Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.  In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.  At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated.  From 0.1 to 10 μmol L^–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal.  When the LPA concentration was 15 μmol L^–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst.  Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2

and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol

L^–1.  The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L^–1 to LPA-10 μmol L^–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L^–1 LPA-treated embryos compared with LPA-10 μmol L^–1 group (P<0.05).  The inner cell mass in 15 μmol L^–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal.  Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells.  The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.  They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L^–1, while 15 μmol L^–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells.  The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L^–1 group and LPA-10 μmol L^–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L^–1 group compared with LPA-10 μmol L^–1 group (P<0.05).  Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L^–1 concentration.  This study references the IVF embryo production and embryonic stem cell research of domestic animals. 

In maize, two root epidermis-expressed ammonium transporters ZmAMT1;1a and ZmAMT1;3 play major roles in high-affinity ammonium uptake.  However, the transcriptional regulation of ZmAMT1s in roots for ensuring optimal ammonium acquisition remains largely unknown.  Here, using a split root system we showed that ZmAMT1;1a and ZmAMT1;3 transcript levels were induced by localized ammonium supply to nitrogen-deficient roots.  This enhanced expression of ZmAMT1s correlated with increases in ¹⁵NH₄⁺ influx rates and tissue glutamine concentrations in roots.  When ammonium was supplied together with methionine sulfoximine, an inhibitor of glutamine synthase, ammonium-induced expression of ZmAMT1s disappeared, suggesting that glutamine rather than ammonium itself regulated ZmAMT1s expression.  When glutamine was supplied to nitrogen-deficient roots, expression levels of ZmAMT1s were enhanced, and negative feedback regulation could subsequently occur by supply of glutamine at a high level.  Thus, our results indicated an ammonium-dependent regulation of ZmAMT1s at transcript levels, and a dual role of glutamine was suggested in the regulation of ammonium uptake in maize roots.

Chenghua (CH) pig and Qingyu (QY) pig are typical Chinese native fatty breeds.  CH pig is mainly distributed in Chengdu Plain, while QY pig is widely distributed throughout the mountain areas around the Sichuan Basin.  There are significant differences in their phenotypic traits, including body image, growth performance, and meat quality.  This study compared several meat quality traits of CH and QY pigs and conducted a genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS).  It was observed that the pH at 45 min (pH_45min, P=5.22e–13), lightness at 45 min (L*_45min, P=4.85e–5), and lightness at 24 h (L*_24h, P=3.57e–5) of CH pigs were higher than those of QY pigs.  We detected 10 699 differentially methylated cytosines (DMCs) and 2 760 differentially methylated genes (DMGs) associated with these DMCs.  Functional analysis showed that these DMGs were mainly enriched in the AMPK signaling pathway, Type II diabetes mellitus, Insulin signaling pathway, mTOR signaling pathway, and Insulin resistance.  Furthermore, 15 DMGs were associated with fat metabolism (ACACA, CAB39, CRADD, CRTC2, FASN, and GCK), muscle development (HK2, IKBKB, MTOR, PIK3CD, PPARGC1A, and RPTOR), or meat quality traits (PCK1, PRKAG2, and SLC2A4).  The findings may help to understand further the epigenetic regulation mechanisms of meat quality traits in pigs and provide new basic data for the study of local pigs.

