Acyl-CoA oxidase 1 is involved in γ-decalactone release from peach (Prunus persica) fruit.

KEY MESSAGE: γ-Decalactone accumulation in peach mesocarp was highly correlated with ACX enzyme activity and natural PpACX1 content. Adding the purified recombinant PpACX1 induced γ-decalactone biosynthesis in cultured mesocarp discs in vitro. Previous gene expression studies have implied that acyl coenzyme A oxidase (ACX) is related to lactones synthesis, the characteristic aroma compounds of peach. Here, we analysed the correlation between γ-decalactone content and ACX enzyme activity in mesocarp of five different types of fully ripe peach varieties. Furthermore, 'Hu Jing Mi Lu' ('HJ') and 'Feng Hua Yu Lu' ('YL'), which have strong aroma among them, at four ripening stages were selected to study the role of ACX in lactone biosynthesis. The result showed that γ-decalactone was the most abundant lactone compound. γ-Decalactone accumulation was highly correlated with ACX enzyme activity. Mass spectrometry (MS) showed that PpACX1 was the most abundant PpACX protein in fully ripe mesocarp of cv. 'HJ'. To further elucidate the function of the PpACX1 protein, the PpACX1 gene was heterologously expressed in a bacterial system and characterized in vitro. MS identification gave the molecular weight of the recombinant PpACX1 as 94.44 kDa and the coverage rate of the peptide segments was 47.3%. In cultured mesocarp discs in vitro, adding the purified recombinant PpACX1 and C16-CoA substrate induced the expected γ-decalactone biosynthesis. Using a sandwich ELISA based on mixed mono- and polyclonal antibodies against recombinant PpACX1, PpACX1 content in mesocarp was found to be highly correlated with γ-decalactone accumulation in mesocarp of five fully ripe varieties and four ripening stages of 'HJ' and 'YL'. This study revealed the vital function of PpACX1 in γ-decalactone biosynthesis in peach fruit.

Genetic diversity of male and female Chinese bayberry (Myrica rubra) populations and identification of sex-associated markers.

BACKGROUND: Chinese bayberry (Myrica rubra Sieb. & Zucc.) is an important subtropical evergreen fruit tree in southern China. Generally dioecious, the female plants are cultivated for fruit and have been studied extensively, but male plants have received very little attention. Knowledge of males may have a major impact on conservation and genetic improvement as well as on breeding. Using 84 polymorphic SSRs, we genotyped 213 M. rubra individuals (99 male individuals, 113 female varieties and 1 monoecious) and compared the difference in genetic diversity between the female and the male populations. RESULTS: Neighbour-joining cluster analysis separated M. rubra from three related species, and the male from female populations within M. rubra. By structure analysis, 178 M. rubra accessions were assigned to two subpopulations: Male dominated (98) and Female dominated (80). The well-known cultivars 'Biqi' and 'Dongkui', and the landraces 'Fenhong' are derived from three different gene pools. Female population had a slightly higher values of genetic diversity parameters (such as number of alleles and heterozygosity) than the male population, but not significantly different. The SSR loci ZJU062 and ZJU130 showed an empirical Fst value of 0.455 and 0.333, respectively, which are significantly above the 95 % confidence level, indicating that they are outlier loci related to sex separation. CONCLUSION: The male and female populations of Chinese bayberry have similar genetic diversity in terms of average number of alleles and level of heterozygosity, but were clearly separated by genetic structure analysis due to two markers associated with sex type, ZJU062 and ZJU130. Zhejiang Province China could be the centre of diversity of M. rubra in China, with wide genetic diversity coverage; and the two representative cultivars 'Biqi' and 'Dongkui', and one landrace 'Fenhong' in three female subpopulations. This research provides genetic information on male and female Chinese bayberry and will act as a reference for breeding programs.

Piriformospora indica confers cadmium tolerance in Nicotiana tabacum.

Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance against biotic and abiotic stresses. In order to confirm the influence of P. indica on growth, proline, malondialdehyde (MDA), chlorophyll, and cadmium (Cd) amounts in Nicotiana tabacum under Cd stress, hydroponics, pot and field trials were conducted. The results showed that P. indica can store Cd in plant roots and reduce leaf Cd content, reduce the concentration of MDA, and increase the proline and chlorophyll content and the activities of catalase, peroxidase, and superoxide dismutase under hydroponic Cd stress. RT-PCR analysis showed that the relative expression level of genes Gsh2, TaPCS1, oas1, GPX, and Hsp70 in colonized plants was 4.3, 1.4, 2.9, 1.7, and 6.9 fold higher than in un-colonized plants respectively. Cd exposure significantly reduced un-colonized plants' agronomic traits compared to P. indica-colonized ones. Our results suggested that P. indica can sequester Cd in roots, so that much less cadmium was transported to leaves, and the increased concentrations of antioxidant enzymes, pigments and proline contents, as well as the higher expression of stress-related phytochelatin biosynthesis genes in P. indica-inoculated plants, may also serve to protect N. tabacum plants against oxidative damage, enhancing Cd tolerance.

Microbial community structure in fermentation process of Shaoxing rice wine by Illumina-based metagenomic sequencing.

BACKGROUND: To understand the role of the community structure of microbes in the environment in the fermentation of Shaoxing rice wine, samples collected from a wine factory were subjected to Illumina-based metagenomic sequencing. RESULTS: De novo assembly of the sequencing reads allowed the characterisation of more than 23 thousand microbial genes derived from 1.7 and 1.88 Gbp of sequences from two samples fermented for 5 and 30 days respectively. The microbial community structure at different fermentation times of Shaoxing rice wine was revealed, showing the different roles of the microbiota in the fermentation process of Shaoxing rice wine. The gene function of both samples was also studied in the COG database, with most genes belonging to category S (function unknown), category E (amino acid transport and metabolism) and unclassified group. CONCLUSION: The results show that both the microbial community structure and gene function composition change greatly at different time points of Shaoxing rice wine fermentation.

Bomidin: An Optimized Antimicrobial Peptide With Broad Antiviral Activity Against Enveloped Viruses.

The rapid evolution of highly infectious pathogens is a major threat to global public health. In the front line of defense against bacteria, fungi, and viruses, antimicrobial peptides (AMPs) are naturally produced by all living organisms and offer new possibilities for next-generation antibiotic development. However, the low yields and difficulties in the extraction and purification of AMPs have hindered their industry and scientific research applications. To overcome these barriers, we enabled high expression of bomidin, a commercial recombinant AMP based upon bovine myeloid antimicrobial peptide-27. This novel AMP, which can be expressed in Escherichia coli by adding methionine to the bomidin sequence, can be produced in bulk and is more biologically active than chemically synthesized AMPs. We verified the function of bomidin against a variety of bacteria and enveloped viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), herpes simplex virus (HSV), dengue virus (DENV), and chikungunya virus (CHIKV). Furthermore, based on the molecular modeling of bomidin and membrane lipids, we elucidated the possible mechanism by which bomidin disrupts bacterial and viral membranes. Thus, we obtained a novel AMP with an optimized, efficient heterologous expression system for potential therapeutic application against a wide range of life-threatening pathogens.

Metaproteomics associated with severe early childhood caries highlights the differences in salivary proteins.

