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Genetic manipulation of plant genes in prokaryotes has been widely used in molecular biology, but the function of a DNA sequence is far from being fully known. Here, we discovered that a plant protein-coding gene containing the CRAL_TRIO domain serves as a promoter in bacteria. We firstly characterized CitPITP1 from Citrus, which contains the CRAL_TRIO domain, and identified a 64-bp sequence (key64) that is critical for prokaryotic promoter activity. In vitro experiments indicated that the bacterial RNA polymerase subunit RpoD specifically binds to key64. We then expanded our research to fungi, plant and animal species to identify key64-like sequences. Five such prokaryotic promoters were isolated from Amborella, Rice, Arabidopsis and Citrus. Two conserved motifs were identified, and mutation analysis indicated that the nucleotides at positions 7, 29 and 30 are crucial for key64-like transcription activity. We detected full-length recombinant CitPITP1 from E. coli, and visualized a CitPITP1-GFP fusion protein in plant cells, supporting the idea that CitPITP1 encodes a protein. However, although exon 4 of CitPITP1 contained key64, it did not demonstrate promoter activity in plants. Our study describes a new basal promoter, provides evidence for neofunction of gene elements across different kingdoms, and provides new knowledge for the modular design of promoters.

Carotenoids are indispensable for both human health and plant survival. Citrus, is one of the fruit crops richest in carotenoid compounds, with approximately 115 kinds of carotenoids; tremendous diversity in carotenoids composition and concentration exists among various species, showing different colors from nearly white to crimson. The carotenoid biosynthetic pathway and the key carotenogenic genes have been identified in citrus; however, the underlying regulatory mechanisms remain unclear. In this study, among the main species of genus Citrus (primitive, wild, and cultivated), we detected carotenoids in flavedo using High-Performance Liquid Chromatography, and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes. Intriguingly, both carotenoid composition and content were generally increased during the evolution of citrus, and the corresponding variations in the promoters were identified, including the gain or loss of critical environmental stress-responsive elements and hormone-responsive elements, which are closely associated with carotenoid enhancement. In addition, pummelo has the most heat-responsive elements, but the Mangshan mandarin does not have this element in the promoters of PSY, which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation. Moreover, the abscisic acid-responsive motif was rich in almost all the seven species, but the ethylene-responsive motif was deficient, which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus. Overall, our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus, which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.

Anisodamine hydrobromide (Ani HBr) has been used to improve the microcirculation during cardiovascular disorders and sepsis. Glycocalyx plays an important role in preserving the endothelial cell (EC) barrier permeability and nitric oxide (NO) production. We aimed to test the hypothesis that Ani HBr could protect the EC against permeability and NO production via preventing glycocalyx shedding. A human cerebral microvascular EC hCMEC/D3 injury model induced by lipopolysaccharide (LPS) was established. Ani HBr was administrated to ECs with the LPS challenge. Cell viability was performed by Cell Counting Kit-8 assay. Cell proliferation and apoptosis were detected by EdU and Hoechst 33342 staining. Apoptosis and cell cycle were also assessed by flow cytometry with annexin V staining and propidium iodide staining, respectively. Then, adherens junction integrity was evaluated basing on the immunofluorescence staining of vascular endothelial cadherin (VE-cadherin). The glycocalyx component heparan sulfate (HS) was stained in ECs. The cell permeability was evaluated by leakage of fluorescein isothiocyanate (FITC)-dextran. Cellular NO production was measured by the method of nitric acid reductase. Ani HBr at 20 μg/mL significantly increased the viability of ECs with LPS challenge, but significantly inhibited the cell viability at 80 μg/mL, showing a bidirectional regulation of cell viability by Ani HBr. Ani HBr had not significantly change the LPS-induced EC proliferation. Ani HBr significantly reversed the induction of LPS on EC apoptosis. Ani HBr reinstated the LPS-induced glycocalyx and VE-cadherin shedding and adherens junction disruption. Ani HBr significantly alleviated LPS-induced EC layer permeability and NO production. Ani HBr protects ECs against LPS-induced increase in cell barrier permeability and nitric oxide production via preserving the integrity of glycocalyx. Ani HBr is a promising drug to rescue or protect the glycocalyx.

Background. The Coronavirus Disease 2019 (COVID-19) outbreak in Wuhan, China, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anisodamine hydrobromide injection (AHI), the main ingredient of which is anisodamine, is a listed drug for improving microcirculation in China. Anisodamine can improve the condition of patients with COVID-19. Materials and Methods. Protein-protein interactions obtained from the String databases were used to construct the protein interaction network (PIN) of AHI using Cytoscape. The crucial targets of AHI PIN were screened by calculating three topological parameters. Gene ontology and pathway enrichment analyses were performed. The intersection between the AHI component proteins and angiotensin-converting enzyme 2 (ACE2) coexpression proteins was analyzed. We further investigated our predictions of crucial targets by performing molecular docking studies with anisodamine. Results. The PIN of AHI, including 172 nodes and 1454 interactions, was constructed. A total of 54 crucial targets were obtained based on topological feature calculations. The results of Gene Ontology showed that AHI could regulate cell death, cytokine-mediated signaling pathways, and immune system processes. KEGG disease pathways were mainly enriched in viral infections, cancer, and immune system diseases. Between AHI targets and ACE2 coexpression proteins, 26 common proteins were obtained. The results of molecular docking showed that anisodamine bound well to all the crucial targets. Conclusion. The network pharmacological strategy integrated molecular docking to explore the mechanism of action of AHI against COVID-19. It provides protein targets associated with COVID-19 that may be further tested as therapeutic targets of anisodamine.

