转Bt Cry1Ac基因抗虫棉花外源基因漂移研究

【目的】研究转Bt Cry1Ac基因棉花外源基因漂移规律,为转基因棉花生态安全性评价提供依据。【方法】2014年在吐鲁番市和2015年在库尔勒市,利用小区试验种植转基因棉花,翌年利用卡那霉素抗性检测结合分子检测的方法检测外源基因的漂移。【结果】在距离转基因棉花小区边缘1~120 m均能检测到含有外源基因Cry1Ac。SGK321种子漂移率最大漂移率是向南方向距离转基因棉花小区边缘1 m处,漂移率为5 %;GK19最大漂移率是向西方向达到了10.09 %。以方向因素和距离因素建立Cry1Ac基因漂移Logistic回归预测模型,方向和距离对种子漂移都有显著影响(P <0.05),但方向和距离两个因子并不能最终决定外源基因的漂移率。【结论】转基因棉花外源基因漂移率受方向和距离的影响,但最终决定外源基因的漂移率,并不是方向和距离联合作用的结果。     

dep1 improves rice grain yield and nitrogen use efficiency simultaneously by enhancing nitrogen and dry matter translocation

The rice cultivars carrying dep1 (dense and erect panicle 1) have the potential to achieve both high grain yield and high nitrogen use efficiency (NUE).  However, few studies have focused on the agronomic and physiological performance of those cultivars associated with high yield and high NUE under field conditions.  Therefore, we evaluated the yield performance and NUE of two near-isogenic lines (NILs) carrying DEP1 (NIL-DEP1) and dep1-1 (NIL-dep1) genes under the Nanjing 6 background at 0 and 120 kg N ha^–1.  Grain yield and NUE for grain production (NUEg) were 25.5 and 21.9% higher in NIL-dep1 compared to NIL-DEP1 averaged across N treatments and planting years, respectively.  The yield advantage of NIL-dep1 over NIL-DEP1 was mainly due to larger sink size (i.e., higher total spikelet number), grain-filling percentage, total dry matter production, and harvest index.  N utilization rather than N uptake contributed to the high yield of NIL-dep1.  Significantly higher NUEg in NIL-dep1 was associated with higher N and dry matter translocation efficiency, lower leaf and stem N concentration at maturity, and higher glutamine synthetase (GS) activity in leaves.  In conclusion, dep1 improved grain yield and NUE by increasing N and dry matter transport due to higher leaf GS activity under field conditions during the grain-filling period.     

Investigation of Aegilopsumbellulatafor stripe rust resistance,heading date,and the contents of iron,zinc, and gluten protein

Aegilops umbellulata (UU) is a wheat wild relative that has potential use in the genetic improvement of wheat.  In this study, 46 Ae. umbellulata accessions were investigated for stripe rust resistance, heading date (HD), and the contents of iron (Fe), zinc (Zn), and seed gluten proteins.  Forty-two of the accessions were classified as resistant to stripe rust, while the other four accessions were classified as susceptible to stripe rust in four environments.  The average HD of Ae. umbellulata was significantly longer than that of three common wheat cultivars (180.9 d vs. 137.0 d), with the exception of PI226500 (138.9 d).  The Ae. umbellulata accessions also showed high variability in Fe (69.74–348.09 mg kg^–1) and Zn (49.83–101.65 mg kg^–1) contents. Three accessions (viz., PI542362, PI542363, and PI554399) showed relatively higher Fe (230.96–348.09 mg kg^–1) and Zn (92.46–101.65 mg kg^–1) contents than the others.  The Fe content of Ae. umbellulata was similar to those of Ae. comosa and Ae. markgrafii but higher than those of Ae. tauschii and common wheat.  Aegilops umbellulata showed a higher Zn content than Ae. tauschii, Ae. comosa, and common wheat, but a lower content than Ae. markgrafii.  Furthermore, Ae. umbellulata had the highest proportion of γ-gliadin among all the species investigated (Ae. umbellulata vs. other species=mean 72.11% vs. 49.37%; range: 55.33–86.99% vs. 29.60–67.91%).  These results demonstrated that Ae. umbellulata exhibits great diversity in the investigated traits, so it can provide a potential gene pool for the genetic improvement of these traits in wheat.     

