国内亚洲2022精品成人| 色播亚洲综合网| 韩国精品一区二区三区| 国产aⅴ精品一区二区三区波| 日韩欧美免费精品| 久久久久久免费高清国产稀缺| 精品欧美一区二区三区在线| 啦啦啦 在线观看视频| 777久久人妻少妇嫩草av网站| 精品一区二区三区四区五区乱码| 国产精品免费一区二区三区在线| 无限看片的www在线观看| 久9热在线精品视频| 欧美黑人精品巨大| 亚洲国产日韩欧美精品在线观看 | av天堂在线播放| 久久热在线av| 国产高清有码在线观看视频 | www日本黄色视频网| 国产1区2区3区精品| 亚洲精品久久国产高清桃花| 日韩成人在线观看一区二区三区| 国内精品久久久久精免费| 色播亚洲综合网| 婷婷精品国产亚洲av在线| 黄片播放在线免费| 狠狠狠狠99中文字幕| 亚洲专区中文字幕在线| 久久午夜综合久久蜜桃| 亚洲狠狠婷婷综合久久图片| 999久久久精品免费观看国产| 成人三级做爰电影| a级毛片a级免费在线| 亚洲精品在线美女| 欧美另类亚洲清纯唯美| 嫩草影院精品99| 色哟哟哟哟哟哟| 亚洲国产精品久久男人天堂| 亚洲九九香蕉| 老汉色∧v一级毛片| svipshipincom国产片| 香蕉丝袜av| 日韩免费av在线播放| 国产又黄又爽又无遮挡在线| 满18在线观看网站| 亚洲精品在线观看二区| 高潮久久久久久久久久久不卡| 欧美黑人巨大hd| 精品久久久久久,| 国产激情偷乱视频一区二区| 久久久久九九精品影院| 国产不卡一卡二|