Fruit shape is an important trait for fruit appearance and commercial value. Diversity of fruit-shape has been utilized in the breeding of pummelo (Citrus maxima), a basic species in Citrus. However, little is known about genetic basis of fruit shape in citrus. In this study, we identified 16 OVATE family protein (OFP) genes in the pummelo genome. Phylogenetically, they were classified into three subfamilies, which was consistent with the classification of their Arabidopsis orthologs. Synteny analysis suggested that segment and tandem duplications were responsible for their expansion in pummelo. Expression pattern analysis of Citrus OFPs (CitOFPs) showed that CitOFP19 had significantly higher expression level in the ovaries of round pummelo than in those of pear-shaped pummelo. Heterologous overexpression of CitOFP19 in tomato resulted in pear-shaped ovary and fruit shape. Taken together, this study characterized OVATE gene family in Citrus genome and assessed the function of CitOFP19.

Carotenoids are important secondary metabolites that may participate in response to extreme environments. Fruit color changes were observed in peaches growing at altitude on the Tibetan Plateau. Here, we qualitatively and quantitatively analyzed 43 kinds of carotenoids in 96 Tibetan peach and 12 cultivated peach fruit samples. Comparative analysis revealed that 25 kinds of carotenoids accumulated at significantly different levels between Tibetan peaches and cultivated peaches. Based on a population structure analysis, the carotenoid levels of Tibetan peaches were divided into two groups, which are mainly affected by the environmental factors light and temperature. The correlation analysis implied that the levels of 9 carotenoids were significantly correlated with altitude. qRT-PCR results showed that PSY, CCD4 and BCH were significantly differently expressed between the low and high altitude Tibetan peaches. In summary, this study showed that the abundant variation in carotenoids was highly associated with high-altitude adaptations in Tibetan peach fruit.

Limiting the scan views of X-ray computed tomography (CT) can make radiation dose reduced efficiently and consequently weaken the damage of ionizing radiation. However, it will degrade the reconstructed CT images. In this paper, we proposed to predict the missing projections and improve the reconstructed CT images by constructing an autoencoder-like generative adversarial network (GAN) with joint loss function. In the generator network, we train an autoencoder-like convolutional neural network (CNN) to generate the missing projections given a sinogram of the limited-view CT projections. For the discriminator network, a CNN is used to classify an input sinogram as real or synthetic one. To produce more realistic images, the joint loss function which includes not only reconstruction loss, but the adversarial loss is employed. While reconstruction loss can capture the overall structure of the missing projections, the latter can pick a particular mode from the distribution and make the results much sharper. After the missing projections have been estimated, we reconstruct the CT images from the completed projections by utilizing conventional filtered back-projection (FBP) method. The experiments prove the capability of our method to achieve a considerable improvement in limited-view CT reconstruction.

Bronze gudgeon, Coreius heterodon, is an endemic and economically important fish in the Yangtze River, whose abundance has declined dramatically because of dam construction, overfishing, and water pollution. The Gezhouba and Three Gorges dams block connection of the bronze gudgeon populations above and below the dams. We collected bronze gudgeon from four sites in the mainstem of the Yangtze River, with one site above the dams and three sites below the dams, and studied genetic structure within and among the samples using 12 microsatellite DNA markers. Differences in indexes of genetic diversity were not significant among all the samples. No recent dramatic decrease of effective population size was inferred for all the samples using the population bottleneck test. Overall and pairwise genetic differentiation showed no significant genetic differentiation. Membership proportions of three genetic clusters inferred using assignment analysis were not significantly different among the samples. These results suggested that the genetic diversity and structure of bronze gudgeon were uniform across the samples. However, the Hardy-Weinberg equilibrium test, fixation index and linkage disequilibrium test indicated genetic subdivision of bronze gudgeon in the upper reach of the Three Gorges Dam. The present study and future studies including tributary samples will provide an important baseline of genetic diversity and population structure of bronze gudgeon in the Yangtze River, which is critical for monitoring and evaluating impacts of the large-scale dams on this species.

Rock carp Procypris rabaudi is a vulnerable endemic fish in the upper Yangtze River. Hatchery release has been carried out as a major stock enhancement strategy for this species. Ten microsatellite loci were chosen to compare genetic variation between one wild population and two hatchery groups to evaluate the potential impacts of hatchery release on the genetic structure of the wild population. Two different models indicated strong evidence of recent bottlenecks in all groups. The hatchery groups were lower in the mean number of alleles per locus, allelic richness, and allelic diversity compared with the wild population. The 80% membership coefficient indicated that 14% of the wild fish could be assigned as hybrids of wild and hatchery fish. Our results suggested that hatchery release will further reduce the natural genetic diversity in the wild population, change the genetic structure of the rock carp population, and may not benefit restoration of this vulnerable fish species.

