香京julia种子在线播放

The BPH strain was originally collected from a rice field in Hangzhou, China. Insects were reared on rice seedlings (rice variety: Xiushui 134) in a walk-in chamber at 26 ± 0.5°C, with a light: dark photoperiod of 16: 8 h and relative humidity of 50 ± 5%.

The amino acid sequences of Drosophila NF-Y homologs were used to screen BPH genomic and transcriptomic databases for homologs. Total RNA was isolated from BPH nymphs using RNAiso plus (Takara #9109) according to the manufacturer’s protocol. For cDNA syntheses, 450 ng of total RNA was reversely transcribed in a 10 μL reaction with HiScript QRT SuperMix (Vazyme #R223-01). The NlNF-YA, NlNF-YB, and NlNF-YC sequences were amplified from cDNA using fidelity DNA polymerase (Toyobo #930700) with NlNF-YA-F/R, NlNF-YB-F/R, and NlNF-YC-F/R primer pairs, respectively (Supplementary Table 1). The PCR product was cloned and the sequence was determined by Sanger sequencing.

To identify the exon-intron construction of NlNF-Y, open reading frames (ORFs) of NlNF-YA, NlNF-YB, and NlNF-YC were used to search a BPH genomic database (Xue et al., 2014). For phylogenetic analysis, 39 NF-Y sequences from 13 species including D. melanogaster, N. lugens, Acyrthosiphon pisum, Apis cerana, Bemisia tabaci, Bombyx mori, Cryptotermes secundus, Halyomorpha halys, Monomorium pharaonis, Nasonia vitripennis, Nicrophorus vespilloides, Tribolium castaneum, and Zootermopsis nevadensis were used for sequence alignment. A phylogenetic tree was constructed with maximum-likelihood method and 1000 bootstraps using the MEGA 7 program (Kumar et al., 2016).

Total RNA was isolated from eggs (n = 100), first-instar (n = 100), second-instar (n = 50), third-instar (n = 50), fourth-instar (n = 30), fifth-instar nymphs (n = 15), and adult females (n = 15), which were laid or ecdysed within 24 h. To examine tissue distribution, we dissected the head (n = 50), antenna (n = 200), tergum (n = 50), leg (n = 100), fat body (n = 30), cuticle (n = 50), and gut (n = 100) from four-instar nymphs for RNA extraction. First-strand cDNA was synthesized from total RNA (450 ng) using HiScript QRT SuperMix (Vazyme #R223-01). The synthesized cDNAs were 10-fold diluted and used as templates for quantitative real-time PCR (qPCR). The qPCR primers for NlNF-Y (Supplementary Table 1) were designed using Primer-Blast.¹ The qPCR was conducted on a CFX96TM real-time PCR detection system (Bio-Rad) using the following conditions: denaturation for 3 min at 95°C, followed by 40 cycles at 95°C for 10 s, and 60°C for 30 s. The ribosomal protein S11 gene (rps11) was used as the internal reference gene (Yuan et al., 2014). The 2^–ΔΔCt method (Ct represents the cycle threshold) was used to measure the relative expression level (Livak and Schmittgen, 2001). Three independent biological replicates with three technical replicates were conducted for each experiment.

The dsRNAs were synthesized using a T7 High Yield Transcription Kit (Vazyme #TR101-02) according to the manufacturer’s instructions with primers containing the T7 RNA polymerase promoter at both ends (Supplementary Table 1). A dsRNA injection was carried out as in our previous study (Xu et al., 2015). Briefly, fourth-instar nymphs were anesthetized with carbon dioxide for 10–15 s. Approximately 150 ng dsRNA was microinjected into the mesothorax using a FemtoJet microinjection system (Eppendorf). To investigate whether disruption of NlNF-YA affected ecdysone signaling activity, fifth-instar nymphs (n = 10 for each of three replicates) were collected to examine the expression levels of NlE74A and NlE75B by qPCR using corresponding primers (Supplementary Table 1). To investigate RNAi efficiency, BPHs (n = 5 for each of three replicates) at 24 h after adult eclosion were collected for qPCR assay.