OBJECTIVE: To investigate the salivary metaproteomic characteristics of the children with and without severe early childhood caries (S-ECC). DESIGN: In this study, we collected unstimulated saliva samples from 34 children (age 3-4 years) with caries free (NC, dmfs (= index of decayed, missing due to caries, or filled tooth surfaces) = 0, n = 23) and with S-ECC (dmfs≥10, n = 11). Salivary proteins were extracted and reduced, and then a Liquid Chromatography/Mass Spectrometry system was used to identify proteins. RESULTS: Nearly 3000 proteins were identified in this study, and about 3.5 % of the proteins originated from human while 86 % were derived from microbes. The salivary protein types in the NC group were statistically greater than those in the S-ECC group (P <0.05). Specifically, the salivary protein types derived from microbes in the NC group were significantly greater than those in the S-ECC group. Three proteins, human lactoferrin, penicillin-binding protein 1C [Burkholderia ubonensis], human alpha-defensin 1 (F28a mutant), were decreased statistically in the NC group compared to the S-ECC group (P < 0.05). Only one protein, 50S ribosomal protein L17 secreted by Haemophilus haemolyticus, was significantly increased in the NC group compared to the S-ECC group. Salivary IgA was the top highest protein in the NC group whereas human lysozyme was the top highest protein in the S-ECC group. CONCLUSIONS: The differential proteins recognized in this study may be conducive for finding a caries biomarker. Understanding the metaproteomic characteristics can help us to control the caries from human origin and microbial origin.

Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage.

Although the ecdysteroid of the silkworm had been studied for decades, the proteome of the prothoracic gland, the primary source of ecdysteroid hormones, has not been studied previously. In the present paper, we utilized a proteomic approach to investigate the fifth instar prothoracic gland during the growth and development of the silkworm, Bombyx mori L. The two-dimensional electrophoresis results showed that the majority of proteins were acidic proteins, especially concentrated in the area of 25-65 kDa, with pI values of between 4 and 7, and the difference was not distinct. When compared with Qiufeng (Japanese strain), the interspecific distinction was larger than the intraspecific distinction, and 19 particular spots, excized from the third, fifth and ninth days of p50 (Chinese strain) and Qiufeng were subjected to MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) analysis. We sorted them into seven catagories: energetics and/or metabolism, storage proteins, protection, lipid metabolism, signal transduction, cell function and unknown function proteins. Of these proteins, arginine methyltransferase is discussed as playing an important role in regulating the activation of ecdysteroidogenesis via transcription or translation.

QTL-BSA: A Bulked Segregant Analysis and Visualization Pipeline for QTL-seq.

In recent years, the application of Whole Genome Sequencing (WGS) on plants has generated sufficient data for the identification of trait-associated genomic loci or genes. A high-throughput genome-assisted QTL-seq strategy, combined with bulked-segregant analysis and WGS of two bulked populations from a segregating progeny with opposite phenotypic trait values, has gained increasing popularities in research community. However, there is no publicly available user friendly software for the identification and visualization. Hence, we developed a tool named QTL-BSA (QTL-bulked segregant analysis and visualization pipeline), which could facilitate the rapid identification and visualization of candidate QTLs from QTL-seq. As a proof-of-concept study, we have applied the tool for the rapid discovery and the identification of genes related with the partial blast resistance in rice. Genomic region of the major QTL identified on chromosome 6, is located between 1.52 and 4.32 Mb, which is consistent with previous studies (2.39-4.39 Mb). We also derived the gene and QTLs functional annotation of this region. QTL-BSA offers a comprehensive solution to facilitate a wide range of programming and visualization tasks in QTL-seq analysis, is expected to be used widely by the research community.

Circulating proteomic panels for diagnosis and risk stratification of acute-on-chronic liver failure in patients with viral hepatitis B.