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.  In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.  At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated.  From 0.1 to 10 μmol L^–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal.  When the LPA concentration was 15 μmol L^–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst.  Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L^–1.  The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L^–1 to LPA-10 μmol L^–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L^–1 LPA-treated embryos compared with LPA-10 μmol L^–1 group (P<0.05).  The inner cell mass in 15 μmol L^–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal.  Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells.  The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.  They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L^–1, while 15 μmol L^–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells.  The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L^–1 group and LPA-10 μmol L^–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L^–1 group compared with LPA-10 μmol L^–1 group (P<0.05).  Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L^–1 concentration.  This study references the IVF embryo production and embryonic stem cell research of domestic animals.      

In vitro and in silico studies of salicylic acid on systemic induced resistance against bacterial leaf blight disease and enhancement of crop yield

Salicylic acid (SA) is an effective elicitor to promote plant defenses and growth.  This study aimed to investigate rice (Oryza sativa L.) cv. Khao Dawk Mali 105 treated with salicylic acid (SA)-Ricemate as an enhanced plant protection mechanism against bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo).  Results indicated that the use of SA-Ricemate as a foliar spray at concentrations of more than 100 mg L^–1 can reduce the severity of BLB  by 71%.  SA-Ricemate treatment also increased the hydrogen peroxide (H₂O₂) content of rice leaf tissues over untreated samples by 39–61%.  Malondialdehyde (MDA) in rice leaves treated with SA-Ricemate also showed an increase of 50–65% when comparing to non-treated samples.  The differential development of these defense compounds was faster and distinct when the SA-Ricemate-treated rice was infected with Xoo, indicating plant-induced resistance.  Besides, SA-Ricemate elicitor at a concentration of 50–250 mg L^–1 was correlated with a substantial increase in the accumulation of total chlorophyll content at 2.53–2.73 mg g^–1 of fresh weight which suggests that plant growth is activated by SA-Ricemate.  The catalase- and aldehyde dehydrogenase-binding sites were searched for using the CASTp server, and the findings were compared to the template.  Chemsketch was used to design and optimize SA, which was then docked to the catalase and aldehyde dehydrogenase-binding domains of the enzymes using the GOLD 3.0.1 Software.  SA is shown in several docked conformations with the enzymes catalase and aldehyde dehydrogenase.  All three catalase amino acids (GLN7, VAL27, and GLU38) were discovered to be involved in the creation of a strong hydrogen bond with SA when SA was present.  In this mechanism, the aldehyde dehydrogenase amino acids LYS5, HIS6, and ASP2 were all implicated, and these amino acids created strong hydrogen bonds with SA.  In field conditions, SA-Ricemate significantly reduced disease severity by 78% and the total grain yield was significantly increased which was an increase of plant height, tiller per hill, and panicle in three field trials during Aug–Nov 2017 and 2018.  Therefore, SA-Ricemate can be used as an alternative elicitor on replacing harmful pesticides to control BLB disease with a high potential of increasing rice defenses, growth, and yield components.     

Quantifying in situ N2 fluxes from an intensively managed calcareous soil using the 15N gas-flux method

Denitrification-induced nitrogen (N) losses from croplands may be greatly increased by intensive fertilization.  However, the accurate quantification of these losses is still challenging due to insufficient available in situ measurements of soil dinitrogen (N₂) emissions.  We carried out two one-week experiments in a maize–wheat cropping system with calcareous soil using the ¹⁵N gas-flux (¹⁵NGF) method to measure in situ N₂ fluxes following urea application.  Applications of ¹⁵N-labeled urea (99 atom%, 130–150 kg N ha⁻¹) were followed by irrigation on the 1st, 3rd, and 5th days after fertilization (DAF 1, 3, and 5, respectively).  The detection limits of the soil N₂ fluxes were 163–1 565, 81–485, and 54–281 μg N m⁻² h⁻¹ for the two-, four-, and six-hour static chamber enclosures, respectively.  The N₂ fluxes measured in 120 cases varied between 159 and 2 943 (811 on average) μg N m⁻² h⁻¹, which were higher than the detection limits, with the exception of only two cases.  The N₂ fluxes at DAF 3 were significantly higher (by nearly 80% (P<0.01)) than those at DAF 1 and 5 in the maize experiment, while there were no significant differences among the irrigation times in the wheat experiment.  The N₂ fluxes and the ratios of nitrous oxide (N₂O) to the N₂O plus N₂ fluxes following urea application to maize were approximately 65% and 11 times larger, respectively (P<0.01), than those following urea application to wheat.  Such differences could be mainly attributed to the higher soil water contents, temperatures, and availability of soil N substrates in the maize experiment than in the wheat experiment.  This study suggests that the ¹⁵NGF method is sensitive enough to measure in situ N₂ fluxes from intensively fertilized croplands with calcareous soils.     