Fourth-instar nymphs (36–48 h after ecdysis, hAE) were microinjected with dsRNAs targeting NlNF-YA (dsNlNF-YA), NlNF-YB (dsNlNF-YB), NlNF-YC (dsNlNF-YC), or GFP (dsGFP). After adult eclosion (24 h), images of the forewings and hind tibiae were captured with a DFC320 digital camera attached to a Leica S8AP0 stereomicroscope using a LAS digital imaging system (v. 3.8). Digital images of forewings (n = 20) and hind tibias (n = 20) were collected for the measurement of forewing size and hind tibia length using ImageJ (v. 1.47).

Fourth-instar nymphs (36–48 hAE) were microinjected with corresponding dsRNAs targeting each gene, and then nymphs were maintained on fresh rice seedings until the fifth-instar stage (within 48 h). Fifth-instar nymphs were microinjected with 20E (1 mg/ml) or distilled water, and the adult eclosion rate was calculated.

Fourth-instar nymphs (36–48 hAE) were microinjected with dsRNAs targeting NlNF-YA, NlNF-YB, NlNF-YC, or GFP. The thoraxes were dissected from adult females (24 h after emergence) for Transmission Electron Microscopy (TEM) as in our previous study (Xue et al., 2013). Briefly, samples were fixed in 2.5% glutaraldehyde overnight at 4°C. After fixation, samples were post-fixed in 1% osmium tetroxide for 1.5 h. Then, samples were dehydrated in a standard ethanol/acetone series, infiltrated and embedded in Spurr medium, and then superthin sections were cut. The sections were stained with 5% uranyl acetate followed by Reynolds’ lead citrate solution and observed under a JEM-1230 transmission electron microscope (JEOL).

Newly emerged females and males (within 12 h after eclosion) were microinjected with dsNF-YA, ds-NF-YB, dsNF-YC, or dsGfp. Then each female was allowed to match with two males in a glass tube. Insects were removed at 5 days after matching, and eggs were counted under a Leica S8AP0 stereomicroscope.

Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). Means were compared using a two-tailed Student’s t-test and log-rank (Mantel-Cox) test at a significance level set at ^∗P < 0.05, ^∗∗P ≤ 0.01, and ^∗∗∗P ≤ 0.001.

We identified three genes encoding BPH NF-Y homologs, NlNF-YA, NlNF-YB, and NlNF-YC, in a BLAST search against BPH genomic (Xue et al., 2014) and transcriptome databases using the D. melanogaster NF-Y proteins as query sequences. The ORFs of NlNF-YA, NlNF-YB, and NlNF-YC were 1002-, 609-, and 1011-bp in length, encoding 333, 202, and 336 amino acid residues, respectively (Supplementary Figures 1–3). Exon-intron construction analysis showed that the NlNF-YA, NlNF-YB, and NlNF-YC ORFs consist of eight, five, and eight exons, located in scaffolds 6450/1657, 3414, and 1809, respectively. A phylogenetic analysis based on NF-Y homologs from 13 species showed that NlNF-YA, NlNF-YB, and NlNF-YC together with their counterparts formed three distinct clusters (Figure 1), indicating that the BPH NF-Y genes we identified were authentic NF-Y homologs.

Spatio-temporal expression of NlNF-YA, NlNF-YB, and NlNF-YC was examined by qPCR. NlNF-YA, NlNF-YB, and NlNF-YC transcripts were readily detected from egg to adult stages, and relatively high levels were detected in eggs laid within 24 h (Egg-24 h) (Figure 2A), indicating that they might play important functions in egg development. Tissue distribution analysis showed that NlNF-YA, NlNF-YB, and NlNF-YC were evenly expressed in head, antenna, tergum, leg, fat body, cuticle, and gut of fourth-instar nymphs (Figure 2B), indicating NlNF-Y genes might be important for BPH growth and development.

To investigate whether NlNF-YA, NlNF-YB, and NlNF-YC played any roles in BPH development, fourth-instar short-wing-destined nymphs were microinjected with corresponding dsRNAs targeting each gene. At 48 h after microinjection, RNAi efficiency was examined by qPCR, which showed that dsRNA treatments significantly down-regulated the expression levels of NlNF-YA, NlNF-YB, and NlNF-YC by 63.7, 99.9, and 82.7%, respectively, relative to the GFP^RNAi treatment (Figure 3A). Notably, NlNF-YA^RNAi, NlNF-YB^RNAi, and NlNF-YC^RNAi caused high mortality, leading to approximately 60, 60, and 40% of nymphs died before adult eclosion (Figure 3B), respectively. The remaining nymphs could successfully molt into adults. However, these adults had moderately smaller and slightly malformed forewings relative to GFP^RNAi BPHs (Figures 4A,B). NlNF-YA^RNAi, NlNF-YB^RNAi, and NlNF-YC^RNAi specifically reduced forewing size but had marginal roles on hind tibiae length (Figure 4C). These data indicated that NlNF-YA, NlNF-YB, and NlNF-YC were essential for normal wing growth in BPHs.

