Wheat (Triticum aestivum) is a major staple food crop worldwide. Genetic dissection of important agronomic traits is essential for continuous improvement of wheat yield to meet the demand of the world's growing population. We conducted a large-scale genome-wide association study (GWAS) using a panel of 768 wheat cultivars that were genotyped with 327 609 single-nucleotide polymorphisms generated by genotyping-by-sequencing and detected 395 quantitative trait loci (QTLs) for 12 traits under 7 environments. Among them, 273 QTLs were delimited to %1.0-Mb intervals and 7 of them are either known genes (Rht-D, Vrn-B1, and Vrn-D1) that have been cloned or known QTLs (TaGA2ox8, APO1, TaSus1-7B, and Rht12) that were previously mapped. Eight putative candidate genes were identified for three QTLs that enhance spike seed setting and grain size using gene expression data and were validated in three biparental populations. Protein sequence analysis identified 33 putative wheat orthologs that have high identity with rice genes in QTLs affecting similar traits. Large r 2 values for additive effects observed among the QTLs for most traits indicated that the phenotypes of these identified QTLs were highly predictable. Results from this study demonstrated that significantly increasing GWAS population size and marker density greatly improves detection and identification of candidate genes underlying a QTL, solidifying the foundation for large-scale QTL fine mapping, candidate gene validation, and developing functional markers for genomics-based breeding in wheat.

L-Isoleucine dioxygenase (IDO) specifically converts L-isoleucine(L-Ile) to 4-hydroxyisoleucine(4-HIL). To obtain IDO with improved activity, a strategy was developed that is dependent on the restoration of succinate-minus E. coli cell growth by the coupling of L-Ile hydroxylation and the oxidation of α-ketoglutarate(α-KGA) to succinate. Five mutants were obtained with this strategy, and the characteristics of IDOM3, which exhibited the highest activity, were studied. The catalytic efficiency, thermal stability and catalytic rate of IDOM3 were significantly improved compared with those of wild-type IDO. Moreover, an efficient method for the biotransformation of 4-HIL by resting cells expressing IDOM3 was developed, with which 151.9 mmol of 4-HIL/L (22.4 g/L) was synthesized in 12 h while the substrates seldom exhibited additional consumption.

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BACKGROUND: Currently, nanobodies are undergoing intensive studies due to their characteristics of nanoscale size, high affinity and specificity, and deep tissue penetration. The development of novel and simplified methods to purify nanobodies is required because high purity and high recovery are the limiting factors for the downstream preparation of nanobodies. RESULTS: In this study, ammonium sulfate precipitation (ASP) and electropositive multimodal chromatography (Capto MMC)were integrated for tag-free nanobody (2D5) purification. ASP was our first choice for protein purification because the process is simple, easy to operate, and has low cost. ASP achieved a 36% reduction in Escherichia coli host cell proteins (HCPs), a 99.1% reduction in E. coli host cell DNA, and an 85.1% reduction in endotoxins with a recovery of 81.7% and a purity enhancement from 6.2% to 73.9%. Capto MMC was seamlessly integrated for 2D5 purification. The recovery of this step was 83.0%. The entire two-step purification platform yielded 2D5 with 397 ppm of HCPs, 198 ppb of DNA, 204 EU/mL of endotoxins, and total protein recovery of 67.8%. The purity of 2D5 was 97.5%. The purification method was also used to purify a nanobody against PD-L1 (KPU) with 97.7% purity, > 95.7% recovery, 223 ppm of HCPs, 634 ppb of DNA and 154 EU/mL of endotoxins. CONCLUSION: The established method had no effect on the affinity of 2D5. A simple and efficient purification platform that integrates ASP and Capto MMC was established, providing a more promising alternative to the conventional affinity chromatography-based nanobody purification strategy. Affinity analysis of 2D5The affinity of antibodies refers to the bond strength between the antibody binding sites and the antigenic determinant; it is one of the fundamental kinetic parameters in the drug discovery J Chem Technol Biotechnol 2020; 95: 246-254

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