Diamide derivatives containing a trifluoromethylpyridine skeleton: Design,synthesis, and insecticidal activity

Diamide derivatives are biologically active molecules that have been widely applied in recent years in research on pesticides, especially insecticides.  Using a simple and environmentally friendly scheme, a series of new diamide derivatives containing a trifluoromethylpyridine skeleton was designed, synthesized, and confirmed by ¹H, ¹⁹F and ¹³C NMR, and HR-MS.  Their insecticidal activities against Plutella xylostella and Helicoverpa armigera were measured and the relationship between structure and activity was investigated.  Eight of the title compounds (D2, D5, D10, D21, D28, D29, D30 and D33) showed 100% activity against P. xylostella at 500 mg L^–1.  One compound, D33, still showed 100% activity against P. xylostella at 100 mg L^–1 and had the lowest LC₅₀ (lethal concentration 50%, 3.7 mg L^–1) among the synthesized compounds.  Molecular docking analysis revealed that D33 could be thoroughly embedded in the active pocket of the ryanodine receptor via hydrogen bonding in a manner similar to the commercial insecticide chlorantraniliprole.     

Biosynthesis of artemisinic acid in engineered Saccharomyces cerevisiae and its attractiveness to the mirid bug Apolygus lucorum

Artemisia annua is an important preferred host of the mirid bug Apolygus lucorum in autumn.  Volatiles emitted from A. annua attract A. lucorum.  Volatile artemisinic acid of A. annua is a precursor of artemisinin that has been widely investigated in the Chinese herbal medicine field.  However, little is known at this point about the biological roles of artemisinic acid in regulating the behavioral trends of A. lucorum.  In this study, we collected volatiles from A. annua at the seedling stage by using headspace solid phase microextraction (HS-SPME).  Gas chromatography-mass spectrometry (GC-MS) analysis showed that approximately 11.03±6.00 and 238.25±121.67 ng h^–1 artemisinic acid were detected in volatile samples and milled samples, respectively.  Subsequently, a key gene for artemisinic acid synthesis, the cytochrome P450 gene cyp71av1, was expressed in engineered Saccharomyces cerevisiae to catalyze the production of artemisinic acid.  After the addition of exogenous artemisinic alcohol or artemisinic aldehyde, artemisinic acid was identified as the product of the expressed gene.  In electroantennogram (EAG) recordings, 3-day-old adult A. lucorum showed significant electrophysiological responses to artemisinic alcohol, artemisinic aldehyde and artemisinic acid.  Furthermore, 3-day-old female bugs were significantly attracted by artemisinic acid and artemisinic alcohol at a concentration of 10 mmol L^–1, whereas 3-day-old male bugs were attracted significantly by 10 mmol L^–1 artemisinic acid and artemisinic aldehyde.  We propose that artemisinic acid and its precursors could be used as potential attractant components for the design of novel integrated pest management strategies to control A. lucorum.     

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research.  However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits.  In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.  However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.  Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’.  The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L⁻¹ sucrose, 8 g L⁻¹ agar, 5 mg L⁻¹  6-benzylaminopurine (6-BA), 2 mg L⁻¹ thidiazuron (TDZ), and 1 mg L⁻¹ 1-naphthlcetic acid (NAA), pH 5.8).  A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L⁻¹) and kanamycin (15 mg L⁻¹).  We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material.  These findings make a significant contribution to apple biology research     

Grain yield,nitrogen use efficiency and physiological performance of indica/japonica hybrid rice in response to various nitrogen rates