Chronic HBV infection (CHB) can lead to acute-on-chronic liver failure (HBV-ACLF) characterized by high mortality. This study aimed to reveal ACLF-related proteomic alterations, from which protein based diagnostic and prognostic scores for HBV-ACLF were developed. Methods: Ten healthy controls, 16 CHB, and 19 HBV-ACLF according to COSSH (Chinese group on the study of severe hepatitis B) criteria were enrolled to obtain the comprehensive proteomic portrait related to HBV-ACLF initiation and progression. Potential markers of HBV-ACLF were further selected based on organ specificity and functionality. An additional cohort included 77 healthy controls, 92 CHB and 71 HBV-ACLF was used to validate the proteomic signatures via targeted proteomic assays. Results: Significant losses of plasma proteins related to multiple functional clusters, including fatty acid metabolism/transport, immuno-response, complement and coagulation systems, were observed in ACLF patients. In the validation study, 28 proteins were confirmed able to separate ACLF, CHB patients. A diagnostic classifier P4 (APOC3, HRG, TF, KLKB1) was built to differentiate ACLF from CHB with high accuracy (auROC = 0.956). A prognostic model P8 (GC, HRG, HPR, SERPINA6, age, NEU, INR and total protein) was built to distinguish survivors from non-survivors in 28 and 90-days follow-up (auROC = 0.882, 0.871), and to stratify ACLF patients into risk subgroups showing significant difference in 28 and 90-days mortality (HR=7.77, 7.45, both P<0.0001). In addition, P8 score correlated with ACLF grades and numbers of extra-hepatic organ failures in ACLF patients, and was able to predict ACLF-associated coagulation and brain failure within 90 days (auROC = 0.815, 0.842). Conclusions: Proteomic signatures developed in this study reflected the deficiency of key hematological functions in HBV-ACLF patients, and show potential for HBV-ACLF diagnosis and risk prediction in complementary to current clinical based parameters.

Proteomic approach to understand the molecular physiology of symbiotic interaction between Piriformospora indica and Brassica napus.

Many studies have been now focused on the promising approach of fungal endophytes to protect the plant from nutrient deficiency and environmental stresses along with better development and productivity. Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we used integrated in-depth proteome analyses to characterize the relationship between endophyte Piriformospora indica and Brassica napus plant highlighting its potential involvement in symbiosis and overall growth and development of the plant. An LC-MS/MS based label-free quantitative technique was used to evaluate the differential proteomics under P. indica treatment vs. control plants. In this study, 8,123 proteins were assessed, of which 46 showed significant abundance (34 downregulated and 12 upregulated) under high confidence conditions (p-value ≤ 0.05, fold change ≥2, confidence level 95%). Mapping of identified differentially expressed proteins with bioinformatics tools such as GO and KEGG pathway analysis showed significant enrichment of gene sets involves in metabolic processes, symbiotic signaling, stress/defense responses, energy production, nutrient acquisition, biosynthesis of essential metabolites. These proteins are responsible for root's architectural modification, cell remodeling, and cellular homeostasis during the symbiotic growth phase of plant's life. We tried to enhance our knowledge that how the biological pathways modulate during symbiosis?

Piriformospora indica promotes growth, seed yield and quality of Brassica napus L.

In current scenario, crop productivity is being challenged by decreasing soil fertility. To cope up with this problem, different beneficial microbes are explored to increase the crop productivity with value additions. In this study, Brassica napus L., an important agricultural economic oilseed crop with rich source of nutritive qualities, was interacted with Piriformospora indica, a unique root colonizing fungus with wide host range and multifunctional aspects. The fungus-treated plants showed a significant increase in agronomic parameters with plant biomass, lodging-resistance, early bolting and flowering, oil yield and quality. Nutritional analysis revealed that plants treated by P. indica had reduced erucic acid and glucosinolates contents, and increased the accumulation of N, Ca, Mg, P, K, S, B, Fe and Zn elements. Low erucic acid and glucosinolates contents are important parameters for high quality oil, because oils high in erucic acid and glucosinolates are considered undesirable for human nutrition. Furthermore, the expression profiles of two encoding enzyme genes, Bn-FAE1 and BnECR, which are responsible for regulating erucic acid biosynthesis, were down-regulated at mid- and late- life stages during seeds development in colonized plants. These results demonstrated that P. indica played an important role in enhancing plant growth, rapeseed yield and quality improvement of B. napus.

A preliminary genetic linkage map of the Pacific abalone Haliotis discus hannai Ino.

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F(1) family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (alpha = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.

Curcumin-supplemented diets increase superoxide dismutase activity and mean lifespan in Drosophila.

