Marek’s disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek’s disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken.  The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines.  An experiment was conducted to elucidate the role of Meq in MD tumor transformation.  RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1.  A small interfering RNA with an interference efficiency of 70% (P<0.01) was transfected into MSB1 cells to knock down the expression of Meq gene.  The cell proliferation, cycle and apoptosis were detected post-Meq knockdown.  The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h (P<0.01), 60 h (P<0.05) and 72 h (P<0.01) post-Meq knockdown.  The cell cycle was unaffected (P>0.05).  B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway.  The activity of caspase-6 was upregulated (P<0.05) significantly and BCL2 gene expression was downregulated (P<0.05) significantly post-Meq knockdown, suggesting cell apoptosis might be induced.  MSB1 cell migration did not exhibit any obvious change (P>0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P<0.05) remarkably post-Meq knockdown.  The Meq knockdown might affect the main features of tumorous cells, including proliferation, apoptosis, and invasion, suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.

Soil alkalinity is a major factor that restricts the growth of apple roots. To analyze the response of apple roots to alkali stress, the root structure and endogenous hormones of two apple rootstocks, Malus prunifolia (alkali-tolerant) and Malus hupehensis (alkali-sensitive), were compared. To understand alkali tolerance of M. prunifolia at the molecular level, transcriptome analysis was performed. When plants were cultured in alkaline conditions for 15 d, the root growth of M. hupehensis with weak alkali tolerance decreased significantly. Analysis of endogenous hormone levels showed that the concentrations of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in M. hupehensis under alkali stress were lower than those in the control. However, the trend for IAA and ZR in M. prunifolia was the opposite. The concentration of abscisic acid (ABA) in the roots of the two apple rootstocks under alkali stress increased, but the concentration of ABA in the roots of M. prunifolia was higher than that in M. hupehensis. The expression of IAA-related genes ARF5, GH3.6, SAUR36, and SAUR32 and the Cytokinin (CTK)-related gene IPT5 in M. prunifolia was higher than those in the control, but the expression of these genes in M. hupehensis was lower than those in the control. The expression of ABA-related genes CIPK1 and AHK1 increased in the two apple rootstocks under alkali stress, but the expression of CIPK1 and AHK1 in M. prunifolia was higher than in M. hupehensis. These results demonstrated that under alkali stress, the increase of IAA, ZR, and ABA in roots and the increase of the expression of related genes promoted the growth of roots and improved the alkali tolerance of apple rootstocks.

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. 

Maize is one of the most important crops worldwide, but it suffers from salt stress when grown in saline-alkaline soil. There is therefore an urgent need to improve maize salt tolerance and crop yield. In this study, the SsNHX1 gene of Suaeda salsa, which encodes a vacuolar membrane Na⁺/H⁺ antiporter, was transformed into the maize inbred line 18-599 by Agrobacterium-mediated transformation. Transgenic maize plants overexpressing the SsNHX1 gene showed less growth retardation when treated with an increasing NaCl gradient of up to 1%, indicating enhanced salt tolerance. The improved salt tolerance of transgenic plants was also demonstrated by a significantly elevated seed germination rate (79%) and a reduction in seminal root length inhibition. Moreover, transgenic plants under salt stress exhibited less physiological damage. SsNHX1-overexpressing transgenic maize accumulated more Na⁺ and K⁺ than wild-type (WT) plants particularly in the leaves, resulting in a higher ratio of K+/Na+ in the leaves under salt stress. This result revealed that the improved salt tolerance of SsNHX1-overexpressing transgenic maize plants was likely attributed to SsNHX1-mediated localization of Na⁺ to vacuoles and subsequent maintenance of the cytosolic ionic balance. In addition, SsNHX1 overexpression also improved the drought tolerance of the transgenic maize plants, as rehydrated transgenic plants were restored to normal growth while WT plants did not grow normally after dehydration treatment. Therefore, based on our engineering approach, SsNHX1 represents a promising candidate gene for improving the salt and drought tolerance of maize and other crops.