Because short-wing BPHs lack hindwings and IFM, we used long-wing BPHs to investigate whether NlNF-YA, NlNF-YB, and NlNF-YC had any effect on IFM development. For this, fourth-instar long-wing-destined nymphs were microinjected with corresponding dsRNAs targeting each gene. NlNF-YA^RNAi, NlNF-YB^RNAi, and NlNF-YC^RNAi gave rise to adults with curved forewings and hindwings (Figure 5A). In line with the phenotype in short-winged BPHs, smaller forewings and hindwings were observed in adults previously treated with either NlNF-YA^RNAi, NlNF-YB^RNAi, or NlNF-YC^RNAi (Figure 4B), although there was no discernable effect on hind tibia length (Figure 4C). Next, these adults were collected for IFM ultrastructure examination using TEM. In GFP^RNAi adults, sarcomeres were clearly divided by Z discs and well-organized (Figure 6). In contrast, NlNF-YA^RNAi, NlNF-YB^RNAi, or NlNF-YC^RNAi led to defective sarcomeres, with deformed Z discs and weakly organized myofibrillar. These events indicated that the NlNF-Y complex might be essential for IFM assembly in BPH.

Given that NlNF-Y genes were evenly expressed across nymphal stages (Figure 2A), we investigated whether knockdown of NlNF-Y genes would affect nymph growth. For this purpose, third-instar nymphs were microinjected with corresponding dsRNAs targeting each gene. Nymphs treated with NlNF-YA^RNAi, NlNF-YB^RNAi, or NlNF-YC^RNAi had an extended fifth-instar duration compared with those treated with GFP^RNAi (Figure 7A), with NlNF-YA^RNAi showing the most profound effect (Figure 7A). In addition, the majority of NlNF-YA^RNAi-treated nymphs died before adult emergence (Figure 7B), indicating that the disruption of the NF-Y complex impaired nymph-to-adult ecdysis.

Given that ecdysone is a hormone that initiates all major developmental transitions from the egg, to larva, to pupa, to adult in insects (Gilbert et al., 2002; Dubrovsky, 2005), we asked whether it mediated the effects of NlNF-YA on adult molting. For this purpose, we first assessed the expression levels of two early ecdysone response genes, NlE74A and NlE75E (Dubrovsky, 2005), in the context of NlNF-YA knockdown. NlNF-YA^RNAi significantly reduced both NlE74A and NlE75E transcripts compared with GFP^RNAi (Figure 7C), indicating that knockdown of NlNF-YA might impair ecdyson signaling activity. Following this observation, we asked whether the addition of ecdysone could rescue the NlNF-YA^RNAi defect. To this end, fourth-instar nymphs were microinjected with dsNlNF-YA, followed by microinjection with 20E when nymphs proceeded to the fifth-instar at 48 h after eclosion. The addition of 20E could partially restore the molting defect caused by NlNF-YA^RNAi (Figure 7B) compared with the addition of water, indicating that NlNF-YA^RNAi decreased adult eclosion at least partially through the ecdysone signaling pathway.

Given that relatively high amounts of NlNF-YA, NlNF-YB, and NlNF-YC transcripts were detected at the early egg stage (Figure 2A), we asked whether NlNF-YA, NlNF-YB, and NlNF-YC contributed to BPH reproduction. Female and male adults at 12 h after eclosion were microinjected with corresponding dsRNAs targeting each gene, and then allowed to mate and to lay eggs for 5 days. BPHs treated with NlNF-YA^RNAi, NlNF-YB^RNAi, and NlNF-YC^RNAi laid substantially fewer eggs than those treated with GFP^RNAi (Figure 8A). In addition, the eggs laid by BPHs previously treated with NlNF-YA^RNAi, NlNF-YB^RNAi, or NlNF-YC^RNAi failed to develop eye pigmentation, a characteristic hallmark of egg development, indicating NlNF-Y genes might be essential for embryogenesis (Figure 8B).