Curcumin is a polyphenolic bioactive compound in turmeric. We examined if antioxidant effects of curcumin are associated with lifespan extension in Drosophila. In this experiment, females and males of Drosophila were fed diets either containing no curcumin (C0) or supplemented with curcumin at 0.5 (C1) and 1.0 (C2) mg/g of diet. The levels of malondialdehyde (MDA), enzyme activity of superoxide dismutase (SOD), and expression of seven age-related genes in females and males were analyzed. We found that C1 and C2 increased mean lifespan by 6.2 % and 25.8 % in females, and by 15.5 % and 12.6 % in males, respectively. Meanwhile, C1 and C2 significantly decreased MDA levels and increased SOD activity in both genders. Diets C1 in females and C2 in males are effective in extending mean lifespan and improving levels of two physiological and biochemical measures related to aging in Drosophila. Lifespan extension of curcumin in Drosophila was associated with the up-regulation of Mn-SOD and CuZn-SOD genes, and the down-regulation of dInR, ATTD, Def, CecB, and DptB genes. The present results suggest that curcumin increases mean lifespan of Drosophila via regulating gene expression of the key enzyme SOD and reducing accumulation of MDA and lipid peroxidation. This study provided new insights for understanding the anti-aging mechanism of curcumin in Drosophila.

Molecular identification of cDNA, immunolocalization, and expression of a putative odorant-binding protein from an Asian honey bee, Apis cerana cerana.

An odorant-binding protein cDNA (Acer-ASP2) was cloned and characterized from antennae of adult workers of an Asian honey bee, Apis cerana cerana F. (Hymenoptera: Apidae). The full-length open reading frame of Acer-ASP2 cDNA was 429 bp, encoding 142 amino acids. Protein signature analyses revealed that it contained six conserved cysteines with an N-terminal signal sequence of 19 amino acids. The deduced protein sequence shares high homology with Amel-ASP2 from the honey bee, Apis mellifera L., and low similarity with odorant-binding proteins from other species of insects. Immunocytochemical localization showed that Acer-ASP2 was concentrated in the lymph of olfactory sensilla, such as sensilla placodea and sensilla trichodea A. Real-time polymerase chain reaction of Acer-ASP2 transcripts showed that Acer-ASP2 was expressed on antennae but not in other general anatomical regions of the body. Temporally, Acer-ASP2 was expressed at a relatively high level in adults during two periods (9 and 27 vs. 1, 15, and 30 days). This timing is correlated with the production of beeswax and searching behavior for nectar/pollen, respectively. Thus, Acer-ASP2 is a species-specific gene that we propose to be involved in the acquisition of odorant molecules from nectar, pollen, and other general odorant sources.

Sequence analysis of functional Apisimin-2 cDNA from royal jelly of Chinese honeybee and its expression in Escherichia coli.

Apisimin is one of the functional peptides from royal jelly. The aim of this study was to analyze and in vitro express a new gene encoding Acc-apisimin-2 from Chinese honeybee (Apis cerana cerana) in Escherichia coli. Ninety-six clones containing apisimin expressed sequence tag (EST) were identified from 8568 effective ESTs of the cDNA library of Chinese honeybee worker heads. The coding region of the matured peptide from one clone containing Acc-apisimin-2 gene was sub-cloned into the prokaryotic expression vector pGEX-4T-2. The recombinant vector then was transformed into E. coli BL21 (DE3) for expression. The expression product was analyzed with SDS-PAGE and Western blot. The total length of the Acc-apisimin-2 cDNA was 379 bp, containing an open-reading frame (ORF) of 237 nucleotides encoding a 78 amino acid residue precursor. The Acc-apisimin-2 gene shared 100% homologies with Am-apisimin from A. mellifera, but 93% and 91% homologies with Aciapisimin from A. cerana indica and the previously reported Acc-apisimin-1 sequence (AY278991) on a nucleotide level, respectively. The GST-Acc-apisimin-2 fusion protein expressed in the recombinant vector was about 31 kDa in size and accumulated up to about 22.1% of the total bacterial proteins. About 50% of the recombinant protein was soluble. The fusion protein purified through affinity chromatography was cross reactive with GST antibody, which confirmed the successful expression of GST-Acc-apisimin-2.