CCCH (C3H) Zinc finger (Znf) transcription factors (TFs), as a novel type of Znf gene, regulate the expression of genes by binding to their mRNAs and play important roles in plant growth and development and abiotic stress resistance.  Longan (Dimocarpous longan) is a tropical/subtropical fruit tree of great economic importance in Southeast Asia.  However, genomic information on C3H and their functions in longan are still unknown.  In this study, a comprehensive analysis of the longan C3H (DlC3H) gene family was carried out.  A total of 49 DlC3H genes in three clades were identified from the longan genome database.  Characteristics of the genes were analyzed with respect to gene structure, motif composition, phylogenetic tree and potential functions.  The analysis of alternative splicing (AS) events suggested that AS events in DlC3H genes were related to the transformation from longan non-embryonic to embryonic cultures.  Promoter analysis indicated that most of the DlC3H genes included cis-acting elements associated with hormones and stresses responses.  Quantitative real-time PCR (qRT-PCR) analysis indicated that 26 of the 49 DlC3Hs, which possess methyl jasmonate (MeJA) and abscisic acid (ABA) responsive cis-acting elements, showed differential expression patterns under treatment with ABA, MeJA and their endogenous inhibitors, suggesting that DlC3Hs might be involved in the ABA and MeJA signaling pathways.  The expression profiles of 17 of the 49 DlC3Hs in non-embryonic callus and three tissues of embryonic cultures showed that only five of the 17 DlC3Hs had the same expression trends as the FPKM trends in transcriptome data; the expression levels of DlC3H07/14/16/36/49 in embryogenic callus and DlC3H04/38 in globular embryos were high, suggesting that they have different functions in embryonic development.  Further, we verified that DlC3H01/03/05/11/19/39 were regulated by sRNAs by a modified 5´ RLM-RACE method.  This study provides the first systematic analysis of C3H genes in longan, and found that C3H genes may be involved in hormone and stress responses, and somatic embryogenesis.  Our preliminary investigation may provide clues to further studies on the characteristics and functions of this family in longan.

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 

DNA methyltransferases (Dnmts) comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns.  In pig, the molecular characterization and tissue expression profile of Dnmt gene family are not clear.  To solve this problem, reverse transcriptase PCR and rapid amplification of cDNA ends were used to clone the sequences of the porcine Dnmt2 and Dnmt3b genes.  Furthermore, the mRNA expression profiles of Dnmt1, Dnmt2, Dnmt3a and Dnmt3b genes from 54 adult tissues and 2 entire fetuses of Rongchang pig were analyzed by quantitative real-time PCR (qRT-PCR).  As a result, the lengths of porcine Dnmt2 and Dnmt3b gene cDNAs were 1 227 and 2 559 bp with cytosine-C5 specific DNA methylase domain, respectively.  The four Dnmt genes were highly expressed in longissimus dorsi muscle (P<0.01).  Dnmt1 is highly expressed in heart (P<0.01) and Dnmt 2 shows its preference in liver and seminal vesicle tissue (P<0.01).  Dnmt3a and Dnmt3b are highly expressed in the two fetus stages (P<0.01).  All these results suggested that each gene has its specific expression profile, and deeper study is required to dig more details between the methylation level and Dnmt family mRNA expressions in different tissues.

Cellulose, a major constituent of plant biomass, is synthesized by a cellulose synthase complex.  It has been demonstrated that MYB genes transcriptionally regulate cellulose synthase in Arabidopsis.  However, little is known about this process in tomato.  Here, two MYB (SlMYB1/2) and three cellulose synthase (CESA) (SlCESA4/5/6) genes were isolated.  SlMYB1/2 and SlCESA4/5/6 accumulation was found to correspond to cellulose accumulation in different tissues of tomato.  Dual luciferase assays indicated that these two MYBs were transcriptional activators that interact with promoters of SlCESA4/5/6.  Moreover, SlMYB2 could also activate promoters of SlMYB1/2, suggesting the possible underlying auto-activation mechanisms for MYB transcription factors.  Transient over-expression of SlMYB1/2 in Nicotiana tabacum up-regulated tobacco endogenous NtCESA genes and increased cellulose accumulation.  The function of SlMYB1/2 was further investigated using stable transformation and the results indicated that N. tabacum lines heterologous expressing SlMYB1/2 displayed a pleiotropic phenotype, long and narrow leaves, with NtCESA induced and significant increase of cellulose.  In conclusion, our data suggest that tomato SlMYB1/2 have transcriptional regulatory roles in cellulose biosynthesis and SlMYB2 was more effective than SlMYB1, which may due to the transcriptional activation by SlMYB2 on SlMYB1 and itself.

