ClNAC100 Is a NAC Transcription Factor of Chinese Fir in Response to Phosphate Starvation.

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between -1519 bp and -589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.

Hybridized Frequency Combs in Multimode Cavity Electromechanical System.

The cavity electromechanical devices with radiation-pressure interaction induced Kerr-like nonlinearity are promising candidates to generate microwave frequency combs. We construct a silicon-nitride membrane based superconducting cavity electromechanical device and study two mechanical modes synergistic frequency combs. Around the threshold of intracavity field instability, we first show independent frequency combs with tooth spacing equal to each mechanical mode frequency. At the overlap boundaries of these two individual mechanical mode mediated instability thresholds, we observe hybridization of frequency combs based on the cavity field mediated indirect coupling between these two mechanical modes. The spectrum lines turn out to be unequally spaced, but can be recognized in combinations of the coexisting frequency combs. Beyond the boundary, the comb reverts to the single mode case, and which mechanical mode frequency will the tooth spacing be depends on the mode competition. Our work demonstrates mechanical mode competition enabled switchability of frequency comb tooth spacing and can be extended to other devices with multiple nonlinearities.

Development of novel polymorphic microsatellite markers for Picea brachytyla.

BACKGROUND: Picea brachytyla is a unique tree species in China. Due to being extensively exploited in the past, it is listed as Vulnerable in the IUCN Red List. It is mainly distributed across the Hengduan and Daba-Qinglin mountains and has been found in other areas including Sichuan Province and Qinghai Province, China. Microsatellites, or simple sequence repeats (SSRs), are widely used in correlational studies of genetic protection. Few markers have been developed for P. brachytyla because of the small number of trees and scholarly resources available for study. METHODS AND RESULTS: The genomic DNA of P. brachytyla was sequenced using the DNBSEQ platform, and unigenes were obtained after assembly and deredundancy. Of the 100 primer pairs screened, we isolated 10 useful microsatellite loci from P. brachytyla genes. The observed and expected heterozygosity values ranged from 0.173 (P24) to 0.788 (P79; mean 0.469) and 0.199 (P87) to 0.911 (P79; mean 0.700), respectively. Polymorphism-information content (PIC) ranged from 0.190 (P84) to 0.904 (P79; mean 0.666). Only P84 and P72 were in a Hardy-Weinberg equilibrium (P > 0.05) in the different P. brachytyla populations. All the levels of linkage disequilibrium (LD) were high for the 10 SSR loci indicating that there were no autocorrelations among the 10 SSR loci. CONCLUSIONS: The novel polymorphic microsatellite markers showed high polymorphism for P. brachytyla. These polymorphic microsatellites can provide a basis for future conservation and genetic research on this rare plant species.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa.

Secondary cell wall (SCW) biosynthesis is the biological process that generates wood, an important renewable feedstock for materials and energy. NAC domain transcription factors, particularly Vascular-Related NAC-Domain (VND) and Secondary Wall-Associated NAC Domain (SND) proteins, are known to regulate SCW differentiation. The regulation of VND and SND is important to maintain homeostasis for plants to avoid abnormal growth and development. We previously identified a splice variant, PtrSND1-A2IR , derived from PtrSND1-A2 as a dominant-negative regulator, which suppresses the transactivation of all PtrSND1 family members. PtrSND1-A2IR also suppresses the self-activation of the PtrSND1 family members except for its cognate transcription factor, PtrSND1-A2, suggesting the existence of an unknown factor needed to regulate PtrSND1-A2 Here, a splice variant, PtrVND6-C1IR , derived from PtrVND6-C1 was discovered that suppresses the protein functions of all PtrVND6 family members. PtrVND6-C1IR also suppresses the expression of all PtrSND1 members, including PtrSND1-A2, demonstrating that PtrVND6-C1IR is the previously unidentified regulator of PtrSND1-A2 We also found that PtrVND6-C1IR cannot suppress the expression of its cognate transcription factor, PtrVND6-C1PtrVND6-C1 is suppressed by PtrSND1-A2IR Both PtrVND6-C1IR and PtrSND1-A2IR cannot suppress their cognate transcription factors but can suppress all members of the other family. The results indicate that the splice variants from the PtrVND6 and PtrSND1 family may exert reciprocal cross-regulation for complete transcriptional regulation of these two families in wood formation. This reciprocal cross-regulation between families suggests a general mechanism among NAC domain proteins and likely other transcription factors, where intron-retained splice variants provide an additional level of regulation.

[Yishen Tongluo Recipe combined with minimally invasive surgery for the treatment of varicocele-associated asthenospermia].

OBJECTIVE: To observe the clinical effect of Yishen Tongluo Recipe (YTR) combined with minimally invasive surgery (MIS) in the treatment of varicocele-associated asthenospermia (of the kidney-deficiency and collateral-obstruction type). METHODS: We randomly divided 116 patients with varicocele-associated asthenospermia and up to the inclusion criteria into three groups and treated them by YTR combined with MIS (low spermatic vein ligation at the external inguinal ring orifice under the microscope) (the YTR+MIS group, n = 39), YTR alone (the YTR group, n = 38), or MIS alone (the MIS group, n = 39). At 12 weeks after treatment, we compared the total effectiveness rate, the percentage of progressively motile sperm (PMS), sperm straight line velocity (VSL), sperm DNA fragmentation index (DFI) and symptoms among the three groups of patients. RESULTS: After treatment, the total effectiveness rate was significantly higher in the YTR+MIS than in the YTR and the MIS group (89.74% vs 63.16% and 64.10%, P < 0.05), and so were the percentage of PMS (31.67 ± 3.93 vs 24.48 ± 3.15 and 25.57 ± 3.37, P < 0.05) and VSL (ï¼»30.19 ± 5.32ï¼½ vs ï¼»27.19 ± 5.48ï¼½ and ï¼»27.55 ± 5.24ï¼½ µm/s, P < 0.05), but the DFI was remarkably lower in the former than in the latter two groups (11.25 ± 8.27 vs 15.87 ± 5.45 and 15.26 ± 4.36, P < 0.05). The symptoms were improved more significantly in the YTR+MIS than in the YTR and MIS groups (P < 0.05). CONCLUSIONS: Yishen Tongluo Recipe combined with minimally invasive surgery, as an effective and safe strategy, can significantly improve sperm quality and sperm DNA integrity in patients with varicocele-associated asthenospermia.