Utilizing the heterosis of indica/japonica hybrid rice (IJHR) is an effective way to further increase rice grain yield.  Rational application of nitrogen (N) fertilizer plays a very important role in using the heterosis of IJHR to achieve its great yield potential.  However, the responses of the grain yield and N utilization of IJHR to N application rates and the underlying physiological mechanism remain elusive.  The purpose of this study was to clarify these issues.  Three rice cultivars currently used in rice production, an IJHR cultivar Yongyou 2640 (YY2640), a japonica cultivar Lianjing 7 (LJ-7) and an indica cultivar Yangdao 6 (YD-6), were grown in the field with six N rates (0, 100, 200, 300, 400, and 500 kg ha^–1) in 2018 and 2019.  The results showed that with the increase in N application rates, the grain yield of each test cultivar increased at first and then decreased, and the highest grain yield was at the N rate of 400 kg ha^–1 for YY2640, with a grain yield of 13.4 t ha^–1, and at 300 kg ha^–1 for LJ-7 and YD-6, with grain yields of 9.4–10.6 t ha^–1.  The grain yield and N use efficiency (NUE) of YY2640 were higher than those of LJ-7 or YD-6 at the same N rate, especially at the higher N rates.  When compared with LJ-7 or YD-6, YY2640 exhibited better physiological traits, including greater root oxidation activity and leaf photosynthetic rate, higher cytokinin content in the roots and leaves, and more remobilization of assimilates from the stem to the grain during grain filling.  The results suggest that IJHR could attain both higher grain yield and higher NUE than inbred rice at either low or high N application rates.  Improved shoot and root traits of the IJHR contribute to its higher grain yield and NUE, and a higher content of cytokinins in the IJHR plants plays a vital role in their responses to N application rates and also benefits other physiological processes.      

OsMas1, a novel maspardin protein gene,confers tolerance to salt and drought stresses by regulating ABA signaling in rice

Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality.  Therefore, developing crops with higher drought and salt tolerance is highly desirable.  This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice.  The OsMas1 protein was localized to the cytoplasm.  The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, and abscisic acid (ABA) treatments in rice.  The OsMas1 gene was introduced into the rice cultivar Zhonghua 11 (wild type, WT).  OsMas1-overexpression (OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance; in contrast, OsMas1-interference (OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses, compared with WT.  OsMas1-OE plants exhibited enhanced hypersensitivity, while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.  ABA, proline and K⁺ contents and superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and photosynthesis activities were significantly increased.  In contrast, malonaldehyde (MDA), hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^-·), and Na⁺ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.  Overexpression of OsMas1 up-regulated the genes involved in ABA signaling, proline biosynthesis, reactive oxygen species (ROS)-scavenging system, photosynthesis, and ion transport under salt and drought stresses.  Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice, which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.     

陆地棉盐胁迫应答基因GhPEAMT1的克隆及功能分析

【目的】磷酸乙醇胺N-甲基转移酶（phosphoethanolamine N-methyltransferse,PEAMT）是植物磷酸胆碱合成的关键酶,而磷酸胆碱是可以增强植物抗性的甘氨酸甜菜碱合成底物胆碱的前体。通过对陆地棉磷酸乙醇胺N-甲基转移酶基因（GhPEAMT1）的克隆、表达模式分析,以及功能验证,探究GhPEAMT1在陆地棉响应盐胁迫中的生物学功能,为棉花耐盐品种的选育提供基因资源。【方法】根据转录组测序数据分析,最终确定GhPEAMT1为耐盐候选基因;通过聚合酶链式反应（PCR）扩增目的基因;通过生物信息学分析基因结构特征、预测蛋白质相对分子质量以及进化关系;利用荧光定量PCR（qRT-PCR）分析耐盐棉花品种早熟长绒7号和感盐棉花品种南丹巴地大花在NaCl胁迫后不同时间点不同组织的表达特征;构建亚细胞定位载体,进行烟草的瞬时转化,确定蛋白质在细胞中的位置;构建基因超表达载体,通过花序浸染法转化拟南芥,分析转基因拟南芥种子在盐胁迫下的萌发率和转基因拟南芥主根长度;利用病毒诱导基因沉默技术（virus induced gene silencing,VIGS）对早熟长绒7号棉花品种进行目的基因沉默,提取棉花叶片的RNA进行实时荧光定量用以检测基因沉默效率,随后用40 g·L^-1的NaCl溶液的处理对照棉花植株（CK）、注射TRV2:00的棉花植株、GhPEAMT1A和GhPEAMT1D沉默成功的棉花植株,测定棉花叶片过氧化氢酶（catalase,CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase,GPX）的活性。【结果】克隆获得GhPEAMT1的2个同源基因GhPEAMT1A和GhPEAMT1D,2个基因的CDS全长分别为1 488和1 485 bp;构建进化树发现,GhPEAMT1与海岛棉同源基因的亲缘关系最近,而与其他物种相比,与可可同源性最高;盐胁迫条件下,GhPEAMT1A和GhPEAMT1D在耐盐品种早熟长绒7号叶片和根中的表达量显著高于感盐品种南丹巴地大花。亚细胞定位显示该基因位于细胞质中;超表达转基因拟南芥后,在100和150 mmol·L^-1 NaCl的MS培养基上,转基因拟南芥的种子萌发率显著高于野生型拟南芥。在50和100 mmol·L^-1 NaCl的MS培养基上,转基因拟南芥的主根长极显著高于野生型拟南芥的主根长;用40 g·L^-1的NaCl溶液处理24 h后,GhPEAMT1A和GhPEAMT1D沉默成功的棉花植株叶片比CK和注射TRV2:00的棉花植株的棉花叶片萎蔫严重,盐胁迫后,与注射空载棉花植株相比,注射TRV2::GhPEAMT1A棉花植株过氧化氢酶（CAT）活性降低、谷胱甘肽过氧化物酶（GPX）活性降低。【结论】GhPEAMT1可以增强拟南芥对盐胁迫的耐性,GhPEAMT1能够响应盐胁迫并起正向调控作用。     