Three groups of gynogenetic diploid bighead carp were successfully obtained by means of artificial gynogenesis. The activation rates of gynogenesis varied from 75.9% to 98.8%, and the frequency of spontaneous diploidization was around 0.4%. Over 2000 normally gynogenetic diploid fry were obtained in three gynogenetic groups. The haploid karyotype consisted of nine metacentric, 12 submetacentric, three subtelocentric chromosomes and 45 arms. The chromosome number was 48 from gynogenetic diploid. The results showed that the genetic material of offspring was maternal. The aneuploid hybrid embryos of bighead carp and Xingguo red common carp with chromosome numbers ranging from 28 to 73 did not survive post hatch, likely the result of incompatibility between the nucleus and the cytoplasm of two parents. Sixty RAPD primers from three groups were used for total DNA amplification of gynogenetic offspring, maternal and 'paternal' fish. A total of 451 bands were amplified from three kinds of samples above. From maternal bighead carp, 256 bands were amplified; however, there were 251 shared bands between maternal and gynogenetic bighead carp. From artificial gynogenetic offspring, two 'paternal' DNA segments without an expression function were found. An UPGMA tree showed that gynogenetic offspring were closely clustered and the genetic identity among them was very high (0.956).

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Three populations of Pelteobagrus vachelli and Pelteobagrus fulvidraco of the Yangtze River were examined by PCR-RFLP analysis of mitochondrial DNA fragments. ND5/6 and D-loop fragments were digested by 10 restriction endonucleases. Significant geographic variations between upstream and mid-downstream populations in the haplotype frequencies and restriction patterns were revealed. This suggested that the diversity of P. vachelli was high; 11 haplotypes were obtained from all the samples. The upstream population shared seven haplotypes and the middle and downstream populations shared another four haplotypes. Among all of the haplotypes, one haplotype was shared in 30 samples of the populations from middle and downstream, but it was not found in the upstream population. Any haplotype found in the upstream population was not detected in the middle and downstream populations. Genetic diversity of P. fulvidraco was low and only five haplotyes were detected from all 60 samples. Phylogenic relationships also indicated that the fishes from upstream and mid-downstream were apparently divided into two populations.

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles ranging from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (P < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F-st distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.

The Poyang Lake is the largest lake and the main nursery area in the middle basin of the Changjiang (Yangtze) River. We compared molecular genetic markers of silver carp among populations of the Changjiang River, the Ganjiang River and the Poyang Lake using the ND5/6 region of mtDNA. Analysis of restriction fragment length polymorphisms (RFLPs) of this region revealed distinct variation between the Ganjiang River and the Changjiang River populations. The Poyang Lake is linked with the Ganjiang River and the Changjiang River. Shared RFLP fragments between the Ganjiang River population and the Poyang Lake population are as high as 61.4%. The value is 47.74% between the populations of the Changjiang River and that of the Poyang Lake. Frequencies of bands peculiar to the Ganjiang River population are the same as in the Poyang Lake population. We conclude that the Poyang Lake silver carp population consists mainly of the Ganjiang River population. The water level of the Poyang Lake outlet, which is higher than that of the Changjiang River in the silver carp spawning season, supports this conclusion.

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser, (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form ofA. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.

About a third of microsatellite primers designed for common carp (Cyprinus carpio) was successfully amplified in silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). These markers, inherited in Mendelian mode, are of potential applications in cypinid genetics.

Microsatellite markers and D-loop sequences of mtDNA from a female allotetraploid parent carp and her progenies of generations 1 and 2 induced by sperm of five distant fish species were analyzed. Eleven microsatellite markers were used to identify 48 alleles from the allotetraploid female. The same number of alleles (48) appeared in the first and second generations of the gynogenetic offspring, regardless of the source of the sperm used as an activator. The mtDNA D-loop analysis was performed on the female tetraploid parent, 25 gynogenetic offspring, and 5 sperm-donor species. Fourteen variable sites from the 1,018 bp sequences were observed in the offspring as compared to the female tetraploid parent. Results from D-loop sequence and microsatellite marker analysis showed exclusive maternal transmission, and no genetic information was derived from the father. Our study suggests that progenies of artificial tetraploid carp are genetically stable, which is important for genetic breeding of this tetraploid fish.

Mature eggs of allotetraploid carp were activated by inactive sperm or crossed with normal sperms of common carp (Cyprinus carpio), crucian carp (Carassius auratus), Chinese blunt snout bream (Megalobrama amblycephala), Hemiculter leucisculus and Pseudorasbora parva. Chromosome counts showed that all offspring of these crosses presented a mode number of 200 chromosomes (4n = 200), and their morphological traits are much like maternal. Microsatelite marker and RAPD patterns between allotetraploid maternal and its offspring, reproduced from different paternal species, were identical. Cytological, morphological and molecular evidences suggested that allotetraploid carp female nucleus would not fuse with any male nucleus and its reproduction mode might be gynogenesis and therefore their offspring are retaining their tetraploidy and give origin to clonal individuals.

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.