Poplar miR472a targeting NBS-LRRs is involved in effective defence against the necrotrophic fungus Cytospora chrysosperma.

The hemibiotroph Colletotrichum gloeosporioides and the necrotroph Cytospora chrysosperma cause poplar foliage and stem disease, respectively, resulting in substantial economic losses. In this study, Populus trichocarpa ptc-miR472a was down-regulated in leaves treated with salicylic acid, jasmonic acid (JA) or bacterial flagellin (flg22). Here, ptc-miR472a and a short tandem target mimic (STTM) of miR472a were overexpressed in P. alba × P. glandulosa, and overexpression lines of miR472a and silenced lines of STTM472a were generated. Compared with the STTM472a and wild type lines, lower reactive oxygen species accumulation was detected in miR472a overexpressing plants treated with flg22, C. gloeosporioides or C. chrysosperma. In addition, the miR472a overexpressing lines exhibited the highest susceptibility to the hemibiotroph, C. gloeosporioides, but the highest effective defence response to the necrotroph, C. chrysosperma. The JA/ethylene marker gene ERF1 was rapidly up-regulated in miR472a overexpressing plants. Furthermore, five phased, secondary, small interfering RNAs (phasiRNAs) were confirmed in the miR472a overexpressing and STTM472a lines, triggering phasiRNAs predicted to enhance NBS-LRR silencing. Taken together, our results revealed that ptc-miR472a exerts a key role in plant immunity to C. gloeosporioides and C. chrysosperma by targeting NBS-LRR transcripts. This study provides a new strategy and method in plant breeding to improve plant disease resistance.

[The Effects of Complex of Benzoquinone on Fermi Resonance].

Fermi resonance phenomenon exists in simple compounds and it also widely exists in vibration spectra of complex. The complex can be formed by adding up simple compounds. As a result, the characteristic parameters of some parts of molecule will make changes, and the molecular spectra have a significant change along with it. Benzoquinone and proline in the solution form charge-transfer complex under certain conditions, but the spectra intensity is weak, our research uses Teflon liquid-core optical fiber technology to gain high quality resonance Raman spectra. We acquire Raman spectra of Benzoquinone and its complex in experiments, and analyze the characteristic parameters of Fermi resonance according to J. F. Bertran quantum theory. The results shows that, because of the formation of complex, Fermi resonance peak of C==0 bond shifts to high wavelength, the spectra intensity decreases, the frequency space increases, the coupling coefficient increases. The explanation is that, in the solution of complex, proline is donor, while benzoquinone is acceptor, the non-bonding electron of N atom which is belong to proline transfers to the pi anti-bonding orbital of benzoquinone, then n-pi* charge transfer complex is produced. That causes the change of molecular energy level, changes the Raman spectra. All these researches provide new idea and clue for spectral line certification and attribution of complex molecules, complexes and polymer.

Genome-wide identification and functional prediction of novel and drought-responsive lincRNAs in Populus trichocarpa.

Protein-coding genes are considered to be a dominant component of the eukaryotic transcriptome; however, many studies have shown that intergenic, non-coding transcripts also play an important role. Long intergenic non-coding RNAs (lincRNAs) were found to play a vital role in human and Arabidopsis. However, lincRNAs and their regulatory roles remain poorly characterized in woody plants, especially Populus trichocarpa (P. trichocarpa). A large set of Populus RNA-Seq data were examined with high sequencing depth under control and drought conditions and a total of 2542 lincRNA candidates were identified. In total, 51 lincRNAs and 20 lincRNAs were identified as putative targets and target mimics of known Populus miRNAs, respectively. A total of 504 lincRNAs were found to be drought responsive, eight of which were confirmed by RT-qPCR. These findings provide a comprehensive view of Populus lincRNAs, which will enable in-depth functional analysis.

Single-base-resolution methylomes of Populus trichocarpa reveal the association between DNA methylation and drought stress.

BACKGROUND: DNA methylation is an important biological form of epigenetic modification, playing key roles in plant development and environmental responses. RESULTS: In this study, we examined single-base resolution methylomes of Populus under control and drought stress conditions using high-throughput bisulfite sequencing for the first time. Our data showed methylation levels of methylated cytosines, upstream 2 kp, downstream 2kb, and repeatitive sequences significantly increased after drought treatment in Populus. Interestingly, methylation in 100 bp upstream of the transcriptional start site (TSS) repressed gene expression, while methylations in 100-2000 bp upstream of TSS and within the gene body were positively associated with gene expression. Integrated with the transcriptomic data, we found that all cis-splicing genes were non-methylated, suggesting that DNA methylation may not associate with cis-splicing. However, our results showed that 80% of trans-splicing genes were methylated. Moreover, we found 1156 transcription factors (TFs) with reduced methylation and expression levels and 690 TFs with increased methylation and expression levels after drought treatment. These TFs may play important roles in Populus drought stress responses through the changes of DNA methylation. CONCLUSIONS: These findings may provide valuable new insight into our understanding of the interaction between gene expression and methylation of drought responses in Populus.