Preparation and efficacy evaluation of Paenibacillus polymyxa KM2501-1 microbial organic fertilizer against root-knot nematodes

Root-knot nematodes (RKNs) cause huge yield losses to agricultural crops worldwide.  Meanwhile, livestock manure is often improperly managed by farmers, which leads to serious environmental pollution.  To resolve these two problems, this study developed a procedure for the conversion of chicken manure to organic fertilizer by larvae of Hermetia illucens L. and Bacillus subtilis BSF-CL.  Chicken manure organic fertilizer was then mixed thoroughly with Paenibacillus polymyxa KM2501-1 to a final concentration of 1.5×10⁸ CFU g^–1.  The efficacy of KM2501-1 microbial organic fertilizer in controlling root-knot nematodes was evaluated in pot and field experiments.  In pot experiments, applying KM2501-1 microbial organic fertilizer either as a base fertilizer or as a fumigant at the dose of 40 g/pot suppressed root-knot disease by 61.76 and 69.05% compared to the corresponding control treatments, respectively.  When applied as a fumigant at the dose of 1 kg m^–2 in field experiments, KM2501-1 microbial organic fertilizer enhanced the growth of tomato plants, suppressed root-knot disease by 49.97%, and reduced second stage juveniles of RKN in soil by 88.68%.  KM2501-1 microbial organic fertilizer controlled RKNs better than commercial bio-organic fertilizer in both pot and field experiments.  These results demonstrate that this co-conversion process efficiently transforms chicken manure into high value-added larvae biomass and KM2501-1 microbial organic fertilizer with potential application as a novel nematode control agent.     

Growth and nitrogen productivity of drip-irrigated winter wheat under different nitrogen fertigation strategies in the North China Plain

Excessive application of nitrogen (N) fertilizer is the main cause of N loss and poor use efficiency in winter wheat (Triticum aestivum L.) production in the North China Plain (NCP).  Drip fertigation is considered to be an effective method for improving N use efficiency and reducing losses, while the performance of drip fertigation in winter wheat is limited by poor N scheduling.  A two-year field experiment was conducted to evaluate the growth, development and yield of drip-fertigated winter wheat under different split urea (46% N, 240 kg ha^–1) applications.  The six treatments consisted of five fertigation N application scheduling programs and one slow-release fertilizer (SRF) application.  The five N scheduling treatments were N0–100 (0% at sowing and 100% at jointing/booting), N25–75 (25% at sowing and 75% at jointing and booting), N50–50 (50% at sowing and 50% at jointing/booting), N75–25 (75% at sowing and 25 at jointing/booting), and N100–0 (100% at sowing and 0% at jointing/booting).  The SRF (43% N, 240 kg ha^–1) was only used as fertilizer at sowing.  Split N application significantly (P<0.05) affected wheat grain yield, yield components, aboveground biomass (ABM), water use efficiency (WUE) and nitrogen partial factor productivity (NPFP).  The N50–50 and SRF treatments respectively had the highest yield (8.84 and 8.85 t ha^–1), ABM (20.67 and 20.83 t ha^–1), WUE (2.28 and 2.17 kg m^–3) and NPFP (36.82 and 36.88 kg kg^–1).  This work provided substantial evidence that urea-N applied in equal splits between basal and topdressing doses compete economically with the highly expensive SRF for fertilization of winter wheat crops.  Although the single-dose SRF could reduce labor costs involved with the traditional method of manual spreading, the drip fertigation system used in this study with the N50–50 treatment provides an option for farmers to maintain wheat production in the NCP.     