    Flixweed (Descurainia sophia L.) is a problematic and widespread weed in winter wheat fields and has been controlled by tribenuron-methyl for more than twenty years in China. In this study, a flixweed accession (Hebei 25, HB25) with an Asp-376-Glu mutation in acetohydroxy acid synthase (AHAS) was identified and purified. The purified HB25 accession (pHB25) developed 758.1-fold resistance to tribenuron-methyl and exhibited obvious cross-resistance to four AHAS-inhibiting herbicides. The resistant/susceptible (R/S) ratios of 50% plant growth reduction (GR₅₀) to herbicides of halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 346.1, 15.7, 8.1 and 7.1, respectively. The reduced AHAS sensitivities to four different AHAS-inhibiting herbicides, which were caused by the Asp-376-Glu mutation, were responsible for the resistance and cross-resistance to AHAS-inhibiting herbicides. The R/S ratios of 50% inhibition of AHAS activity (I₅₀) to tribenuron-methyl, halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 844.5, 532.9, 74.5, 13.3 and 5.5, respectively. The results of AHAS activity in vitro were highly correlated with that of whole-plant response experiments.

The western flower thrips, Frankliniella occidentalis (Pergande) is a highly invasive pest that is able to exploit many crops across a wide range of environmental conditions. Five full-length cDNAs of heat shock protein (HSP) genes (Fo-HSP90, Fo-HSP70, Fo-HSP60, Fo-HSP40 and Fo-HSP28.9) were cloned from F. occidentalis, and their expression profiles were investigated under conditions of thermal stress and insecticide exposure, and at different stages during development, using real-time quantitative PCR. All five gene sequences showed high similarity to homologs in other species, indicating the conserved function of this gene family. HSP60 represents an informative phylogenetic marker at the ordinal taxonomic level within Insecta, but HSP90, which has two homologous copies in Hymenoptera, was not informative. The expression of Fo-HSPs under thermal stress suggests that Fo-HSP90, Fo-HSP70, and Fo-HSP28.9 are inducible by both cold and heat stress, Fo-HSP40 is only heat-inducible, and Fo-HSP60 is thermally insensitive. There were two patterns of cold induction of Fo-HSPs: one is from 0 to 4°C and the other is around -8°C. All five Fo-HSPs genes were induced by exposure to sublethal concentrations of the insecticide avermectin. The expression of the five Fo-HSPs during different developmental stages suggests that they all play a role in development of F. occidentalis.

The rice blast, caused by fungus Magnaporthe oryzae, is a major constraint to the world food security. Hyphal growth is the foundation of fungal development and proliferation of fungi. To investigate genes involved in hyphal growth of this fungus, digital gene expression tag profiling was used to compare a previously generated temperature-sensitive mutant which defect at hyphae growth and reduction on pathogenicity, with its related wildtype strain. 416 genes were detected as differential expression, 178 of which were specifically expressed in Guy-11 but down-regulated expression in the mutant. Functional classification analysis revealed the phenotype mutation may be mainly caused by a defection in translational and vacuolerelated processes. The results and the protocol used will improve our knowledge on morphogenesis and promote the further study on M. oryzae pathogenesis.

Spermatogonial stem cells (SSCs) are the key to maintaining production of the sperms and healthy offsprings, and also treating breeding livestock’s reproductive damage and infertility. MicroRNAs act a decisive role in regulating gene expression in many cells and tissues, including in processes such as proliferation, self-renewal, differentiation, and apoptosis of stem cells. However, the miRNA mechanism in regulation of SSCs is still unclear. Here, high-throughput sequencing was used to identify specific miRNAs. We confirmed that miR-21-5p was concentrated in both goat and mouse SSCs, and enhanced the proliferation and antiapoptotic ability of SSCs. In vivo experiments have shown that miR-21-5p resisted the damage of the chemotherapy drug Busulfan to germ cells, ameliorated Busulfan-induced testicular dysfunction, and maintained spermatogenesis. Further RNA-seq and target gene prediction revealed that SPRY1 and FASLG are targets of miR-21-5p, thereby activating downstream signaling pathways such as MAPK/ERK, PI3K-AKT, and apoptosis. In summary, miR-21-5p is crucial for the self-renewal and maintenance of SSCs. This study provides new avenues for treating breeding livestock’s reproductive damages, infertility, oligospermia, and other conditions.