T6V#2S·6AL and T6V#4S·6DL translocation chromosomes developed from the cross of wheat and different Dasypyrum villosum accessions have good powdery mildew (PM) resistance, but their pairing and pyramiding behavior remains unclear.  Results in this study indicated that the pairing frequency rate of the two differently originated 6VS chromosomes in their F₁ hybrid was 18.9% according to genomic in situ hybridization (GISH); the PM resistance plants in the F₂ generation from the cross between T6V#4S·6DL translocation line Pm97033 and its PM susceptible wheat variety Wan7107 was fewer than expected.  However, the ratio of the resistant vs. the susceptible plants of 15:1 in the F₂ generation derived from the cross between the two translocation lines of T6V#2S·6AL and T6V#4S·6DL fitted well.  Plants segregation ratio (homozygous:heterozygous:lacking) revealed by molecular marker for T6V#4S·6DL or T6V#2S·6AL in their F₂ populations fitted the expected values of 1:2:1 well, inferring that the pairing of the two alien chromosome arms facilitates the transmission of T6V#4S·6DL from the F₁ to the F₂ generation.  A quadrivalent was also observed in 21% of pollen mother cells (PMCs) of homozygote plants containing the two pairs of translocated chromosomes.  The chromosome pairing between 6V#2S and 6V#4S indicates that it will be possible to obtain recombinants and clarify if the PM resistance determinant on one alien chromosome arm is different from that on the other.  

We evaluated the effects of neutral detergent soluble fiber (NDSF) and sucrose supplementation on ruminal fermentation, microbial synthesis, and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC). The experiment had a 2×2 factorial design with two dosages of sucrose, low (ca. 0.26 g d-1, low-sucrose) and high (ca. 1.01 g d-1, high-sucrose), and two dosages of supplied NDSF, low (1.95 g d-1, low-NDSF) and high (2.70 g d-1, high-NDSF). Interactions between NDSF and sucrose were detected for xylanase activity from solid fraction and apparent disappearance of neutral detergent fiber (NDF) and hemicellulose, with the lowest values observed for high-NDSF and high-sucrose treatment. Supplemental NDSF appeared to increase the molar proportion of acetate and reduce that of butyrate; however, the effects of supplemental sucrose on VFA profiles depended upon NDSF amount. There was a NDSF×sucrose interaction for the production of methane. High-NDSF fermenters had lower ammonia-N production, greater daily N flow of solidassociated microbial pellets and total microorganisms, and greater microbial synthesis efficiency compared with low- NDSF fermenters. Supplementation with NDSF resulted in an increase in 16S rDNA copies of Ruminococcus flavefaciens and a reduction in copies of Ruminococcus albus. Supplementation with sucrose tended to increase the 16S rDNA copies of R. albus from liquid fraction, but did not affect daily total microbial N flow and cellulolytic bacterium populations from solid fraction. These data indicate that the effects of the interaction between NDSF and sugars on ruminal fermentation and fiber digestion should be taken into account in diet formulation. Ruminal fermentation and metabolism of sugars warrant further investigation.

Mature-green tomato fruit (Solanum lycopersicum cv. Zhenfen 202) were exposed to different UV-C irradiation at 2, 4, 8, and 16 kJ m-2 and then stored under the dark at 14°C and 95% relative humidity (RH) for 35 d. Of these four doses, UV-C irradiation at 4 and 8 kJ m-2 significantly increased total phenolic contents in present tomato fruit by 21.2 and 20.2%, respectively. Furthermore, UV-C irradiation at 4 or 8 kJ m-2 promoted the accumulation of total flavonoids and increased the antioxidant activity. 2 or 16 kJ m-2 UV-C irradiation also enhanced antioxidant activity, but to a lesser extent. Seven phenolic compounds, viz., gallic acid, (+)-catechin, chlorogenic acid, cafferic acid, syringic acid, p-coumaric acid, and quercetin in tomato fruit were identified and quantified by HPLC. Gallic acid was the major phenolic compound in tomato fruit and significantly correlated with antioxidant activity. 4 or 8 kJ m-2 UV-C irradiation significantly increased the contents of gallic acid, chlorogenic acid, syringic acid, p-coumaric acid, and quercetin. The optimum dose of UV-C irradiation in terms of increased phenolic compound content and enhanced Antioxidant activity was determined to be 4 or 8 kJ m-2.