Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.  Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects.  However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research.  We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system.  After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.  The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene.  Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting.  RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes.  Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P_24h=0.31, P_48h=0.96, P_72h=0.24, P_96h=0.17, and P_120h=0.38) on cell proliferation of PK15 cells.  These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment

Soybean is one of the most important food crops worldwide.  Like other legumes, soybean can form symbiotic relationships with Rhizobium species.  Nitrogen fixation of soybean via its symbiosis with Rhizobium is pivotal for sustainable agriculture.  Type III effectors (T3Es) are essential regulators of the establishment of the symbiosis, and nodule number is a feature of nitrogen-affected nodulation.  However, genes encoding T3Es at quantitative trait loci (QTLs) related to nodulation have rarely been identified. Chromosome segment substitution lines (CSSLs) have a common genetic background but only a few loci with heterogeneous genetic information; thus, they are suitable materials for identifying candidate genes at a target locus.  In this study, a CSSL population was used to identify the QTLs related to nodule number in soybean.  Single nucleotide polymorphism (SNP) markers and candidate genes within the QTLs interval were detected, and it was determined which genes showed differential expression between isolines.  Four candidate genes (GmCDPK28, GmNAC1, GmbHLH, and GmERF5) linked to the SNPs were identified as being related to nodule traits and pivotal processes and pathways involved in symbiosis establishment.  A candidate gene (GmERF5) encoding a transcription factor that may interact directly with the T3E NopAA was identified.  The confirmed CSSLs with important segments and candidate genes identified in this study are valuable resources for further studies on the genetic network and T3Es involved in the signaling pathway that is essential for symbiosis establishment. 

Cotton plants are recalcitrant with regards to transformation and induced regeneration.  In the present study, 5-enolpyruvylshikimate-3-phosphate (EPSPS), a glyphosate resistant gene from the bacterium Agrobacterium sp. strain CP4, was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacterium-mediated transformation of shoot apex.  Primary transformants were initially screened using a 0.26% glyphosate spray and subsequently by PCR analysis.  Five out of 4 000 transformants from T₁ seeds were obtained resulting in an in planta transformation rate of 0.125%.  Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance.  Transgene insertion was analyzed by Southern blot analysis.  Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR, Western blot, and ELISA methods.  Transgenic T₃ plants were resistant to as much as 0.4% of glyphosate treatments in field trials.  Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars.  Since this method does not require sterile conditions, the use of specialized growth media or the application of plant hormones, it can be conducted under the greenhouse condition.

Zinc (Zn) deficiency is widespread among citrus plants, but information about the mechanisms for Zn deficiency response in these plants is scarce. In the present study, different navel orange (Citrus sinensis (L.) Osbeck) leaves with various yellowing levels were sampled in our experimental orchard, and upon estimation of nutrient contents, Zn deficiencies were diagnosed as mild, moderate, and severe. Further analysis of chlorophyll content, photosynthetic characteristics, antioxidant enzyme activities, and expression levels of Zn/Iron-regulated transporter-like protein (ZIP) family genes were conducted in the sampled Zn-deficient leaves. The results showed that chlorophyll contents and net photosynthetic rate (P_n) seemed to decrease with reduced Zn contents. In addition, comparison of severe Zn-deficient and normal leaves revealed that activities of peroxidase (POD) and catalase (CAT) increased significantly, whereas that of Zn-containing enzymes such as Cu/Zn superoxide dismutase (Cu/Zn-SOD) significantly reduced with decreasing Zn contents. As expected, expression of the ZIP family genes, ZIP1, ZIP3, and ZIP4, was induced by Zn deficiencies. These results deepen our understanding of Zn deficiency in citrus plants as well as provide useful preliminary information for further research.

    The vacuolar proton pump ATPase (V-H⁺-ATPase), which is a multi-subunit membrane protein complex, plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. In this study, three genes encoding V-H⁺-ATPase subunits B (ScVHA-B, GenBank: JF826506), C (ScVHA-C, GenBank: JF826507) and H (ScVHA-H, GenBank: JF826508) were isolated from the halophyte Suaeda corniculata. The transcript levels of ScVHA-B, ScVHA-C and ScVHA-H were increased by salt, drought and saline-alkali treatments. V-H⁺-ATPase activity was also examined under salt, drought and saline-alkali stresses. The results showed that V-H⁺-ATPase activity was correlated with salt, drought and saline-alkali stress. Furthermore, V-H⁺-ATPase subunits B, C and H (ScVHA-B, ScVHA-C and ScVHA-H) from S. corniculata were introduced separately into the alfalfa genome. The transgenic alfalfa was verified by Southern and Northern blot analysis. During salt and saline-alkali stresses, transgenic linevacuolar proton pump, salt tolerance, saline-alkali tolerance, alfalfa
s carrying the B, C and H subunits had higher germination rates than the wild type (WT). More free proline, higher superoxide dismutase (SOD) activity and lower malondialdehyde (MDA) levels were detected in the transgenic plants under salt and saline-alkali treatments. Moreover, the ScVHA-B transgenic lines showed greater tolerance to salt and saline-alkali stresses than the WT. These results suggest that overexpression of ScVHA-B, ScVHA-C and ScVHA-H improves tolerance to salt and saline-alkali stresses in transgenic alfalfa.