Identification and functional characterization of intermediate-size non-coding RNAs in maize.

BACKGROUND: The majority of eukaryote genomes can be actively transcribed into non-coding RNAs (ncRNAs), which are functionally important in development and evolution. In the study of maize, an important crop for both humans and animals, aside from microRNAs and long non-coding RNAs, few studies have been conducted on intermediate-size ncRNAs. RESULTS: We constructed a homogenized cDNA library of 50-500 nt RNAs in the maize inbred line Chang 7-2. Sequencing revealed 169 ncRNAs, which contained 58 known and 111 novel ncRNAs (including 70 snoRNAs, 27 snRNAs, 13 unclassified ncRNAs and one tRNA). Forty of the novel ncRNAs were specific to the Panicoideae, and 24% of them are located on sense-strand of the 5' or 3' terminus of protein coding genes on chromosome. Target site analysis found that 22 snoRNAs can guide to 38 2'-O-methylation and pseudouridylation modification sites of ribosomal RNAs and small nuclear RNAs. Expression analysis showed that 43 ncRNAs exhibited significantly altered expression in different tissues or developmental stages of maize seedlings, eight ncRNAs had tissue-specific expression and five ncRNAs were strictly accumulated in the early stage of leaf development. Further analysis showed that 3 of the 5 stage-specific ncRNAs (Zm-3, Zm-18, and Zm-73) can be highly induced under drought and salt stress, while one snoRNA Zm-8 can be repressed under PEG-simulated drought condition. CONCLUSIONS: We provided a genome-wide identification and functional analysis of ncRNAs with a size range of 50-500 nt in maize. 111 novel ncRNAs were cloned and 40 ncRNAs were determined to be specific to Panicoideae. 43 ncRNAs changed significantly during maize development, three ncRNAs can be strongly induced under drought and salt stress, suggesting their roles in maize stress response. This work set a foundation for further study of intermediate-size ncRNAs in maize.

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori.

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2'-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.

Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli.

Glutamate decarboxylase (GAD) has the potential of converting L-glutamate to gamma-aminobutyric acid (GABA), which is an important non-proteinogenic amino acid that has a potential use as food additive or dietary supplement for its physiological functions. A novel pyridoxal 5'-phosphate (PLP)-dependent glutamate decarboxylase (LsGAD) was cloned from GRAS (generally recognized as safe) Lactobacillus senmaizukei by genome mining and efficiently expressed in Escherichia coli BL21. The LsGAD displayed excellent temperature property, pH property and kinetic parameters compared with the probe LbGAD and the other GADs. By increasing the copy number of the LsGAD encoding gene, the expression level of LsGAD and the biosynthesis yield of GABA were increased, which was near to 2 times of that was expressed in single copy. These results established a solid foundation for increasing the added value of L-glutamate and the biosynthesis of GABA.

Molecular investigation of "Candidatus Mycoplasma haemobos" in goats and sheep in central China.

Hemoplasma "Candidatus Mycoplasma haemobos" infections in cattle have been reported in East Asia, Europe, and South America, whereas same cases were documented in buffalo and cattle in Southern China. From April 2018 to May 2018, a mycoplasma epidemic was reported in the mountainous area of central China; Boophilus microplus has also been documented, causing severe haematuria in goats and sheep. The infected animals slowly recovered after diminazene aceturate and praziquantel treatment. To determine whether the hemoplasma caused this infection, 67 blood samples (42 from goats, 25 from sheep) and 132 B. microplus samples were collected for PCR and sequence analysis. The results showed that 19 out of the 42 goat blood samples, 10 out of the 25 sheep blood samples, and 70 out of the 132 B. microplus samples (53%) tested positive for "C. M. haemobos". This study provides molecular evidence of "C. M. haemobos" infections in goat and sheep, and that B. microplus harbours "C. M. haemobos".

Experimental infection of BALB/c mice with a caprine Pestivirus H isolate.