Reduction of N2O emissions by DMPP depends on the interactions of nitrogen sources (digestate vs. urea) with soil properties

The inhibition of nitrification by mixing nitrification inhibitors (NI) with fertilizers is emerging as an effective method to reduce fertilizer-induced nitrous oxide (N₂O) emissions.  The additive 3,4-dimethylpyrazole phosphate (DMPP) apparently inhibits ammonia oxidizing bacteria (AOB) more than ammonia oxidizing archaea (AOA), which dominate the nitrification in alkaline and acid soil, respectively.  However, the efficacy of DMPP in terms of nitrogen sources interacting with soil properties remains unclear.  We therefore conducted a microcosm experiment using three typical Chinese agricultural soils with contrasting pH values (fluvo-aquic soil, black soil and red soil), which were fertilized with either digestate or urea in conjunction with a range of DMPP concentrations.  In the alkaline fluvo-aquic soil, fertilization with either urea or digestate induced a peak in N₂O emission (60 μg N kg^–1 d^–1) coinciding with the rapid nitrification within 3 d following fertilization.  DMPP almost eliminated this peak in N₂O emission, reducing it by nearly 90%, despite the fact that the nitrification rate was only reduced by 50%.  In the acid black soil, only the digestate induced an N₂O emission that increased gradually, reaching its maximum (20 μg N kg^–1 d^–1) after 5–7 d.  The nitrification rate and N₂O emission were both marginally reduced by DMPP in the black soil, and the N₂O yield (N₂O-N per NO₂^–+NO₃^–-N produced) was exceptionally high at 3.5%, suggesting that the digestate induced heterotrophic denitrification.  In the acid red soil, the N₂O emission spiked in the digestate and urea treatments at 50 and 10 μg N kg^–1 d^–1, respectively, and DMPP reduced the rates substantially by nearly 70%.  Compared with 0.5% DMPP, the higher concentrations of DMPP (1.0 to 1.5%) did not exert a significantly (P<0.05) better inhibition effect on the N₂O emissions in these soils (either with digestate or urea).  This study highlights the importance of matching the nitrogen sources, soil properties and NIs to achieve a high efficiency of N₂O emission reduction.     

Cotton maturity and responses to harvest aids following chemical topping with mepiquat chloride during bloom period

Early maturity, complete defoliation and boll opening are essential for the efficient machine harvesting of cotton.  Chemical topping, involving one extra application of mepiquat chloride (MC) in addition to its traditional multiple-application strategy, may be able to replace manual topping.  However, it is not known whether this chemical topping technique will influence maturity or cotton responses to harvest aids.  In this 2-yr field study, we determined the effects of the timing of chemical topping using various rates of MC on boll opening percentage (BOP) before application of harvest aids (50% thidiazuron·ethephon suspension concentrate, referred to as TE), and the defoliation percentage (DP) and BOP 14 days after TE application.  The results indicated that late chemical topping (near the physiological cutout, when the nodes above white flower is equal to 5.0) significantly decreased BOP before TE by 5.9–11.2% compared with early (at peak bloom) or middle (seven days after peak bloom) treatments in 2019, which was a relatively normal year based on crop condition.  Also, a high MC rate (270 g ha^–1) showed a significantly lower (22.0%) BOP before TE than low (90 g ha^–1) or medium (180 g ha^–1) rates.  In 2020, which was characterized by stronger vegetative growth in the late season, the late chemical topping reduced the number of leaves before TE application relative to early or middle treatments, but had lower DP (23.2–27.2%) 14 days after TE application.  The high MC rate showed a leaf count before TE application that was similar to the low and medium rates, but it showed the most leaves after TE and much lower (15.0–21.7%) DP in 2020.  These results suggest that late timing of chemical topping and a high MC rate decreased the sensitivity of leaves to harvest aids.  Further analysis indicated that the late chemical topping mainly affected the leaf drop from the mainstem and fruiting branches where the late regrowth occurred, and the high MC rate reduced leaf shedding from these parts and also from the vegetative branches.  In conclusion, chemical topping with MC during the bloom period affected cotton maturity and responses to harvest aids in different ways according to the crop condition.  To avoid the risks of delayed maturity and poor defoliation after the application of harvest aids, chemical topping should not be performed too late (i.e., near the physiological cutout) by using MC at more than 180 g ha^–1.  The optimum timing of chemical topping probably varies from peak bloom to around seven days later, and the safest MC rates for chemical topping should be less than 180 g ha^–1.     

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.