Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis

Somatic embryo (SE) regeneration is an ideal experimental system to realize rapid propagation of excellent clones and genetic improvement for perennial gymnosperms. In the present study, genes encoding the miRNA166 precursor were identified and LamiR166a was successfully transformed into the gymnosperm Larix leptolepis (L. leptolepis) and five LamiR166a over-expressed embryonic cell lines were screened out as stable embryo masses. As expected, the targets of miR166a, LaHDZ31-34, were all down-regulated in transgenic lines according to qRT-PCR results. The results showed that the percentage of normal SEs with 4–7 cotyledons was 77.0?% in wild type (WT) lines, but was reduced to 60.3?% in the pSuper::MIR166a lines with “cup-shaped” embryos comprised 7.0?% of WT and 20.7?% of transgenic embryos. Microscopic observation further showed that the intermediate region surrounded by the cotyledons was larger than in the control, with no upward bulge of the shoot apical meristem (SAM). The expression pattern of the two meristem marker genes CLAVATA (CLV) and WUSCHEL-related homeobox (WOX) were investigated. The results showed that the expression levels of WOX were three times higher in transgenic lines than in WT samples, which suggest that miR166a may indirectly regulate SAM development by directly affecting WOX expression. Besides, overexpression of LamiR166a clearly increased the rooting rate and promoted lateral root formation in L. leptolepis seedlings. These results may provide new insights into the regulatory role of miR166 in gymnosperms, and also new applications for forestry production in practice.     

Site-directed mutagenesis of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Alternaria</Emphasis> sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

The α-l-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-l-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp²⁵², Asp²⁵⁷, Asp²⁶⁴, Glu⁵³⁰, Arg⁵⁴⁸, His⁵⁵³, and Trp⁵⁵⁵) and may form the hydrophobic pocket in stabilizing donor (Trp²⁶¹, Tyr³⁰², Tyr³¹⁶, and Trp³⁶⁹) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp²⁵⁷. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.     

Functional role of metalloproteins in genome stability

Cells contain a large number of metalloproteins that commonly harbor at least one metal ion cofactor. In metalloproteins, metal ions are usually coordinated by oxygen, sulfur, or nitrogen centers belonging to amino acid residues in the protein. The presence of the metal ion in metalloproteins allows them to take part in diverse biological processes, such as genome stability, metabolic catalysis, and cell cycle progression. Clinically, alteration of the function of metalloproteins in mammals is genetically associated with diseases characterized by DNA damage and repair defects. The present review focuses on the current perspectives of metal ion homeostasis in different organisms and summarizes the most recent understanding on magnesium, copper, iron, and manganese-containing proteins and their functional involvement in the maintenance of genome stability.     

Iterative Usage of Fixed and Random Effect Models for Powerful and Efficient Genome-Wide Association Studies

False positives in a Genome-Wide Association Study (GWAS) can be effectively controlled by a fixed effect and random effect Mixed Linear Model (MLM) that incorporates population structure and kinship among individuals to adjust association tests on markers; however, the adjustment also compromises true positives. The modified MLM method, Multiple Loci Linear Mixed Model (MLMM), incorporates multiple markers simultaneously as covariates in a stepwise MLM to partially remove the confounding between testing markers and kinship. To completely eliminate the confounding, we divided MLMM into two parts: Fixed Effect Model (FEM) and a Random Effect Model (REM) and use them iteratively. FEM contains testing markers, one at a time, and multiple associated markers as covariates to control false positives. To avoid model over-fitting problem in FEM, the associated markers are estimated in REM by using them to define kinship. The P values of testing markers and the associated markers are unified at each iteration. We named the new method as Fixed and random model Circulating Probability Unification (FarmCPU). Both real and simulated data analyses demonstrated that FarmCPU improves statistical power compared to current methods. Additional benefits include an efficient computing time that is linear to both number of individuals and number of markers. Now, a dataset with half million individuals and half million markers can be analyzed within three days.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.