To data, small animal Pestivirus H infection models have not been established. In order to develop a new infection model, BALB/c mice were inoculated with Pestivirus H strain HN1507. The virus-inoculated mice displayed nasal discharge and fever clinical signs. Histopathological changes in Pestivirus H-infected mice included alveolar septa thickening and alveolar atrophy in the lungs from 1 to 11 days post-inoculation (PI). Furthermore, we observed tracheal epithelial cell abscission and inflammatory cell infiltration in the tracheas from 1 to 9 days PI, infiltration of eosinophils in the spleens from 1 to 9 days PI, intestinal villi abscission and lysis of epithelial cells in the intestines from 1 to 11 days PI. The results of virus isolation showed that Pestivirus H replicated well in the lungs, tracheas, spleens, and intestines of infected BALB/c mice, and peak viral titers were observed 3 days PI. RT-PCR and immunofluorescence results were in agreement with the virus isolation results; however, the hearts of infected mice from 1 to 3 days PI were positive while virus isolation results were negative. To the best of our knowledge, this is the first study reporting Pestivirus H detection in BALB/c mice. Our findings indicated that Pestivirus H strain HN1507 was pathogenic to BALB/c mice and caused clinical signs and histopathological lesions in Pestivirus H-infected BALB/c mice.

Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity.

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD+-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg-1, which was close to four times the activity of the probe. Meanwhile, the kcat/ Km value of LhDMDH was 28.80 S-1·mM-1, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.

Genome-Wide Identification and Characterization of microRNAs in Developing Grains of Zea mays L.

The development and maturation of maize kernel involves meticulous and fine gene regulation at transcriptional and post-transcriptional levels, and miRNAs play important roles during this process. Although a number of miRNAs have been identified in maize seed, the ones involved in the early development of grains and in different lines of maize have not been well studied. Here, we profiled four small RNA libraries, each constructed from groups of immature grains of Zea mays inbred line Chang 7-2 collected 4-6, 7-9, 12-14, and 18-23 days after pollination (DAP). A total of 40 known (containing 111 unique miRNAs) and 162 novel (containing 196 unique miRNA candidates) miRNA families were identified. For conserved and novel miRNAs with over 100 total reads, 44% had higher accumulation before the 9th DAP, especially miR166 family members. 42% of miRNAs had highest accumulation during 12-14 DAP (which is the transition stage from embryogenesis to nutrient storage). Only 14% of miRNAs had higher expression 18-23 DAP. Prediction of potential targets of all miRNAs showed that 165 miRNA families had 377 target genes. For miR164 and miR166, we showed that the transcriptional levels of their target genes were significantly decreased when co-expressed with their cognate miRNA precursors in vivo. Further analysis shows miR159, miR164, miR166, miR171, miR390, miR399, and miR529 families have putative roles in the embryogenesis of maize grain development by participating in transcriptional regulation and morphogenesis, while miR167 and miR528 families participate in metabolism process and stress response during nutrient storage. Our study is the first to present an integrated dynamic expression pattern of miRNAs during maize kernel formation and maturation.

Genome mining and motif truncation of glycoside hydrolase family 5 endo-ß-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

Two novel glycosyl hydrolase family 5 (GH5) ß-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9KM. The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant ß-mannanases potential additives for use in the food and feed industries.

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori.

Small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs (four small nucleolar RNAs and five unclassified ncRNAs) that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm-152, exhibited converse expression pattern with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.

Expression analysis and binding experiments of chemosensory proteins indicate multiple roles in Bombyx mori.

Chemosensory proteins (CSPs) are a family of small soluble proteins that, in addition to the odorant-binding proteins (OBPs), are involved in chemical communication. To understand the physiological function of the 16 known CSPs in the silkworm Bombyx mori, we investigated the expression patterns in different tissues and developmental stages using quantitative real-time RT-PCR (Q-PCR) and Western blot analysis. The results indicated that most CSPs were widely expressed in embryos, larvae, pupae and adults but were developmentally regulated. Such broad spatial and temporal expression was inconsistent with a specific association with chemosensory function. We conclude that CSPs are multifunctional proteins that are involved in diverse cellular processes and that can play non-chemosensory as well as chemosensory roles. Binding experiments revealed different binding characteristics of CSP1 and CSP2, with retinal being the best ligand, suggesting a putative function of these CSPs as carriers.