Effects of different mulching and fertilization on phosphorus transformation in upland farmland.

In the present study, the impact of different soil surface mulching, fertilization on phosphorus mineralization and bio-availability of spring maize at various growth stages and soil layers (0-20 and 20-40 cm soil layer) were evaluated. The results indicated that the contents of total P and Olsen-Phosphorus (Olsen-P) in the soils of 0-20 cm soil layer were significantly higher than those in the 20-40 cm soil layer at different stages. The addition of organic fertilizer significantly increased the soil total P and Olsen-P content in the 0-20 cm soil layer. The different surface mulching, no mulching (NM), gravel mulching (GM) and film mulching (FM) were significantly affected by the content of Olsen-P in both soil layers during the critical growth period of spring maize. The Ca10-P contents in both soil layers were the maximum in terms of the inorganic phosphorus content in soils with different surface mulching and different fertilization. Surface mulching significantly affected the transformation of inorganic phosphorus in different soil layers of dry-land farmland, and accelerated the increase of Ca2-P content (first phosphorus source) in 0-20 cm soil layer by GM and FM. In addition, phosphorus combined with inorganic nitrogen fertilizer increased Ca8-P (second Olsen-P source) to a certain extent, and reduced the relative content of Ca2-P (first phosphorus source). Compared with phosphate (P), nitrogen and phosphorus (NP) treatments, manure and nitrogen and phosphorus (MNP) treatments increased the contents of Ca2-P (first phosphorus source) and Ca8-P (second effective phosphorus source), while it reduced the insoluble phosphorus source (O-P) content.

OTUD6A promotes prostate tumorigenesis via deubiquitinating Brg1 and AR.

Ovarian tumor (OTU) subfamily deubiquitinases are involved in various cellular processes, such as inflammation, ferroptosis and tumorigenesis; however, their pathological roles in prostate cancer (PCa) remain largely unexplored. In this study, we observed that several OTU members displayed genomic amplification in PCa, among which ovarian tumor deubiquitinase 6A (OTUD6A) amplified in the top around 15-20%. Further clinical investigation showed that the OTUD6A protein was highly expressed in prostate tumors, and increased OTUD6A expression correlated with a higher biochemical recurrence risk after prostatectomy. Biologically, wild-type but not a catalytically inactive mutant form of OTUD6A was required for PCa cell progression. In vivo experiments demonstrated that OTUD6A oligonucleotides markedly suppressed prostate tumorigenesis in PtenPC-/- mice and patient-derived xenograft (PDX) models. Mechanistically, the SWI/SNF ATPase subunit Brg1 and the nuclear receptor AR (androgen receptor) were identified as essential substrates for OTUD6A in PCa cells by a mass spectrometry (MS) screening approach. Furthermore, OTUD6A stabilized these two proteins by erasing the K27-linked polyubiquitination of Brg1 and K11-linked polyubiquitination of AR. OTUD6A amplification exhibited strong mutual exclusivity with mutations in the tumor suppressors FBXW7 and SPOP. Collectively, our results indicate the therapeutic potential of targeting OTUD6A as a deubiquitinase of Brg1 and AR for PCa treatment.

[Recombinant expression of truncated exonuclease â § and its application in in vitro DNA recombination].

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×105 U/mg. In a 10 µL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×106 cfu/µg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×104 recombinant clones per µg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.

Cloning and molecular characterization of rutin degrading enzyme from tartary buckwheat (Fagopyrum tataricum Gaertn.).

Rutin and quercetin, abundant in tartary buckwheat, have physiological and pharmacological functions and play roles in abiotic stress tolerance in plant. Rutin degrading enzymes (RDE) are the key enzymes for rutin metabolism. However, the RDE coding sequence information has not been available. In this study, a 1515-bp coding sequence of RDE was cloned from tartary buckwheat (named FtRDE) using 5' and 3' RACE, based on the FtRDE protein sequence. The recombinant RDE (rRDE) expressed in P.pastoris with glycosylation modification degraded rutin into quercetin and the Glu171 and Glu382 were indispensable residues for catalytic activity. FtRDE was highly expressed in seed filling stage and response to ABA and MeJA, confirmed by qRT-PCR and FtRDE promoter activity analysis in mesophyll protoplast. This study provided a new approach for the large-scale preparation of RDE by heterologous expression and production of quercetin by hydrolyzing rutin, and could be helpful for understanding the FtRDE function under stress conditions.